PECTINS AND PECTINASES
Progress in Biotechnology ~~
Volume 1 New Approaches t o Research on Cereal Carbohydrates (Hill and Munck, Editors) Volume 2 Biology of Anaerobic Bacteria (Dubourguier et al., Editors) Volume 3 Modifications and Applications of Industrial Polysaccharides (Yalpani, Editor) Volume 4 lnterbiotech '87. Enzyme Technologies (Blaiej and Zemek, Editors) Volume 5 In Vitro Immunization in Hybridoma Technology (Borrebaeck, Editor) Volume 6 lnterbiotech '89. Mathematical Modelling in Biotechnology (Blafej and Ottova, Editors) Volume 7 Xylans and Xylanases (Visser et al., Editors) Volume 8 Biocatalysis i n Non-Conventional Media (Tramper et al., Editors) Volume 9 ECB6: Proceedings of the 6th European Congress on Biotechnology (Alberghina et al., Editors) Volume 10 Carbohydrate Bioengineering (Petersen et al., Editors) Volume 11 Immobilized Cells: Basics and Applications (Wijffels et al., Editors) Volume 12 Enzymes for Carbohydrate Engineering (Kwan-Hwa Park et al., Editors) Volume 13 High Pressure Bioscience and Biotechnology (Hayashi and Balny, Editors) Volume 14 Pectins and Pectinases (Visser and Voragen, Editors)
Progress in Biotechnology 14
PECTINS AND PECTINASES Proceedings of an International Symposium, Wageningen, The Netherlands, December 3-7, 1995
Edited by J. Visser Section of Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, Wageningen, The Netherlands
A.G.J. Voragen Department of Food Science, Section of Food Chemistry and Food Microbiology, Wageningen Agricultural University, Wageningen, The Netherlands
ELSEVIER Amsterdam - Lausanne - New York - Oxford - Shannon Tokyo 1996
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Published by: Elsevier Science B.V. P.O. Box 21 1 1000 AE Amsterdam The Netherlands
Library o f Congress Cataloging-in-Pub
ication Data
P e c t i n e s and pec inases : p r o c e e d i n g s o f an i n e r n a t i o n a l svmDosium. . . Wageningen, t h e N e t h e r l a n d s , December 3-7, 1995 / e d i t e d by J. V i s s e r . A . G . J . Voragen. rn -- (~.P .r nI=.n r o a a i, ,n, h i n t o r h n n l sn vnyQy 8 ,. l a ) rn . r-.... Includes b i b l i o g r a p h i c a l references. ISBN 0-444-82330-1 ( a l k . p a p e r ) 1 . Pectin--Congresses. 2. Pectinase--Congresses. I.V i s s e r , J. 11. Voragen. A . G. J. 111. S e r i e s . TP248.P4P43 1996 664'.25--d~20 96-36458 CIP
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ISBN 0-444-82330-1 01996 Elsevier Science B.V. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher, Elsevier Science B.V., Permissions Department, P.O. Box 521,1000 A M Amsterdam, The Netherlands. No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances i n the medical sciences, the Publisher recommends th'at independent verification of diagnoses and drug dosages should be made. Special regulations for readers in the USA - This publication has been registered with the Copyright Clearance Center Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01293, USA. Information can be obtained from the CCC about conditions under which photocopies of parts of this publication may be made in the USA. All other copyright questions, including photocopying outside the USA should be referred to the copyright owner, Elsevier Science B.V., unless otherwise specified. This book is printed on acid-free paper. Printed in the Netherlands
PREFACE The Intemational Symposium on Pectins and Pectinases held in Wageningen, The Netherlands from December 3-7, 1995 was a very stimulating and succesful meeting. Attended by 190 participants representing 25 different countries, the Symposium provided the platform for many multidisciplinary interactions. Scientists working in applied fields such as pectin manufacturing, enzyme production and food processing often met for the first time with molecular biologists and X-ray crystallographers who have in recent years started to discover how interesting pectin degrading and modifying enzymes are and how complex the substrate is on which these enzymes act. Progress in pectin and pectinase research has been most prominent in two areas over the past 5 years viz in analyzing and elucidating the complex chemical structure of pectin and in unraveling the mode of action and the structure of various pectin degrading enzymes as well as in cloning the corresponding genes. It also became evident that we still know far too little about the biosynthesis of pectin and the role of pectin in plant cell wall architecture. The Symposium was further highlighted by an initiative of IPPA to honour Prof. Walter Pilnik for all his activities in the past in the interest of pectin research and of the association of pectin producing industries. This was done by awarding a talented, junior scientist in the field to present the so-called Walter Pilnik Lecture. This award, which will be given every second year, was given to Dr Maureen McCann. The editors of this volume hope that the Proceedings of this Symposium will not only reflect the present status of research in this area but that it will also remain a useful reference book.
Wageningen, July 11, 1996. J. Visser A.G.J. Voragen
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O R G A N I Z I N G COMMITTEE A. G.J. Voragen
(chairman)
M.A. Kusters-van Someren
(secretary) J. Visser
(treasurer)
Dept of Food Science Section of Food Chemistry and -Microbiology Wageningen Agricultural University Section of Molecular Genetics of Industrial Microorganisms Wageningen Agricultural University Section of Molecular Genetics of Industrial Microorganisms Wageningen Agricultural University
G. Beldman
Dept of Food Science Section of Food Chemistry and -Microbiology Wageningen Agricultural University
J. Benen
Section of Molecular Genetics of Industrial Microorganisms Wageningen Agricultural University
H.A. Schols
Dept of Food Science Section of Food Chemistry and -Microbiology Wageningen Agricultural University
SCIENTIFIC ADVISORY COMMITTEE W. Pilnik
Wageningen, The Netherlands
(honorary chairman) P. Albersheim A.B. Bennett H.P. Heldt-Hansen J. Jenkins M. Rinaudo J. Robert-Baudouy K. Roberts J. SOderberg J.F. Thibault A.J. Vroemen
Athens, USA Davis, USA Copenhagen, Denmark Reading, UK Grenoble, France Villeurbanne, France Norwich, UK Copenhagen, Denmark Nantes, France Seclin, France
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ACKN OW LED G EMENT S The Organizing Committee of the International Symposium on Pectins and Pectinases acknowledges the following organizations for their support. Without their contribution this Symposium would not have been possible. Citrus Colloids Ltd. Copenhagen Pectin A/S Cooperatieve Suiker Unie U.A. Dalgety S.F Ltd - Food Technology Center Danisco Ingredients Dionex B. V. Gist-brocades France S.A. Groupe Danone Herbstreith & Fox K.G. Hercules European Research Center B. V. IPPA Koninklijke Nederlandse Chemische Vereniging Landbouwuniversiteit Wageningen Cluster Biomoleculaire Wetenschappen Nederlandse Unilever Bedrijven B. V. Novo Nordisk A/S Obipektin A. G. Quest International Systems Bio-Industries VLAG The Graduate School for Advanced Studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences -
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xi CONTENTS.
STRUCTURE, PHYSICAL AND CHEMICAL PROPERTIES OF PECTINS
Complex pectins: Structure elucidation using enzymes. H.A. Schols and A.G.J. Voragen.............................................................................................
3
Physicochemical properties of pectins in solution and gel states. M. Rinaudo...........................................................................................................................
21
Interactions of pectins with multivalent cations: Phase diagrams and structural aspects. M.A. K Axelos, C. Garniel; C.M.G.C. Retiard and J. -F: Thibault ..........................................
35
An hypothesis: The same six polysaccharides are components of the primary cell walls of
all higher plants. f? Albersheim, A. G. Darvill, M.A. 0 'Neill, H.A. Schols and A. G.J. Voragen ........................
47
Acetylation of rhamnogalacturonan I and homogalacturonan: Theoretical calculations. M. Kouwijzel; H. Schols and S. Pkrez ...................................................................................
57
The pectic polysaccharide rhamnogalacturonan I1 is a major component of the polysaccharides present in fruit-derived products. I? Pellerin, 7: Doco, S. Kdal, I? Williamsand J-M. Brilloiret ...............................................
67
Partial characterization of xylogalacturonans from cell walls of ripe watermelon fruit: inhibition of endopolygalacturonase activity by xylosylation. L. Yu and A.J. Mort ..............................................................................................................
79
PECTIN BIOSYNTHESIS AND BIOLOGICAL EFFECTS OF (DEGRADED) PECTIN
Plant cell wall architecture: the role of pectins. M. C. McCann and K. Roberts ..............................................................................................
91
Cell free synthesis of the pectic polysaccharide homogalacturonan. D. Mohnen, R.L. Doong, K. Liljebjelke, G. Frnlish and J. Chan ........................................
109
Biosynthesis in vitro of pectic ( 1 + 4)-R-D-galactan. L.S. Brickell at1dJ.S.G. Reid ..............................................................................................
127
Cell wall pectins: From immunochemical characterization to biological activity. P Van Cutsem and J. Messiaen ..........................................................................................
13 5
xii
Methyl-esterification, de-esterification and gelation of pectins in the primary cell wall. R. Goldberg, C. Morvan, A. Jairneaii and M. C. Jarvis .......................................................
15 1
Contribution of pectins on health care.
H. Yamada .........................................................................................................................
173
The role of polygalacturonase, PGIP and pectin oligomers in hngal infection. F: Cervone, G. de Lorenzo, B. Aracri, D. Bellincampi, C. Caprari, A.J. Clark, A. Desiderio, A. Devoto, F: Leckie, B. Mattei, L. Nirss and G. Salvi ...................................
191
Biologically active pectin oligomers in ripening tomato fruits.
E. Melotto, L. C. Greve and J.M. Labavitch ........................................................................
207
IDENTIFICATION, MODE OF ACTION AND 3-D STRUCTURE OF PECTINASES Kinetics and mode of action of Aspergilhrs tiiger polygalacturonases. J.A.E. Benen, H. C.M. Kestel; L. Parenicovci and J. Msser ..................................................
22 1
New enzymes active towards pectic structures. G. Beldman, M. Muttel; M.J.F:Searle-van Leeintien, L.A.M. van den Broek, H.A. Schols and A. G.J. Voragen .........................................................................................
23 1
The Bsubunit of tomato fruit polygalacturonase isoenzyme 1 defines a new class of plant cell proteins involved in pectin metabolism: AroGPs (Aromatic amino acid rich Glyco Proteins). D. DellaPenna, C. Watson,Jl? Liir and D. Schirchman......................................................
247
Characterisation of RG degradation products of new RGases using RG-rhamnohydrolaseand RG-galacturonohydrolase. M. Muttel; C.M. G. C. Renard, G. Beldnian, H.A. Schols and A. G.J. Voragen ......................
263
The effect of glycosylation of endopolygalacturonasesand polygalacturonase inhibiting proteins on the production of oligogalacturonides. C. W! Bergmann, B. Cook, A.G. Darvill, I? Alhersheim, D. Bellincampi and C. Caprari .....275 Envinia pectate lyase differences revealed by action pattern analyses. S.Bartling, P Derkx, C. Wegener and 0. Olsen .................................................................
283
Functional implications of the three-dimensional structures of pectate lyases. F: Jurnak, N. Kita, M. Garrett, S.E. Heffron, R. Scavetta, C. Boyd and N. Keen .................295
xiii
MOLECULAR GENETICS AND REGULATION OF PECTINASE BIOSYNTHESIS IN SAPROPHYTIC AND PHYTOPATHOGENIC MICROBIAL SYSTEMS
Regulation of pectinase biosynthesis in Eminia chrysanthemi. N. Hugouvieux-Cotte-Pattat, S. Reverchon, W Nassel; G. Condemine and J. Robert-Baudouy .............................................................................................................
311
Molecular genetic and biochemical aspects of pectin degradation in Aspergillus. J. Benen, L. Parenicova,M. Kusters-van Someren, H. Kester and J. Ksser ........................
33 1
Expression of polygalacturonase and pectinesterase in normal and transgenic tomatoes. G. nicker andJ. Zhang ......................................................................................................
347
Role of pectin methylesterase in tomato fruit ripening and quality attributes of processed tomato juice. A.K. Handa, D.M. Tiemann, K.K. Mishra, B. R. Thakur and R.K. Singh .............................
355
Molecular characterization and expression of CoIletoh.ichum lindemiithianum genes encoding endopolygalacturonase. S. Centis, K Hugouvieux, J. Foiirniec C. Lnfiite, M. 7: Esquerre-Eigaye and B. Dirmas ...........................................................................................................................
369
Cloning of genes encoding pectin-degrading enzymes in Azospirillum irakense. M.A. Bekri, J. Desail; L. van Lonimel andJ. Vnriderleyden ................................................
377
Transgenic potatoes that express an Erwinia pectate lyase isoenzyme.
C. Wegenel; S. Bcrrtling, J. Webel; S. Hoffnintiri-Beiiiiirig and 0. Olsen ..............................
385
Pectins and pectolytic enzymes in relation to development and processing of green beans (Phaseolus vulgaris L.). K. Recourt, T Stolle-Sniits, J.M. Lnats, J. G. Beekhriizen, C.E.M. Ehbelaal; A.G.J. Voragen, H.J. Wichers and C. van Dijk ....................................................................
399
APPLICATIONS. A) DEVELOPMENTS IN PECTIN MANUFACTURING AND APPLICATIONS
Rheological methods to characterize pectins in solutions and gels.
H.-U. Endress, C. Doschl-Volle andK. Dengler .................................................................
407
Effects of extrusion-cooking on pectin-rich materials. J.-I? Thibault,M.-C. Ralet, M.A. I.:Axelos and G. Della VXle ............................................
425
xiv
Pectin degradation in UF-membrane reactors with commercial pectinases. C. Dinnella, A. Stagni, G. Lanzarini, E Alfaiii, M. Cantarella and A. Gallijiuoco...............439
B) APPLICATION OF PECTINASES IN BEVERAGE, FOOD, FEED AND NOVEL TECHNOLOGIES
Application of pectinases in beverages. C. Grassin andP Fauquembergzie .....................................................................................
453
Application of tailormade pectinases. H.P Heldt-Hansen, L. !F Kofod, G. Biidolfen, PM. Nielsen, S. Hiittel and i? Bladt ............ 463 Cations increase activity and enhance permeation of pectinesterase in ultrafiltration.
L. Wicker............................................................................................................................
475
Production, characterization and application of rhamnogalacturonase. H. Hennink, H. Stam andM.G. van Oort............................................................................
485
Effects of a new canning process on cell wall pectic substances, calcium retention and texture of canned carrots. H. Siliha, K Jahn and K. Gierschner.................................................................................
495
SHORT COMMUNICATIONSBASED ON POSTER PRESENTATIONS. STRUCTURE, PHYSICAL AND CHEMICAL PROPERTIES OF PECTINS
Isolation and sequential extraction of cell wall polysaccharides from soy meal. M.M.H. Huisman, H.A. Schols and A. G.J. Voragen............................................................
5 11
Modelling a pentasaccharide fragment of rhamnogalacturonan I. M. Broadhurst, S. Cros, R. Hofsniann, W Mackir and S. Perez ..........................................
5 17
Influence of some cations on the reaction of apple pectin with ammonia in homogeneous media. P Denev and Chl: Kratchatiov ...........................................................................................
527
Heavy metals binding by pectins: selectivity, quantification and characterisation. KM. Dronnet, C.M. G.C. Renard, M.A. !L Axelos and J. -E Thibault ....................................
535
Quantitative Raman spectroscopy. Prediction of the degree of esterification in pectins. S.B. Engelsen and L. Norgaard ..........................................................................................
54 1
xv
Polysaccharides from Chorisia speciosu St. Hill. E.B. Beleski-Carneiro, M.R. Sierakowski,J.L.M.S. Ganter;S,F: Zawadzki-Baggio and F: Reicher ....................................................................................................................
549
Rigid and flexible pectic polymers in onion cell walls. M.-A. Ha, B. W Evans, D.C. Apperley at1dM.C. Jarvis ......................................................
561
Changes in pectic polysaccharides during elaboration of table olives. A. Heredia, R. GuillCn, C. Sanchez, A. JimCnez andJ. Fernandez-Bola2os ........................
569
Pectins from different tissue zones of apple: characterisation and enzymatic hydrolysis.
P Massiot. A. Baron at1dJ.E Drilleau ...............................................................................
577
Investigations of the influence of various cations on the rheological properties of high-esterified pectin gels. S. Neidhart, C. Hannak and K. Gierschner.........................................................................
5 83
Changes in molecular weight and carbohydrate composition of cell wall polyuronide and hemicellulose during ripening in strawberry fruit. I.:Nogata, K. Yoza, K. Kusirnioto and H. Ohf a ...................................................................
59 1
Autoclave extraction of sugar beet pulp yields gel-forming pectic hairy regions. A. Oosterveld, G. Beldman, J.M. de Bruijii at1dA.G.J. Voragen .........................................
597
Pectins in mild alkaline conditions: l3-elimination and kinetics of demethylation. C.M.G.C. Renard and J. -I? Thibairlt ..................................................................................
603
Potentiometric titration of poly(a-D)galacturonic acid. D. Rudan-Tasi? and C. Klojirtar ........................................................................................
609
Structural studies of a pectic polysaccharide from Plantago major L. A.B. Samuelsen, E.H. Cohen, B.S. Pairlsen and J.K. Wold..................................................
6 19
Structural characterization of a novel rhamnogalacturonan I1 with macrophage Fc receptor expression enhancing activity from the leaves of Patiax ginseng C.A. Meyer. K.-S. Shin, H. Kiyohara*T Matsunloto atid H. Yaniada......................................................
623
Structural features of pectic polysaccharides of red beet (Beta vzrlgaris conditiva). G.R. Strasser;D.E. Wechsler and R. Aniado .......................................................................
63 1
Isolation and physicochemical characterisation of xylose-rich pectic polysaccharides from wheat straw. R. Sun, J.M. Lawther and WB. Batiks ................................................................................
637
Chemical synthesis of oligosaccharides related to arabinogalactan-proteins(AGPs). J.E Valdor and W Mackie ..................................................................................................
645
xvi Structural features of pectic substances during growth and ripening of apples. D.E. Wechslel: G.R. Slrasser and R. Aniado .......................................................................
65 1
PECTIN BIOSYNTHESIS AND BIOLOGICAL EFFECTS OF (DEGRADED) PECTIN Metabolism of pectin in the gastrointestinal tract. G. Dongowski and H. Anger ..............................................................................................
659
Cell wall properties of transgenic tobacco plants that express a yeast derived acid invertase in their vacuole. S. Hoffmann-Benning, R. Ehrwald, L. Willniitzeratid J. Fisahn .........................................
667
Pectic polysaccharide from roots of Glycyrrhiza iiralensis:Possible contribution of neutral oligosaccharide in the galacturonase-resistant region to anti-complementary and mitogenic activities. H. Kiyohara, N. Takenioto, J. -E Zhao, H. Kmuaniiira and H. Yantada................................
673
Immunologically active polysaccharides from cell suspension of Heliarithiis 1805. M. Kratchnnova, M. Zlieva, E. Pnvlova, A . Pavlov and N. Markova ...................................
679
Pectins and pectinases in stem rust-infected wheat. M. Mierair, B. GraeJtier;A . J. Mort atid B.M. Moerschhacher...........................................
687
Bioactive fragments from pea pectin. 0. Zabotiti, N. Ibragimova, D. Ayiipova, 0. Gzirjatm), K Lozovaya, G. Beldmati and A. Voragen .................................................................................................................
693
atitniiis
IDENTIFICATION, MODE OF ACTION AND 3-D STRUCTURE OF PECTINASES Stereochemistry of hydrolysis of glycosidic linkage by three Aspergillus polygalacturonases. 19 Bieb, J.A.E. Benen, H.C.M. Kester; K. Heinrichova andJ b s e r ..................................
705
Pectin methyltransferases from suspension-cultured cells and seedlings of flax (Linum usitatissimiim L,), l? Bruyant, M.I? Briiymt-Vannier; 7: Boirrlard, A . Gnirdi~iet-Schaiininti, B. Thoiron atid C. Morvan .................................................................................................
711
Pectinases from Rhizopzis sp. efficient in enhancing the hydrolyzation of raw cassava starch: Purification and characterization. L. Chitradon, t! Pooiipairoj, t! Mahakhan, b! Kitpreechavatiichatid N. Lotong.. ...............71 5
xvii
Isolation, characterization and immuno localization of orange fruit acetyl esterase. T.M.I. E. Christensen, J.E. Nielseri and J.D. Mikkelseri .......................................................
723
Enzyme-mediated substrate immunolocalization of polygalacturonic acid within barley epidermal cell walls utilizing endopolygalacturonase of Cochliobobrs sativus and a monoclonal antibody specific for the enzyme. R.l? Clay, C. W Bergmarin and M.S. Firller ........................................................................
13 1
Influence of glucose and polygalacturonic acid on the synthesis and activity of the polygalacturonase from the yeast strain SCPP. A. Gainvors and A. Belarbi ................................................................................................
739
Pectin lyase from Fusariiim oxysporiini f. sp. radicis lycopersici: purification and characterization. M.A. Guevara, M. T Gonzdez-fat;ir andP Estivez ............................................................
747
Enzymic release of ferulic acid from sugar beet pulp using a specific esterase from Aspergillus niger. PA. Kroon, C.B. FaiiJds9C. BredJon orid G. WilJianison...................................................
761
Characterization of some endo-polygalacturonases from Sclei'otinia sclerotiorum. M.B. Martel, R. Letoltblori andM. Fivre ...........................................................................
769
Analysis of the interaction between PGIP from Phaseohis viilgaris L. and fungal endopolygalacturonases using biosensor technology B. Mattei, G. Salvi, C. Caprari, G. De Lorenzo, L? Crescenzi and E Cervone .....................
775
Rhamnogalacturonan a-L-rhamnopyranosyl-( 1 -+4)-a-D-galactopyranosyluronide lyase, a new enzyme able to cleave RG regions of pectin. M. Mutter; I.J. Colqiihoiiii, G. Reldninir, H.A. Schols and A.G.J. Vorngen..........................
783
Purification and characterisation of galactose-induced pectinases from the exo-l mutant strain of Neiirospora crassci. L.B. Crotti, J.A. Jorge, H.E Ereriri aridM.L. TM. Polizeli .................................................
787
Acetyl esterases ofdspergilliisn i p : purification and mode of action on pectins. M.J.E Searle-van Leeimen, J . 2 Mncken, D. Schipper; A.G.J. Vorngenand G. Beldman ........................................................................................................................
793
A polygalacturonase inhibitor of Dieflenhachia niacirlata. A . Chitre arid N. I.:Shastri ..................................................................................................
799
Multiple forms of carrot exopolygalacturonase.
E. Stratilova, M. Dziirova and D. Mi.slovic.?ova...................................................................
807
xviii
MOLECULAR GENETICS AND REGULATION OF PECTINASE BIOSYNTHESIS IN SAPROPHYTIC AND PHYTOPATHOGENIC MICROBIAL SYSTEMS Primary structure and characterization of an exo-polygalacturonase from Aspergillus tubingensis. H.C.M. Kestec M.A. Kirsters-van Someren, K Miiller and J. Ksser ....................................
8 17
pgaE from Aspergillus niger encodes a fourth polygalacturonase. Molecular cloning and biochemical characterisation of the gene product. L. Parenlicova, J.A.E. Benen, H.C.M. Kester andJ. Ksser ................................................
825
Identification of a seventh endo-pectate lyase of the phytopathogenic bacterium Erwinia chrysanthemi. C. Pissavin, J. Robert-Baiidoiiyand N. Hiigorivieiix-Cotte-Pattat.......................................
83 1
Synthesis of new methyl esters of 3-deoxy-D-erythro-2-hexulosonicacid (KDG) analogs, inducers of the expression of pectinase genes in bacteria Erwinia chrysanthemi. F: Alessi, G. Condemine,A. Doirtheaii, J. Robert-Baiidoiiy and D. Anker ...........................
845
Pectin methylesterase B of Erwinia chrysanthemi, the first pectinase characterised as a membrane lipoprotein. FE. Shevchik, G. Condeniine,N. Hirgoii~iieiix-Cotte-Pattatand .J. Robert-Baiidoiry ........... 837 Production of pectinases from Rhizopris sp. in solid substrates. L. Chitradon,I? Mahakhan, I? Poonpairoj, I? Kitpreechavanich and N. Lotong .................853 Endo-polygalacturonase of the yeast Klriyveromyce.~rnarxianirs is constitutive, highly active on native pectin and is the main extracellular protein. R.F: Schwan, R.M. Cooper andA.E. Wheals ......................................................................
861
Polygalacturonase and pectinmethylesterase activities during growth of Helianthirs annirirs 1805 cell suspension. M. Ilieva, M. Kratchanova, E, Pavlovc~,T Diniovcr and A. I’avlov .....................................
869
Differential expression of Erwinia chryscrnthenii strain 3937 pectate lyases in pathogenesis of African violets: importance of low iron environmental conditions. C. Mascbirx, N. Hiigoiivieirx-Cotte-Pattat nnd D. Expert...................................................
875
Regulation of polygalacturonases in two isolates of Fiisariiini oxysporiim fsp. radicis Iycopersici (FORL). B. Patiiio, M.L. Posada, M. 7: Gonzdez-JaPn, M.J. Martinez and C. Vazqirez ....................
88 1
Endo-pectinase production by intraspecific hybrids of Aspergillus sp. CH-Y- 1043 obtained by protoplast fusion. S.Solis, E. Flores-Sanchez and C. Hiiitrdn ........................................................................
893
xix
Candida boidinii - a new found producer of pectic enzymes complex. E. Stratilova, E. Breierova and R. Vadkertiovci...................................................................
899
Cloning, sequence and expression of the gene coding for rhamnogalacturonase (RHG) of Aspergillus aculeatus: a novel pectinolytic enzyme. M.E.G. Suykerbuyk, PJ. Schaap, H. Stant, K Musters and J. Ksser ..................................
907
Pectinase secretion by Aspergillus FP- 180 and Aspergillus niger N-402 growing under stress induced by the pH of culture medium. B.A. Trejo-Aguilal;J. h s e r and G. Agiilar 0 ...................................................................
9 15
Selection of a constitutive hyper-pectinolytic mutant from a Penicillium strain. N. HadjTaieb, M. Ayadi and A. Gargouri ..........................................................................
921
APPLICATIONS. A) DEVELOPMENTS IN PECTIN MANUFACTURING AND APPLICATIONS
Production of hypocaloric jellies of grape juice with sunflower pectin. M.L. AlarcLTo-Silva, H. Gil Azitiheira, M.I.N. Jatnrcirio, M. C.A. Leitco and TC. Curado .......................................................................................................................
931
Influence of microwave pretreatment of fresh orange peels on pectin extraction. M. Kratchanova, E. Pavlova, I. Patrchev and Chi: Kratchatiov ..........................................
94 1
Properties of pectinesterase from Penicilliimifellutatiuni Biourge and new developments in pectin applications. KL. Aizenberg, S.A. Syrchin, S.A. Sedina K N . Vasil 'ev, L.N. Shitikareiiko, RI. Demchenko and 19 N. Htte ............................................................................................
947
B) APPLICATION OF PECTINASES IN BEVERAGE, FOOD, FEED AND NOVEL TECHNOLOGIES
Enzymatic maceration of apple parenchyma: modelling of the degradation. A. Baron, R Massiot, C. Ella Missatig and J.E Drilleaii ....................................................
957
Enzymatic treatment in the extraction of cold-pressed lemon peel oils. L. Coll, D. Saiira, J.M. Ros, M. Moliner and J. Laencitia...................................................
963
Immobilized pectinase efficiency in the depolymerisation of pectin in a model solution and apple juice. C. Dinnella, A. Stagni, G. Lanzarini aiidM. Lniis ..............................................................
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Pectinases in wood debarking. M. Ratto and L. Piikari ......................................................................................................
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Oligouronides of pectins in membrane reactor by enzymatic degradation of pectins from Citrus peel. A preliminary study. J.M. Ros, D. Saura, L. Coll, M. Molirier mid J. Laenciim. ......................... .........................
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STRUCTURE, PHYSICAL AND CHEMICAL PROPERTIES OF PECTINS
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J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All fights reserved.
Complex Pectins: Structure elucidation using enzymes H.A. Schols and A.G.J. Voragen Wageningen Agricultural University, Department of Food Science, Bomenweg 2, 6703 HD Wageningen, The Netherlands
Abstract
A pectic fraction, retained by ultrafiltration of the juice from enzyme treated apple tissue and resistant to further enzymic degradation, was isolated and characterized using chemical and enzymic methods. The fraction was termed MHR (modified hairy regions) and this fraction was characterized by a high arabinose content, next to a high rhamnose to galacturonic acid ratio and a high acetyl content and smaller proportions of xylose and galactose. Rhamnogalacturonase (RGase), an enzyme able to hydrolyze galacturonic acid-(1-,2)rhamnosyl linkages within the rhamnogalacturonan backbone of MHR was used to obtain both oligomeric and polymeric degradation products. These RGase-oligomers consist of a tetrameric or hexameric backbone of alternating rhamnose and galacturonic acid residues with a galactose residue substituted at C-4 of part of the rhamnose moieties. Next to the subunit from which these oligomers were released, two other subunits were recognized: a highly methyl esterified xylogalacturonan segment and residual stubs of the backbone rich in branched arabinose side chains. Comparison of the MHR with non-modified pectic hairy regions of apple cell wall, isolated in a mild and defined way, revealed great resemblance indicating that the modifications of the MHR during enzymic liquefaction were only minor. Analogous MHR fractions could be isolated from potato fibre, pear, carrot, leek, and onion tissue. Finally, an adapted model is presented for the prevailing population of apple MHR having the highest molecular weight. The universal validity of this model for pectic hairy regions from other plant sources is discussed.
1. INTRODUCTION Plant cell walls form a single continuous extracellular matrix through the body of the plant and the walls of many cells together form the skeleton of plant tissues. The cell matrix consists of various types of polysaccharides, proteins and lignin in varying amounts, organized in such a way that the cell wall is chemically rather stable and physically robust. Primary walls can best be described as reinforced, multi-component gels [1]. Cell walls control cell growth by influencing cell size and shape, but also act as a pre-existing structural barrier to invasion of micro-organisms [2]. When infection or wounding of the wall occurs,
the cell may respond by thickening, lignification or suberization of the cell, production of phenolic acids and extensins, pigments, etc. [ 1]. When edible parts of plants are considered; the quality attributes of fresh fruits and vegetables (e.g. ripeness, texture) and their processing characteristics in the manufacture of foods (juices, nectars, purees, preserves) are determined to a large extent by the cell walls of plant raw materials. These cell walls also influence the extractability of important constituents of plant raw materials like sugar, oil, proteins, etc. Detailed knowledge of the major constituents making up the cell walls (e.g. pectic substances, hemicelluloses, cellulose, and structural proteins) and the ultra-structure of the cell wall is important to control and improve processing and utilization of plant products. One of the important constituents of plant cell walls is formed by the group of pectic substances [3], which has in common that all polymers contain a high proportion of galacturonic acid residues. Pectins are extracted on industrial scale from suitable plant (waste) materials e.g. apple pomace and citrus peel and used by the food industry due to their ability to form gels under certain circumstances and to increase the viscosity of drinks. They are also widely applied as stabilizers in acid milk products. Food nutritionists are interested in pectins in foods because they are considered as dietary fibre and have been shown to lower blood cholesterol levels. Interest is also directed to their pharmaceutical activities [4]. From the above, it may be clear that scientists from various disciplines are interested in the precise structure of both extracted pectins as well as pectic substances in plant cell walls. In the following, a short summary will be given of the existing literature on the structure of pectic substances, and recent studies on the structure elucidation of highly ramified regions of pectins using enzymes will be reported.
2. PECTIC SUBSTANCES The pectic polysaccharides are probably the most complex class of wall polysaccharides [1] and comprise a family of acidic polymers like homogalacturonans, rhamnogalacturonans with several neutral polymers like arabinans, galactans and arabinogalactans attached to it [1,3,5]. The pectin consists of a backbone, in which "smooth" t~-D-(1--,4)-galacturonan regions are interrupted by ramified rhamnogalacturonan regions, highly substituted by neutral sugar rich side chains. It is suggested by De Vries et al [6] that only up to 10% of the galacturonosyl residues are included in the ramified (hairy) regions while these regions carry almost all of the neutral sugar residues. Individual classes of the pectic substances will be discussed below.
2.1 Homogalacturonans Homogalacturonan segments are defined as polymers consisting predominantly of ot-(1--,4)linked galacturonosyl residues [2]. Pure homogalacturonans have been rarely reported [1], although one of the reasons might be the poor solubility of these polymers [2]. On the other hand, more and more indications are found that homogalacturonans are covalently linked to RG-I and other cell wall polymers [1,6,7]. Homogalacturonans are usually extracted from plant material by mild acid treatment [7,8]. Depending on the extraction method used, some modification of the polymer may occur. Uninterrupted homogalacturonan regions with a degree of polymerisation (DP) of approximately 70-100 have been isolated from various plant
tissues like carrot [9], apple, beet and citrus [7,10]. An important feature of galacturonans is the esterification of the galacturonic acid residues with methanol and/or acetic acid. The DM is defined as the number of moles of methanol per 100 moles of galacturonic acid. Pectins are called high methoxyl pectins when the value for DM is 50 or higher. In the other cases, the pectin is called low methoxyl pectin. For native apple pectins a random distribution of the methoxyl groups over the galacturonan chain was found [11-13]. For commercially extracted pectins, the distribution was found to be slightly different since the relative amounts of mono-, di-, and trigalacturonides in endo PGdigests suggested small blocks of unesterified galacturonosyl residues [14]. Kiyohara et al. [14] found galacturonic acid oligomers up to octamer after PG-digestion of homogalacturonans from Angelica acutiloba Kitagawa, suggesting a more blockwise distribution. Acetyl groups are usually only present in low amounts in pectins from e.g. apple and citrus, but are present in much higher amounts in pectins from sugar beet [15] and potato [16].
2.2 Rhamnogalacturonan I and II The group of Albersheim devoted a high number of publications to RG-I type of pectic substances which is conveniently reviewed by O'Neill et al. [5]. RG-I is the major polysaccharide solubilized from suspension-cultured sycamore cell walls after treatment with PG and was found to represent 7-14 % of the cell wall [5]. The RG-I polymer is composed of alternating rhamnose and galacturonic acid residues. The length is unknown, but it could contain as many as 300 rhamnose and 300 galacturonic acid residues [2]. Polymers containing this backbone are present in most if not all higher plant cell walls [5]. Next to the rhamnose and galacturonic acid residues in the backbone, RG-I is composed of side chains containing arabinofuranosyl-, galactopyranosyl- and minor quantities of fucopyranosyl residues [5]. Next to RG-I, the group of Albersheim also described rhamnogalacturonan II (RG-II). Characteristic for RG-II is the presence of rare sugars like 2-O-methyl-fucose, 2-0methyl-xylose, apiose, aceric acid, 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) and 3deoxy-D-lyxo-2-heptulosaric acid (DHA), next to the more common sugar residues rhamnose, fucose, arabinose, galactose, galacturonic acid and glucuronic acid [2]. 2.3 Other galacturonic acid-containing plant cell wall polysaccharides The presence of xylogalacturonans in which terminal xylose is linked directly to the galacturonosyl residues has been reported for mountain pine pollen [ 17], soy beans [18], and kidney beans [19,20]. The presence of terminal xylose residues linked directly to galacturonic acid moieties in pectic substances from apple has also been suggested [21,22]. Apiogalacturonan regions are proposed to be present in pectins extracted from eel grass [23] and duckweed [24]. Side chains containing glucuronic acid, galacturonic acid linked to galactose or rhamnose residues, and galactose side chains linked via a galacturonosyl residue to the C-4 of rhamnose in the backbone have been described to be present in pectic fragments from leaves and roots of several plants having an anti-complementary activity [25].
3. PECTIC ENZYMES Pectic enzymes are classified according to their mode of attack on the galacturonan part of
the pectin molecule. The main classes of pectic enzymes are pectin methylesterases (PE), polygalacturonase (PG), pectin- and pectate lyases (PL, PAL)[26-29]. Recently, in analogy to the enzymic degradation of the homogalacturonan regions of pectin, a whole array of enzymes specific for the degradation of rhamnogalacturonan regions within the pectin molecule has been recognised and these enzymes will be discussed in more detail in this book by Beldman et al [30]. The availability of these novel enzymes, next to the known pectic enzymes, offer new opportunities to use them as analytical tools in revealing the structure of oligo- and polysaccharides [31,32]. In contrast with frequently used chemical degradation methods, which usually have a poor selectivity, these enzymes act in a defined way. To be able to recognize different structural units within the polymer, endo-acting types of enzyme are preferred, although accessory enzymes might be essential as well [30]. In this study, we used purified polygalacturonase, pectin methylesterase, rhamnogalacturonan hydrolase and rhamnogalacturonan acetylesterase from fungal origin to study the structure of polymeric pectin fragments, remaining after degradation of apple tissue by a technical enzyme preparation.
4. PECTIN FRAGMENTS, RESISTANT TO ENZYMIC DEGRADATION DURING FRUIT PROCESSING During the preparation of apple juices either by direct pressing of the pulp or by the use of enzymes, it was found that significant amounts of polymeric pectic fragments were present in the enzyme-treated juices [33]. The amount and sugar composition of the fragments depend strongly on the enzyme mixture used. Obviously, the technical enzyme preparations solubilized high amounts of cell wall polysaccharides in the juice, but were lacking in enzyme activities to degrade the solubilize fragments further. Due to their relatively high concentration in the juice (4 g/L), the polysaccharides may lead to problems during processing and storage of the juice (concentrate). This was an incentive to study the enzyme resistant pectic fragments and to compare the structural features with those found for unmodified pectic molecules in the apple cell wall. This knowledge was considered to be essential in understanding the role of plant cell wall polysaccharides during processing and facilitating the introduction of new tailor-made enzymes and to develop new applicationdirected strategies in fruit and vegetable processing.
4.1 Modified Hairy Regions The enzyme-resistant fraction was in first instance characterized by its sugar composition" the fraction was rich in arabinose, while next to galacturonic acid (21 mol %) also rhamnose, xylose and galactose residues were present in significant amounts. A degree of methylation (DM; calculated on uronic acid content) of 40% and a degree of acetylation (DA) of 60% was calculated [34]. Such a high content in acetyl groups was not reported before in apple tissue. Another remarkable characteristic of the fraction was the high rhamnose:galacturonic acid ratio. The sugar composition revealed that the fragments resembled the so-called "hairy regions" as described by De Vries et al. [6], although De Vries did not mention the presence of acetyl groups. Since the fraction was isolated after treatment of the entire cell wall with various polysaccharide-degrading enzymes present in the technical enzyme preparation used,
which might have caused modifications, our fraction was designed MHR (modified hairy regions) further on. MHR also has nearly the same sugar composition and sugar linkage composition as the above mentioned RG-I. However, due to the fact that we used a quite unconventional way of extraction and since RG-I is considered to consist of strictly alternating rhamnose and galacturonic acid residues [5] we chose to use the term MHR rather than RG-I. The MHR fraction was characterized by its sugar linkage composition and it was found that the arabinose residues were less branched as mentioned in literature, probably due to debranching during the liquefaction process. Other bindings were quite representative for apple pectins [35] and RG-I type of polymers from several sources [5]. Attempts to degrade MHR by specific chemical methods like fl-elimination, treatment with metallic lithium, etc, did not result in more structural information. Also, purified enzymes like polygalacturonases, lyases, xylanases and galactanases available at that time, were not able to degrade MHR further. However, assaying more than 40 different crude enzyme preparations resulted in the recognition of one preparation which was able to hydrolyze linkages within the backbone of MHR. The enzyme responsible for this activity, rhamnogalacturonan hydrolase (RGase), was isolated from this preparation (Pectinex Ultra SP, obtained from Aspergillus aculeatus) and was further characterized [30,36]. HPSEC analysis of the RGase digest of saponified MHR revealed that only part of the glycosidic bonds (ca 4%) could be hydrolysed by RGase resulting in a mixture of polymeric material and oligomers of ca 1-2 kDa. These oligomers could be separated conveniently by high-performance anion-exchange chromatography (HPAEC) using pulsed amperometric detection (Figure 1)[37]. The structure of the oligomeric reaction products was elucidated by NMR spectroscopy following preparative HPAEC [37,38]. The structures found are summarized in Table 1. It can also be seen that the backbone of all oligomers consists of alternating rhamnose and galacturonic acid residues, with a galactose residue linked to C-4 of (part of) the rhamnose moieties. It was shown that Table 1 Structures of identified oligomers, obtained after degradation of apple MHR-S by RGase. a - R h a p - ( 1 --* 4 ) - a - G a l p A - ( 1 -~ 2 ) - a - R h a p - ( 1 --, 4 ) - G a l p A a - R h a p - ( 1 ~ 4)-ot-GalpA-(1 --* 2 ) ~ a _ R h a p _ ( 1
4)-GalpA /3-Galp-(1 ~ 4) / /3-Galp-(1 ~ 4 ) - a - R h a p - ( 1 --~ 4 ) - a - G a l p A - ( 1 -* 2 ) - a - R h a p - ( 1 -* 4 ) - G a l p A
fl-Galp-(1 ~ 4 ) - a - R h a p - ( 1 -* 4)-ot-GalpA-(1 --~ 2 ) ~ a _ R h a p _ ( 1
4)-GalpA /3-Galp-(1 ~ 4) / a - R h a p - ( 1 ~ 4)-ot-GalpA-(1 ~ 2)-ot-Rhap-(1 ~ 4 ) - a - G a l p A - ( 1 --~ 2 ) - a - R h a p - ( 1 ~ 4 ) - G a l p A a - R h a p - ( 1 ~ 4 ) - a - G a l p A - ( 1 ~ 2 ) - a - R h a p - ( 1 ~ 4)-ot-GalpA-(1 --~ 2 ) ~ a _ R h a p _ ( 1 /3-Galp-(1 ~ 4 ) " /3-Galp-(1
4)-a-Rhap-(1
4)-GaipA
4)-ot-GalpA-(1
2 ) x ' a - R h a p - ( 1 --* 4 ) - a - G a l p A - ( 1 ---, 2)-o~-Rhap-(1 ~ 4 ) - G a l p A /3-Galp-(1 ~ 4) t
/3-Galp-(1 ---, 4 ) - a - R h a p - ( 1 ---, 4 ) - a - G a l p A - ( 1 ---, 2 ) - a - R h a p - ( 1 ---, 4 ) - a - G a l p A - ( 1 ---, 2 ) ~ a _ R h a p _ ( 1 /3-Galp-(1 ~ 4) /
4)-GalpA
/3-Galp-(1 --, 4 ) - a - R h a p - ( 1 ~ 4)-tr-GalpA-(1 ---, 2)~ot_Rhap_( 1 ~ 4 ) - a - G a l p A - ( 1 ---, 2 ) ~ a _ R h a p _ ( 1 /3-Galp-(1 ---, 4) I
/3-Galp-(1 ---, 4) /
_..r
4)-GalpA
1000 900 8OO ..-..
700
cJ Z
IV
v} c o n
600 ~E
c_
..
VIII
C3 < (3_
500 ~r-400 "~ 300 200 100
0
5
15 2'0 215 Retention time (min)
10
30
3S
Figure 1. Elution profile on HPAEC of apple MHR-S after treatment with RGase at 30~ and pH 5.0 for 24h [37].
RGase was only active on MHR after removal of the acetyl groups either chemically or enzymatically. This indicates that these acetyl groups are positioned in the alternating rhamnose-galacturonic acid sequences from which the oligomers are released. All available information at that time could be accommodated in a hypothetical model [34]. Most of the galactose residues were present terminally linked to rhamnose moieties within the rhamnogalacturonan backbone and it was assumed that most of the terminally linked xylose residues were linked directly to galacturonic acid residues present in the backbone. The relatively long arabinan side chains were rather linear, probably caused by the enzyme treatment. Due to the rhamnose:galacturonic acid ratio found, the backbone was thought to consist of alternating rhamnose : galacturonic acid sequences in addition to segments rich in galacturonic acid. Obviously these galacturonic acid rich segments were resistant towards pectic enzymes.This first model had quite some limitations, due to the fact that it was quite speculative over the arrangement of the various building units (e.g. methyl esters, acetyl groups, xylose side chains) over the molecule. Further research was directed to reveal the structure of MHR in more detail.
4.2 Three different suhunits of apple MHR Analysis of the MHR by high-performance size-exclusion chromatography (HPSEC) revealed three distinct populations to be present, which were isolated on a preparative scale. The composition of the populations differed mainly in the relative proportion of rhamnose, xylose and the amount of methyl esters and acetyl groups, although the general characteristics were rather similar (Table 2). However, degradation studies with RGase showed that, in contrast
Table 2 Sugar composition (mol%) of the fractions of MHR isolated by chromatography over Sephacryl $200 and $500 [34]. Sugar
MHR
Rha Ara Xyl Man Gal Glc GalA OMe OAc
6 55 8 0 9 1 21 42 60
Rha:GalA
A
B
C
5 50 11 0 10 0 24 28 55
6 59 5 0 13 0 17 84 57
10 47 3 0 7 0 33 100 21
0.29
0.21
0.35
0.30
with the populations A and B, population C could not be degraded by RGase. Investigation of the high molecular weight degradation products of MHR after RGase treatment was hindered by their co-elution with non-degraded molecules of the population C. For this reason, the saponified high molecular weight MHR population A, representing ca 60% of MHR, was further used in structural studies. It was degraded by RGase and the digest was separated by SEC on a Sephacryl $200 column (Figure 2). Three distinct populations could be recognised varying in the ratio between uronic acids and neutral sugars.
1so! 100-
50-
/
i
ji O-
. i
i
0.2
III
, l
I
0.4
),
,
016
|
!
O.g
~| I
I
1.0
KAy
Figure 2.-Size-exclusion chromatography on Sephacryl $200 of MHR population A after degradation with RGase: , uronic acids; .... , neutral sugars [39].
10 Table 3 Sugar composition (mol %) of MHR population A and fractions I-IV, obtained after Sephacryl $200 size-exclusion chromatography of the digest of MHR population A with RGase [39]. Sugar Rha Ara Xyl Man Gal Glc GalA DM DA Xyl:GalA
MHR pop.A
I
II
III
IV 23 11 0 0 29 0 37
5
4
1
3
50 11 0 10 0 24 28 55
8 38 0 4 5 41
84 3 0 3 3 6
81 1 0 5 3 7
0.5
0.9
0.5
0.16
0
The sugar composition of both the starting material and the fractions obtained is shown in Table 3. Fraction IV represented the typical RGase oligomers as was demonstrated by HPAEC (not shown). Fractions II and III covered a broad molecular weight range and were both rich in arabinose residues, next to smaller amounts of rhamnose, xylose, galactose, glucose and galacturonic acid. Apparently, these fractions represent arabinans, connected to some residual stubs of the pectic backbone having a rhamnose:galacturonic acid ratio resembling that of the starting material. Surprisingly, fraction I consisted mainly of xylose and galacturonic acid residues ( > 80%). Since in the original MHR mixture 70% of the xylose residues were terminally linked, and 20% of all galacturonic acid residues present were branched through C-3 [2], it was assumed that fraction I represents a xylogalacturonan segment. A similar xylogalacturonan fraction was obtained from non-saponified MHR population A after incubation by RGase in combination of RGAEase (rhamnogalacturonan acetylesterase). RGAEase has been shown to be specific for MHR and to remove 70% of the acetyl groups present in MHR esterified to the galacturonic acid residues [40]. HPSEC revealed a similar elution pattern of the digest as compared to the RGase of chemically saponified MHR. To determine homogeneity in composition and charge, both xylogalacturonan fractions were further chromatographed on a DEAE Sephacryl Fast Flow column. For the xylogalacturonan originating from the saponified MHR population A, all material essentially eluted in one (Figure 3a), suggesting that the xylogalacturonan molecules were quite homogeneous. The minor quantities of rhamnose, galactose and arabinose probably originated from connected rhamnogalacturonan fragment remaining after RGase action. When the xylogalacturonan originating from the RGase/RGAEase digest of the non-saponified MHR population A was eluted over the anion-exchange column, three different populations could be recognized (Figure 3b). Even the last eluting population, representing 68% of the polysaccharides, was much less retained on the anion-exchange material as compared to the chemically saponified
11
a
/-
150 -
100
20
-
Z /
1.0 50-
0
00
60.~
2.0
o
40
< O Z 1.0
20 |
0
, 0
, 100
,
, 200
,
, 300
,
, 400
,
,
,
0.0
500 v o l u m e (ml)
Figure 3. Anion-exchange chromatography on DEAE Sepharose of fraction I of the Sephacryl $300 fractionation of a) saponified MHR population A after degradation with RGase and after b) degradation of the non-saponified MHR population A with RGase and RGAEase: , uronic acid; .... , neutral sugars; thin line, NaOAc gradient [39].
12 Table 4. Sugar composition (mol %) of the xylogalacturonan fractions obtained after DEAE Sepharose anion-exchange chromatography of the Sephacryl $300 fractions I originating from RGase degraded saponified apple MHR population A (1-a and l-b) and from RGase/RGAEase treated non-saponified apple MHR population A (2-a, 2-b, and 2-c). Sugar
1-a
1-b
2-a
2-b
2-c
Rha Ara Xyl Man Gal Glc GalA
3 5 34 2 3 1 52
2 5 25 3 3 4 58
3 11 26 4 5 9 42
3 14 22 2 4 5 50
5 6 34 1 4 3 47
Xyl:GalA
0.7
0.4
0.6
0.4
0.7
,
xylogalacturonan (Figure 3a). This can be explained by the fact that in population A still a considerable number of methoxyl groups are present (28 moles per 100 moles of galacturonic acid) being esterified to the carboxyl group of the galacturonic acid residues. The presence of three different xylogalacturonan populations indicates that the methyl esters are not equally distributed over the various xylogalacturonan molecules and that distinct groups of esterified xylogalacturonan molecules exist. Obviously, fraction 2-a is more methyl esterified as compared to fractions 2-b and 2-c, since the interaction with the anion-exchanger is lower. The sugar composition of all fractions is presented in Table 4 and although minor differences can be observed, their structures are believed to be in principle the same. Fraction 2-c, which is supposed to be representative for the methyl esterified xylogalacturonans of apple MHR, was further investigated by various ~H- and ~3C-NMR spectroscopic methods [39]. It was confirmed that all xylose residues were terminally fllinked to C-3 of the galacturonic acid residues, present in a high molecular weight c~-(1--,4)galacturonan. Both the methoxyl groups and xylose side chains appeared to be randomly distributed over the back bone and from the two-dimensional NMR spectra, a degree of methylation of 39% was calculated. 4.3 A new hypothetical model for apple MHR The identified structures of the degradation products obtained after degradation of MHR population A by RGase are used to propose a new hypothetical model of MHR population A. MHR population A is considered to consist of xylogalacturonan segments (subunit I); rhamnogalacturonan stubs rich in arabinan side chains representing subunit II, and of RGase oligomers as released from the rhamnogalacturonan regions (subunit III). On weight basis, the ratio of subunits I, II, and III was estimated to be 2:3:1. The relative proportion of the three subunits was calculated using molecular weights of 80, 20-30, 8-12, and 1-1.5 kDa for the subunits I, II and III respectively (HPSEC). It was calculated that in one molecule of MHR population A, there is only one xylogalacturonan segment present, next to 5-6
13
Figure 4. Hypothetical structure of apple pectin and of the prevailing population of MHR isolated herefrom. SR, smooth regions; HR, hairy regions. Subunit 1,xylo-galacturonan; subunit 11, stubs of the backbone rich in arabinan side chains; subunit In, rhamnogalacturonase oligomers. The distribution of acetyl groups is not presented, but there is evidence [36,40] that the major part of the acetyl groups are located within subunit 111. No information is available on the presence of methyl esters in subunit 11.
14 segments of subunit II, while on average 12 RGase oligomers are present. It is speculated that the length of the xylogalacturonan backbone might be 75-100 galacturonosyl units. Variations were found for the degree of xylose substitution (40-90%) and also differences in methyl esterification were observed. The xylogalacturonan fraction which was investigated by NMR had a DM value of ___ 40%. By anion-exchange chromatography using a DEAE-column calibrated with pectins with known DM, also a DM value around 40% was calculated for this fraction. For the other two xylogalacturonan fractions, DM values of approximately 70 and 85-90 were calculated. The length of the arabinan side chains present in subunit II may vary between 30 and 50 arabinose residues and several of these chains might be connected to the rhamnogalacturonan backbone which consists of only 8-10 sugar residues (ratio of rhamnose to galacturonic acid is 0.2 to 0.4). The arabinans might be connected directly to a rhamnose residue in the backbone or via one or more galactose residues [41]. Based on these findings, an updated model of apple MHR population A is presented in Figure 4. The sequence in which the various subunits are arranged is purely speculative. On the other hand, whereas RGase is characterized to be a typical endo-acting enzyme (based on HPSEC analysis), the release of RGase oligomers is not representative for an endo-attack. This might indicate that the regions of alternating rhamnose and galacturonic acid residues within the MHR backbone are not extremely long, and probably are interrupted with other subunits resistant to hydrolysis by RGase.
4.4 Apple hairy regions When the new model for MHR was proposed, the question arose to which extend the model could be used for pectins as present in the apple cell wall. It was realized that the MHR fraction might originate from pectic materials located in different sites of the cell wall structures of apple having different structures. It was also not known exactly to which extend the structure of the 'native' hairy regions were changed by the enzymic liquefaction process. Using mild extraction conditions, various pectin populations have been isolated" pectins extractable by cold buffer, hot buffer, chelating agents and by cold alkali [42]. Degradation of the various pectins by purified PG and PE resulted in different populations of hairy regions (HR). The sugar composition and the sugar linkage composition of the various HR fractions resembled that of MHR, although the relative proportion of galactose was somewhat higher. Degradation by RGase resulted in exactly the same set of typical RGase-oligomers as was described for the MHR. Both the absolute amount of the oligomers released as the ratio between the various RGase-oligomers differed for the various HR-fractions: the amount of oligomers released and the degree of branching increased when the pectins were extracted under more harsh conditions. Fractionation of the RGase digest revealed the presence of fragments having a backbone of rhamnose and galacturonic acid (ratio 1:5) being rich in arabinose (50-70%), comparable to the subunit II found for MHR population A. Some other fractions were clearly enriched in xylose and galacturonic acid, indicating the presence of xylogalacturonan segments (subunit I) [42]. It is concluded that the hairy regions isolated from the different pectin extracts consist of principally the same building blocks as MHR, although the arrangement of these blocks might vary. Treatment with RGase resulted in the release of RGase-oligomers from all HR fractions, but the elution behaviour of the remaining polymers varied significantly. It is suggested that the RGase oligomers in some hairy regions are located in the extremities of
15 the molecules, whereas in other hairy regions they are thought to be distributed more randomly over the molecule. This confirms De Vries's view that pectins can be characterized as consisting of repeating units, consisting of repeating units [43]. In addition, it can be stated that the distribution of the latter repeating units over the pectin molecules is rather diverse. 4.5 MHR from various plant materials To investigate whether MHR-fragments are unique for apple tissue or can be found in cell walls of plant material of other origin, the same liquefaction process was used to degrade the cell wall polysaccharides of a variety of other plant materials like leek, onion, carrot, pear and potato [44]. From all juices obtained, a similar MHR fraction could be isolated, although variations were found with respect to arabinose and xylose content which were rather low in some cases. Typical characteristics of all MHR fractions isolated were the high ratio between rhamnose and galacturonic acid residues and a high acetyl content, similar to those found for apple MHR. Galactose residues were present in all MHR (10-30 mol %) of which 35-70% appeared to be terminally linked. All isolated MHR fractions had a broad molecular weight distribution; minor differences were found for the sugar composition of the various Mw populations. In all cases, the DA value decreased and the DM increased with decreasing molecular weight. Rhamnogalacturonase degraded all MHR fractions in a similar fashion as compared to apple MHR and HR resulting in the release of the same characteristic RGaseoligomers. It is considered that the model as presented in Figure 4 might also be valid for the MHR fractions isolated from other plant materials. However, the ratio between the subunits I, II, and III may vary. Especially the presence of the xylogalacturonan subunit seems to depend on the origin of the MHR. The model might also be valid for pectic fractions from above mentioned above or other sources as described in the literature, although the different building blocks were not always recognized as such by the authors.
5. IS A GENERAL MODEL FOR PLANT CELL WALL PECTIC SUBSTANCES FEASIBLE? One of the challenges for scientists is to express their findings in a model which has a more general validity. De Vries [43] stated that all polysaccharides containing galacturonic acid may be constructed of the building blocks: homogalacturonan, apiogalacturonan, xylogalacturonan, rhamnogalacturonan, and galactogalacturonan. As illustrated in this chapter, enzymes capable to split within these regions facilitate a more closer view into the quite complex ramified regions of pectins, and even might enable us to propose a more general model for pectic substances originating from plant cell walls. Our results obtained for apple pectic substances and the modified hairy regions of plant material from several other sources suggest that the model for apple MHR, as proposed in Figure 4, may be used as a starting point to develop such a model. Principal differences in relative amounts between the various subunits may exist, which is substantiated by the fact that no indications were found in our study for the presence of RG-II or apiogalacturonan type of polymers in apple, although Pellerin et al [45] found relatively high proportions of RG-II in apple and grape juice. Suggestions for major variations, which might be present
16
Table 5. Suggested subunits present in most pectic substances, next to possible variations within an individual subunit.
subunit
diversity based on:
homogalacturonan [8,461
* length of the homogalacturonan sequences between individual rhamnose residues * degree and distribution of methyl esterification * degree and distribution of acetyl esterification
RG-I
* nature of neutral sugars (and acidic sugars?) present in side chains * length, sugar, and linkage composition and degree of branching of side chains * distribution of side chains over the alternating rhamnose-(1--,4)-galacturonic acid backbone
I51
rhamnogalacturonan * rhamnose:galacturonic acid ratio [39] * relative proportion of neutral sugars (mainly arabinose and galactose) 9 length, degree of branching and distribution of side chains 9 degree and distribution of acetyl esterification (and methyl esterification?) RG-II
[5]
* proportion of common and rare sugars like Ome-xylose, KDO, DHA * number, type and distribution of uronic acids in the (side) chain * attachment and distribution of RG-II chains over the pectic molecule * presence and position of ferulic acid?
xylogalacturonan I39]
* degree of xylose substitution * degree of methyl esterification (and acetylation?) * distribution of substituents over the backbone
apiogalacturonan [23,24]
* * * *
degree of apiose substitution length of apiose chains methyl esterification? distribution of substituents over the backbone
within one given subunit, are listed in Table 5. In literature, ramified pectic molecules are sometimes termed RG-I, but also the more general term rhamnogalacturonan is used. Since it became clear that RG-I is used for pectic segments which have a strictly alternating sequence of rhamnose and galacturonic acid [5], the more general name "rhamnogalacturonan" might be used for pectic fragments having a low rhamnose:galacturonic acid ratio. To be able to differentiate between rhamnogalacturonans and homogalacturonans having individual rhamnose moieties in between long sequences of galacturonic acid, we propose to consider fractions having a ratio varying between 0.05 and 1 as rhamnogalacturonans. Segments of the backbone of at least 20 galacturonic acid residues with only one solitary rhamnose residue can then be termed homogalacturonans. Although the definition of rhamnogalacturonan would include RG-II type of polysaccharides, it is recognized that the term RG-II is used for a rather characteristic segment of pectic molecules.
17 Due to the rare sugars present in RG-II, no confusion is expected to occur.
6 SIGNIFICANCE FOR FUTURE PECTIN RESEARCH Although much research has been directed to reveal the structure of pectins as they occur in the plant cell wall and pectins used as food ingredient, our understanding of structurefunction relationships is rather limited. Until now, pectins were usually characterized by their galacturonic acid and neutral sugar content, the degree of esterification and their molecular weight distribution. However, in food applications it was found that two pectins having same chemical characteristics (e.g galacturonic acid content, degree of methylation, etc), might differ significantly in their physical properties. This may suggest that the distribution of the methyl esters over the molecule might be more important than the absolute amount of ester groups [46,47]. The same might be true for native pectins: the length, nature and attachment of the side chains and the distribution of the various subunits within the hairy regions, and the distribution of ramified regions over the pectin molecule might be more important than the absolute amount and types of neutral sugars. As demonstrated in this paper, an important step to recognize individual subunits within a complex polysaccharide can be made by using purified and well characterized enzymes. As demonstrated throughout this symposium book, numerous pectic enzymes belonging to the same category (e.g polygalacturonases, lyases, rhamnogalacturonases) are being studied intensively at this moment and will be available in the near future to study differences in the fine structure of chemically identical polysaccharides. Essential in this approach is also the possibility to differentiate between the numerous, structurally quite similar degradation products by various supplementary chromatographic techniques and to have access to advanced spectroscopic methods like NMR- and MS-techniques to identify these oligosacchaxides unequivocally.
7. REFERENCES 1 2 3 4 5 6 7 8
A. Bacic, P.J. Harris, and B.A. Stone, The Biochemistry of Plants, Vol 14, Carbohydrates, Academic, London, 1988, pp. 297-369. M. McNeil, A.G. Darvil, S.C. Fry, and P. Albersheim, Ann. Rev. Biochem., 53 (1984) 625663. A.G.J. Voragen, W. Pilnik, J-F. Thibault, M.A.V. Axelos, C.M.G.C. Renard, in A.M. Stephen (Ed) Food polysaccharides and their applications, Marcel Dekker Inc., New York, 1995, pp. 287-339. H-U. Endress, in R.G. Walter (Ed), The Chemistry and Technology of pectin, Academic, London, 1991, pp. 251-268. M. O'Neill, P. Albersheim, and A.G. Darvill, in P.M. Dey (Ed.), Methods in Plant Biochemistry, Vol. 2, Carbohydrates, Academic, London, 1990, pp. 415-441. J.A. De Vries, F.M. Rombouts, A.G.J. Voragen, and W. Pilnik, Carbohydr. Polym., 2 (1982) 25-33. V. Zitko, and C.T. Bishop, Can. J. Chem., 44 (1966) 1275-1282. J.-F. Thibault, C.M.G.C. Renard, M.A.V. Axelos, P. Roger, and M-J. Cr6peau, Carbohydr. Res., 238 (1993) 271-286.
18 9 H. Konno, Y. Yamasaki, and K. Katoh, Phytochem., 25 (1986) 623-627. 10 T.P. Kravtchenko, M. Penci, A.G.J. Voragen, and W. Pilnik, Carbohydr. Polym., 20 (1993) 195-205. 11 J. De Vries, M. Hansen, J. Soderberg, P.E. Glahn, and J.K. Pedersen, Carbohydr. Polym., 6 (1986) 165-176. 12 J.A. De Vries, F.M. Rombouts, A.G.J. Voragen, and W. Pilnik, Carbohydr. Polym., 3 (1983) 245-258. 13 J.A. De Vries, A.G.J. Voragen F.M. Rombouts, and W. Pilnik, Carbohydr. Polym., 4 (1984) 3-14. 14 H. Kiyohara, J.-C Cyong, and H. Yamada, Carbohydr. Res., 182 (1988) 259-275. 15 F.M. Rombouts, and J.-F. Thibault, Carbohydr. Res., 154 (1986) 177-187. 16 A.G.J. Voragen, H.A. Schols, and W. Pilnik, Food Hydrocolloids, 1 (1986) 65-70. 17 O. Bouveng, Acta Chemica Scand., 19 (1965) 953-963. 18 T. Kikuchi, and H. Sugimoto, Agr. Biol. Chem., 40 (1976) 87-92. 19 Y. Matsuura, Nippon NOgeikagaku Kaishi, 58 (1984) 253-259. 20 Y. Matsuura, Nippon NOgeikagaku Kaishi, 58 (1984) 1111-1115. 21 A.J. Barrett, and D.H. Northcote, Biochem. J., 94 (1965) 617-627. 22 J.A. De Vries, F.M. Rombouts, A.G.J. Voragen, and W. Pilnik, Carbohydr. Polym., 3 (1983) 193-205. 23 Y.S. Ovodov, Pure Appl. Chem., 42 (1975) 351-369. 24 D.A. Hart, and P.K. Kindel, Biochem. J., 116 (1970a) 569-579. 25 X.B. Sun, T. Matsumoto and H. Yamada, Carb. Polymers, 25 (1994) 117-122. 26 W. Pilnik, and A.G.J. Voragen, in P.F. Fox (Ed), Food Enzymology, Vol. 1, Elsevier Applied Science, London, 1991, pp. 303-336. 27 W. Pilnik, and F.M. Rombouts, in G.G. Birch, N. Blakebrough, and K.J. Parker (Eds), Enzymes and Food Processing, Applied Science Publishers LTD, London, 1981, pp. 105-128. 28 J.K. Burns, in R.G. Walter (Ed), The Chemistry and Technology ofpectin, Academic, London, 1991, pp. 165-188. 29 T. Sajjaanantakul, and L.A. Pitifer, in R.G. Walter (Ed), The Chemistry and Technology of pectin, Academic, London, 1991, pp. 135-164. 30 G. Beldman, M. Mutter, M.J.F. Searle-van Leeuwen, L.A.M. van den Brock, H.A. Schols,and A.G.J. Voragen, in J. Visser et al (Eds), Pectins and Pectinases, Elsevier, Amsterdam, 1996. 31 A.G.J. Voragen, H.A. Schols, and H. Gruppen, in F. Meuser, D. J. Manners, and W. Seibel (Eds.), Plant Polymeric Carbohydrates, Royal Society of Chemistry, Cambridge, UK, 1993, pp 3-15. 32 J. An, L. Zhang, M.A. O'Neill, P. Albersheim, and A.G. Darvill, Carbohydr. Res., 264 (1994) 83-96. 33 H.A. Schols, P.H. in 't Veld, W. van Deelen, and A.G.J. Voragen, Z. Lebensm. Unters. Forsch., 192 (1991) 142-148. 34 H.A. Schols, M.A. Posthumus, and A.G.J. Voragen, Carbohydr. Res., 206 (1990) 117-129. 35 J.A. De Vries, C.H. den Uijl, A.G.J. Voragen, F.M. Rombouts, and W.Pilnik, Carbohydr. Polym., 3 (1983) 193-205. 36 H.A. Schols, C.C.J.M. Geraeds, M.J.F. Searle-van Leeuwen, F.J.M. Kormeling, and A.G.J. Voragen, Carbohydr. Res., 206 (1990) 105-115. 37 H.A. Schols, A.G.J. Voragen, and I.J. Colquhoun, Carbohydr. Res., 256 (1994) 97-111. 38 I.J. Colquhoun, G.A. de Ruiter, H.A. Schols, and A.G.J. Voragen, Carbohyclr. Res., 206 (1990) 131-144. 39 H.A. Schols, E.J. Bakx, D. Schipper, and A.G.J. Voragen, Carbohydr. Res., 279 (1995) 265279.
19 40 M.J.F. Searle-van Leeuwen, L.A.M. van den Broek, H.A. Schols, G. Beldman, and A.G.J. Voragen, Appl. Microbiol. Biotechnol., 38 (1992) 347-349. 41 J.M. Lau, M. McNeil, A.G. Darvill, and P. Albersheim, Carbohydr. Res., 168 (1988) 245-274. 42 H.A. Schols, E. Vierhuis, E.J. Bakx, and A.G.J. Voragen, Carbohydr. Res., 275 (1995) 343360. 43 J.A. De Vries, in G.O. Philips, D.J. Wedlock, and P.A. Williams (Eds.), Gums and Stabilizers for the Food Industry 4, IRL Press, Oxford, 1988, pp. 25-29. 44 H.A. Schols and A.G.J. Voragen, Carbohydr. Res., 256 (1994) 83-95 45 P. Pellerin, T. Doco, S. Vidal, P. Williams, and J-M. Brillouet, in J. Visser et al (Eds), Pectins and Pectinases, Elsevier, Amsterdam, 1996. 46 T.P. Kravtchenko, Studies on the structure of industrial high methoxyl pectins, Ph.D. Thesis, Wageningen Agricultural University, The Netherlands, 1992. 47 P-E. Glahn and C. Rolin, in G.O. Philips, D.J. Wedlock, and P.A. Williams (Eds.), Gums and Stabilizers for the Food Industry 8, IRL Press, Oxford, 1996, in press.
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J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All fights reserved.
21
Physicochemical properties of pectins in solution and gel states M. Rinaudo CERMAV- CNRS BP 53 - 38041 Grenoble C@dex 9 (France)
Abstract
This paper concerns the main properties of water soluble pectins in sol and gel states. First of all, the methods of purification and characterization are discussed. The method of steric exclusion chromatography equipped with different detectors is demonstrated as the most useful to determine the macromolecular characteristics of these polymers ;the role of aggregation is pointed out. The worm like chain behaviour of the pectins is developped allowing to predict the dimensions of the chain as soon as the persistence length is known. The experimental data are compared with the theoretical prediction obtained from conformational analysis. The polyelectrolyte properties of pectins are breefly exposed ; specially the role of the carboxylic groups distribution along the chain is demonstrated to controll the electrostatic properties. The viscometric behaviour depends on the ionic concentration and on the nature of the counterions in relation with electrostatic repulsions. Then, the ionic selectivity is discussed and related to the mechanism of crosslinking with divalent counterions. The sol-gel transition is then examined for LM and HM pectins and the mechanisms described in these two cases. The physical properties of the gels are related to the microstructure of the polymers and few data are examined.
1. I N T R O D U C T I O N
Pectins is a general term for a group of natural polymers based on polymerized galacturonic acid partly esterified with methanol. In addition these polymers must be considered as copolymers due to existence of neutral sugar branched zones. [1]. Some uronic acid units may also be esterified on 0-2 or 0-3 position with acetic acid. The pectins occur in the cell wall of higher plants and control at least partly the mechanical properties, the ion exchange properties and the swelling of the cell walls.
22 In situ, pectins may form a 3-D network based on different mechanisms of interaction ; they play an important role on cohesion of cell walls but also in recognition reaction [2, 3]. The commercial samples of pectins mainly used as food additives represent modified forms of the natural polymers due to the conditions of extraction. Nevertheless, it is usually recognized two categories of pectins :the high methoxyl pectins (HM) with a degree of methylation DM>50% forming gels at low pH in presence of saccharose to reduce the water activity and the low methoxyl pectins (LM with DM<50%) forming gel in presence of calcium [4]. This paper will not described the chemical structure of pectins which is a difficult problem [1] even if the physical properties in solution and ability to form gel must be directly related with the distribution of the units along the chain. The functional properties of pectins are not only related to the neutral sugar content (up to 15 %) but also to the distribution of structural blocks having very different contibutions.
2. STRUCTURE
AND MOLECULAR CHARACTERISTICS
Pectins are heteropolysaccharides with axial-axial e~(1, 4 ) - D galactopyranosyluronic acid units condensed in the 4C1 conformation and interrupted by ~-(1,2)-L rhamnopyranosyl residues. The structure is based on blocks of galacturonan partly esterified (the smooth zones) and blocks of highly ramified rhamnogalacturonan regions (hairy zones). The exact structure depends on the sources but also on the methods used to isolate the pectins [1,5]. Few data are given in Table 1. Table 1 Average composition for some pectins source materials Source % pectins % galacturonic (dry matter) acid Sunflower head 10-25 % 90 Citrus peel 20-30 % 85 Sugar beet pulp 15-25 % 50 Apple pomace 15-25 % 75 Potato fiber 15 % 50
DM 30-40 75-80 60 75-80 30
In the galacturonan block, the distribution of methoxyl groups is also very important for physical properties but also enzymic suceptibility ;it was analyzed by different authors using different technics [6-9]. Specially the presence of acetyl groups in sugar beet, sunflower and potato pectins is claimed to inhibit the gelation and limit their utilization. On the opposite, ferulic acid attached to neutral sugar unit in spinach or sugar beet pectins was used to crosslink pectins [10]. Concerning the structure analysis of pectins, the average compositions are usely determined :the galacturonic acid content (AGA%), the degree of esterification (DE% including the degree of methylation DM and the degree of acetylation DA), the neutral sugar yield and composition obtained by complete hydrolysis and chromatographic analysis. The AGA and DE can be obtained by different technics
23 but we proposed a conductimetric method [11] combined with 1H nmr for the methoxyl or acetyl determination. Then, the macromolecular characterization is necessary to obtain :the molecular weight distribution of the polymeric material and the average molecular weights. For this purpose, the first important condition is to get a perfectly molecular soluble material which means to avoid aggregation and/or take off insoluble material. This point was previously discussed [12]. The polysaccharide must be isolated preferentially as a sodium salt form to be fully soluble in water or in presence of some NaCI used to screen electrostatic interactions. The best way then to characterize the pectins is the steric exclusion chromatography equipped with a multidetection and particularly a light scattering detector [13]. SEC can be performed using a neutral eluent such as NH4 NO3 or Na NO3 O.1M ; a minimum molar concentration is needed to screen the electrostatic exclusion on the porous columm material [14-21]. This technic gives the molecular weight distribution and the average molecular weights (Mn, Mw). The Mark-Houwink parameters allow to relate the intrinsic viscosity [~] with the viscometric average molecular weight Mv through the relation :
[11] = KIVI~
(1)
Few sets of K,a parameters were given in references 17 and 22 ; K and a are parameters which may depends on the fine structure i.e. rhamnose content, DE... when they play on the stiffness of the chain. Nevertheless, it seems that the viscometry must be used in carefully defined conditions to avoid aggregation which often surestimates the viscosity ;in that conditions, [~] is related to the physical properties of the solution (tickening properties) but not directly to Mv. Whatever is the technic used to determine the molecular weights on pectins, it is necessary to eliminate aggregates often mentionned in the litterature [13, 19, 2123] ; for this, ultracentrifugation was used but it seems that filtration through hydrophilic membranes with very small pores (as low as 0.1 or 0.05 l~m) is easier. At the moment, one recommends to determine the molecular characteristics of pectins using SEC chromatography equipped with a differential refractometer, a multiangle laser light scattering detector and a viscometer as previously described [25]. This technique needs no calibration with the usual molecular weight standards such dextrans and pullulans... In our work, we clearly demonstrated the presence of aggregates even not eliminated by filtration on 0.05 l~m pore membranes ; this causes always overestimated values of Mw when obtained in static light scattering. In an exemple previously given, when one gets Mw = 70,000 in static light scattering, SEC shows that 85% of the materials has only Mw = 34,800, even after filtration through 0.05 l~m membranes [13].
24 3. STIFFNESS OF THE CHAINS
As discussed in the last 3 years, polysaccharides behave in solution under a worm like chain [26] ; t h e local stiffness of the chain is characterized by a persistance length (Ip) ;the larger Ip is, the larger the chain deviates from the gaussian behaviour in the usual molecular weight range of these natural polymers [27]. This makes difficult to use the relations given in litterature for synthetic polymers and specially to relate [n] with the chain dimensions. The Flory constant (~) and the Mark-Houvink coefficient (a) are directly dependent on the persistence length and in fact, on the number of Kuhn segments in the chain. Due to the ionic charge on the backbone, it is also necessary to take into account the intra and interchain electrostatic repulsions. Considering the isolated chains this is introduced in the model we developped when an electrostatic contribution (le) modifies the intrinsic persistence length Ip and an electrostatic excluded volume parameter ael is considered. Then the radius of giration RG of a polymeric chains at a given salt concentration is : RG= RO,O (IPlple)l/2 (Zel
(2)
RG,e is the radius of giration in the 0-state assumed to correspond in polyelectrolyte systems to the value extrapolated to infinite salt concentration. The relation of Benoit-Doty for high molecular weights gives : 2 Lip aG,e= 3
(3)
with L the contour length of the molecule (here L = M.b/mo with b = 4.3 A ~ the length of the repeat unit, mo the mass of the repeat unit and M the molecular weight of the considered chain) [28]. Then, the dimensions of a chain can be predicted whatever are the solution conditions if the intrinsic persistence length (Ip) is known ; Ip is a parameter reflecting the local structure and up to now, few tentatives to calculate Ip from conformational analysis exist in the litterature. The conformational analysis was developped recently by different authors first on the disaccharide units to investigate the role of the charge on C-6 position on the conformation [29-32]. From this study, the 21 or right-handed 31 helices were described as most problable conformations [30,31]. These conformations were also demonstrated from x-ray diffraction [33] or on the basis of circular dichro'fsm [34]. From these data, the repeat unit in the axial-axial conformation has a 4.3 A ~ length which will be used to characterize the electrostatic properties. From conformational analysis it appears that there is no signifiant influence of the methoxyl group on the conformational behaviour [31,32] ; only the role of the carboxylic group on the long range electrostatic interaction must be important
25 specially in dilute aqueous solution and it will explain the role of pH and DM on the properties. From disaccharide analysis, few authors predict the conformation of the poly a-D galacturonan as well as the role of the charge density [35,36] ; they determine the persistence length Ip that will allow us to explain the behaviour in solution. Considering the chain dimensions, for very long gaussian chain, one predicts : 2
= N b2 Coo
(4)
in which N rigid units of length b form a gaussian chain ; Coo, the characteristic ratio 2 reflects the short range interactions and the average value of the mean squared end-to-end distance (with
2 = R~/6).
Assuming a worm like chain
model as discussed previously for other polysaccharides, one can write 2 = 2 Lip
(5)
in wich L is the contour length (L=Nb). From the relations (4,5), it comes : In Coo ~ 2""b
(6)
From reference 35, it seems that the parameter Coo passes through a minimum for 50% of charged group ; t h e same group also demonstrated the role of the rhamnose content on the flexibility of the chain. The experimental results were compared with the prediction and relativity good agreement was obtained [18]. Recent data indicate a relative good agreement between theoritical prediction for a polygalacto-galacturonic acid in vacuum and experimental data (Coo=24 i.e. Ip~50A ~ [36]. Nevertheless, it seems that up to now, the persistence length calculated are often larger than the experimental value. For homogalacturonan, it should be reasonable to find a value in the range of Ip obtained for a guluronic rich alginate (Ip~100A ~ [27]. Some experimental values obtained for Ip are given in Table 2. A still usual method to characterize the behaviour of polysaccharides is to determine the flexibility parameter B introduced by Smidsrod and Haug [38] ;for pectins, B varies between 0.072 and 0.017 indicating a relatively stiff molecules [18]. Corresponding to this rigidity, for a given molecular weight, pectins may be considered as a good thickening additive in absence of interchain interactions.
26 Table 2 Some of the values given for the persistence length of pectins Reference Ip (nm) remarks 37 15-28 - depending on DM with a minimum DM ~ 50% -light scattering experiment 13, 24 9 - SEC with light scattering detector 18 4.2-17 -intrinsic viscosity measurments 19 20 -light scattering 20 17 and 31 -light scattering 21 7 - SEC + light scattering on homogalacturonan 22 2-2.5 - SEC + light scattering 42 4.5 - Viscosity and radiation scattering
4. POLYELECTROLYTE
PROPERTIES
Due to the presence of uronic acids, pectins are polyelectrolytes ;the electrostatic properties depend directly on the charge parameter (;L) related to the average distance between 2 charged groups on the chain. The charge parameter is given by : ve 2 - DLkT
(7)
with v the number of ionic charged on a molecule of contour length L ; e is the electronic charge, D the dielectric constant of the solvent, kT the Boltzmann term. For a homogalacturonan under sodium form one can determine ;L = 1.65. Then in the case of pectins, the properties will depend on the average distribution of the methoxyl groups (reflected by DM) as discussed by Kohn [39], but also on the mode of distribution of the carboxylic groups (blokwise or random) [40] and on the length of the block (or the degree of polymerisation ) [41]. It is important to consider the effective value of ;L and not the average on the total chain. The influence of structural characteristics is specially clear on the activity coefficient of calcium counterion. This behaviour is also related to the ability of pectins to form gels in presence of divalent counterions. The activity of counterions whatever is the valency is imposed by the local charge parameter (;L) ;few experimental data were discussed previously [40, 41].
2? For the homogalacturonan, the activity coefficient of sodium is 0.54 but that of calcium 0.12, in very dilute solution, indicating a dimer formation. The activity coefficient 7 is directly imposed by ;L with 9 1
7 - 2z;C
(8)
One of the main characteristic of polyelectrolyte is the pK of the - COOH function ; as usually with polyelectrolyte only the intrinsic pK (pKo) extrapolated to zero charge characterizes the polymer [41] ; one gets 3.30 which is in same range as other carboxylic polymers ;the apparent values of pK (pKa) depends on the charge distribution, on the polymer concentration, on the ionic strength of the solution and on the nature of the counterions. The pK (a) is directly related with the effective local charge density ; one can write 1-(z e ~ (a) pKa = pH + log ~ = PKo - 0.434 kT
(9)
with (z, the degree of dissociation or neutralization and ~ (a), the electrostatic potential at the minimum distance of approach of the chain (a). A pH-induced conformational transition during neutralization was mentionned (43). In absence of external salt, strong electrostatic interchain interactions exist in solution causing very large increase of the reduced viscosity and the formation of a pseudo electrostatic 3D network ;this was recently discussed [24]. It implies the salt sensitivity of the viscosity which decreases when neutral salt is added due to a screening effect on the long range electrostatic repulsions. It is now admitted that the intrinsic viscosity in monovalent electrolyte (concentration Cs) is given by the folloving relation : [I~]CS ----[1'1]oo + S Cs-1/2
(10)
[~]o~ is the intrinsic viscosity extrapolated to infinite salt concentration. The slope S of the dependence [11] (Cs 1/2) is related to the stiffness of the molecule (Ip or B). The larger Ip is, the lower is the salt sensitivity on viscosity. The viscosity of sodium pectinate was previously examined by Pals and Hermans [44]. In salt excess (NaCI 0.1 M), the viscosity of a pectate sodium form solution (q) is related to the polymer concentration (c) and the molecular weight (or [T1] = KM a) by the relation : q-~o
~= qo
C [11] + k'(c[TI])2 + B(c[11])n
(11)
qo is the viscosity of the solvent, k' is the Huggins constant (k'= 0.3-0.5) and n is usually in the range of 3-4 when no chain interactions exist in the solution. One
28 obtains a unique curve when log ~sp (at zero shear rate) is plotted as a function of log C[T1] for different solutions with different concentrations or/and different molecular weights.
5. IONIC SELECTIVITY No specific ionic selectivity is really admitted in pectins with monovalent counterions due to the relativity low charge parameter ; a very interesting behaviour is observed when divalent counterions are considered. Specially, it was demonstrated that when DM<50% the activity coefficient of magnesium is much larger than that of calcium. The transport parameters (f) were found following the order [45] : f Ba< f Sr<< f Mg With different pectins, one found that the activity coefficient of calcium has a value half that of magnesium ;this is interprated as the basis of a dimer formation in presence of calcium. The specific interaction of calcium was described as the eggbox model first proposed for polyguluronate in which oxygen atoms coordinated to calcium [46]. Recently, the comparative behaviour of Mg and Ca with homogalacturonan was reexamined [47]. This ionic selectivity observed in solution is directly related with the ability to form gels [48]. From circular dichro'(sm it is shown that stronger interaction exists with calcium compared with sodium counterions [41,48].
6. GELATION IN PRESENCE OF CALCIUM
This mechanism of gelation is the most commonly investigated (Table 3). When DM is less than 50%, gels are formed as soon as the polymer concentration is larger than a limit (C>C*, the overlap concentration C* is in the range of [11]-1) in the presence of calcium by the egg-box mechanism first suggested for alginates [4850]. The gelation depends on the distribution of the carboxylic groups in addition of the average DM [48] and on the pH [51-53]. General behaviours describing physical gels were developped previously [54,55]. The gel strengh depends on the method used to prepare the gels, and specially the way the pectins were deesterified ; an exemple on sugarbeet pectins gives very clear data on the role of DM and compares enzyme treated and chemically modified pectins [56]. The two methods enhanced gelling ability but chemically modified pectins had a grainy texture, and became more brittle due to skrinkage ;the enzymatically modified pectins gave no skrinkage. The role of pectins treatment on gel stiffness was also demonstrated on sugar beet and citrus pectins [57].
20 The rheological behaviour in the range of LM pectin was analyzed and the solgel diagram established [59] for different stoichiometric ratios. In their paper, these authors determined the gel times for sodium pectate during calcium-induced gelation and the variation of the gel time with polymer concentration, stoichrometric ratio and temperature. Table 3 Comparison of pectin gelation Conditions Mechanism
HM Low water activity (65% sacharose) pH<3.5 H- bonding/Hydrophobic
LM Presence of multivalent counterions (Ca +2) Calcium bridge
The role of the different structural parameters were also examined ;the effect of the charge distribution seems to indicate that a block with a minimum of seven nonesterified residues along each of the two chains involved in the junctions are needed to stabilize the network (60). This group also established a phase diagram relating the polymer concentration or the overlap parameter (C [T1] ) tO the stoichiometric ratio R (R = 2 [Ca 2+] / [Coo-]) ;they define the sol-gel transition and the domain for high R and C were syneresis exist [60-63]. Concerning the sol-gel transition, using a scalling approach of this phenomenum, it was shown that the percolation model can describe the properties of the gelation transition [48, 62]. On acidic pH, the calcium pectate gel is less cooperative (more thermoreversible) but becomes stronger at neutrality. The cooperative junction zone formation is stabilized when DM decreases and carboxylic blocks allow the egg box mechanism to be effective [50, 55, 64]. A schematic representation of the gel formed is given in Figure 1. The more cooperative is the calcium fixation, the more rigid is the gel. We recently demonstrated that the elastic modulus at a constant weight concentration of pectin is directly proportional, as a first approximation, to the galacturonic acid yield. At end, it is important to mention that calcium pectate gel beads were compared with calcium alginates gel beads for all entrapment uses [65, 66] ;in this work, the authors determined the pore size of the beads by size exclusion chromatography using dextran standards and other solutes. 7. HIGH METHOXYL
PECTINS GELATION
When the ionic charge density is low or DM high and when the pH is decreased to reduce the carboxylic dissociation, the pectins form gels usually in presence of a water soluble solute added to reduce the water activity. The junction zones seem to be stabilized by hydrophobic bonds between methyl ester groups as well as hydrogen bonds [67]. For these HM gels, the gel strength is lowered when pH increases and in the same time, one gets a gradual lowering of the setting temperature.
30
(a)
/~jl
('
'
I
I
12
12
,
Ca+2
,,
~ "~c'.," ,-,o
o ,,~, - , .,,;,o
#~'-o
o~-.o,
7
(b) H30 + .
.
.
.
SUCrOSe
/
,
H
I Figure 1. Mechanisms of crosslinkage of pectins (a) LM in presence of Ca +2 (b) HM in acid medium.
The gelation characteristics for HM pectins are mainly imposed by the pH (in the range 2.0 -3.5 in presence of sucrose 60-65%). Generally the setting time increases when DM values increases from 30 to 50 (role of steric interference of the mehtyl ester groups with hydrogen bonding) ; from DM 50 to 70, the setting time decreases due to increase of the hydrophobic interaction [58]. The yield stress of these gels also show a large dependency with DM and pass through a maximum in the range of 72% [51]. Some characteristics of these gels are given in Tables 4 and
31 5. More informations are given in references 1 and 4. The conformation of the polymeric chain forming the junction zones where described previously [33]. Table 4 Characteristics of gelation of HM pectins [1,4, 68] Type Setting Temperature Setting time (s) rapid set medium rapid set slow set
(~
85-90 75-80 60-70
90-105 110-140 170-225
DM 71-75 67-70 63-66
Table 5 Effect of DM on pectin gel strength under low water activity conditions *[50] DM Yield Stress (N) 27 0.55 38 1.45 69 6.1 * 60% v/v ethylene glycol; 1% w/v pectin ; pill .9 ; 25~ Whatever is the mechanism of interchain interactions to form a three dimensional network, the basis is the formation of a cooperative junction whose stability depends on the specific energy of the linkages and the number of units cooperatively bound, as well as the number of chains involved. 8.
CONCLUSION
This paper is a short review on the physico-chemical behaviour of pectins in solution. These polysaccharides behave as worm like chain characterized by a persistence length in the range of 10nm. The role of the rhamnose content must have some importance on the stiffness of the molecules but it is not clearly demonstrated up to now. The macromolecular characterization can be obtained by gel permeation chromatography if the partly insoluble or aggregated materials is carefully taken off.The pectins can be also characterized by their effective and average charge parameter (~.) ;the difference between these two values is directly related to the carboxylic groups distribution along the chain. The blockwise or random distribution of the carboxylic groups which controls the interaction with calcium counterions also controls the ability to stabilize a gel. The HM and LM pectins give two very different types of gels ;the mechanisms of stabilization of the junction zones in the two cases are described and few characteristics given. The different molecular characteristics (DE, distribution of methoxyl or acetyl substituents, neutral sugar content or rhamnose content) play an important role on the kinetic of gelation, mechanical properties of the gel formed and also on the experimental conditions to form the stronger gels. All these points were briefly discussed.
32 9.
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
REFERENCES
W.Pilnik, A.G.J. Voragen in Adv. Plant Cell. Biochem. Biotechnol., JAI Press Ltd, Volume 1 (1992) 219. H.A. Schols, M.A. Posthumus, A.G.J. Voragen, Carbohydr. Res., 206 (1990) 117. M.C. Jarvis, Plant Cell and Environment, 7 (1984) 153. P. Reymond, S. Gr0nberger, K. Paul, M. MLiller, E.E. Farmer, Proc. Natl. AcadSci (USA), 92 (1995) 4145. C.Robin, J. De Vries, in Food Gels, Edit. P.Harris. Elsevier applied Sciences Chap. 10 (1990) 401. C.D. May, Carbohydr. Polym., 12 (1990) 79. J.A. De Vries, M. Hansen, J.Soderberg, P.E. Glahn, J.K. Pedersen, Carbohydr. Polym., 6 (1986) 165. H. Grasdalen, O.E. Bakoy, B. Larsen, Carbohydr. Res., 184 (1988) 183. J.F. Thibault, C.M.G.C. Renard, M.A.V. Axelos, P. Roger, M.J. Crepeau, Carbohydr. Res., 238 (1983) 271. E. Westerlund, P. Aman, R.E. Andersson, R. Andersson, Carbohydr. Polym., 14 (1991) 179. F.M. Rombouts, J.F. Thibault, in "Chemistry and Function of Pectins" ACS Symposium series N~ Edit M.L. Fishman, J.J. Jen, (1986) 49. J.F. Thibault, M. Rinaudo, Proc. Int. workshop on "Plant Polysaccharides, Structure and Function" Nantes, France (1984) 214. M. Rinaudo, J. Appl. Polym. Sci. Applied Polymer Symposium, 52 (1993) 11. A. Malovikova, M. Rinaudo, M. Milas, Carbohydr. Polym., 22 (1993) 87. H.G. Barth., J. Liquid Chromatography, 3 (1980) 1481. M.L. Fishman, D.T. Gillespie, S.M. Sondey, R.A. Barford, Agricultural and Food Chemistry, (1989) 584. M.L. Fishman, Y.S. El Atawy, S. M. Sonday, D.T. Gillespie, K.B. Hicks, Carbohydr. Polym., 15 (1991) 89. H.A.Deckers, C. Olieman, F.M. Rombouts, W. Pilnik, Carbohydr. Polym., 6 (1986) 361. M.A.V. Axelos, J.F.Thibault, Int. J. Biol. Macromol., 13 (1991) 77. R.C. Jordan, D.A. Brant, Biopolymers, 17 (1978) 2885. H.D. Chapman, V.J. Morris, R.R. Selvendram, M.A. O'Neill, Carbohydr. Res., 165 (1987) 53. D.Hourdet, G. Muller, Carbohydr. Polym., 16 (1991) 113. D.Hourdet, G. Muller, Carbohydr. Polym., 16 (1991) 409. G. Berth, H. Dautzenberg, G. Rother, Carbohydr. Polym., 25 (1994) 177 ;25 (1994) 187 and 25 (1994) 197. A.Malovikova, M.Milas, M.Rinaudo, R.Borsali, in "Macroions Characterization from dilute solution to complex fluids", ACS Symposium series 548, Edit. K.S.Schmitz, (1994) 297. B.Tinland, J.Mazet, M.Rinaudo, Makromol. Chem. Rapid. Commun. 9 (1988) 69. M.Rinaudo, Polymer Bull., 27 (1992) 585. M.Rinaudo in Macromolecules 1992, Edit. J. Kahovec. VSP, (1993) pp. 207. E.Fouissac, M.Milas, M.Rinaudo, R.Borsali, Macromolecules, 25 (1992) 5613. C.Gouvion, K. Mazeau, I.Tvaroska, J. Mol. Struct., 344 (1995) 157-170. A. Di Nola, G. Fabrizi, D. Lamba, A.L. Segre, Biopolymers, 34 (1994) 457. S. Cros, C. Herv6 du Penhoat, N. Bouchemal, H.Ohassan, A.Imberty, S.Perez, Int. J. Biol. Macromol., 14 (1992) 313. J.R. Ruggiero, R. Urbani, A. Cesaro, Int. J. Biol. Macromol., 17 (1995) 205. M.D. Walkingshaw, S.J. Arnott, J. Mol. Bio., 153 (1981) 1055 and 153 (1981) 1 {%7K
33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54. 55 56 57 58 59 60 61 62
63 64 65 66 67 68
E.R. Morris, D.A. Powell, M.J. Gidley, D.A. Rees, J. Mol. Biol., 155 (1981) 507. J.R. Ruggiero, R. Urbani, A. Cesaro, Int. J. Biol. Macromol., 17 (1995) 213. B.Boutherin, K.Mazeau, I.Tvaroska, Carbohydr. Polym. Submitted (1995). I.G.Plashchina, M.G.Semenova, E.E.Braudo, V.B. Tolstoguzov, Carbohydr. Polym., 5 (1985) 159. O.Smidsrod, A.Haug, Biopolymers, 10 (1971) 1213. R.Kohn, Pure and Applied Chemistry, 42 (1975) 371. J.F.Thibault, M.Rinaudo, Biopolymers, 24 (1985) 2131. G.Ravanat, M.Rinaudo, Biopolymers, 19 (1980) 2209. M.A.V. Axelos, J. Lefebvre, J.F. Thibault, Food Hydrocolloids, 1 (1987) 569. A. Cesaro, A. Ciana, F. Delben, G. Manzini, S. Paoletti, Biopolymers, 21 (1982) 431. D.T.F. Pals, J.J. Hermans, J. Polym. Sci., 3 (1948) 897 and J. Polym. Sci., 6 (1948) 733. J.F.Thibault, M.Rinaudo, Biopolymers, 25 (1986) 455. D.A. Rees, E.J. Welsh, Angew. Chem. Int. Ed. Engl., 16 (1977) 214. A. Malovikova, M.Rinaudo, M.Milas, Biopolymers, 34 (1994) 1059. J.F. Thibault, M. Rinaudo, British Polym. J., 17 (1985) 181. J.F.Thibault, M. Rinaudo, in "Chemistry and function of Pectins", ACS Symposium series n~ Edit. M.L. Fishman. J.J. Jen. (1986) pp. 61. M.Rinaudo in "Biogenesis and Biodegradation of plant cell wall polymers" ACS Symposium series n~ Edit :N.G. Lewis, M. Paice, (1989) pp. 324/ G.T. Grant, E.R. Morris, D.A. Rees, P.J.C. Smith, D. Thom. FEBS Lett 32 (1973) 195. E.R.Morris, M.J.Gidley, E.J. Murray, D.A.Powell, D.A.Rees, Int. J. Biol. Macromol., 2 (1980) 327. M.A.F.Davis, M.J.Gidley, E.R.Morris, D.A.Powell, D.A.Rees, Int. J. Biol. Macromol., 2 (1980) 330. M.J.Gidley, E.R.Morris, E.J.Murray, D.A.Powell, D.A.Rees, Int. J. Biol. Macromol., 2 (1980) 332. M. Rinaudo in "Food hydrocollo'~'ds :Structures, Properties and Functions" Edit. :K. Nishinari, E. Doi, Plenum Press, NY (1994) 21. M.Rinaudo, J. Intelligent Material Systems and Structures, 4 (1993) 210. J.A. Matthew, S.J. Howson, M.H.J. Keenan, P.S. Belton, Carbohydr. Polym. 12 (1990) 295. G. Williamson, C.B. Faulds, J.A. Matthew, D. Archer, V.J. Morris, G.J. Brownsey, M.J. Ridout, Carbohydr. Polym. 13 (1990) 387. R. Whistler, J.R. Daniel in "Food Additives" Edit A.L. Branen, P.M. Davidson, S. Salminen, Marcel Dekker NY (1990) 395. D.Durand, C.Bertrand, A.H. Clark, A.Lips, Int. J. Biol. Macromol., 12 (1990) 14. M.A.V. Axelos, J.F. Thibault in "The chemistry and Technology of Pectin" Academic Press (1991) 109. C. Garnier, M.A.V. Axelos, J.F. Thibault, Food Hydrocollo'l'ds 5 (1991) 105. M.A.V. Axelos, C. Garnier, J.F. Thibault in "The Living cell in its 4 dimensions" Proceedings of 47 ~ R~union de la division de chimie physique de la Soci~t~ Frangaise de Biophysique" Gif sur Yvette France. Edit G. Paillotin. American Institute of Physics (1991) 569. C. Garnier, M.A.V. Axelos, J.F. Thibault, Carbohydr. Res 240 (1993) 219. Ph.Debongnie, M.Mestagh, M.Rinaudo, Carbohydr. Res., 170 (1987) 137. P. Gemeiner, L. Kurillova, O. Markovic, A. Malovikova, D. Uhrin, M. Ilavsky, V. Stefuca, M. Polakovic, V. Bales, Biotechnol. Appli. Biochem. 13 (1991) 335. L. Kurillova, P. Gemeiner, M. Ilavsky, V. Stefuca, M. Polakovic, A. Welwarbova; D.T. Toth, Biotechnol. Appl. Biochem. 16 (1992) 236. D. Oakenfull, A. Scott, J. Food Sci 49 (1984) 1093. G.H. Joseph, W.E. Baier, Food Technol., 3 (1949) 18.
This Page Intentionally Left Blank
J. Visserand A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996Elsevier ScienceB.V.All rights reserved.
35
Interactions of pectins with multivalent cations" Phase diagrams and structural aspects M.A.V. Axelos, C. Garnier, C.M.G.C. Renard, J.-F. Thibault
Institut National de la Recherche Agronomique (INRA) Centre de Recherches Agro-Alimentaires Rue de la G~raudi~re, BP 1627 44316 Nantes c~dex 03, France
Abstract Despite it complexity the addition of multivalent cation in aqueous solutions of pectins is best illustrated in the phase diagram with variables: the added cation concentration and the pectin concentration. Two transitions can be detected: a phase separation always occurs when the electrolyte concentration increases separating the diagram in two main regions; in addition, in the lower, one-phase region, there may be a sol-gel transition. The position of these two transitions depends strongly on the charge density of the polymer (degree of esterification, pH) and the nature of the cation. The other relevant parameters investigated were the ionic strength and the temperature. Beside this qualitative approach structural investigations have been done by small angle X-ray and neutron scattering on pectin in presence of calcium ions. While the overall polymer conformation remains unchanged, the junction zones in the gel phase, may be viewed as rod-like cylinders whose cross-section increases with the calcium concentration from 3 to 14/~ indicating that, under this experimental conditions, large bundles of chains are formed.
1. I N T R O D U C T I O N
Much attention has been paid to the pectin-calcium system because of its application in the food industry as gelling agent. A large body of the litterature on this subject is devoted to the gel formation as function of calcium concentration, pH and temperature [1-8]. There were also some reports on the
35 interactions of pectins with other cations such as copper, zinc, cadmium, or nickel [9-14]. In a more general scheme, pectins interact strongly with cations as is expected for a polyanionic polymer in presence of opposite charges [15-19]. For these polyanions, a massive addition of monovalent salt is needed to precipitate the polymer. In contrast only a small amount of multivalent salt is necessary to get a phase separation. Sometimes gelation takes place in the one-phase region giving rise to an homogeneous gel, and to a gel with syneresis above the phase separation. The existence and relative position of all these different physical states is of considerable practical importance and must be illustrated through the determination of a phase diagram. A phase diagram, added salts versus polymer concentration, is an operative way to visualise easily the phase separation curve and the sol-gel transition line for a given polymer-cation system. In this paper we report a systematic study of the phase diagrams of pectin/calcium and pectin/copper mixtures. The relevant parameters investigated were the polymer charge density through the degree of esterification, the ionic strength, the pH and the temperature. Beside this approach structural investigations were carried out in polymer solutions and gels by small angle X-ray and neutron scattering [20-21]. Neutron scattering allows the determination of the local polymer flexibility or the persistence length whatever the ionic environment. This experimental technique has the great advantage of allowing the determination of this characteristic length directly at the same length scale that is between 10 and 500/~. In this work we study the effect of the degree of esterification on the persistence length of pectins in presence of 0.1M NaC1 by applying the Kratky method to the data [22]. X-ray scattering enhances the cation-rich zones and thus allows the determination of the network crosslinks, the so-called junction zone or egg-boxes [23]. Changes in the structure of these junctions have been followed as function of the calcium added and in presence of 0.1M NaC1.
2. E X P E R I M E N T A L SECTION
2.1 Samples The source materials were commercial pectins apple A30 and citrus pectin C73 kindly supplied by Unipectine (France) and Copenhagen Pectin Factory (Denmark) respectively. Polygalacturonic acid samples (named SR) were obtained by acid hydrolysis of a fully de-esterified citrus pectin as previously described [24]. Citrus pectins with different degree of esterification (DE) were obtained by controlled acid de-esterification [8]. The DE were determined by titrimetry, and the galacturonic acid (GalA) content was measured by the m-hydroxybiphenyl method [25]. The structural charge density parameter ~, introduced by Lifson and Katchalsky [26], is given by: ~ = e2/bDkT(1-DE/100) were e is the electron charge, kT the Boltzman term, b
37 the length of the monomeric unit (4.35/~), and D the dielectric constant of the solvent. The intrinsic viscosities were determined on the pectin solutions in 0.1M NaC1, pH 7 at 25~ by the double Huggins-Kraemer extrapolation. The chemical characterisation of the samples used are listed in Table 1. The samples were dissolved in distilled water with gentle stirring overnight. The acidic form of the samples was obtained by percolating these solutions through a strong H § exchanger. The pH of pectin solutions was adjusted with 0.1M NaOH. The solutions were filtered through a 0.8 ~tm filter. Concentrations were calculated by determination of the dry matter.
Table 1 Characteristics of the samples sample GalA(%dm) C73 76.3 C44 79.2 C40 80.9 C32 93.5 C28 92.7 A30 76.8 SR 100
DE 73 44 40 32 28 28 0
[hi (L/g) 0.562 0.362 0.329 0.141 0.271 0.282 0.086
0.44 0.90 0.97 1.09 1.16 1.16 1.61
2.2. Phase diagrams The pectin solutions were mixed at 70~ with hot CaC12 or CuC12 solutions for 3 minutes and then poured into sealed tubes. After standing at least 48 hours at the setting temperature, the tubes were tilted and phase diagrams were determined by visual inspection. In the one phase region, when the sample was seen to flow easily, it was said t h a t the system was still a sol. When the meniscus was seen not to deform under it own weight, the system was considered a gel. The sol-gel transition was taken at the onset of meniscus deformation when the tube is held horizontal. Syneresis and precipitation were detected by the presence of water at the gel surface or by the existence of large turbid aggregates which could be centrifugated. In the case of precipitation by monovalent salts (NaC1), the phase separation was t a k e n at the initial break points in the resulting optical density versus NaC1 concentration curves obtained at 600 nm. This method has been also used to detect the phase separation with CuC12 at very low polymer concentrations.
2.3 Small angle scattering Small angle X-ray scatteri~ng measurements were performed using the synchrotron radiation of the DCI storage ring at LURE (Universit~ d'Orsay,
38 France).The collected data, on beam D24, covered the scattering vector q range from 0.4 to 0.006 A -1, q = (4~sin0/2)/~, where 0 was the scattering angle and ~ the wavelength. Small angle neutron scattering measurements were carried out with the PACE diffractometer at the Laboratoire L6on Brillouin, (CE Saclay, France). The q range observed was 3.4 10 -3 to 0.2 A -1. Samples were prepared in deuterated instead of ordinary water to achieve a suitable value for the neutron contrast factor.
3. R E S U L T S AND DISCUSSION
3.1 P h a s e s e p a r a t i o n Precipitation by a monovalent salt was found to require very high electrolyte concentration: for the C73 sample at pH 4 and 20~ [NaC1]precip" = 0.37 M and was independent of the polymer concentration in the range studied. No gel phase was observed. This result is in good agreement with what has been obtained for a large number of weakly charged synthetic polymer and known as the so-called type-H systems [15]. High methoxyl pectins must be soluble in water only because of the presence of some charged groups and this solubility is reduced by screening the charge allowing attractive interactions between neutral monomers which may induce phase separation [27]. 0.0030
A
=S
0.0020
o 0.1M N a C I
v
o
0.0010
Y ...B._.~.~
0.0000
9w a t e r
Y ------~''''~-'-'~ i
i
I
i
i
2
4
6
8
10
Polymer c o n c e n t r a t i o n (gl L)
Figure 1. Influence of the ionic strength on the phase separation curve of the C73 sample at pH 7, 20~ in presence of CuC12. In contrast in presence of calcium or copper the phase separation took place for low values of the added salts and almost in stoichiometric proportion with the CO0- concentration. As shown in Figure 1 the addition of monovalent
39 salts (0.1M NaC1) have led to a decrease in the critical concentration of copper and thus to a large increase of the upper biphasic domain. The same results were obtained with calcium for a more charged pectin. These results may be interpreted as above by a decrease of the polymer solubility and in this case resolubilisation by an excess of sodium cannot be expected. The effect of the nature of the divalent cation is very pronounced as illustrated in Figure 2 on sample A30. Pectins were found to be much more sensitive to copper than to calcium. A scale of affinity towards divalent cations can be easily obtained this way [18]. This result corroborates what has been measured by pH titration upon addition of increasing amount of cations [28,29], where the order of decreasing selectivity was: Pb -- Cu >> Zn > Cd = Ni > Ca . This scale does not follow the size of the radius of the cations but is in agreement with the sequence of complex stability of Irving-Williams [30].
0.007 0.006 ~ . 0.005 =E u~ 0.004
9 calcium
.o 0.003
[] C o p p e r
Ir
m
o
0.002 0.001 0
I
I
t
I
I
2
4
6
8
10
P o l y m e r c o n c e n t r a t i o n (g/L)
Figure 2. Influence of the nature of the divalent cation on the phase separation curve for sample A30 in NaC1 0.1M, pH 7, 20~ In Figure 2 it must be noticed also that the shape of the phase separation curves were different for Cu and for Ca in the low concentration range. A relatively large amount of calcium ions are required for precipitation while only a very small amount of copper ions are needed. This observation has been attributed to the existence of an equilibrium constant between free and bound cations which will be much less important for Ca t h a n for Cu [18]. The same feature has been observed, in the same solvent whatever the DE as it is shown in Figures 3 and 4, and also in water. Concave curvatures are always found with calcium while straight lines are obtained in presence of copper. In Figures 3 and 4 the polymer concentration was expressed in mole of carboxyl groups in order to take into account the difference in the charge density between the samples. With copper (Figure 3) all the experimental phase separation points fall almost on a same line t h a t is to say t h a t the total concentration of copper required for precipitation depends only on
40
the number of carboxyl groups carried by the polymer. This result implies that the copper/pectin interaction is independent of the nature of the units surrouding
0.0040 0.0035 0.0030 A
0.0025
o
0.0015
C73
0.0020
C44 A
A30
[]
0.0010 0.0005
0.0000 0
I
I
I
I
I
0.01
0.02
0.03
0.04
0.05
COO- (M)
Figure 3. Influence of the DE on the phase separation curve for pectins at pH 7, in 0.1M NaC1, 20~ in presence of CuC12.
a given carboxyl group. In the case of calcium the experimental data cannot be reduced to a single curve. More and more calcium is required to get the precipitation as the DE increases for a given number of COO-. This behaviour indicates that the number of successive carboxyl groups along the backbone, and not only the total number, must be taken into account to explain the pectin/calcium interactions. 0.012 0.01 0.008
S
[] c 2 8
=-- 0.006 o
" c40
0.004
c48
0.002 ]
~
I
I
I
0.01
0.02
0.03
0.04
0.05
COO- (M)
Figure 4. Influence of the DE on the phase separation curve for pectins at pH 7, in 0.1M NaC1, 20~ in presence of CaC12.
41 Such a result may be put in parallel with the determination of the bound cations obtained by ion-specific electrode or dual-wavelength spectrophotometric method, and analysed in terms of cooperativity [7,29,31].
3.2 Sol-gel t r a n s i t i o n The sol-gel transition has been determined visually, with calcium and copper, for different pectins under different external conditions. As shown in Figure 5 for sample C44 the homogeneous gel phase is situated between the two transition lines. The extension of this phase was found to depend mainly on the DE, temperature and nature of the cation. With calcium the amount of cation required to get a gel increased with the degree of esterification and above 50% it became impossible to get a gel [8]. 0.009 0.008 A 0.007 0.006 .ooo = 0.004 .o 0
~
0.003 0.002 0.001
Ca
~
O
~
0
2
~
Cu
o
[]
,
,
,
,
.
,
I
4
6
8
10
12
14
16
Polymer concentration (g/L)
Figure 5. Sol-gel transition curve for sample C44 in 0.1M NaC1, pH 7, 20~ with copper (0) and calcium (O) and phase separation curve with copper (m) and cal ci um (O). On the opposite, with copper, gels were obtained more easily for high methoxyl pectins. In general the extension of the gel phase was larger with calcium than with copper. Contrary to the phase separation curve, the sol/gel transition is very sensitive to the temperature: more cations are required to get a gel phase when the temperature increases and thus the extension of the gel phase decreases [8]. The sol/gel transition as determined above is well reproducible but overestimates the real amount of cation at the transition. Gelation is a transition from liquid to solid during which the polymeric systems suffers dramatic modifications on their macroscopic viscoelastic behavior. The whole phenomenon can be thus followed by the evolution of the mechanical properties through dynamic experiments. The behaviour of the complex shear modulus G*(co) reflects the distribution of therelaxation time of the growing clusters. At the gel point the broad distribution of
42 the relaxation times gives rise to a power-law frequency variation of G*(r G* = G' + iG" with G' ~ G " ~ r ~ [32]. The critical behaviour of the mechanical properties of the cross-linking polymers near the gelation threshold is of particular interest for the question of the universality of such systems [33]. In case of calcium previous rheological studies on the A30 sample have shown t h a t the percolation theory can describe the properties of gelation transition accurately, leading to the expected value for the critical exponent A = 0.7 [34]. In case of copper some rheological experiments carried out at a given polymer concentration and increasing amount of cations indicates t h a t copper/pectin systems in the one-phase domain behave as a viscoelastic liquid rather t h a n a viscoelastic solid referred to as true gel (G'(r = G o when r with G Othe equilibrium shear modulus)[35]. Despite the lack of experimental data the range in cation and polymer concentration in which true gels may be observed seemed very limited. These results corroborate the strength of the binding of copper by pectins evidenced by the properties of the phase separation curves.
3.3 P o l y m e r c o n f o r m a t i o n The persistence length was evaluated from the transition between the scattering behaviour of an ideal coil at low q with I(q) - q2 and the scattering behaviour of a rod at large q with I(q) - q-1. A Kratky plot, I(q) x q2 versus q, consists of two regions separated by a break. The region at low q is horizontal if the conformation of the polymer is Gaussian, while the region at large q will be a straight line if the polymer chain is represented by a rod. The break point q* can be related to the persistence length Lp through equation : Lp = 6/~ / q* The persistence length of pectins, in condition of charge screening (0.1M NaC1) were estimated from data as in Figure 6.
0,00007 0,00006 0,00005 0,00004 o" 0,00003 0,00002 0,00001 0
9
0
0,05
0,1 q 1~-1)
0,15
Figure 6 . Kratky plot of sample C44 in 0.1M NaC1
|
0,2
43 Depending on the DE the Lp values are in the range 45 to 75/~. These values are satisfactorily coincident with the theoretical value obtained recently by molecular modeling: Lp = 135 /~ for homogalacturonan chain [36] instead of 400 /~ as previously computed [37]. From molecular modeling it is claimed t h a t methyl groups have no influence on the polymer conformation so the change in the persistence length with DE in 0.1 M NaC1 could be attributed to differences in the polymer/solvent interaction. In this range of q values, the determination of the persistence length is insensitive to the polydispersity of the sample which is a great advantage in contrast to results obtained from ligth scattering: Lp between 170 to 3 9 0 / ~ depending on the DE [38] or Lp = 310 or 170 ,~ [39]. A detailed discussion on this particuliar point is reported in this book by M. Rinaudo. Pectins are less flexible than pullulan, Lp about 15 ,~ [22], or amylose in 0.1 M KC1 Lp = 17/k [40], as flexible as xanthan in its disordered conformation: Lp = 50 /~ [41] and much more flexible than xanthan in its ordered state Lp = 350/~ [42].
3.4 Junction
zone structure
Assuming t h a t the scattering mainly comes from the electronic contrast between the calcium and the solvent or the polymer rather than between the polymer and the solvent, the measured intensity allows to determine the structure of the cation-rich zones. As the cation concentration increased the shape of the intensity curve changed from the one of the polymer with I(q) - q-1 for q > q* to a single straight line with I(q) ~ q-1.3 at high calcium concentration. This behaviour indicates the presence of rod-like structures with a size distribution of the cross-section radii. The average size of the cross-section radius of these structures must be determined through a Guinier plot LnqI(q) versus q as shown in figure 7.
5
S I
0
0,005
q (.~.1)
I
I
0,01
0,015
Figure 7. Guinier plot for a rod-like particle. Sample C40 in 0.1M NaC1 (6 10 .3 g/L) with calcium (6 10 .3 mol/L)
44 From the slope in figure 7, for example, a radius of 20/~ may be determined for this sample. A more complete treatment of the data as proposed by J.M. Guenet [43] leads to junction zone structures with limited size of their radii between 30 to 10 /~ for the maximum and minimum radius respectively with a number average radius of 20 A. In such structures about 10 chains may be assembled since the radius of one isolated chain is about 5 /~, measured by the same method. This calculation is very crude and does not take into account the effective volume occupied by the calcium ions and problems of steric hindrance which will necessitate molecular modeling. The length of the junction zones were not determined by this method because their length is larger than the maximum size reached by small angle X-ray scattering.
CONCLUSION
The comparison of phase diagrams obtained with different cations led to the conclusion that the binding energy between the cation and the polymer is preponderant on both sol -gel transition and phase separation. As the binding energy decreases, more cations are required to get a gel and the gel phase domain increases. It must be pointed out also that the non-ionic interactions are to be taken into account to understand the phase separation. From a structural point of view the junction zones in pectin gels with calcium will be formed by the assembly of chains with a size distribution of their radii leading to 2 or 3 chains for the smallest one and more than 20 for the biggest one.
REFERENCES
1 2 3 4 5 6 7 8 9
D.A. Rees, Carbohydr. Polym., 2 (1982) 254. J.F. Thibault and M. Rinaudo, Biopolymers, 25 (1986) 455. C.D. May, Carbohydr. Polym., 12 (1990) 79. C. Rolin and J. De Vries, Pectin in Food Gels, P. Harris (ed.) Elsevier Applied Science, London and New York 1990, p. 401. D. Durand, C Bertrand, A.H. Clark and A. Lips, Int. J. Biol. Macromol., 12 (1990) 14. M.A.V. Axelos, J. Lefebvre, C.G. Qiu and M.A. Rao, The Chemistry and Technology of Pectin, Academic Press 1991. E.E. Braudo, A.A. Soshinsky, V.P. Yurvey and V.B. Tolstoguzov, Carbohydr. Polym., 18 (1992) 165. C. Gamier, M.A.V. Axelos, J.F. Thibault, Carbohydr. Res., 240 (1993) 219. G. Manzini, A. Cesaro, F. Delben, S. Paoletti and E. Reisenhofer, Bioelectrochem. Bioenerg.,12 (1984) 443.
45
10 S. Deiana, G. Micera, G. Muggiolu, C Gessa, A. Pusino, Colloids and Surfaces, 6 (1983) 17. 11 J. Mattai and J.C.T. Kwak, Macromolecules, 19 (1986) 1663. 12 R. Kohn, Carbohydr. Res., 160 (1987) 343. 13 B.M. Nair, N.-G. Asp, M. Nyman and H. Persson, Food Chem., 23 (1987) 295. 14 P. Debongnie, M. Mestdagh, A. Domard and M. Rinaudo, Food Hydrocoll., 5 (1991) 109. 15 A. Ikegami and N. Imai, J. Polymer. Sci., 56 (1962) 133. 16 C. Allain and L. Salom~, Macromolecules, 23 (1990) 981. 17 R. Rahbari and J. Francois, Polymer, 33 (1992) 1449. 18 M.A.V. Axelos, M.M. Mestdagh, J. Francois, Macromolecules, 27 (1994) 6594. 19 L. Piculell, B. Lindman, Adv. in Colloid and Interface Science, 41 (1992) 149. 20 O. Glatter and O. Kratky (eds.), Small Angle X-ray Scattering, Academic Press, London, 1982. 21 J des Cloizeaux and G. Jannink (eds.), Les polym~res en solution: leur mod~lisation et leur structure, Les Editions de Physique, France, 1987. 22 Y. Muroga, Y. Yamada, I. Noda, M. Nagasawa, Macromolecules, 20 (1987) 3003. 23 G.T. Grant, E.R. Morris, D.A. Rees, P.J.C. Smith and D. Thom, FEBS Lett., 32 (1973) 195. 24 J.-F. Thibault, C.M.G.C. Renard, M.A.V. Axelos, P. Roger and M.J. Crepeau, Carbohydr. Res., 238 (1993) 271. 25 J.-F. Thibault, Lebensm.-Wiss. Technol., 12 (1979) 247. 26 S. Lifson and A. Katchalsky, J. Polym. Sci., 13 (1954) 43. 27 J.F. Joanny and L. Leibler, J. Phys. France, 51 (1990) 545. 28 P. Debongnie, Ph.D., Louvain la Neuve, Belgique, 1991 29 V. Dronnet, C.M.M.G.C. Renard, M.A.V. Axelos and J.F. Thibault, Carbohydr. Polym., to be published. 30 F.A. Cotton and G. Wilkinson, Basic Inorganic Chemistry, Wiley, NY, 1976. 31 C. Gamier, M.A.V. Axelos, J.F. Thibault, Carbohydr. Res., 256 (1994) 71. 32 D. Stauffer, Phys. Rep., 54 (1979) 1. 33 D. Stauffer, A. Coniglio, M. Adam, Adv. Polym. Sci., 44 (1982) 103. 34 M.A.V. Axelos and M. Kolb, Phys. Rev. Lett., 64 (1990) 1457. 35 M.A.V. Axelos, M.M. Mestdagh, unplubished results 36 S. Cros, C. Garnier, M.A.V. Axelos, A. Imberty, S. Perez, Biopolymers (1996) 37 B.A. Burton and D.A. Brant, Biopolymers, 22 (1983) 1769. 38 I.G. Plashchina, M.G. Semenova, E.E. Braudo and V.B. Tolstoguzov, Carbohydr. Polym., 5 (1985) 159. 39 H. D. Chapman, V.J. Morris, R.R. Selvendran, M.A. O'Neill, Carbohydr. Res. 165 (1987) 53. 40 P. Roger and P. Colonna, Carbohydr. Res. 227 (1992) 73. 41 M. Milas, M. Rinaudo, R. Duplessix, R. Borsali, P. Lindner, Macromolecules, 28 (1995) 3119. 42 B. Tinland and M. Rinaudo, Macromlecules, 22 (1989) 1863. 43 J.M. Guenet, J. Phys. II France, 4 (1994) 1077.
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J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
47
An Hypothesis: The Same Six Polysaccharides are Components of the Primary Cell Walls of All Higher Plants P. Albersheim l, A.G. Darvill 1, M.A. O'Neill l, H.A. Schols 2, and A.G.J. Voragen 2 1 Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, 220 Riverbend Road, Athens, GA 30602-4712, USA 2 Wageningen Agricultural University, Department oi~Food Science, Bonamweg 2, 6703 HD Wageningen, The Netherlands
PRIMARY AND SECONDARY CELL WALLS The walls of growing plant cells are called primary cell walls. So, too, are the walls of most of the cells of the succulent tissues of plants, such as the walls surrounding the spongy parenchyma cells of leaves and fruit. Polysaccharides generally constitute 90 to 100% of the structural polymers of primary cell walls. When a cell grows, the bonds between existing wall polysaccharides are broken, and as the wall expands, newly synthesized wall polysaccharides are inserted between existing ones. We believe this process involves the breaking and formation of numerous covalent and non-covalent bonds. In this way, cells can elongate many times their length without weakening the wall. Furthermore, the fine structures of some of the of primary cell wall polysaccharides undergo defined changes as the cells pass through different stages of development [ 1-9]. All secondary cell walls develop from primary cell walls. Cells no longer grow once lignin is added to their walls. Lignification, which is a key step in the conversion of a primary cell wall into a secondary cell wall, results in terminal differentiation of the encased cell. Indeed, many cells with lignified walls die. The totipotency of plant cells is limited to cells enveloped in primary walls. THE HYPOTHESIS
We hypothesize that the fundamental processes of cell wall expansion are conserved in all higher plants, that is, growth of the cells of all higher plants requires the synthesis and insertion of the same polysaccharides by the same procedures. If this hypothesis is correct, then all primary cell walls have a common set of structural polysaccharides. The commonality of the "primary cell wall polysaccharides" hypothesis does n o t require that (i) the common polysaccharides be present in all cell walls in the same proportions, (ii) the polysaccharides be
48 distributed equally throughout a wall, or that (iii) the homologous polysaccharides of the walls of different cells have identical structures. This hypothesis also (iv) does not preclude the primary cell walls of selected tissues from containing additional wall polysaccharides or proteins that have specialized functions. It is the purpose of this essay to address the question of whether all primary cell walls contain a common set of structural polysaccharides. Proteins are recognized by their functions and cellular locations as much as by their amino acid sequences. For example, cytochrome C is present in fungi, plants, and animals, but this protein has strikingly different amino acid sequences in different organisms. Nevertheless, scientists have no trouble identifying cytochrome C from its heme group and its characteristic absorption spectra, and by recognizing that the protein is located in mitochondria and has the appropriate biochemical activity. Of course, sequencing cytochrome C would reveal it contains 26 invariant amino acids out of a total of 104 amino acids. Even the same enzymes isolated from different tissues of the same organism can have different amino acid sequences (isozymes) and/or different points of attachment and different structures of carbohydrate side chains (glycoforms). Of course, the catalytic activities of enzymes greatly facilitate their identification. The fine structures of at least three of the polysaccharides of primary cell walls vary in a defined manner from tissue to tissue within a plant and from plant species to plant species, in some ways mimicking the structural variation of proteins. Moreover, the structures of the cell wall and extracellular polysaccharides of suspension-cultured sycamore cells have not changed in an observable manner in 35 years. The structural characterization of each primary cell wall polysaccharide is still a challenging research project even if the same polysaccharide from another tissue or plant has already been characterized. There is no standardized or automated procedure for elucidating the structures of polysaccharides as there are for polynucleotides and proteins. Furthermore, the structure of a polysaccharide cannot be confirmed by determining the nucleotide sequence of a gene. On the other hand, carbohydrate scientists do not have problems identifying polysaccharides even when the polysaccharide in question is a member of a structurally diverse family such as the peptidoglycans, teichoic acids, and lipopolysaccharides of bacteria, and the glycosaminoglycans of animals. For example, differences in their glycosyl compositions make it an easy matter to distinguish between heparin, chondroitin sulfate, and hyaluronic acid even though all three of these extracellular matrix glycosaminoglycans are acidic molecules built up from disaccharide subunits. Polysaccharides are generally identified from their glycosyl-residue and glycosyl-linkage compositions, and the identity is confirmed by determining the sequence and modes of attachment of the glycosyl residues of oligosaccharide subunits. Susceptibility to cleavage by a well-characterized, homogeneous enzyme is also used to identify polysaccharides. For example, homogalacturonan is the only known substrate of endopolygalacturonase.
THE POLYSACCHARIDES OF PRIMARY CELL WALLS Cellulose.--Most biologists know that cellulose (1--~4-1inked ~-D-glucan) is a polysaccharide component of all primary and secondary cell walls. Indeed, plant cell walls are
49 often referred to as cellulosic walls even though cellulose accounts for only about 20% of primary cell walls, and in some monocotyledons, may account for as little as 4% [10-13]. All primary cell walls depend on cellulose microfibrils for tensile strength. In order for primary cell walls to grow, the cellulose microfibrils must move relative to one another and the microfibrils must be kept from combining to form large, intractable aggregates of the type present in secondary cell walls. The cellulose/hemicellulose network.--The cellulose microfibrils of primary cell walls are prevented from aggregating because their surfaces are coated with hemicelluloses, polysaccharides that bind tightly to the surface of cellulose microfibrils [14]. Two herrdcelluloses--xyloglucan and arabinoxylan--are components of all primary cell walls, although the relative amounts of the two hemicelluloses varies from plant to plant. For example, xyloglucan accounts for about 20% and arabinoxylan about 5% of the walls of suspension-cultured sycamore cells, while xyloglucan makes up only about 5% and arabinoxylan about 40% of the walls of suspension-cultured maize cells [ 15]. It is possible that small amounts of a third hemicellulose, a glucomannan or galactoglucomannan, is a component of primary cell walls, as mannose invariably accounts for about 1% of primary cell walls and mannose is not a component of the six ubiquitous polysaccharides. Hemicelluloses bind tightly, via multiple hydrogen bonds, to the surface of cellulose microfibrils. It is believed that the hydrogen-bonding to the cellulose microfibrils by the cellulose-like backbone of a hemicellulose chain is eventually sterically interrupted by carbohydrate side chains of the hemicellulose [14]. Furthermore, it has been proposed that a portion of a hemicellulose chain that is sterically prevented from binding to one cellulose microfibril will form, if possible, a multiple hydrogen-bond attachment to another cellulose microfibril, thereby cross-linking the microfibrils. The cross-linking of cellulose microfibrils by hemicelluloses creates a cellulose/hemicellulose network, one of the two polysaccharide networks that endow primary cell walls with many of their physical and biological properties. The pectin network.--The second polysaccharide network present in primary cell walls is composed of pectic polysaccharides. The pectin network appears to coexist with the cellulose/hemicellulose network, that is, both networks appear to be able to share the same space [16-19]. However, the proportions of the two networks appear to vary from location to location within a single cell wall as well as from the primary wall of one type of cell to the primary wall of a another type of cell [9,20-22]. There are three pectic polysaccharides in all primary cell walls that have been studied; these are rhamnogalacturonan II, rhamnogalacturonan I, and homogalacturonan. Rhamnogalacturonan II...Endopolygalacturonase-solubilized rhamnogalacturonan fl (RG-I/) is a low molecular weight (--4.8 kDa) complex polysaccharide (11 different sugars in more than 20 different linkages) [23]. The structure of RG-II is highly conserved, as apparently identical RG-II molecules have been obtained following endopolygalacturonase treatment of the primary cell walls of rice, onion, Douglas fir, sycamore, grape, and apple, and strong evidence of the presence of this polysaccharide has been obtained in other cell walls [24-28]. RG-II has a 'homogalacturonan' backbone composed of about nine 1--~4-1inked ~-D-galactosyluronic acid
50 residues [29]. The number of residues in the backbone may depend on the particular glycosidic bond of homogalacturonan that the endopolygalacturonase cleaves when solubilizing RG-II; homogalacturonan is believed to be covalently linked to the O-1 and/or 0-4 of the terminal reducing and non-reducing galactosyluronic acid residues of the galacturonan backbone of RG1I. Four different, complex side chains are attached to 0-2 or 0-3 of four of the backbone residues [23]. These side chains sterically prevent endopolygalacturonase from cleaving the backbone, which explains why intact RG-II is released from cell walls by endopolygalacturonase. Indeed, RG-II is highly resistant to glycanase digestion [30,31]. RG-I and RG-II both contain rhamnosyl and galactosyluronic acid residues, but these polysaccharides are not structurally related. Rhamnogalacturonan l.--Rhamnogalacturonan I (RG-I) has a backbone composed of as many as 100 repeats of the disaccharide [-+2)-tX-L-rhamnosyl-(1-+4)-tx-n-galactosyluronic acid-(1---~] [32,33]. Arabinosyl- and galactosyl-rich side chains are attached to 0-4 of the rhamnosyl residues, although the proportion of rhamnosyl residues with attached side chains varies from -20% to --80% depending on the source of the polysaccharide. Indeed, this type of variation occurs even in the family of RG-I isolated from the walls of a single cell type [34]. Furthermore, the side chains can vary in size from a single glycosyl residue to 50 or more glycosyl residues [35,36]. Occasionally the side chains are terminated by fucosyl, glucuronosyl, or 4-O-methyl glucuronosyl residues [37]. Xylosyl residues always account for about 1% of RG-I [37], but their locations in the molecule have not been ascertained; the xylosyl residues may be attached to the galactosyluronic acid residues of the backbone. Much remains to be learned about the structures and distribution of the side chains of this large family of polysaccharides. Primary cell walls also contain arabinan, galactan and two forms of arabinogalactan: type I arabinogalactan with a 1-+4-1inked 13-n-galactan backbone and type 11 arabinogalactan with a 1-->3-1inked B-n-galactan backbone [38,39]. Most of the arabinan and galactan and some of the arabinogalactan of primary cell walls is present in the wall as covalently attached side chains of RG-I. On the other hand, type I and type II arabinogalactans can be isolated from primary cell walls unattached to other cell wall components, although type ]I arabinogalactan is the quantitatively predominant component of arabinogalactan proteins, more often referred to as AGPs [3,38-42]. AGPs are not considered structural components of primary cell walls, as they are water soluble and are frequently associated with the plasma membrane [3,43-45]. The question remains as to whether the type I and II arabinogalactans are the seventh and eighth polysaccharides of primary cell walls or are innate parts of RG-Is and AGPs. Indeed, arabinan and galactan might be the ninth and tenth polysaccharides of primary cell walls. The answer probably lies in whether arabinan, galactan, and the arabinogalactans are synthesized in conjunction with the synthesis of RG-I and the polypeptides of AGPs or whether they are synthesized independently and then added in the wall to the RG-I backbone and AGP polypeptides. The fact that the more abundant side chains on RG-I appear to be composed of single galactosyl or arabinosyl residues [35] while other side chains vary in size from small oligosaccharides to polysaccharides [35] does not indicate whether RG-I, with its side chains attached, is fully synthesized in the endomembrane system. The 'free' arabinan, galactan, and
51 arabinogalactan molecules may arise by cleavage of RG-I side chains. This question might be answered by isolating RG-I from the Golgi and determining whether it possesses the normal complement of arabinose- and galactose-containing side chains. Homogalacturonan.--Homogalacturonan, a homopolymer of 1-->4-1inked (X-Dgalactosyluronic acid residues, is renowned for its ability to form gels, a property widely utilized in the food industry and in all likelihood a property that determines some of the functions of pectin in primary cell walls [19,46,47]. Many (--70%) of the carboxyl groups of the galactosyluronic acid residues of primary cell wall homogalacturonan are methyl-esterified. The degree of methyl-esterification is variable [19,46,47]. It is possible that the degree of esterification of homogalacturonans decreases in proportion to the amount of time the homogalacturonan is resident in the wall. The variously esterified forms of homogalacturonan appear to be concentrated in specific regions of primary cell walls [48]. For example, the middle lamella appears to contain sparsely methyl-esterified homogalacturonan [49-53]. Homogalacturonans that are less than --50% methyl-esterified readily form gels, especially in the presence of calcium, which is present in primary cell walls. The fewer the methyl ester groups and the more the distribution of methyl esters is in blocks, the greater the propensity of the homogalacturonan chains to form gels [46,47].
The pectin network revisited.--The importance of the interconnections of the pectic polysaccharides to the integrity of the pectin network has been highlighted by the recent discovery that RG-II is present in primary walls as a mixture of monomers and dimers [54]. The dimers are covalently cross-linked by borate diesters [55,56]. If single molecules of homogalacturonan are covalently attached to both RG-I and RG-II, the covalently cross-linked RG-II dimers would explain how the network of the three types of pectic polysaccharides is covalently connected and covalently cross-linked. A portion of each of these polysaccharides can be solubilized from purified primary cell walls by treatment of the walls with pure endopolygalacturonase, even though this enzyme only cleaves homogalacturonan chains. This fact constitutes the principal evidence supporting the apparently widely held view that the three pectic polysaccharides are covalently connected. The covalent connections could be accomplished if, for example, the reducing end of RG-I is glycosidically linked to the non-reducing end of homogalacturonan and the reducing end of homogalacturonan is glycosidically linked to the non-reducing end of the galacturonan backbone of RG-II. This explanation appears to be generally accepted even though endopolygalacturonase would release all three polysaccharides even if only a single homogalacturonan chain were attached to either RG-I or RG-II but not to both polysaccharides. Other polysaccharides of primary cell walls.--A complex mixture of enzymes including endopolygalacturonase, pectin methylesterase, and/or pectin lyase solubilizes a mixture of polysaccharides from the primary cell walls of fruits [57-64]. Food scientists have referred for some 15 years to this mixture of polysaccharides as the 'hairy region' to describe the highly branched character of the polysaccharides in the fraction and to emphasize the contrast to unbranched homogalacturonan. The recent discovery of rhamnogalacturonan hydrolase [65,66], which selectively cleaves the backbone of RG-I, led to the realization that the hairy
52 region is largely composed of RG-I. This observation was confirmed by the glycosyl-residue and glycosyl-linkage compositions of the fraction and by the demonstration that treating the hairy region with rhamnogalacturonan hydrolase generates fragments of the RG-I backbone with long side chains of branched arabinan. Selected tissues of some plants contain xylogalacturonan [67], a seventh primary cell wall polysaccharide. Studies of the 'hairy regions' of apples led to the isolation of xylogalacturonan [61]. Xylogalacturonan was solubilized when the hairy region polysaccharides were treated with rhamnogalacturonan hydrolase, which cleaves the backbone of RG-I but does not cleave xylogalacturonan [66]. This result suggests that xylogalacturonan and RG-I are connected covalently in the wall. Different populations of xylogalacturonan have been isolated that have ratios of xylosyl residues to galactosyluronic acid residues ranging from 0.4-0.9, and the proportion of methyl-esterified galactosyluronic acids also ranges from 40-90% [61,62,68]. Xylogalacturonan may have a specialized function associated with storage tissues of reproductive organs, as the cell walls of peas, soybeans, watermelon, and pine pollen have all been shown to contain xylogalacturonan [60,67,69-71 ]. Certain tissues of some plants contain other polysaccharides in their primary cell walls. Apiogalacturonan, which is another possible 'seventh' polysaccharide, has a homogalacturonan backbone with mono- and di-apiosyl side chains [72,73]. Apiogalacturonan was first detected in the primary cell walls of duckweed (Lemna spp), an aquatic monocot [72,73], and has recently been identified in the cell walls of a sea grass (Zosteracea), another aquatic monocot [74,75]. The walls of the leaf sheaths (coleoptiles) of cereals (Gramineae) contain a 3- and 4linked 13-D-glucan (mix-linked glucan) [76,77]. There is evidence that the mixed-linked glucan serves as a reserve energy source for the coleoptiles in addition to its possible structural function. Up to 5% of the mass of some primary cell walls is made up of a structural glycoprotein, such as the heavily glycosylated, hydroxyproline-rich 'extensin' [78]. Primary cell walls also contain catalytic amounts of more than 100 proteins that together may account for 1% of primary cell walls. Cations, which account for 1-5% of the mass of primary cell walls, probably contribute to the physical properties of the wall through their interactions with the wall polysaccharides. The properties of apiogalacturonan, xylogalacturonan, mixlinked glucan, and structural glycoproteins undoubtedly contribute specialized functions that are only required in selected tissues of some plants. CONCLUSION This essay was written in an attempt to explain our overview of primary cell walls and to reach consensus on the nomenclature of primary cell wall polysaccharides. We present evidence supporting the hypothesis that cellulose, xyloglucan, arabinoxylan, homogalacturonan, RG-I, and RG-II are the six polysaccharides common to all primary cell walls of higher plants. In many cells, these six polysaccharides account for all or nearly all o f the primary wall polysaccharides. Like the physically interacting proteins that constitute the electron transport machinery of mitochondria, the structures of the six apparently ubiquitous polysaccharides of primary cell walls have been conserved during evolution. Indeed, we hypothesize that the common set of six structural polysaccharides of primary cell walls have been structurally
53 conserved because they physically interact while endowing the walls with closely related mechanisms for cell enlargement, cell differentiation, and resistance to pests. ACKNOWLEDGMENTS This research was supported in part by the United States Department of Energy (DOE) grant DE-FG05-93ER20115 (to AGD) and by the DOE-funded (DE-FG09-93ER20097) Center for Plant and Microbial Complex Carbohydrates. REFERENCES
10
11 12 13 14 15 16 17 18 19
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59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78
F. Liners, T. Gaspar and P. Van Cutsem, Planta, 192 (1994) 545. F. Liners and P. Van Cutsem, Protoplasma, 170 (1992) 10. P. Marty, R. Goldberg, M. Liberman, B. Vian, Y. Bertheau and B. Jouan, Plant Physiol. Biochem., 33 (1995) 409. A. Geitmann, Y.-Q. Li and M. Cresti, Protoplasma, 187 (1995) 168. M.A. O'Neill, D. Warrenfeltz, K. Kates, P. Pellerin, T. Doco, A.G. Darvill and P. Albersheim (1996) in preparation. M. Kobayashi, T. Matoh and J. Azuma, Plant Physiol. (1996) in press. T. Ishii, Carbohydr. Res. (1996), in press. H.A. Schols, M.A. Posthumus and A.G.J. Voragen, Carbohydr. Res., 206 (1990) 117. A.G.J. Voragen and W Pilnik, In: J.R. Whitacker and P.E. Sonnet (eds.) Biocatalysis in Agricultural Biotechnology, A CS Symposium Series, Volume 389, American Chemical Society, Washington, DC, 1989. L. Yu and A. Mort, In: J Visser (ed.) Pectins and Pectinases, Elsevier, Amsterdam, 1996. R.M. Weightman, C.M.G.C. Renard and J.-F. Thibault, Carbohydr. Polym., 24 (1994) 139. H.A. Schols, E.J. Bakx, D. Schipper and A.G.J. Voragen, Carbohydr. Res., 279 (1995) 265. H.A. Schols, E. Vierhuis, E.J. Bakx, and A.G.J. Voragen, Carbohydr. Res., 275 (1995) 343. H.A. Schols and A.G.J. Voragen, carbohydr. Res., 256 (1994) 83. H.A. Schols, A.G.J. Voragen and I.J. Colquhoun, Carbohydr. Res., 256 (1994) 97. H.A. Schols, C.C.J.M. Geraeds, M.F. Searle-van Leeuwen, F.J.M. Kormelink and A.G.J. Voragen, Carbohydr. Res., 206 (1990) 105. M. Mutter, I.J. Colquhoun, H.A. Schols, G. Beldman and A.G.J. Voragen, Plant Physiol., 110 (1996) 73. H.O Bouveng, Acta Chem. Scand., 19 (1965) 953. H.A. Schols and A.G.J. Voragen, In: J Visser (ed.) Pectins and Pectinases, Elsevier, Amsterdam, 1996. Y. Matsuura, Nippon N6geikagaku Kaishi, 58 (1984) 253. Y. Matsuura, Nippon N6geikagaku Kaishi, 58 (1984) 1111. T. Kikuchi and H. Sugimoto, Agr. Biol. Chem., 40 (1976) 87. D.A. Hart and P.K. Kindel, Biochemistry, 9 (1970) 2190. D.A. Hart and P.K. Kindel, Biochem. J., 116 (1970) 569. R.G. Ovodova, V.E. Vaskovsky and Y.S. Ovodov, Carbohydr. Res., 6 (1968) 328. J. Webster and B.A. Stone, Aquatic Bot., 47 (1994) 39. D.J. Nevins, D.J. Huber, R. Yamamoto and W.H. Loescher, Plant Physiol, 60 (1977) 617. Y. Kato and D.J. Nevins, Carbohydr. Res. 147 (1986) 69. M.J. Kieliszewski and D.T.A. Lamport, Plant J., 5 (1994) 157.
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J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All fights reserved.
57
Acetylation of rhamnogalacturonan I and homogalacturonan: Theoretical calculations Milou Kouwijzer a, Henk Schols b and Serge P6rez a aIng6nierie Mol6culaire, Institut National de la Recherche Agronomique, Nantes, France bLevensmiddelenchemie en -microbiologie, Landbouwuniversiteit Wageningen, Nederland
Abstract The possible confirmation of experimental data through theoretical calculations on the exact position of acetyl groups at the galacturonic acid residues in pectin backbones was investigated. With MM3(92) it was calculated that acetyl groups at both 02 and 03 of galacturonic acid in the backbone of rhamnogalacturonan I (RG-I) and homogalacturonan are energeticallyfavourable, where the most important contribution comes from an acetyl group at 02. The stabilising energies appeared to arise from interaction of the acetyl groups with both neighbouring rhamnose and galacturonic acid residues. The attachment of galactose at rhamnose in RG-I had no significant effect on the calculated results. Also, the presence of acetyl groups did not alter the conformational behaviour of the backbones very much; stable helices could be build in agreement with earlier experimental or theoretical reports.
INTRODUCTION Generally, low amounts of acetyl groups are found in native pectins. However, in (e.g.) sugar-beet pectin about 35 acetyl groups are found on every 100 galacturonosyl residues [1]. Although it is generally assumed that the acetyl groups are esterified with the galacturonic acid present in the pectin backbone, different reports have been published on their exact position. Keenan et al. presented a ~3C-NMR study of sugar-beet pectin [2]. They concluded that both of the available ring positions (02 and 03) of glycosyluronic residues can be occupied. In contrast with this, Komalavilas and Mort determined in the rhamnose-rich portion of pectin (from cell walls isolated from carrot, cotton, tobacco and tomato, after treatment with anhydrous hydrogen fluoride and NMR analysis of the resulting disaccharides) acetyl groups uniquely at the 03 position of galacturonic acid [3]. According to them, the possibility that 0 2 of the galacturonic acid residue was acetylated in the original polymer seems unlikely. Schols, Posthumus and Voragen characterised pectins isolated from apple juice produced by the liquefaction process [4]. They concluded that acetyl groups were found at 0 2 and/or 03. Lerouge et al. isolated rhamnogalacturonan I from the walls of suspension-cultured sycamore cells [5]. A combination of 1H-NMR spectroscopy and periodate oxidation provided strong evidence that every galacturonic acid residue in the backbone repeating unit is O-acetylated at either C2 or C3. Finally, Ishii isolated acetylated rhamnogalacturonan oligomers from bamboo shoot cell walls by an enzyme preparation and characterised them [6]. Fast atom bombardment (FAB-MS) and NMR spectroscopy revealed that acetylation occurred primarily at 02, but sometimes at both 0 2 and 03. In this paper we report a theoretical (molecular mechanical) study on subunits of rhamnogalacturonan I (RG-I) and homogalacturonan (HG) to investigate whether it was possible to confirm experimentally obtained results. Also, the conformational behaviour of the pectin backbones is discussed.
58 2. M E T H O D S
For all energy calculations the molecular mechanics program MM3(92) [7,8] was used. This has been widely used in carbohydrate modelling and has been shown to give a correct description of ring geometries, anomeric equilibria and linkage torsion angles with suitable accuracy [9,10]. The total energy takes into account bond stretching, angle bending, torsions (and cross terms), van der Waals interactions and electrostatics through dipole-dipole interactions from stored bond moments. Since the energies of isolated molecules were calculated, and not in an aqueous environment, the formation of intramolecular hydrogen bonds is largely overestimated [ 11 ]. To decrease this effect, a dielectric constant of 78.5 was used, which diminishes all electrostatic interactions. All calculations were performed on Silicon Graphics Indy workstations. The atomic numbering and the torsion angles of interest are shown in Figure 1. The definitions of the glycosidic torsion angles in a disaccharide A(1---)x)B are q0 = O(O5A--C1A-O1A--CXB) and /g=O(C1A--O1A-CXB-'C(x+I)B). The signs of the torsion angles are in agreement with the IUPAC-IUB conventions [ 12].
Figure 1: Atomic numbering and torsion angles of interest for GalA(1---~2)Rha (left) and GalA(1--)4)GalA (right, plots made with PLUTON [13]). For clarity, most of the hydroxyl hydrogen atoms are not shown. First, minimum energy conformations of ~-L-rhamnose (Rha), ~-D-galacturonic acid (GalA) and the latter with an acetyl group attached to 0 2 and/or 03 were calculated. These were used as building blocks for several oligosaccharides, together with a 13-D-galactose structure which was taken from the MONOBANK database [ 14]. Then, the potential energy for the Rha(1---~4)GalA disaccharide was calculated as a function of the two glycosidic torsional angles to determine the favoured conformations of this link. The same was done for the GalA(1---~2)Rha and the GalA(1---~4)GalA disaccharides. To validate that the presence of acetyl groups does not change these favoured conformations significantly, the calculations were repeated for the corresponding disaccharides with two acetyl groups attached to the galacturonic acid residue(s). These maps were later used to build stable, integer helices.
59 Since the energy is calculated in an empirical rather than an quantummechanical way, every molecule has its own zero point. Therefore, energies of different conformations of a molecule can be compared, but not energies of different molecules. To be able to compare energies of oligosaccharides with and without acetyl groups, the energies of galacturonic acid with and without an acetyl group attached to 0 2 and/or 03 were recalculated with the hydrogens at O 1 an 0 4 replaced by a methyl group. The torsion angles around the C1-O1 and the C4-O4 bonds were taken the same as in the favoured conformation calculated for the disaccharides previously. Finally, a number of oligosaccharides was constructed, shown in Schemes 1 and 2, with aid of the polysaccharide building program POLYS [ 15]. The conformations of the constituting monosaccharides were as calculated before, and all glycosidic linkages were in the calculated lowest energy conformation. The energy of each oligosaccharide was calculated. On the basis of these energies and the energy differences between the methylated galacturonic acids with and without acetyl groups, a prediction could be made for the energy of the oligosaccharides with one or more acetyl groups attached to the galacturonic acid residues marked in bold face in Schemes 1 and 2. These predictions are only valid when there is no net interaction between the acetyl group(s) and the neighbouring residues in the oligosaccharides. Therefore, the difference between these predicted energies and the truly calculated energies for the acetylated oligosaccharides indicates whether the presence of an acetyl group has a (de-)stabilising effect. 1) Rha( 1--->4)GalA( 1---~2)Rha( 1--->4)G a l a ( 1--->2)Rha(1--->4)G a l a ( 1--->2)Rha( 1---~4)GalA 2) Rha( 1--->4)GalA( 1--->2)Rha(1---->4)G a l a ( 1---~2)Rha( 1---~4)GalA( 1--->2)Rha( 1---~4)GalA 4
1" 1
Gal 3) Rha( 1---)4)GalA( 1---)2)Rha( 1~ 4 ) GalA( 1---)2)Rha( 1~4)GalA 4 4
1" 1
Gal
1" 1
Gal
4) GalA( 1---~2)Rha( 1---->4)G a l a ( 1--->2)Rha( 1--->4)G a l a ( 1--->2)Rha( 1--->4)GalA 4 4
1"
1"
1
1
Gal
Gal
Scheme 1. RG-I oligomers used in the calculations. For the monomers marked in bold face, acetyl groups were attached to 02 and/or 03 in subsequent calculations.
60
1) GalA( 1---)4)GalA( 1---)4)GalA( 1---)4)G a l a ( 1---~4)GalA( 1---)4)GalA( 1---)4)GalA 2) GalA( 1---)4)GalA( 1---~4)GalA( 1---)4)GalA( 1---)4)GalA( 1---)4)GalA( 1---)4)GalA 3) GalA( 1---~4)GalA( 1---~4)GalA( 1---~4)GalA( 1---)4)GalA( 1---)4)GalA( 1---~4)GalA Scheme 2. HG oligomers used in the calculations. For the monomers marked in bold face, acetyl groups were attached to 0 2 and/or 03 in subsequent calculations.
3. R E S U L T S A N D D I S C U S S I O N
After the minimisation of the monosaccharides, potential energy maps were calculated for the disaccharides GalA(1---)2)Rha, Rha(1---)4)GalA and GalA(1---)4)GalA, and for these disaccharides with two acetyl groups attached to the galacturonic residues. They are given in Figures 2-4. Although there are some differences between the maps with and without acetyl groups, the overall appearance and the position of the minimum does not change significantly for the GalA(1----)2)Rha and the Rha(1---)4)GalA disaccharides. In the subsequent calculations, for the GalA(1---~2)Rha disaccharide the conformation t o = 8 0 ~ ~ = - 1 6 0 ~ was used; for the Rha(1---~4)GalA disaccharide to = - 8 5 ~ ~ = 150 ~ For the non-acetylated GalA(1---~4)GalA disaccharide we calculated to = 80 ~ ~ = 100 ~ as the global minimum; the local minimum at tp = 100 ~ ~ = 170 ~ is found 0.5 kcal/mol higher in energy. For the acetylated form of this disaccharide, the order of these two minima is found to be reversed, and the energy difference is now 1.5 kcal/mol. Therefore, we have chosen to use the conformation to = 100 ~ ~ = 170 ~ for this link in the subsequent calculations, whether there are acetyl groups attached to the galacturonic residues or not. For the Gal(1---~4)Rha link we used the calculated minimum energy conformation to = -110 ~ ~ = 160 ~ 360
'
360
240
bl
24O ,r 120
120
J
0 a
i | | t i
| | | l l
120
i
|
240 ~
|
I
I
0
|
360
'
0 b
....
i ' ' ' ' ' l ' ' ' ' '
120
240
360
~p
Figure 2. Potential energy maps of a) GalA(1---)2)Rha as a function of the glycosidic dihedrals, b) the same disaccharide with the galacturonic acid acetylated at 0 2 and 03. The conformation used to build integer helices is indicated.
6l
360
360
240
240
120
120
|
0
i | , | j , , , , ,
120
a
I ' '
240
' ' '
' ' ' ' ' 1 '
360
0 b
99
. . . .
120
I
240
. . . . .
360
99
Figure 3. Potential energy maps of a) Rha(1---~4)GalA as a function of the glycosidic dihedrals; b) the same disaccharide with the galacturonic acid acetylated at 0 2 and 03. The conformations used to build integer helices are indicated. 360
360
4ol %
4ol 120
120
0
0 0
a
120
240
99
360
0
b
120
240
360
99
Figure 4. Potential energy maps of a) GalA(I~4)GalA as a function of the glycosidic dihedrals; b) the same disaccharide with the galacturonic acid acetylated at 0 2 and 03. The conformations used to build integer helices are indicated. In this way we constructed the oligosaccharides shown in Schemes 1 and 2, and methylated galacturonic acid residues with one or more acetyl groups. The energies of the non-acetylated oligosaccharides were calculated, and with the energy differences between the methylated galacturonic acid residues predictions were made for the acetylated oligosaccharides. These were compared with the actually calculated energies; the results are presented in Tables 1 and 2. Since the results for each compound do not differ more than 0.1 kcal/mol from the others, the average values are given. The energy differences in the last two lines of the Table 1 do decrease with 0.4 or 0.5 kcal/mol, however, for compound 4 in Scheme 1 when the first galacturonic acid is omitted in the oligosaccharide. Apparently, the interaction of the acetyl group at 0 3 with not only the nearest neighbour is of significant importance. As mentioned before, the calculated energies are those of isolated molecules. To decrease the influence of intramolecular hydrogen bonds formed i n v a c u o but which are not often found
62 in aqueous solutions, a high dielectric constant was used. Unfortunately, this does not only affect the formation of hydrogen bonds, but all electrostatic interactions. Therefore, all calculations (except the minimisation of the monosaccharides and the determination of the glycosidic angles in the disaccharides) were repeated with a dielectric constant of 4. This did diminish most figures, but the overall tendency seen in Tables 1 and 2 did not change. Table 1 <Ecalc - Epred> (kcal/mol) for the RG-I oligomers given in Scheme 1 with the different substituents at the monomers marked in bold face .........................
02-H,
O2-H, O3-H O2-Ac, O3-H O2-H, O3-Ac O2-Ac, O3-Ac
03-H
........
O2-Ac, O3-H O2-H,O3-Ac O2-Ac,O3-Ac -1.1 -2.3 -1.8 -3.0
-1.1 -0.6 -1.8
-0.7 -0.7 -1.3 -1.5
-1.9 -2.3 -2.5 -3.1
Table 2 < E c a l c - Epred> (kcal/mol) for the HG oligomers given in Scheme 2 with the different substituents at the monomers marked in bold face O2-H, O3-H O2-Ac, O3-H O2-H, O3-Ac O2-Ac, O3-Ac O2-H, O3-H -1.1 -0.9 -2.0 O2-Ac, O3-H -1.1 -2.1 -2.0 -3.1 O2-H, O3-Ac -0.9 -2.0 -1.8 -2.9 O2-Ac, O3-Ac -2.0 -3.0 -2.9 -4.0
..........
........
-..-~
-.
........................
-~-~- .......
: ....................
: .........
-
.....................
-~--.---::::::-.-~
.................
-~-~-
....................
~ = ~ .
...........
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...........
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: ............................
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.
.
.
.
.
.
.
.
.
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.
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.
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.
From the low-energy conformations calculated for the acetylated disaccharides, helical backbone models could be build and optimised to the nearest integer helix with the POLYS program [ 15]. The A conformation of the GalA(1---~2)Rha disaccharide combined with either the A or the B conformation of the Rha(1---~4)GalA disaccharide resulted in the RG-I helices shown in Figure 5. The AA type is a left-handed three-fold helix with an advance per repeat, h, of 6.75 ,~; the AB type is two fold, h is here 7.48 ,~. These are in close agreement with calculations on helical conformation of the RG-I backbone with the galacturonic acids not acetylated, but methylated at 06 [16]. Since these authors found that methylation does not influence the conformational behaviour significantly, the agreement between their and our results is not surprising. When our conformation B' is used, a helix closely resembling the AB helix is calculated: two folded with h equal to 7.53/k. Two stable helices were generated by optimising acetylated HG from the two low-energy conformations to integer helices. The results are shown in Figure 6. The A type is a two-folded helix with h equal to 4.14 ,~. This may be compared with a proposed 2-folded helix with h equal to 4.35/k for calcium gels (on the basis of circular dichroism, calcium stoichiometry and competitive inhibition) [17,18]. The B type is a right-handed three-fold helix with h equal to 4.45/k; in close agreement with results from fibre diffraction performed on sodium and calcium pectate gels [19,20], which yielded right-handed three-folded models with h equal to 4.3 ,~. Also, our results are in good agreement again with calculations on methylated homogalacturonan [21 ].
63
Figure 5. Acetylated RG-I helices generated from the low-energy conformations of the disaccharides (see Figures 2 and 3). Left the AA type with n = -3 and h = 6.75 .~; right the AB type with n = 2 and h = 7.48 .~.
64
Figure 6. Acetylated HG helices generated from the low-energy conformations of the disaccharide (see Figure 4). Left the A type with n = 2 and h = 4.14/k; right the B type with n = 3 and h = 4.45 ~.
65
4. C O N C L U S I O N S On the basis of our energy calculations we conclude that acetyl groups at both 0 2 and 0 3 of galacturonic acid in the backbone of rhamnogalacturonan I and homogalacturonan are energetically favourable. The favourable influence of an acetyl group at 0 2 is usually somewhat bigger than at 0 3 . This is in good agreement with Keenan et al. [2], Schols et al. [4] and Ishii [6], but less with Komalavilas and Mort [3] and Lerouge et al. [5]. Next to this, we found that the stabilising energy arises from interaction of the acetyl groups with both neighbouring rhamnose and galacturonic acid. The substitution of galactose at rhamnose in the rhamnogalacturonan backbone did not have a significant effect on the calculated results. Finally, the acetylation of galacturonic acid in RG-I and H G did not lead to significantly different helical models of these backbones. Integer helices with low-energy conformations could be build in nice agreement with other experimental or theoretical results.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
A.G.J. Voragen, W. Pilnik, J.-F. Thibault, M.A.V. Axelos and C.M.G.C. Renard; in Food Polysaccharides and Their Applications, Ed. A. M. Stephen; Marcel Dekker Inc., New York 1995; 287 M.H.J. Keenan, P.S. Belton, J.A. Matthew and S.J. Howson; Carbohydr. Res. 138 (1985) 168 P. Komalavilas and A. Mort; Carbohydr. Res. 189 (1989) 261 H.A. Schols, M.A. Posthumus and A.G.J. Voragen; Carbohydr. Res. 206 (1990) 117 P. Lerouge, M.A. O'Neill, A.G. Darvill and P. Albersheim; Carbohydr. Res. 243 (1993) 359 T. Ishii; Mokuzai Gakkaishi 41 (1995) 561 MM3(1992), QCPE, Creative Arts Building 181, Indiana University, Bloomington, IN 47405, USA N.L. Allinger, Y.H. Yuh and J.H. Lii; J. Am. Chem. Soc. 111 (1989) 8551 S. P6rez, A. Imberty and J.P. Carver; Adv. Comput. Biol. 1 (1994) 146 A.D. French and M.K. Dowd; J. Mol. Struct. (Theochem) 286 (1993) 183 M.L.C.E. Kouwijzer and P.D.J. Grootenhuis; J. Phys. Chem. 99 (1995) 13426 IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN), Eur. J. Biochem. 131 (1983) 5 A.L. Spek; Acta Crystallogr. A46 (1990) C34 S. P6rez and M.M. Delage; Carbohydr. Res. 212 (1991) 253 S.B. Engelsen, S. Cros, W. Mackie and S. P6rez; Biopolymers (1996) in press S. Cros, C. Garnier, M.A.V. Axelos, A. Imberty and S. P6rez; Biopolymers (1996) in press E.R. Morris, D.A. Powell, M.J. Gidley and D.A. Rees; J. Mol. Biol. 155 (1982) 507 D.A. Powell, E.R. Morris, M.J. Gidley and D.A. Rees; J. Mol. Biol. 155 (1982) 517 M.D. Walkinshaw and S. Arnott; J. Mol. Biol. 153 (1981) 1075 M.D. Walkinshaw and S. Arnott; J. Mol. Biol. 153 (1981) 1055 S. Cros, A. Imberty and S. P6rez; Int. J. Biol. Macromol. 14 (1992) 313
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J. Visser and A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V.All rights reserved.
67
The Pectic Polysaccharide Rhamnogalacturonan II is a major Component of the Polysaccharides present in Fruit-derived Products. Patrice Pellerin, Thierry Doco, St6phane Vidal, Pascale Williams and Jean-Marc Brillouet
Institut National de la Recherche Agronomique, Institut des Produits de la Vigne, Laboratoire des Polym~res et des Techniques Physico-Chimiques 2 Place Viala, F-34060 Montpellier Cedex, France
Abstract
Rhamnogalacturonan II (RG-II) was present in three fruit-derived products, a red wine, an apple juice and a tomato juice, at proximate respective concentrations of 100, 200 and 30 mg/L. Total polysaccharides were recovered by ultrafiltration and RG-II was isolated by sizeexclusion and anion-exchange chromatography. The composition of the three isolated fractions included the rare monosaccharides, apiose, 2-O-methyl-L-fucose, 2-O-methyl-D-xylose, Kdo, Dha and aceric acid that are characteristic of RG-II. Glycosyl-linkage analyses of neutral and acidic sugars were in accordance with the known structure of this complex pectic polysaccharide. Our results indicate that RG-II is released in an undegraded form from pectins during fruit processing and that its concentration increases with the use of liquefying enzymes.
1. INTRODUCTION
Pectins, a family of polysaccharides present in all plant cell walls, are composed of three galacturonic acid-rich polysaccharides, homogalacturonan, rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II). Rhamnogalacturonan II, the smallest and most complex pectic polysaccharide [1], can be isolated after treatment of plant cell walls by fungal endopolygalacturonase [2]. Its presence has been reported in the cell walls from sycamore (Acer pseudoplatanus), douglas fir (Pseudotsuga menziesii) [3], rice (Oryza sativa) [ 4], onion
68 a-D-GalA p -(I--->4)- lOt-D-GalA p -(I---~4)] -~-D-GalA p -(I--->4)- ~-D-GalA p n v
9
v
d~
3
3
2
2
1"
I"
1"
1"
2 fl-D-Dha p 5
2 Kdo p 5
1 fl-L-Araf
1
1" a-L-Rhap
1 fl-D-Api f 3'
1" ~L-Rhap 3
I Ac-O---> 3-~L-AceAf 2
I" 1 Ac-O--->Ot-L-Fucp -(I--)2)- ot-D-Gal p 2 4
I
1"
1
I"
Me
1 fl-D-Api f 3' 1
Ot-D-GalAp-(l-->2)-i~L-Rhap-(3<--- I)-]}--D-GalAp 4
I'
I ot-D-Xylp -(l--->3)-ot-L-Fucp 2 4
I Me
I' 1 ot-L-Ara p 2
I" I ~D-GIcA p 2
I" I ot-D-Gal p
$
1 ct-L-Rha p 2
1" n=4to8
1 ,6-L-Araf
Figure 1. The model structure of plant RG-II [ 1, 13] with four oligoglycosidic side-chains linked to the homogalacturonan backbone.
69
(Allium cepa) [5], kiwi fruit (Actinidia deliciosa) [6], Bupleurum falcatum roots [7] and Arabidopsis thaliana [8]. The structure of RG-II is extremely complex with twelve different glycosyl residues, including several rare characteristic monosaccharides such as apiose [2], 2-O-methyl-L-fucose [2], 2-O-methyl-D-xylose [2], acetic acid (3-C- carboxy-5-deoxy-L-xylose) [9], Kdo (3-
deoxy-D-manno-octulosonic acid) [10] and Dha (3-deoxy-D-lyxo-heptulosaric acid) [11] and common monosaccharides involved in unusual glycosidic linkages as for example a fully substituted rhamnopyranose. This structure [ 1,12,13] built on a backbone of seven to twelve 1,4-1inked a-D-galacturonosyl residues carrying four oligogycosidic side-chains (Figure 1) is amazingly conserved in all the plants where RG-II has been studied. The exact sites of attachment of the four oligoglycosyl side-chains to the homogalacturonan backbone remain to be determined. The polysaccharide composition and structure of pectic polysaccharides present in fruitderived products have been the aim of numerous studies [14-19]. However, the attention of scientists was focused on the analysis of rhamnogalacturonan I or so-called pectic hairy regions. As far as we know, the presence of RG-II in fruit juices has not been reported. RG-II has been purified from a red wine by the authors by size-exclusion and ionexchange chromatography [20] and found as one of the main polysaccharides in wine but also in grape must directly after grape berry pressing [21]. Composition and glycosyl-linkage analyses indicated that the RG-II from grape and wine presented all the structural features of the known model.
2. EXPERIMENTAL
2.1. Fruit-derived products used as source of pectic polysaccharides
A red wine was obtained from Carignan noir grapes (Vitis vinifera) harvested in 1991 at the INRA-Pech Rouge Experimental Station. Mature grapes were stemmed and crushed before fermentation (7 days at 28 ~
in presence of total grape berry cell wall material. The insoluble
material was finally eliminated by pressing, 5 g/hL SO2 was added and the obtained red wine stored at 12~ Golden delicious apples (Malus domestica) and tomatoes (Solanum lycopersicum) were purchased at the local market. Fruits were peeled and sliced before treatment (4 h at 45 ~ with 0.1% Pectinex|
Ultra Sp-L (Novo Ferment) in presence of 9 mM ascorbic acid. The
insoluble material was then eliminated by centrifugation and the obtained juices dialysed against distilled water.
70 2.2. Recovery of total polysaccharides and RG-II purification
Total polysaccharides were recovered in the ultrafiltration retentates on a Carbosep M5 membrane (Tech-Sep, MWCO 20 kDa) in the case of the red wine, or on a Centricon 30 membrane (Amicon, MWCO 30 kDa) in the case of the apple and tomato juices. RG-II purification from the total polysaccharide concentrates included, if necessary, several chromatography steps: - anion-exchange chromatography on a 5 x 80 cm DEAE-Macroprep (Bio-Rad) column
equilibrated in 40 mM sodium citrate pH 4.6, elution being obtained with a NaC1 stepwise elution. - size-exclusion chromatography on a 1.6 x 95 cm Sephacryl S-200 HR (Pharmacia) column equilibrated in 50 mM sodium acetate buffer pH 5, containing 50 mM NaC1. - affinity chromatography on a 5 x 70 cm Concanavalin A-Ultrogel (Sepracor) column equilibrated in 50 mM sodium acetate buffer pH 5.6, containing 150 mM NaC1, 1 mM CaC12, 1 mM MnC12 and 1 mM MgC12. This last step was used to separate the wine RG-II preparation from contaminating yeast mannoproteins.
2.3. Analytical methods
Molecular weight distributions were examined by high performance size-exclusion chromatography (HPSEC) as described [22] on two serial Shodex OHpak KB-803 and KB805 columns (0.8 x 30 cm; Showa Denko, Japan) with a OHpak KB-800P guard column (0.6 x 5 cm), equilibrated at 1 mL/min in 0.1 M LiNO3. The system was calibrated with a pullulan calibration kit (Showa Denko). Glycosyl-residue compositions were determined by GC-CIMS analysis of the per-Otrimethylsilylated methyl glycosides [23] separated on a fused-silica DB-1 capillary column. The identity of each peak was confirmed by CIMS on a HP-5989 MS Engine (HewlettPackard). Glycosyl-linkages were determined by GC-EIMS of the partially methylated alditol acetates. RG-II samples (2 mg) were methylated using sodium methyl sulfinyl carbanion and methyl iodide in dimethyl sulfoxide [24] followed by reduction of the uronosyl groups with lithium triethylborodeuteride (Superdeuteride |
Aldrich) [23,25]. Methylated and carboxyl-
reduced samples were then submitted to acid hydrolysis, NaB H4 reduction and acetylation, partially methylated alditol acetates being analysed by EIMS on two fused-silica capillary columns (DB-1 and DB-225) [20].
71
P800
I
P200
I
PSO P20
PS
a
s I
I
I
,
I
I
'-a
o t4..l
%
] I
,
j
I,,
I
# l I
I
13
I
I
17
I
I_
21
Time (min) Fig. 2: HPSEC profiles of three RG-II fractions purified from red wine (a), apple juice (b) and tomato juice (c) on Shodex OHpak KB columns. Elution times and Mw (kDa) of the pullulan standards are shown.
72 3. RESULTS AND DISCUSSION
3.1. Purification of RG-II from fruit-derived products The total polysaccharide preparations from wine, apple and tomato juices were analysed in our HPSEC system. The obtained profiles (Figure 2) were all characterized by the presence of a main sharp peak eluted at the same elution volume (18.2 to 18.6 min) as a previously purified wine RG-II [20]. In fact, the presence of rhamnogalacturonan II was detected in the three polysaccharide preparations and it could be easily purified by following the presence of its characteristic sugars (2-O-methyl-L-fucose, 2-O-methyl-D-xylose, apiose, aceric acid, Kdo and Dha) during polysaccharide fractionation by anion-exchange or size-exclusion chromatography. We finally obtained three purified RG-II fractions which gave narrow symmetrical peaks in HPSEC (Figure 2) with respective elution times of 18.29, 18.66 and 18.21 min, and corresponded to respective proximate concentrations of 100, 200 and 30 mg/L in the red wine, the apple and the tomato juices.
3.2. Composition analysis The glycosyl-residue compositions of the three purified fractions (Table 1) were very similar with a predominance of galacturonic acid, rhamnose and arabinose. The presence in the three purified fractions of the rare monosaccharides characteristic of RG-II (e.g. 2-O-methylL-fucose, 2-O-methyl-D-xylose, apiose, Kdo, Dha and aceric acid) was confirmed by GCCIMS analysis. The molar ratios corresponded approximately to the known structure of the RG-II molecule (Figure 1) and to previously published data for RG-II from sycamore [26], rice [4], arabidospis leaves [8] and Pectinol [ 12]. However, slight differences were observed between our three preparations. For example, the rhamnose content was high in the wine RG-II, arabinose being abundant in apple and galactose in tomato juice RG-II preparations. These discrepancies with the theoretical model are more likely to arise from polysaccharide contaminants such as arabinan in the case of the apple juice or arabinogalactan in the tomato juice than from an heterogeneity in the structure of the RG-II molecule. The amounts of Kdo and Dha were low in the apple-juice RG-II, aceric acid being in all samples lower than the theoretical value expected (3.5 mol %). In fact, these acidic sugars are known for their acid-lability and for their uneasy quantitative determination [8,11].
73 Table 1 Glycosyl-residue composition (mol %) of RG-II isolated from fruit-derived products Sugar residue Rhamnose
Red Wine
Apple Juice
Tomato Juice
19.8
16.6
12.9
2-O-CH3-Fucose
3.4
3.8
3.5
Fucose
2.6
4.0
2.7
2-O-CH3-Xylose
2.7
2.3
2.7
Apiose
5.9
8.3
7.5
Arabinose
11.0
17.8
13.2
Galactose
7.9
9.5
12.3
Aceric acid
1.8
1.4
1.8
35.1
31.8
34.1
Galacturonic acid Glucuronic acid
2.7
1.9
3.5
Kdo
4.1
0.8
3.1
Dha
3.0
1.8
2.7
3.3. Giycosyl-linkage analysis The RG-II samples were permethylated according to Hakomori [24] followed by reduction of their methyl-esterified carboxyl groups before the hydrolysis, reduction and acetylation steps. This procedure released the C-6 of uronic acids as acetylated and thus allowed an unambiguous identification of the galactosyl methyl ethers arising from galacturonic acid [25]. The galactosyl residues from RG-II are found as 2,3,4,6- and 3,6methyl ethers while methyl ethers arising from the uronosyl residues are found as 6-O-acetyl6,6'-dideuterated-hexitols. The identification of all the methyl ethers and the presence of dideuterated residues were confirmed by GC-EIMS analyses. The glycosyl-linkage compositions of our three RG-II preparations were similar (Table 2) and corresponded to the relative sugar molar ratios obtained from compositional (TMS derivatives) analyses indicating that methylation was complete. Almost all the methyl ethers obtained could be attributed to known residues of the RG-n molecule (Figure 1) and our data were in accordance to that previously reported for RG-II from different plant origins [3,8,12,20,26]. The relative molar ratios of these "characteristic" methyl ethers (calculated on the basis of 1 residue of 3,4-1inked fucose) were almost all in stoichiometric amounts (Table 3), confirming thus that our three preparations corresponded to the accepted model for RG-II.
74 Table 2 Glycosyl-linkage compositions (mol %) of RG-II isolated from fruit-derived products Glycosyl residue Rha
Linkage
Red Wine
Terminal 2-1inked 3-1inked 2,4-1inked 2,3-1inked 2,3,4-1inked Total
2-O-CH3-Fuc
Terminal
Fuc
3,4-1inked
Total Total 2-O-CH3-Xyl
Terminal Total
Api
3'-linked 2,3,3'-linked Total
Ara
Terminal 2-1inked 3-1inked 4 or 5-1inked 2,3-1inked Total
Gal
Terminal 3-1inked 3,6-1inked 3,4-1inked 2,4-1inked
Aceric acid b
2-1inked
Total Total GalA b
Terminal 4-1inked 3,4-1inked 2,4-1inked 2,3,4-1inked Total
GlcA b
Terminal 2-1inked
Apple Juice
Tomato Juice
7.0 6.1 4.8 0.8 4.9
3.1 5.2 3.1 2.4 4.5
4.4 1.9 3.8 0.2 0.5 6.2
(23.6) a
(18.3)
(17.0)
3.2
2.5
2.7
(3.2)
(2.5)
(2.7)
3.6
3.4
3.4
(3.6)
(3.4)
(3.4)
2.8
4.6
2.5
(2.8)
(4.6)
(2.5)
6.3 2.6
6.7 -
4.8 3.2
(8.9)
(6.7)
(8.0)
6.6 0.8 3.5
7.6 2.7 2.3 3.5
6.6 2.6 0.5 0.8 1.0
(10.9)
(16.1)
(11.5)
4.3 1.4 3.3 0.4 2.9
5.6 1.9 3.1 1.2 4.1
1.3 1.3 4.0 (6.6)
(12.3)
(15.9)
1.9
0.7
0.6
(1.9)
(0.7)
(0.6)
10.9 9.5 6.4 5.8 3.2
10.2 9.6 6.6 4.5 2.1
10.4 8.8 5.5 5.7 3.3
(35.8)
(33.0)
(33.7)
0.4 2.3
2.4
0.9 3.8
Total (2.7) a Relativemole percent of each parent sugar family within total sugars,
(4.7) (2.4) b 6,6'-dideuterated ether
75 However, slight discrepancies between our actual results and the expected data were observed as for instance the high amounts obtained for all the rhamnosyl methyl ethers. Terminal arabinose/3,4-1inked fucose ratios were close to 2 in the three preparations as initially reported for the wine RG-II [20] and confirmed by the recent discovery [27] of an additional terminal arabinofuranose residue on the aceric acid-containing oligosaccharide (Figure 1). Acetic acid could be identified by GC-EIMS as being 2-1inked indicating that this marker sugar of RG-n is present in fruit-derived RG-II's with the same linkage as in sycamore [28]. The molar ratios of galacturonosyl residues to 3,4-1inked fucose showed that the structural organization of the fruit RG-II was consistent with the model given in Figure 1. In fact, our data indicated that the average dp of the homogalacturonan backbone was close to 8 with 3 GalA in terminal non-reducing position (two of them being linked to the 2,3,4-1inked rhamnose), 3 GalA unsubstituted in the backbone (4-1inked residues) and 4 carrying the oligoglycosyl side chains either on position 2 (3,4-1inked residues) or 3 (2,4-1inked residues) [131.
Table 3 Relative molar ratios (calculated on the basis of 1 residue of 3,4-1inked fucose) of methyl ethers characteristic for RG-II in the preparations isolated from fruit-derived products Glycosyl residue
Linkage
Red Wine
Apple Juice
Tomato Juice
Rha
Terminal 2-1inked 3-1inked 2,3,4-1inked
2.0 1.7 1.3 1.4
0.9 1.5 0.9 1.3
1.3 0.6 1.1 1.8
2-O-CH3-Fuc
Terminal
0.9
0.7
0.8
Fuc 2-O-CH3-Xyl
3,4-1inked Terminal
1.0 0.8
1.0 1.3
1.0 0.8
Api
3'-linked
1.8
1.9
1.4
Ara
Terminal
1.9
2.2
1.9
Gal
Terminal 2,4-1inked
0.4 1.1
1.3 0.9
1.6 1.2
Aceric acid
2-1inked
0.5
0.2
0.2
GalA
Terminal 4-1inked 3,4-1inked 2,4-1inked
3.0 2.6 1.8 1.7
2.9 2.8 1.9 1.3
3.1 2.6 1.6 1.7
GlcA
2-1inked
0.7
0.7
1.1
76 Some additional methyl ethers, although already reported in other RG-II preparations, could not be attributed to any known residue of the molecule. It is the case of the 2,3,4-linked galacturonic acid (also reported in sycamore, Pectinol [ 12,26] and wine [20] RG-II's), of the 2,3-1inked arabinose (also found in sycamore [27] and wine [20]), of the 3,4-1inked galactose (also detected in Pectinol [26], douglas fir [3] and wine [20]) or of the 2,3,3'-linked apiose (found only in wine [20]). The presence of these unexplained residues in RG-II from several plant origins suggests the possibility of having additional linkages whose identification requires further investigations. The low amounts in 3- and 3,6-1inked galactosyl, in 3- and 5-1inked arabinosyl residues may be due to the presence of contaminating arabinogalactans and arabinans in apple and tomato juice RG-II preparations. Dha and Kdo being destroyed under the acidic conditions used to cleave glycosidic linkages [ 11] their methyl ethers could not be analyzed in this study.
4. C O N C L U S I O N
This study is the first report of the presence of rhamnogalacturonan II in fruit-derived products With the exception of the RG-II from wine [20]. Our RG-II preparations correspond very closely to the described model [ 1,13], confirming the conservation of its structure among plant cell walls. The complexity of the structure and composition of RG-II with several rare sugars uneasy to identify may be one possible explanation of why this fascinating molecule remained undetected in apple juices for such a long time. It is noticeable that RG-II remained in an apparently undegraded form after treament with pectinase-containing enzyme preparations (e.g. Pectinex | Ultra Sp-L). The use of liquefying enzymes during fruit processing induces an enrichment of the derived products in RG-II at the expense of other pectic polysaccharides like homogalacturonan or RG-I. The RG-II concentration in fruit-derived products seems to be related to the extent of liquefaction of insoluble material. The particular case of the apple juice used in the present study which was obtained after almost complete liquefaction of the fruits and contained approximately 200 mg/L of RG-II is significant in this respect. The exact relation between RG-II concentration and cell wall solubilization during enzymic fruit processing has to be stated more precisely. We believe that RG-II will be the focus of more studies in the near future, especially to determine the consequences of its presence in fruit-derived products.
77 5. A C K N O W L E D G M E N T S
Many thanks are due to Mr. Jean-Paul Lepoutre (Laboratoire des ArSmes et des Substances Naturelles, IPV, INRA, Montpellier, France) for EIMS and CIMS analyses and to Dr. Malcolm O'Neill (Complex Carbohydrate Research Center, The University of Georgia, USA) for his help in the identification of TMS derivatives.
6. A B B R E V I A T I O N S
RG-II, Rhamnogalacturonan II; Kdo, 3-deoxy-D-manno-octulosonic acid; Dha, 3-deoxy-
D-lyxo-heptulosaric acid; aceric acid, 3-C- carboxy-5-deoxy-L-xylose; TMS, per-Otrimethylsilylated methyl glycosides.
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J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
79
Partial c h a r a c t e r i z a t i o n of x y l o g a l a c t u r o n a n s f r o m cell walls of ripe w a t e r m e l o n fruit: inhibition of e n d o p o l y g a l a c t u r o n a s e activity by x y l o s y l a t i o n Liying Yu and Andrew J. Mort Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater OK 74078-3035 USA Abstract In our studies of pectins from cotton suspension culture cell walls, we consistently find a 50% methylesterified homogalacturonan (HG) region, resistant to endopolygalacturonase (EPG) digestion, associated with the rhamnose-rich pectic region commonly known as RGI. Since saponification does not enable the EPG to digest this HG and it contains approximately one Xyl residue per three GalA residues, it is likely that its resistance to digestion is caused by the xylose. Only small amounts of the Xyl-substituted HG can be obtained from the cotton cell walls. Large amounts of Xyl-substituted HG are present in the cell walls of watermelon, and we have used it to characterize the effects of xylosylation on the activity of EPG and to produce fragments of the xylogalacturonan for characterization. The action of EPG on the xylogalacturonan solubilized from watermelon walls by 0.1 M NaOH, which contains about one Xyl for every seven GalA residues, produces fragments ranging in the length of their HG backbone from one up to at least 26 residues. Apart from the expected GalA 1_3 oligomers that one would obtain from the limit digest of pectic acid, all of the fragments contained one Xyl per three or four GalA. Thus, it appears that the Xyl residues are clustered within the substrate(s) and do provide protection from digestion by EPG.
1.
INTRODUCTION
When cotton suspension culture cell walls are treated with pure endopolygalacturonase (EPG), large amounts of GalA oligomers are released, but only a small amount of the rhamnose-rich rhamnogalacturonan that is called rhamnogalacturonan I (RGI) by many is released. A subsequent treatment of the residual walls with cellulase or endoglucanase solubilizes around 40% of the rhamnogalacturonan along with xyloglucan and cellulose fragments [ 1]. Compositional analysis of the rhamnogalacturonan, which adsorbs to a DEAE Sephadex column, shows it to contain a ratio of GalA : Rha of around 1.8 : 1. The well known RGI pectic fragment has a repeating disaccharide backbone of GalA-Rha, yielding a ratio of GalA : Rha of 1 : 1. It appears, therefore, that we are isolating a complex of RGI and some homogalacturonan (HG). Even after alkaline deesterification this RGI-HG complex is resistant to EPG digestion [2]. Schols et al. [3, 4] have obtained a similar pectic fraction by digestion of fruit walls with a commercial enzyme mixture and called it modified hairy region (MHR). We hypothesized that the resistance to digestion by EPG of what we called RGI-HG complex was due to the presence of short HG segments interspersed with short RGI segments and that these HG segments were too short to be substrates for the EPG. By using HF a t - 2 3 ~ the glycosyl linkages of Rha residues can be cleaved without cleaving those of GalA [5]. Thus, we expected to be able to obtain these short HG fragments ending in Rha after an HF treatment under these conditions. Since only small quantities of the RGI-HG complex could be obtained from the cotton walls, we investigated other sources from which we might be able to obtain large enough quantities of the short HG oligomers ending in a Rha residue for structural chatracterization and use as standards. Watermelon fruit produce a large amount of a relatively uniform cell type in the mesocarp. Our initial approach for obtaining the oligomers was to treat the intact walls directly in HF and then to purify the desired oligomers. To make the oligomers suitable for high resolution anion-exchange chromatography, we saponified them. However,
80 the oligomer mixture was very difficult to resolubilize after saponification, neutralization, and drying. To circumvent these steps, we decided to saponify the cell walls before the HF treatment. The saponification treatment alone solubilized a considerable portion of the pectin, and it was used to provide information on the presence of xylose in HG regions of pectins and the influence of Xyl on EPG action.
2. MATERIALS AND METHODS 2.1.
Preparation and treatment of mild-alkali-extract
Watermelon cell walls, prepared as follows, were kindly provided by Dr. Niels O. Maness of the Department of Horticulture and Landscape Architecture of Oklahoma State University. Ripe watermelon mesocarp tissues were placed on ice, diced into small pieces, and then homogenized on ice in Tris-saturated phenol to give enzymically inactive watermelon cell walls [6]. The solids were collected on two layers of mira cloth and washed with water until the smell of phenol was gone. The crude cell walls were further washed with chloroform : methanol (1 : 1, V/V) and acetone until a fluffy consistency was obtained. The acetone-washed cell wall residue was dried in an oven at 60 ~ and stored in a brown bottle. To prepare the mild-alkali-extract, dry watermelon cell walls were suspended in a solution of 0.1 N NaOH, and allowed to react with stirring at room temperature for 15 minutes. A pH of 13, as indicated by pH paper, was kept constant during this period by addition of 0.1 N NaOH. To ensure complete reaction, the treatment was continued overnight at 4 ~ The soluble portion was separated by centrifugation at 10,000 RPM for 20 minutes in a Sorval GSA rotor. The insoluble portion was washed twice with water. The supernatants were combined and, after neutralization to pH 7.0 with acetic acid, dialyzed against distilled water and freeze dried. Dry mild-alkali-extract was digested with excess EPG (approximately 0.05 units/mg sample) from Megazyme (Sydney, Australia) by suspending in 50 mM NH4Ac buffer, pH 5.2, and incubating at room temperature with very gentle stirring for at least two days. A few drops of toluene were added to inhibit bacterial growth. Xyl residues were removed from mild-alkali-extract and EPG-digestion-resistant fragments by treatment with anhydrous HF at-12 ~ using the procedure described by Mort et al. [7]. The monosaccharide composition of all the samples was determined by GLC analysis of the trimethylsilyl methyl glycoside derivatives [ 1]. Determination of the degree of methylesterification of pectin was performed by the method of Maness et al. [8].
2.2.
Ion-exchange chromatography of unlabeled oligosaccharides
Ion-exchange chromatography was performed on a Dionex Bio-LC system (Dionex Corporation, Sunnyvale, CA) using a semi-preparative Carbopac PA 1 anion-exchange column (9 • 250 mm) with a continuous permanganate post-column detector [9]. Gradient elution of different fragments was accomplished with NH4Ac buffer, pH 5.2, with a flow rate of 2 ml/min. Samples were injected into the system equilibrated at 30 mM NH4Ac (97% water, 3% M NH4Ac), and the sample components were eluted after a 2-minute lag period using a gradient of NH4Ac from 30 mM to 400 mM over 13 minutes, then to 650 mM over 30 minutes, and to 1000 rnM over 15 minutes, with a final 5-minute hold at 1000 mM to wash the column. The initial conditions were then reestablished and held until a stable baseline was obtained prior to injection of subsequent samples.
2.3.
Gel filtration chromatography
Samples were fractionated by size on stainless steel columns packed with TSK-GEL ToyoPearl gel filtration medium, (Supelco Inc., Bellefonte, PA) in 50 mM NH4Ac, pH 5.2. For HW-55 S (fractionation range for dextrans: 1000-200,000 daltons) and HW-50 S (fractionation range for dextrans: 500-20,000 daltons), the column was 10 • 500 mm and the flow rate was 1 ml/min. For HW-40 S (fractionation range for dextrans: 100-7000 daltons),
81 the column was 22 x 500 mm and a flow rate of 2 ml/min was used. We used a Dionex reagent pump and Valco injector. The eluate was monitored with a Shodex RI-71 refractive index detector. Pooled fractions were lyophilized several times to remove the NH4Ac.
2.4.
Fractionation of labeled oligomers by ion-exchange chromatography
GalA oligomers were labeled with 2-aminopyridine (2-AP) at their reducing end by the condensation reaction described by Maness et al. [ 10]. The 2-AP labeled oligosaccharides were chromatographed on a CarboPac PAl00 HPLC anion-exchange column (4 • 250 ram) using a Dionex Bio-LC Carbohydrate System connected to a RF-535 variable excitation and emission wavelength fluorescence detector (Shimadzu, Kyoto, Japan). Labeled oligosaccharides were detected using an excitation wavelength of 290 nm, and an emission wavelength of 350 nm. The elution buffer consisted of solvent A (water), and solvent B (M phosphate buffer, pH 7.0, 250 mM NaC1). A flow rate of 1 ml/min was used. Samples were injected into the system equilibrated with 50 mM phosphate buffer and sample components were eluted after a 3-minute lag period using a gradient of phosphate buffer from 50 mM to 270 mM over 47 minutes, to 350 mM over 40 minutes, to 430 mM over 65 minutes, then to 500 mM over 5 minutes, with a final 5-minute hold at 500 mM to wash the column. The initial conditions were then maintained until a stable baseline was obtained prior to injection of the next sample.
2.5.
Gas chromatography and mass spectrometry (GC-MS)
The glycosyl-linkage composition of mild-alkali-extract was determined by GC-MS (HP 5989B Engine, Hewlett Packard, DE) of the partially methylated alditol acetates performed mainly as described by [2]. One to three gl of sample dissolved in ethyl acetate was separated on a DB-225, fused-silica, capillary column with a capillary injection system used for oncolumn injection and analyzed by GC-MS using the EI mode. The temperature program was as follows: Injection at 80 ~ hold for 4 minutes at 160 ~ increase to 220 at 2 ~ and hold for 10 minutes. The linkages of the different sugar residues were identified based on their mass fragmentation patterns and retention times relative to standards.
2.6. 1H.NMR
spectroscopy
1H spectra of samples were recorded on a Varian Unity Plus 600 MHz n.m.r, spectrometer by Dr. Feng Qiu (Department of Chemistry, CUNY College of Staten Island). Samples were lyophilized twice from DaO to eliminate exchangeable protons. 3.
3.1.
R E S U L T S AND D I S C U S S I O N
Solubilization of pectin by mild alkali
The sugar compositions of the watermelon cell walls and the alkali extract, as determined by methanolysis and trimethylsilylation, are shown in Table 1. The degree of methylesterification of the total wall GalA was about 45%. The alkali extract accounted for about 15% by weight of the cell walls, and about 28% of the GalA. It contained predominantly GalA (about 76 mole% of the sugar), but was also rich in Xyl, Ara, Gal, and Rha. The composition suggests the presence of both HG regions and RGI-like regions with Ara- and Gal-containing sidechains. The high Xyl content indicates the presence of xylan or a xylogalacturonan [ 11 ]. Because we did not fractionate the extract in any way, we do not know if the various pectic regions are covalently linked together or separate from each other.
3.2. Generation and partial fragments of pectin
characterization
of EPG-digestion-resistant
The mild-alkali-extract was digested to completion with excess EPG at room temperature for at least 48 hours. Figure 1 shows the separation of the products on a Dionex PAl ionexchange column. The limit digest of pectic acid (nonesterified HG) by this enzyme is mono-,
82 di-, and tri-galacturonic acid [ 12]. The repeating disaccharide backbone of rhamnogalacturonan is resistant to digestion. Any xylogalacturonan present should be protected from digestion in regions with a high density of xylose residues [ 11]. Table 1 Mole percent of sugars in watermelon cell walls and in the mild-alkali-extract of watermelon cell walls Material
Ara
Rha
X ) , I GalA
Man
Intact walls 10.1 4.2 15.8 62.4 0.8 Alkali extract after dialysisb 7.1 3.2 11.0 76.1 0.3 WallResidue 12.9 3.9 19.6 46.9 0.8 awt% (weight percent sugar) was calculated from the sum of the GC for a known weight of sample. b(12,000 cut off membrane)
1.2
, 0.6 0.8
o
t t
0.4_~ 0.2 o
A
S
Jl]L[I. - ' " II i111
,,
20
wt% a wt(m~;)
6.7 7.2 9.1 weights
8.0 33.8 200 1.4 51.4 30 5.2 32.3 145 of sugar detected by
[0 z
, ko.o
:
-"
,~
f= P G - d i g e s t i o n - r e s i s t a n t I i ~ [l-.'lJ'llfrag ments collecting region -
10
Glc
C
'
0
Gal
30
40
Time(min)
50
60
70
~
0.4 0.2
o
Figure 1. Chromatogram of the completely EPG-digested alkali extract on a semipreparative PA 1 column. Table 2 shows the sugar compositions of the various fractions indicated in Figure 1. Fraction A, Fraction B, and Fraction C were found to be predominantly mono-, di-, and trigalacturonic acid by co-chromatography on the PAl column with standard oligomers and by co-migration on high performance capillary zone electrophoresis [12] (data not shown). All fractions contained Rha, GalA, Ara, and Gal indicating the presence of rhamnogalacturonan throughout the entire effluent from the column. All fractions also contained significant amounts of Xyl. The material eluting in the region labeled EPG-digestion-resistant fragments contained a high ratio of GalA to Rha indicating that it was not predominantly rhamnogalacturonan even though it was resistant to EPG digestion. The high Xyl content suggests the presence of EPGresistant xylogalacturonan fragments.
83 Table 2 Glycosyl compositions (mole%) of the different fractions from PAl anion-exchange chromatography of the mild-alkali-extract treated by EPG Material
Ara
Rha
Xyl
GalA
Man
Gal
Glc
Unbound sugars 24.8 Fraction A 4.8 Fraction B 3.0 Fraction C 1.2 EPG-digestion-resistant fragments 7.6
11.2 0.2 0.8 0.9 4.8
27.0 3.4 1.2 1.1 16.6
7.9 84.5 92.1 94.7 68.0
1.3 0.7 0.3 0.4 trace
22.0 6.0 2.6 1.1 5.0
5.7 0.3 trace 0.7 trace
Schols et al. [ 11 ] have reported a high molecular weight EPG-resistant xylogalacturonan from apple which had a degree of substitution of its GalA residues with xylose of about 0.7. Since we were interested in determining if xylosylation could be the cause of the EPG resistance of the cotton HG in the RGI-HG complex, we wanted to know how high a degree of xylosylation was necessary to provide EPG resistance. If we assume (in the simplest case) that all of the Rha in the EPG-digestion-resistant fragments was in a rhamnogalacturonan with a strictly repeating GalA-Rha disaccharide backbone, we can estimate how much of the GalA was in the HG backbone. Subtracting 4.8% from the total GalA leaves 63.2 mole% of the sample. If all of the Xyl was in the HG region there would be 3.8 residues of GalA per Xyl, or a degree of substitution of the GalA residues of only 0.26. Since the EPG we used is not able to hydrolyze tri-galacturonic acid [13, 14], and requires four contiguous non-methylesterified GalA residues within partially esterfied pectins in order to act [12, 15], it is likely that four adjacent GalA residues without Xyl substitution in HG segments are needed by the enzyme for activity. The 4 : 1 molar ratio of GalA and Xyl in the EPG-digestion-resistant fragments (Table 2) is in good agreement with this assumption. To estimate the lengths of the HG backbone of the oligosaccharides protected from EPG digestion, these oligomers were treated with HF a t - 1 2 ~ to remove the Xyl residues, thus converting them into oligogalacturonans. Since the HF treatment is not totally selective, there is some degradation of the GalA chains. Also, the rhamnogalacturonan would be completely converted into mono- and disaccharides [ 16], as would any xylan. The oligosaccharides were derivatized with 2-aminopyridine and separated using a PAl00 anion-exchange column with fluorescence detection. The chromatogram resulting from the EPG-digestion-resistant fragments (Figure 2) was compared to that for a similarly labeled sample of undegraded pectic acid in which we could resolve oligomers of up to --70 GalA residues. Short oligomers could be identified by co-chromatography with commercial standards, and the identity of the longer ones inferred by assuming that the samples contained only a homologous series of GalA oligomers. Stretches of GalA residues of up to at least 25 were detected in the EPG-resistant fragments, indicating that oligomers of at least 25 residues were protected from the EPG. To obtain a more pure Xyl-GalA-containing fraction for determination of how the Xyl residues are attached to the HG, the EPG-digestion-resistant fragments from the PAl column (Figure 1) were further fractionated on an HW-50 S gel filtration column (data not shown). RGI, which is resistant to EPG digestion and is thought to be a quite high molecular weight region of pectin, especially when it still contains its sidechains of arabinans and galactans, eluted predominantly in the void volume. The sugars eluting in the fractionation range were predominantly Xyl and GalA in a ratio of around 1 : 3. The glycosyl-linkage composition of the EPG-digestion-resistant fragments obtained in the fractionation range of the HW-50 S column was determined by GC-MS of the partially methylated alditol acetate derivatives after reduction of the GalA residues to galactose. Three major products were obtained and identified as 2,3,4-tri-O-methyl- 1,5-di-acetyl-xylitol, 2,3,-di-O-methyl- 1,4,5,6-tetra-acetyl-galactitol, and 2-O-methyl-1,3,4,5,6-penta-acetyl-galactitol. This is consistant with the oligomers having a 14 linked backbone of GalA residues with some single terminal Xyl residues attached at 0-3.
84 0.6
0.14 ~0.12
,r"l
0.1
_, ',
5
, oo81111Ill i i ~
,~oo6 ~0.04 . _
1111 IIiill - ," /
-~ 0.02 m 0
05
O~ I,
:.,,., =
io4: ~"z.:
: o3
.-'
, o2~~o
a o
', 20
-0.02 0
20
40
60
01
'-
80 100 120 Time(rain)
140
,.'*' , , u .
0
160
Figure 2. Chromatogram on a PA 100 column of the 2-aminopyridine-labeled oligomers from the EPG-resistant fragments after de-xylosylation with HF at-12 ~ 3.3. Isolation of a small xylogalacturonan fragment To obtain small xylogalacturonan fragments for further characterization, the EPG digest of the total alkali extract was first separated by gel filtration chromatography on HW-55 S to remove the rhamnogalacturonan (Figure 3).
0.5
x Q} "0
Vi
0.4
-: 0.3 ._~ or 0.2 t._ Nm Q)
"0.1 0
10
I
20
I
30
40
I
'
'
'
'
50
60 Time(rain) Figure 3. Chromatogram of the EPG-digested total alkali extract on an HW-55 S gel filtration column. Fractions were pooled as indicated in the figure and analyzed for their sugar compositions (Table 3). The sugar compositions clearly show that most of the neutral sugars were concentrated in the large fragments that eluted close to the void volume of the column (Fraction
85 1 and Fraction 2), and that the material (Fraction 3) eluting in the later part of the fractionation range of the column and in the included volume was composed mainly of GalA and Xyl residues. Table 3 Glycosyl compositions (mole%) of the different fractions from gel filtration HW-55 S chromatography of the mild-alkali-extract treated by EPG Material
Ara
Rha
Xyl
GalA
Man
Gal
Glc
Alkali extract Fraction 1 Fraction 2 Fraction 3
7.1 35.9 17.5 2.0
3.2 12.9 6.3 1.1
11.0 10.5 12.6 12.8
76.1 24.9 45.0 81.1
0.3 0.3 0.4 0.5
7.2 15.3 16.6
1.4 1.1 1.6
1.8
1.4
From its sugar composition, we can infer that Fraction 1 was similar to the 'Hairy region' of pectin in apple [ 11] containing both RGI-like polysaccharide regions with arabinan and galactan sidechains and an HG with Xyl as a single unit in high density along the GalA backbone. Fraction 2 contained less Rha, but more GalA (the molar ratio of GalA to Rha was 7). The molar ratio of GalA residues to Xyl was 3.5. This fraction probably corresponds to a fraction containing smaller RGI-like polysaccharide regions but richer in xylogalacturonan fragments protected from EPG by Xyl residues. Fraction 3 from the HW-55 S column contained mainly GalA and Xyl. This fraction contained xylogalacturonan fragments plus mono-, di-, and trigalacturonic acid oligomers from the limit digest of the non-protected regions of HG. Fraction 3 from the HW-55 S column was further separated by gel filtration on an HW-40 S column (Figure 4) to search for the smallest xylogalacturonan fragment and to remove the purely GalA oligomers.
0.12 -
void volume
0.1 X
|
0.08
|
0.06
"0 C =,=,,,
Vi
== 0.04
In N'=
0.02
0
[
]
20
40
60
80
100
Time(min)
Figure 4. Chromatogram on HW-40 S column of the low molecular weight fraction (Fraction 3) from the HW-55 S column chromatography of the EPG-digested alkali extract.
86 The sugar compositions of the pooled fractions from the HW-40 S column are shown in Table 4 . Fractions 1 and 2 contained both GalA and Xyl, whereas, Fractions 3, 4 and 5 contained mainly GalA and were found to contain tri-, di-, and mono-galacturonic acid, respectively. Fraction 6 contained only a small amount of sugar and was assumed to be salt. Because GalA-containing oligomers elute earlier from gel filtration columns in low ionic strength buffers than those of the same size composed of neutral sugars [ 17], we expected that the oligomers in peak 2 would be relatively small, and that even those in the void volume (peak 1), would be quite small since they eluted late from the HW-55 S column. Unfortunately, there was not very good resolution between peaks 1 and 2. Table 4 Glycosyl compositions (mole%) of the different fractions from gel filtration HW-40 S of Fraction 3 from HW-55 S Material Fraction Fraction Fraction Fraction Fraction
Ara
Rha
Xyl
GalA
Man
Gal
Glc
4.5 2.3 0.7 0.7 0.3
3.1 0.7 0.2 0.2 0.2
23.8 22.2 3.9 1.6 0.8
63.8 74.1 93.8 95.6 96.1
0.5 0.7 0.8 0.5 0.4
4.4 1.8 0.7 1.3 1.3
trace 1.1 1.2 0.2 0.9
1 2 3 4 5
To isolate a small pure xylogalacturonan fragment, Fraction 2 from the HW-40 S gel filtration chromatography was further separated on a Carbopac PAl anion-exchange column (Figure 5). Two major fractions and a number of minor components were resolved by the PA 1 column chromatography. Four fractions as indicated in Figure 5 were collected and analyzed for their sugar compositions (Table 5 ). From the sugar compositions all four fractions appeared to be xylogalacturonan fragments containing from 3 to 4 GalA residues per Xyl.
m
/
0.8
I
/
I
/
I
/
GalA3
.
80 m~
I
/
80.6
100~
I
/
I
/
60
s"
o 0.4
/ / .~ /
,?,
0.2
I
I
I
I
/
//
0
10
20
40
m
20
~'P
I
/
o
==
I
30
40
50
60
70
o
Tirne(min)
Figure 5. Chromatogram on a PAl ion-exchange column of the material in Fraction 2 of the HW-40 S chromatography illustrated in Figure 4.
87 Table 5 Glycosyl compositions (mole%) of the different fractions from PAl anion-exchange chromatography of Fraction 2 of HW-40 S separation Material
Ara
Rha
X~r
GalA
Glc
Fraction A Fraction B Fraction C Fraction D
1.8 1.2 1.7 3.2
2.7 0.6 2.1 1.9
22.6 18.5 24.9 21.7
70.4 78.7 70.1 73.2
2.0 1.0 1.1 trace
Fraction B was chosen for further characterization because it was the most abundant. After labeling of Fraction B with ANTS, it migrated on capillary electrophoresis at around the same rate as ANTS-labeled GalA6. Since, under the conditions we used [ 12], the migration rate of oligosaccharides is thought to be inversly proportional to their radii of gyration, the oligosaccharide in Fraction B probably contained 5-7 sugar residues.
3.4. NMR spectroscopy of a xylogalacturonan fragment The 1D 1H spectrum of Fraction B of Figure 5 is shown in Figure 6. A gradient COSY spectrum (not shown) was used to assist in partial assignment of the signals. Two distinct sets of signals characteristic of 13-Xyl were observed along with those expected for an a-1,4 linked oligogalacturonan. The chemical shifts of the Xyl residues were compared to those published for a di-arabinosylxylotetraose produced from a xylan [2]. The Xyl exhibiting a signal at 4.52 ppm for H-1 had chemical shifts for all of its other protons close to those for the terminal 13-Xyl in the oligosaccharide from xylan mentioned above. The other [I-Xyl with its H-1 signal at 4.64 ppm had all of its other proton chemical shifts 0.05-0.1 ppm downfield of the fI-Xyl found in the xylan derived oligomer. The nature of the differences between the Xyl residues is not known. Schols et al. [11] reported multiple sets of terminal [~-Xyl signals in the xylogalacturonan of apple MHR. We also identified most GalA chemical shifts in the xylogalacturonan fragment. The chemical shifts agree closely with those in tri-GalA ([ 18] and [Mort and Ryan, unpublished]). By comparing the sum of the peak areas of the o~and 13H-1 resonances of the reducing GalA to that for the H-1 resonances for all the other GalA, it appears that the oligomer contains 4-5 GalA residues and probably one Xyl residue. 4.
CONCLUSIONS
Mild alkali extraction of watermelon cell walls solubilizes pectins rich in rhamnogalacturonans and xylogalacturonans. The Xyl residues in some of the xylogalacturonan regions are distributed far enough apart that EPG can digest them to produce fragments ranging in the length of their HG backbones from one up to at least 26 residues. Small fragments containing only GalA and Xyl can be purified from the EPG digest of the alkali extract. These were found to be a(1-4) linked GalA oligomers with 13-Xyl residues attached to some of the 0-3 positions. 5.
ACKNOWLEDGMENTS
This research was supported in part by DOE grant DE-FG02-93ER20102 and NSF grant EHR-9108771. It has been approved for publication by the director of the Oklahoma Agricultural Experiment Station. We wish to thank Dr. Margaret Pierce for providing valuable comments on the manuscript.
88
ppm
5.00
4.00
Figure 6. 1H NMR spectrum of the xylogalacturonan fragment found in Fraction B from the PA 1 chromatography illustrated in Figure 5. 6.
REFERENCES
1 X. Qi, B.X. Behrens, P. West and A.J. Mort, Plant Physiol., 108 (1995) 1691. 2 H. Gruppen, R.A. Hoffmann, F.J.M. Kormelink, A.G.J. Voragen, J.P. Kamerling and J.F.G. Vliegenthart, Carbohydr. Res., 233 (1992) 45. 3 H.A. Schols, M.A. Posthumus and A.G.J. Voragen, Carbohydr. Res., 206 (1990) 117. 4 H.A. Schols and A.G.J. Voragen, Carbohydr. Res., 256 (1994) 83. 5 A.J. Mort, P. Komalavilas, G.L. Rorrer and D.T.A. Lamport, in H.F. Linskens and J.F. Jackson (eds.), Plant Fibers, Springer-Verlag, Berlin (1989) 37. 6 D. Huber, Phytochemistry, 30 (1991) 2523. 7 A.J. Mort, F. Qiu and N.O. Maness, Carbohydr. Res., 247 (1993) 21. 8 N.O. Maness, J.D. Ryan and A.J. Mort, Anal. Biochem., 185 (1990) 346. 9 J. Thomas and A.J. Mort, Anal. Biochem., 223 (1994) 99. 10 N.O. Maness, E.T. Miranda and A.J. Mort, J. Chromatogr., 587 (1991) 177. 11 H.A. Schols, E.J. Bakx, D. Schipper and A.G.J. Voragen, Carbohydr. Res., 279 (1995) 265. 12 A.J. Mort and E.M.W. Chen, Electrophoresis, (in press). 13 J.F. Thibault, Carbohydr. Polym., 3 (1983) 259. 14 Z. Zhang, M.L. Pierce and A.J. Mort, Electrophoresis, (in press). 15 E.M.W. Chen and A.J. Mort, Carbohydr. Polym., (in press). 16 P. Komalavilas and A.J. Mort, Carbohydr. Res., 189 (1989) 261. 17 A.J. Mort, B.M. Moerschbacher, M.L. Pierce and N.O. Maness, Carbohydr. Res., 215 (1991) 219. 18 S.B. Tjan, A.G.J. Voragen and W. Pilnik, Carbohydr. Res., 34 (1974) 15.
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J. Visserand A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996ElsevierScienceB.V.All rights reserved.
91
Plant cell wall architecture: the role of pectins
M. C. McCann and K. Roberts
Department of Cell Biology, John Innes Centre, Colney lane, Norwich, NR4 7UH, UK.
Abstract
In order to elucidate the role of pectin in cell wall architecture, we have established a suite of techniques applicable at the single cell wall level, including the fast-freeze deep-etch rotary-shadowed replica technique, the use of immunogold probes for specific pectic epitopes, and Fourier Transform Infrared microspectroscopy. We have used these techniques on both isolated pectic polymers and pectins in situ within the cell wall in a wide variety of biological systems. Pectins are chemically heterogeneous, localised in particular cell-wall domains and developmentally-regulated. Screening for appropriate pectin and pectinase mutants using FTIR microspectroscopy offers a possible approach to determine the structural and regulatory functions of these molecules during plant growth and differentiation.
1. PECTIN F O R M S A S T R U C T U R A L N E T W O R K W I T H I N THE CELL WALL
In order to understand how cell wall architecture translates into the mechanical and rheological properties of whole tissues and organs, a minimal requirement is to find out how the three relatively invariant cell wall polysaccharide classes, cellulose, cross-linking glycans, and pectins, are put together in space. Our initial structural studies relied on first developing rapid-freeze, deep-etch, rotary-shadowed replica methods and then applying these to a very simple primary wall system, onion parenchyma cells (1). By conventional EM (Electron Microscopy) fixation, dehydration and resinembedding, the cell wall appears as a fuzzy zone in the electron microscope with little defined structure, and walls of very different composition can have a remarkably similar appearance (2). The fast-freeze, deep-etch, rotaryshadowed replica technique has several important advantages over conventional EM techniques; no chemical fixatives or dehydrants, are used, so
92 the wall is as close to the in vivo state as possible; no specific stains are necessary, all of the molecules present are visualised at high resolution: the three-dimensional molecular arrangement of the wall is preserved and can be visualised by the use of stereo pairs of micrographs.
Fig 1. Electron micrograph of a platinum/carbon replica prepared by the fastfreeze, deep-etch, rotary-shadow replica technique printed in reverse contrast. Cell walls of onion parenchyma have an elaborate structure with many thin fibres bridging between thicker cellulosic microfibrils. Scale bar represents 200nm.
Isolated wall polymers can also be visualised by spraying the polymers in glycerol onto a freshly cleaved surface of mica, drying them down in vacuo, and then rotary-shadowing the preparation with platinum/palladium and carbon (3). It is also possible to adsorb polymers to plastic-filmed gold grids, to immunolabel with a colloidal-gold-conjugated secondary antibody and then to negatively stain to see if the distribution of particular epitopes is uniform along the polymer (3) (Fig 2). By combining our images of these walls and their constituent polymers during chemical extraction, with data from immunogold labelling of thinsectioned material, we were able to construct a simple structural model of the primary cell wall of onion (Fig 3).
93
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Fig 2 Immunogold negative staining, with a monoclonal antibody (JIM 5 (13)) that recognises a relatively unesterified pectic epitope, of rhamnogalacturonans extracted from onion cell walls. Arrows indicate 5 nm colloidal gold particles. Scale bar represents 200nm. Cellulosic microfibrils of 5 to 12 nanometres diameter are spaced 20 to 40 nanometres apart. Xyloglucans, which range up to 400 nanometres in length, hydrogen-bond to two or more cellulose microfibrils, cross-linking them to form a network. The onion wall is 100 nm thick and so there is only room to accommodate 3 or 4 layers of microfibrils between the plasma membrane and the middle lamella. The middle lamella is the region of interface of two walls from neighbouring cells and, in onion, is composed mainly of calcium-cross-linked pectins. A second network of pectins that is independent but coextensive with the cellulose/xyloglucan network, limits the porosity of the wall to about 10 nanometres. Immunogold labelling with monoclonal antibody probes shows the presence of pectic epitopes throughout the cell wall (3), and the relative independence of the pectin network is implied by the relative ease with which it is extracted by chemical agents which break Ca2+ and ester bonds. Using the fast-freeze, deep-etch, rotary-shadowed replica technique, we have demonstrated that the removal of the pectin network does not seem to' affect the structural integrity of the cellulose-hemicellulose network but does increase the pore size of the meshwork of fibrils (1). It has also been shown that the pectin matrix
94 determines wall porosity using FRAP (Fluorescence recovery after photobleaching) to examine macromolecular transport of fluorescentlyderivatised dextrans and proteins of graded sizes across soybean cell walls (5). There is evidence for the presence of a large proportion of galactose in the onion cell wall (6) and a recent NMR study (7) suggests that this neutral galactan may be further involved in limiting porosity, by forming short flexible rods, anchored at one end that protrude into the pores of the network. NMR spectroscopy obtained as either solution-state or solid-state spectra can be used to determine the relative mobilities of different wall polymers and side-chains within the wall: neutral pectins such as galactans are highly mobile within the wall environment and these may further restrict access of regulatory enzymes and molecules to the cellulose-xyloglucan network (7).
MIDDLE LAMELLA
PRIMARY CELL WALL
PLASMA MEMBRANE
Fig 3 An extremely simplified and schematic representation of how three broad classes of polymer are arranged in the onion cell wall (taken from McCann and Roberts 1991 (4)). Although simplistic, the sizes and spacings of the polymers are based on direct measurements of native walls (1) and are drawn to scale. Scale bar represents 50nm.
Our 'two network' model of the primary wall receives support from a variety of indirect observations. For example it has been shown that when a cell wall is regenerated by a carrot protoplast a h o m o g a l a c t u r o n a n / rhamnogalacturonan shell is laid down first, through which the cellulose/ hemicellulose network is later intercalated (8). Further evidence that pectin may form an independent network is seen in the fact that walls from suspension-cultured cells of totnato (Lycopersicon esculentum VF 36),
95 adapted to growth on 12~tM 2,6-dichlorobenzonitrile (DCB), a cellulose synthesis inhibitor, make a pectin-rich wall that virtually lacks a cellulosexyloglucan network (9). The formation of one network has been prevented, while the other is still able to provide a cell wall with at least some of its usual functions. The cells have a reduced growth rate and their unusual cell walls differ from those of non-adapted cells by having both reduced levels of hydroxyproline (about one-third that of non-adapted walls), and a much higher proportion of homogalacturonan and rhamnogalacturonan-like polymers. More recent studies (10) further support the notion that Ca2+bridged pectates comprise the major load-bearing network in DCB-adapted cells since both tomato and tobacco cells grown on DCB can be lysed by treatment with the divalent cation chelator, cyclohexanediaminetetraacetic acid (CDTA). In the absence of suitable cell wall mutants, DCB-adapted tomato cells provide an opportunity to characterise the pectin network of the plant cell wall. It should be noted that synthesis and secretion of hemicellulose is not inhibited but, in the absence of a cellulose framework for it to stick to, most of the xyloglucan secreted remains in soluble form in the cells' culture medium (9, 10) while other non-cellulosic polysaccharides and other uronic-acid-rich polymers predominate in the wall. Infrared spectroscopy is a well-established technique in the spectroscopist's arsenal which has only recently been applied to biological samples following the advent of rapid Fourier Transform data acquisition technology that permits subtraction of water absorbance. In the spectrometer an infrared source emits radiation with a range of frequencies which is then passed through the sample. Particular chemical bonds in the sample absorb radiation of specific frequencies from the beam, and this is plotted out as an absorbance spectrum against frequency (11). The real power of this technique is in the attachment of the microscope accessory where the beam is diverted to pass through a sample sitting on the microscope stage and this permits us to define precisely the area of a single cell wall as small as 10 by 10 microns from which infrared data can be obtained (12). In the infrared region 1200-900 cm-1, pectins have a similar profile to that of polygalacturonic acid, and can easily be distinguished from non-pectic polysaccharides. When the pectins are treated with base, the ester linkage will be destroyed, and in such cases, two absorptions may be seen at about 1600 and 1420 cm-1 (antisymmetric and symmetric COO- stretches): these two peaks are then diagnostic for pectin in salt form. The most diagnostic peak in the infrared spectra of cell walls is the peak centred at about 1745 cm-1, arising from the ester carbonyl stretching associated with pectin. Its exact frequency and bandshape reveal the type (saturated-alkyl or aryl esters) a n d / o r environment of the ester groups.
96
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Fig 4 FTIR spectra of walls of DCB-adapted and n o n - a d a p t e d tomato suspension cells, onion parenchyma cell walls, and polygalacturonic acid (Sigma). a = ester peak, b = free acid stretches from pectins, y axis is absorbance, x axis is wavenumber (frequency inverse). The Fourier Transform Infrared (FTIR) spectrum obtained from nonadapted tomato cell walls is very similar to that from the onion parenchyma cell wall (both contain cellulose, xyloglucan and pectin) although there is more protein in the tomato walls (amide stretches at 1550 and 1650 cm-1) (Fig 4). In DCB-adapted tomato cell walls, the spectrum more closely resembles that of either purified pectins or of a commercial polygalacturonic acid sample from Sigma with peaks in common at 1140, 1095, 1070, 1015 and 950 cm-1 in the carbohydrate region of the spectrum as well as the free acid stretches at 1600 and 1414 cm-1 and an ester peak at 1725 cmq. An ester band at 1740 cm-1 is evident in both onion parenchyma and non-adapted tomato cell wall samples. It is possible that this shift in the ester peak simply reflects the different local molecular environment of this bond, but it is also possible that a different ester is made in the DCB-adapted cell walls, as phenolic esters absorb around 1720 cm-1 whilst carboxylic esters absorb at 1740 cm-1. The
97 carboxylic acid stretches at 1600 and 1414 cm-1 are more prominent in the adapted walls, indicating a much higher proportion of unesterified pectin in these walls. These results are consistent with chemical analyses which indicate that unadapted walls contain about 20% uronic acid, whereas in adapted walls this component rises to about 40-45% of the dry weight. Using conventional electron microscopy techniques, the appearance and thickness of the adapted walls are very similar to unadapted walls (2). We infer from this that conventional EM stains such as uranyl acetate and lead citrate show some specificity for pectic polymers and that neutral molecules such as cellulose and xyloglucan are not well imaged by these stains. The ability of the adapted cells to regulate the thickness of the walls they make in the absence of a normal cellulose/xyloglucan network raises the question of what factors normally limit the thickness of a cell wall, which seems to be under relatively strict developmental control and is genotypespecific. Two possibilities exist: either the width is determined by an independent mechanism which acts on both networks or the width is determined by one network that in turn regulates the other. As wall thickness is conserved in the absence of the cellulose/xyloglucan network it seems very likely that, by default, the pectin ( a n d / o r other non-cellulosic wall components) are involved in its determination. Replicas of the tomato cell walls are very similar to those of onion parenchyma cell walls but replicas of the DCB-adapted walls did not show the structure of the walls clearly. The principle components of the adapted walls are shorter thinner fibres which seemed to form a gel-like structure with little evidence of long cellulosic microfibrils characteristic of the unadapted cells. It is possible that such a gel will bind water more strongly and reduce the amount of etching that takes place, resulting in a less well-defined replica (2).
2. PECTINS ARE SPATIALLY LOCALISED IN CELL WALL DOMAINS Even within the relatively simple onion cell wall, different pectin subpopulations can be distinguished by their sugar composition (6), FTIR spectra (12), antibody affinities and length distributions (Na2CO3-extractable pectins are larger molecules than CDTA-extractable pectins) (4). Using two monoclonal antibodies that recognise relatively methyl-esterified and unesterified pectic epitopes (13), we have shown the variation of pectic polysaccharide localisation between species, between tissues, and between domains within a single wall. In some species, onion (2), tomato, and sugar beet (13), the interface regions between cells, ie the middle lamella and the cell corners, are rich in relatively unesterified pectins which may function in cell-cell adhesion and play an important structural role in tissue integrity. Cell corners, in particular, may act as joists in the scaffolding function of the wall, bearing much of the mechanical load of the tissue (Jeronomidis, pers. comm.). In Zinnia leaves, although all of the cell-walls contain methyl-esterified pectin,
98 this is localised to an outer region of the wall in the mesophyll and palisade cells. The outer layer of esterified pectin may restrict cell-cell adhesion so that air spaces can develope (14). Pathogens also tend to attack cell corners, and the complex mixture of pectins and arabinogalactan proteins found there may have a role in enmeshing the invading organism as well as signalling defense mechanisms to operate through the release of small pectic fragments. Within a plant, different cell types such as cortex, epidermis, phloem sieve tubes or xylem each have different proportions of wall components and therefore presumably different architectures. Cell walls also display different surface markers, arabinogalactan proteins, that reflect early developmental cell patterning and cell positions within the plant (15). As well as tissuespecific differences that arise from the cell's developmental history, the wall is also locally modified in structure and composition at different regions of the cell surface. Even around a single cell, modifications occur that distinguish between transverse and longitudinal walls, for example in sieve tube elements there is a preferential digestion of end walls. We know that mechanisms for polarised secretion of wall components exist such that one wall of a cell may have a different composition from the wall on the opposite side of the cell. In carrot root cap cells, wall thickness, composition and distribution of pectic epitopes differ between the wall facing the soil and that facing the epidermal cell (4). Some substances such as waxes are only secreted to the outer epidermal face. Three times as much wall material must be deposited on the outer epidermal face to allow the same change in length for both surfaces of the cell as the cell wall on this surface is three times thicker. Within a single wall there are zones of different architecture; the middle lamella, plasmodesmata, casparian strips, thickenings, channels, pitfields and the cell corners, and there are also domains across a wall where the degree of pectin esterification is modified. Immunogold labelling with an antibody to unesterified pectin, showed that three zones could be defined across the width of even the simple onion parenchyma wall: a distinct layer of rigid rod-like polymers in the middle lamella, a fuzzy zone next to this also labelled, and then a region nearest to the plasma membrane which does not label (3). The size of micro-domains in the wall compared with the sizes of the polymers that must be accommodated in these regions implies that mechanisms must exist for packaging and positioning of large molecules. For example, unesterified pectins can range up to 700nm in length and yet, in some cell types, are accommodated in a middle lamella of 10 to 20 nm width, and so must at least be constrained to lie parallel to the plasma membrane. Pectic gels made from extracted pectins have hundred-fold greater volumes than the cell walls from which they are extracted. This micro-diversity within walls is now changing our view of the wall from a homogeneous and uniform building material to a mosaic of different wall architectures, a tesselated structure, in which the tiles have unique properties that each contribute to the multifunctional properties of the apoplast.
99 3. PECTINS ARE MODIFIED D U R I N G CELL G R O W T H
A major goal in cell wall research is to understand the molecular basis of cell elongation since it is the cell wall that places constraints on both the size and shape of the cell (16). ,Expansion can occur either isodiametrically or by elongation to generate a range of specialised cell shapes, and the rate of expansion must somehow be coupled to the rate of cell division. Given the spatial complexities of wall architecture, the problem is not simply how coordinate regulation of wall polysaccharide synthesis and deposition is effected in a single cell, but how two or three different architectures (eg at tissue boundaries where two longitudinal walls of different tissue type and one transverse wall meet and adhere) can be synchronously reorganised such that shearing between adjacent cells does not occur. Another major question is how the necessary re-orientation of cellulose and other wall polymers is achieved to establish the direction in which the cell will elongate. From our simple model, one prediction that can be made is that changes in both networks, the cellulose/xyloglucan network and the pectin network, are demanded during cell growth. Cell shape is essentially predetermined by the net orientation of cellulose microfibrils within the cell wall and this in turn appears to depend on the net orientation of cortical microtubules within the cell. In the switch from isodiametric to elongation growth, transversely oriented cortical microtubule arrays may determine the orientation of newly deposited cellulose microfibrils. What happens to other wall polymers when cellulose microfibrils become oriented? Polarisers can be inserted in the path of the IR beam to determine whether band frequencies of specific functional groups are oriented transversely or longitudinally with respect to the long axis of the cell (17). Polarised FTIR microspectroscopy shows that both cellulose and free acid stretches attributable to pectin are oriented transversely to the direction of cell elongation during growth of both carrot and tobacco suspension cells (17, 18). The polarised spectra of epidermal tissue in an elongating carrot stem shows that an extreme case of polarisation of molecules exists in the epidermis with virtually every peak polarised including those associated with pectic polysaccharides and proteins (17). This may reflect the putative role of epidermal tissue to act as the major constraint in organ growth. Supportive evidence for the idea of oriented pectin deposition in epidermal tissue comes from indirect immunofluorescence microscopy of glancing sections of pea stem epidermis using the antibody to unesterified pectin. Images show a clear banding pattern of the pectic epitope transverse to the long axis of the cell (17). Other tissue types have distinctive spectra in which only some components are polarised. In the elongating cortex of carrot stem, peaks in the carbohydrate region 1200 to 900 and from protein are polarised, while the stele has polarised phenolics, protein and a dramatically different carbohydrate profile (17). It seems that the organisation of cell wall architecture during elongation is tissue-specific and may be species-specific, reflecting tissuespecific and species-specific differences in composition. As the wall remains the same thickness and the microfibril spacing is
100 also maintained, it is clear that a cell that has elongated 20-fold will have only 1/20th of the original wall material remaining in the final elongated wall. It is therefore probable that the polarised peaks in the spectra arise from newly deposited wall components during growth. However, we cannot deduce the absolute orientation of these molecules with respect to the long axis of the cell without more detailed information on their conformations. One of the most common modifications to pectins is methyl esterification of the carboxylic acid functional groups resulting in screening of the negative charges used for calcium cross-linking. In walls of tobacco suspension cells, the proportion of total esters is initially about 50% in dividing cells, rises to 78% during maximal elongation and then drops to 68% at stationary phase after elongation has stopped (18). Methyl esters account for only one half of total ester in tobacco cells during the cell division phase. However, the proportion of methyl esters rises to about two-thirds during elongation and the increase in esterification is accounted for entirely by methyl esters. The proportion of methyl ester does not decline at stationary phase, so the decrease in total ester must be due to loss of a different ester. An increase in methyl ester accompanies cell elongation but loss of non-methyl esters is associated with cessation of expansion.
Table 1 Proportions of uronic acid and methyl and non-methyl esters in tobacco suspension cell walls (mol%)
Tobacco Cells
Uronic Acid
GalA
Total ester Methyl ester Other ester
Dividing Elongating Elongated
Day 5 9 16
26 19 21
53 78 68
25 51 48
28 27 20
The mole fraction of uronic acid in polymers in the walls of unadapted cells decreases slightly during the culture period. However, there is a large increase in the mass of all polymers during cell expansion and elongation. The proportion of total GalA esters also increases transiently from 65% to 80% during maximal elongation of maize coleoptiles (19). This transient increase is a result of formation of an unidentified ester, whereas methyl esters decreased slightly in proportion throughout elongation. GLC-MS analysis shows that only a proportion of the ester can be accounted for by
101 methyl esters in both cell types. In tobacco, the proportion of the unidentified esters remains unchanged as the cells transit from division to expansion, but, as in maize coeoptiles, decreases in these esters accompany culmination of growth events. Carrot suspension cells also show a decrease in esterification after elongation (17). The alterations in methyl and total esterified uronic acids measured by GLC-MS are consistent with results from immunolabelling of thin sections with monoclonal antibodies that recognise specific pectic epitopes. Immunolabelling density with JIM 5, a monoclonal antibody that recognises a relatively unesterified pectic epitope, is almost abolished in all walls during elongation of unadapted cells whilst labelling with JIM 7, a monoclonal antibody that recognises a relatively methylesterified pectic epitope, is increased (18). Although JIM 7 labels walls of cells at stationary phase reflecting the lack of change in methyl ester content, JIM 5 labelling density is substantially increased by only a 10% decrease in total ester (18). Hence the pectin in the wall is close to the limit of pectin esterification at which the antibody will still bind and de-esterification is due to loss of other esters implying the activity of a novel esterase. Methyl esterification during elongation must occur uniformly along newly synthesized pectin molecules as no JIM 5-reactive epitopes are available for antibody binding. It seems likely that fine control of gel matrix properties can only be achieved if positions of esterification on long pectin molecules (some range up to 700nm in length) (3) are very precisely defined. At maximum elongation, it appears that the unesterified pectin is secreted into the medium and that therefore an entire pectic network is being replaced (Fig 5). This raises some interesting questions. How does t h e n e w network of more methyl esterified pectin become inserted into the wall? Is the whole network replaced before elongation can occur or are there domains that are capable of wall loosening? What happens in tissues, where the 'older' unesterified pectin must move into the middle lamella? The pectic network in the wall is replaced by newly-synthesized highly-esterified pectins, and older un- or de-esterified pectins may contribute to the increase in surface area of the middle lamella region during growth. Modifications in the degree of esterification of polygalacturonic acid and the size, frequency and conformation of junction zones could influence the fixed-charge density and/or porosity of the pectin gel (20). Loosening of the pectin network concomitant with wall expansion may involve changes in pectin gel rheology w h i c h influence m e t a b o l i s m of the cellulose/xyloglucan network by the fine control of pectin methyl esterification, calcium ion and proton concentration, and the activity of pectin methylesterase (PME) (21). The enormous number of cell wall enzymes may include as yet uncharacterised transesterases and pectin transglycosylases.
102
Fig 5 Immunogold labelling on thin sections of low-temperature embedded cell walls from 9-day old tobacco cells with JIM 5, a monoclonal antibody that recognises a relatively unesterified pectic epitope. Cell walls of elongating cells label very weakly, but material that is being secreted into the culture medium labels strongly. The old part of the wall is labelled but new wall material is not. Measurements of yield stress on gels of isolated pectins have shown that the optimum degree of esterification (d.e.) for maximum gel strength is around 70% (22). The gel strength is not dependent on divalent cation concentration, and may be a result of non-covalent interactions with ester groups helping to stabilise interchain junctions. Morris et al (1980) note a sharp reduction in gel strength at around 80% d.e. which they speculate is due to disruption of these non-covalent interactions. Maximal elongation of both tobacco cells and maize coleoptiles occurs at 80% d.e. of pectin. Whilst isolated pectin gels are clearly abiotic systems, gelation characteristics of pectins in the wall may well be greatly modified by the presence of other components, and the isolated pectin gels were made under conditions of low water activity, nevertheless, aggregation of pectins by non-ionic interchain associations has also been documented by gel permeation chromatography (23) in aqueous pectin solutions, and as a contributing mechanism to network formation in calcium pectate gels (24). High-performance size exclusion chromatography measurements (25) and direct visualisation in
103
the electron microscope (3) have also demonstrated aggregation of isolated pectins. It is possible that disruption of the non-covalent interactions, which may be analogous to tertiary interactions in protein folding, allows increased accessibility of expansins (26) or transglycosylases (27) or hydrolases (28) to the cellulose-xyloglucan network in the cell wall as well as providing a suitable ionic environment for relevant enzymes. A 10% reduction in esterification results in a gel of optimum strength (22). By stationary phase, unadapted tobacco cells have 70% d.e. Further, the complexity and diversity in pectin structure and localisation in cell walls of different species, tissues, and in different wall domains around a single cell (3) may reflect a functional diversity in the fine control of gel rheology. Given the variety in cell wall composition between tissues, and even between walls bordering a single cell (4), it is important to demonstrate that data obtained from bulk chemical analysis is applicable to the walls of all cells in the population sampled. Using FTIR microspectroscopy and electron microscopy, two methodologies which permit analysis at the level of a single cell wall, we have shown that the observed changes in esterification at different growth stages occur, however, in the entire cell population, thus validating the conclusions from the bulk analysis (18).
4. PECTINS M A Y H A V E I M P O R T A N T ( U N K N O W N ) D E V E L O P M E N T A L FUNCTIONS
The classical picture of plant development consists of the production of new cells in specialized regions of cell proliferation, the subsequent coordinated expansion or elongation of these cells and finally, depending on their position with respect to neighbours, a process of cell specialization or differentiation. During growth, it seems that there is replacement or reorganisation of existing networks with newly synthesized polymers that may confer new rheological properties on the wall architecture, to produce architectures that are capable of elongation or expansion, and it seems probable, given the complex spatial localisation patterns of pectins and their heterogeneous nature, that specific changes in pectic composition are necessary for some differentiation events. The Z i n n i a mesophyll cell system is unique in higher plants, in offering controllable semi-synchronous cell differentiation (29). Gentle grinding of Zinnia leaves in a mortar and pestle releases isolated mesophyll and palisade cells leaving sheets of epidermis and the vasculature, that can be separated by centrifugation. If these single cells are incubated in a 1:1 ratio of auxin to cytokinin then first the microtubules bundle up and align, then cellulose is laid down in secondary cell wall thickenings, and finally, after about 4 days, lignin is deposited in the thickenings, and finally the cell autolyses to form a tracheary element (30). About 60% of the mesophyll cells will synchronously trans-differentiate to tracheary elements making the
104 Zinnia system very useful for looking at developmentally-regulated changes
in wall architecture (14). Changes in wall architecture occur during isodiametric cell expansion, cell elongation, and the thickening of a growing wall to its mature thickness, and so it is important to define whether the synthesis and secretion of particular cell-wall molecules correlates with differentiation events or with general cell expansion. Cells are stimulated into producing wall polysaccharides upon subculture, so we used a non-inductive medium, in which the cells only expand, for comparison with inductive medium at all times.
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Fig 6 Immunodot-blots of culture-medium aliquots sampled during the time-course of differentiation of Z i n n i a mesophyll cells to tracheary elements and dotted on to nitrocellulose. I = Inductive medium, N = Noninductive medium. The JIM 7 epitope dries in a series of concentric rings on the nitrocellulose, indicating a mixed population of pectins. During the timecourse, the rhamnose content of inductive culture medium increases dramatically compared with non-inductive medium.
105 Cells in liquid culture are known to secrete extracellular polysaccharides and proteins, and so analysis of the culture medium provides a convenient "snapshot" of which soluble polymers are newly synthesized (14). Aliquots of culture medium sampled during the time course of differentiation were analysed by immunodot-blots on nitrocellulose. The methyl-esterified pectic epitope, recognised by the JIM 7 antibody, is present in both inductive and non-inductive medium, but the pectins are synthesized and secreted at greater concentration in inductive medium than in non-inductive medium (14). However, pectins are a heterogeneous group of cell-wall polymers, and methyl esterification is a common modification to the newly-synthesized polysaccharides. The JIM 7-reactive epitope dries on dot-blots in a reproducible series of concentric rings with consistent marked differences between the rings present in the inductive and non-inductive medium suggesting that during the drying of the dots some chromatographic separation of a mixture of pectins occurs on the nitrocellulose. Sugar and linkage analysis shows that a branched rhamnogalacturonan (4-GalA, 2Rhm, 2,4-Rhm) with short side-chains (no Type I arabinogalactan detected) increases in amount during the time-course, with the rhamnogalacturonan being over ten-fold more abundant than in the non-inductive medium. Rhamnogalacturonan I has also been shown to be a developmental marker for the non-hair-forming cells in epidermal cells of Arabidopsis roots (Hahn, pers. comm.). In the meristematic region of the root, the epitope is present only in the epidermis and only in specific cells.
5. F U T U R E D I R E C T I O N S
International collaboration has allowed us access to a set of selected for by sugar analysis (31), that will allow the phenotypic effects of subtle changes in sugar and lignin monomer composition to be explored (collaboration with Dr W-D Reiter, Connecticut). We have recently used FTIR spectroscopy in conjunction with Principal Component Analysis to show that a rapid screen using FTIR spectroscopy (2 minutes per spectrum) is feasible to select for cell-wall mutants. Although many polysaccharides have absorbances in the so-called fingerprint region of the spectrum, where many complex vibrational modes overlap and peaks cannot be assigned uniquely, in this region the spectra constitute speciesspecific and tissue-specific fingerprints of cell walls, reflecting even subtle differences in composition. Ester and free acid peaks are well-resolved in the spectrum, making FTIR spectroscopy a possible approach to screen for pectin or pectinase mutants. The recent development and application of methodologies sensitive at the single cell wall level has shown that traditional bulk analytical techniques average out important intrinsic heterogeneity in sampled populations. By exploring the diversity of cell walls using novel cryopreservation techniques for electron microscopy and non-invasive
Arabidopsis mutants,
106
vibrational spectroscopies, it should become possible to correlate macroscopic properties with the presence of specific architectures, leading to predictive models for the macroscopic properties of cell wall-derived products. With a large spectral database, statistical methods such as principal component and cluster analysis can be used to determine if correlations exist between spectral features and, for example, taxonomic classification, rheological properties, or tensile strength. Many hundreds of different proteins are present in the cellwall, and many of these may interact with pectins. By using a mutant approach, we hope to address the functions of these proteins and the consequences for pectin structure and function.
6. R E F E R E N C E S
1. McCann, M.C., Wells, B. and Roberts, K. (1990) J. Cell Sci. 96, 323-34. 2. Wells, B., McCann, M.C., Shedletzky, E., Delmer, D. and Roberts, K. (1994) J. Microscopy, 173, 155-164. 3. McCann, M.C., Wells, B. and Roberts, K. (1992) J. Microscopy 166, 123-136. 4. McCann, M.C. and Roberts, K. (1991) In: The Cytoskeletal Basis of Plant Growth and Form (ed. C.W Lloyd), pp 109-129. Academic Press. 5. Baron-Epel, O., Gharyl, P. K. and Schindler, M. (1988) Planta 175, 389-395. 6. Redgwell RJ, Selvendran RR. (1986) Carbohydrate Research 157, 183-199. 7. Foster, T.J., Ablett, S., McCann, M.C. and Gidley, M.J. (1995) Mobilityresolved NMR spectroscopy of cell walls. Biopolymers, in press. 8. Shea, E.M., Gibeaut, D.M. and Carpita, N.C. (1989) Planta 179, 293-308. 9. Shedletzky, E., Shmuel, M., Delmer, D.P. and Lamport, D.T.A. (1990) Plant Physiol 94, 980-987. 10. Shedletzky, E., Shmuel, M., Trainin, T., Kalman, S. and Delmer, D.P. (1992) Plant Physiol. 100, 120-130. 11. Williams DH, Fleming I. (1980) In Spectroscopic Methods in Organic Chemistry, edn 3, pp. 35-73. McGraw Hill, New York. 12. McCann, M.C., Hammouri, M.K., Wilson, R.H., Belton, P.S. and Roberts, K. (1992)Plant Physiology 100, 1940-1947. 13. Knox, J.P., Linstead, P.J., King, J., Cooper, C. and Roberts, K. (1990) Planta 181,512-521. 14. Stacey, N.J., Roberts, K., Carpita, N.C., Wells, B. and McCann, M.C. (1995) Dynamic changes in cell surface molecules are very early events in the differentiation of mesophyll cells from Zinnia elegans into tracheary elements. The Plant Journal, in press. 15. Pennell, R.I., Janniche, L., Kjellbom, P., Scofield, G.N., Peart, J.M. and Roberts, K. (1991) The Plant Cell 3, 1317-1326. 16. McCann, M.C. and Roberts, K. (1994) J. Experimental Botany, 45, 1683-1691. 17. McCann, M.C., Stacey, N.J., Wilson, R. and Roberts, K. (1993) J. Cell Sci. 106, 1347-1356 18. McCann, M.C., Shi, J., Roberts, K. and Carpita, N.C. (1994) The Plant Journal, 5(6), 773-785.
107 19. Kim, J-B. and Carpita N.C. (1992) Plant Physiology 98, 646-653. 20. Carpita, N.C. and Gibeaut, D.M. (1993) The Plant Journal 3, 1-30. 21. Ricard, J. and Noat, G. (1986)Eur. J. Biochem. 155, 183-190. 22. Morris, E. R., Gidley, M. J., Murray, E. J., Powell, D. A., and Rees, D. A. (1980) Internatl. J. Biol. Macromolec. 2, 327-330. 23. Davis, M. A. F., Gidley, M. J., Morris, E. R., Powell, D. A. and Rees, D. A. (1980)Int. J. Biol. Macromol. 2, 330-332. 24. Gidley, M. J., Morris, E. R., Murray, E. J., Powell, D. A. and Rees, D. A. (1980) Int. J. Biol. Macromol. 2, 332-334. 25. Fishman, M. L., Gillespie, D. T., Sondey, S. M. and Barford, R. A. (1989) J. Agric. Food Chem. 37, 584-591 26. McQueen-Mason, S., Durachko, D. M. and Cosgrove, D.J. (1992) Plant Cell 4, 1425-1433. 27. Fry, S. C., Smith, R. C., Renwick, K. F., Martin, D. J., Hodge, S. K. and Matthews, K.J. (1991) Biochem. J. 282, 821-828. 28. Taiz, L. (1984) Annu. Rev. Plant Physiol. 35, 585-657. 29. Chasan, R. (1994) The Plant Cell 6, 917-919 30. Fukuda, H. and Komamine, A. (1980)Plant Physiol. 52, 57-60. 31. Reiter WD, Chapple CCS, Somerville CR. (1993) Science 261, 1032-1035.
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J. Visser and A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
109
Cell free synthesis of the pectic polysaccharide homogalacturonan Debra Mohnen, Ron Lou Doong, Karen Liljebjelke, Gregory Fralish and Jinlene Chan.
Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, 220 Riverbend Rd., Athens, GA 30602, USA
Abstract
Polygalacturonate 4-a-galacturonosyltransferase (PGA-GalAT) (EC 2.4.1.43), the enzyme that synthesizes the pectin polysaccharide homogalacturonan (HGA), has been identified in microsomal membranes isolated from tobacco (Nicotiana tabacum L. cv Samsun) cell suspension cultures. The radiolabeled nucleotide sugar substrate, UDP[14C]galacturonic acid (UDP-GalA), is synthesized by epimerization of UDP-[14C]glucuronic acid using crude particulate preparations from radish roots. Incubation of tobacco membranes with UDP-GalA results in a time-dependent incorporation of [14C]-galacturonic acid into a chloroform-methanol-precipitable and 65% ethanol-insoluble product. The synthesis of HGA in microsomal membranes occurs at a pH of 7.8, a temperature of 25 to 30~ an apparent Km for UDP-GalA of-8.9 laM and a Vmax of-150 pmol min-1 mg-1 protein.. Treatment of the product with base to hydrolyze ester linkages followed by digestion of the base-treated product with a homogeneous endopolygalacturonase results in cleavage of 34% to 89% of [14C]-labeled product into components that co-chromatograph with mono-, di-, and tri-galacturonic acid, indicating that a large portion of product contains contiguous 1,4-1inked ~-D-galactosyluronic acid residues. The product synthesized in cell free tobacco membranes has a molecular mass of-105,000 daltons based on dextran standards. Approximately 45% to 67% of the galacturonic residues in the synthesized HGA appear to be esterified since base-treatment prior to EPGase-fragmentation is required in order to obtain a maximal yield of mono-, di-, and tri-galacturonic acid. Comparison of the sensitivity of base-treated and pectin methylesterase-treated products to fragmentation by EPGase indicates that at least 40% of the base sensitive linkages are methyl esters. Conditions have been established that allow for up to 90% of the activity in detergentdispersed membranes to be recovered as soluble enzyme.
1. INTRODUCTION
Pectin is a major polysacchatide component of the plant primary cell walls that is thought to be important for cell wall strength (20,49,73), wall ion exchange and sieving properties, cell-cell adhesion (49,77), and cell-cell communication (reviewed in (13,15,55). Pectin is also an important food fiber and is ,an economically important nutritional and gelling agent in foods. The pectic polysaccharides are thought to play important roles in cell growth and
110
development. Pectins are among the first polysaccharides deposited during cell division (72) and oligosaccharide fragments of the pectic polysaccharides have been shown to be signal molecules in plant development and in plant defense reactions (13,15,55,68). It is remarkable that, despite their importance for the development and survival of the plant, and despite their economic value, very little is known about how the pectic polysaccharides are biosynthesized (29,33). It can be estimated, based on the complexity of the structures of the pectic polysaccharides homogalacturonan (HGA), rhamnogalacturonan-I (RG-I) and rhamnogalacturonan II (RG-II) (1,35,42,43,62,63,82), that at least 41 different enzymes, including glycosyltransferases, methyltransferases, and acetyltransferases, are required for the immediate biosynthesis of pectin. Here we describe our current progress in identifying and characterizing the enzyme polygalacturonate 4-~-D-galactosyluronic acid transferase (called PGA-GalAT in this paper) that synthesizes HGA. We initiated our study of pectic polysaccharide biosynthesis by starting with HGA since approximately' ~60% of pectin consists of HGA (53). HGA also appears to be the first, or among the first, pectic polysaccharides synthesized in the Golgi (75), and HGA is the source of biologically active oligogalacturonides (13). In addition, the HGA portion of pectin appears to be functionally important in m u r o due to its ability, in the presence of Ca ~, to form junction zones that result in the cross-linking of pectin in the wall (10). This paper begins with a brief description of pectin structure and an overview of the general mechanism of cell wall polysaccharide biosynthesis. This is followed by a summary of previous research on PGA-GalAT and a description of a facile method to synthesize UDP[14C]-galacturonic acid. Finally, the paper ends with a summary of our work on the identification, partial characterization, and initial solubilization of the homogalacturonan biosynthetic enzyme PGA-GalAT.
1.1. Pectin structure
The primary walls of growing plant cells are composed of ~90% carbohydrate and 10% protein (51). Carbohydrate in the primary wall is present predominantly as cellulose, hemicellulose, and pectin. The pectic polysaccharides, are defined as a group of cell wall polymers containing o~-l,4-1inked D-galactosyluronic acid residues (62,76). Pectic polysaccharides are a major component of the primary cell wall of dicots (22-35%), are abundant in gymnosperms and non-graminaceous monocots, and are present in reduced amounts (~10%) in the primary walls of the graminaceae (27,62). The pectic polysaccharides that appear to be present in all primary plant cell wall are HGA, RG-I and RG-II (62). Each of these pectic polysaccharides contains galacturonic acid. RG-I is a family of large, complex, branched polysaccharides with a molecular weight between 105 and 106 daltons. The backbone of RG-I, an alternating disaccharide of [---)4)-o~-D-GalpA(1 ~2)-c~-L-Rhap-1 ~ ] , is substituted at approximately half of the rhamnosyl residues at C-4 with oligosaccharide side chains consisting mostly of arabinosyl and galactosyl residues. Galactosyluronic acid may also be a component of some of the side chains. RG-II is a very complex polysaccharide of ~30 glycosyl residues and has a backbone of ~-l,4-1inked Dgalactosyluronic acid with structurally complex side chains attached to C-2 and/or C-3. Some of these side chains contain c~-linked and/or B-linked galacturonic acid. There are several other galacturonic acid containing glycans that may be components of the pectic
111 polysaccharides, but whose general distribution in the primary wail is less clear. These include apiogalacturonans (30), xylogalacturonans (2,62,71), root mucilage "pectin" (79), and the carbohydrate portion of arabinogalactan proteins (25,27). The pectic polysaccharide that contains the bulk of galacturonic acid in the wall, however, is HGA. Homogalacmronans are chains of ct-1,4-1inked D-galacturonic acid residues. Although HGA is a relatively simple linear homopolymer of 1,4-1inked o~-D-galacturonic acid, its biosynthesis is likely to be complicated by the fact that much of the HGA in the plant cell wall is partially methyl-esterified at the C-6 carboxyl (4,58) and there is also evidence that some HGA contains acetyl esters at C-2 and/or C-3 (35,67) and may also contain other unidentified esters (9,50).
1.2. General mechanisms of plant cell wall polysaccharide biosynthesis At least two types of enzymes are involved in the synthesis of plant cell wall polysaccharides. One type catalyzes the production of the substrates for polysaccharide synthesis, the nucleoside diphosphate sugars (NDP-sugars) (23,27,36), while the second group catalyzes the synthesis of the oligosaccharide/polysaccharide chain. The latter group, in addition to consisting of glycosyltransferases that synthesize the bulk of the polysaccharides, could also include enzymes that form the primer for polysaccharide synthesis (46,47,74). The polymerization of wall polysaccharides is hypothesized to occur in three steps: chain initiation, chain elongation, and chain termination (17,33). Protein, lipid, or polysaccharide primers for chain initiation have been suggested, but no detailed mechanism has yet been determined for chain initiation of cell wall polysaccharides (17,33). The direction of polysaccharide elongation has been shown, at least for the elongation of B-1,3and 13-1,3:l,4-glucans in ryegrass (32), to occur by addition of new residues to the nonreducing end. Although this agrees with the conventional direction of polysaccharide synthesis (17), determination of the actual direction of elongation for many polysaccharides remains to be determined (33,65). Little is known about polysaccharide termination, however, it has been hypothesized that the rate of membrane vesicle movement and fusion with the plasma membrane may play a role in determining chain length (17). All glycosylation reactions in plants, which includes synthesis of wall matrix polysaccharides and glycoproteins, are believed to be catalyzed by membrane-bound enzymes (17). Cellulose is believed to be polymerized and deposited in the wall by a plasmamembrane-localized cellulose synthase complex (16). The pectic and hemicellulosic wall polysaccharides, on the other hand, are apparently synthesized in the Golgi apparatus and subsequently moved via vesicles to the cell surface, as shown by in vivo and in vitro labeling studies (61) and by antibody localization of the polysaccharide products (56,75). In the ER and Golgi apparatus it has been suggested that, as in animals, nucleotide sugar translocators transfer the substrates for polymerization (NDP-sugars) to the lumen of the ER and Golgi where the glycosyltransferases catalyze polymerization (17). Many different glycosyltransferase activities involved in higher plant wall biosynthesis have been identified in cell free membrane fractions, but in only a few cases has glycosyltransferase activity been retained in detergent-solubilized preparations, and in even fewer cases have any purified polypeptides been identified as plant cell wall glycosyltransferases (29,33).
112 1.3. Previous Research on Biosynthesis of o~-l,4-1inked homogalacturonan The immediate substrate for polygalacturonate synthesis is UDP-galacturonic acid (81). UDP-GalA has been isolated from plants (59) and several pathways for its formation have been documented (23,31). In the 1960s, Hassid and colleagues synthesized oligo/polygalacturonic acid using cell free extracts (45,80,81). When radiolabeled UDPGalA was added to particulate fractions from mung bean, tomato, or turnip, a TCA or ethanol insoluble and hot water soluble material was synthesized (45,80). This material, when deesterified, was completely hydrolyzed to D-galacturonic acid by a polygalacturonasecontaining crude enzyme preparation from Penicillium, and yielded unsaturated digalacturonic acid after partial degradation with exopolygalacturonic acid transeliminase (80). In addition, treatment of the biosynthesized material with fungal pectinase released mono-, di- and tri-galacturonic acid (45). The cell free HGA-synthesizing enzyme preparation from mung bean (Phaseolus aureus) was subsequently shown to have an apparent Michaelis constant of 1.7 }aM for UDP-GalA, required 1.7 mM MnC12 and 0.4 M sucrose for maximum activity, and, at a substrate concentration of 35 }aM, catalyzed the polymerization of GalA residues at a rate of 4.7 nmoles per mg protein per minute (81). Attempts to solubilize the enzyme(s) by digitonin treatment or saponification resulted in total loss of activity (81). More recently, Cumming and Brett (14) reported limited solubilization of PGA-GalAT using LDAO, however, no follow up of this work has been published. In spite of the early success in identifying a putative PGA-GalAT activity in cell free extracts, there has been little further published work on this enzyme (7,14) and there have been no reports of the purification, characterization, or cloning of the gene encoding this glycosyltransferase. Part of the reason may be that since there is no commercially available source of the radiolabeled precursor sugar, UDP-GalA, it must by synthesized (52), a time consuming and/or expensive process. The available evidence supports a HGA biosynthesis model in which UDP-galacturonic acid is a substrate for synthesis of HGA in the ER/Golgi, and HGA is subsequently methylesterified in the ER/Golgi, perhaps by an enzyme complex (40). The same particulate fraction from mung bean tha~ 1 contained t~:e PGA-GalAT activity was shown to contain an enzyme that transferred the C-labeled methyl groups from S-adenosyl-L-methionine to the carboxyl groups of polygalacturonic acid (38). The model that HGA is first synthesized and subsequently methylated is supported by the inability of plant extracts to incorporate UDPmethyl-D-galacturonic acid into HGA (81) and by data demonstrating that the formation of HGA is a prerequisite for methylation while the rate of HGA formation from UDP-GalA is independent of the methylation process (39).
2. PRODUCTION OF RADIOLABELED UDP-GALACTURONIC ACID
The substrate for HGA synthesis is UDP-D-galacturonic acid (UDP-GalA) (81). UDP-GalA has been isolated from plants (59) and several pathways for its synthesis in planta have been reported (31). In vitro studies on pectin biosynthesis have been hampered since radiolabeled UDP-galacturonic acid is not commercially available. Previous researchers have used several
113 methods to synthesize 3H-labeled or 14C-labeled UDP-GalA (24,52,60,80). We have found that the enzymatic 4-epimerization of commercially available, radiolabeled UDP-Dglucuronic acid (UDP-GlcA) using particulate preparations from radish roots (Raphanus sativus) (52) gives a reproducible yield of radiolabeled UDP-GalA (44). Radish roots are an effective and inexpensive source of UDP-GlcA 4-epimerase. The UDP-GlcA epimerase activity in radish root preparations is stable for at least one and a half years when stored at -80~ Furthermore, the yield of UDP-GalA obtained using radish root particulate preparations was stable throughout a five hour epimerization reaction (44). This stability of UDP-GalA suggests that few, if any, UDP-sugar degradative enzymes (i.e. phosphatases and phosphodiesterses) (66,69) were active under the conditions used. Maximum epimerization (39%) of UDP-GlcA to UDP-GalA occurs after one hour of incubation of radish root extracts with UDP-GlcA. The UDP-[14C]GalA generated by 4-epimerization of UDP-[laC]GlcA is purified by high-performance anion-exchange chromatography (HPAEC) over a CarboPac PAl column (Dionex, Sunnyvale, CA) using a 50-950 mM linear gradient of ammonium formate. The UDP-GalA co-fractionating peak collected after HPAEC and desalted by lyophilization represents the synthesized UDP-[14C]GalA (44). Approximately 16% of the starting UDP[14C]GlcA can be recovered as UDP-[14C]GalA. This yield corresponds to a--40% epimerization rate and a ~40% recovery of homogeneous UDP-GalA from the reaction products after fractionation and desalting. Several lines of evidence have been used to establish that the 262 nm absorbing-peak that appears in radish root extracts incubated with UDP-GlcA, and separated by HPAEC, is indeed UDP-GalA (44). The UV-absorbing peak has a retention time identical to authentic UDP-GalA during HPAEC. Glycosyl-residue composition analysis demonstrates the presence of galacturonic acid in radish root extracts incubated with UDP-GlcA, as expected if UDP-GalA is produced from UDP-GlcA. No galacturonic acid was detected in radish root extracts to which no exogenous UDP-GlcA was added. Matrix- assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) of the putative UDP-GalA fraction gives a m/z of 597.7, the expected mass of the (M-H)- of UDP-GalA. Synthesized UDP-[14C]GalA migrates in the same position as authentic UDP-GalA when analyzed by thin-layer chromatography. Finally, the UDP-[14C]GalA produced can be used to synthesize HGA in vitro using membranes preparations from tobacco suspension culture cells (18). Success in synthesizing HGA confirms that the described method can be used to produce UDP-[14C]GalA suitable as a substrate for pectin synthesis.
3. CELL-FREE SYNTHESIS OF HOMOGALACTURONAN
A full understanding of the role of pectin in plant development requires elucidation of the mechanisms that regulate pectin biosynthesis (6). Our strategy for studying the biosynthesis of HGA was to 1) establish a PGA-GalAT assay that would allow detection of synthesized HGA, 2) characterize the enzyme in microsomal membranes, 3) characterize the product synthesized by the enzyme in microsomal membranes, and 4) solubilize the enzyme and characterize the solubilized enzyme and its product:
114 3.1 Strategy for identifying PGA-GalAT PGA-GalAT catalyzes the transfer of galacturonic acid (GalA) from UDP-GalA to HGA. Since each of the pectic polysaccharides HGA, RG-I, and RG-II contain galacturonic acid, each of these polysaccharides could become 14C-labeled in biosynthesis reactions using UDP[14C]galacturonic acid as substrate. The proportion of [14C]galacturonic acid that would theoretically be incorporated into HGA compared to the other pectic polysaccharides was therefore determined. Table 1 shows the relative distribution of galacturonic acid in the pectic polysaccharides from sycamore cell suspension cultures, an extensively structurally characterized pectin (62). From this analysis it was calculated that the bulk of the galacturonic acid (-87%) from UDP-[14C]GalA would be incorporated into HGA.
Table 1 Distribution of galacturonic acid (GalA) in the pectic polysaccharides of sycamore cell suspensions % GalA in Distribution of Pectic Polysaccharide % Total Wall % of Pectin Polymer GalA (%) 100 87-91 Homogalacturonan 24 57-69 Rhamnogalacturonan I 7-14 20-33 17 5-9 Rhamnogalacturonan II 4 10-11 31 5 The percentage of homogalacturonan, rhamnogalacturonan I, and rhamnogalacturonan II in the total wall polysaccharides and in pectin of sycamore cell suspensions was taken from O'Neill et al. (62). The relative distribution of galacturonic acid in pectin was calculated by multiplying the % of the polysaccharides in pectin by the % galacturonic acid in each polysaccharide. The relative distribution of galacturonic acid values were totaled and the percentage of galacturonic acid in homogalacturonan, rhamnogalacturonan I, and rhamnogalacturonan II was calculated.
Incorporation of a similar high percentage of galacturonic acid into HGA in tobacco would be expected since the glycosyl residue compositions of pectin from sycamore (21,22) and tobacco (5,34) are similar. In addition, Table II shows a comparison of the glycosyl residue composition of pectin released from the cell walls of Nicotiana tabacum cv Samsun cell suspensions (the variety of tobacco used for the work described in this paper) with the pectin released from sycamore cell suspensions (21). Both sources of pectin were released by treatment of the walls with endopolygalacturonase. The overall glycosyl composition profiles of sycamore and tobacco pectin are similar. However, the tobacco pectin has a greater mole percentage of galacturonic acid than sycamore pectin and a smaller mole percentage of arabinose and galactose. Nevertheless, since tobacco pectin has an even greater mole percentage of galacturonic acid than sycamore pectin, it would be expected that at least 87% of the galacturonic acid would be incorporated into HGA in tobacco. The strategy chosen to identify PGA-GalAT was, therefore, to assay for the incorporation of UDP-[14C]GalA into a polymeric product.
115 Tobacco suspension cultures were chosen for the proposed studies since suspension cultures produce large amounts of pectin and are good sources of glycosyltransferases. In addition, the type of pectin present varies with the stage of growth in culture (83), allowing the effect of growth stage on PGA-GalAT expression to be studied. We are also interested in studying PGA-GalAT in tobacco since thin cell-layer explants from tobacco respond to a1,4-1inked oligomers of galacturonic acid by a change in organogenesis (48,54). A long term goal is to study the role of pectin polysaccharide biosynthesis in the regulation of in vitro organogenesis of tobacco explants by oligogalacturonides (3,48).
Table 2 Glycosyl residue composition of pectic polysaccharides" from tobacco and sycamore Glycosyl residue Normalized mole % Sycamorebcell suspensions Tobacco cell suspensions Arabinose 15.2 2.6 Fucose 1.2 1.0 Galactose 11.6 1.4 Galacturonic acid 56.0 74.2 Glucuronic acid 5.0 7.5 Mannose 1.8 0 Rhamnose 7.4 6.4 Xylose 1.5 0.5 unknownc 6.5 aPectic polysaccharides were released from total walls by digestion with endopolygalacturonase ~Data from (21), glycosyl residues <1% of total are not shown CAn unidentified uronic acid
3.2. Establishment of an assay for PGA-GalAT activity An assay that is specific for the incorporation of UDP-[14C]GalA into HGA is required in order to identify PGA-GalAT. The method of BolweU et al. (7) was modified for use as an assay to detect HGA with a degree of polymerization > 7 (18). Following incubation of reactions containing UDP-[14C]GalA, reaction buffer, and tobacco membranes for defined intervals, oligogalacturonides (DP of 7 to 23) are added as carrier, and the reactions are stopped with chloroform:methanol (3:2) to solubilize membrane lipids and to precipitate the synthesized product (18). The pellets are washed twice with aqueous 65% ethanol to remove sucrose and unincorporated UDP-GalA. Aqueous 65% ethanol gives the maximum recovery of small oligogalacturonides in the pellets while completely solubilizing UDP-GalA. Using the PGA-GalAT assay, oligogalacturonides of DP 7 are largely retained in the pellets, while UDP-GalA and mono-, di-, and tri-galacturonic acid are solubilizcd. Tetra- to hexagalacturonic acid are partially retained. Thus, the described precipitation and washing method (18) serves as a procedure to assay for the incorporation of galacturonic acid into HGAs with a degree of polymerization of (DP) > 7. The percentage of 14C-recovered in the final pellets of 20-30 minute reactions using this procedure ranged from 2.5-20% of the UDp-14C-GalA added.
116
3.3. Synthesis of homogalacturonan in cell free membranes The incubation of UDP-[14C]GalA with tobacco microsomal membranes results in a time-dependent incorporation of radioactivity into precipitable product (18). When membranes are heated to 60~ for 15 minutes prior to incubation with UDP-[14C]GalA, the PGA-GalAT activity is destroyed, providing evidence that the time dependent production of product is enzyme-catalyzed. The reaction proceeds at an optimal rate at pH 7.8 and at temperatures ranging from 25~176 The enzyme in cell free membranes has an apparent K m of 8.9 + 3.5 (mean + SEM) for the enzyme-catalyzed incorporation of UDP-[14C]GalA into [14C]GalA-labeled product and a Vmax of 150 + 5 pmol min-1 mg-1 protein (mean 5: SEM) (18). Since it had been reported that 1.7 mM MnC12 stimulates PGA-GalAT activity in Phaseolus aureus membranes (81), we tested the effect of MnC12 concentration on PGAGalAT activity in tobacco microsomal membranes. Maximal PGA-GalAT activity was obtained at 0.25 mM MnC12 yields. Monovalent salts were also found to stimulate PGAGalAT activity with both NaC1 and KC1 giving approximately 76% stimulation of activity at 25 mM.
4. CHARACTERIZATION OF THE PRODUCTS SYNTHESIZED BY PGA-GALAT
IN MICROSOMAL MEMBRANES
4.1. Scheme for characterizing synthesized homogalacturonan An enzyme that specifically hydrolyzes r homogalacturonan, a homogeneous EPGase from Aspergillus niger, was used to establish that the synthesized [14C]-labeled product contained 1,4-1inked o~-D-galactosyluronic acid residues. Hydrolysis of polygalacturonic acid using this EPGase generates mono- di- and tri-galacturonic acid (11,64). Thus, treatment of synthesized HGA product with EPGase was expected to generate [14C]-labeled mono-, di- and tri-galacturonic acid. HGA in cell walls, however, is partially methyl-esterified (26,62) and is, therefore, partially resistant to EPGase treatment since EPGase requires several adjacent non-methylesterif'led galacturonic residues in order to fragment HGA (12,26). The possibility therefore existed that the tobacco microsomal membranes contained sufficient endogenous substrate (e.g. S-adenosylmethionine) to methylesterify the synthesized HGA (39,41) and, that such a product might not fully susceptible to fragmentation by EPGase. To test this possibility, the synthesized product was treated with base at 4~ to hydrolyze esters (26) and the amount of mono-, di-, and trigalacturonic acid released by EPGase digestion of intact and base-treated product was analyzed. This strategy yielded information on both the amount of [14C]GalA incorporated into HGA, and the proportion of HGA that was potentially esterified.
4.2. Sensitivity of product to cleavage by endopolygalacturonase The PGA-GalAT assay precipitates HGA larger than a trimer (18), thus, any HGA product completely hydrolyzed by EPGase treatment would be lost when re-precipitated in
117 the assay. The digestion of the synthesized product with EPGase followed by reprecipitation of the product in the PGA-GalAT assay resulted in only 82% of the radioactivity being recovered in the pellet compared to the re-precipitated control (18). These results revealed that at least 18% of the synthesized product contained t~l,4-galacturonic acid linkages. Treatment of the product with base to de-esterify galacturonic acid followed by cleavage of the base-treated product by EPGase resulted in release of 44% of the radioactivity in the base-treated pellet as apparent mono-, di- and tri-galacturonic acid. These results suggest that at least 44% of the synthesized product recovered is HGA and that a significant amount of product (~47%) is esterified.
4.3. Thin layer chromatography homogalacturonan
evidence that
the synthesized
product is
The synthesized product was further analyzed by TLC and autoradiography (18). UDPGalA and oligogalacturonides of DP of 2 to 10 were separated by TLC in the following order: UDP-GalA ran just behind the solvent front, followed by galacturonic acid and, sequentially, oligogalacturonides of DP 2 through 10. Commercially available polygalacturonic acid and oligogalacturonides of DP >11 remained at the origin. Intact radio-labeled product synthesized by tobacco microsomal membranes remained at the origin, as expected for polygalacturonic acid. Synthesized product that was base-treated ran in a similar fashion. Product that was treated with EPGase, however, ran at two locations on the TLC. Approximately 20% of the EPGase-treated product ran with a similar retention time as mono-pentagalaturonic acid. The remainder of the product remained at the origin. Product that was base-treated and subsequently treated with EPGase yielded the greatest amount of radioactivity that co-migrated with mono-pentagalacturonic. Quantitation of the radioactivity in three separate TLC plate experiments demonstrated that 34-50% of the base+EPGAse treated product runs as galacturonic acid and/or small oligogalacturonides, suggesting that at least 34-50% of the product is HGA. The fact that treatment of the synthesized product with base increased the amounts of oligogalacturonides released following EPGase treatment suggests that some of the synthesized product (--45%) is esterified.
4.4. High performance anion-exchange chromatography evidence that the synthesized product is homogalacturonan Another approach to characterize the [14C]GalA-labeled products synthesized by tobacco microsomal membranes was to filter the products through a 0.2 pm nylon filter to remove particulates, fractionate the products in the filtrate by high performance anion-exchange chromatography (HPAEC) over a Dionex CarboPac PAl column, collect the fractions, and determine the amount of radioactivity in each fraction. Fractionation of the intact products in this fashion revealed that only ,-10% of the radioactive product passed through the nylon filter, while the remaining 90% was retained on the filter. This result suggested that the synthesized HGA formed aggregates that did not pass through a 0.2 pm filter. Such an aggregation of HGA could be caused by cations (Mn ++) in the synthesis buffer and/or the dehydrating action of the organic solvents. A cation-dependent aggregation of the synthesized HGA was shown to be responsible since resuspension of intact product in 10 mM EDTA enabled ~60% of the product to pass through a 0.2 pm filter. The aggregation of the
118 synthesized product by cations adds further support that the product contained HGA since the aggregation of oligo- and poly-galacturonic acid in the presence of divalent cations is well documented (57). A larger percentage of the base-treated synthesized product was able to pass through the 0.2 )am filter (15-55% depending on the experiment). Treatment of oligogalacturonides with alkali is known to dissociate aggregates (57). Separation of the base-treated synthesized product by HPAEC revealed that most of the radioactivity eluted with retention times similar to oligo- and polygalacturonic acid. Approximately 36% of the EPGase-treated synthesized product passed through the 0.2 )am and was separated by HPAEC. The bulk of the radioactivity (58%) on the column eluted with retention times identical to authentic mono-, di-, and tri-galacturonic, suggesting that at least 21% of the initial product was HGA. Finally, 77% of the base+EPGase-treated synthesized product passed through the f'dter with 83% of the radioactivity eluting as mono-, di-, and tri-galacturonic acid upon HPAEC. These results showed that at least 64% of the product was homogalacturonan and that a large part of the homogalactur0nan (-67%) was esterified. Qualitatively similar results were obtained in a total of four experiments with an average of 24% of the HPAEC recovered-EPGasetreated product, and 52% of the Base+EPGase treated product, co-fractionating with authentic mono-, di-, and tri-galacturonic acid. Taken together the results confirm that a major part of the synthesized product consists of ~-l,4-1inked galacturonic acid, and thus, demonstrates that PGA-GalAT has been identified in tobacco cell free membranes. In all four HPAEC experiments there was a significant increase (49% on average) in the amount of total product that could be enzymatically hydrolyzed into mono- to tri- galacturonic acid following de-esterification with base. This suggests that on average-49% of the HGA synthesized was esterified. The amount of the base-labile linkage in the synthesized product that could be attributed to a methyl ester was examined by comparing the abilities of base-treatment and treatment with pectin methylesterase (PME) to render the product sensitive to fragmentation by EPGase (18). Base-treatment is not specific for the type of ester linkage while treatment with PME is relatively specific for the type of ester linkage cleaved. PME most readily hydrolyzes methylesters at the C6 carboxyl but can also hydrolyze ethyl esters at a reduced (--6-16%) rate (64). The radiolabeled product was synthesized, treated with base, EPGase, Base+EPGase, PME, and PME+EPGase, and intact and treated product was filtered and separated by HPAEC. The results of intact, base-, EPGase-, and Base+EPGase-treated product were similar to those described above. Treatment of product with purified EPGase allowed 54% of the product to pass through the falter with 42% of the product eluting as mono-, di- and tri-galacturonic acid. Treatment of product with base+EPGase resulted allowed 92% of the radioactive product to pass through the falter with 89% of total product being converted to mono-, di- and tri-galacturonic acid. Taken together these results suggest that --89% of the product synthesized was HGA and that ~53% of the HGA was esterified. Treatment of product with PME+EPGase allowed 54% of the product to pass through the filter with 53% of the radioactive product co-fractionating with authentic mono-, di- and trigalacturonic acid. Thus, a significant portion of the esterified HGA (-40%) appeared to be methylesterified. The possibility that synthesis of the HGA in tobacco membranes might be limited by the lack of methyl donor was ruled out since the addition of exogenous S-adenosylmethionine, a methyl donor known to function in the methylesterification of pectin (41,70), did not
119 stimulate the rate of synthesis, nor did it increase the amount of product produced. Thus, it appears that the synthesis of pectin in tobacco results in a partially methylesterified HGA.
4.5 The product synthesized in microsomai membranes is large The relative mass of the intact and treated synthesized product was determined by gel f'lltration chromatography over a Superose 12HR 10/30 column (18). The intact product eluted as a single peak with an apparent molecular mass of ~105,000 daltons compared to dextran. Product that was treated with base or PME eluted with an apparent molecular mass of ~201,000 daltons. We hypothesize that the apparent increased mass of the base- and PMEtreated product compared to intact product is due to different interactions of intact or treated product with the column matrix, including hydrophobic interactions (esterified product) or charge effects (base- and PME- treated product). In either case, however, the apparent mass of the synthesized product is large (-105,000 daltons). If the entire 105,000 daltons corresponds to homogalacturonan (i.e. there is no non-HGA primer and no RG-II- or RG-Ilike sugars are covalently attached to HGA), the HGA would have a degree of polymerization of approximately 600 galactosyluronic acid residues. Product treated with base+EPGase or PME+EPGase had a much smaller apparent mass, a mass equivalent to mono-, di- and tri-galacturonic acid. EPGase-treated product co-eluted with authentic mono-, di- and tri-galacturonic acid, as well as eluting at a slightly later retention time. We conclude that the EPGase-treated product is a mixture of mono-, di- and tri-galacturonic acid and small oligomers of esterified galacturonic acid. Such esterified galacturonic oligomers may interact with the column matrix resulting in a later elution time. The drastic reduction in the size of the intact, base-treated, or PME-treated product upon EPGase-treatment c o n f m s that the product synthesized in tobacco microsomal membranes is largely polymeric HGA.
5. SOLUBILIZATION OF PGA-GALAT
PGA-GalAT, like most cell wall synthases, appears to be a membrane protein. The purification of a membrane protein requires solubilizing the membrane with a concentration of a detergent that allows the formation of protein-detergent or protein detergent-lipid micelles that retain enzymatic activity. The establishment of optimal solubilization conditions is an empirical process. Our initial strategy involved screening eleven detergents including both zwitterionic (CHAPS, CHAPSO, Sulfobetaine SB12) and nonionic (n-Octylglucoside, n-dodecylmaltoside, deoxyBigCHAPS, BIGCHAPs, Triton X- 100, Triton X- 114, Thesit and Genapol X-080) detergents. In all cases, the addition of a 10xCMC concentration of the detergents to microsomal membranes (i.e. dispersed membranes) resulted in a drastic (2499.5%) inhibition of PGA-GalAT compared to the activity in microsomal membranes. In addition, almost no PGA-GalAT activity (<4.3%) was recovered in the soluble fractions following centrifugation of the dispersed membranes. These results suggested that breakage of the membrane system was inhibiting PGA-GalAT activity under the conditions used and implied that either 1) the PGA-GalAT activity was dependent upon an enzyme complex
120 which was being disassembled by the introduction of detergent, 2) the detergent was directly inhibiting the PGA-GalAT, 3) an essential ion or cofactor was being diluted or lost, or 4) the assay conditions were not optimized for the solubilized enzyme. The last possibility was tested first by varying several parameters of the reaction and solubilization conditions to determine their effect on the activity of solubilized PGA-GalA. It was found that two parameters were particularly important for obtaining appreciable solubilization of PGAGalAT: the addition of EDTA to the solubilization buffer and the use of primer consisting of oligogalacturonides of ~DP of 7-23. We have currently established conditions that give up to 90% solubilization of PGA-GalAT activity in detergent-dispersed membranes representing 25-30% of the PGA-GalAT activity in cell free membranes. Our future efforts are aimed increasing the yield of solubilized PGA-GalAT, characterizing the solubilized enzyme and its product, and purifying solubilized PGA-GalAT.
6. CONCLUSIONS
We have identified polygalacturonate 4-ot-galacturonosyltransferase activity in cell free membrane preparations from tobacco cell suspension cultures. Similar activities have previously been reported in Phaseolus aureus (mung bean) (38,45,80,81), Acer pseudoplatanus (sycamore maple) (7), and Pisum sativum (pea) (14). However, in the earlier studies the nature of the product was not fully characterized. The incubation of tobacco microsomal membranes with UDP-[14C]GalA reported here results in a time dependent production of a product that consists of at least 34-89% ot-l,4-1inked homogalacturonan and has a molecular mass of--105,000 daltons compared to dextran standards. The maximum yield of product occurs within 20 minutes at a pH of 7.8, at a temperature between 25-30~ and with an apparent k m of--8.9 pM and a Vma x of 150 pmo1 min -1 mg -1 protein. The pH optimum of 7.8 reported here is significantly higher than the previously reported pH optimum of 6.0 (81). A pH of 7.2-7.8 agrees with the operational pH of several other Golgi localized glycosyltransferases (8,28,66). The apparent k m of--8.9 pM is similar to the km of 1.7 pM reported for mung bean PGA-GalAT (81) but differs significantly from the k m of 0.77 mM reported for sycamore maple PGA-GalAT (7). Pre-treatment of the product with base to remove ester linkages increased the extent of fragmentation by EPGase revealing that at least 45-67% of the HGA synthesized was esterified. Comparison of the sensitivity of base-treated and PME-treated product to cleavage by EPGase revealed that -.40% of the esterified galacturonic acid was methylesterified. The result that only ~40% of the esterified product was methyl-esterified suggested the presence of another, unidentified ester. McCann et al. have independently found similar evidence for a unique ester in tobacco cell walls (50). Upon analysis of mature walls of tobacco cell suspension cultures, they found that 53% of the galacturonic acid (presumably from HGA) was esterified and that only 47% of the esterified galacturonic acid was methylesterified (50). The remaining ester was not identified. The fact that the HGA synthesized in our study was partially (33-55%) unesterified suggests that, at least in tobacco cell suspension cultures, pectin is synthesized in a partially esterified, rather than a ft~y
121 esterified, form. The possibility, however, that an endogenous PME may have contributed to the presence of unesterified galacturonic acid residues can not be excluded. That the product obtained in this work was only partially-esterified also suggests that synthesis of HGA is not directly linked to methyl esterification. This conclusion is consistent with the observation that the rate of HGA formation by PGA-GalAT is independent of the methyl esterification process (39). Thus, our data support a model for pectin biosynthesis in which a HGA chain is first synthesized and then, at least partially, methylesterified. Such a mechanism of synthesis is also consistent with the observation of Schaumann et al. who compared the levels of PME, pectin methyltransferase, and the degree of pectin esterification in Linum usitatissimum, and concluded that, at least during certain times of development, pectin is synthesized with a low degree of methylesterification (70). Additional support for the proposed synthesis model comes from localization studies using antibodies with epitopes directed against pectic polysaccharides (reviewed in (19,75)). Such studies have supported prior biochemical results (37,61) demonstrating that HGA is first synthesized as an unesterified chain, the HGA is subsequently at least partially methylesterified, and finally the more complicated pectic polysaccharides (e.g. RG-I) are synthesized (19,78). Confirmation of this hypothetical pattern of synthesis will require the isolation and localization of the glycosyltransferases responsible for pectin biosynthesis. Our success in retaining PGAGalAT activity in detergent-solubilized enzyme should facilitate the purification of PGAGalAT.
ACKNOWLEDGMENTS
We thank Hans Peter Heldt-Hansen for the gift of cloned pectin methylesterase, and Carl Bergrnann for the gifts of endopolygalacturonase from Fusarium moniliforme, the homogeneous endopolygalacturonase from Aspergillus niger, and the purified cloned pectin methylesterase. We also thank Parastoo Azadi and Russell Carlson for performing glycosyl residue composition analyses and our colleagues at the Complex Carbohydrate Research Center for their helpful discussions. This work was supported by a University of Georgia Research Foundation Faculty Research Grant, by NRI Competitive Grants Program/USDA Award #94-37304-1103 and, in part, by U.S. Department of Energy-funded (DE-FG0593ER-20097) Center for Plant and Microbial Complex Carbohydrates.
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126 71. Schols, H.A., Vierhuis, E., Bakx, E.J., and Voragen, A.G.J. (1995): Different populations of pectic hairy regions occur in apple cell walls. Carbohydr~es. 275:343360. 72. Shea, E.M., Gibeaut, D.M., and Carpita, N.C. (1989): Structural analysis of the cell walls regenerated by carrot protoplasts. Planta, 179:293-308. 73. Shedletzl~, E., Shmuel, M., Trainin, T., Kalman, S., and Delmer, D. (1992): Cell wall structure in cells adapted to growth on the cellulose-synthesis inhibitor 2,6dichlorobenzonitrile. Plant Physiol. 100:120-130. 74. Smythe, C., Caudwell, F.B., Ferguson, M., and Cohen, P. (1988): Isolation and structural analysis of a peptide containing the novel tyrosyl-glucose linkage in glycogenin. EMBO J. 7:2681-2686. 75. Staehelin, L.A. and Moore, I. (1995): The plant Golgi apparatus: structure, functional organization and trafficking mechanisms. Annu~ev.Plant Physiol.Plant Mol.Biol. 46:261-288. 76. Stephen, A,M. (1983): Other plant polysaccharides. In: The Polysaccharides,Vol.2, edited by G.O. AspinaU, pp. 97-193. Academic Press, New York. 77. Stephenson, M.B. and Hawes, M.C. (1994): Correlation of Pectin Methylesterase Activity in Root Caps of Pea with Root Border Cell Separation. Plant Physiol. 106:739745. 78. Stoddart, R.W. and Northcote, D.H. (1967): Metabolic relationships of the isolated fractions of the pectic substances of actively growing sycamore cells. Biochem.J. 105:45-59. 79. Tomoda, M., Suzuki, Y., and Satoh, N. (1979): Plant mucilages. XXIII. Partial hydrolysis of Abelmoschus-mucilage M and the structural features of its polysaccharide moiety. Chem.Pharm.Bull. 27:1651-1656. 80. Villemez, C.L., Lin, T.-Y., and Hassid, W.Z. (1965): Biosynthesis of the polygalacturonic acid chain of pectin by a particulate enzyme preparation from phaseolus aureus seedlings. Proc.Natl.Acad.Sci.USA, 54:1626-1632. 81. Villemez, C.L., Swanson, A.L., and Hassid, W.Z. (1966): Properties of a p01ygalacturonic acid-synthesizing enzyme system from Phaseolus aureus seedlings. Arch.Biochem.Biophys. 116:446-452. 82. Whitcombe, A.J., O'Neill, M.A., Steffan, W., Albersheim, P., and Darvill, A.G. (1995): Structural characterization of the pectic polysacchride, Rhamnogalactuionan-II. Carbohydr_Res. 271:15-29. 83. York, W.S., Darvill, A.G., McNeil, M., Stevenson, T.T., and Albersheim, P. (1985): Isolation and characterization of plant cell walls and cell wall components. Methods Enzymol. 118:3-40.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
127
Biosynthesis in vitro of pectic (1--->4)-13-D-galactan Laura S Brickell arid J S Grant Reid Department of Biological and Molecular Sciences, University of Stirling, Stirling FK9 4LA, Scotland, United Kingdom
Abstract Membrane preparations from mung bean (Vigna radiata) hypocotyls catalysed the incorporation of label from UDP-14C-galactose (UDP-Gal) into a polymeric product which released only 14C-galactose on acid hydrolysis. The product was characterised as (1---~4)-13-Dgalactan by hydrolysis using two pure enzymes, a novel exo-(1--->4)-13-D-galactanasefrom the cotyledons of germinated Lupinus angustifolius seeds [ 1] and an endo-(1---~4)-13-D-galactanase from A spergillus niger, followed by TLC separation and quantitative digital autoradiography of the labelled digestion products. Digital autoradiographic methods were used also to show that other UDP-Gal utilising enzyme activities, including UDP-Gal-4-epimerase and phosphodiesterases, were present in the catalytic membrane preparations, and competed significantly with the galactan synthase for the UDP-Gal substrate. The galactan synthase had an apparent pH optimum at pH 6.5 in the presence of Mg +2. Galactan synthase activity peaked two days after the seeds were set out to germinate, and this was followed by a marked increase in the (1---~4)-13-D-galactan content of the tissue cell walls.
I. INTRODUCTION Linear (1---~4)-13-D-galactan is a common structural feature of the side-chains of pectins from many sources. The galactan may be unsubstituted or may form the core of the so-called type I arabinogalactans, which carry short side-chains of (1---~5)-linked L-arabinofuranosyl residues attached mainly to C-3 [2,3]. In the cotyledons of certain lupin seeds, galactan has a storage function. The cell walls of the storage mesophyll cells are massively thickened [4], and consist mainly of (arabino)galactan attached, apparently, to a rhamnogalacturonan core [5,6,7]. After germination most of the galactose- and arabinose-containing polysaccharides are mobilised from the cotyledonary mesophyll walls, leaving a galactose-depleted, rhamnose- and uronic acid-rich residual wall [4]. The mobilisation of galactan occurs in the absence of any endo-cleaving galactanase and is catalysed mainly, if not exclusively, by a novel exo-(1 --->4)-13D-galactanase [1]. This enzyme catalyses the cleavage of single galactose residues consecutively from the non-reducing ends of (1 ---)4)-13-D-galactan and (1 ---)4)-13-1inkedgalactooligosaccharides, and does not hydrolyse other plant cell wall polysaccharides [1 ]. An enzyme with similar properties has since been isolated from ripening tomato fruits, and is believed to be ripening-associated [8]. It is now our intention to make use of the natural amplification of galactan in lupinseed cotyledons to investigate the enzymatic mechanism of (1---~4)-13-D-
128 galactan biosynthesis in the developing seeds. The work described here was carried out to develop suitable analytical procedures using a convenient non-seasonal source of plant tissue. The choice of mung bean (Vigna radiata) hypocotyls was based on earlier observations that membrane preparations from that tissue catalysed the efficient incorporation of label from UDP-~4C-galactose into a polymeric product which gave ~4C-galactose on acid hydrolysis but was not characterised further [9]. Membrane preparations from cultured flax cells also catalyse the synthesis of polymeric material hydrolysing to galactose [10]. 2. MATERIAL AND METHODS
2.1. Enzyme preparation. Mung beans, purchased from a local supermarket, were soaked overnight and planted in moist vermiculite. The plantlets were normally harvested and weighed 3 days after planting, and ground (without cotyledons) with sand in the presence of 50 mM Tris-HCl pH 7.5 containing 1 mM EDTA. After a brief centrifugation at 1000g to remove sand and grossparticulate material, homogenates were routinely centrifuged at 5000g and 40,000g. The 40,000g pellet was resuspended, normally in 50 mM Tris-HCl, pH 7.5 (0.3 ml per 50 seedlings). 2.2. Routine assay conditions. Membranes (50 lal in a total assay volume of 100 pl) were incubated with UDP-Gal (0.1 raM) and MgSO4 (10 raM) in 25 mM Tris-HCl buffer pH 7.5, for 10 or 60 rain. Reactions were stopped by heating at 100~ for 3 min. Lupin galactan (0.1 mg) was added as a 0.1% solution, methanol was added to give a final concentration of 70% by volume, and the tubes were capped, heated at 70~ for 5 min and centrifuged (13000g; 5 min). Supematants were discarded or retained for analysis. Pellets were washed twice more with 70% methanol at 70~ and the supernatants were discarded. The final pellets were either dissolved in preparation for scintillation counting, or were suspended in water and freeze dried in preparation for analysis. 2.3. Analysis of pellets and su~matanls. Pellets were prepared for scintillation counting by dissolution in concentrated hydrochloric acid (15 lal, 70~ 10 min). The solution was then dissolved in scintillation fluid (Emulsifier Safe, Packard) and counted in a Packard Tri-Carb 2000CA scintillation spectrometer. Samples of supematants were dissolved directly in the scintillation fluid. All counts were corrected to dpm. Freeze-dried pellets were resuspended by heating at 100~ in 50 mM ammonium acetate buffer, pH 4.5, and digested exhaustively either with the pure exo(1---~4)-13-D-galactanase from germinated lupin seeds, prepared according to Buckeridge and Reid [1] or with a pure commercial endo-(1---~4)-fJ-D-galactanase from Aspergillus niger (Megazyme). Aliquots of the centrifuged galactanase digests or, when appropriate, the first 70% methanol supematants were subjected to liqid scintillation counting and were examined by thin layer chromatography (TLC) and digital autoradiography [11].
2.4. Analysis of seedlings for cell wall (l-->4)-13-D-galactan. Each batch of seedlings (without cotyledons) was ground in 24% (weight/volume) KOH to which a few crystals of NaBH 4 had been added, and the mixture was centrifuged
129 (15000g, 30 min). Ethanol (3 volumes) containing sufficient glacial acetic acid to neutralise the KOH was added to the supernatant. The precipitate formed on chilling was collected by centrifugation as above, washed three times with 70% (by volume) ethanol and freeze dried. The freeze dried material was dissolved in water, the volume noted, and an aliquot subjected to exhaustive digestion with the lupin exo-(1---~4)-13-D-galactanase[1 ]. Any galactose released was determined quantitatively [12].
3. RESULTS
3.1 Preparation of the membranes. Mung bean tissues were ground in a mortar with sand in the presence of extraction buffer and l mM EDTA. Incorporation of label from UDP-Gal into polymeric products was routinely assayed by incubating membranes prepared from mung bean hypocotyls with UDP~4C-Gal (0.1 mM) and Mg § (10 mM). After heat-deactivation of the enzyme, lupin galactan was added as carrier and any label in macromolecular products (70% methanol-insoluble) was determined. Optimisation of protocols for membrane isolation led to the routine adoption of a very simple procedure, involving a "clean-up" spin at 5000g, and recovery of a 40,000g pellet which contained over 80% of the total galactan synthase activity. Very little activity was recovered either in a subsequent 100,000g pellet or in the final supematant (Table 1). Incorporation of up to 15% of the UDP-Gal substrate was obtained routinely. Table 1 Galactan synthase activity distribution using two centrifugation protocols. Post-1000g supernatants were centrifuged as indicated. Centrifugations were at 4~
Sedimentation
Incorporation (% of activity* recovered)
4-step protocol 10,000g 40,000g
100,000g supernatant
3O 59 3.0 7.7
3-step protocol 5,000g 40,000g supernatant
14 81 5.7
*Calculated using total counts in macromolecular material - products not characterised.
130
3.2. Analysis of macromolecular products. Our standard incorporation assays contained resuspended particulate enzyme, labelled UDP-Gal (0.1 mM) and Mg § (10 mM) in resuspension buffer (Tris, pH 7.5). After incubation, reaction mixtures were heated briefly to 100~ and soluble lupin galactan was added, to ensure the precipitation of small amounts of galactan formed in the enzyme reaction and dissolved during the heating step. Precipitation of macromolecular products was achieved by adding methanol to a final concentration of 70%. The pellet was freed of soluble labelled products, including residual UDP-Gal, by repeated extraction with hot 70% methanol and was then analysed for labelled (1---~4)-13-D-galactan. The supernatant was analysed for soluble labelled products. Pellets were analysed for total incorporation of label by scintillation counting. They were also hydrolysed with hot trifluoroacetic acid (TFA). When the TFA hydrolysates were separated by TLC and the chromatograms were examined by digital autoradiography [ 11 ], the only labelled compound detected comigrated exactly with galactose. To determine their (1 ---~4)13-D-galactan content, pellets were digested with the pure exo-(1---~4)-13-D-galactanase from germinated lupin seeds, and with the endo-(1 ---~4)-D-galactanase from A spergillus niger, under conditions optimised to give complete digestion of the lupin galactan carrier. With each enzyme, digestion brought over 90% of the radioactivity originally in the pellet into solution. The structure of the remaining insoluble material was not investigated. When the soluble digests were separated by TLC and analysed quantitatively by digital autoradiography, the labelled saccharides comigrated exactly with, and were in the same proportions as, those formed from the carrier galactan. Moreover when time-course digestions were carried out with the endo-galactanase, the labelled oligosaccharide intermediates present at each time-point corresponded, qualitatively and quantitatively, with those from the lupin galactan carrier (Fig.
1). O
9
9
0
0.17 0.25 0.50
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0
.
....
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, ~.'~,
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Figure 1. Time course digestion, using A niger endo(1-M)-13-D-galactanase, of the pellet containing labelled biosyntheticproductand carrier lupin galaetan. Samples taken at different times were separated by TLC. The plates were subjected to digital autoradiography to reveal labelled compounds, and were then charred with H2SO4 to reveal lupin galaetan digestion products. A: Galaetan digestion products. B: Digital autoradiogram. Ga = galaetose; Ga2-Ga4 = (1---M)-I~-galactobiose-galaetotetraose.
Also the pattern of release of labelled galactose brought about by the exo-galactanase corresponded exactly to that observed for the carrier galactan. At the end of the enzyme incubations, less than 1% of the dissolved radioactivity remained at the origin of the TLC,
131 apparently inaccessible to enzyme digestion. Thus over 90% of the label incorporated from UDP-Gal into macromolecular products was (1--~4)-13-D-galactan and, at least under the conditions of our routine incorporation assay, the radioactivity solubilised by endo- or exogalactanase digestion gave an accurate estimate of the amounts of (1 --}4)-13-D-galactan formed. 3.3. Stability of the enzyme and substrate under assay conditions Fig. 2 shows a progress curve for the incorporation of label from UDP-Gal under the conditions of our routine assay. Clearly the rate of incorporation was high initially, but decreased very quickly, essentially to zero after 60 minutes. Constant rates of incorporation were observed only over short times, but plots of incorporation versus enzyme amount were acceptably linear for 10 minute incubations. The observed (Fig. 2) decrease in incorporation rates at longer incubation times was not due to depletion of the substrate by the galactan synthase reaction, since the maximum incorporation observed (Fig. 2) corresponded to less than 15% of the added substrate. Furthermore the decrease in reaction rate was not due to equilibrium being reached, since glycosyl transfers from high-energy sugar nucleotide substrates to polysaccharide acceptors are essentially irreversible. Thus the enzyme was possibly unstable under assay conditions, or else the substrate was being removed in competing reactions which gave 70% methanol-soluble products. The data in Fig 3 demonstrate clearly that the enzyme was highly unstable at the assay temperature (30~ in the absence of substrate, exhibiting a half life time of about 10 minutes (log plot not shown). ~-,
8000
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~gs
10000
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6000
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8000
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Figure 2. Progress curve for the incorporation of label from UDP-Gal.
2000 o
0
10 20 30 40 50 Enzyme pre-incubation (rain)
60
Figure 3. Stability of the galactan synthase activity. Samples were pre-incubated at 30~ prior to assay.
However it is possible that the stability of the enzyme was enhanced under the conditions of the routine assay, because of the presence of its substrate. To investigate the possibility of substrate depletion due to competing reactions, the supematants corresponding to each of the time-course points were separated by TLC, and the chromatograms subjected to digital autoradiography. The results (Fig 4) showed that the labelled UDP-Gal substrate was depleted rapidly, with the increase of a major labelled compound of higher migration, and a minor slower-moving component (Fig 4). The minor component comigrated with unlabelled UDPGlc, and resulted presumably from the action of a UDP-Gal 4-epimerase present in the
132 membrane preparation. There was, however, no transfer of labelled glucose residues to polysaccharides, as witnessed by the absence of labelled glucose in TFA hydrolysates of our 70% methanol-insoluble pellets. The fast-moving labelled compound was not resolved from galactose and galactose-l-phosphate. The latter would be a product of phosphodiesterase action on UDP-Gal, the former of phosphatase action on Gal-l-P. The time-course of depletion of the UDP-Gal substrate corresponded closely with the observed time-dependent decrease in the rate of galactose incorporation. Thus it is probable that enzyme instability and substrate depletion both contributed to the observed (Fig. 2) tailing off of incorporation with time.
"
"*: " "
,
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,
;
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Figure 4. Digital autoradiogram of supematants from time-points of Fig. 2. Lanes 1 - 10 = incubation times 0, 5, 15, 20, 30, 45, 60, 90, 120 min. Lane 11 = labelled UDP-Gal as reference.
3.4. Apparent pH optimum of the galactan synlhase When the activity of the enzyme was assayed at a range of pH values between pH 5.0 and pH 9.5 in Tris (pH 6.3 to 9.5) and MES (pH 5.0 - 6.5) buffers (Fig 5) there was a fairly sharp apparent optimum of label incorporation at about pH 6.5. The same result was obtained using PIPES buffers, pH 5.5 - 7.5. Analysis ofpellets confirmed that (1-->4)-13-D-galactan was the main product over the range covered by the Tris buffer. Examination of the Tris-buffer supernatants by TLC and digital autoradiography showed appreciable substrate depletion. However it was relatively constant at pH values at or below pH 7.5, indicating that the apparent optimum at pH 6.5 probably did reflect the variation of galactan synthase activity with pH. Above pH 7.5 substrate depletion increased, and was almost total at pH 9.5. The decrease in labelled UDP-Gal over this range was accompanied by a corresponding increase in the labelled compound which comigrated with UDP-GIc. 3.5. Galactan synthase in relation to (1--~4)-~D-galactan levels in tissue cell walls Mung bean seedlings at different stages of development were treated with 24% (by weight) KOH to extract non-cellulosic polysaccharides as fully as possible. After neutralisation of the extracts, the polysaccharides were isolated by ethanol precipitation, and
133 their (1---~4)-13-D-galactan contents were determined by digestion with the lupin exo-(1---~4)-13-Dgalactanase with quantitative determination of the D-galactose released. Further samples of the same seedlings were used to prepare membranes, which were tested for galactan synthase activity. At day 1, when the seeds had not completed germination but were fully imbibed, radicles were removed for extraction. By day 2 radicles were approximately 0.3 cm long: thereafter seedlings elongated linearly, reaching 5.8 cm at day 6. The galactan content of seedlings (Fig 6) was very low at days 1 and 2, but then increased almost linearly up to 130 n moles galactose equivalent at day 6. Galactan synthase activity was very low at day 1, then peaked at day 2, just before the beginning of the increase in galactan content. From day 3 galactan synthase paraUed galactan content. 4 DISCUSSION We have confirmed earlier observations (9) that membrane preparations from mung bean seedlings catalyse the formation of a galactan from UDP-Gal. Using novel enzymatic approaches we have shown further that the predominant (c. 90%) polysaccharide formed is a (1---~4)-13-D-galactan. Under the conditions of the incorporation assay adopted as standard in this work, there is significant conversion of UDP-Gal into soluble products, notably compounds comigrating on TLC with GaI-1-P and/or Gal, and with UDP-GIc. Furthermore the synthase activity is unstable at the assay temperature. The enzyme activity shows an apparent pH optimum at pH 6.5. Galactan synthase activity in membrane preparations from seedlings at different stages of development correlates positively with the content of (1---}4)-13D-galactan in the seedling cell walls. In future work we shall optimise the assay conditions to enhance enzyme stability, and to minimise other enzymatic reactions competing for UDPGal, and shall attempt the solubilisation and purification of the enzyme from mung bean and developing lupin seeds. The data in Fig. 6 allow a comparison of the rate of galactan accumulation in mung bean tissues in vivo, with rates of galactan synthesis in vitro. There are numerous reasons why such a comparison may not be valid. Galactan may be turned over rapidly, so that true synthesis rates may exceed accumulation rates significantly. Also our assay was not optimised and is unlikely to reflect maximum rates achievable in vitro. Nonetheless it is encouraging to note that accumulation rates in vivo (130 nmol galactose equivalent per seedling in 4 days 1.4 n mol per hour) are similar to synthesis rates in vitro (300 dpm per seedling per 10 min 0.78 n mol per hour). The galactan synthesised in vitro from UDP-Gal by flax membranes (10) was partially characterised. Part of it was water-soluble and part required NaOH to bring it into solution. The size-fractionated water and NaOH extracts with the highest associated radioactivity were subjected to methylation analysis to determine the predominant linkages in the total (as opposed to labelled) polysaccharide included in these fractions. Results indicated that watersoluble fractions contained a preponderance of (1---~4)-galactosyl linkages, whereas NaOH soluble fractions contained branched galactans. If the in vitro synthesised labelled flax polymers had the same solubility and structural characteristics, then the water-soluble labelled galactan was probably (l~4)-linked. Our results provide some support for this conclusion, since the flax enzyme exhibited two pH optima for (total) galactan synthesis, one of them (at pH 6.5) identical with the apparent pH optimum for (1---}4)-13-galactan synthesis in our experiments.
134 8000
d~
Figure 5. Incorporation at different pH values. Tris-HCl buffers were used over the range pH 6.3 - 9.5 and MES buffers from pH 5.0 - 6.5. (10 min assays)
6000
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~'.o s'.s ,r 6'.s 7'.0 7'.s 8'.0 #.s s'.o sis pll
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Figure 6. Galactan - - o - - synthase activity in relation to (1---}4)-13-D1so ~ ~, galactan content in the o~ cell walls of mung bean "~ seedlings
//
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Jw , - - - - o , ///
,
I
3
2
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i
i
!
4
5
6
7
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seedling age (days) 5 REFERENCES
4 5 6 7 8 9 10 11 12
M.S. Buckeridge and J.S.G. Reid, Planta, 192 (1994) 502 M. McNeil, A.G. Darvill, S.C. Fry and P. Albersheim, Ann. Rev. Biochem., 53 (1984) 625 A.G.J. Voragen, W. Pilnik, J.-F. Thibault, M.A.V. Axelos and C.M.G.C. Renard. In A.M. Stephen (ed.), Food Polysaccharides and their Applications, Dekker, New York, 1995 L.A. Crawshaw and J.S.G. Reid, Planta, 160 (1984) 449 B. Carr6, J.-M. Brillouet and J.-F. Thibault, J. Agr. Food Chem., 33 (1985) 285 M.T. AI-Kaisey and Wilkie, K.C.B, Carbohydr. Res., 227 (1992) 147 N.W.H. Cheetham, P.C.-K. Cheung and A.J Evans, Carbohydr. Polymers, 22 (1993) 37 A.T. Carey, K. Holt, S. Pieard, R. Wilde, et al., Plant Physiol., 108 (1995) 1099 J.M. McNab, C.L. Villemez and P. Albersheim, Biochem. J., 106 (1968) 355 F. Goubet and C. Morvan, Plant Cell Physiol., 34 (1993) 1297 M. Becker, C. Vincent and J.S.G. Reid, Planta, 195 [1995] 331 M. Edwards, Y.J.L. Bowman, I.C.M. Dea, J.S.G. Reid, J. Biol. Chem., 263 (1988) 4333
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
135
Cell wall pectins 9 from immunochemical characterization to biological activity P. Van Cutsem and J. Messiaen D6partement de B!ologie, Facult6s Universitaires de Namur, rue de Bruxelles 61, B-5000 Namur, Belgium
Abstract
The plant cell wall contains high and low affinity sites for the adsorption of bivalent cations. High affinity sites constitute a significant amount of the constitutive homopolygalacturonic acids (PGA). Monoclonal antibodies have been raised to PGA and they recognize oligomers with degrees of polymerization (DP)>_9 under a calcium-induced conformation. Immunocytochemistry indicates the presence of this acidic pectin at cell corners mainly, unless deesterification of the primary wall. The same pectic fragments with DP_>9 trigger the activation of defense mechanisms in plant cells by a signal transduction pathway involving calcium and phosphoinositides. The binding of fragments by the antibodies suppresses the response, which means that the epitope recognized by the antibody is part of the pectic signal perceived by plant cells.
1. ION EXCHANGE PROPERTIES OF THE CELL WALLS The galacturonic acids of a plant cell wall mainly belong to smooth chains of homopolygalacturonic acid (PGA) and to hairy regions of rhamnogalacturonan I (RGI). In green plants, other uronic acids can be found in hemicelluloses. Provided they are not methylesterified, all these carboxylic acids deprotonate at the more or less acidic pH of wall water. The electrostatic charges of these polyanions are then compensated by cations ultimately derived from the environment. The adsorption of cations obeys several rules. The first of them is the electroselectivity : higher charged cations interact electrostatically with anions much more than monovalent cations. The hydration of the ions also plays a prominent role : the large hydration sphere of magnesium keeps the ion at a higher distance from the negative charges of the pectin than an other bivalent cation such as calcium. Other factors are involved, such as the heterogeneity and/or the local geometry of the adsorption sites, the contribution of nearby
136
ligands, the charge distribution over the molecule in the case of organic cations, etc... It is therefore not surprising to observe that plant cell walls discriminate precisely between cations adsorbed from the surrounding solution. It is possible to accurately quantify the selectivity of this cell wall adsorption by performing ion exchange isotherms. The procedure consists in equilibrating isolated cell walls with renewed solutions of two competing cations. After several changes, the cell walls are removed, dried, weighed, and the cation content of their acidic extracts tested by atomic absorption spectrophotometry. In absence of any selectivity, the amount of each cation adsorbed in the cell wall must be exactly proportional to the amount of each cation present in the treatment solution. For example, the proportions of two bivalent cations adsorbed in the cell wall must be the same as in the equilibrium solution. In other words, the ion exchange isotherm must be diagonal. Such a study has been performed on a model plant system, the Nitella flexilis cell wall [ 1, 2, 3 ]. This freshwater alga has giant internodal cells whose easily isolated cell walls constitute a simplified model of higher plant cell walls : it has no lignin and its pectin is not methylesterified. Isolated cell walls are cut in pieces and distributed in different lots over the whole exchange isotherm to reduce variability between experimental points.
1.0 1 mN
o e~
10 m N O
0.5
0
0.5 1.0 Copper in solution
Fig. 1. The proportion of uronates that bind copper in the isolated Nitella cell wall is plotted as a function of the fraction of copper in a mixed solution of copper and calcium.
137
The results of a typical isotherm between chlorides of copper and calcium ions is shown on Fig. 1. The strong departure of the curves from the ideal diagonal exchange reflects the selectivity of the wall uronates for Cu 2+. For example, when the solution contains 5% Cu 2+ and 95% Ca 2+ chlorides, about 40% of the ion exchange sites of the wall bind copper. It is possible to calculate the selectivity coefficient of this exchange for each point of the isotherm and to express its logarithm as a function of the amount of cation in the cell wall. Unexpectedly, the plot of this coefficient displayed on Fig. 2.a shows that the selectivity is all but constant : it decreases linearly when more uronic acids release calcium for copper, with a break when about the first 30% of the negative charges are compensated by the preferred cation. This is a first indication of an heterogeneity in the pool of uronic acids of the walls, and suggests that there may exist two types of exchange sites. A similar selectivity curve is observed for a K + - Ca 2+ exchange (Fig. 2.b).
2.5
2.0 .
z
2.0 1.5 lmN
I
10mN
1.5 @
1.0
I
i
i
I
i
0
t
10m i
i
i
10t
i
0.5
1.0 cellwall N Cu2+ .,
0
i
i
i
i
i
0.5
i
I
i
I
m cell wall
N Ca2+
1.0 --"
Fig. 2. The logarithm of the selectivity coefficients of a calcium-copper (a) or a calcium-potassium (b) exchange isotherm is plotted as a function of the proportion of the preferred ions adsorbed in the cell walls.
2. EPR SPECTRA
Since cupric ions are paramagnetic, it is possible by electron paramagnetic resonance (EPR) to obtain information on the status and the environment of the Cu 2+ ions adsorbed on uronic acids [4, 5]. Nitella cell walls with uronate charges compensated to 9 or 100% with copper in equilibrium with mixed copper and zinc chloride solutions had their EPR spectra recorded at two different temperatures, 93 and 293 ~ (Fig. 3.a, b).
138
a
/T
b
=93K
/T
l~cu2+ : 0"09
Ul
NZn2+ = 0.91
[!
=93K
NCu2+ = 1.00
w Fig. 3. EPR spectra of Nitella flexilis cell walls with cupric ions on 9% (a) or 100% (b) of the uronates. The spectra have been recorded at two temperatures (arbitrary intensities). The sharp small peaks indicate g = 2.0028.
When little copper was present in the walls, the lineshape of the spectra was well resolved and did not change much on thawing. This is characteristic of ions immobilized on their adsorption sites. The complete substitution of zinc for cupric ions (Fig. 3b) modifies the lineshape that is now strongly affected by increasing the temperature. This indicates a higher mobility of the last ions adsorbed on sites of lower affinity, as deduced from the exchange isotherms. Strongly bound cations would be predominant at low coverage; a second population of more weakly adsorbed cations, preserving some mobility at room temperature, would develop as the Cu 2+ content increases.
a
b
100G H
Fig. 4. The EPR spectra at 93 ~ of cell walls saturated with copper have been best fitted to theoretical lineshapes assuming only one type (a) or two types (b) of exchange sites for the adsorption of the ions. Vertical arrows at g = 2.0028.
139
The EPR spectra of cell walls saturated with copper has been fired to the numerical solutions of the spin hamiltonian describing the EPR lineshape of cupric ions. Two simulations have been performed. The first one (Fig. 4.a) considers that all uronic acids of the cell walls are similar : the best fit is rather poor. The second one assumes existence of two populations of exchange sites with different parameters. In this case, the optimization is much better and confirms the existence of two different types of uronic acids in the cell wall (Fig. 4.b). As the copper content of cell walls increases, the Cu 2+ ions distribute between the two types of uronates : most of them on high affinity sites first, at low copper contents; more on low affinity sites afterwards. The percentage of the uronic acids that bind Cu 2+ with a high affinity is plotted on Fig. 5 as a function of the relative amount of copper in the walls. We can see that about 30% of the uronic acids consist of high affinity sites.
l l00 IZ
I
I
I
I
I
I
i
I
i
I
9
2.0
T -
-
I
i
i
i
0
Z
Zn2+
0
1.5
50
1.0
i "
i
I
I
I
i
50
I
I
I
-- Total N CuZ+
I
100
Fig. 5. Distribution of cupric ions in the cell wall between high affinity CuI and low affinity CUli exchange sites.
2.38
2.39
2.40
I
2.41
g#
Fig. 6. The logarithm of the selectivity coefficient of the exchange in the wall is plotted as a function of a magnetic parameter of copper adsorbed on high affinity sites.
These high affinity sites determine most of the selectivity of the ion exchange. This is deduced from Fig. 6 in which the selectivity of the whole ion exchange (ln KN) is plotted as a function of gill, a magnetic parameter of cupric ions adsorbed on high affinity sites. This parameter is particularly sensitive to the degree of covalence of the bound between copper and its
140
ligands. We can thus conclude that the shape of the exchange isotherms is largely determined by the selectivity of the high affinity sites for the cations. The biochemical nature of these high and low affinity sites can be deduced from the literature. The high affinity sites (about 30% of all exchange sites, Fig. 5) have EPR spectra characteristic of homopolygalacturonic acid polymers [6]. The walls also comprise about 40% of RGI and hemicellulosic uronates [7, 8]. The remaining 30% thus represent low affinity homopolygalacturonic acids. In other words, the homopolygalacturonic acids of the cell walls distribute in two equal amounts of high and low affinity exchange sites. This is compatible with the dimer formation predicted by the egg box model between pectin chains in the 2~ helical conformation [9, 10]. According to this model, we would associate the high affinity sites with the inner faces of the dimers.
2. MONOCLONAL ANTIBODIES TO PGA Knowing the importance of PGA for the ion exchange in the cell wall, we considered the production of monoclonal antibodies (Ab) to homopolygalacturonic acid. We produced one IgG1 hybridoma, named 2F4, that appeared to be specific of PGA [ 11 ]. Its specificity strongly depends on the nature of the cations present during the tests. A calcium/monovalent cation ratio of about 1/150 must be kept constant during all the steps of the ELISA. Higher and lower ratios strongly decrease the binding of the antibodies. The substitution of calcium by magnesium annuls the recognition of pectin by the antibody (Fig. 7) as well as the addition of EDTA to the buffers (not shown). This is strong evidence that the Ab binds a calcium-induced supramolecular conformation of PGA. This supramolecular conformation corresponds to the formation, under low calcium concentrations, of dimerized chains of polygalacturonates in 2~ helical symmetry, according to the egg box model. The internal faces of these dimers that bind calcium cooperatively would correspond to the high affinity sites deduced from ion exchange and EPR data. In this model, the external faces of the dimers would bind monovalent cations. The fact that higher calcium contents lower the binding of the Ab, suggests that the additional calcium, adsorbed on low affinity sites (Fig. 2b), modifies or masks the conformation of the high affinity sites that contain the relevant epitope. The nature of this modification is unclear : multimerization of preformed dimers [ 10], change of helicity of the pectate chains ... ?
141 0.8
1.2 0.6 t-
t'3
~
0.8
O (D (:1.
,_
t--"
"Q
"O t-
0.4
<
11 0
"~ 0.4 <
o
0.0
Fig. 7 Recognition of pectin by the 2F4 Ab in an ELISA test. C a 2+ has been replaced by Mg 2+ at different steps of the test.
0.2
0.0 RI0 R20 R30 R40
B30 B40 PGA
Fig. 8 Absorbance of an ELISA test where the 2F4 binds pectins with different degrees of methylesterification (R%, random or B%, block distribution)
The esterification of the carboxylic groups of pectins prevents the fixation of calcium ions and thus their dimerization under a form recognizable by the antibodies [ 12]. Randomly deesterified pectins with a DE of less than 30% bind the antibodies, whereas sequentially distributed uronates are recognized up to a DE of 40% (Fig. 8). This confirms the independent observation that calcium ions are tightly bound by randomly deesterified pectins if their DM<30% and up to a higher DM if their distribution is blockwise [ 13]. This clearly indicates that the sequence distribution of galacturonates is of paramount importance for the pectic chains to achieve the calcium-induced conformation recognized by the antibodies. The binding of the antibody is size-dependent. Only the preincubation of the antibodies with oligopectates of degree of polymerization (DP) > 9 inhibits the binding to pectin immobilized in the wells of an ELISA test (Fig. 9.a, b). The difference between dimerized DP8 and DP9 oligomers lies in the fact that dimerized DP9 could accommodate five calcium ions between their two chains whereas DP8 could only four, which is apparently insufficient for the complexes to resist thermal agitation.
142
The association of two DP9 chains creates more than one epitope per dimer since after incubation with the 2F4 antibodies, the complexes precipitate on centrifugation. In other words, the 2F4 epitope must be much smaller than dimerized DP9.
0.8 ~, 120 r O eO .O <
100
0.6
DP 9 ~) DP 9
"6 80
0.4
60
0.2
0.0
s
40
<
0
_=
10 0
101
102 ~tM/L
Fig. 9. The 2F4 antibodies preincubated with oligopectates of DP<9 are not inhibited and bind pectin in an ELISA test (a). Longer oligomers and PGA inhibit the Ab. N Inh :control with non inhibited antibodies. Even at higher concentrations, shorter DPs do not bind the antibodies (b).
4. IMMUNOCYTOCHEMISTRY OF PECTINS
Immunogold localization of the pectic epitope has been performed on different types of cells: cell suspensions, roots, shoots, meristems, coleoptiles, pollen grains, protoplasts from different species : carrot, sugar beet, tobacco, oat ... The pattern of labeling was always the same : polygalacturonic acid was essentially located on the material expanded at three-way junctions between cells or lining intercellular space, but was not found in primary walls. No epitope could be located close to the plasma membrane (Fig. 10.a). Middle lamellae far from junction zones and walls of meristematic cells were never labeled. However, since pectins can be methylesterified and/or acetylesterified, sections were treated on grid with an orange peel methylesterase to remove the methyl groups or with NaOH to remove both methyl and acetyl groups. After the enzymatic treatment, all the primary walls of most of the samples bind the
143 gold label (Fig. 10.b), except sugar beet cells that are well known to contain acetylated pectins and thus need an alkaline treatment to de-acetylate their pectins. By differential treatment of the samples, it is thus possible to discriminate between these three types of pectins :acidic, methylesterified and acetylesterified.
Fig. 10. Intercellular junction zones of carrot cells grown in suspension have been observed in electron microscopy after immunogold labeling with the 2F4 antibody. (a) no treatment of the sections prior to labeling :the gold particles are restricted to the center of the junction zones; (b) enzymatic (pectin methyl esterase) deesterification of the E.M. grids before labeling : t h e deesterified pectins present in the primary walls now bind the probe. Scale bars = 1 ktm.
The differentiation of cells occurs concomitantly to modifications of wall components. The nature of the pectins of the walls changes under the action of enzymes, among which esterases, secreted between the apical meristematic cells and the more basal differentiated cells. The apposition of new layers of pectins with different compositions at the inner surface of the walls is another mechanism by which the cells adapt their immediate environment. Using the 2F4 antibody, we have observed, in plant suspensions as well as in tissues, a third mechanism involved in wall modification. Numerous invaginations of the
144
plasma membrane internalized labeled wall material into cytoplasmic vesicles that finally fused with vacuoles. Several vacuoles with differentially labeled material at apparently different stages of degradation could be found in the same cells, all facts that strongly suggested the existence of a turn-over of pectins. Early during the infection of a plant by bacterial or fungal pathogens, pectolytic enzymes are secreted by the invading microorganisms. The wall pectins are deesterified, which allows the subsequent action of polygalacturonases and pectate lyases that depolymerize the pectins. The action of these enzymes may finally end up in tissue maceration. When infected tissues are labeled with the 2F4 probe, gold particles are detected across the walls, up to the plasmalemma. This situation is never observed with healthy plants in which no calcium-induced pectic dimers norm'ally contact the membranes of living cells. The perception by the plant cells of large amounts of pectic fragments at their outer membrane surface is then interpreted as the presence of a lethal menace. The plant cells respond by a drastic reorientation of their metabolism towards defense reactions. These events are mediated by signal transduction chains that link perception at the plasmalemma to nuclear and enzymatic responses [14].
5. SIGNAL TRANSDUCTION INITIATED BY PECTIC FRAGMENTS
Reports describing the elicitation of defense reactions by pectic oligomers appeared more than ten years ago [ 15, 16, 17]. The tx-l,4-oligogalacturonides were described as endogenous signaling molecules that triggered the production of mechanical barriers, hydrolytic enzymes or specific chemicals such as phytoalexins that impaired pathogen growth. Other studies revealed the implications at much lower concentrations of pectic fragments in morphogenesis : inhibition of auxin action [ 18], reorientation of morphogenesis... [ 19,20,21 ]. Little if any information was available on the very first step of the transduction process 9the binding of the fragments to receptors. Since the preincubation of active oligomers with the 2F4 antibody suppressed all [Ca2+]i increase and the cytoplasmic acidification (not shown), and since the PAL response of elicited cells was a linear function of the recognition of the pectic fragments by the 2F4 antibody (Fig. 11 a, b), we concluded that the 2F4 epitope was part of the supramolecular conformation that elicited defense responses in plant cells. This would explain the size dependency of the defense response of
145 plants to pectic fragments, similar to the one observed with the 2F4 antibody 9 fragments must be long enough to adopt the Ca2+-induced supramolecular conformation recognized by the plant cell receptors. However, efforts to detect receptors to pectic fragments have until now remained unsuccessful.
a c-"
20"
[]
0
b --:t-
No elicit or 9With elicitor
0
t _
El.
>,~
20
15
15 .
. m
m
. m
o 6
._1
(~
10
10 O
0
c
c r .
c m
5
0 m
0 v
E
0
E v 0
0
LO
0 ~
Ca/Na ratio
0 0
0
'
0
9
I U3
0
' 9
I
'
C,O
I",.
0
0
.
2 F4 MoAb response (abs. 405 nm)
Fig. 11. The PAL enzymatic activity of carrot cells has been measured in the presence of pectic fragments of DP>9 9(a) controls and treatments have been performed in solutions with varying Ca 2§ to Na + ratios; (b) the PAL activity is expressed as a function of the recognition by 2F4 antibodies of the pectic fragments incubated in the same salts solutions as in (a).
How pectic signals were transduced was unknown, but information suggested that cytosolic free C a 2+ might be involved [22]. Fluorescence ratio imaging has then been used to follow the evolution of free calcium concentrations ([Ca2+]i) after stimulation of carrot protoplasts by oligogalacturonides [23]. It appeared that the [Ca2+]i underwent a prolonged increase, only when pectic fragments with a DP>9 were applied in the presence of a CaZ+]Na+ equivalent ratio of 1/150, whereas the use of low calcium to sodium ratios or short
146
oligomers did not result in any cytosolic calcium mobilization. The plasma membrane depolarized concomitantly, and was followed by a cytosolic acidification. A series of defense genes was transcribed specifically within six hours in treated cells but not in control cells incubated with the CaE+/Na+ salts only, with short oligomers or with DP 9-16 oligomers in a too low CaZ+/Na+ ratio. The PAL catalytic activity peaked 24h after the start of the test [24, 25]. We then used a pharmacological approach to try to decipher the signal transduction pathway that linked perception at the cell periphery to transcription in the nucleus. It first appeared that G proteins were involved, since pertussis (PTX) and cholera (CTX) toxins, GDP-[3-S and GTP-'f-S modulated calcium mobilization and PAL catalytic activation. Lithium and neomycin that both interfere with the phosphoinositide metabolism, delayed the [Ca2+]i increase and impaired PAL catalytic activation. Calcium channel blockers, verapamil and nifedipine, blocked all responses whereas the simple application of a calcium ionophore, Br-A23187, induced PAL transcription and enzymatic activation. The depolarization of the membrane by 80 mM KC1 induced a much slower [Ca2+]i increase, incompatible with the kinetics observed when pectic fragments were used. This strongly suggests that calcium penetrates through receptor-operated rather than voltage-gated calcium channels. A bit further in the transduction cascade, the u.v. activation of caged IP3 electroporated in protoplasts triggered a peak of [Ca2+]i and a full PAL activation. All these results point to the involvement, in response to pectic signals, of a phosphoinositide transduction pathway similar to the one existing in animal cells. Calmidazolium, a calmodulin inhibitor, did not alter [Ca2+]i mobilization, but suppressed membrane depolarization, cytosolic acidification, PAL transcription and enzymatic activation. Calcium-calmodulin complexes are classically known to interfere with protein kinases and we hypothesized that they favored the phosphorylation of the plasmalemma proton pump which was then inhibited and led to the observed cytosolic acidification, membrane depolarization and potassium release. Staurosporine, a kinase inhibitor, also suppressed PAL transcription and enzymatic activation, which indicates that the phosphorylation of still unknown (transcription) factors happened in response to stimulation by pectic fragments. However, another staurosporine-insensitive but salicylic acid responding pathway led to the transcription of pathogenesis related (PR) genes [26].
147
Verapamil Nifedipine wall
Alkalinisation
Br-A23187 PTX ,
(s
t~
f,)CTX
~L) ~
GTPrS
(~"~ ~
~
GDPflS
FC
It+
~
"2 - ~
Vanadate
K+
(~Ne~
*
plasma,emma Ill|
cyto,,asm
i:...
:? ~
~
-
9
~
~!:~ :1:::::~:~ i ~ ' ~ . ~ J.< ....
IsP.sensitivel ~I- , ~ [SP-inse~tiVe.I I Kinase I" ~ l pathway !
IPAL
~
'
. ~
.
]t
"--"
)
(~~~~!
IPAL, 4CL... genes I
..
' (~
IPR protein genesl.<_(~
' CaM I Inhibitors ~
d~
Acidification ~1 + Depolarisation Salicylic acid
I I Pa prot,,i, I
Fig. 12. Tentative model of the signal transduction chain that links the perception of pectic fragments to defense responses in carrot cells. Abbreviations : al]7, heterotrimeric G protein; CaM, calmodulin; 4CL, 4coumarate-CoA ligase; CTX, cholera toxin; FC, fusicoccine; GDP-[]-S and GYP-yS, guanosine 5'-O-(2-thiodiphosphate) and guanosine 5'-O-(3-thiotriphosphate); IP3, 1,4,5-inositol trisphosphate; PAL, phenylalanine ammonia-lyase; PLC, phospholipase C; PR, pathogenesis related; PTX, pertussis toxin; Rc, receptor; SP, staurosporine. Activation and inhibition are symbolized by + and respectively.
This linear scheme of signal transduction (Fig. 12) from hypothetical membrane receptors to [Ca2+]i and IP3 increases, calcium-calmodulin interaction, kinases activation and gene transcription is clearly an oversimplification of the reality : several receptors must exist that are connected to different transduction cascades that activate a series of defense genes. Cross-talking between the pathways further complicates the picture. However, this represents a starting model on which to elaborate more refined hypotheses.
148
6. CONCLUSIONS
Two important properties of plant pectin, ion exchange selectivity and signaling ability, are essentially determined by its homopolygalacturonic acid component. Since pectins are synthesized and secreted under a largely esterified form, plant cells can modulate these properties by in situ targeting of pectinesterases. Once deesterified, the pectins bind cations selectively. Calcium, the prevailing cation in the apoplastic fluid, induces the dimerization of pectic fragments with DPs>9. These pectic fragments may be solubilized from the wall by pectinolytic enzymes released by invading pathogens. Soluble fragments in a calcium-induced conformation may then bind plasmalemma receptors and trigger specific responses. The central point in this process is the release from the walls of acidic fragments of adequate length and the induction by calcium of a supramolecular conformation recognized by specific receptors. The precise description of this conformation certainly deserves further detailed investigation.
7. REFERENCES
1. 2. 3. 4.
P. Van Cutsem and C. Gillet, Plant and Soil 62 (1981) 367 P. Van Cutsem and C. Gillet, J. Exp. Bot. 33 (1982) 847 P. Van Cutsem and C. Gillet, Plant Physiol. 73 (1983) 865 P. Van Cutsem, M.M. Mestdagh, P.G. Rouxhet and C. Gillet, Reactive Polymers 2 (1984) 31 5. P. Van Cutsem, C. Gillet, M.M. Mestdagh and P.G. Rouxhet, Collect. Colloq. Semin., Inst. Fr. Pet. 42 (1985) 171 6. P. Debongnies and M.M. Mestdagh, Carbohydr. Res. 170 (1987) 137 7. H. Morikawa and M. Senda, Plant Cell Physiol. 15 (1974) 1139 8. L. T a i z , J.-P. Metraux and P.A. Richmond, in O. Kiermayer (ed.) Cytomorphogenesis in plants, Springer Verlag, Wien, New York (1981) 231 9. G.T. Grant, E.R. Morris, D.A. Rees, P.J.C. Smith and D. Thom, FEBS Letters 32 (1973) 195 10. D.A. Powell, E.R. Morris, M.J. Gidley and D.A. Rees, J. Mol. Biol. 155 (1982) 517 11. F. Liners, J.-J. Letesson, C. Didembourg and P. Van Cutsem, Plant Physiol. 91 (1989) 1419 12. F. Liners, J.-F. Thibault and P. Van Cutsem, Plant Physiol. 99 (1992) 1099
149
13. J.F. Thibault and M. Rinaudo, Biopolymers 24 (1985) 2131 14. P. Van Cutsem and J. Messiaen, Acta Bot. Need. 43 (1994) 231 15. M.G. Hahn, A.G. Darvill and P. Albersheim, Plant Physiol. 68 (1981) 1161 16. E.A. Nothnagel, M. McNeil, P. Albersheim and A. Dell, Plant Physiol. 71 (1983) 916 17. D.F. Jin and C.A. West, Plant Physiol. 74 (1984) 989 18. C. Branca, G. De Lorenzo, F. Cervone, Physiol. Plant. 72 (1988) 499 19. S. Eberhard, N. Doubrava, V. Marf~, D. Mohnen, A. Southwick, A. Darvill and P. Albersheim, Plant Cell 1 (1989) 747 20. V. Marf/t, D.J. Gollin, S. Eberhard, D. Mohnen, A. Darvill and P. Albersheim, Plant J 1 (1991) 217 21. D. Bellincampi, G. Salvi, G. De Lorenzo, F. Cervone, V. Marf/t, S. Eberhard, A. Darvill and P. Albersheim, Plant J. 1 (1991) 217 22. F. Kurosaki, Y. Tsurusawa and A Nishi, Phytochemistry 26 (1987) 1919 23. J. Messiaen, N.D. Read, P. Van Cutsem and A.J. Trewavas, J. Cell Science 104 (1993) 365 24. J. Messiaen and P. Van Cutsem, Plant Cell Physiol. 34 (1993) 1117 25. J. Messiaen and P. Van Cutsem, Plant Cell Physiol. 35 (1994) 677 26. J. Messiaen, Ph. D. Thesis, University of Namur, Belgium, ISBN 2 87037 198 5 (1994)
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J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V.All rights reserved.
METHYL-ESTERIFICATION, DE-ESTERIFICATION OF PECTINS IN THE PRIMARY CELL WALL.
151
AND GELATION
R. Goldberga, C. Morvanb, A. Jauneall b and M.C. Jarvisc a: Institut Jacques Monod, Universit6 Paris VII 75251 Paris cedex, France. b: SCUEOR, URA 203-CNRS, Universit6 de Rouen, 76821 Mont-Saint-Aignan cedex, France. c: Chemistry Department, Glasgow University, Glasgow G12 8QQ, Scotland. Abstract
This paper deals with the enzymes controlling the extent and pattern of methylesterification in pectins within the primary walls of plant cells. It also reviews the consequences of methyl-esterification for gel formation within the cell wall and for the resistance of the wall to mechanical stress. Methyl ester groups are added to pectic galacturonans by pectin methyltransferase (PMT) enzymes during pectin synthesis. Later, within the cell wall, some methyl esters may be removed by pectin methylesterase (PME) enzymes. These enzymes activities were examined in three systems, in each of which growing or dividing cells were compared with inactive cells: suspension-cultured cells of flax, mung bean hypocotyls and poplar cambium. In each system the pectins in the walls of actively growing or dividing cells were more highly methyl-esterified than those of inactive cells. Microsomal PMT activity was characterised throughout the growth cycle of suspension-cultured flax cells. The total PMT activity was maximal during the phase of rapid growth and declined when growth ceased. It was stimulated by exogenous pectins, the optimum type depending on pH. Several basic, neutral and acidic isoforms were solubilised. A number of PME isoforms were present in all three plant systems. In mung bean hypocotyls and poplar stems, neutral isoforms predominated in active cells and basic isoforms in non-growing cells. Three mung bean PMEs were characterised and the most basic one sequenced. It is suggested that as the cells pass beyond the stage of active growth, the pectins are less methyl-esterified at the point when they are exported into the wall and causing stronger bounding of the basic PME isoforms which predominate at that growth stage. The interactions of non-esterified pectic carboxyls with cations control the gelation of pectins and the mechanical properties of the gels. A new 'cable' model is presented for the structure of calcium pectate gels at the high concentrations typical of cell walls. The cable model is based on conformational analysis of galacturonans by solid-state NMR, and incorporates not only the accepted 21 helical 'egg-box' structures but also 31 helical and intermediate regions. Similar NMR experiments on cell walls revealed still more complex gels with methyl-esterified chain regions participating in both junction zones and inter-junction segments. Because of this structural complexity the stability of cation binding covers a wide range within and between cell walls. The chelating agents most often used to extract pectins have a high enough atfinity for calcium ions to remove them completely from cell walls, although imidazole is weaker than the rest.
152 SIMS microscopy of flax hypocotyls showed that calcium ions, bound to low-ester galaeturonan segments, were concentrated in the epidermal cell walls and particularly at the tricellular junctions. Since the trieellular junctions are stressed by turgor pressure and contain only these pectins, they are a good example of in m u r o load-bearing by mechanically strong pectate gels. However low-ester pectins with a high affinity for calcium ions ~so appear to stiffen the Vigna epidermal cell wall longitudinally and may contribute to the cessation of growth as the hypocotyl matures. 1. INTRODUCTION The molecular structures of the pectic group of polysaccharides have been widely reviewed [ 1,2]. They share a core chain of galacturonie acid residues interspersed with rhamnose and substituted with a variety of side-chains. Pectins are generally assumed to be block eopolymers, since pure galacturonans (homogalaeturonans) and a variety of distinctive branched polymer blocks have been isolated by slight depolymerisation or, more often, by extraction under slightly depolymerising conditions, and residues characteristic of all these blocks have been found in large undegraded pectic polymers [3-9]. Well-characterised branched blocks include RGI and RGII [1]. The more abundant RGI [1, 10, 11] is approximately equivalent to the 'hairy regions' ofVoragen et al [2] which are themselves made up of subunits including both galactansubstituted and arabinan-substituted rhamnogalaeturonans, and xylogalaeturonans [12,13]. RGII is a complex fragment containing apiose, acerie acid, KDO and O-methylated sugars [1,14]. Undegraded pectins can contain more than 50% of neutral side-chains by mass, but because these side-chains are flexibly coiled, [15, M.A. Ha, D.C. Apperley & M.C. Jarvis unpublished] most of the length of the molecule is comprised of stiff, straight galacturonan segments with the branched blocks making up short, densely bushy regions between them. The structure and physical properties of pectic polymers show exceptionally wide variation, even within a single cell wall. It seems reasonable to assume that this structural diversity is the result of diverse biosynthetic systems and corresponds to a diversity of stressbearing functions in different parts of the cell wall. Much of the variation is in the proportions of blocks of differem types and in the length of the galactan and arabinan side-chains. However the number and distribution of free, unesterified galacturonate earboxyl groups within the galaeturonan regions are equally variable [2]. Because galacturonoyl esterifieation controls the charge density of the pectic molecule as a whole it is probably the dominant influence on the overall shape of the polymer and its capacity for aggregating with other pectic molecules to form gels. In this review we focus on the biosynthetic mechanisms by which the methyl-esterification of the galacturonan chains in pectins is controlled. We go on to examine the structure of the pectic gels that are formed and their relationship with the stresses carried by specific parts of the cell wall.
153 2. BIOSYNTHESIS AND TURNOVER OF PECTIC METHYL ESTERS
The classical picture of the biosynthesis of pectins is depicted in fig.1. It is commonly accepted that the polygalacturonic backbone is polymerised in the cis Golgi cistemae, methylesterified in the medial Golgi and substituted with side-chains in the trans Golgi [16]. Methyl-estedfication results from the activity of pectin methyltransferases (PMT) which transfer methyl groups from S-adenosylmethionine to a polygalacturonic acceptor [17]. Unestertfied galaeturonan blocks may then be generated in muro through the activity of cell wall bound pectin methylesterases (PME) cleaving ester bonds on consecutive galacturonate residues along the chain [18]. However, there are several observations that cannot be explained by such a scheme: UDPG~IA
Io GA
o n
o
~
.
SAM~ 9
I
l e methylated GA I
golgi
9
'
1
PME
,~.'-~
l
CW, cell wall PM, plasma membrane GA, galacturonic acid SAM, S-adenosylmethionine PMT,pectinmethyltransferase PME,pectinmethylesterase
b .'fully methylated c : not methylated
Figure 1. Hypothetic biosynthesis of pectic polymers -Smooth regions contain esterified and unesterified blocks but also regions with randomly distributed methyl groups. How are these generated? What is the exact degree of esterification of the pectins exported into the apoplast? -Most cell walls are able to deesterify exogeneous pectin very rapidly but in vivo these cell wails still contain large amounts of methyl-esterified pectins. How can the substrate and the enzymes coexist inside cell walls? -In most plant materials that have been examined, several PME isoforms have been extracted from the cell walls. What is their respective function? How is their synthesis regulated? -Immunolocalisation experiments have shown that some Golgi vesicles can be labelled with the JIM 5 antibody, which is known to recognize low-ester pectins [ 19]. This raises the question of the action pattern of the pectin methyltransferases. Can they produce pectins with different
154 degrees of methylation? Compared with the PMEs, very little information is available on these enzymes. The aim of the first part of this paper is therefore to take stock of our present knowledge of the two activities involved in methyl turnover. We have investigated the respective amounts of esterified and unesterified pectins during growth processes and, in parallel, the activities of PMT and PME.Three different growing plant systems were examined: flax cell suspension cultures [20]; mung bean hypocotyls, which are known to exhibit a well defined growth gradient [21]; and inner bark tissues (cambium and phloem derivatives) from poplar stems. In each case we have compared active cells (dividing or elongating) and resting cells in order to uncover possible relationships between pectin structures and growth potential. 2.1 Pectins from active and resting cells The data reported in table 1 show that the pectin fraction in active cells is rich in methylester[fled galacturonans, whereas the resting cells contain mostly low-ester acidic pectins.
Table l. Pectins from active and resting cells. Two different biological materials were investigated: flax cell suspensions atter 6 (actively dividing cells) and 14 (resting cells) days and mung bean hypocotyl segments sectioned in the elongating zone (active cells located in the upper part of hypocotyls of 3 days old seedlings) and atter cessation of elongation (resting cells located in the lower part ofhypocotyls). CEC, cation exchange capacity; GA, galacturonic acids. -1 CEC and GA expressed as kteq mg cell walls. Active cells flax
Resting cells
mung bean
flax
mung bean
CEC
550
628
855
772
Esterified GA
335
672
345
378
Unesterified GA Esterified GA
1.64
0.93
2.48
2.04
These acidic molecules might result from either the lack of PMT activity or the action of PME, known to be present in most plant cell walls. Pectin methyltransferases and pectin methylesterases extracted from active andresting cells were therefore characterized.
155
2.2 Pectin methyltransferases from active and resting cells of flax. The possibility that the initial degree of methyl-esterification might be controlled by the properties of the methyltransferase enzymes was examined by partial characterisation of these enzymes in suspension-cultured cells of flax. Pectin methyltransferases being enzymes characteristic of the Golgi apparatus [22], microsomes were fractionated daily for ten days from suspension-cultured flax cells and incubated in the presence of ~4C-SAM, the universal donor of methyl groups.
With endogeneous pectic polysaccharides as substrates, the pectin methyltransferase activity was measured as radioactivity linked to oxalate-soluble polysaccharides aiter extensive washing of microsomes with 1M ethanolic NaC1. Figure 2 shows that the rate of methylestefification of pectic substances was maximal on days 4 and 6; these maximum activities were observed within this period in at least five independent experiments. On the other hand, little activity was noted in young cells before day 2, and in old cells after day 9. In other words during the stationary phase the newly synthesised pectins remained unesterified because of the lack of pectin methyltransferase activity. 40
120 100
~ -I
80
~-'
3O
.15
••••i••
20
e-
._~
6O
. . . . . . . .
40
"'I
20 ko 0
5
10
15
Age of cultures (days)
Figure 2: Pectin methyltransferase activity of supension-cultured cells of flax. ( 9 Variation in fresh weight of filtered cells. (A)Total activity was measured by monitoring methylation in vitro from SAM. 1 AU: 1000 dpm In these experiments the pectin methyltransferase activity in vitro was generally only a few picokatal g-i of protein, due mainly to the limited amount of SAM and endogeneous polysaccharide substrate. However the addition of exogeneous pectic substrate increased the incorporation of radioactivity up to twelve-fold. At neutral pH, the stimulation was larger in the presence of highly methylated pectin than in the presence of polygalacturonic acid. At pH 5.5, on the other
156 hand, it was greatest in the presence of low-methylated pectins and this treatment gave the greatest stimulation observed. Preliminary experiments indicated good solubilisation of the enzymes using Triton-Xl00 detergent [23], allowing further investigation on the pectin methyltransferase properties. The apparent Km for the methyl donor SAM was estimated to 0.05-0.5 lxM depending on the pectic acceptor and the fractionation step. The apparent Km for the pectic aceeptor was in the range 15 mM of carboxylic acid. Several isoforms were identified: (1) basic (pH range 8.5-9) which accounted for 40-45% of the total activity, (2) neutral (pH range 6.5-7.5), representing 35-40% of the total activity and (3) acid forms (pH range 4.5-6). Fractionation by size-exclusion gel chromatography (Pharmacia Sephacryl $200) showed that several polypeptides were active (MMa > 150 000 bearing 27 _+5 % of the total activity; MMa 100-120 000 with 25+_5% of the activity; MMa 50-80 000 with 12 +3% of the activity; and MMa < 10 000 accounting for 35+5% of the total activity). Thus the isoforms of pectin methyltransferase are numerous and varied in their physical properties. The basic isoforms which were the largest single group would be expected to interact electrostatically with their pectic substrates, and the strength of the interaction will increase with the density of negative charges on the pectic galacturonan chain. It may be significant that at the optimum pHs for trans-methylation onto high-ester and low-ester pectins, pH 7 and pH 5.5 respectively, about half of the carboxyl groups were ionised in each case. 2.3 Pectinesterases from active and resting cells. When cell-wall fragments are incubated in molar NaC1, ionically bound proteins are released into the incubation medium. All investigated crude cell extracts deesterified Citrus pectin (Table 2) but the deesterification rates were clearly higher when the enzymes were still bound to the cell walls, indicating a major loss of activity during the solubilization process.
Table 2. PME activity solubilized from cell walls of active and resting cells. Activities as neq. H +. mg protein-1 Active cells Cell suspensions Hypocotyls
12.6 12.5
resting cells 16.2 34
All cell-wall extracts contained several PME isoforms differing in their isoelectric points. The isoenzyme patterns changed significantly during cell ageing. As illustrated in Fig. 3, in both poplar stems and mung bean hypocotyls, basic isoforms became prevalem in mature, resting cells whereas in young, growing cells, neutral isoforms were predominant.
157 70
~
v'
,
!
60 !" a~
50
-
4O
-
30
-
,
>~
_-
Ill'
Iz
Ds
.9~ . ~
...
0
r~
~.7(~%; :
:~0 ~10.
~:
i ,,
A
B
C
D
may
hypocotyl levels
december
march
Figure 3. PME isoform patterns in cell wall extracts from active and resting cells. a: cell wall extracts from successive segments (A, B, C and D) sectioned along mung bean hypocotyls and exhibiting decreasing elongation rates; u, 13, and 7 are the main PME isoforms present in the extracts, their pI are respectively around 7.5, 8.5 and above 9.1. b: cell wall extracts obtained from poplar cambium and inner bark tissues during cambial active (may) and rest (december and march) periods; 1, 2 and 3 represent the activity of 3 groups of PME isoforms with pI around 5-6, 7.5 and above 9.1. Activities expressed as percent of total PME activity present in each extract. In flax cells, young cells as well as mature cells contained both neutral and basic isoforms, the proportions of which varied slightly. Acidic isoforms were also detected. Some properties of the different isoforms extracted from mung bean hypocotyl cell walls (eaUed respectively PEa, PEI3 and PET, ot for the neutral isoforrn, 13 for the PME with a pI around 8.5 and 7 for the most basic one) are reported in Table 3. The three esterases differed not only in their pI but also by their Mr, their pH optimum and the ionic strength necessary for their solubilizatiorL Table 3. Properties of mung bean puritied PME isoforms Enzyme
pI
Mr (Da)
PEa PEI3 PET
7.5 8.5 above 9.5
47,000 34,000 35,000
optimal pH 5.6 to 8. 7.6 5.6
solubilized with 0.2 M NaC1 1.0 M NaC1 0.4 M NaC1
When comparing the two basic isoforms PE[5 and PET, it can be noted that the more basic one, PET, surprisingly requires a lower ionic strength for its solubilization than PEI3. The
158 activities of the three isoforms exhibited different kinetic parameters and different ionic sensitivity (Table 4, figure 4). The neutral PME which predominates in young active cells was the least affected by the pH and the ionic composition of the microenvironment and was readily solubilised. Table 4. Kinetic parameters of mung bean PME isoforms. Assays performed at pH 7.6 in 150 mM NaCI; values between brackets have been obtained at pH 5.6. Enzymatic fractions
Km Vmax Vmax / Km (10.3 M methyl groups) (tteqH+ min" mg protein")
PEot PEI3 PF_q
1.32 0.23 11.8
'~-
~e
0.20 (0.11) 1.74 (0.17) 0.12 (0.65)
MgCl 2
800
_ ~--" _
700
t~
~oo c-
300
~
"E
200
r
0.27 0.40 1.43
--o--
y
600
400
c
0
0
50
1O0
150
200
0
50
1 O0
150
200
salt c o n c e n t r a t i o n (mM)
Figure 4. Rates of pectin hydrolysis as a function of salt concentration at pH 7.6; rates as meq H §min-1 lag protein". (e) PEa, (11) PEI3 and (o) PEY, a for the neutral isoform, 13for the PME with a pI around 8.5 and Y for the most basic one. Differences were also observed in the internal amino acid sequences of the three isoforms. Figure 5 shows the sequences of the oligopeptides obtained after tryptic hydrolysis of the three purified isoforms. Only one N-terminal sequence (from PEy) could be obtained, the two others being blocked. Using oligonucleotide primers deduced from the N-terminal and one of the internal oligopeptides of PET, a eDNA fragment was obtained by PCR, cloned and sequenced [24]; all the PET peptides could be aligned with the aminoacid sequence of the mature protein deduced from the eDNA. The comparison of the amino acid sequence of PEY with other known pectin methylesterases from tomato [25], Petunia [26], Arabidopsis [27,28] and Brassica napus [29] shows that a glycosylation site is present in all cases and that all primary structures were
159 most homologous at the C-terminal region. This corresponds to a domain, the 'esterase-like domain' of Albani [29] that specifies the pectinmethylesterase function for the proteins. In contrast, the N-terminal regions are less conserved between species. In several cDNAs this region is relatively long and seems to be more susceptible to post-translational N-glycosylation. It has been suggested that this N-leader sequence may have a role in targeting the protein towards the apoplasm and also in inactivating or stabilising the protein during export. All the cloned and sequenced cDNAs corresponded to basic proteins. Figure 5. Partial peptidic sequences of peptides resulting from tryptic cleavage PME isoforms. The peptides were separated by reverse-phase HPLC. N-terminus (N-ter) was obtained with the entire PE T
PEa XGAYFENVEVI
PEI3 TVSEAUDA
PET
XNLMXVGDGI
ENVEVP
TVTEAVASAPDNGK YVIYVK
HQAVALR
DITFQNTAGPSK
VGADQSVINR
GVTFENSAGPSK
HQAVALR
IDAFQDTLYAHSNR
XYSR
PWK
EDPNQNTGTSIQ
NYLGR
TYLGR
TVIMQSSID
XLFTAQGR
EYQNTGPGAGTSNR
VNWPGYHIITSAAEASK
VTWPGYR
VITDAREAR TTTIITASR
PE T N-terminus DVKANVVAQDGSGSGI~KTVTEAVA
It would be very interesting to obtain sequence data on neutral PMEs. In spite of this lack of information it can be suggested, taking into account the important differences in the ionic and substrate-binding characteristics of the three mung bean isoforms, that they may also progress differently along the polygalacturonic backbone. Basic isoforms have been shown to act in a bloekwise manner, de-estedfying their way along the galacturonan chain while remaining attached to it by virtue of their positive charge, so that they give rise to acidic blocks that can aggregate in the presence of calcium ions [18]. Marcovic [18] demonstrated that in contrast, fungal PMEs deesterify randomly. If we hypothesise that neutral plant PMEs might also act randomly, lacking the tendency to remain ionically bound to acidic galacturonans, we can propose the following scheme to explain the observations on mung bean hypocotyls (Fig. 6). In young cells, at the top of the hypocotyl, neutral PME isoforms predominate. Their action might be restricted either by their localisation away from the substrate, or by the initial structure of their substrate e.g. a particularly high DE. In older cell walls, the activity of basic isoforms, in particular PE~/ whose catalytic efficiency is high at acidic pH, increases and generates free carboxyl groups.
160
-r
Pectin o o . . . . . o r 9 9 *. 9
neutral PMF /\
~H~0434MH~I40~
\
/~
alkaline PME /x
/\
8 ~. ~ 8 ~ 8
/\
I
-~ ~176
J Ii o0..ooooooooooooo
. . . . . . . . . . . . . .
Figure 6. Hypothetical action pattern of neutral and alkaline PME isoforms. 9 methylated galacturonic units; O unesteritied galacturonic acids; EPG, endopolygalacturonase.
These results show that both the pectin methylesterases and the pectin methyltransferases are heterogeneous groups of enzymes, so that there is ample opportunity for the developmental control of methylesterification by the balance between the different isoforms of the two enzymes and by their response to environmental conditions and to the charge density of the existing substrate. In particular, it appears that the high level of methylesterification of pectins in young tissues is brought about by a relatively high total methyltransferase activity in the Golgi, producing pectins that are already too heavily esterified to be attacked by the neutral PME isoforms that predominate in these tissues. Galacturonan chains with a random distribution of carboxyl groups show less tendency to aggregate, which is compatible with the high extensibility of the young cell walls. This example illustrates the complexity of the enzymic mechanisms needed to control the methyl ester content of pectins, and their potential for spatial and developmental regulation. 3. GEL FORMATION BY PECTINS WITHIN THE CELL WALL The quantity and distribution ofnon-esterified carboxylate residues in pectins control the binding of counterions, and thus affect the propensity of the pectins for forming aggregates, gels and precipitates. Indeed the percentage of non-esterified residues is more informative about the properties of pectins than the percentage of esterified residues (DE)" increasing the DE from 60% to 80% halves the mean charge-density and the cation-binding capacity. Cations mediate what is almost certainly the principal form of crosslinking between pectic molecules, although covalent bonds of various suspected kinds [30-35], and probably others as yet unknown, can also link pectic molecules together. We will concentrate here on galacturonans. The capacity of rhamnogalacturonans to gel in the presence of cations is uncertain. Lolium RGI at high
161 concentration formed a gel with calcium ions [A. Chesson and M.C. Jarvis, unpublished] but this may have been due to a small proportion of galacturonan present. When contaminating galacturonan segments were excluded the aflSfity of flax RGI for calcium ions was relatively low [C. Morvan and M.C. Jarvis, unpublished]. At present, therefore, there is no clear evidence that the branched segments of pectins participate in the calcium-mediated junction zones, although they may well be capable of other forms of cross-linking.
3.1 Chain conformation in galacturonan gels Shared cations will align galaeturonan chains alongside one another if the chain conformation allows holes at regular intervals for the cations, matched to their size and preferred coordination geometry, and adjacent to negative charges. The range of conformations possible for the pectic (1,4)-ot-D-galacturonosyl linkage has been explored by modelling and proton NMR in disaccharides [36-40] and the polysaccharide itself [41]. It is centred on the 21 conformation which has a 180~ rotation between galacturonosyl residues. With little change in energy or chain length, polymers starting in this conformation can twist in either direction to give a fight-handed (31) or a lett-handed (32) helix with three residues per turn, or slightly further to give a 41 helix. Insertion of a single a(1,2)-L-rhamnosyl residue introduces a sharp kink in the chain [41, 42]. Thus a rather wide range of linkage conformations is sterically possible and direct experimental evidence is needed to distinguish those that give stable aggregates with any cation.
Figure 7. The 'egg-box' structure for the junction zones of dilute calcium pectate gels: two galacturonan chains in the twofold (21) helical conformation with calcium ions (shaded circles) locked between them. For gels of pectate with calcium ions the 21 helical 'egg-box' structure (Figure 7) [43,44] has become widely accepted, but the evidence supporting it is derived mainly from gels of <10 g/1 polymer content rather than the much more concentrated gels (ca. 200 g/l) found in primary cell-walls. Indeed when the 'egg-box' model first appeared [43] its originators suggested that when the dilute gels they studied were dehydrated there was a conformational transition to the right-handed (31) helical form that had been characterised by Walkinshaw and Arnott [45,46] in X-ray diffraction studies on unusually crystalline fibres of calcium pectate (figure 8). More recent, detailed information about calcium coordination from XAFS [47] supports the idea that this conformational change takes place in gels of low and decreasing water coment. Direct experimental data, therefore, are needed to identify the conformers present at biologically relevant pectate concentrations. Conformational data of this type can be obtained by solid-state 13C NMR spectrometry. The chemical shifts (peak positions) of the carbons on either side of any glycosidic linkage are sensitive to the conformation of the linkage.
162
0
0
0
0
0
0
0
0
0
0
0
0
Figure 8. The Walkinshaw & Arnott model [46] for solid calcium pectate, with the galaeturonan chains in the right-handed (31) helical conformation. This application of 13C NMR is quite different from the use of the nuclear Overhauser effect to derive conformations from solution-state proton NMR data [36-38]. The relationship of 13C chemical shiit to conformation is not simple and until recently was known only on an empirical basis. However a theoretical description based on the anomeric effect [48] has now been established [49]. This theoretical development allowed the distinctive spectral features of all the major conformations of pectate to be defined (Table 5). The chemical shiits assigned to the 31 helix were derived from spectra of the solid Ca-, H-, Na- and Me-forms of galacturonan and guluronan, in which this helical form had been unambiguously established by X-ray crystallography. The chemical shiits for the 21 helix were similarly based on the spectrtun of solid guluronate. The spectral assignments for the lett-handed (32) helix and the right-handed form intermediate between the 21 and 31 helices were based on theoretical grounds alone, no standards being available. Table 5. Solid-state 13CNMR data for galacturonans
Egg-box (21 helical) Ca form 31 helical- H,Me,Na forms 31 helical Ca form Intermediate 21/31 helical, Ca 32 helical form
C- 1, ppm 100 101 101 94-100 102-105
C-4, ppm 77-78 80-81 80-81 78-80 78-80
Once these spectral assignments had been established it was possible to determine which of the possible conformational forms were present in calcium pectate gels [50]. Suprisingly, there was evidence for all of them (Figure 9).The 21 -31 conformational transition between the gel and the dry solid was observable in both directions (figure 10) but involved only some of the galacturonan chains. Both conformational forms were present in the gel and in the solid: only the proportions changed. The solid also showed spectral features corresponding to fight-handed helices intermediate between the 31 and 21 forms. There were also signs of leit-handed helices. these probably resulted from the twisting of chains in the junction zones towards the righthanded (31) helix, since chain segments between the junction zones would then have to twist in the opposite direction.
163
Figure 9. The cable model for the structure of concentrated calcium pectate gels. 'Egg-box' dimers link single-chains segments (top leit) and are themselves linked together by larger aggregates of either 'egg-box' or 3] helical chains (lower fight)
A
/%f
. ..... .;.. . . . . . . . .
..............
L_
_
~ % J
l/
I00
90
80
.. ,,, 70
PPM
B
ioo
9o
80
70.
PPM
mqJ:.lmq.,Jlmqmq~ml..Im, lmqj.qmqmllmqm~lm~l..In.llml..Imqmq~"q 220 200 180 160 140 120 I00 80 60 40 20 0 PPM
Figure 10. solid-state 13C NMR spectra of calcium pectate in the solid (A) and gel (B) forms. Inset: resolution-enhanced spectra. The gel concentration was 290 g/1.
164 The name 'cable structure' is proposed for the model in Figure 9, based on an unusual feature of the structure: it contains two levels of aggregation. First, single chains join in places to form dimeric 'egg-box' junction zones. These two elements are sufficient to form a gel and may well be all that is present in the dilute gels on which the 'egg-box' model was originally based. Secondly, in concentrated gels the 'egg-box' dimers also function as inter-junction segments between junction zones with four or more chains in the 31 and 21 conformations. The cable model also has a remarkable capacity to change its structure in response to its chemical environment (cations, dehydration), without leaving the gel state.
3.2 Pectin gels in cell walls Many features of the cable structure for pectate can also be observed in the solid-state ~SC NMR spectra of intact cell walls. However the interpretation of cell-wall spectra is complicated by overlaps between the peaks ~om galacturonans and those from other polysaccharides. The pectic C-4 peak at 80 ppm, characteristic of 31 helical galacturonans, can be distinguished in the well-resolved spectra from hydrated cell walls (Figure 11), although there may be interference from C-4 of 13(1,4)-linked galactans when these are abundant. The 77 ppm peak assigned to the 'egg-box' conformation is obscured by overlap with signals from cellulose and other polymers. This problem can be overcome by making use of the proton spin-spin relaxation time T2, which increases with chain flexibility and thus is greater for pectins than for cellulose or most bemiceHuloses. A T2-based subspectnan of the pectins was isolated and showed that both 21 and 31 helical conformations were present in the galacturonans of hydrated onion cell walls [51]. The intermediate forms (C-I, 94-100 ppm) have been observed in dry cell walls from a number of species. Any signals from the leli-handed helix (C-1 > 101 ppm) would be obscured by C- 1 of cellulose and other polysacchaddes. 120
100
8o ~ 60
,"
20
. . . . . . . . . . . .
180
170
160
150
140
130
120
110
100
,,,
.............
90
;.
80
,.,
70
60
50
40
30
20
10
0
ppm
Figure 11. Solid-state 13C NMR spectrum of hydrated cell walls from Citrus peel, with high content of methyl-estedfied pectin.
165 An exceptionally long proton T2 was found for the pectic methyl group in the spectrum of onion cell walls [51]. Unusually slow cross-polarisation also marked these methyl groups as highly mobile in the cell walls of a number of species, even in the absence of water (unpublished). They were much more mobile than the methyls of acetyl or rhamnosyl residues. This is attributable to their freedom to rotate even when the rest of the molecule is immobile, a characteristic of methyl groups in a hydrophobic microenvironment. Sugar-acid gels, the kind formed by highly esterified pectin in jams, have a hydrophobic string of methyls in the core of their junction zones, which are oligomeric aggregates of 31 helical pectin chains. From the rapid rotation of the pectic methyl groups and the dominance of the 31 helical form (C-4, 80 ppm) in Citrus cell walls which have highly esterified pectin (Figure 11) it seems likely that their internal water activity is locally low enough to permit the formation of a similar type of junction zone between some of the pectin chains. Much of the methyl-esterified galacturonan in hydrated onion and other cell walls, however, was much more mobile. It eross-polarised so slowly that it did not appear in conventional CP-MAS spectra. Spectra for this highly mobile material, which also included galactan and arabinan side-chains of RG1, were recovered by allowing a longer time for crosspolarisation and correcting for Tip decay [51].Similar spectra have been recorded by Foster et al [15] by singie-pulse excitation of~3C, a quite different NMR technique for observing polymers in states intermediate between liquid and solid. These methyl-esterified galacturonate residues were so mobile that they must have been present in single-chain inter-junction segments, not junction zones. Mobile non esterified residues associated with monovalent counterions (C-6, 177 ppm) have also been observed and may be present in the same chains. Thus pectins in muro contain most elements of the cable model but have additional features due to estedfication (acetyl- as well as methyl-) and branching. The ionic junction zones are similar to those of calcium pectate gels in vitro but also contain methyl-esterified junctions, and most of the single chains probably have a relatively high degree of methyl-esterification.
3.3. Binding of cations by pectin gels Given the remarkable variety of single and aggregated chains that make up a pectic gel in vivo or even in vitro, there must be a correspondingly wide variety of cation-binding sites with different affinities for the cations. While electrostatic energy, as expressed in the theory of Manning [52,53], is sufficient to account for much of the behaviour of dissolved pectic polymers at high dilution, the binding strength increases as aggregates and then gels form at higher polymer concentration. This cooperative mode of cation binding may be described by the Ca/Mg selectivity coefficient [53] or by adjusting the Manning linear charge-density parameter ~ to include all the free charges of a bundle of chains [54]. Concentrated pectate gels with a low content of divalent counterions bind calcium ions tenaciously (Figure 12). Might a small number of residual calcium ions, uncomplexed by the standard chelating pectin extractants like CDTA, be responsible for the retention of significant amounts of pectins against any attempts to extract them chemically? Comparison of Figure 12 with Table 6 shows that this is unlikely. CDTA and EDTA chelate calcium ions so effectively that their removal is essentially quantitative, as has been shown by total digestion and cation analysis of the CDTA residue [55].
166
5O
E ._~ 40 __o t~ 0
"
:30
Pectate - o - Pectin
r 0
~
20
L_ :3
~
10
-3
I
t
-4
-5
,
I
I
-6
-7
-8
log [free Ca]
Figure 12. Binding of calcium ions by pectate and apple pectin, measured by equih'brium dialysis against citrate to buffer the concentration of free calcium ions at low levels.
These extractants have an undesirable tendency to bind to extracted pectins, however [56]. Presumably they form ternary complexes with pectate and calcium. Imidaz~le has been suggested as an alternative [56] but Table 6 shows that it does not bind calcium ions very strongly. It may be advisable to repeat the extraction wkh imidazole a number of times in sequence. Citrate is another possible chelating extractant [57] but k has not yet been demonstrated whether k can be readily removed from pectic solutions. When extracting pectic polymers with chelating anions it is important to use a cation whose pectate salts are soluble at high concentrations. K § is superior to Na + in this respect and, judging by the recovery of galacturonans from anion-exchange resins, NH4 + may be better still [58]. Table 6. Free calcium concentrations in equilibrium with common complexing agents. A low flee calcium concentration implies effective complexation, whether the complex formed is soluble or insoluble. The data were derived from either stability constants (soluble complexes) or solubility products (insoluble complexes).
CDTA EDTA EGTA Ckrate, pH 6.5 Ammonium oxalate Imidazole
[free Ca 2+1 1014 M 10 12 M 10 12 M 10 1~ M 10 .8 M 10 -6 M
167 Until this point it has been implicitly assumed that calcium is the dominant counterion for pectic polymers in the cell wall. The cations associated with cell walls after their isolation are almost irrelevant to this question because both cations and natural chelating agents like citrate are redistributed on an overwhelming scale when the cell membranes are broken. An indirect estimate of the cell wall cations was derived by analysis of the cations in apoplastic fluid centrifuged from living Pisum and Vigna hypocotyl segments (Table 7). Equlibration of similar solutions with the eeU wall suggested that calcium was the cation bound to the largest number of pectic carboxyl groups but that sodium and magnesium ions were also present in substantial quantity. In an elegant approach to the same question, McDougaU and co-workers [59] have come to similar conclusions after non-aqueous extraction of fast-frozen tissue. Table 7. Cation concentrations in cell-wall fluid centrifuged from Vigna hypocotyls Cation
mM
Caz+ Mg 2+ Na +
1 2 10
K+
3
Some divalent cations such as Cu2+ and Pb> form very stable complexes with pectate, but are unlikely to be present at sufficient concentration in the apoplast of plants to form a major fraction of the counterions associated with the pectic fraction in vivo. The A13+ion may deserve closer examination, as it is certainly able to displace Ca2+ from cell walls and reaches substantial concentrations in plant roots under some conditions [60,61 ]. alumim'um is not usually considered to be freely translocated, however. Basic peptides with their negative charges spaced at a similar interval to galacturonans (0.43 nm or a small multiple thereof) can in principle have a very high affinity for pectate [62,63], but the extensins that are associated with the most insoluble pectic fractions [64] do not appear to have this type of structure. The possibility that the nonextractable pectic polymers in most cell walls are very strongly complexed with some cation other than Ca2§ cannot be ruled out, but there is little evidence to support it at present.
3.4 Spatial variation in pectin structure and cation binding A more direct approach is to image calcium ions within the cell wall. X-ray analysis can be used for ion mapping within biological tissues [65] but with relatively poor sensitivity it requires long exposure times with high beam currents that damage the organic matrix. Consequently the published data are usually in the form of EDS spectra rather than elemental mapping. SIMS microscopy however is capable of locating cations within biological samples with a resolution of 100-300 nm [66]. Figure 13 (A and B) illustrates that, as expected, calcium is located principally in the cell walls while potassium is intracellular. The binding of calcium ions to the cell wall avoids any problems of ion displacement during the preparation of the sample. Developing flax hypocotyls show a complex, developmentally related spatial pattern of calcium accumulation in the cell walls (Figure 13 A and C). This pattern must reflect local variations in the affinity of the cell-wall pectins for calcium ions, since the apoplast of the hypocotyl is continuous and freely permeable for cations to equilibrate within it.
168 Figure 13. Ion distribution in transverse section of flax hypocotyl. SIMS microscopy. (A), distribution of calcium (4~ and (B), potassium (39K) in the mature zone of flax hypocotyl constituted of cells which had stopped their elongation. (C), distribution of calcium in young flax hypoeotyl constituted of elongating cells. (D), small areas of calcium accumulation in the angles of the intercellular spaces.
i
3~
2:-'.
4~ ~
c(
2: 2r L~ i" L~
45 41 :37 33 25 21 !7 i:~:
i'
L
J
i
ic !
' 52 45 42 39 3E: 33 3~ 27 24 21 18 15 12
:
! i'--
I
j4!
,,
i
i37
t
','32 !25
12~ i
12g 'IE
I t
" (i iiiii i
The false-colour scale indicates the ion-signal intensities (cps for an acquisition time of 7 min for A, B and D; or 10 rain for C). E, epidermis; CP, cortical parenchyma; A, air-space. In the epidermis, one observes an accumulation of calcium in the trieellular junction (arrow head) and in the tangential wall (open arrow). In young flax hypocotyl, the calcium-signal intensities were lower than in mature flax hypocotyl (compare A and C).
169 The most conspicuous concentrations of calcium in the cell-walls of the flax hypocotyi were in the epidermal and subepidermal layers, especially at the tricellular junctions (figure 13 D), where these were filled with pectic polymers [67]. Open tricellular junctions with intercellular spaces had smaller areas of calcium accumulation where the walls of each pair of cells diverged. These sites were occupied by relatively linear pectic polymers with a low degree of esterification, which could be visualised with gold-labelled endopolygalacturonase [68] and were extractable by chelation of calcium with CDTA. Similar pectic polymers are located in the corresponding sites in other plant tissue, as established by susceptibility to polygalacturonase [69], by labelling with the 2F4 antibody which recognises low-ester, calcium-bound galacturonan [70], and by preferential labelling of low-ester galacturonan with the JIM5 rather than the JIM7 antibody [ 19,71 ]. 3.5 Pectic polymers under stress
The characteristic nature of the pectins of the tricellular junctions is only one feature of the complex disposition of varying types of pectins throughout the cell wall, but it is suitable for more detailed examination for three reasons. Firstly, the pectic polymers concerned are close in structure to the linear commercial galacturonans whose physical chemistry is best understood, so that we can expect the 'cable model' to apply. The elastic modulus of galacturonan gels with Ca 2+ depends on the proportion of the polymer participating in junction zones. Thus as the polymer charge density and the Ca 2+ concentration increase, the gel becomes more rigid until, at the extreme, it consists entirely of junction zones and collapses to a precipitate [72,73]. Branching and the insertion of rhamnose residues are likely to reduce the formation of ionic junction zones, so linear, low-ester, high-calcium gels may be expected to show the greatest rigidity. Secondly, these pectins are the only polymers present in the tricellular junctions, so the question of redistribution of stresses between them and microfibrils does not arise. Thirdly, the mechanical stresses carried by the pectins at the tricellular junctions can be calculated precisely because they are directly related to turgor, originating from its tendency to make the cells spherical. At any cell comer the stress exerted by turgor can be resolved into one component FT in the plane of each cell wall, stretching the walls as expected, and a fimher, radial component FC that tends to pull the tricellular junction apart and delaminate the walls next to it (Figure 14). It is this second component FC that is carded by the specialised pectate gel in the tricellular junction, and by the pectins that form a gel continuous with it across the thickness of the cell wall [74]. Where intercellular spaces are absent this stress is surprisingly large and is given by the equation: FC = 2FTcos[p/2(1 - 2/n)] where FC is the radial force separating the cells at the corners, FT is the tangential force along the side walls due to turgor and n is the number of cell sides in section. It can be shown that in normal cells with n<6, FC >FT [74]. Introducing an intercellular space by rounding the cell comers reduces the stress but concentrates it at the point where the walls of each pair of cells diverge. Thus the pectic polymers that can be expected to gel with the greatest rigidity are concentrated precisely where the well-characterised radial stress borne by the pectic fraction is greatest.
170
&
&
&
Figure 14. Distribution of turgor-induced stresses at a tricellular junction. FT is the componem of the stress in the plane of each cell wall and FC is the radial force separating the cells at the comers. This example may give us more confidence in interpreting the lower methyl-esteriiication of inactive cells (Section 2) in relation to the dgidification of the wail that brings growth to a halt. There is one problem. Currem models of the primary walls of dicot cells [75,76] suggest that it is the cellulose-xyloglucan network, not the pectic fraction, that is primarily responsible for resisting the expansion of the cell. However there is circumstantial evidence that calcium pectate gels contribute to the longitudinal rigidity ofhypocotyls [77,78]. For example the plastic extensibility of soybean epidermis increased reversibly on partial removal of calcium ions [77]. These observations appear to be real even if we cannot at present interpret them in terms of the load-bearing architecture of the cell wall. Perhaps pectins control slippage between microfibril layers within the wall structure. Wherever the rigidifying pectins are located, the changes that occur in their methylesterification pattern with maturity - reduced total esterification and an increased proportion of non-esterified blocks- are those that would be expected to increase the rigidity of the gel. This was illustrated by in vivo N M R measurements of polymer rigidity within the walls of growing and mature celery collenchyma cells, an analogue of the outer epidermal cell wall of seedlings ([79]; K.M. Fenwick, M.C. Jarvis and D.C. Apperley, unpublished). In growing collenchyma there were considerable amounts of heavily methyl-esterified galacturonan, showing high mobility in the kHz frequency range. When growth had ceased, low-ester galacturonans predominated and these were much more rigid. Considerable progress has been made in understanding the enzymology underlying this process, as outlined in the first part of this paper. In contrast the biosynthetic mechanisms by which linear, low-ester pectic polymers are selectively targeted to the comers of the cell wall remain to be established, although there may be clues in the sensitivity of the individual isoforms of PMT and PME to the local physical environment (e.g. charge density) within the Golgi and within the wall. Also, we cannot rule out the possibility that mechanical factors may act directly: stretching orients pectins and facilitates their ordered aggregation, thus affecting the type of structure that forms and its charge density. It can safely be assumed that the interplay of mechanical and biosynthetic factors is complex and delicately controlled: intricacy of structure implies intricacy in synthesis, and pectins within the cell wall are certainly intricate.
171 4. R E F E R E N C E S 1. O~eill, M., Albersheim, P., and Darvill, A. G. (1990) Meth. in Plant Biochem. 2, 415. 2. Voragen, A. G. J., Thibault, J. F., Axelos, M. A. V., and Renard, C. M. G. C.(1995) in Food Polysaccharides (Stephen, A. M. and Dea, I. Eds.), pp. 287-339, Marcel Dekker. 3. Aspinall, G. O., Begbie, R., Hamilton, A., and Whyte, J. N. C. (1967) J. Chem. Soc. C 1065. 4. Aspinall, G. O., Gestetner, B., Molloy, J. A., and Uddin, M. (1968) J. Chem. Soc C 2554. 5. Jarvis, M. C., Threlfall, D. R., and Friend, J. (1981) Planta 152, 93. 6. Selvendran, R. R. (1985) J. Cell Sci., Suppk 2, 51. 7. Massiot, P., Rouau, X., and Thibault, J. F. (1988) Carbohydr. Res. 172, 229. 8. Renard, C. M. G. C., Voragen, A. G. J., Thibault, J. F., and Pilnik, W. (1991) Carbohydr. Polym. 16, 137. 9. Rihouey, C., Morvan, C., Borissova, I., Jauneau, A., Demarty, M., and Jarvis, M. (1995) Carbohydr. Polym. 24, in press. 10. McNeil, M., Darvill, A. G., and Albersheim, P. (1980) Plant Physiol. 66, 1128. 11. McNeil, M., Darvill, A. G., and Albersheim, P. (1982) Plant Physiol. 70, 1586. 12. Schols, H. A. and Voragen, A. G. J. (1994) Carbohydr. Res. 256, 83. 13. Schols, H. A., Voragen, A. G. J. and Colquhoun, I. J. (1994) Carbohydr. Res. 256, 97. 14. Spellman, M. W., McNeil, M., Darvill, A. G., Albersheim, P., and Dell, A. (1983) Carbohydr. Res. 122, 131. 15. Foster, T. J., Ablett, S., Mccann, M. C., and Gidley, M. J. (1996) Biopolymers, in press. 16. Zhang, G.F., and Staehlin, L.A. (1992) Plant Physiol. 99, 1070. 17. Kauss, H., Swanson, A.L., Arnold, R., and Odzuck, W. (1969) B. B. A. 192, 55. 18. Markovic,O., and Kohn, R. (1984) Experientia, 40, 842. 19..Knox, J.P, Linstead, P.J., King, J., Cooper, C., and Roferts K. (1991) Planta, 181,512. 20. Schaumann, A., Bruyant-Vannier, M.P., Goubet, F., and Morvan C. (1993) Plant Cell Physiol. 34, 891. 21. Goldberg, R., and Prat, R. (1982) Plant Cell Physiol. 23, 1145. 22. Vannier, M.P., Thoiron B., Morvan, C. and Demarty M. (1992) Biochem. J., 286, 863. 23. Bruyant-Vannier, M.P., Gaudinet-Schaumann, A., Bourlard ,T., and Morvan, C. (1996) Plant Physiol. Biochem., 34, (4) in press. 24. Bordenave, M., Breton, C.E., Goldberg, R., Huet, J.C. and Pernollet, J.C. (1996) Accession X94443 EMBL Databank 25. Hall, L.N. Bird, C.R., Picton, S., Tucker, G.A., Seymour, G.B., and Grierson, D. (1994) Plant Mol. Biol., 25, 313. 26. Mu, J.H., Stains, J.P., and Kao, T.H. (1994) Plant Mol. Biol., 25, 539. 27. Richard, L., Qin, L.X., Gadal P., and Goldberg, R. (1994) FEBS Letters, 355, 135. 28. Richard, L., Qin, L.X., and Goldberg, R. (1996) Gene in press. 29. Albani, D., Altosaar, I., Arnison, P.G., and Fabijanski, S.F. (1991) Plant Molec. Biol. 16, 501. 30. Renard, C. M. G. C., Schols, H. A., Voragen, A. G. J., Thibault, J. F., and Pilnik, W. (1991) Carbohydr. Polym. 15, 13. 31. Jarvis, M. C., Threlfall, D. R., and Friend, J. (1981) Planta 152, 93. 32. Fry, S.C. (1986) Ann. Rev. Plant Physiol. 37, 165. 33. Rombouts, F. M. and Thibault, J. F. (1986) Carbohydr. Res. 154, 177. 34. Kim, J. B. and Carpita, N. C. (1992) Plant Physiol. 98, 646. 35. Brown, J. A. and Fry, S. C. (1993) Plant Physiol. 103,993. 36. Hricovini, M., Bystricky, S. and Malovikova, A. (1991) Carbohydr. Res. 220, 23. 37. Di Nola, A., Fabrizi, G., Lambda, D., and Segre, A. L. (1994) Biopolymers 34, 457. 38. Cros, S., Herve du Penhoat, C., Bouchemal, N., Ohassan, H., Imberty, A., and Perez, S. (1992) Int. J. Biol. Macromol. 14, 313.
172 39. Gouvion, C., Mazeau, K., Heyraud, A., Taravel, F. R., and Tvaroska, I. (1994) Carbohydr. Res. 261,187. 40. Ruggiero, J. R., Urbani, R., and Cesaro, A. (1995) Int. J. Biol. Macromol 17, 205. 41. Ruggiero, J. R., Urbani, R., and Cesaro, A. (1995) Int. J. Biol. Macromol 17, 213. 42. Rees, D.A. and Wight, N.J. (1969) Biochem. J. 115, 431. 43. Morris, E. R., Powell, D. A., Gidley, M. J., and Rees, D. A. (1982) J. Mol. Biol. 155, 507. 44. Powell, D. A., Morris, E. R., Gidley, M. J., and Rees, D. A. (1982) J. Mol. Biol. 155, 517. 45. Walkinshaw, M. D. and Arnott, S. (1981) J. Mol. Biol. 153, 1055. 46. Walkinshaw, M. D. and Arnott, S. (1981) J. Mol. Biol. 153, 1075. 47. Alagna, L., Prosperi, T., Tomlinson, A. G., and Rizzo, R. (1986) J. Phys. Chem. 90, 6853. 48. Kirby, A. J. The Anomeric Effect and Related Stereoelectronic Effects at Oxygen, Springer, Berlin 1983. 49. Jarvis, M. C. (1994)Carbohydr. Res. 259, 311. 50. Jarvis, M. C. and Apperley, D. C. (1995) Carbohydr. Res. 275, 131. 51. Ha, M.A., Evans, B.W., Apperley, D.C. and Jarvis, M.C., this volume. 52. Goldberg, R., Morvan, C. and Roland, J.C. (1986) Plant Cell Physiol. 27, 417. 53. Goldberg, R., Morvan, C., Herve du Penhoat, C. and Michon, V. (1989) Plant Cell Physiol. 30, 163. 54. Thibault, J.F. and Rinaudo, M. (1986) Biopolymers 24, 2131. 55. Jarvis, M.C. (1982) Planta 154, 354. 56. Mort, A.J., Moerschbacher, B.M., Pierce, M.L. and Maness, N.O. (1991) Carbohydr. Res. 215, 219. 57. Jarvis, M. C., Logan, A. S., and Duncan, H. J. (1984) Physiol. Plantarum 61, 81. 58. Cheng, L. and Kindel, P. K. (1995) Anal. Biochem. 228, 109. 59. MacDougaU, A.J., Parker, R. and Selvendran, R.R. (1996) Plant Physiol., in press. 60. Blamey, F. P. C. and Dowling, A. J. (1995) Plant And Soil 171,137. 61. Ostatekboczynski, Z., Kerven, G. L., and Blamey, F. P. C. (1995) Plant And Soil 171, 41. 62. Cassab, G. I. and Varner, J. E. (1988) Ann. Rev. Plant Physiol. and P.lant Mol. Biol. 39, 321. 63. Bystricky, S., Kohn, R., Sticzay, T., and Blaha, K. (1986) Coll. Czech. Chem. Commun. 51, 1772. 64. Qi, x.Y., Behrens, B.X., West, P.R. and Mort, A.J. (1995) Plant Physiol. 108, 1691. 65. LazofD. and L/iuchli A. (1991). Planta, 184: 327. 66. TheUier M., Ripoll C., Quintana C., Sommer F., Chevallier P. and Dainty J., (1993). Meth. Enzymol., 227: 535. 67. Rihouey, C., Jauneau, A., Cabin-Flaman, A., Demarty, M., Lefebvre, F. and Morvan, C. (1995). Plant Physiol. Biochem. 33,497. 68. Cabin-Flaman, A., Jauneau, A., Morvan, C. and Demarty, M. (1993). J. Microsc. 171, 117. 69. J.C. Roland and B. Vian, (1981) J. Cell Sci. 48, 333. 70 Liners, F. and Van Cutsem, P. (1992) Protoplasma 107, 10. 71. Roy S., Jauneau A. and Vian B (1994). Plant Physiol. Biochem., 32: 633. 72. Gamier, C., Axelos, M.A.V. and Thibault, J.F. (1993) Carbohydr. Res. 240, 219. 73. Axelos, M.A.V., Gamier, C. and Thibault, J.F., this volume. 74. Jarvis, M.C. (1992) Planta 187, 218. 75. McCann, M. C. and Roberts, K. (1994) J. Exp. Bot. 45, 1683. 76. Gibeaut, D. M. and Carpita, N. C. (1994) Faseb J. 8, 904. 77. Virk, S.S. and Cleland, R.E. (1988) Planta 176, 60. 78. Prat, R., Goeissaz, M.B. and Goldberg, R. (1984) Plant Cell Physiol. 25, 1459. 79. Jarvis, M. C. and Apperley, D. C. (1990) Plant Physiol. 92, 61.
J. Visserand A.G.J. Voragen(Editors),Pectinsand Pectinases 9 1996ElsevierScienceB.V.All rights reserved.
173
C o n t r i b u t i o n of p e c t i n s on h e a l t h c a r e H. Yamada Oriental Medicine Research Center, The Kitasato Institute 5-9-1, Shirokane, Minato-ku, Tokyo 108, Japan
Abstracts
Pectins have been shown to possess a variety of pharmacological activities such as several immunostimulating activity, anti-metastasis activity, anti-ulcer activity, anti-nephrosis activity, cholesterol decreasing effect and so on. Pectins are also applicable for drug delivery and as the vaccine for typhoid fever. Pharmacological activities which were observed in pectins and pectic polysaccharides seem to be dependent on the fine carbohydrate structures. Many pharmacological activities have been appeared in the ramified region, but some high molecular weight rhamnogalacturonan II like region also showed Fc-receptor up-regulation for macrophages. Even if natural pectin had no activity, chemical and enzymic modifications may provide useful product for health care. Present observations suggest that application of pectin on health care brings many possibilities of benefits for human being.
1. I N T R O D U C T I O N
Many plants have long been known to have medicinal effect therefore have been used as medicines especially in traditional medicines. Even today, about 75% of the world popuration still relies on plant, plant extracts and other tools of traditional medicines in basic health need. While incidences of numerous globally rampant infections had been reduced by the development of the modern medicines such as antibiotics, some chronic disorders of endogenous nature instead surfaced during the second half of the 20th century [1]. Furthermore, pressing medical problems such as non-specific, constitutional or psychosomatic diseases have also increased. Disillusion with modern medicine has on occation brought about by severe adverse effects of synthetic compounds. Because of these circumstances traditional medicinal herbs have began to attract worldwide attention. Herbal extracts contain substances with both low and high molecular weight. Although biologically active substances of the former compounds in medicinal herbs including Kampo (Japanese herbal) medicines have been studied, they can not account for all of the clinical effects achieved. Pharmacological activities have been observed in fractions with high molecular weights from boiled water extracts of the medicinal herbs. Of the high molecular weight substances, various pharmacological
174 activities have been observed in pectic polysaccharides and pectins isolated from plants containing medicinal herbs [2-6] as shown in Table 1. Therefore pectic polysaccharides and pectins are possible to use as medicines and for other health care. Table 1 Pharmacological activity of pectins isolated from plants containing medicinal herbs Immunostimulating activity 9Complement activating activity 9Mitogenic activity 9Fc receptor up-regulation on macrophages (enhancing activity of immune complex clearance) 9Stimulation of macrophage phagocytosis Anti-ulcer activity Anti-metastasis activity Anti-nephritis activity and anti-nephrosis activity Hypoglycemic activity Cholesterol decreasing effect Applications : Vaccine for typhoid fever Drug delivery (arabinogalacan)
Pectic substance refers to a group of closely associated polysaccharides from the primary cell walls and intracellular region of higher plants [7], and are probably the most complex class of polysaccharides in plant cell wall components. Pectins, which are the major plant cell wall components, and generally comprise a large quantity of esterified and unesterified galacturonans with a small of rhamnogalacturonans substituted neutral oligo and/or polysaccharide chains. Pectins have been used as important food fiber, ubiquitous nutritional factor and gelling agent in food. Pectic polysaccharides comprise a family of rhamnogalacturonans and several neutral oligosaccharides (arabino- and galactooligosaccharides), and polysaccharides (arabinans, galactans, arabinogalactans) which are believed to be covalently attached to the rhamnogalacturonan backbone primarily through the rhamnopyranosyl residues [8]. They are usually extracted from walls with water or aqueous solution. By digestion with pectinase [endo-a-(1---)4) polygalacturonase], rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II) have been obtained, and characterized their detailed carbohydrate sequences by Albersheim group [9]. RG-I is probably similar part as hairy region or ramified region of pectins referred by other groups, and is composed of rhamnogalacturonan core substituted with neutral carbohydrate side chains such as arabinan, galactan, arabinogalactans and related oligosaccharides. RG-II is composed of rare sugars such 2-methylfucose, 2methylxylose, apiose, aceric acid, KDO (2-keto-3-deoxyocturosonic acid) and DHA
175
(3-deoxy-D-lyxo-2-heptulosaric acid) in addition to galacturonic acid, glucuronic acid, rhamnose and arabinose. Pharmacological activities, which were obseved in pectins and pectic polysaccharides, seemed to be dependent on the region of fine carbohydrate structures [3, 6]. The present paper deals with pharmacological activities of pectins and pectic polysaccharides and discuss the role of pectins in health care. 2. ANTI-METASTASIS ACTIVITY Prostate cancer is the most commonly diagnosed cancer in U.S. men and is the second leading cause of male cancer deaths [10]. Approximately 50% of patients diagnosed with prostate cancer have disease that has or will metastasize to the skeltal system. At present, there is no effective curative therapy and very little palliative therapy for patients with metastatic disease [11]. The process of tumor cell metastasis requires that cells depart from primary tumors, in_vade the basement membrene, travers through the blood stream, interact with the vascular endothelilum of the target organ, extravasate, and proliferate to form secondary tumor colonies [12]. It is generally that many stages of this metastatic cascade involve cellular interactions mediated by cell surface components, such as carbohydrate binding proteins, which include galectins [13]. Tumor galactose-binding proteins mediate cellular recognition by linking oligosaccharides with terminal D-galactoside residues on adjacent cells. Platt and Raz tested the effect of citrus pectin (CP) on modulation of the lung colonization of B16-F1 melanoma cells, and found that co-injection of modified citrus pectin (MCP), which was prepared by alkaline treatment and mild acid hydrolysis of the pectin, with B16 melanoma cells resulted in a marked inhibition (>90%) of their ability to colonize the lungs of the mice receiving the injection, however natural pectin resulted in a significant increase in the appearance of tumor colonies in the lung [14]. Oral administration of MCP also inhibited spontaneous metastatic colony formation of rat prostate adenocarcinoma MATLyLu cells [11]. MCP inhibited MAT-LyLu cell adhesion to rat endothelial cells in time and dose-dependent manners. From these observations, they concluded that oral intake of MCP acts as a potent inhibitor of spontaneous prostate carcinoma metastasis in the rats [11]. Because galectin-3 is present in human prostate adenocarcinoma cell line and MCP interferes with cell-cell interactions mediated by cell surface galectin-3 molecules [11], it was suggested that the inhibition of cancer metastasis might be caused by the action of galactose-rich ramified region in MCP against cell-cell interactions.
3. IMMUNOSTIMULATING ACTIVITY
3.1. Fc-receptor up-regulation on macrophages Combination of circulating antigens and antibodies to form immune complexes is a normal phenomenon, and normally this immune complex eliminates by
176 reticuloendothelial system. But, if the excecssive quantities of are formed, immune complex deposits to several tissues, in which case the complement system is activated, resulting in anaphylatoxin formation and then several inflammations occur. Therefore it has been considered that this deposition of immune complex may be one of the causes of auto-immune disease development. A primary function of mononuclear phagocytic cells is to bind immune complex throuogh Fc and complement receptors, followed by subsequent endocytosis and degradation. Therefore the binding of immune complex to these cells is an important functional paramater for immune complex clearance, and the enhancing substance for this binding has a possibility that is able to treat auto-immune diseases. We developed a new photometoric microassay for immune complex binding to macrophages in a homologous system [15]. Thioglycollate elicited murine peritonial macrophages from ICR mice were cultured with or without test samples. Thereafter macrophage monolayer was washed with PBS then GAG (glucose oxidase-anti-glucose oxidase complex as a model immune complex) solution was added and incubated at 4~ After the incubation, the macrophages were washed with PBS to remove unbound GAG followed by solubilization of bound GAG to macrophages with a detergent, Nonidet P-40. Then the bound immune complexes to macrophages were measured by the enzyme activity used colorimetric determination. When the ability of immune complex clearance through Fc receptor of macrophages was tested by this assay, acidic pectic polysaccharide, bupleuran 2IIb, from Bupleurum falcatum was found to have a potent activity [16]. Bupleuran 2IIb remarkably enhanced GAG binding through Fc receptor of macrophages by dose dependent manner (Fig. 1). When bupleuran 2IIb was 0.50 u~ 0
0 0.25 m Bupleuran 2IIb 0 ~
[-I Bupleuran 2IIc
.
!
50 Concentration (~g/ml)
!
100
Fig. 1 Immune complex binding activity of pectic polysaccharides (Reproduced with permission from reference 6. copyright 1994
Carbohydratepolymer).
177 administrated to the mice, immune complex was clearanced from the circulating blood by dose dependent manner [17]. However, bupleuran 2IIb did not affect to the carbon clearance. These results suggest that bupleuran 2IIb specifically potentiate function of macrophage in clearance of immune complex through Fc receptor (FcR). Bupleuran 2IIb, which is a homogenous polysaccharide, and has a molecular weight of 23,000, consists of highly methyl esterified and less esterified galacturonan regions, ramified region and KDO containing region [18-20]. The ramified region consists of rhamnogalacturonan core which is substituted with several n e u t r a l carbohydrate side chains such as arabino- and galactooligosaccharides, arabinogalactan or arabinopyranan. KDO-containing region resembles to rhamnogalacturonan II (RG-II) which was first reported in plant cell wall by Darvill et al. [21]. This region consisted of galacturonan substituted with rare sugars in plant cells such as KDO, DHA, apiose, aceric acid, 2-methylfucose and 2-metylxylose. When bupleuran 2IIb was treated with endo-polygalacturonase, the enzyme resistant ramified region, PG-1, showed a potent activity, but the activities of KDO-containing region and oligogalacturonides were weak or negligible (Fig. 2). This result suggest that the ramified region consisting of rhamnogalacturonan core with neutral carbohydrate chains is important for the binding activity. Scatchard analysis indicated that bupleuran 2IIb enhanced FcR expression on macrophage cell surface but did not increase affinity of the receptors (Fig. 3). Bupleuran 2IIb-stimulated cells showed enhanced expression of both FcRI and FcRII m-RNA, which were measured as PCR products [16]. When macrophages were incubated with bupleuran 2IIb, a rapid increase of intracellular Ca 2+ level was observed by a fluorescence image analysis using the calcium-sensitive dye, Fura-2 [22]. However, signal transduction through protein kinases C and A did not involve to the expression of activity. These observations suggest that bupleuran 2IIb may be bind to the macrophage through the specific polysaccharide receptor for this ramified region, then this stimulation enhances FcR gene-expression by through signal transduction due to the increase of intracellular Ca 2+,and then FcR protein may be up-regulated on the macrophage surface (Fig. 4). Although roots of Panax ginseng C. A. Meyer have been used as a valuable Chinese medicine, an immune complex clearance enhancing polysaccharide (GL4IIb2') has been isolated from hot water extract of ginseng leaves (Fig. 5) [23, 24]. GL-4IIb2' has a molecular weight at 8000-10000, and was resistant against endoa-(l~4)-polygalacturonase. GL-4IIb2' consists of plant rare component sugars such as 2-methylfucose, 2-methylxylose, DHA, KDO and aceric acid which are characteristic in RG-II. Structure of GL-4IIb2' was similar as rhamnogalacturona II, but it was rich in a-Rha-(1-~5)-Kdo side chain, and has some additional linkages and relatively higher molecular weight compared with RG-II (Fig. 6). Up to now, no pharmacological activity has been reported in RG-II. Therefore this is a first pharmacological activity observed in RG-II-like structure.
178
Percent of Bound GAG Control (vehicle) Bupleuran 2IIb PG-1 (ramified region) PG-2 (KDO containing region) PG-3 (oligogalacturonides)
N=4 (1001~g/ml)
I L
! 100
I 200
300
Fig. 2 Immune complex binding enhancing activity of Carbohydrate fragments obtained by endo-polygalacturonase digestion
15
9 Bupleuran 2lib (lO01~g/ml) 0 Control
cO
o i-d 10 X v
0
10
20
30
40
Bound GAG(ng/105 cells) Fig. 3 Scatchard analysis of immune complex binding to the macrophages incubated with bupleuran 2IIb (reproduced with permission from reference 6. copyright 1994
Carbohydrate Polymer).
179
300"
Immune Complexes ] I Immure Complexes ; Clearance FcR up-regulation
[ FcR 'J
i mRNA
!
BUPleuran 2lib
]
250
Stimulation Proteinous Binding nSlte
=' Recognition =
,
?
|DNA !
200 150
[Signal Transduction !
Macropha~les
GL-411b2'
I:k
!
A
100.
i
501
.
.01
Fig. 4 FcR up-regulation and immune complexes clearance by bupleuran 2IIb
GI.-411b-2
GL-411b-2
RG-II walls upon digestion with
endo-PGa~
|
60 Relatively strong macrophage FcR expression enhancing activity
Structural feature
100
Released from p r i m a ~ cell
of endo-PGase
dp Pharmacological activity
.
Fig. 5 Effect of GL-4IIb2' and periodate oxdization product on FcR mediated immune complex binding to macrophages
Obtained without treatment
Preparation
.
.1 1 l0 Concentration (mg/ml)
[ Rha-(1 ->5)-KDO
60 - 30 Not reported Ara-(1 9 -->5)-DHA ]
5"1 1 "1 [ AceA containing nonasaccharide ] "1 : $
: :3 Ara f( 1~ 2)-Rha-(1 -~2),,'Ara-(1~ 4)iGal-(1 ~ 2)-AceA-(1 ~3)-Rha-(1 -,3')-Api
:
9
o. = , ,
,, , , . . , ,
...,p
: 2 ' T
1 2-O-Methyl-Fuc
........... ' .: : : Rha-(1 -~2)iAra-(1-~4)!Gal-(1 -,2)-AceA-(1-,3)-Rha-(1 -~3')-Api RG-II
i
i
.......... ~ 1
2-O-MethyI-Fuc
Fig. 6 Structural differences between GL-4IIb2' and RG-II
180
3.2 Complement activating (anti-complementary) activity The complement system consists of over 20 serum proteins including 9 complement components (C1-C9) and their regulators. The complement proteins are activated by a cascade machanism of classical or alternative pathways. The classical pathway is activated by the binding of C1 to the Fc region of immune complexes containing IgM and IgG antibodies and is followed by further activation. On the other hand alternative pathway is directly activated from C3 by some activators such as lipopolysaccharide, and is follwed by further activation. Complement activation appears to be intrinsically associated with several immune reactions such as the activation of macrophages and lymphocytes, immunopotentiation and so on [25]. Several complement activating polysaccharides have been discovered in the medicinal herbs such as the roots of Angelica acutiloba Kitagawa, the leaves of Artemisia princeps PAMP, the roots of Bupleurum falcatum L, the roots and leaves of Panax ginseng, the roots of Glycyrrhiza uralensis Fisch et DC, berries of Viscum album var. album (L.) and so on [3, 26, 27]. Six kinds of complement activating pectins (AR-2IIa, 2IIb, 2IIc and 2IId) and pectic arabinogalactans (AGIIa, AGIIb-1) have been purified from the hot water extract of the roots ofAngelilca acutiloba [28-30]. AGIIa and AR-2IId showed the most potent complement activating activity. AGIIb-1 showed moderate activity and others were weak. These four pectins commonly consisit of over 90% a-(1-~4)linked galacturonan and a small amount of ramified region which contains rhamnogalacturonan core with neutral carbohydrate side chains such as arabinan, galactan and arabinogalactan which are assumed to be linked to position 4 of rhamnose [30]. Digestion with endo-a-(1-->4)-polygalacturonase after deesterification gave ramified region as an enzyme resisitant fraction and several oligogalacturonides [30]. The ramified region from each pectin had a more potent complement-activating activity than the corresponding original pectin, but the oligogalacturonides had weak or negligible activities (Table 2) [30]. These results suggest that for AR-2IIa, 2IIb, 2IIc and 2IId, ramified region might be essential for expression of the activity since each ramified region from four pectins showed similar potent activity. Because (l~3,6)-linked long ~galactosyl chains and oligosaccharides containing ~-(1-~6)-galactose, which were obtained from ramified regions, showed potent or significant activity, for AR-2IIa, 2IIb, 2IIc and 2IId, the neutral carbohydrate side chains attached to the rhamnogalacturonan core might be essential for expression of the activity (Fig. 7) [31]. Although AR-2IIa~-IIc activated complement through the classical pathway but not the alternative pathway, all ramified regions (PG-1) activated complement through both pathways. AR-2IId had a different methyl-ester distribution in the galacturonan region compared to the other three pectins [32]. These results suggest that this galacturonan moiety may modulate the activation of an alternative pathway by the ramified region, and this modulation may be controlled by the distribution of the methyl-ester on the polygalacturonan moiety. Both arabinogalactans, AGIIa and AGIIb-1, were characterized to contain arabino~-3,6-galactans by structural analysis [28, 29]. These carbohydrate structures seem to be common for complement activation by pectins and pectic polysaccharides. Acidic arabinogalactan from berries of Viscum album also strongly activated
181
complement [27]. Table 2 Anti-complementary activity of enzymic digestion products from the pectins of
Angelica acutiloba Concentration (pg/mL) 1000 500 100
Product
Product
Inhibition of TCHs0 (%)
Inhibition of TCHso (%) AR-2IIa Original PG-1 PG-2 PG-3 AR-2IIb Original PG-1 PG-2 PG-3
59.5 82.5 38.0 19.2
30.8 74.5 23.8
0.6 25.0 2.0
62.6 82.2 22.0 4.8
31.8 74.5 2.0 1.5
2.0 25.5
Concentration (~g/mL) 1000 500 100
AR-2IIc Original PG-1 PG-2 PG-3 AR-2IId Original PG-1 PG-2 PG-3
54.3 84.5 28.5 1.8
29.2 82.8
3.0 41.8
84.5 48.8 33.0 n.d. a
85.0 81.8 10.0 n.d.
52.5 40.0 n.d.
"Not determined. PG-1, ramified region; PG-3, oligogalacturonides
/
lO0
I RG core I
Gal-~3Gal--,3Gal O
h~ o3 .i--i
f
50-
\
r 03
g
Gal~6Gal-~6Gal Gal~6Gal~6Gal~6Gal Gal~6Gal~Rha Gal-~6Gal-~6Gal~Rha Gal--,6Gal~Rha-~Rha
.~
O
0 0
!
!
!
100
200
300
Concentration (mg/ml)
6
$
6
Gal
Gal
Gal
Gal
Gal
Gal
$
Gal
(reproduced with permission from reference 6. copyright 1994
Carbohydrate Polymer).
Figure 7. Complement activating potency of the ramified region and its neutral oligosaccharides from the pectins of Angelica acutiloba
182 3.3 Other immunostimulating activity Wagner et al. obtained immunostimulating pectic polysaccharides from plant cell culture of Echinacea purpurea [5]. From the extracellular polysaccharide mixture, acidic arabinogalactan (Echinacea-polysaccharide II) was purified [33]. Echinacea-polysaccharide II, which has a molecular weight at 75,000, was effective in activating macrophages to cytotoxicity against tumor cells [34] and in vitro as well as in vivo against microorganisms such as Leishmania enriettii and Candida albicans. This polysaccharide induced macrophages to produce tumor necrosis factor (TNF-a), interleukin-1 (IL-1), interferon-~2 and oxygen radicals. Echinacea-polysaccharide II consists of arabino-3,6-~-galactan part, rhamnogalacturonan part and arabinan part [5], therefore it is suggested that the polysaccharide may be certain ramified region of pectic polysaccharide. Compounds of plant origin which modify immunological responses have also been shown to influence natural cytotoxicity against tumor cells. Viscum album extracts (Iscador-M| contain a component which strongly increases the cytolytic activity of peripheral blood mononuclear cells (PBMCs) from human [35]. The rapid formation of conjugates between effector cells and tumor cells in the presence of V. album extracts appears to involve bridging by V. album rhanmogalacturonan which enhances the cytotoxicity of human NK cells [36]. Pre-incubation of NK cells with the rhamnogalacturonan did not activate the ~lling potential. Therefore, a synergistic effect of NK cell-tumor cell bridging by the rhanmogalacturonan, together with the activation of NK cytotoxicity by physiological response modifiers such as interleukin-2 might offer a new basis for the effective treatment of cancer [36]. Mitogenic polysaccharide was obtained form the roots of Glycyrrhiza uralensis [26]. Endo-a-(1-~4)-polygalacturonase digestion indicates, this mitogenic polysaccharide has a pectic nature and the enzyme resisitant ramified region showed more potent mitogenic activity than the original polysaccharide. This mitogenic polysaccharide also contained RG-II like region. Several arabino-3,6-~-galactans, which were obtained from Panax notoginseng and Saposhinikovia divaricata, were observed to activate the reticuloendothelial system (RES) in vivo by carbon clearance test [37, 38]. Pectins from Glycyrrhiza uralensis, Euchommia ulmoides and Angelica acutiloba also had RES activating activity, and arabino-3,6-~-galactan rich pectin showed more potent activity [39-41]. 4. ANTI-ULCER ACTIVITY During a study of the polysaccharides from Chinese herbs, potent anti-ulcer activity was observed in the acidic polysaccharide fraction (BR-2) from Bupleurum falcatum, and the active polysaccharides, bupleuran 2IIb and 2IIc were purified [42]. Hundred mg/kg ofbupleuran 2IIb and 2IIc showed the significant anti-ulcer activity against HCl-ethanol induced ulcerogenesis in mice (Fig. 8). This activity was almost same with positive control, sucralfate. The activity of bupleuran 2IIc was higher than that of sucralfate. Bupleuran 2IIc, which has a molecular
183 weight of 63,000, consists of 85.8% of galacturonan region comprising of 70% of a-(l~4)-linked galacturonic acid, 30% of carboxymethylated galacturonic acid and branched galacturonic acid [43]. Bupleuran 2IIc also contained ramified region which consisted of rhamnogalacturonan core and several arabino- and galactooligosaccharide side chains attached to either 2-1inked rhamnosyl residue through 4-1inked galacturonic acid or 2-1inked rhamnose directly in the rhamnogalacturonan core. RG-II like region was also contained in bupleuran 2IIc as a minor region [43, 44]. The oral administration of BR-2 at doses 50 to 200 mg/kg prevented the formation of gastric lesions induced by HCl-ethanol by dose dependent manner [45]. The intraperitoneal and the subcutaneous administrations of BR-2 also dose dependently reduced this gastric lesion. These results suggested that BR-2 exerts through not only a local action but also a systemic action in the stomach. BR-2 also inhibited a variety of acute and chronic experimental ulcer models such as ethanol induced ulcer, indomethacinHC1 induced ulcer, pyrolus ligated ulcer, water-immersion stress ulcer and acetic acid induced ulcer by oral administration [45]. The collective results suggested that the major mechanism of muc0sal protection by BR-2 may be due to its anti-secretory activity on acid and pepsin, its increased protective coating and its radical scavenging effects but not involved in the action of endogenous prostagrandins and mucus synthesis [45, 46]. The ramified region seemed to be one of active site in bupleuran 2IIc. Therefore anti-polysaccharide antibody was made by immunization of its ramified region (PG-1) to the rabbits. Then highly sensitive ELISA method using the purified antibody was developed in order to detect the active polysaccharide. This method is very useful for quality control of the active polysaccharide and for study of absorbtion to the body and pharmacodynamics of the polysaccharide [47]. In this ELISA system, anti-bupleuran 2IIc-PG-1 antibody, which was purified by Protein G-Sepharose, was coated as first antibody on the microtiter plate, and the ramified region was detected by the biotynylated anti-bupleuran 2IIc-PG-1, which was purified by both Protein-G Sepharose and bupleuran 2IIcPG-1 immobilized Sepharose, and the enzyme labelled streptavidin. Bupleuran 2IIc-PG-1 at concentrations greater than lng/well could be measured by this two site sandwich ELISA method. Immunohistograph showed that lymph-follicle in payer's patch and liver both were stained with anti-bupleuran 2IIc-PG-1 antibody specific IgG after oral administration of bupleuran 2IIc. Bupleuran 2IIc-PG-1 was also detected in the liver homogenate one week after oral administration by sandwich ELISA method (Fig. 9). These results indicate that at least a part of bupleuran 2IIc was absorbed to the body after the oral administration. Ginseng leaves also contained an unique anti-ulcer polysaccharide [48, 49]. The most active anti-ulcer polysaccharide, GL-BIII, had a molecular weight at 16,000, and 56.6% of neutral sugar and 33.1% of uronic acid were contained. As major component sugars, rhamnose, arabinose, galactose, galacturonic acid and glucuronic acid were detected. Although most of phamacologically active
184
" " I "
30 [
]
100 mg/kg, p.o. mean + s.e.m. (n=8) ** p<0.01 *** p<0.001
20 ~D
-~,,4 m
~
0.20
***p < O.Ol (n = 6) 0
0.10
N
?:',.,o%/
0
0.00
Control Fig. 8 Effects of bupleuran 2IIb and 2IIc on HC1/ethanol induced gastric lesions in mice (Reproduced with permission from ref. 6. copyright 1994 Carbohydrate Polymer)
bupleuran 2IIc
Fig. 9 Distribution of orally administrated bupleuran 2IIc in mouse liver (after 1 week)
pectins consist of ramified region, galacturonan region and RG-II like region, GL-BIII was endo-polygalacturonase resistant and contained only ramified region which consists of r h a m n o g a l a c t u r o n a n core attached long neutral carbohydrate side chains composed of 6-1inked glucosyl, 2-1inked mannosyl and other sugar residues and short side chains composed of arabinosyl and galactosyl residues [50]. Oral administration of GL-BIII showed a potent anti-ulcer activity against HC1/ethanol induced gastric ulcer of mice by dose dependent m a n n e r (Table 3).
Table 3 Effect of GL-BIII on HCl/ethanol-induced gastric lesions in mice Treatment (p.o.) Control GL-BIII
Dose (mg/kg)
N
-
8
12.5
8 8 8 8
25 50 100
Lesion Index (min)
Inhibition (%)
21.4 +_3.1 17.4 + 2.6 12.1 +_2.1" 7.5 _+ 1.7"* 4.8 + 1.2"**
18.7 43.5 64.9 77.6
Expressed as mean + S.E.; *P<0.05, **P<0.01, ***P<0.001.
-
185 5. ANTI-TUMOR ACTIVITY The polysaccharide fraction from the roots of Angelica acutiloba Kitagawa was found to have a potent antitumor activity against ascitic form of Sarcoma-180, IMC carcinoma, and MethA fibrosarcoma as well as the solid tumor MM-46 tumor [51]. Purified active polysaccharide, AR-4E-2 was characterized to contain rhamnogalacturonan moiety in which 2,4-disubstituted rhamnose residues were attached to 4-substituted galacturonic acid through position 2 of rhamnose. AR-4E-2 also contained highly branched 3,5-arabinan and (l~4)-galactan [51]. 6. ANTI-NEPHROSIS ACTIVITY Salviae miltiorrhizae radix (SMR) has been used in China for the treatment of various renal diseases or lesions in blood circulation. Guoji et al. reported that SMR suppressed urinary protein excretion and improved levels of serum albumin, cholesterol and lipid peroxide in rats which had aminonucleoside (puromycin)induced nephrosis [52]. They purified the active component in a hot-water extracts of SMR, and certain acidic polysaccharide containing 80% galacturonic acid, probably pectin, was found to reduce urinary protein excretion in rats with the experimental nephrosis by oral or intramuscular administration [52]. Electron microscopical analysis also revealed that the extent and the severity of lesions of the epithelial cells in glomerulus were significantly less in the rats treated with this acidic polysaccharide. Because the effect on experimental nephrosis was decreased by a reduction of carboxyl groups, it was suggested that pectin may stabilize and restore the glomerular basement membrene structure by its polyanionic nature due to galacturonan region, and this may contribute to the improvement of puromycin-induced nephrosis.
7. VACCINE F O R TYPHOID F E V E R
Capsular polysaccharide (Vi) is both an essential virulence factor and protective antigen of Salmonella typhi [53]. Vi is a linear homopolymer of (1--)4)-a-DGalpANAc, variably O-acetylated at C-3 as shown in Fig. 10 [54]. Field trials in Nepal and in Republic of South Africa showed that a single injection of Vi conferred about 70% protection against typhoid fever in older children and in adults [55]. Its protective action is to elicit a critical level of serum antibodies and the Vi conjugates elicited significantly higher levels of serum antibodies than did Vi alone. Because high molecular weight of Vi causes the conjugates to be poorly soluble and standardization is very difficult, Szy et al. attempted to apply citrus pectin as an immunogen instead of Vi [55]. Pectins were mainly composed of (1-~4)-linked a-D-galacturonic acid with small of neutral sugars. Therefore, they prepared O-acetylated pectin (OAcPec) which has different structure from Vi only in that its C-2 is substituted with OAc rather than NAc (Fig. 10). Pectin did not react with Vi antiserum in double immunodiffusion, but OAcPec formed a
186 line of identity with Vi. OAcPec is not immunogenic as like Vi, but OAcPec conjugated to tetanus toxoid elicited Vi antibodies in mice, and reinjection elicited a booster response. These observations indicates that the use of pectin as an immunogen for prevention of a systemic infection caused by capsulated pathogen (S. typhi) provides a novel approach to improve the preparation and immunogenicity of polysaccharide-based vaccines [55].
/ r
Vi antiserum "x~
COOH 0
o
C~176
o
Capsular polysaccharide O-acetylated pectin (OAcPec) (Vi) of Salmonella typhi Vaccine: OAcPec-tetanus toxoid conjuage Fig. 10 Cross reactivity of Vi antiserum against Vi and OAcPec 8. A P P L I C A T I O N FOR HEPATIC D R U G DELIVERY
Delivery of diagnostic or therapeutic agents to hepatocytes has often been achived by attachment of the agent to carrier molecules that bind the asialoglycoprotein receptor [56]. Pectins consist of ramified region which substituted arabinans and/or arabinogalactans. Groman et al. demonstrated that intravenous injection of radiolabelled arabinogalactan (4mg/kg) from the tree Larix occidentalis in rats resulted in 52.5% of the dose being present in the liver, while prior injection of asialofetuin (100mg/kg) reduced hepatic radioactivity to 3.54% [57]. When the tritiated arabinogalactan was injected, radioactivity cleared from the liver with a half-life of 3.42 days [57]. The arabinogalactan produced no adverse reactions in single intravenous dose (mouse, 5000 mg/kg) and repeat dose toxcity studies (rats, 500 mg/kg/day, 90days). This arabinogalactan had highly branched structure and consisted mainly of terminal 6-1inked and 3,6-disubstituted ~-galactopyranosyl and terminal a-arabinofuranosyl and aarabinopyranosyl residues. Therefore it indicates that arabinogalactan is suitable as carrier for delivering diagonostic or therapeutic agents to hepatocytes via the asialoglycoprotein receptor. 9. C O N C L U S I O N S
Results presented herein indicates that pectins and pectic polysaccharides
187 involve in several pharmacological activities. Each pharmacological activity of the pectins may depend on their fine chemical structure of characteristic structural units such as galacturonan, ramified region (rhamnogalacturonan core substituted arabinan and/or arabinogalactan) and rhamnogalacturonan II (RG-II) like region in pectin molecules (Table 4). Many pharmacological activities have been appeared in the ramified region therefore detailed structural analysis of neutral carbohydrate side chains will be required in order to elucidate exact essential carbohydrate sequence for the expression of activity. Most of pharmacologically active pectins contained RG-II like region [19] therefore it also has a possibility to involve in some phamacological activity. Even if natural pectin had no activity, chemical and enzymic modification of pectin may provide useful product for health care. Present observations suggest that application of pectins on health care brings many possibilities of benefits for human being.
Table 4 Pharmacological activities of structural unit in pectin ramified region hairy region RG-II RG-I Vaccine for Typhoid fever Drug dilivery Anti-metastasis Anti-ulcer (roots of Bupleurum falcatum) (leaves of Panax ginseng) Immunostimulating activity 1) complement activation 2) FcR up-regulation (roots of Bupleurum falcatum) (leaves of Panax ginseng) 3) Macrophage phagocytosis (activation of RES) 4) anti-tumor activity Anti-nephrosis Cholesterol decreasing
galacturonan region
188 10. R E F E R E N C E S
10 11 12 13 14 15 16 17 18
H. Yamada, Asia Pacific J. Phamacol., 9 (1994) 209. G. Franz, Planta Med., 55 (1989) 493. H. Yamada and H. Kiyohara, H. M. Chang (ed.), Abstracts of Chinese Medicine, vol. 3, pp. 104, The Chinese University of Hong Kong, N. T. shatin, Chinese Medicinal Material Research Center, 1989. R. Srivastava and D. K. Kulshreshtha, Phytochemistry, 28 (1989) 2877. H. Wagner, Pure Appl. Chem., 62 (1990) 1217. H. Yamada, Carbohydr. Polymers, 25 (1994) 269. D. W. James JR, J. Preiss and A. D. Elbein, G. O. Aspinal (ed.) The polysaccharides, vol. 3, pp. 142, Academic Press, London, 1985. A. Bacic, P. J. Harris and B. A. Stone, The Biochemistry of Plants, vol. 14, pp. 309, Academic Press, London, 1988. M. McNeil, A. G. Darvill, P..~nnan, L. E. Franzen and P. Albersheim, V. Ginsburg (ed.), Methods in Enzymology, vol. 83, Academic Press, New York, 1982. P. A. Wingo, T. Tong and S. Bolden, CA Cancer J. Clin., 45 (1995) 8. K. J. Pienta, H. Naik, A. Akhtal, K. Yamazaki, T. S. Replogle, J. Lehr, T. L. Donat, L. Tait, V. Hogan and A. Raz, J. Natl. Cancer Inst., 87 (1995) 348. E. C. Kohn, Anticancer Res., 13 (1993) 2553. A. Raz and R. Lotan, Cancer Metastasis Rev. 6 (1987) 433. D. Platt and A. Raz, J. Natl. Cancer Inst., 84 (1992) 438 T. Matsumoto, M. Tanaka, H. Yamada and J. C. Cyong, J. Immunol Methods, 129 (1990) 283. T. Matsumoto, J. C. Cyong, H. Kiyohara, H. Matsui, A. Abe, M. Hirano, H. Danbara and H. Yamada, Int. J. Immunopharmacol., 15 (1993) 683. H. Yamada, Folia Pharmacol. Jpn., 106 (1995) 229 (in Japanese). H. Yamada, K. S. Ra, H. Kiyohara, J. C. Cyong and Y. Otsuka, Carbohydr. Res., 189 (1989) 209. M. Hirano, H. Kiohara and H. Yamada, Planta Med., 60 (1994) 450. T. Matsumoto, M. Hirano, H. Kiyohara and H. Yamada, Carbohydr. Res., 270 (1995) 221. A. G. Darvill, M. McNeil and P. Albersheim, Plant Physiol., 62 (1978) 473. T. Matsumoto and H. Yamada, J. Pharm. Pharmacol., 47 (1995) 152. X. B. Sun, T. Matsumoto and H. Yamada, Phytomedicines, 1 (1994) 225. K. S. Shin, H. Kiyohara, T. Matsumoto and H. Yamada, Abstracts of 17th Japanese Carbohydr. Symp., pp. 216 (1995) Kyoto, Japan. T. G. Egivang and A. D. Befus, Immunology, 51 (1984) 207. J. F. Zhao, H. Kiyohara, H. Yamada, N. Takemoto and H. Kawamura, Carbohydr. Res., 219 (1991) 149. H. Wagner and E. Jordan, Phytochemistry, 27 (1988) 2511. H. Yamada, H. Kiyohara, J. C. Cyong and Y. Otsuka, Molec. Immunol., 22 ~
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(1984) 295. H. Yamada, H. Kiyohara, J. C. Cyong and Y. Otsuka, Carbohydr. Res., 159 (1987) 275. H. Kiyohara, J. C. Cyong and H. Yamada, Carbohydr. Res., 182 (1988) 259. H. Kiyohara, J. C. Cyong and H. Yamada, Carbohydr. Res., 193 (1989) 201. H. Kiyohara and H. Yamada, Carbohydr. Polym., 25 (1994) 117. H. Wagner, H. Stuppner, W. Schafer and M. Zenk, Phytochemistry, 27 (1988) 119. B. Luettig, C. Steinmtlller, G. E. Gifford, H. Wagner and M. L. Lohmann Matthes, J. Nail. Cancer. Inst., 81 (1989) 669. E. A. Mueller, K. Hamprecht and F. A. Anderson, Immunopharmacol., 17 (1989) 11. E. A. Mueller and F. A. Anderer, Immunopharmacol., 19 (1990) 69. K. Ohtani, K. Mizutani, S. Hatono, R. Kasai, R. Sumino, T. Shiota, M. Ushijima, J. Zhou, T. Fuwa and O. Tanaka, Planta Med., 53 (1987) 166. N. Shimizu, M. Tomoda, R. Gonda, M. Kanari, N. Takahashi and N. Takahashi, Chem, Pharm. Bull., 37 (1989) 1329. M. Tomoda, N. Shimidzu, M. Kanari, R. Gonda, S. Arai and Y. Okuda, Chem, Pharm. Bull., 38 (1990) 1667. M. Tomoda, R. Gonda, N. Shimidzu and M. Kanari, Phytochemistry, 29 ( 1990 ) 3091. N. Shimidzu, M. Tomoda, R. Gonda, M. Kanari, A. Kubota and A. Kubota, Chem, Pharm. Bull., 37 (1989) 3054. H. Yamada, X. B. Sun, T. Matsumoto, K. S. Ra, M. Hirano and H. Kiyohara, Planta Med., 57 (1991) 555. H. Yamada, M. Hirano and H. Kiyohara, Carbohydr. Res., 219 (1991) 173. M. Hirano, H. Kiyohara, T. Matsumoto and H. Yamada, Carbohydr. Res., 251 (1994) 145. X.B. Sun, T. Matsumoto and H. Yamada, J. Pharm. Pharmacol., 43 (1991) 699. T. Matsumoto, R. Moriguchi and H. Yamada, J. Pharm. Pharmacol., 45 (1993) 535. M.H. Sakurai, T. Matsumoto, H. Kiyohara and H. Yamada, submitted. X.B. Sun, T. Matsumoto and H. Yamada, Planta Med., 58 (1992) 445. X.B. Sun, T. Matsumoto and H. Yamada, Planta Med., 58 (1992) 432. H. Kiyohara, M. Hirano X. G. Wen, T. Matsumoto, X. B. Sun and H. Yamada, Carbohydr. Res., 263 (1994) 89. H. Yamada, K. Komiyama, H. Kiyohara, J. C. Cyong, Y. Hirakawa and Y. Otsuka, Planta Med., 56 (1990) 182. Y. Guoji, J. Kajihara, S. Kirihara, K. Kato and H. Abe, Phytotherapy Res., 8 (1994) 337. J.D. Robbins and J. B. Robbins, J. Infect. Dis., 47 (1984) 436. K. Heyns and G. Kiessling, Carbohydr. Res., 3 (1967) 340.
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S. C. Szu, S. Bystricky, M. Hinojosa-Ahumada, W. Egan and J. B. Robbins, Infect. Immun., 62 (1994) 5545. 56 D. K. F. Meijer, G. Molema, R. W. Jansen and J. F. Mollenaar, Claasseu (ed.) Trend in Drug Research, vol. 13, pp. 303 (1990) Elsevier, Amsterdam. 57 E. V. Groman, P. M. Enriquez, C. Jung and L. Josephson, Bioconjugate Chem., 5 (1994) 547.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
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The role of polygalacturonase, PGIP and pectin oligomers in
fungal infection F. Cervone, G. De Lorenzo, B. Aracri, D. Bellincampi, C. Caprari, A.J. Clark, A. Desiderio, A. Devoto, F. Leckie, B. Mattei, L. Nuss and G. Salvi Dipartimento di Biologia Vegetale, Universit& di Roma "La Sapienza", Piazzale Aldo Moro 5, 00185 Roma, Italy. Abstract
The interaction between fungal endopolygalacturonases and a plant cell wall PGIP (PolyGalacturonase-lnhibiting Protein)in plant-pathogen recognition is being investigated. This protein-protein interaction has been shown to favour the formation of oligogalacturonides able to elicit plant defense responses. A single mutation in the endopolygalacturonase gene of Fusanum moni/iforme abolishes the hydrolytic activity but does not affect the elicitor activity of the enzyme and its ability to interact with PGIP. Accumulation of pgip mRNA in different race-cultivar interactions (either compatible or incompatible) between Colletotrichum lindemuthianum and Phaseolus vulgaris has been followed by Northern blot and in situ hybridisation analyses. Rapid accumulation ofpgip mRNA correlates with the appearance of the hypersensitive response in incompatible interactions, while a more delayed increase, coincident with the onset of lesion formation, occurs in compatible interactions. PGIP exhibits a modular structure: its amino acid sequence can be divided into a set of 10.5 leucine-rich tandemly repeated units (LRR=leucinerich repeats), each derived from modifications of a 24-amino acid peptide. A LRR structure has been observed in several proteins implicated in protein-protein interactions and in the extracellular domain of a cloned Arabidopsis receptor-like protein kinase (RLK5); a LRR structure has also been observed in the products of several resistance genes recently cloned. A plasma membrane-associated high molecular weight protein cross-reacting with an antibody prepared agaist PGIP is being purified in our laboratory. We suggest that PGIP may belong to a class of receptor complexes specialized for defense against microbes. 1. INTRODUCTION Plants are continually exposed to a vast array of potential phytopathogenic fungi; nevertheless, plants resist to most of them by blocking fungal development soon after penetration. Resistance against pathogens can be distinguished in resistance at the species level (non-host resistance) and resistance at the cultivar level (race-cultivar resistance). Plants lack a circulatory system and antibodies and have evolved a defense mechanism that is distinct from the vertebrate immune
192
system. In contrast to animal cells, each plant cell is capable of defending itself by a means of a combination of constitutive and induced defense mechanisms. The effectiveness of the plant defense responses against a pathogen lies both in the magnitude and in the rapidity of their onset. Plants have evolved the ability to perceive the presence of a pathogen (recognition), to transmit this information inside the contact cell and to neighbouring cells and to induce a number of reactions both Iocalised at the infection site and at a systemic level which work in concert to block the infection [1]. It it now generally accepted that in both non-host and race-cultivar resistance the recognition is mediated by signal molecules (elicitors) produced by the pathogen and complementary plant "receptor" molecules. Signals and "receptors" interact at the contact surfaces between the two organisms to start the signal transduction pathway that leads to the activation of the various plant defense responses. In spite of intense research, there are only a few cases where the interactions can be defined in molecular terms. This chapter will deal with the role of fungal polygalacturonases in infection and damage of the plant tissue, and, also, with the role of polygalacturonase, PGIP and oligogalacturonides in the recognition steps leading to the rejection of a potential pathogenic fungus by a plant. 2. POLYGALACTURONASE
2.1. The role of polygalacturonase in pathogenicity. One of the barriers against phytopathogenic fungi is the plant polysacchariderich cell wall. The vast majority of fungi need to breach these barrier to gain access to the plant tissue, and to this purpose secretes a number of enzymes capable of degrading the wall polymers. When fungi are grown on plant cell wall material in vitro, pectic enzymes are invariably the first enzymes to be secreted, followed by hemicellulases and cellulases [2,3]. The action of pectic enzymes and in particular of endopolygalacturonase on cell walls appears to be a prerequisite for wall degradation by other enzymes [4]. Only after pectic enzymes have acted on their substrates, the cellulose-xyloglucan framework, which is normally embedded in the pectin matrix [5], becomes accessible and inducers for cellulase and hemicellulase can be released. Endopolygalacturonases exhibit a great variety of isoenzymatic forms [6]. The molecular basis of this polymorphism has been elucidated only for a few enzymes. For example, the endopolygalacturonase secreted by the phytopathogenic fungus Fusarium moniliforme consists of four molecular mass glycoforms, which derive from a single endopolygalacturonase gene product [7]. The occurrence of multiple isoforms, each of which may in turn comprise multiple glycoforms, may have a physiological significance. Redundancy of the components of the offence arsenal may allow to accomodate pathogenesis in a variety of different conditions and hosts, as well as protect the fungus from losses of pathogenicity functions. Different forms of the same enzyme may differ in terms of stability, specific activity, pH optimum, substrate preference or degradation kinetics, and types of oligosaccharides released. They may also differ in the mechanisms or extent of their induction in planta.
193
Purified pectic enzymes capable of cleaving the o~-l,4-glycosidic bonds of homogalacturonan in an "endo" manner cause plant tissue maceration (cell separation). These enzymes also cause injury and death of unplasmolysed plant cells. It has been proposed that cell death results from a physical weakening of the wall caused by degradation of pectic polysaccharides, such that the wall can no longer resist the pressure exerted by the protoplast [8]. The most convincing evidence supporting this theory is that plasmolysed cells are not killed when treated with purified pectic enzymes and that cell wall degradation, maceration and cell death appear simultaneously with no spatial and temporal gaps [9]. Against, there are instead the arguments that cellulose and hemicellulose, and not the pectic polymers, are the load-bearing components of primary cell walls [10], and therefore that degradation of the pectic polysaccharides is unlikely to weaken the wall to such an extent that protoplasts would burst. The role of endopolygalacturonase in pathogenicity has been questioned by Scott-Craig et al. [11], who described the generation of a specific endopolygalacturonase mutant of Cochliobolus carbonum by homologous integration of a plasmid containing an internal fragment of the gene. Pathogenicity on maize of the mutant was qualitatively indistinguishible from that of the wild-type strain, indicating that in this compatible interaction the endopolygalacturonase is not required. Nevertheless the possibility that the mutated strain produces in planta another form of endopolygalacturonase was not ruled out.
2.2. The role of polygalacturonase in eliciting the plant defense responses Active plant defense responses include the rapid death ~of the plant cells which first come into contact with the pathogen (hypersensitive response), a rapid oxidative burst, cross-linking and strengthening of the plant cell wall, the induction of the phenylpropanoid pathway and the synthesis of lignin, the synthesis and the accumulation of antimicrobial compounds named phytoalexins, the synthesis of hydroxyproline-rich glycoproteins (HRGPs) and fungal wall degrading enzymes (chitinases, glucanases). Many different molecules, termed elicitors, have been shown to induce defense responses when applied to plant tissues [10,12]. Pectic enzymes with an "endo" mode of action behave as elicitors. The ability of a Rhizopus stolonifer endopolygalacturonase to elicit a phytoalexin biosynthesis-related enzyme (casbene synthase) in castor bean seedlings was described by Lee and West [13]. The authors suggested that the elicitor activity of the enzyme was mediated by the products released from the plant cell wall and demonstrated that the catalytic activity of the endopolygalacturonase was necessary for its eliciting activity [14]. Since this initial observation, the number of reports on the elicitor activity of fungal endopolygalacturonases in different systems has steadily increased. Fungal endopolygalacturonases have been shown to elicit different responses such as the accumulation of phytoalexins [15,16], the synthesis of lignin [17,18] and the production of 13-1,3-glucanase [16,19]. Elicitor activity of endopolygalacturonases suggests that during an attempted invasion the enzymes may play two antithetical roles: as efficient fungal aggression tools, or potential signal molecules (elicitors). The early timing of endopolygalacturonase production is compatible with both functions.
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3. OLIGOGALACTURONIDES 3.1. The role of oligogalacturonides in eliciting the plant defense responses The available evidence indicates that fungal endopolygalacturonases are not directly responsible for the induction of plant defense responses, but are rather "pre-elicitors" that release from the plant cell wall the true elicitors [20], the oligogalacturonides. The first evidence that a galacturonide-rich fraction released from the walls induces accumulation of the phytoalexin glyceollin in soybean was given by Hahn et al. [21], who named the active component of the fraction "endogenous elicitor". Since then, oligogalacturonides with degree of polymerization between 10 and 15 have been reported to elicit a variety of defense responses, including the accumulation of phytoalexins [22], the synthesis of endo-13-1,3glucanase [23] and chitinase [24], the synthesis of lignin [17,25] and elicitation of necrosis [26], as well as the accumulation of PGIP [27]. In carrot suspensioncultured cells, oligogalacturonides induced the activation of PAL, a key enzyme in phenylpropanoid and lignin biosynthesis [28]. Responses elicited in carrot cells by oligogalacturonides appeared to be a consequence of a transcriptional activation of several defense genes [29]. The mechanisms by which oligogalacturonides act as signals for activation of defense responses remain unknown. Several rapid responses occurring at the plant cell surface may be part of the transduction of the oligogalacturonide signal. Oligogalacturonides have been shown to be internalised through a rapid receptormediated endocytosis in soybean suspension-cultured cells [30] and to induce, within 5 minutes, transient stimulation of cytoplasmic Ca++ influx, K+ efflux, cytoplasmic acidification and depolarization of plasma membrane of tobacco cultured cells [31,32]. The elevation of cytosolic free Ca++ levels induced by oligogalacturonides has been also observed in carrot protoplasts [33]. Interestingly, the increase of external pH may stabilise in vivo the elicitor-active oligogalacturonides, since the lower activity exhibited by endopolygalacturonase at higher non optimal pH values would prevent their degradation to shorter inactive products. Oligogalacturonides have been shown to generate H20 2 production in cucumber [34], soybean [35-37] and castor bean [17]. In plants, production of reactive oxygen species and processes associated with this production, such as the oxidative cross-linking of the cell wall and lipid peroxidation, have been proposed to contribute to both programmed cell death and rapid activation of defense responses [38]. Oligogalacturonides also enhance the in vitro phosphorylation of a 34 kD protein associated with plasma membranes of potato and tomato (pp34) [37,39,40]. Oligogalacturonides bind Ca ++ forming intermolecular complexes named "egg boxes". Conformational analysis has established that the "egg box" conformation requires oligopectate fragments with a degree of polymerization higher than 10 [41]. On the basis of the correlation between the degree of polymerization required for these intermolecular conformations and the elicitor activity, it has been suggested that oligogalacturonide- Ca++ complexes are the active molecular signals [42,43].
195
4. PGIP The isolation of a polygalacturonase inhibitor from P. vulgads was reported in the early 70s [44]. Since this initial report, the occurrence of PGIPs has been reported in a variety of dicotyledonous plants, including bean, cucumber, pea, green pepper, tomato, apple, pear, peach, oranges, alfalfa [10 and references therein] and, more recently, soybean [45] and raspberry [46]. PGIPs have also been identified in the pectin-rich monocotyledonous plants Allium cepa and A. porrum [47]. Some of these inhibitors are predominantly ionically bound to the plant cell wall while others are readily extracted from it with dilute buffers. PGIPs are typically effective against the endopolygalacturonases of fungi and ineffective against other pectic enzymes either of microbial or fungal origin [48]. PGIP from bean hypocotyls in vitro protected bean cell walls against degradation by endopolygalacturonase of C. lindemuthianum [49]. Similarly, PGIP prepared from tomato cell walls in vitro protected these walls from degradation by a complex enzyme mixture produced in culture by Fusanum oxysporum f.sp. lycopersici [2]. Some observations suggest a correlation between presence of PGIP and resistance of plants to fungi. In a study on the distribution of PGIP in various tissues of P. vulgads, levels of PGIP in hypocotyls were shown to increase six to nine-fold during seedling growth [50]. Although susceptibility tests were not performed, the increase in PGIP levels may be correlated with the increase in resistance occurring in bean hypocotyls when primary leaves begin to develop [51]. Increasing susceptibility of ripening pear fruits to Dothiorella gregada and Botdtis cinerea correlated with a decline in the concentration of PGIP [52]. In raspberry fruits, the level of PGIP was maximal in immature green fruit, which are more resistant to fungal attack, and decreased in mature more susceptible fruits [46]. 4.1. PGIP regulates the activity of fungal endopolygalacturonases Oligogalacturonides of chain length varying between 10 and 15, which are transiently produced by the action of the endopolygalacturonase on homogalacturonan, are elicitors of defense responses; shorter oligomers have little or no elicitor activity [10,12]. Thus, endopolygalacturonases release elicitor-active oligogalacturonides, but also degrade them into inactive oligomers. This implies that an extensive degradation of the cell wall homogalacturonan negatively affects the eliciting activity of the endopolygalacturonase. PGIP modulates the endopolygalacturonase activity in vitro in such a way that the balance between release of elicitor-active oligogalacturonides and depolymerization of the active oligogalacturonides into inactive molecules is altered and the accumulation of elicitor-active molecules is favoured [53]. 4.2. PGIP is specialized for interaction with other macromolecules Endopolygalacturonase and PGIP form a specific, reversible, saturable, high affinity complex [54]. The amino acid sequence of the PGIP exhibits characteristics of significant internal homology [55]. The internal homology domain spans about 258 amino acids, from position 69 to 326 and exhibits a modular structure: it can be divided into a set of 10.5 tandemly repeating units, each derived from modifications of a 24-amino acid peptide. The alignment between the 10.5 segments shows a periodic distribution of a few amino acids rather than a recurrent sequence of
196
several different amino acids. The repeat element has regularly spaced Leu residues (LRR=leucine-rich repeats) Over thirty proteins from bacteria to human contain leucine-rich tandem repeats similar to those found in the PGIP [56 and references therein]. The consensus sequences for the repeating units are surprisingly similar considering the range of organisms and the widely divergent functions of the different proteins. The great similarity in the structure of these proteins indicates a strong selection pressure for conservation of this structure, which is likely to have been attained by a series of unequal cross-overs of an oligonucleotide sequence coding for a prototype leucine-rich building block. The similarity may also indicate an evolutionary conservation between the proteins, or may reflect the coincidental and convergent evolution of a protein domain. Since a common feature among these proteins appears to be that of membrane association and/or protein binding, a domain comprised of tandem leucine-rich repeats may represent a structure specialized to achieve strong interactions between macromolecules. The crystal structure of one LRR protein, the RNAse inhibitor, has revealed that leucine-rich repeats correspond to 13-o~structural units. This units are arranged for a parallel 13-sheet with one surface exposed to solvent so that the protein acquires an unusual non-globular shape, which may be responsible for proteinbinding functions [57]. 4.3. The structure of PGIP is similar to that of proteins involved in signal transduction Many of the proteins with LRR are involved in signal transduction pathways. In this regard, the similarity between PGIP and a cloned A. thaliana receptor-like protein kinase (RLKS) [58] is of particular interest. The RLK5 protein exhibits a three-domain molecular topology similar to all of the known receptor tyrosine kinases, with an N-terminal putative extracellular domain, a central putative transmembrane domain, and a C-terminal domain homologous to the catalytic core of serine-threonine kinases. The homology of RLK5 with PGIP is, however, limited to the putative extracellular receptor-like domain. The catalytic domain of RLK5 is homologous to that present in the deduced protein of other cloned genes (ZmPK1, RLK1, RLK4) [58]. However, the putative extracellular domains of the ZmPK1, RLKland RLK4 deduced proteins differ from that of RLK5 or PGIP and appear to be related to the products of Brassica SLG (S Locus Glycoprotein) and SRK (S Locus Receptor Kinase) genes [59]. These two genes are physically linked to each other and linked to the S locus controlling selfincompatibility in Brassica. The SLG product is a secreted glycoprotein, while SRK is a transmembrane receptor protein kinase; SLG and the extracellular receptor domain of SRK isolated from the same S haplotype share a high level (>80%) of sequence identity [59]. Since both the SLG and SRK proteins are required for the self-incompatibility response, a model has been proposed in which SRK, acting in combination with SLG, couples the recognition event at the pollen-stigma interface to a cytoplasmic phosphorylation cascade that leads to pollen rejection [59,60]. At a cellular and genetic level there are many similarities between self incompatibility and the incompatible defense response. The fact that proteins can work together to determine the outcome of an interaction suggest that PGIP could
197
FUNGAL TIP
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,,
2.~.
~......~
Phyt~
rl
ns 4 " " ~ ~ ~ ..
ase
CYTOPLASM
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Figure 1. Schematic representation of the possible role of PGIP in signalling at the interface between plants and fungi. correspond to the SLG part of the SLG-SRK partnership and thus a SRK-like PGIP may exist. The analogy between the recognition system involved in pollen-stigma interactions and that involved in plant-pathogen interactions at the cellular and genetic levels [55,61], and the relation between PGIP and the extracellular domain of RLK5, raises the possibility that PGIP acts as a secreted "receptor" involved in recognition between plants and fungi. A PGIP-related class of two-component (secreted receptor/transmembrane receptor-kinase) signalling systems, similar to that controlling self-incompatibility in Brassica, may be present on the plant cell surface (Figure 1). This class would differ from the Brassica system in the features of the receptor component and/or the type of catalytic kinase domain, and might be implicated in different aspects of plant-fungus recognition, such as that leading to non-host resistance or to race-cultivar specificity. PGIP may therefore represent an SLG counterpart in a class of receptor/receptor kinase complex of the kind invoved in Brassica self-incompatibility [55]. 4.4. PGIP is related to the products of the resistance gene. After decades of intense research several plant resistance genes (R) which participate in the gene-for-gene hypothesis have been cloned. In these cases resistance is only expressed when a plant that contains a specific R gene
198
Ancestrai ieucine- rich- repeat protein
~ Piasmamembrane
PGIP ?
, [
PGIP
Transduction domain
Figure 2. A hypothesis on the evolution of PGIPs and of the plant cell surface LRR two-element receptor complexes. recognises a pathogen that has the corresponding avirulence (avr) gene. With one exception the cloned genes code for LRR proteins [62-64] and references therein). On the basis of sequence analysis these genes can be grouped in different classes [65]. One class includes the tobacco gene N against the tobacco mosaic virus, the Arabidopsis bacterial resistance genes RPS2 and RPM1 [62], and the flax gene L6 [63]. A common characteristic of the proteins encoded by these genes is, in addition to the presence of a LRR domain, the presence of a putative nucleotide binding site (NBS) similar to that found in Ras proteins and in the 13subunit of the ATP synthase. Another class, including the tomato gene Cf-9 for resistance to Cladosporium fulvum, is characterised by a LRR N-terminal protein domain, which is correlated to PGIP [55], and a hydrophobic putative transmembrane C-terminal domain [64,66]. Interestingly, the Cf-9 product was found to be homologous to PGIPs also in the protein regions outside the leucine-rich repeat domain. Cf-9 clearly shares an evolutionary relation with PGIPs [67]. These findings suggest that there is more than just a simple analogy between the genes encoding PGIP and genes encoding resistance proteins. The pgip genes could belong to a super-family of genes, which includes the resistance genes, specialised for recognition of foreign molecules. A hypothesis on the evolution of PGIPs and of the plant cell surface LRR two-element receptor complexes is presented in Figure 2.
199
4.5. Accumulation of pgip mRNA correlates with hypersensitive response in race-cultivar interactions One gene encoding PGIP of P. vulgaris has been cloned and characterized [68].The pgip gene of P. vulgaris predicted a 342 amino acid polypeptide including a 29 amino acid signal peptide for secretion and four potential glycosylation sites. By Northern blot analysis, a 1.2 kb transcript was detected in suspension-cultured cells and to a lesser extent, in leaves, hypocotyls, and flowers [68]. Using the cloned gene as a probe, we have demonstrated that accumulation of pgip transcripts is induced in suspension-cultured bean cells following addition of elicitoractive oligogalacturonides and fungal glucan to the medium [27]. We have also shown that pgip mRNA accumulated in P. vulgaris hypocotyls in response to wounding or treatment with salicylic acid. Accumulation of pgip mRNA has also been followed in different race-cultivar interactions (either compatible or incompatible) between C. lindemuthianum and P. vulgaris by Northern blot and in situ hybridisation analyses. Rapid accumulation of pgip mRNA correlated with the appearance of the hypersensitive response in incompatible interactions, while a more delayed increase, coincident with the onset of lesion formation, occurred in compatible interactions. In incompatible interactions, the accumulation of the pgip mRNA was higher in epidermal cells proximal to the site of infection and within a few cell layers of parenchymal cells immediately below. These data indicate that PGIP expression is regulated upon the early race-specific recognition event in a manner similar to that observed for the known defense-related genes. The 5' flanking sequences of the P. vulgaris pgip gene have also been sequenced. Sequence analysis has revealed the presence of a 7-bp sequence that exhibits one nucleotide mismatch relative to the sequence found in the promoters of several elicitor- and UV light-inducible plant genes and identified by in vivo footprinting as an elicitor-inducible motif [69,70]. A region from nt -404 to nt -359 shared significant similarity with a region present in the promoters of two potato wound-inducible genes, pin2 and win2 [69,71]. The presence of these regions in the pgip promoter is particularly interesting, given the characteristics of inducibility of pgip expression by elicitors and wounding. We have undertaken the functional analysis of the pgip promoter by expressing different pgip-~-glucuronidase (GUS) gene fusions in transfected tobacco protoplasts and transgenic tobacco plants obtained by Agrobacterium-mediated transformation. 4.6. A PGIP-like protein is associated with plasma membranes Years ago we showed that fungal endopolygalacturonases were able to bind to protoplasts of P. vulgaris suggesting the presence of a polygalacturonase-binding protein at the external surface of cells [72,73]. By using an antibody raised against PGIP we have detected now the presence of a putative PGIP-like protein of about 100 kD in a bean plasma membrane preparation (Figure 3A). We have not been able yet to associate any activity with the plasma membrane PGIP-like protein. By Northern blot analysis we have also detected, in cell suspension cultures of P. vulgaris, the presence of 2.9 kb mRNA that hybridizes with a pgip-specific probe (Figure 3B).
200 1
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kD 100 .............................
3
4 A
B
Kb 2.9- ~ii~i~i:~ 9 1.3
j
Figure 3. A. Western blot analysis of membrane fractions from P. vulgaris probed with a PGIP-specific antibody. 1) PGIP purified from P. vulgaris; 2) and 3) membranes; 4) supernatant of the membrane preparation. B. Northern blot analysis of total RNA extracted from hypocotyls of P. vulgaris and hybridised to a radioactive pgip-specific probe. 5. FACTORS AFFECTING POLYGALACTURONASE-PGIP INTERACTION 5.1. A single mutation affects enzyme activity but not elicitor and PGIP binding abilities of endopolygalacturonase The sequence encoding the endopolygalacturonase of Fusarium moniliforme [74] was cloned into the E. col#yeast shuttle vector Yepsecl for secretion in yeast [75]. The recombinant plasmid (pCC6) was used to transform Saccharomyces cerevisiae strain $150-2B; transformed yeast cells were able to secrete endopolygalacturonase activity in the culture medium. The enzyme (wtYendopolygalacturonase) was purified, characterized, and shown to possess biochemical and biological properties similar to those of the endopolygalacturonase purified from F. moniliforme. The wtY-endopolygalacturonase was able to macerate potato medullary tissue disks and to elicit glyceollin in soybean cotyledons; moreover, it was inhibited by the PGIP purified from P. vulgaris. The sequence encoding endopolygalacturonase in pCC6 was subjected to site-directed mutagenesis. Three residues in a region highly conserved in all the sequences known to encode polygalacturonases [11,74] were separately mutated: His 234 was mutated into Lys, Ser 237 and Ser 240, respectively into Gly. Each of the mutated sequences was used to transform S. cerevisiae The mutated enzymes produced by the transformed yeast cells were purified and characterized. Replacement of His 234 with Lys abolished the enzymatic activity, confirming the biochemical evidence obtained by Cooke et al. [76] and Rexov~-Benkov& et al. [77] that a His residue is critical for the catalytic activity of the enzyme. Interestingly, the inactive enzyme carrying the mutation His 234->Lys elicited glyceollin accumulation in the soybean cotyledon assay, suggesting that this biological activity of endopolygalacturonase is not due solely to its ability to release oligogalacturonides from the plant cell wall. Replacement of either Ser-237 or Ser-240 with Gly reduced the enzymatic activity to 48% and 6%, respectively, of the wtY-endcpolygalacturonase. The interaction between the variant enzymes and the PGIP purified from P. vulgaris was investigated using a biosensor based on surface plasmon resonance (BIAlite*)
201
(Mattei et al., this book). The three variant enzymes were still able to interact and bind to PGIP with association constants comparable to that of the wild type enzyme. 5.2. A family of PGIPs is expressed in P. vulgaris L. In the attempt to elucidate the role of PGIP in plant resistance to fungi, different pgip-related genes are being characterized. It has been shown that a small family of pgip genes, likely clustered on chromosome 10, is present in the genome of P. vulgafis [78] and several pgip-related clones have already been isolated in our laboratory. Polymorphisms were found in the lengths of the fragments that hybridised to the pgip probe [78]. Characterization of all pgip members could give a significant contribution to the role of PGIP and correlated proteins in the communication between plant and pathogen.The structural and functional analyses of these clones are in progress in our laboratory. In collaboration with MOGEN (The Netherlands), we have obtained transgenic tomato plants expressing high levels of P. vulgafis PGIP. A chimeric gene has been constructed possessing the structural beanpgip-1 gene under the control of the CaMV 35S promoter, that allows constitutive and high-level expression in most plant tissues. The chimeric gene was introduced into tomato plants by A. tumefaciens-mediated transformation. The transgenic plants widely varied in terms of PGIP levels in the tissues; very high expression (levels from 60 to 100 folds higher than those of untransformed tomato plants) were observed in some of them. In the plants analysed, levels of PGIP well correlated with levels of transcripts of the inserted pgip gene. The PGIP purified from tomato transgenic plants exhibited a specificity different from that of PGIP purified from P. vulgafis (Figure 4).The possibility that a family of PGIPs with different specificities is expressed in P. vulgaris is being investigated.
100
~
100
o
o
o~..l
F
x~ 50
~
PGA. niger
50 PG A. niger
PG F. monili~rme
-" PG F. moniliforme 0 A
200 PGIP (ng)
400
0
20
40
60
80
PGIP (ng)
Figure 4. Inhibition of A. niger and F. moniliforme endopolygalacturonase by PGIP purified fom tomato transgenic plants (A) and from bean (B).
202
6. CONCLUSIONS
Specificity in plant-fungus interactions is likely to be determined by recognition steps involving pathogen-derived signals and complementary sensor (receptor) molecules of plant origin. Both signals and receptors are thought to play their roles at the contact surfaces between the two organisms. A while ago, we suggested that a clever strategy of the plant would be to recognize, as fungal signals, those factors that are required for basic compatibility and therefore have to be maintained by the microorganism during evolution for successful parasitism [6]. As possible signals in plant-fungal interactions, we suggested the endopolygalacturonases. The interaction between fungal endopolygalacturonase and PGIP has the requisites for functioning in a perception mechanism that leads to incompatibility. Both molecules are synthesized very early during an attempted infection and physically interact to give rise to the formation of oligogalacturonides that act as elicitors of several defense responses [54]. The recent cloning of several resistance genes has shed some light how the plant recognize molecules from pathogenic microorganisms. The products of all the isolated resistance genes share the common characteristic of being LRR proteins. It seems therefore that plants have selected this special structure for their immunological performances. PGIP is the first LRR protein characterized in plants and clearly shares an evolutionary relation with some of the resistance gene products. For example, the Cf-9 product is homologous to PGIPs not only in the leucine-rich repeat region but also outside. The PGIP gene could therefore belong to a super-family of genes, which includes the resistance genes and has the role of recognising non-self molecules. The PG-PGIP relationship offers the opportunity to dissect a known signalling interaction using powerful molecular techniques. One pgip gene of P. vulgaris has been characterized but the evidence points to there being a family of expressed genes and hence a family of PGIP proteins which could well differ in their specificities and in their expression patterns. It is essential to the elucidation of the endopolygalacturonases-PGIP interaction to discover how many PGIP proteins are involved, under what circumstances they are expressed and their specifities upon expression. 7. A C K N O W L E D G E M E N T S
This work was supported by the Ministero delle Risorse Agroindustriali e Forestali (MIRAAF) and by The European Community Grants CHRX-CT93-0244 and AIR 3-CT94-2215. 8. REFERENCES
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j. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
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B i o l o g i c a l l y A c t i v e P e c t i n O l i g o m e r s in R i p e n i n g T o m a t o Fruits Eunice Melotto a, L. Carl Greve b, and J.M. Labavitchb aDepartamento de Botanica, Escola Superior de Agricultura Luiz de Queiroz, Universidade de Sao Paulo Piracicaba bpomo1ogy Department, University of California, Davis, CA 95616
Abstract
Recent work has shown that pectic oligosaccharides extracted from tomatoes are capable of promoting ripening in mature green tomato fruit explants (11). A water soluble, ethanolinsoluble extract of autolytically-inactivated tomato pericarp tissue will elicit a transient increase in ethylene biosynthesis when applied to pericarp discs from mature green fruit. Treatment of Na2CO3.soluble but not chelator-soluble pectin from tomato with pure tomato polygalacturonase 1 generates oligomers that are similar to those extracted from ripening fruit. The endogenous concentration of these oligomers is low, but in excess of that necessary to promote ethylene biosynthesis in this system (29). A major difficulty in assigning a regulatory role to such pectic oligomers is that at the onset of ripening in tomato fruit little evidence of polygalacturonase activity exists. However our work demonstrating the presence of neutral sugar-rich acidic oligomers suggests that other enzymatic activities capable of degrading pectic substances exist in developing tomatoes. The recent report (22) of a rhamnogalacturonase-type activity in developing fruit supports this supposition. The possibility of a role for pectic oligomers in fruit ripening regulation will be discussed.
INTRODUCTION Oligosaccharides as potential regulatory signals. In the 1970s Ayers, Albersheim and colleagues showed that oligosaccharides derived from the cell wall of the fungus Phytophthora megasperma sojae could elicit the production of phytoalexins from tissues of its soybean host and suggested that these carbohydrates represented endogenous signals for turning on host defenses (2,3). Since that time a considerable amount of research has shown that the addition of various carbohydrates (mono-,oligo, and polysaccharides) to various parts (cells, tissues, organs) of assorted plant species can activate various aspects of development. Initially, the interactions of pathogens and their hosts were studied perhaps because cell wall breakdown is so often evident in these situations (14). That these interactions lead to elicitation of the production of pathogenesis-related proteins, hydroxyproline-rich glycoproteins, phytoalexins, lignin, ethylene and other potential
208 aspects of host defenses has also been clearly demonstrated (reviewed in 15). The possibility that carbohydrate elicitors could regulate "non-pathological" aspects of plant development has also been examined. Studies have suggested a regulatory role in morphogenesis (at least in tissue culture), cell elongation, ethylene synthesis, and fruit ripening. More recent work has shown that oligosaccharides can promote ion pumping, production of active oxygen species, and phosphorylation of proteins; responses proposed to be components of pathways for transduction of plant hormone signals (reviewed in 31). Specific binding of the heptaglucoside elicitor derived from P. megasperma cell walls to soybean membranes has been demonstrated with the use of techniques analogous to those employed to identify receptors for plant hormones (13). Although much work remains, it is reasonable to propose that cell wall-derived oligosaccharides can be endogenous regulators of plant development. Cell wall breakdown is certainly a feature of fruit ripening (12, 16, 21, 24, 25) and thus it is easy to propose a source for active oligosaccharides. Ripening is a complex array of processes that together characterize the terminal stages of fruit development. Fruits like the tomato are classified as climacteric because ripening is preceded by dramatic increases in ethylene synthesis and respiration, with all of the ripening-related events (alterations of sugar and organic acid concentrations, changes in color, aroma and flavor, and flesh softening) following in a relatively short time (19). While ethylene promotes ripening and ripening involves the differential expression of many genes, not all of them are ethylene-responsive. Thus other regulatory factors must interact with ethylene to coordinate the ripening process. The cellular event(s) that promotes the initial increase in ethylene production is unknown. Each of the individual ripening events has been studied extensively (7). Of all fruits, the ripening of the tomato has probably received the most attention. This is particularly true of the softening phenomenon where examination of cell wall breakdown - including analysis of polysaccharide changes and production of putative cell wall-degrading enzymes - has been extensive (reviewed in 7, 16). It is clear that the breakdown of pectic polysaccharides, apparently catalyzed, at least in part, by the combined action of PG and PE is a feature of this cell wall metabolism. However, studies of the ripening of transgenic tomatoes with altered PG or PE production (17, 27, 35) make it equally clear that softening is not solely dependent on pectin breakdown. Other aspects of cell wall component breakdown, including digestion of xyloglucan (25) and pectic galactan (21), may also be involved. The question of how (mechanistically) tissue softening is linked to cell wall metabolism remains unanswered. While ripening-related changes are dramatic in tomato and clearly mark the onset of cellular senescence they do not occur simultaneously throughout the fruit. It is easy to follow the development of red pigment as an indicator of ripening, and each tomato variety displays its own pattern of pigmentation change which can be perturbed by environment and growing season (19). The 'Castlemart' variety (the subject of this report) begins its reddening in the center (columella and locules) and then a wave of color sweeps up through the outer pericarp, proceeding from the blossom to stem ends. Over the past several years we have used discs cut from the outer pericarp of tomatoes to study several metabolic aspects of tomato fruit ripening (10). While disc excision causes a wound response that must be allowed to dissipate before testing can begin, the use of
209 discs reduces the tissue variability that comes because ripening is not uniform throughout the fruit and provides several uniform experimental "units" from each fruit used. Discs cut from the outer pericarp of MG fruits will individually ripen and show the typical ripening characteristics (color change, ethylene synthesis, and softening - including changes in wall components and hydrolytic enzymes) of intact fruit. Nevertheless, discs cut from the blossom end of the MG fruit "ripen" a bit sooner than those cut from the stem end of the same fruit (unpublished observations). Clearly there are aspects of the regulation of ripening that depend more on position and developmental time than on the specific biochemical pathways that are routinely studied at the whole fruit or whole fruit extract levels. Our current concept of the way tomato fruit ripening is promoted and coordinated relies on the observations that (a) treatment of mature fruits with ethylene stimulates them to ripen and (b) an increased ethylene production is the first of the characteristic ripening changes one can measure (7, 8). Furthermore, tomato fruit whose ethylene production is largely blocked by transgenic modification of its synthesis show retarded ripening that can be reversed by ethylene application (26). These studies further demonstrate the importance of ethylene in regulation of tomato ripening but also raise several questions. Some facets of ripening (e.g., the production of the mRNA for PG). occur in spite of the reduced ethylene synthesis and fruits whose ripening off the plant is delayed, ripen normally when left attached (26). Ethylene production of intact fruit does not begin simultaneously throughout the organ. It apparently begins in the fruit's central tissues and proceeds to the outer pericarp (9). The duration of an intact fruit's increased ethylene production is longer than that of individual ripening pericarp discs (10), further suggesting that the ethylene production pattern of the intact fruit is a composite integration of the patterns of individual fruit parts/sectors. Finally, it is not clear what signals the fruit cells to begin producing ethylene. In earlier work we showed that oligosaccharides produced by acid hydrolysis of citrus pectin could stimulate the ethylene production and ripening of tomato pericarp discs (11). In this report we describe the isolation of neutral sugar-containing pectin oligomers from ripening tomato fruits and show that these can promote aspects of ripening in isolated discs. Our observations thus far are consistent with the idea that one component of the regulation of ripening in tomato fruits is pectic oligosaccharides.
MATERIALS AND METHODS Tomato fruits (Lycopersicon esculentum Mill. var. 'Castlemart') were collected from vines grown in the field at the University of California, Davis. Pericarp discs were cut from surface sterilized MG stage fruit (10). Droplets (10 ul) of test solutions (see below) were applied to the cut surface of discs and disc ethylene production was measured as described previously (11). The amounts of test materials used were based on colorimetric assay (6) of uronic acid content. Small sections (0.5 cm 3) of pericarp tissue from B stage fruit were cut and placed immediately into boiling 95% (v/v) ethanol and refluxed for 15 min. The ethanol was then decanted and the boiled tissue was homogenized in cold water. The sample was
210 centrifuged (680g, 20 mm) and the supernatant (water-soluble fraction) was saved for analysis and subsequent use. The pellet was then sequentially extracted with CDTA and Na2CO3, as previously described (12, 29) Tomato oligomers. The water-soluble fraction was made 80% (v/v) with ethanol, stored in the cold (4~ overnight, and precipitated materials were collected by centrifugation. The precipitates were air dried to remove ethanol and stored until needed. Preparations were redissolved and analyzed by analytical HPLC by a modification (29) of the technique of Hotchkiss et al. (23). Our assessment of the degree of polymerization of individual oligomeric peaks was based on the relative retention time of a galacturonic acid octomer (a gift from the Complex Carbohydrate Research Center, Athens, Georgia; Spiro et al. [33]). The G7 and G12 citrus pectin oligomers used in earlier work (11) were also analyzed in this fashion. In some cases individual oligomer peaks were collected for testing. For the tomato extracts, this further purification was accomplished by collection of individual peaks following preparative scale HPLC, followed by concentration and desalting using YCO5 ultrafiltration. For the more plentiful citrus preparations, the initial separation was based on anion exchange chromatography in an imidazole-HC1 gradient (29). Uronic acid peaks were identified and the middle portions of individual peaks were pooled and ultrafiltered. The CDTA- and Na2CO3-soluble pectins were also used as test materials. Extracts were dialyzed against water and freeze-dried. They were then tested directly or after digestion with purified tomato PG1 ( treatment at 37 ~ in sodium acetate, pH 4.5). Qualitative analysis of selected fractions was based on GLC separation of alditol acetates (5). Alditol acetates from uronosyl residues were prepared by methanolysis followed by reduction (4).
RESULTS AND DISCUSSION Previous work in our laboratory (11) showed that acidic oligosaccharides from citrus pectin could promote ethylene production and ripening (accelerated red color development) when applied to excised tomato pericarp discs. Furthermore, the acceleration of ripening was not due solely to the promotion of ethylene synthesis. Red color development at the inner (cut) tissue surface to which the treatment was applied was greater in response to oligomer treatment than was the response to a treatment with the ethylene precursor, ACC. Thus pectin oligomers have the potential of serving as ripening regulators. We (29) asked if analogous pectin oligomers were present in ripening tomato tissues in order to extend our examination of the potential endogenous role of oligomers as ripening regulators. The effect of a water-soluble, uronic acid-containing fraction extracted from B tomato fruit tissue on ethylene production of pericarp discs cut from MG tomatoes is shown in Figure 1. As little as l~zg of uronic acid in the extract can cause a doubling of ethylene production. Figure 2c shows the HPLC separation of the components in this active extract and compares it to the G7 and G12 preparations (Figs. 2a,b) used in earlier work. While this side-by-side comparison does not provide proof of the similarity of the active tomato and citrus preparations, it certainly suggests that each represents a series of
211
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Fig. 1. Promotion of ethylene synthesis by MG pericarp discs following treatment with water or varying concentrations (uronic acid equivalents) of B fruit water soluble fraction. Bars indicate SEs for the means of measurements of sets of 8 discs/teatment fr wt, Fresh weight. acidic oligosaccharides having similar size distributions. Based on HPLC elution of an octomer of galacturonic acid, the range of DPs represented by the tomato extract is 4 to 12. When peaks were integrated and quantified in relation to the octomer standard (Table I), it becomes clear that the cleanly resolved peaks represent only a small portion of the uronic acid in the preparations. This is particularly true for the tomato extract. Only 2 % of its uronic acid is represented by the roughly-identified, oligomer peaks. Can we be confident, then, that the oligosaccharides shown by HPLC are the active components in our extracts? Two lines of evidence suggest that the answer is yes. When the oligomercontaining tissue extract is subjected to gradient anion exchange chromatography, a heterogeneous population of uronic acid-containing materials is indicated (Fig. 3). HPLC analysis of pooled fractions from this analysis revealed oligomers only in fractions 26-41, and only those fractions promoted disc ethylene synthesis (data not shown). When individual peaks were collected following preparative scale HPLC, those chromatographing in the DP 7-10 range have ethylene-inducing activity (in relationship to their uronic acid content) greater than that of the unfractionated fruit extract (Fig. 4). We conclude, therefore, that the activity of the extract (Figure 1) is primarily due to its oligomer content. Huber and O'Donoghue (25) have reported that pectins extracted from tomato cell walls that have been prepared in Tris-buffered phenol (to remove the potential for autolytic digestion by residual, active PG) do not contain oligomers such as those we described above. While we have taken care to inactivate PG (boiling tissue pieces for 15 minutes in ethanol) and have found no active PG or cell wall autolytic activity following this treatment, we recognize that our treatment is probably less complete than theirs. Because the oligomer population we have described is quite small, we have to accept that active PG might have been present below our level of detection. If so, the oligomers we have described could have been generated in vitro. We do not think that this is the case, however. The preparation that Huber and O'Donoghue used in their analyses was a
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z'8
3'2
Fig. 2. HPLC ion exchange gradient separation of samples (25 ug of uronic acid) of smaller (G7) citrus pectin oligomers (a), larger (G12) citrus pectin oligomers (b), and the B. fruit extract (c). Detection was by PAD. For each sample the peak co-eluting with the standard galacturonate octamer was designated peak 8, and the remaining peaks were numbered consecutively.
6
225 9 =
150
;
75
'0 II
4
8
~2 Relentton
6
20
~,,
Ttme ( m,n I
CDTA extract of cell walls. That extract (based on the work of Carrington et al. [12]) would have included the uronic acid we have identified as water soluble (and containing active oligomers) and wall-bound, chelator-soluble polymers containing uronide not included in our analysis. Thus, our oligomer-containing fraction would have been diluted to 40% of its initial concentration (to 0.8% of the uronic acid, based on the data in Table I and Fig. 4) in the material they analyzed. The extract was then subjected to gel filtration (Sepharose CL-2B-300) and fractions containing lower molecular weight uronic acids (in the range of 100,000 daltons and lower, according to manufacturer's specifications) were pooled for subsequent oligomer separation on Bio-Gel P-4. Fractions from this separation were then assayed for uronic acid, and none was found in the column's included volume. Given the low concentration of pectin oligomers we report in our extracts, and the fact that they, apparently, began their serial analysis with 500 ~zg of galacturonic acid equivalents, we would be surprised if that analysis had revealed tomato fruit oligomers. It was sufficient to identify pectin oligomers in extracts of ripening avocados, however. One might ask why we are stressing this point. The answer is simple. The report of Huber and O'Donoghue has been interpreted by many to be an
213
Table 1. Distribution of uronic acids in acid oligomers resolved by HPLC gradient ion-exchange chromatography (Fig. 2) Quantitation is based on comparison of integrated areas with the peak area for a known amount of galacturonic acid octomer (quantitation is approximate because of uncertainties with PAD detection of different sized oligomers). Samples(25 ug, galacturonic acid equivalents) of G7, G12, and B extract were chromotographed. Yield is the sum of the uronic acid measured in all oligomer peaks for a given sample expressed as a percentage of the 25 ~g injected. Oligomer peak numbers are shown in Figure 2 and the values shown are in ng of galacturonic acid equivalents. Peak No. Sample
4
5
6
7
8
9
10
11
12
13
G7
902
1190
694
485
347
274
232
201
104
90
14
G12
462
563
619
614
463
341
295
286
189
129
B extract
50
80
93
106
67
42
26
23
12
Yield 18.00
83
16.70 2.00
Table II. Carbohydrate compositions (weight percentage) of individual oligomer peaks purified (QAE-Sephadex or HPLC ion-
exchange separation, respectively)from mixtures of citrus pectin oligomers or B fruit extracts Compositions shown are for peaks whose biological activity is described in Figure 4. Uronic acid values are based on colorimetric assay. Proportions of neutral sugars were determined by GC and adjusted so that totals equal 100%. In fact, some oligomers (G7: peaks 8, 9 and 10. B extract: peak 10) produced small (less than 1% of the total integrated area), unknown peaks in the GC chromatograms. Component Source
Galacturonic Acid
Rha
Ara
Xyl
Man
Gal
Glc
Citrus oligomers Peak 8
93.9
I. 1
2.0
0.2
0.3
1.0
1.4
Peak 9
95.5
0.9
1.9
0.1
0.2
0.5
0.8
Peak 10
86.7
2.5
3.3
0.8
1.7
1.9
3.2
Peak 7
71.1
6.2
3.3
1.2
4.5
4.6
9.1
Peak 8
58.2
5.8
3.1
1.8
2.4
15.0
13.8
B extract
Peak 9
55.1
13.2
4.4
2.6
5.0
11.8
7.9
Peak 10
56.4
10.1
5.9
1.5
2.9
18.5
4.7
Peak 11
60.8
3.3
3.3
1.9
3.5
14.5
12.8
indication that pectin oligomers are not present in ripening tomato pericarp and, thus, that any consideration of a role for them in regulation of ripening is folly. We do not accept that oligomer presence can be ruled out by the described analysis. We accept that if they are endogenous (i.e., were not produced by PG that remained active after boiling pericarp in ethanol), the presence and biological activity we have reported here do not prove, p e r s e , that pectin oligomers are endogenous ripening regulators, and this point will be examined further. How might these oligomers be produced in the fruit? The easiest answer is that limited PG action on cell wall pectins generates them, and we have tested this point in vitro. CDTA- and Na2CO3-soluble pectins and PGA (included as a positive control) were incubated with purified tomato PG1. Surprisingly, only the carbonate-soluble material and PGA were digested (based on HPLC analysis of reaction mixtures and generation of
214
,0!!I
50 A .--I
do
1 t
::L (#1 '10 (.3
t~
20 tO 1.,,. 10
0 r 0
9
|
"
20
|
"
|
40
-
60
' i
-
80
Fraction
l
100
120
Number
Fig. 4. Ethylene production by MG pericarp discs treated with 50 /~g (uronic acid equivalents) of partially purified G7 citrus oligomers (a) or 30/~g of individual B fruit oligomers purified by HPLC. Water was used for the control. Peak numbers correspond to those shown in Fig. 2. Bars indicate SEs for the means of measurements of 8 discs/treatment.
30
Control (water)
--.---e,-----
20
-----O m
G7
.... g--"
Peak 8
.... O---
Peak 9
- - "dr" -
P e a k 10
16
e"
e-
=T= ..-" T'o.
"':';:"' "'" / ~'~"o
1 e-
14
----e---
Control (water)
---0-----
Breaker extract
.... m---
Peak 7
----.O'--
Peak 8
- - -z~--
P e a k 10
- - .t- -
P e a k 11
"
12
,c."
i 10 i
r162
sI
.~'/" ~r"
4
T
0
1
2
Mourn after
3
Fig. 3. QAE-Sephadex gradient separation of the B fruit extract. An 18 mg (uronic acid equivalents) sample of extract was dissolved in 20 ml of 125 mM imidazole-HC1 buffer (pH 7.0) and applied to the column. The column was then eluted with 50 ml 125 mM buffer followed by a 125 mM to 1.5 M buffer gradient (500ml), and, finally, 50 ml of 1.5 M buffer. Fractions of 5 ml were collected and assayed for uronic acids. Groups of fractions (26-41, 45-50, 53-75 and 84-100) were pooled, concentrated by ultrafiltration and analyzed by HPLC.
4
treatment
215 Fig. 5. Effect of PG1 digestion on the ethylene synthesis-inducing activity of CDTA-soluble tomato pectin (a), Na2CO3-soluble tomato pectin (b) and polygalacturonic acid (c). Controls were treated with solutions of heatinactivated PG1. Treatment doses were 10/tg of uronic acid equivalents. The line legends shown in panel a apply to all panels. Bars indicate SEs for the means of measurements of sets of 8 discs/teatment, fr wt, Fresh weight.
'i PG-treated --tl--
.'~ o.o
L_
Not digested
"'"" t~~176
OI r
.~ oi
10
t-
In
o C
~
0
""
1
2 Hours
""'"'t
3 after
4 treatment
5
6
reducing sugars; data not shown). The digested substrates, but not the undigested material, showed ethylene inducing activity in our pericarp disc assay system (Fig. 5). We have not yet compared the structures of the extracted and PGl-generated active oligomers. If the endogenous material is a participant in the initiation of ripening, however, it is unlikely the fruit PG is responsible for its origin. Several studies (e.g.; 8, 20) have shown that tomato fruit synthesis of PG follows (by as much as a day or more) the increase in fruit ethylene production that is accepted as a marker of ripening's onset. Of course, ripening is not a simultaneous, whole fruit phenomenon. It is a developmental event the passes through the fruit (its different tissue areas, perhaps even from cell to
216 cell) in a wave and so it is possible that an undetectable bit of ripening-initiating PG preceeds the rise in ethylene production as the first cells begin to ripen. However, even though studies have shown wave-like patterns of PG gene activity (30) and protein (34) in ripening tomatoes, we are not aware of data indicating that this PG "appearance" preceeds increased synthesis of ethylene. However, Schols et al. (32) have identified a pectin hydrolase produced by cultures of the fungus Aspergillus aculeatus and identified it as an RGase because it hydrolyzes the linkage between rhamnosyl and galacturonosyl residues in rhanmogalacturonans, and Gross et al. (22) have recently reported the presence of RGase activity in MG tomato fruits. Studies of in vitro RGase action on complex pectin substrates show that the enzyme often needs assistance from an assortment of "pectin processing" glycosidases to cleave the pectin backbone and that the breakdown products that result are relatively neutral sugar-rich (1, 32). MG and ripening tomato fruits contain a substantial variety of glycosidases (36). When we analyzed the oligomers in our B stage fruit extract we found them to contain an assortment of the neutral sugar residues that are normally associated with complex pectins (Table II). It is conceivable, therefore, that tomato RGase action (perhaps supported by glycosidases) could explain the presence of the oligomers we have described, and that limited pectin hydrolysis could lead to the initiation of ripening. On the other hand, recent work has shown that pectic oligomers can be transported over limited distances in tomato plants (28) and work in our lab has shown that limited pectin synthesis in tomato fruits even after ripening has begun (18). This means that the hydrolytic origin for endogenous, active pectin oligomers of tomato fruits is only one of several possible explanations for the presence we have reported here. Our laboratory is continuing its examination of the possibility that oligosaccharides contribute to the regulation of tomato fruit ripening. Figures and tables from Melotto et al. (1994) are reprinted with permission from Plant Physiology. Abbreviations: ACC, 1-aminocyclopropane-l-carboxylic acid; B, breaker stage (beginning to ripen); CDTA, trans-l,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid; DP, degree of polymerization; G7, set of smaller pectin oligomers generated by acid hydrolysis of G12, set of larger citrus pectin oligomers; MG, mature green stage (ready to ripen); PE, pectin methylesterase; PG, polygalacturonase; PGA, polygalacturonic acid; RGase, rhamnogalacturonan hydrolase.
REFERENCES An J., L. Zhang, M.A. O'Neill, P. Albersheim, A.G. Darvill. 1994. Isolation and structural characterization of endo-rhamnogalacturonase-generated fragments of the backbone of rhamnogalacteronan I. Carbohydr. Res. 264:83-96.
217 2. Ayers, A.R., J. Ebel, F. Finelli, N. Berger, P. Albersheim. 1976. Plant Physiol. 57:751-759. 3. Ayers, A.R., B.S. Valent, J. Ebel, P. Albersheim. 1976. Plant Physiol. 57:766-774. 4. Bhat, U.R., H. Mayer, A. Yokota, R.I. Hollingworth, R.W. Carlson. 1991. J. Bact. 173:21552159. 5. Blakeney, A.B., P.J. Harris, R.J. Henri, B.A. Stone. 1983. Carbohydr. Res. 113:291-299. 6. Blumenkrantz, N., G. Asboe-Hansen. 1973. Anal. Biochem. 54:484-489. 7. Brady, C.J. 1987. Ann. Rev. Plant Physiol. 38:155-178. 8. Bardy, C.J., G. MacAlpine, W.B. McGlasson, Y. Ueda. 1982. Aust. J. Plant Physiol. 9:171-178. 9. Brecht, J.K. 19897. HortScience 2:476-479. 10. Campbell, A., M. Huysamer, H.U. Stotz, L.C. Greve, J.M. Labavitch. 1990. Plant Physiol. 94:1582-1589. 11. Campbell, A., J.M. Labavitch. 1991. Plant Physiol. 97:706-713. 12. Carrington, C.M., L.C. Greve, J.M. Labavitch. 1993. Plant Physiol. 103:429-434. 13. Cheong, J.J., M.G. Hahn. 1991. Plant Cell. 3:137-147. 14. Cooper, R.M. 1984. In: (Woods and Jillis, eds.) "Plant Diseases: Infection, Damage and Loss." Blackwell, Oxford, pgs. 13-27. 15. Darvill. A.G., P. Albersheim. 1984. Ann. Rev. Plant Physiol. 35:234-275. 16. Fischer, R.L., A.B. Bennet. 1991. Ann. Rev. Plant Physiol. and Mol. Biol. 42:675-703. 17. Giovannoni, J.J., D. DellaPenna, A.B. Bennett, R.L. Fischer. 1989. Plant Cell 1:53-63. 18. Greve, L.C., J.M. Labavitch. 1991. Plant Physiol. 97:1456-1461. 19. Grierson, D., M. Kader. 1986. In: (Atherton and Rudich, eds.). "The Tomato Crop - A Scientific Basis for Improvement" Chapman and Hall, london, New York. pgs. 241-280. 20. Grierson, D., G.A. Tucker. 1983. Planta 157:174-179. 21 Grierson, D., G.A. Tucker. 1983. Planta 157:174-179. 22. Gross, K.C., D.A. Starrett, H-J. Chen. 1995. Acta Hort. 398:121-130. 23 Hotchkiss, A.T., K.B. Hicks. 1990. Anal. Biochem. 184:200-206. J 24. Huber, D.J. 1983. J. Amer. Soc. Hort. Sci. 108:405-409. 25. Huber, D.J., E.M. O'Donoghue. 1993. Plant Physiol. 102:473-480. 26. Klee. H.J. 1993. Plant Physiol. 102:911-916. 27. Kramer, M., R. Sanders, H. Bockan, C. Waters, R.E. Sheehy, W.R. Hyatt. Postharv. Biol. technol. 1:241-255. 28. MacDougall, A.J., N.M.Rigby, P.W. Needs, R.R. Selvendran. 1992. Planta 188:566-574. 29. Melotto, E., L.C. Greve, J.M. Labavitch. 1994. Plant Physiol. 106:575-581. 30. Montgomery, J., V. Pollard, J. Deikmen, R.L. Fischer. 1993. Plant Cell 5:1049-1062. 31. Ryan, C.L., E.E. Ramer. 1991. Ann. Rev. Plant Physiol. and Mol. Biol. 42:651-674. 32. Schols, H.A., C.C.J.M. Gereads, M.F. Searle van Leeuwen, M.F. Kormelink, A.G.J. Voragen. 1990. Carbohydr. Res. 206:105-115. 33. Spiro, M.D., K.A. Kates, A.L. Koller, M.A. O'Neill, P. Albersheim, A. Darvill. 1993. Carbohydr. Res. 247:9-20. 34. Tieman, D.M., A.K. Handa. 1989. Plant Physiol. 90:17-20. 35. Tieman, D.M., R.W. Harriman, G. Ramomohow, A.K. Handa. 1989. Plant Cell 4:667-679. 36. Wallner, S.J., J.E. Walker. 1975. Plant Physiol. 55:94-98.
This Page Intentionally Left Blank
IDENTIFICATION, MODE OF ACTION AND 3-D STRUCTURE OF PECTINASES
This Page Intentionally Left Blank
J. Visser and A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All fights reserved.
Kinetics and mode of action of
221
AspergiUus niger polygalacturonases
Jacques A.E. Benen, Harry C.M. Kester, Lucie Parenicovfi and Jaap Visser. Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, Dreyenlaan 2, 6703 HA Wageningen, The Netherlands.
Abstract Endo-polygalacturonases I and II (PGI and PGII) isolated from recombinant A. niger were characterized with respect to pH optimum, activity on polygalacturonic acid (pga), mode of action and kinetics on oligogalacturonates. Vmax and Km values using pga as a substrate at the optimum pH 4.1 were calculated as 500 U/mg and 0.15 mg/ml and 2000 U/mg and 0.15 mg/ml for PGI and PGII, respectively. Mode of action analysis revealed a random cleavage pattern for PGII while for PGI multiple attack on a single chain was observed. For PGII a partial subsite map was obtained. Site directed mutagenesis of His223 of PGII with subsequent analysis of the mutated PGII revealed that His223 is essential for catalysis.
Introduction Polygalacturonases from primarily fungal origin have been studied since several decades. Nowadays many genes encoding polygalacturonases, both exo- and endo-acting, from numerous different species have been cloned. Despite the large number of genes available and the long record of polygalacturonase studies most studies were directed at the purification of the enzymes and a limited characterization comprising mostly whether the enzyme is exo- or endo-acting, the activity on polygalacturonic acid and the pH, temperature and ionic strenght optima while very few studies were carried out toward the understanding of the mode of action, determined by the characteristics of the individual subsites of the enzymes. First studies addressing this problem via the determination of the number of subsites of an Aspergillus niger polygalacturonase and identification of catalytically important residues of this enzyme were described by Rexov~i-Benkovfi [1, 2]. Unfortunately, these studies have found only little follow up by other research groups [3]. Therefore detailed knowledge about 'subsite architecture' of these industrially important enzymes is scarce. Bussink et al. [4] and Kusters-van Someren et al. [5] have shown that in A. niger for both polygalacturonases (PGs) and pectin lyases (PLs) families of genes are present: seven
222 PG and six PL encoding genes were identified [4, 5]. The occurrence of families of genes encoding different enzymes raises the possibility of a concerted action of the enzymes of one or both families in the degradation of pectin. In order to clarify the role of the individual enzymes in the pectin degradation a comparative study in this respect was initiated. For this, individual genes were fused with the pkiA promoter of the glycolytic pyruvate kinase gene that allows expression of the individual genes under conditions where all other pectinases are repressed. Careful analysis of mode of action, kinetic parameters and subsite affinities of the enzymes on model and natural substrates will reveal the role of the individual enzymes in pectin degradation. Here we report on the characterization of recombinant PGI and PGII, the two most abundant PGs in the commercial Rapidase preparation [3] and of a site specific mutated PGII in which His223 was changed into Ala.
Materials and Methods Molecular biology. All DNA manipulations were performed using standard techniques. Promoter-gene fusions were constructed as described by Kusters-van Someren et al. [6]. Site directed mutagenesis of His223 of PGII was carried out in essentially the same way. PCR generated DNA fragments were checked for undesired mutations by sequence analysis. Transformation of A. niger NW228 (pyr, prtF) with appropriate plasmids was done as described before [7]. Growth and purification. A. niger strains transformed with either the pki-pgaI or pla'-pgalI fusion were grown in batch in 1L flasks containing 350 ml minimal medium according to Pontecorvo et al. [8] supplemented with Vishniacs spore element solution, 0 . 1 % yeast extract, 70 mM NH4C1 and 4 % fructose as a carbon source. Cultures were inoculated with 1 • 106 spores/ml and grown at 30 ~ in a rotary shaker for 18 hrs. Mycelium was separated from the medium by filtration over a nylon membrane. The filtrate was adjusted to pH 6.0 and loaded onto a DEAE-Sepharose Fast Flow column pre-equilibrated at pH 6.0 (10 mM Bis-Tris/HC1). Elution was performed with a linear gradient, 0-600 mM NaC1, in 10 mM Bis-Tris pH 6.0. SDS-PAGE of individual fractions demonstrated that the enzymes were pure after this separation. Fractions containing the enzyme were pooled and dialysed against 50 mM Na-acetate pH 4.5 and stored at either -20 ~ or 4 ~ until use. Mode of action and kinetics. Routine polygalacturonase assays were performed in a reaction mixture containing 50 mM Na-acetate pH 4.2 and 0.25 % w/v pga at 30 ~ The release of reducing sugars was determined according to Stephens et al. [9]. For determination of pH optima the 50 mM Na-acetate buffer was replaced by Mcllvain buffers. For the determination of kinetic parameters and for the mode of action of the enzymes reaction products were analysed on a Dionex BioLC/high-performance chromatography system using a Carbo Pac PA-100 anion-exchange column (25 cm x 4 mm) with pulsed amperometric detection. The samples loaded were eluted with a linear gradient of 0.15-0.60 M Na-acetate in 0.1 M NaOH at 1 ml/min in 22 min. Products were quantitated via calibration mixes containing oligogalacturonates with DP 1-8 (G1-G8) at 0.1 mM each and via an internal standard of 0.1 mM glucuronic acid (eluting between G1 and G2) with 50 pl injections.
223
Results and Discussion The PGI and PGII produced from strains transformed with the promoter gene fusion are in all respects tested identical to those enzymes obtained from the wild type strain when grown on pectic substances. For both PGI and PGII the pH optimum is 4.1-4.2 in 50 mM Na-acetate buffer, 30 ~ All further kinetic analyses were performed under these conditions. PGII. Using pga as a substrate Km and Vmax of PGII were calculated as 0.15 mg/ml and 2050 U/mg, respectively. The high Vmax and low Km demonstrate that pga is a good substrate for PGII. By following the product formation as a function of time (Fig. 1) it was demonstrated that PGII is a randomly cleaving endo polygalacturonase. The progression of substrates is characterised by an initial transient increase of higher oligogalacturonates which are gradually converted to oligomers with lower DP, eventually resulting in a mixture of G1, G2 and G3. The rather strong transient increase of G4 and G5 is not a result of transglygosylation, since PGII is an inverting enzyme (see Biely et al. elsewhere in this volume), but is merely due to the slow hydrolysis of these compounds as will be discussed below.
0.4
G1 IS
G2
G3
G4
0.3 90 min
~- 0.2 0.1 0.0
_
0
J A 5
-
-
~ _ . _ . _ . ~ _ A
10
15
-
35 min 10 min I 20
minutes
Figure 1. HPLC analysis of product progression during hydrolysis of 0.25 % polygalacturonate by PGII. Aliquots were withdrawn from the reaction mixture at timed intervals and reactions were stopped by raising the pH of the sample to pH 8.0 by mixing with 1 volume 25 mM Na-phosphate pH 9.5. G1 to G5 indicate the oligogalacturonates with corresponding degree of polymerization. The vertical axis shows the responce of the pulsed amperometric detector and the horizontal axis the elution time. Times of sampling are indicated above the trace.
224
Table I. Mode of action of PGII. Bond cleavage frequencies (BCF) in percentage for oligogalacturonates. The reducing or end of the products is indicated with a solid circle. The position of cleavage is indicated with a solid triangle. DP
BCF (%)
4 5
6
o
o
o o
Products
o
o
o
9
100
o o
o o
o o
9 o
9
67 33
dimer
o o o
o o o
o o o
9 o o
9 o
35 57 8
dimer trimer
9
A
In order to estimate the number of subsites, the binding affinities, the location of the active site and the cleavage patterns reduced and non reduced oligogalacturonates of DP 4 to 6 were used as substrates and the resulting products analysed by HPLC and TLC. Table I lists the bond cleavage frequencies for PGII. G4 is exclusively split in 1-3 mode while reduced G4 was not hydrolysed. G5 is cleaved in the 1-4 and 2-3 mode at 67 % and 33 % respectively, while reduced G5 is only split into reduced G2 and G3. The reduced G6 is not cleaved in the 1-5 mode, while reduced G2 and reduced G3 are readily formed. The non-reduced G6 is cleaved in 1-5, 2-4 and 3-3 modes yielding equimolar product pairs as was also seen for the cleavage of G4 and G5. These data demonstrate that cleavage of the glycosidic bond occurs from the reducing end. In time course experiments using different oligogalacturonates at several concentrations the turnover numbers and Km values were estimated for each oligomer in the individual binding modes and used for the calculation of the thermodynamic parameters of PGII according to Thoma et al. [10] and Hiromi et al. [11]. In Table II the data are presented. An approximation of the intrinsic rate constant, kint, was calculated from Vmax on pga (kint = kcat). From kint and Km and the turnover number for each binding mode the binding energies for each mode were calculated which were in turn used for the calculation of the individual subsite affinities listed in Table III. Since G4 is the smallest substrate used in this study, it is not possible to obtain information of the subsites at positions -3 to 1.
225 Table II, Kinetic and thermodynamic parameters of PGII using oligogalacturonates as substrates. Kp was calculated from Kp=[ko/Km]/kint while kint was obtained as described in the text. AG was calculated from -AGp=RTlnKp + 10 kJ/mole. Mode indicates the cleavage mode DP
Mode
Km • 10-6 (M)
ko
ko/Km x 10-6 Kp
(s -1)
(M -1 s-1)
(M -1)
AGp (kJ/mole)
4
G3 + G1 22
103
4.7
8048
-22.6
5
G4 + G1 13 G3 + G2 25
315 159
24.2 6.4
41438 10960
-26.7 -23.4
G5 + G1 40 G4 + G2 16 G3 + G3 31
148 386 56.5
3.7 24.1 1.8
6336 41267 3082
-22.0 -26.7 -20.2
Table III. Subsite affinities Ai for PGII (i denotes the subsite number). Subsites with a '-' prefix are located towards the non-reducing end of the substrate while subsites with a ' + ' prefix are located towards the reducing end. The active site is located between subsites -1 and + 1. Subsites -3 to + 1 were determined as one value. Subsite (Ai) Affinity (kJ/mole)
-4 +4.2
-3 / + 1 +22.6
+2 +0.8
The subsite map and the data on reduced oligomers, which showed formation of reduced G2 on reduced G5, indicate that the number of subsites is 5, stretching from position-4 to 1. The sum of the individual turnover numbers for each oligomer also shows that the rate of hydrolysis of the oligomers with DP4 and DP5 is much slower than of those with higher DP which is reflected in the product progression curves in Fig. 1. Rexov~i-Benkov~i [1] studied an A. niger endopolygalacturonase which might have been the same as the PGII described here; the cleavage pattern described is very much the same as found here and the pH optimum is exactly the same. The number of subsites for that enzyme was found to be four which is not in agreement with the number found for PGII. However, bearing in mind that in the study of Rexovgt-Benkovgt the individual binding modes of each oligogalacturonate were not addressed and therefore no subsite map was obtained the additional fifth subsite might have been overlooked.
226 PGI.
In a similar way as for PGII the kinetics for PGI were addressed. Using pga as a substrate Km and Vmax were 0.15 mg/ml and 500 U/mg, respectively. Fig. 2 shows the product progression upon pga hydrolysis. There is a strong increase of G1, G2 and G3 which is accompanied by a transient increase of G4 and G5 while there is only a small increase of oligomers with DP higher than 5. The transient increase of G4 and G5 is not due to transglycosylation since like PGII, PGI is also an inverting enzyme (see Biely et al. elsewhere in this volume). The profiles of PGI suggest that the enzyme after first random cleavage of the polymer substrate degrades the higher oligomers formed preferentially via hydrolysis of terminal residues at the reducing end. The latter was demonstrated by hydrolysis of reduced oligogalacturonates while the former was investigated in more detail by analysing the mode of action and cleavage rates on defined oligomers ranging from DP 4 to 8 . PGI hydrolyses G4 mainly to G1 and G3 at equimolar amounts as expected and a small amount is digested into G2 (Results not shown). G5 is cleaved to G1 and G4 in again equimolar amounts and to almost the same extent to G2 and G3 also in equimolar amounts (Results not shown). Thus, for G4 and G5, PGI is not much different from PGII, only the bond cleavage frequencies being slightly different. Upon hydrolysis of G6 to G8 the formation of equimolar product pairs is not observed anymore. With G6 as substrate G1 is formed at least twice as fast as G5 while G4 is formed IS G2
0.14 0.12 0.10 0
0.08
G3 G4 G5
0.06 0.04
J
0.02
IS
0.00 0
G2 G3 G4 G5 I
10
I
15
10 min I
20
minutes
Figure 2. HPLC analysis of product progression during hydrolysis of 0.25 % polygalacturonate by PGI. Aliquots were withdrawn from the reaction mixture at timed intervals and reactions were stopped by raising the pH of the sample to pH 8.0 by mixing with 1 volume 25 mM Na-phosphate pH 9.5. G1 to G5 indicate the oligogalacturonates with corresponding degree of polymerization. The vertical axis shows the responce of the pulsed amperometric detector and the horizontal axis the elution time. Times of sampling are indicated above the trace.
227 faster as G2 (Fig 3). A similar behavior was observed by Robyt and French [12, 13] for cxamylase. They demonstrated that this type of product ratios, that differ from the beginning of the reaction, are due to multiple attack on a single chain. A ratio-plot according to Robyt and French [13] for G1 and G5 formation from G6 supported that PGI exibits multiple attack on G6 (not shown). The fact that still G5 is formed upon hydrolysis of G6 demonstrates that not all G6 bound in the G5-G1 mode is cleaved in a repetitive way. Thus, only a fraction of the substrate bound in this mode is subject to repetitive attack. The ratio between cleavage in this way and normal cleavage into G1 and G5 is determined by the dissociation constant of the G5 generated upon cleavage and the first order rate constant that is responsible for the shift of the bound G5 into the G4-G1 mode. Since still a considerable amount of G5 is formed, the dissociation constant of G5 and the first order 'shift' rate constant are of the same magnitude. However, from G7 and G8 as substrates it is clear that the ratio between the dissociation constant and 'shift' rate constant is completely in favor of shift when DP equals 6 or 7. With G7 as a substrate quite large amounts of G1 are formed while there is no detectable formation of the corresponding G6, only a rapid accumulation of G5 is observed. Similarly with G8 as a substrate quite large amounts of G1 and G2 are formed while there is no detectable formation of the corresponding G7 and G6, only again a rapid accumulation of G5 occurred which indicates that the multiple attack ceases when DP is down to 5 or 4, hence when dissociation is favored over shift. The previous data allow a clear interpretation of Fig. 2. PGI initially hydrolyses pga in a random endolytic way generating higher oligomers which is accompanied by rapid multiple attack of the higher oligomers in an exolytic way to yield mainly monomers and dimers from the reducing end. Therefore no transient accumulation of higher oligomers takes place. The appearance of transient G5 and G4 is due to the fact that the multiple attack of the higher oligomers stops when DP is 4 or 5 and these product are released. The transient accumulation of the tetramer partly originates from the particular binding mode of the higher oligomers. At present we are working on a kinetic model that describes the action pattern of PGI. It has been suggested that the multiple or single attack can be distinguished based on the relative rate of accumulation of mono- di- and trimers upon hydrolysis of polymeric substrate. At first glance this appears to be valid when comparing for example the mode of action of PGI and PGII (see Fig. 1 and Fig. 2). In a comparative study of three polygalacturonases by Pasculli et al [14] the enzymes were classified according these criteria while the rate of accumulation was by far not as clearcut differing as found for PGI and PGII. The observed profiles in that study can also be explained by assuming different rates of hydrolysis of the smaller (DP 4-6) oligogalacturonates. If for instance the rate of hydrolysis of G4 and G5 of PGII would have been higher then the progression curves would have more resembled those of PGI. The only way to discern between multiple or single attack is by analysis of the stochiometry of product pairs upon hydrolysis of oligomeric substrates at initial stages of reaction.
228 40 o
,.i-,
(D c
gl
30
.,.-~
(/) 0
20
g5
E 0c -
~c -
g4 g2 g3
10 ~n-'~ 0
T 5
I I0
I 15
I 20
I 25
I 30
minutes
Figure 3. HPLC analysis of product progression during hydrolysis of 0.5 mM hexagalacturonate by PGI. Aliquots were withdrawn from the reaction mixture at timed intervals and reactions were stopped by raising the pH of the sample to pH 8.0 by mixing with 1 volume 25 mM Na-phosphate pH 9.5. G1 to G5 indicate the oligogalacturonates with corresponding degree of polymerization.
PGII His223Ala. Apart from mode of action and kinetics of wild type enzymes structure function relationships of these industrially important enzymes is of high interest to provide the necessary knowledge for genetic engineering of desired properties. As a first approach the identification of catalytically important residues was addressed in conjunction with the elucidation of the three dimensional structure [15]. Rexovfi-Benkov~i and Marckov~t [2] obtained evidence for the possible involvement of a histidine in catalysis of a fungal endopolygalacturonase. The same results were obtained for PGII using diethyl pyro-carbonate as a modifying agent in the presence or absence of substrate (Kester, unpublished). However chemical modification studies can never give conclusive evidence for the participation of the modified residue in catalysis or binding. The method to dissect the role of individual residues is site directed mutagenesis. Upon alignment of 25 endo- and exo-polygalacturonases taken from the swiss.prot. database only one histidine appeared to be conserved throughout. The conserved histidine at position 223 in PGII was changed into alanine, a small apolar residue. The mutation has a dramatic effect on Vmax using pga as substrate which decreases from 2050 U/mg to 10 U/mg; the Km did not change significantly, however. The mutation also affected the pH optimum of the enzyme as is shown in Fig. 4. The pH optimum narrows down to one pH unit. Since the polygalacturonate concentration used is well above the Km the apparent velocities can roughly be regarded as Vmax values at each pH. So, the fall and rise of the activity within one pH unit strongly suggests that catalysis is governed by only one ionisable group in the mutated enzyme, a glutamate or an aspartate. Thus, the
229
100 >,
80
60
>
9 40
~
"~9 rr
20 0
I
I
I
I
2.5
3
3.5
4
I
I
I
I
4.5
5
5.5
6
pH
Figure 4. Relative activities of wild type PGII and His223Ala mutated enzyme as a function of pH. Wild type enzyme, solid circles; His223Ala mutated enzyme, open circles. 0.30 0.25
' -
IIs
gl.
g3
0.20 0:=L
G
0.15
IS
0.10 0.05 0.00
g2 j~
0
I
5
i
I
10
,
!
15
!
20
minutes
Figure 5. Selected HPLC elution profile of products obtained after incubation of 0.25% polygalacturonate with PGII, upper trace, and PGII H223A, lower trace, respectively, demonstrating the effect of the mutation on catalysis. G1 to G3 indicate the peaks of the corresponding oligogalacturonates. IS indicates the internal standard, glucuronate. The vertical axis shows the pulsed amperometric detector response while the horizontal axis shows the retention time.
230 strong effect on Vmax, the absence of an effect on Km and the striking effect on the pH optimum, the shift in the direction expected, provide evidence for the involvement of His223 in catalysis rather than binding. This is again affirmed by the analysis of the products formed after hydrolysis of pga. In case of an effect on binding it is expected that the product distribution is different from the wild type enzyme however in case of involvement in catalysis only a slight effect, if any, on the product distribution is expected. In Fig. 5 two selected HPLC profiles are presented showing the product distribution when pga is hydrolyzed to the same extent by wild type PGII and mutated PGII. Apart from a small change in G1 contribution there is hardly any change in product distribution. A small effect on G1 is to be expected since the active site histidine is located at the junction of subsites -1 and + 1. These data demonstrate that His223 is involved in catalysis and not in binding.
Acknowledgement This work was financially supported by the European Community grant no. AIR2-CT941345.
References 1) Rexov/t-Benkov~i, L. (1973) Eur. J. Biochem. 39, 109-115. 2) Rexovfi-Benkov~t, L. and Marckov~i, M. (1978) Biochem. Biophys. Acta 523, 162-169. 3) Kester, H.C.M. and Visser, J. (1990) Biotechn. Appl. Biochem. 12, 150-160. 4) Bussink, H.J.S., Buxton, F.P., Fraaye, B.A., de Graaff, L.H. and Visser, J. (1992) Eur. J. Biochem. 208, 83-90. 5) Harmsen, J.A.M., Kusters-van Someren, M.A. and Visser, J. (1990) Curr. Genet. 18, 161-166. 6) Kusters-van Someren, M.A., Flipphy, M.J.A., De Graaff, L.H., van den Broek, H.C., Kester, H.C.M., Hinnen, A. and Visser, J. (1992). Mol. Gen. Genet. 234, 113-120. 7) Goosen, T., Bloemheuvel, G., Gysler, C., de Bie, D.A., van den Broek, H.W.J and Swart, K. (1987) Curr. Genet. 11,499-503. 8) Pontecorvo, G. Roper, J.A., Hemmons, L.J., MacDonald, K.D. and Bufton, A.W.J. (1953) Adv. Genet 5, 141-238. 9) Stephens, B.G., Felkel, H.J.Jr. and Spinelli, W.M. (1974) Anal. Chem. 692-696. 10) Thoma, J.A., Rao, G.V.K., Brothers, C. and Spradlin, J. (1971) J. Biol. Chem. 246, 5621-5635. 11) Hiromi, K., Nitta, Y., Numata, C. and Ono, S. (1973) Bioch. Biophys Acta 302, 362375. 12) Robyt, J.F. and French, D. (1967) Arch. Biochem. Biophys. 122, 8-16. 13) Robyt, J.F. and French, D. (1970) Arch. Biochem. Biophys. 138, 662-670. 14) Pasculli, R., Geraeds, C., Voragen, F and Pilnik, W. (1991) Lebensm. Wiss. u. Technol. 24, 63-70. 15) Schrtiter, K.-H., Arkema, A., Kester, H.C.M., Visser, J. and Dijkstra, B.W. (1994) J. Mol. Biol. 243,351-352.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All fights reserved.
231
New enzymes active towards pectic structures G. Beldman, M. Mutter, M.J.F. Searle-van Leeuwen, L.A.M. van den Broek, H.A. Schols and A.G.J. Voragen.
Wageningen Agricultural University, Department of Food Science, Bomenweg 2, 6703 HD Wageningen, The Netherlands.
Abstract
Microbial pectinases have been used for fruit and vegetable processing for already more than half a decade. With respect to application as well as to fundamental research, most attention has been paid to those enzymes acting towards the 'smooth' homogalacturonan part of the pectin molecule (i.e. polygalacturonase, pectin lyase, pectate lyase, pectin methyl esterase). More recently, enzymes active towards the 'hairy' rhamnogalacturonan part of pectin gained attention, since it was found that in juice processing those structures foul the ultrafiltration membranes used in a final clarification step. Two different rhamnogalacturonases (RGases A and B) were identified and purified from an A~pergillus aculeatus preparation, using apple pectic hairy regions (MHR) as substrate. Based on the structure of the products, RGase A was identified as a hydrolase, splitting the ot-GalAp-(l-2)-o~-Rhap linkage in rhamnogalacturonan, while RGase B appeared to be a lyase splitting the ot.-Rhapct-(1-4)-GalAp linkage by 13elimination. Rhamnogalacturonan oligosaccharides were used to identify and purify two other novel enzymes with high specificity towards rhamnogalacturonan fragments: a rhamnogalacturonan rhamnohydrolase and a rhamnogalacturonan galacturonohydrolase. As an accessory enzyme for the RGases, rhamnogalacturonan acetyl esterase (RGAE) was discovered in the same A. aculeatus preparation. This enzyme appeared to be specific for the de-acetylation of MHR and essential for the degradation of MHR by RGases A and B. The same enzyme (RGAE) could be purified from A. niger, together with two other esterases: a feruloyl esterase (FAE) and an acetyl esterase (PALE) specific for the removal of one type of acetyl group present in the 'smooth' regions of sugar-beet pectin. Finally, the A. aculeatus preparation was found to contain an enzyme releasing the dimer 13Xylp-(1-3)-GalAp from a soluble soy cell wall polysaccharide. The enzyme was partially purified and appeared to be active towards saponified MHR and gum tragacanth as well. It was concluded that this enzyme degraded the xylogalacturonan part in MHR by an exo-fashion.
232 1. INTRODUCTION Enzymatic liquefaction is a relatively new process for the production of juices from fruits and vegetables [ 1]. Essentially the process is as follows: the material is crushed to obtain a pulp which is treated with a combination of pectinases and cellulases. After a certain incubation time, the material becomes a liquid and the juice can be recovered by decantation. Subsequently, a clear juice is obtained by ultrafiltration. A serious problem in this process is the fouling of the ultrafiltration membrane, causing a reduced flux rate. For apple processing, the material responsible for this effect has been isolated and extensively characterized [2-4]. It appeared to consist mainly of ramified pectic 'hairy' regions (MHR), which were not degraded by the pectolytic enzymes present in the technical pectinase preparation.
2. RHAMNOGALACTURONAN DEGRADING ENZYMES FOR FRUIT AND VEGETABLE PROCESSING MHR was used as a substrate to identify and purify novel enzymes able to degrade this structure. A first endo-acting enzyme was found in an A3pergillus aculeatus preparation (Pectinex Ultra SP-L, Novo Nordisk Ferment, Dittingen, Switzerland), purified and identified as a rhamnogalacturonase acting on the rhamnogalacturonan part of MHR [5]. Subsequently this enzyme has been recognised by several other investigators [6, 7]. A second enzyme from the same A. aculeatus strain, which was able to degrade MHR was identified more recently [referred to in ref. 8]. Both enzymes have been cloned in A. oryzae and named RGaseA and RGaseB, respectively [8]. The crude A. aculeatus preparation was able to degrade MHR as such, however the purified RGases were only active towards MHR, after a chemical saponification of the substrate (MHR-S), specifically after removal of the acetyl esters. From this observation it was concluded that also a rhamnogalacturonan acetylesterase (RGAE) should exist in the original preparation. Indeed such enzyme could be purified from it [9].
2.1 MODE OF ACTION OF RGASES A AND B FROM A S P E R G I L L U S A C U L E A T U S High-performance anion-exchange chromatography (HPAEC) of the reaction products from MHR-S showed that RGaseA and B acted differently towards this substrate (Fig. 1). RGaseB produced a series of oligosaccharides with longer retention times than those obtained with RGaseA. The latter products were already isolated and identified by Schols et al. [ 10]. Using the same isolation procedure, we also purified the degradation products from MHR-S made with RGaseB, and identified their structures by NMR spectroscopy. Fig. 2 shows the primary structures of both types of products. The oligosaccharides made by RGaseA are known to consist of a backbone of alternating rhamnose and galacturonic acid residues, with
233 rhamnose at the non-reducing side and a galacturonic acid at the reducing side. The rhamnose residues are either branched or not branched with a galactose residue.
oo
rt
RGase A .
0
.
.
.
.
.
.
.
.
l , l
10
20
30
40
Retention time (min)
Fig. 1. High-performance anion-exchange chromatography of the reaction products from MHR-S, produced by RGasesA and B.
The products obtained with RGaseB contained essentially the same building blocks, but in this case the rhamnose residue was located at the reducing side and a unsaturated galacturonic acid residue was found at the non-reducing side [ 11 ]. Backbone lengths of 4 to 10 units were identified. The conclusion from this work is that RGaseA is a hydrolase, cleaving the backbone of the rhamnogalacturonan part in MHR between a galacturonic acid and rhamnose unit, while RGaseB is a lyase cleaving the rhamnogalacturonan between a rhamnose and a galacturonic acid residue. Lyase activity of RGaseB could be confirmed by measuring the increase of A235of the reaction mixture (Table 1). Using this method, it could be shown that RGaseB was very
234
RGase
A
RGase
( t i t (- ") )
B
| (
n=l -4
)
ct-Rhae-(l-4)-~
H
ct-GalAe-(l-2)-(x-Rha e ot-us-GalAe-(1-2)-ct-Rhae
B-Gale-( 1-4)-ot-Rhae
Fig. 2. Oligomeric reaction products from MHR-S, produced by RGases A and B. specific for the degradation of rhamnogalacturonan structures, either present in MHR-S or as linear oligosaccharides with a DP>18, which were isolated from a beet pulp hydrolysate [ 12]. No activity was found towards highly methylated pectin or polygalacturonic acid, neither at pH 6 or pH 8, both in the presence or absence of Ca z+. So it could be concluded that RGaseB was different from the already known pectin and pectate lyases.
Table 1. Lyase activity ofRGase B toward various substrates (U mg ~ ), determined from the increase in A235 using an e of 4800 M 1 cm ] Substrate MHR-S Linear RG oligomers pectin DM 92.3% PGA n.d., not determined" a 50 mM NaOAc pH 6 b, 20 mM Tris.HCl pH 8; c, 1 mM CaCI2;
pH 6 a
pH 8b
- Ca
+ Ca c
- Ca
+ Ca c
8.8 3.9 0 0
9.8 n.d. 0 0
10.6 n.d. 0 0
11.8 n.d. 0 0
235
Blank RGAE RGaseA Comb. rr #,',
'
........
....
/~
....
-.
j:
~,.~,.~"
-..
/
"'~, .
....... I
~.~':
2O
..~"
. . . . -.-- . . . .
.-~"" ~.
"\
1
25
"~..--1..
30
Time
....j.I
35
(m~n)
Fig. 3. High performance size exclusion chromatography of MHR after degradation with RGAE from A. aculeatus, RGaseA and a combination of these enzymes. The importance of the aforementioned rhamnogalacturonan acetylesterase for the degradation of MHR by RGaseA is presented in Fig. 3. From the high performance size exclusion chromatogram of MHR it can be seen that essentially no degradation occurs when RGaseA or RGAE are used alone. Only the combination of RGaseA and RGAE is able to degrade MHR extensively. With respect to substrate specificity, a high preference of RGAE for acetyl esters in a rhamnogalacturonan was observed (Table 2). No activity was found towards other acetylated polysaccharides such as beet pectin or acetylated xylan, nor towards smaller substrates such as triacetin and acetylsalicylic acid. From MHR about 70% of the acetyl groups could be removed after prolonged incubation. Table 2. Acetyl release by RGAE from different substrates" i
Substrate
Activity (mU/mg)
MHR
Acetyl content (w/w) 4.4
921
Release (% of total) 42 (70) b
Beet pectin Xylan Triacetin Acet~,l salicylic acid
3.0 13.3 81.0 30.0
0 0 0 0
0 0 0 0
' 20 h, 30 ~
pH 5.0.
b after prolonged incubation
236
-
B-Galactosidase
(B-Gal-ase)
-
Rhamnogalacturonan rhamnohydrolase (RG-Rha-ase)
-
Rhamnogalacturonan galacturonohydrolase (RG-GalA-ase)
(Van de Via et al., 1 9 9 4 )
(Mutter et al., 1994)
(Mutter et al., in preparation)
, B-Gal-ase
"
-
..
,.T_.]. " "
-
RG-GalA-ase
/
/ 0
< RG-Rha-ase
Fig. 4. Degradation of rhamnogalacturonooligosaccharides by several exo-acting enzymes. For symbols see Fig. 2. 2.2 RHAMNOGALACTURONOOLIGOSACCHARIDES AS SUBSTRATES TO IDENTIFY NOVEL EXO-ACTING ENZYMES IN ASPERGILLUS ACULEATUS Rhamnogalacturonooligosaccharides were also used to identify and purify new enzymes which are able to degrade these structures by an exo-fashion. All essential enzymes for their degradation have been found (Fig. 4). A rather non-specific 13-galactosidase was purified from an A. niger preparation by Van de Vis et al. [ 13]. This enzyme could split off the galactose side chain from the branched oligosaccharides. The resulting linear oligosaccharide was used to identify and purify a new rhamnogalacturoan rhamnohydrolase (RG-Rha-ase) from A. aculeatus, which cleaves off the rhamnose unit located at the non-reducing end of an oligosaccharide [ 14]. The resulting new oligosaccharide was again used to purify another new enzyme from the same source: the rhamnogalacturonan galacturonohydrolase (RG-GalA-ase), which removes the galacturonic acid unit from the non-reducing end of this oligosaccharide [Mutter et al., in preparation]. Although we do not know their role in enzymatic fruit processing, in which complex mixtures of technical pectinases are being used, these enzymes have been found to be very useful for the identification of hydrolysis reaction products from rhamnogalacturonan, as presented elsewhere in this proceedings [Mutter et al.]. Next to the RG-rhamnohydrolase, an other rhamnose releasing enzyme, active towards p-nitrophenyl-c~-rhamnoside has been purified from
237 for the presence of RGAE. This time we focused on the identification of acetyl esterases in general, using, next to MHR, also other acetylated substrates. In the rhamnogalacturonan part of MHR the acetyl groups are probably linked to the 0-2 and/or 0-3 of the galacturonosyl residue [4] (acetylated pectic 'hairy' regions). As a substrate of which the acetyl groups are located at the 'smooth' homogalacturonan regions, a beet pectin (a gift fromGrindsted, Denmark) was taken. Table 4 shows the sugar composition as well as the degrees of acetylation and methylation of these polysaccharides. From the high galacturonic acid content and the relatively low amount of rhamnose and arabinose, it can indeed be concluded that this beet pectin consists mainly of'smooth' regions with a high degree of esterification. Table 4. Sugar composition (mol%) and degree of acetylation (DA) and methylation (DM) of the pectic substrates. Rha Ara Xyl Man Gal Gluc GalA DA DM
Beet pectin 2 8 tr. tr. 5 tr. 83.8 34 6O
MHR 6 55 8 9 1 21 60 42
A crude A. niger preparation (Rapidase C80, Gist brocades, Seclin, France), was fractionated by column chromatography, using Bio Gel P10, DEAE-Bio Gel A, Cross-linked alginate, Bio Gel HTP, Bio Gel P 100 and Mono-Q (Searle-van Leeuwen et al, in preparation; see also elsewhere in these proceedings). On the DEAE-Bio Gel column three acetyl esterase peaks were detected, based on the activity towards MHR (Fig. 5). The activity of these fractions, named AEI, II and III, was determined toward several acetylated substrates (Table 5). AEI showed a relatively high activity towards beet pectin, acetylated xylan and triacetin, and a relatively low activity towards MHR. Compared with AEI, AEII was very active towards MHR, and had a much lower activity towards the other substrates. For AEIII no clear substrate preference could be observed. Since it was only a minor fraction, no further purification of AEIII was attempted. Making use of the chromatographic methods mentioned above, two different acetyl esterases could be purified from AEI. These esterases were named PAE and FAE. A third esterase was purified from AEII and this esterase was named RGAE, since it appeared to be similar to the
238 the same source. This enzyme was named pnp-rhamnohydrolase [ 14]. Again high specificity of either enzyme for one type of substrate was observed (Table 3). Next to activity towards pnpct-rhamnoside, the pnp-rhamnohydrolase was also active towards naringin and hesperidin, but was not able to act on rhamnogalacturonan-like substrates in which the rhamnose unit is ct-1,41inked to an ct-GalA, such as in RG oligomers, RG polymers, MHR or MHR-S, in galactosylated or degalactosylated form. RG-rhamnohydrolase on the other hand was only active towards RG-like structures and showed no activity when other rhamnosides, as mentioned in Table 3, were used as substrates.
Table 3. Activity of pnp-rhamnohydrolase and RG-rhamnohydrolase towards various Rha-containing substrates. Substrate
Type of linkage involving Rha
pnp-Rha Naringin Hesperidin ot-Solanin a-Chaconin
ct- 1 c~-1,2 to B-Gluc a-l,6 to B-Gluc c~-1,2 to B-Gal ct- 1,2 and c~- 1,4 to B-Gluc
RG-oligo degal. RG oligo RG-hexamer degal. RG hexamer MHR HR-Saponified degal. MHR-S RG-poly degal. RG-poly
c~-1,4 to ct- alA
pnprhamnohydrolase (U/m~) 2.3 2.2 2.2 0 0
RGrhamnohydrolase (U/m~;) 0 0 0 0 0
0 0 0 0 0 0 0 0 0
3.2 32.9 6.1 52.6 2.5 12.9 57.6 3.0 24.3
3. THE ACETYLESTERASES FROM ASPERGILLUS NIGER From the previous paragraphs it is clear that the crude A. aculeatus enzyme preparation contains a whole set of enzymes able to degrade rhamnogalacturonans, including the acetyl esters present in this substrate as isolated from apple (MHR). Rhamnogalacturonan acetyl esterase (RGAE) is an important accessory enzyme for the RGases A and B ofA. aculeatus. A. niger, an other important production organism for pectolytic enzymes, was also investigated
239
Activity " Protein (E280) 1.50
NaCI
MHR
(M)
--e--
Acetylesterase 1.50
AEII
II
AEI
~j
towards
.....
m I
!
1.00
|
1
"~
I: i. I
I
~1 I,
P. il I! i \
0.50
_..=
:1 I; :1 I"
E i\
:1
I~
,
AEIII
:
--
n /I
..-
l_l
.-"
200
250
:
J
l -1 o.so
1
o.oo
"?
0.00 0
50
100
150
Fraction
300
number
Fig. 5. DEAE-chromatography of a desalted crude enzyme preparation from A. niger.
Table 5. Acetyl release (nmol/ml) from acetylated substrates by acetyl esterases at several stages of purification. Conditions protein concentration 2 lag/ml in 20 mM piperazine buffer pH 6.0, 20 h incubation. Solid and dotted lines indicate from which fraction the esterase has been purified. Substrate
Beet pectin
MHR
Acetyl xylan
Triacetin
Substrate concentration (%)
0.4
1.0
0.2
0.4
AEI AEII ................ AEIII PAE FAE RGAE .................. Total acetyl
398 146 195 443 0 6 1452
97 1800 100 267 0 1820 2598
489 169 238 177 590 0 1894
447 136 136 33 1963 0 55000
240 RGAE from A. aculeatus, based on molecular weight and high specificity for MHR (see elsewhere in these proceedings). The FAE was active towards acetyl xylan (with a low molecular weight) and triacetin. In an additional experiment it could be shown that FAE also released ferulic acid from an endoxylanase hydrolysate of sorghum cell walls. It was therefore named feruloyl acetyl esterase (FAE). The PAE was the only one of the three purified esterases from A. niger with activity towards beet pectin. In addition, some activity towards MHR and acetylated xylan was observed. From beet pectin only about 30% of the acetyl groups could be removed by PAE, while just like for the RGAE from A. aculeatus, about 70% of the acetyl esters in MHR were hydrolysed by A. niger RGAE. Fig. 6 shows a schematic structure of the rhamnogalacturonan part of MHR, in which the acetyl groups are located at the 0-3 as well as at the 0-2 and 0-3 positions of the galacturonic acid residue. This could be concluded from some preliminary NMR studies in which several structurally different acetyl esters could be assigned. After treatment with RGAE the overall pattern of the NMR spectrum remained unchanged, indicating that RGAE does not preferentially attack an acetyl ester at a specific position. This would include that all ester groups are equally degradable by RGAE. In beet pectin we are dealing with a homogalacturonan in which the acetyl groups are probably located at the 0-2 and/or the 0-3 positions (Fig. 6). From NMR spectra, essentially two different resonances from acetyl groups could be distinguished. Upon incubation with PAE one of these signals disappeared. It was concluded that only one type of acetyl groups could be removed by this esterase, probably either at the 0-2 or the 0-3 position and/or either present as a single branch point of a galacturonic acid residue or at a double branched galacturonic acid residue. Until now it is not clear how the acetyl esters are distributed over the different OH-groups of the galacturonic acid residues and which of the acetyl groups is being released by PAE. This is still under investigation. However, these results explain at least partially why PAE can only release 30% of the total acetyl groups in beet pectin, probably due to its high selectivity. This in contrary to the unselective action of RGAE towards MHR, resulting in a much higher degree of deacetylation (70%) upon extensive treatment with this enzyme.
241
RGAE: random removal of A c - g r o u p s
in MHR
( PAE: specific removal
of either 0 - 2 or 0 - 3 in beet pectin
B_Gale_(l_4)_ct_Rhae
linked A c - g r o u p s
~--.l
o~-GalAe-(1-2)-ot-Rhar
H
r
e
Fig. 6. Points of attack of RGAE on acetyl esters in H R (upper part) and possible points of attack of PAE on acetyl esters in beet pectin (see text for explanation).
4. ENZYMATIC DEGRADATION OF A SOLUBLE 'HAIRY' PECTIC POLYSACCHARIDE FROM SOY An area of application of pectinases, other than in fruits and vegetable processing, is the use in the production of pure protein from dehulled and defatted soy meal [ 15]. A soluble polysaccharide from soy (SPS) tends to bind to the soy proteins, complicating the purification of this protein. SPS was prepared from defatted soy meal by a protease treatment, after which the insoluble part was treated with a pectinase. The obtained product was again separated in a soluble and insoluble fraction, from which the soluble fraction was ultrafiltered. The obtained high molecular weight material was designated as SPS. In Table 6 the sugar composition of SPS is presented. The material contained a considerable amount of galacturonic acid, xylose, fucose, galactose and a small amount of rhamnose. From a methylation analysis after reduction, it was concluded that at least a considerable part of the galacturonic acid residues was branched, and that about 30% of the xylose residues were terminal. Also a considerable portion of the rhamnose and fucose residues appeared to be terminal. SPS could be degraded by the crude A. aculeatus preparation Pectinex Ultra-SP, giving a product consisting mostly of the monomeric sugars xylose, galactose, fucose, arabinose and
242 galacturonic acid. Next to these products also an unknown peak was observed upon HPAEC analysis (Fig. 7). This unknown product was isolated by preparative HPAEC and identified by NMR spectroscopy. It turned out to be a dimer of xylose 13-(1-3)-linked to galacturonic acid (Fig. 8). Tabel 6. Sugar composition and some structural aspects of SPS Mol %
Structural information (methylation analysis)
Ara Rha Xyl Gal
3 4 24 8
100% terminal 40% terminal 30% terminal Gal/GalA 40% 1,3,41inked
Man Fuc GalA
1
DM" DA b
11 49
72% terminal Gal/GalA 40% 1,3,41inked
21 7
a degree of methylation b degree of acetylation
The enzyme responsible for the production of this dimer from SPS was partially purified by a series of chromatographic steps, using the following columns: DEAE-Sepharose, cross-linked alginate, Q-Sepharose, Phenyl-Sepharose, Bio Gel HTP and Mono-Q. Two fractions were characterized further: the fraction after the HTP column (HTP2) and a purer fraction, obtained after the Mono-Q column (Q2). The HTP2 fraction produced no other products than galacturonic acid and the dimer 13-Xylp-(1,3)-GalAp (Fig. 7). Several electophoretic methods (SDS-PAGE, native PAGE and IEF) showed that fraction Q2 was almost pure, some faint contaminating protein bands were found (Fig. 9). The major band upon SDS-PAGE was at 42 kDa. IEF indicated an isoelectric point at pH 4.3 for the most prominent band. It was observed that the dimer 13-Xylp-(l,3)-GalA t, together with monomeric galacturonic acid, was not only released from SPS by the action of this enzyme, but also from MHR and gum tragacanth. The activity increased after alkaline saponification of the substrates. It is known that one of the subunits of apple H R is a xylogalacturonan, consisting of a galacturonan backbone, of which a part of the galacturonic acid residues is substituted at the 0-3 position with a 13-xylose [16]. The formation of the dimer 13-Xylp-(1,3)-GalAp confirms the presence, as well as the structure of this subunit in MHR.
243
IA
Dimer
(D
0
5
10
15 20 Time (min.)
25
30
Fig. 7. High-performance anion-exchange chromatography of the reaction products from alkali saponified SPS, produced by the crude A. aculeatus preparation (Ultra-SP) and the partially purified fraction HTP-2.
Ho~O~o
J---o o
H
Is'
I~-Xylp-(1-3)-GalAp(cdl~) Fig. 8. The structure of the unknown dimer, released from SPS by the crude A. aculeatus preparation or fractions thereof.
244 Based on HPSEC, it was observed that we are dealing with an enzyme that degrades xylogalacturonans in an exo-manner, releasing the dimer and galacturonic acid. Also beet pectin and polygalacturonic acid were degraded by this enzyme, giving galacturonic acid as the only product from the start of the reaction. It can be concluded that the enzyme is an exogalacturonase, which is not hindered by side-chains of xylose.
SDS-PAGE
kDa
Native
31 20.1 14
IEF
pH
kDa 699
-
440
-
8.157.35
-
6,85
-
140 -
5.85
-
5,20
-
67-
4.55
94
67 43
PAGE
232 -
4.55 --
..~:~:......
3.50 -
Fig. 9. Electrophoresis of the dimer releasing enzyme fraction Q2.
5. CONCLUSIONS As a very simplified model, we can consider the pectin molecule to be build up of a 'smooth' homogalacturonan part, next to a 'hairy' region containing subunits of a rhamnogalacturonan and, in the case of for instance apple, a xylogalacturonan [4, 16]. Much is already known about the wide range of enzymes able to degrade the homogalacturonan part of the pectin molecule. Here we show that in analogy to the degradation of the smooth homogalacturonan regions, a whole array of enzymes is present in A.spergillus preparations, specific for the degradation of hairy regions of pectin.
6. ACKNOWLEDGEMENTS The authors wish to thank Dr. Ian Colquhoun (AFRC, Norwich, England) and Dr. Joke Venekamp (TNO, Zeist, The Netherlands) for their NMR spectroscopic analysis, leading to the
245 primary structures of the oligosaccharides derived from MHR-S by the RGases and of the dimer 13-Xylp-(1,3)-GalAp, respectively. We thank Dr. Dick Schipper (Gist brocades, Delft, The Netherlands) for NMR spectroscopic analysis of the RGAE treated acetylated pectic substrates. Ir Jean-Paul Vincken (WAU, The Netherlands) is acknowledged for useful discussions.
7. R E F E R E N C E S
1. W. Pilnik and A.G.J. Voragen In: J.J.Jen (Ed.): Quality factors of fruits and vegetables. Chemistry and Technology. ACS American Chemical Society Symposium series 405 (1989) ' 250. 2. H.A. Schols, M.A. Posthumus and A.G.J. Voragen, Carbohydr Res 206 (1990) 117. 3. H.A. Schols, E. Vierhuis, E.J. Bakx and A.G.J. Voragen, Carbohydr Res 275 (1995) 343. 4. H.A.Schols, Structural characterization of pectic hairy regions isolated from apple cell walls, Thesis Wageningen Agricultural University (1995). 5. H.A. Schols, C.C.J.M. Geraeds, M.F. Searle -van Leeuwen, F.J.M. Kormelink and A.G.J. Voragen, Carbohydr Res 206 (1990) 105. 6. J. An, L. Zhang, M.A. O'Neill and P. Albersheim, Carbohydr Res 264 (1994) 83. 7. M. Sakamoto, Y. Shirane, I. Naribayashi, K. Kimura, N. Morishita, T. Sakamoto and T. sakai, Eur. J. Biochem 226 (1994) 285. 8. L.V. Kofod, S. Kauppinen, S. Christgau, L.N. Andersen, H.P Heldt-Hansen, K D6rreich and H. Dalboge, J Biol Chem 268 (1994) 29182. 9. M.J.F. Searle-van Leeuwen, L.A.M. Van den Broek, H.A. Schols, G. Beldman and A.G.J Voragen, Appl Microbiol Biotechnol 38 (1992) 347. 10 H.A. Schols, A.G.J. Voragen and I.J. Colquhoun, Carbohydr Res 256 (1994) 97. 11. M. Mutter, I.J. Colquhoun, H.A. Schols, G. Beldman, and A.G.J. Voragen, Plant Physiol 110 (1996) 73. 12. C.M.G.C. Renard, M.Mutter, H.A. Schols, A.G.J.Voragen and J-F. Thibault, Int. J Biol Macromol (in press). 13. J.W. Van de Vis, Characterization and mode of action of enzymes degrading galactan structures of arabinogalactans, Thesis Wageningen Agricultural University (1994). 14. M. Mutter, G. Beldman, H.A. Schols and A.G.J. Voragen, Plant Physiol 106 (1994) 241. 15. H. Gurtler, H.A.S. Olsen, M Schulein, J.L. Adler-Nissen, G.W. Jensen and S. Rijsgaard, UK Patent Application GB 2115820. 16. H.A. Schols, E.J. Bakx, D. Schipper and A.G.J. Voragen, Carbohydr Res 279 (1996) 265.
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J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All fights reserved.
247
The flSubunit of Tomato Fruit Polygalacturonase Isoenzyme 1 Defines a New Class of Plant Cell Proteins Involved in Pectin Metabolism: AroGPs (Aromatic Amino Acid Rich Glyco Proteins) Dean DellaPenna a, Colin Watson, JiPing Liu and David Schuchman Department of Plant Sciences, University of Arizona, Tucson, Arizona, USA, 85721 aAs of March 1, 1996: Department of Biochemistry/200, University of Nevada, Reno, Nevada, USA, 89557-0014
Abstract Understanding the biochemical and molecular mechanisms governing the synthesis, regulation, structure and function of plant-encoded cell wall modifying enzymes, particularly those involved in pectin metabolism, has been a long term goal of research in my laboratory. The ripening tomato fruit has proven to be a highly tractable system for this work and several laboratories have focused on studying a single, ripening-induced pectin degrading enzyme, tomato fruit polygalacturonase or PG. This chapter first reviews the field of tomato fruit PG and then focuses on our recent work with the 13subunit of tomato PG isoenzyme 1. Two PG isozymes can be isolated from ripe fruit, PG2 and PG1, the latter of which is formed by the association of a catalytic PG2 polypeptide with an ancillary glycoprotein, the l~subunit. Multiple lines of evidence suggesting the 13subunit plays an important role in regulating polyuronide degradation have prompted us to clone the 13subunit and study its structure, expression and function. 13subunitmRNA accumulates to high levels in developing fruit and is to a large degree temporally separated from PG2 expression. The 13subunit is encoded as a large precursor protein whose mature domain is composed almost entirely of the novel repeating motif FTNYGxxGNGGxxx in which the phenylalanine residues are post-translationally modified. Results from Bsubunit antisense experiments have conclusively demonstrated that the protein plays a major role in restricting PG2 catalytic activity in vivo during fruit ripening. We have isolated the complete tomato 13subunit gene family, related genes from Arabidopsis and identified homologous sequences in a number of dicots and monocots, suggesting that Bsubunit structure and function may be evolutionarily conserved in plants. We propose the tomato 13 subunit as the archetypal member of a new class of plant cell wall proteins, AroGPs (for Aromatic Amino Acid Rich GlycoProteins). AroGPs are defined by: 1) their overall protein sequence homology and common precursor structure, 2) a conserved 14 amino acid repeating motif in the mature AroGP protein and 3) a high percentage of aromatic amino acids in the mature AroGP protein. Based on the available data, AroGPs appear to be specifically expressed in PG-producing tissues, where they presumably interact with cell wall components and/or catalytic enzymes to regulate cell wall enzymatic activities in vivo.
1. B A C K G R O U N D 1.1. Pectin and Cell Wall Structure Plant cell walls provide the obvious functions of structural support and integrity and can vary tremendously in size, shape, composition and structure depending on cell type, age and function within the plant body. Despite this diversity, plant cell walls are composed of only three major classes of polysaccharides: cellulose, hemicellulose and pectins. Pectins, or polyuronides, are imbedded throughout the cell wall matrix and are particularly abundant in the middle lamella region. Pectins generally account for 10-30% of the cell wall dry weight and
248 collectively constitute a complex group of polysaccharides whose primary component is dgalacturonic acid with a number of other sugars being present as minor constituents. Polyuronides, especially those of the primary plant cell wall, have been the focus of extensive structural analyses in recent years which has provided a detailed characterization of the linkages and composition of "smooth and hairy" pectin blocks, both of which can be present in a single pectin polymer. Such studies have greatly increased our understanding of structural aspects of pectins and their role in cell wall architecture (reviewed in 4).
1.2. Pectin modifications during growth and development Although polyuronides are clearly important structural components of the cell wall they are also a dynamic class of molecules that are constantly being modified during the plant life cycle and are intimately involved in many aspects of plant growth and development. The principal plant enzymes mediating polyuronide modifications are pectin methylesterases, which remove methyl ester groups from the polymer, and polygalacturonases (PGases), which cleave the polymer chain in either an endo or exo fashion. During the past several years efforts by many laboratories in studying plant mediated polyuronide degradation have focused on the structure/regulation/function of a single catalytic PGase activity produced in large amounts during tomato fruit ripening. This system has been targeted due to the large tissue mass, high levels of developmentally regulated PGase activity and extensive physiological and biochemical background. These studies have greatly increased our understanding of the molecular regulation of pectin modification during fruit ripening and led to molecular genetic experiments to test the function of PGase in fruit ripening (reviewed in 8 and 10). However, developmentally-regulated, tissue specific polyuronide modifications by endogenous plant enzymes occur throughout the plant body and the analysis of PGase activity and function in non-fruit tissues is now being critically addressed. In recent years a substantial number of reports have described developmentally regulated, tissue specific PGase activities or cDNAs (many of which show significant homology to tomato fruit PG) in a variety of non-fruit tissues including leaf abscission zones, root cap border cells, newly initiated lateral roots and developing pollen and growing pollen tubes (1, 3, 11, 13, 21, 23). Particularly notable is the Sambucus nigra abscission zone in which two isozymes were identified and characterized that are analogous in their size, structure and sequential appearance to PG isozymes in tomato fruit (see below). These combined reports clearly demonstrate that developmentally regulated, tissue specific, pectin modifying activities/genes are expressed in a variety of cell and tissue types where extensive modification of cell wall structure or loss of cell wall adhesion occurs and suggest a broad role for pectin degrading enzymes in plant growth and development. Furthermore, the fact many non-fruit PG cDNAs show significant homology to tomato fruit PGase suggests that non-fruit PGase activities may be regulated in a fashion similar to tomato fruit PGase. Ongoing work with tomato fruit PGase may therefore provide insight into the regulation/function of PGase activities in other plant tissues.
1.3. PG and pectin degradation during tomato fruit ripening During the ripening of climacteric fruits such as tomato, an increase in the level of chelator-soluble polyuronides, and a corresponding decrease in their molecular size is well correlated with a dramatic increase in extractable endo-PGase activity. The catalytic PG protein responsible for these changes is probably the best characterized pectin degrading enzyme in plants and a variety of molecular and biochemical approaches have conclusively demonstrated that it is indeed the primary enzymic activity responsible for cell wall polyuronide degradation in ripening tomato fruit (10, 22). The catalytic PG polypeptide is encoded by a single gene (the PG gene) whose expression and function have also been extensively studied in recent years (reviewed in 11). The PG gene is transcriptionally activated at the onset of ripening and both PG mRNA and protein accumulate to high levels during the ripening process (7, 8, 11). Although there is only a single gene for the catalytic PG polypeptide, the total PG activity
249
A
B
1
2
1
3 STD ..
~..
PG2~
kD
~. ,,.~
~
-66
~-45
13-Subr ~
9. . . . . . o :
Figure 1. SDS-PAGE and Immunoblot Analysis of Purified PG1 and Separated PG2 and ~Subunit Proteins. (A) Proteins were resolved by SDSPAGE and visualized by Coomassie blue staining. Lane 1, 4 ~tg purified PG1; Lane 2, 2 ~tg purified PG2; Lane 3, 2 ~tg purified 13subunit.
'~,-31 (B) Proteins were resolved by SDSPAGE, blotted and PG2 polypeptides detected by reaction with anti-PG2 antibodies. Lane 1, 2 lxg purified PG1; Lane 2, 1 lxg purified PG2; Lane 3, 1 lxg purified fisubunit. isolated from ripe tomato fruit is attributable to a mixture of several closely related, posttranslationally derived isoenzymes, PG1, PG2A and PG2B (2, 6). The PG2A and PG2B isoenzymes (herein referred to as the PG2 isoenzyme) accumulate late in the ripening process and are each composed of a single catalytic PG polypeptide differing only in degree of glycosylation (2, 6). The PG 1 isoenzyme accumulates first during ripening and is thought to be composed of one or two catalytic PG2 polypeptides tightly associated with an ancillary glycoprotein, the 13subunit protein (13). The level of PG1 produced during ripening is apparently determined by the level of 13subunit protein present in the fruit tissue (8, 10, 17).
1.4. The [~subunit modifies the activity of the catalytic PG protein The [~subunit has been purified from PG 1 by ourselves and others and is a heat stable, acidic, heavily glycosylated protein with an apparent molecular mass of 37-39 kD (19, 26). No enzymatic activity has been identified for the protein. The 13subunit can be extracted from the cell walls of both green and ripe tomato fruit by high salt buffers (13, 14, 18, 19, 20), and in the latter case is associated with PG2 polypeptide(s) in the form of PG 1. Purified 15subunit can also associate with and convert PG2 in vitro into an isoenzyme that closely resembles PG 1 ( 13, 14, 24). Biochemical studies have shown that in vivo and in vitro formation of PG1 by the association of PG2 with the 13-subunit alters the biochemical and enzymic properties of the associated catalytic PG2 polypeptide including its pH optima, response to cations and thermal stability (summarized in Table 1). This later property provides a convenient assay for the levels of PG 1 and PG2 in total cell wall protein extracts.
Table 1: Selected biochemical properties of PG2 and PGI Isozyme
Mol. wt. *50% * 100% pH NaC1 kD inactive inactive optima pI Optima PG 1 --100 79~ 90~ 3.6 7.0 300 mM PG2 45/46 57oC 63oC 4.4 8.0 200 mM 9Heat inactivations are 5 min at the given temperature. Data are from Knegt et al. 1988.
........
250
Figure 2: EDTA soluble polyuronides, PG1/PG2 levels and size fractionation profiles of polyuronides isolated from ripening wild-type fruit. 0, 7 and 20 days after harvest are mature green, mid-ripe and over-ripe stages, respectively.
80 ~
60
~ ~40 2O
Upper panel: EDTA soluble polyuronides were extracted from identically treated tissues and measured. Note the increase in polyuronide solubility during ripening.
0
PG1-
Middle panel: Cell wall proteins were isolated, 10 ggm of each resolved by non-denaturing polyacrylamide gel electrophoresis and PG1 and PG2 isoforms detected by activity staining.
PG20
~
8
~4
_~ ,
3 7 11 20 Days After Harvest
Wild-typePolyuronideSizeProfile ~/,,0Days ~ Dyi7s 2~Days
g2
20
40 60 FractionNumber
80
Lower panel: One mg of chelatorsoluble polyuronides from the indicated stages was size-fractionated on a Sepharose CL4B column, and the uronic acid content of column fractions determined. 0, 7 and 20 day old fruit contain no PG, PG1 only and PG1 and PG2, respectively. Note the similarity of 7 and 20 day profiles despite the large amount of PG2 activity in the latter.
1.5. Several lines of data suggests that the Bsubunit regulates PG activity Several independent lines of physiological, biochemical, and molecular evidence have been put forth to support the hypothesis that PG1 is the active isoenzyme in vivo and have implicated the gsubunit as playing an important role in regulating pectin metabolism. These include the observation that only PG 1 can be extracted when maximal pectin solubilization and depolymerization are observed in both wild-type fruit and tin fruit expressing an inducible PG2 transgene (7). Subsequent accumulation of high levels of extractable PG2 activity in both tissues is not accompanied by further pectin solubilization or depolymerization (9). Furthermore, the in vivo biphasic loss of PG activity during heat treatment of intact fruit tissue mimics the in vitro heat inactivation profile of mixtures of PG1 and PG2 isoenzymes, suggesting that the PG1 complex exists in vivo (17). Finally, analysis of transgenic tomato plants constitutively expressing an antisense PG2 transgene has shown that the residual 1% PG enzyme activity extracted from ripe fruit is exclusively in the form of PG 1 and is sufficient for wild-type levels of pectin solubilization to occur during ripening. Based on this result, it was
251 proposed that only very low levels of PG 1 were required for normal pectin solubilization (22). These combined studies suggest that the presence of extractable PG 1 activity is correlated with pectin solubilization and in some cases with pectin depolymerization during tomato fruit ripening and therefore implicate the Bsubunit protein as an important factor in regulating or restricting the catalytic PG2 protein in vivo (7, 9, 13, 14). However, the data supporting this thesis is largely correlative in nature and definitive proof is lacking regarding the in vivo existence of PG 1, the contribution of the various isoenzymes to ripening-associated polyuronide degradation, and the physiological consequences of PG-dependent polyuronide degradation. 1.6. Conclusions and Directions from Prior Work on Tomato Fruit PG Despite the brevity of the above discussion, it is clear from previous studies that the large increase in PG activity during ripening is the primary enzymatic activity responsible for polyuronide degradation during tomato fruit ripening. Results in transgenic systems have suggested that a single isozyme, PG1, may be the physiologically active PG isoform in vivo. As PG1 is a complex of PG2 and the [~subunit, these data implicate the [~subunit protein as playing an important function in regulating pectin degrading enzyme(s) and hence in the process of pectin solubilization and depolymerization. A clearer molecular and biochemical understanding of the fruit ~subunit, its role in determining PG isoform levels, and the role of related proteins in other non-fruit tissues that contain PGase activities was an obvious and necessary next step in furthering our knowledge of PG function and polyuronide modifications during fruit ripening. To extend our understanding of the assembly, activity, and physiological function of the individual PG isoforms during tomato fruit ripening, and directly address the role of the Bsubunit in pectin metabolism we purified the [~subunit from PG 1 and isolated eDNA clones that encode the protein (26). The remainder of this chapter reviews our progress to date in studying the structure, expression and function of the 13subunit (25, 26, 27). Finally, we describe additional members of the tomato 13subunit gene family, related genes in other plant species and propose that the 13subunit defines a new class of cell wall proteins which we call AroGPs for Aromatic Amino Acid Rich G__lycoProteins_. 2. ISOLATION AND C H A R A C T E R I Z A T I O N OF A BSUBUNIT cDNA CLONE 2.1. Purification of the [3subunit Protein of Tomato Fruit PG1 When analyzed by SDS-PAGE, purified PG 1 was found to contain several polypeptides ranging in size from 37 to 45 kD, as shown in Figure 1A, lane 1. The 44 and 45 kD polypeptides reacted strongly with antiserum raised against purified PG2 (Figure 1B, lanes 1 and 2), and their size and immunoreactivity are consistent with their being the catalytic PG2 polypeptides. Using cation exchange chromatography in urea containing buffers, proteins in the purified PG1 sample could be further separated into two species: the PG2 polypeptides and the ~subunit polypeptide(s) (Figure 1A, lanes 2 and 3, respectively). The [~subunit protein does not react with anti-PG2 antibodies (Figure 1B, lane 3), indicating that it is immunologically distinct from the catalytic PG2 polypeptides. Protein sequencing of purified [3subunit and of several proteolytically derived ~subunit peptide fragments yielded the sequences shown in Table 2. Blank cycles were consistently obtained at specific cycles for each peptide which, in retrospect, invariably correspond to phenylalanine residues in the eDNA sequence for the protein (refer to Table 2 and bold Phe residues in Figure 3). Moreover, amino acid composition analysis of purified ~subunit protein agrees with that deduced from the ~subunit eDNA with the exception of a severe underestimate of the phenylalanine content of the mature protein (3 residues versus 23 from eDNA sequence). These data indicate that most of the phenylalanine residues in the mature ~subunit protein are posttranslationally modified in an as yet undetermined fashion.
252
Table 2. Summary of Protein Sequence Data from the Amino Terminus and Internal Proteolytic Fragments of Purified Tomato Fruit ~-Subunit. Peptide Name
Peptide Sequence
Amino terminus
NH 2- Glu-Lys-His-Ser-Gly-Asp-Ile-His-[ .9 ]-Ala-Thr-Tyr
Lys-C
NH 2- Asn-Gly-Asn-Gly-Ala-Asn-Gly-Gln-[ ? ]-Val
Glu-C- 1
NH 2- Ala-Asn-Ala-Gly-Asp-Gln-Tyr-[ ? ]
Glu-C-2
NH 2- Asn-His-[ ? ]
Arg-C
NH 2- Gly-Ser-Pro-Arg-Asp-Asn-Lys-[ ? ]-Asp-Asn-Tyr-Ala
Underlined sequences indicate amino acid sequences used for the generation of degenerate primers. Bracketed question marks represent blank cycles from the Edman degradation reaction. Additional sequence was obtained after blank cycles in all cases except the Glu-C-1 and Glu-C-2 peptides.
2.2 Cloning of a Fruit BSubunit cDNA and Analysis its Primary Sequence Purification and characterization of the [~subunit permitted the subsequent isolation of eDNA clones encoding the protein. Using oligonucleotides derived from the data in Table 2, numerous [~subunit eDNA clones were isolated and characterized from a green fruit eDNA library, pBsub2.2, the longest eDNA clone encodes a 69-kD protein containing 630 amino acids, more than twice that required to encode the mature gsubunit protein. Included in this open reading frame are all of the protein sequences shown in Table 2, confirming that the eDNA encodes the ~subunit protein. A hydropathy profile of the encoded 69-kD precursor protein is shown in Figure 3. The ~subunit precursor contains at least four distinct protein domains: a 30 a.a. amino-terminal signal sequence, a 78 a.a. amino-terminal propeptide, the mature protein domain, and a large (25 kD) carboxyl-terminal propeptide domain. The presence of a signal sequence is consistent with the protein being targeted to the endomembrane system, as would be expected for a cell wall protein. The predicted 31.5 kD unglycosylated mature domain has a calculated pI of 4.9 and contains all six identified N-linked glycosylation consensus sequences (Figure 3, branched structures), consistent with the reported heavy glycosylation of the mature ~subunit protein. The most striking structural feature of the primary sequence of the mature ~subunit protein domain is the presence of a novel repeating 14-amino acid motif with the general consensus: FTxYGxxxN(x)4_6, where the Phe and Tyr residues are invariable and Thr, Gly and Asn occur with a greater than 50% frequency at positions 2, 5, and 9, respectively. Within the core consensus region there is a strong bias for specific amino acids or amino acid group at certain "x" positions in the motif, such as Ser, Thr, or Asn at position 3, Gly at positions 8, 10, and 11, and charged or uncharged polar amino acids at positions 6, 7, 12, 13, and 14. Inclusion of these residues yields a core consensus of FTNYGxxGNGGxxx where "x" is most often a charged or uncharged polar amino acid. This motif accounts for almost the entire mature protein domain (Figure 3, boxed region) and is not found in other domains of the precursor protein. The five posttranslationally-modified phenylalanine residues discussed earlier are all located in position 1 of this motif (bold phenylalanine residues in Figure 3).
253
5-
~o' I~ "- "31 r
u., l / ,,,"~'~
"5 - i f ' / " ' / * -'~ 100
~,~t,r YY
....
' "l
~,a.latai
I
' ' 1'
Y Y YY Matu.re~-subunitprotein ~ 200 300 400 A m i n o Acid N u m b e r
i ,L~~,1 ,,,,
q,r * ~r","~T,'I ..........
,I
{ ~ } ] 500
600
Mature [$-Subunit Protein Domain Sequence .M2-EKH S ~ D
IH GVNT VNS NK DQS SGK NLH VQK DQY NGE GST DQK ENH SET DDT EAN TDV HIN
L M-cooH Core Consensus
FTNYGxxGNGGxxx
LAS
Figure 3. Hydropathy Profile and mature protein primary sequence of the deduced 13Subunit Precursor Protein. 9Upper panel: The hydropathy profile of the entire 69 kD precursor protein is shown. The abscissa is amino acid residues and the ordinate, positive values indicate hydrophilic. The black and hatched rectangles at the bottom of the figure denote the calculated signal sequence and amino-terminal propeptide domains, respectively. The mature and carboxyl-terminal domains are labeled. N-linked core glycosylation consensus sites are depicted by branched structures. Lower panel: Optimal alignment and consensus sequence of the mature [~-subunit protein repeating motif. The entire contiguous sequence of the mature domain (amino acids 109-397) is shown from top to bottom and left to right. Periods represent gaps in the alignment. Shadowed residues indicate amino acids that occur with a high frequency at a given position. Bold phenylalanine (F) residues are those which yielded blank cycles in protein sequence analysis. The boxed area defines the region of the protein from which the core consensus, FTNYGxxGNGGxxx was derived. The bold amino acids of the core consensus occur with a 50% or greater frequency.
2.3. [3Subunit and PG Expression Are Separated during Fruit Development In addition to structural studies, we have analyzed the expression patterns of ~subunit and PG mRNA during wild-type tomato fruit development (Figure 4). ~subunit and PG differ dramatically in their temporal regulation during wild-type fruit development. [~subunit mRNA is detectable as early as 10 DAP and increases gradually during development to its highest level at 30 DAP, just prior to ripening. During the following 5-day period, ripening is initiated and [~subunit mRNA decreases below detectable limits, while PG mRNA increases to its highest level. A more detailed analysis of the ripening period, using ethylene production as a marker, indicated that ~subunit and PG expression do overlap slightly early in ripening, however, PG protein is not detected until [~subunit mRNA levels decrease (27, results not shown). One likely explanation for the near complete temporal separation of 13subunit and PG expression is that ~subunit expression occurs early in fruit development to allow transport, attachment and localization of the 13subunit protein to specific regions of the cell wall in the absence of PG2.
254
~o~| 9
13-Sub-
...
PG-
.
~.
o
.
!i iI
(2.3 kb)
(1.9 kb)
,,o~~ Fruit(DAP) 0 15 20 25 30 35 40 ..
.
mW "
~
"
,
m
~.~.: :o:.7
~~:~
9
:~i~i ~(~
:9 9149 ~99149 i9~9149149
I:)21 -
(1.0 kb)
Figure 4. RNA Blot Analysis of [~Subunit, PG, and D21 Expression during Fruit Development and Ripening. Total RNA (25 ~tg) isolated from the indicated tomato tissues was probed with either a 13subunit eDNA clone, a cDNA for the catalytic PG polypeptide, or a eDNA for the constitutively expressed mRNA D21. Identical specific activities were used in each hybridization and all blots were exposed for 8 hr.
2.4. Analysis of BSubunit Function Using Transgenic Systems Despite extensive prior studies of the structure, regulation, and function of the catalytic PG protein during tomato fruit ripening, it is still unclear if PG 1 isoenzyme formation occurs in vivo or whether PG1 and/or PG2 alone is sufficient for pectin solubilization and dcpolymerization in vivo. To test the hypothesis that the B subunit is involved in pectin depolymcrization or solubilization in vivo and that the formation of PG1 is required for PG activity in vivo, several independently transformed tomato 13subunit antisense lines with substantially reduced levels of B subunit protein in mature green fruit were generated. Total cell wall proteins isolated from several independently transformed lines were assayed to determine their immunologically detectable levels of Bsubunit and PG proteins as well as the extractable PG 1 isoenzyme activity. Those lines with less than 1% of their total PG activity as PG 1 in ripe fruit were analyzed in greater detail with respect to pectin solubilization and depolymerization during fruit ripening. The antisense line TA8, in which <1% PG1 was detected, contains two copies of the antisense transgene, which segregate as a single locus (102 kanamycin resistant [Kan r] to 33 kanamycin sensitive [KanS]) Due to these combined attributes TA8 was selected for further analysis. 2.5. E x t r a c t a b l e PG Isoenzyme Levels During Control 1 and TA8 Ripening. Total cell wall proteins isolated from developmental stages MG (42 DAP) through Br+ 10 fruit (approximately 53 DAP) were assayed by differential heat inactivation to determine the relative levels of PG1 and PG2 in control fruit and TA8 fruit during ripening. Figure 5A shows that PG1 activity in control 1 fruit was first detectable at the Br+2 stage where it constituted 80% of the total PG activity. A s ripening progressed in control 1 fruit, PG 1 activity
255 increased very slightly, whereas PG2 activity increased almost five-fold. In contrast, Figure 5B shows that very little PG1 activity was detected in TA8 fruit at any ripening stage and that overall PG activity was not significantly different from control fruit. Figure 5C shows the developmental accumulation of lycopene during ripening of control 1 and TA8 fruit was almost identical and indicates that the overall ripening process in TA8 was not adversely affected by the down regulation of 13subunit expression. Similarly, plants expressing an antisense PG2 transgene and accumulating less than 1% of normal PG levels have also been shown to ripen normally as determined by lycopene accumulation and ethylene production rates (22).
A e~
B
Control 1
O
,,8
4
4 Total A " " ---o-- PG2 ---o-- PG1
Total Activity --..o-- PG2 /
/
r
-o ~.2 ~E E 1 1-
-
&
n
MG
m
M
l
U
n
i
n
i
Br +2 +5 +7 Developmental Stage
C 100
~. 80 ~60
m
~
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+10
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. . ~. Br
=
.
+2
.
e
+5
-
e
+7
9
Developmental Stage
-
e
+I0
LvcoDene Production --o-- Control1 -.--B-- TA8
(9 r
O
"20 9
MG
,
9
i
i
i
i
i
Br +2 +5 +7 Developmental Stage
i
,
+10
Figure 5. Polygalacturonase Isoenzyme Activities and Lycopene Production During Control 1 and TA8 Fruit Ripening. (A) and (B) Total cell wall proteins were isolated from tissues of control 1 in (A) and TA8 in (B) at the indicated stages, and the PG1, PG2, and total PG activities were determined by differential heat inactivation. Each time point is the average of at least two separate extractions assayed in duplicate. The developmental stages are MG, mature green; Br, breaker stage, (time of first external color development) and +2, +5, +7, and + 10 are days after breaker. C) Lycopene production during ripening of control 1 and TA8 fruit.
256
17 Control 1
t~
o 120 E
IITA8
.
Figure 6. EDTA-Soluble Polyuronide Levels in Control 1 and TA8 fruit During Ripening. Enzymatically inactive cell walls were isolated from pericarp tissue at the indicated time points for both control 1 and TA8 and the level of EDTA-soluble polyuronides was determined. Bars indicate the standard deviation from the mean. Abbreviations for developmental stages are as given in Figure 5.
if) "0
9 80
C 0
-Q 40 0
t-121
w
0
MG
Br +2 +5 +7 +10 Developmental Stage
t/} I1)
-'gt- 150
w
O
=3 --& a~ 1 0 0
"~15 o
O
Or)
4:50 I--.
0
TA8-23
~c 5
TA8-48
o t_
~ I1.
0
10
15 20 25 Fraction Number
30
Figure 7. EDTA-Soluble P o l y u r o n i d e s and Gel Filtration Analysis of EDTASoluble Polyuronides Isolated from TA8-23 and TA8-48 Br+7 Fruits. The panel inset shows the levels of EDTAsoluble polyuronides isolated from fruit cell walls of TA8-23 ("wild-type" sibling) and TA848 (homozygous for antisense gene) at the Br+7 ripening stage. Bars indicate the standard deviation from the mean. The graph shows the chromatographic profile of 1 mg of EDTAsoluble polyuronides isolated from TA8-23 and TA8-48 resolved on a Sepharose CL4B column.
2.6. Polyuronide Solubilization and Depolymerization in Control and Antisense Fruit To address whether the 6subunit regulates or restricts the action of PG in solubilizing or depolymerizing pectin, or whether modifying Bsubunit expression affects pectin chemistry at all, the physical characteristics of EDTA-soluble cell wall polyuronides were analyzed from transgenic and control fruit tissues. Figure 6 illustrates the solubilization of polyuronides as ripening progresses in TA8 and control 1 fruit. Though the pattern of polyuronide solubility during ripening of TA8 was similar to control 1, the absolute amount of polyuronides extracted from TA8 cell walls throughout ripening was significantly higher as compared to control 1. Similar increases were observed in multiple independent transformants (results not shown). These results indicate that in wild-type fruit, the 6subunit plays an important role in limiting the amount of cell wall pectins that are solubilized during ripening. In addition to increasing pectin
257 solubilization, inhibition of 13 subunit expression in antisense lines was associated with a statistically significant increase in the depolymerization of EDTA-soluble polyuronides at later ripening stages (results not shown). Increased depolymerization of EDTA-soluble polyuronides was also observed in multiple independently derived antisense lines (data not shown) and suggests that in addition to restricting pectin solubilization, the 13subunit also plays a role in limiting pectin depolymerization during wild-type fruit ripening. In the 13subunit antisense line studied in greatest detail (TA8), both phenotypes (increased polyuronide solubility and increased depolymerization) cosegregated with the antisense 13subunit transgene in T2 progeny plants (Figure 7). These results clearly demonstrate that elevated levels of EDTA-soluble polyuronides during ripening and the increased depolymerization of these polyuronides later in ripening are specific, genetically transmittable phenotypes associated with reduced 13subunit protein levels brought about by antisense inhibition of 13 subunit expression during fruit development. The reader is referred to reference 25 for a more complete discussion of these experiments.
Model for BSubunit Action P rote ct i o n/R estri cti on
BSubunit O PG2 1
O 1 PG1 Activity
Figure 8. Model for flsubunit mediated restriction of P G 2 activity. In the model shown, Bsubunit interacts with pectic polymers and/or other cell wall components and therby sterically access of PG2 from those regions the polymer (as indicated by grey circles surrounding the 13subunit) PG2 can only act on susceptible sites in the pectic polymer not occupied by Bsubunit. Note that PG 1 formed in this model by association of PG2 and Bsubunit is inactive.
I Pectic
Polymer
2.7. Conclusions From Antisense Studies The data from 13subunit antisense lines further defines Bsubunit function in vivo by showing that the presence of the 13subunit protein in the cell wall during ripening limits, but is not required for, PG2 activity in vivo (refer to Figure 8). These data are most consistent with 1) the 13subunit protecting or limiting access of the catalytic PG protein to susceptible pectin sites, and 2) PG2 alone being responsible for pectin solubilization and depolymerization in vivo. Two mechanisms for limiting PG2 activity can be proposed that are not necessarily mutually exclusive. An indirect mechanism where interaction of the 13subunit with pectin would exclude PG2 from those regions of its pectic substrate or a direct mechanism where interaction of Bsubunit with PG2 would restrict PG2 mobility to other areas of the cell wall. Bearing in mind the strong affinity of PG2 for the 13subunit in vitro, we would predict that the latter mechanism would be irreversible in vivo. These experiments have not excluded the possibility of PG1 formation in vivo, but suggest that if formed such a complex would not possess catalytic activity as judged by pectin solubilization and depolymerization. It is important to stress that the formation of such an inactive complex may still represent an important control point in the regulation of pectin metabolism.
258 3. BSUBUNITS P R O T E I N S IN O T H E R TISSUES AND BSUBUNIT GENES IN O T H E R PLANTS
3.1. [~-subunit and PGase Expression in Non-Fruit Tissues of Tomato After a six day exposure (versus 8 hours for fruit tissues), ~subunit mRNA could be detected in northern analysis wild-type of root, leaf and flower tissues (results not shown). Though it was expressed at much lower levels, the non-fruit 13subunit rnRNA was identical in size to that in fruit. Immunoblot analysis also indicated a protein identical in size to the fruit 13subunit was present in cell wall protein extracts of roots, leaves and flowers (results not shown). The amount of protein detected was highest in floral tissue (-10% of fruit levels), while root tissue contained the lowest amount. Developmental studies of floral tissues indicated 13subunit antigen was present at all stages (Figure 9). A second larger protein was also consistently detected at the 5mm bud stage and beyond and decreased in size transiently during pollination (flower 2 stage). Surprisingly, western analysis of the same extracts with tomato fruit PG2 antibody detected a protein that was identical in size to tomato fruit PG2 and specifically expressed at the 5 mm and flower 1 stage, just prior to pollination and the decrease in size of the larger ~subunit antigen. The natrue of this PG2 antigen is being further investigated but the expression of proteins immunologically related to fruit PG2 and ~subunit in floral tissues suggests that an association of catalytic PGases and "~subunit-like" proteins may also occur in this tissue.
Bud Length(mm) "~2 ~.
i.
4
3 ..
.
..:
PG2 m
Flower Stage
5 9
1
..~".
9
..
3
4
5
:;:....~,,~::~:~....i.:.:.i:
......~
~~~
2
..
,.
~ 0
:.
:
.
..
9
~:
Figure 9. Western analysis of 13subunit and PG2 antigen levels during tomato fl o w e r development. Cell wall proteins from developing flowers were analyzed for 13subunit and PG2 antigens. Bud length is in millimeters. Flower stage: 1, green, fully developed; 2, yellow and opening; 3, fully opened; 4, petals folded forward; 5, petals faded. The larger antigen is marked by the arrow, an asterisk marks transient size decrease at pollination.
Figure 10. Tissue specific expression of ~-subunit proteins in floral tissues. Stage 3 (fully opened) flowers were collected, dissected and cell wall proteins (5 ~tgm) from the indicated organs isolated and analyzed for ~-subunit antigen. Note the high level of expression in stigma/style and anthers/pollen and restriction of the larger antigen to stigma/style tissues. PG1 lane, 1 ~tg of purified fruit PG 1 protein.
259 We analyzed floral tissues in more detail to determine if ~subunit expression was constitutive or tissue specific. The former result would imply a structural role for the protein while the latter might support a role in regulating or targeting PG or other cell wall activities in this non-fruit tissue. Western analysis of dissected flower parts demonstrated that expression of the immunologically detectable ~subunit antigens are largely restricted to stigma/style tissue and to a lesser extent anthers/pollen (Figure 10). The absence of gsubunit from ovary tissue is particularly noteworthy as the fertilized ovary gives rise to the fruit, a tissue where the fruit [~subunit is highly expressed in a developmentally regulated fashion. Stigma/style tissue exclusively contain the larger [~-subunit antigen at a very high level, while ~subunit protein similar in size to that in fruit are produced at lower levels in anthers/pollen. In fruit tissue [~subunit and PG2 expression are temporally separated during development. The results in Figures 9 and 10 suggest a similar situation may occur in floral tissues where PGases are largely restricted to pollen (1, 3) while production of "[~subunit-like" proteins occurs at its highest level in stigma/style tissues. Such data argues further for an analogous functional role for "[~subunit-like" proteins in regulating or targeting PG activity in floral tissues, potentially to activate or target pollen produced PGases during growth of the pollen tube through the transmitting tissue of the style. Developmental, spatial, structural and functional characterization of ~subunit expression in floral and vegetative tissues is ongoing and may yield insight into the role of 13subunit like proteins in non-fruit tissues such as flowers.
3.2 Isolation and Analysis of Multiple Tomato [~subunit Genomic Clones and Related cDNAs from Arabidopsis. DNA gel blot analysis of tomato genomic DNA digested with a variety of enzymes and probed with the full length [~subunit eDNA insert showed a relatively simple banding pattern. All genomic fragments cosegregate and map to a single locus at the top of chromosome 5 (< 1 centiMorgan resolution). These results suggest that the [~subunit is encoded by a single gene or small number of tightly linked genes. In order to gain further information about the ~subunit gene, its promoter and any related genes in tomato, we isolated and analyzed four gsubunit genomic clones. Southern analysis (data not shown) of the genomic clones using fruit [~subunit eDNA subfragments and oligonucleotides indicates that they can be classified into two groups. The first group encodes the entire fruit gsubunit gene (tomato gene 1 in Figure 11) and several kb of 5' and 3' flanking region. The second group of clones spans a region of >20kb that is not contiguous with group 1 clones and contains two closely related gsubunit genes in tandem (tomato genes 2 and 3 in Figure 11). The coding regions of all three genes have been sequenced and this data is summarized in Figure 11. All three tomato genes encode precursor proteins of approximately the same size and contain a single intron of variable size at the same location. Tomato Genes 2 and 3 show 91% and 84% amino acid identity, respectively, at the protein level to the fruit gsubunit protein (tomato gene 1) with the carboxyl domains of each gene showing higher identity than the mature protein domains. The mature domains of both tomato gene 2 and 3 exhibit the 14 amino acid repeating motif shown in figure 4 for the fruit gsubunit with minor variations being restricted largely to "x" residues of the repeating motif consensus FTNYGxxGNGGxxx. In contrast, the consensus glycosylation sites in genes 2 and 3 are more numerous and not conserved in their placement relative to those in gene 1. Preliminary RNAse protection analysis indicates that Gene 2 is expressed at high levels in floral tissue and in fruit tissue at levels near that of Gene 1. Gene 1 is expressed at high levels in fruit tissue, as expected, and at much low levels in floral tissues. Expression of Gene 3 has not been observed in either tissue and it may represent a pseudo gene or an inducible gsubunit gene that responds to stress, pathogen ingress or other stimuli. When the Arabidopsis Expressed Sequence Tag (EST) Database was searched with the tomato fruit gsubunit protein sequence two related cDNAs were identified (Figure 11). eDNA 2 is near full length and has been completely sequenced, eDNA 1 has also been sequenced but currently lacks approximately 100 amino acids of coding region. The two Arabidopsis cDNAs are 81% identical at the protein level and have lower identity to the protein encoded by tomato gene 1, 64 and 63% for eDNA 1 and eDNA 2, respectively. However, both cDNAs encode
260 proteins that contain the repeating 14 amino acid core mature protein motif, FTxYGxxxN(x)4_6, with F, Y and N being 100% conserved. The other residues in the repeating motif show more variation than the tomato proteins, as reflected in the overall identity of the Arabidopsis cDNA 1 and cDNA 2 proteins tomato gene 1 protein. As with tomato genes 2 and 3, the carboxyl domain of both Arabidopsis proteins shows greater identity to tomato gene 1 than does their mature protein domains. However, it is important to stress that the size, precursor protein structure, amino acid identity and conserved, repeating motif structure contained in the two Arabidopsis cDNAs make it clear that they are homologs of the tomato 13subunit protein. Finally, genomic southern analysis using the fruit 13subunit cDNA as a probe has identified homologous sequences in a wide variety of monocots and dicots suggesting that the 13subunit is an evolutionarily conserved cell wall protein found in most, if not all higher plants. % homology with AroGP1
Tomato Gene I (AroGP1)
100%
Tomato Gene 2 (AroGP2)
91%
Tomato Gene 3 (AroGP3)
84%
Arabidopsis cDNA1
64%
Arabidopsis cDNA2
63%
Structural Domains/Homology
YYY
BBrfff/I
YYY
Mature protein oomain 100J~ 200 300
Y YYYY
~ 400
Y Y
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"
I
Y
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o
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78%/94%
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,
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Figure II: Diagrammatic representation of the tomato Bsubunit gene family members and related cDNAs in Arabidopsis thaliana. The four domains of the respective precursor proteins are coded as in Figure 4. The large triangles represent introns. "Y"s represent the position of glycosylation consensus sequences. Tomato Gene 1 is the fruit 13subunit cDNA. Percentages underneath each mature and carboxyl domain indicate the respective identity to the mature and carboxyl domains of Tomato Gene 1. 3.3. Conclusions from Structural Studies We propose that the tomato fruit 13 subunit is the archetypal member of a new class of plant cell wall proteins, which we have named AroGPs, for Aromatic Amino Acid Rich GlycoProteins. AroGPs have the following characteristics: 1) AroGPs are encoded as a large four domain precursor protein that is more than twice the size of the mature AroGP protein. The precursor contains a large carboxyl terminal extension. 2) The mature AroGP protein domain is composed almost entirely of a repeating 14 amino acid protein motif of the sequence FTxYGxxxN(x)4_6 where F and Y are 100% conserved. 3) The mature domain contains a very high percentage of the aromatic amino acids phenylalanine and tyrosine (> 10% on a molar basis).
261 With regard to structural considerations, AroGPs somewhat resemble plant structural cell wall proteins such as hydroxyproline rich glycoproteins, glycine rich proteins, and proline rich proteins which also contain repetitive amino acid motifs and in some cases posttranslationally modified amino acids such as hydroxyproline and isodityrosine. However, AroGPs differ from these classes of proteins in several respects. First, although AroGPs do contain a repeating amino acid motif, the minimum repeating unit is much longer and shows much greater variability than those found in HRGPs, PRPs and GRPs. Second AroGPs are expressed at high levels in specific tissues (fruit or flowers) at precise developmental times. Other structural proteins have tissue and cell type specific expression of individual gene family members but as a group are generally expressed throughout the plant. Finally, and most importantly, unlike structural proteins such as PRPs and GRPs, AroGPs can bind tightly to and either directly or indirectly affect the activity of another protein with enzymatic activity (for the fruit AroGP this protein is PG). Based on the available data we propose AroGPs represent a new class of plant cell wall proteins specifically expressed in various PG-containing tissues where they interact with cell wall components and catalytic enzymes to regulate cell wall enzymatic activities in vivo. Some questions we are addressing in current and future research include: where and when are the individual tomato gene family members expressed, how similar are the proteins encoded in tomato and other species, what is the function of AroGPs in nonfruit tissues, what is the nature and structural consequence of the modified phenylalanine residues in the protein, and finally, do AroGPs interact with and regulate other catalytic proteins besides PGases? Such studies will further address our basic questions of cell wall enzyme structure, function and regulation in higher plants. 5. REFERENCES
1. 2. 3. 4. 5. 6. 7.
Alan R.L. and Lonsdale D.M. (1992) Plant Mol Biol 20:343-345. Ali, Z.M., and Brady, C.J. (1982). Aust. J. Plant Physiol. 9, 155-169. Brown S.M. and Crouch M.L. (1990) Plant Cell 2:263-274 Carpita N. and Gibeaut D.M. (1993) Plant Journal 3:1-30 DellaPenna, D., and Bennett, A.B. (1988) Plant Physiol. 86, 1057-1063. DellaPenna, D., Kates, D.S., and Bennett, A.B. (1987) 85:502-507. DellaPenna, D., Lashbrook, C.C., Toenjes, K., Giovannoni, J.J., Fischer, R.L., and Bennett, A.B. (1990). Plant Physiol. 94, 1882-1886. 8. Fischer RL and Bennett ABB (1991) Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:675603. 9. Giovannoni, J.J., DellaPenna, D., Bennett, A.B., and Fischer, R.L. (1989) Plant Cell 1, 53-63 10. Giovannoni, J.J., DellaPenna, D., Bennett, A.B., and Fischer, R.L. (1990) Annu. Rev. HortSci. 108, 405-409. 11. Hawes, M.C. and Lin H.J. (1990) Plant Physiology 94:1855-1859. 12. Kalaitzis, P., Koehler, S.M. and Tucker, M.L. (1995) Plant Molecular Biology 28:647656. 13. Knegt E., Vermeer E. and Bruinsma J. (1988) Physiol Plant 72:108-114. 14. Knegt, E., Vermeer, E., Pak, C., and Bruinsma, J. (1991). Physiol. Plant. 82, 237-242. 15. Osteryoung, K.W., Toenjes, K., Hall, B., Winkler, V., and Bennett, A.B. (1990) Plant Cell 2, 1239-1248. 16. Peretto R., Favaron F., Bettini V., De Lorenzo G., Marini S., Alghisi P., Cervone F. and Bonfante P. (1992) Planta 188:164-172. 17. Pogson, B.J., and Brady, C.J. (1993) Postharvest Biol. Technol. 3, 17-26. 18. Pogson, B.J., Brady, C.J., and Orr, G.R. (1991) Aust. J. Plant Physiol. 18, 65-79. 19. Pressey, R. (1984a) Eur. J. Biochem. 144:217-221. 20. Pressey, R. (1984b) Hortscience 19:572.
262 21. Pressey, R. (1988). Reevaluation of the changes in polygalacturonases in tomatoes during ripening. Planta 174, 39-43. 22. Smith, C.J.S., Watson, C.F., Morris, P.C., Bird, C.R., Seymour, G.B., Gray, J.E., Arnold, C., Tucker, G.A., Schuch, W., Harding, S., and Grierson, D. (1990). Plant Mol. Biol. 14, 369-379. 23. Taylor, J.E., Webb, S.T.J., Coupe, S.A., Tucker, G.A. and Roberts, J.A. (1993) J of Expt. Botany 44:92-98. 24. Tucker, G.A., Robertson, N.G. and Grierson, D. (1980) Eur. J. Biochem. 112:119. 25. Watson, C.J., Zheng L.S. and DellaPenna D. (1994) The Plant Cell. 6:1623-1634. 26. Zheng L.S., Heupel R. and DellaPenna D. (1992) Plant Cell 4:1147-1156. 27. Zheng, L.S., Watson C. F. and DellaPenna D. (1994) Plant Physiology. 105:1189-1195.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
263
Characterisation of RG degradation products of new RGases using RG-rhamnohydrolase and RG-galacturonohydrolase' M. Mutter', C.M.G.C. Renard b, G. Beldman', H.A. Schols', A.G.J. Voragen" "Wageningen Agricultural University, Department of Food Chemistry, Bomenweg 2, 6703 HD Wageningen, The Netherlands, fax +31 317 484893 t'Laboratoire de Biochimie et Technologic des Glucides, Centre de Recherche Agroalimentaire, Institut National de la Recherche Agronomique, B.P. 1627, 44316 Nantes c6dex 03, France lFinancial support was from Novo Nordisk A/S (Copenhagen, Denmark)
Abstract Linear rhamnogalacturonan (RG) fragments (RGO's) isolated from an acid hydrolysate of saponified sugar beet pulp were treated with the enzymes RG-hydrolase and RG-lyase. Major tools in the characterisation of the formed degradation products were the exo-acting enzymes RG-rhamnohydrolase and RG-galacturonohydrolase. These exo-enzymes were used to prepare a series of standards of RG oligomers and furthermore to confirm structure assignments, made using high-performance anion-exchange chromatography (HPAEC). The RG-hydrolase was active toward RGO's when the degree of polymerisation (DP) was 12 or higher, while the RG-lyase was only active when the DP was 14 or higher. The alternating RG sequences have to be at least 16 to 18 units long to produce similar oligomers as RG-hydrolase and RG-lyase liberate from apple modified hairy regions (MHR). 1. I N T R O D U C T I O N Schols et al. (1990a) were the first to describe the enzyme rhamnogalacturonase (RGase), able to degrade the hairy (ramified) regions of pectin. Several papers from other workers have been published dealing with RGase activity (Matsuhashi et al., 1992; D0sterh6tt et al., 1993; An et al., 1994 and Sakamoto et al., 1994). Since that time, a set of various enzymes, all with high specificity toward hairy regions of pectin and 'no activity toward homogalacturonan regions, has been found in the authors' laboratory. Searle-van Leeuwen et al. (1992) described an RG-acetylesterase; an RG-rhamnohydrolase was found by Mutter et al. (1994); and recently a newly found RGase, previously called RGase B, turned out to be an RG
264 tx-L-rhamnopyranosyl-(1---~4)-ot-D-galactopyranosyluronide lyase, abbreviated RG-lyase (Mutter et al., 1996). RGase as described by Schols et al. (1990a) is now termed an RG ot-Dgalactopyranosyluronide-(1---~2)-ct-L-rhamnopyranosyl hydrolase, abbreviated RG-hydrolase. A fitth enzyme in this series is an RG-galacturonohydrolase (Mutter et al., in preparation) specific for the removal of the terminal nonreducing GalA unit of RG chains. This enzyme contains no activity toward homogalacturonan structures at all. The discovery of these enzymes enables a better structural characterisation of the hairy (ramified) regions of pectin, as already demonstrated by Schols et al. (1990b) and also of native plant cell wall pectin (Schols et al., 1995). In this study we show how the two exo-enzymes of the above described series, the RG-rhamnohydrolase and the RG-galacturonohydrolase, can be used as tools in the characterisation of unknown RG fragments. These unknown fragments were the products of RG-hydrolase or RG-lyase action toward linear RG oligomers (RGO's), which were produced by acid hydrolysis of sugar beet pulp. 2. M A T E R I A L S
AND METHODS
RG oligomers Sugar beet pulp was saponified and then hydrolysed with 0.1 N HCI at 80 ~ for 72 h. Linear RG oligomers of DP 6 to 18, abbreviated RGO 6 to 18, were isolated by ion-exchange chromatography and size-exclusion chromatography by Renard et al. (1995). Saponified apple MHR was treated with RG-hydrolase, and the branched RG-hydrolase MHR oligomers were isolated using size-exclusion chromatography as described in Mutter et al. (1994). A similar procedure was carried out with RG-lyase, and the branched RG-lyase MHR oligomers were isolated as described in Mutter et al. (1996).
Enzymes RG-hydrolase and RG-lyase from Aspergillus aculeatus were purified using the method of Schols et al. (1990a) and of Kofod et al. (1994) respectively. A 13-galactosidase from Aspergillus niger was purified essentially according to Van de Vis (1994). Purification of an RG-rhamnohydrolase from Aspergillus aculeatus of the type described by Mutter et al. (1994) involved dialysis, DEAE Sepharose fast flow at pH 4.25, SP Sepharose fast flow at pH 4.25, Q Sepharose high performance at pH 6.0, and finally Chelating Sepharose high performance loaded with Cu 2+ ions. In the last purification step, the RG-rhamnohydrolase was separated from an RG-galacturonohydrolase. These purification steps and the characterisation of the RGgalacturonohydrolase will be discussed in detail elsewhere.
265
Incubations with enzymes All substrates (varying between 0.018 and 0.05% w/v) were incubated in 50 mM sodium acetate buffer pH 5.0, containing 0.01% w/v sodium azide, at 40 ~ for 24 h. RGO's were treated with 2.6 lag RG-galacturonohydrolase per mg substrate. When RGO's were sequentially treated with the exo-enzymes to form smaller oligomers, the RGgalacturonohydrolase and the RG-rhamnohydrolase were used in amounts between 2.4 and 2.8 lag and between 9 and 18 lag per mg substrate respectively. RGO's were incubated with 0.18 lag RG-hydrolase and with 0.42 lag RG-lyase per mg substrate. Subsequent incubation of the RG-hydrolase/RG-lyase digest with the exo-enzymes was carried out with 6 lag of RGgalacturonohydrolase and with 16 lag RG-rhamnohydrolase per mg substrate. Removal of Gal from the RG-hydrolase MHR oligomers was performed as described in Mutter et al. (1994). From the RG-lyase MHR oligomers the Gal was removed using 9.3 lag 13galactosidase per mg substrate. Derhamnosylation of the degalactosylated RG-hydrolase MHR oligomers was carried out as described in Mutter et al. (1994).
Analysis using HPAEC HPAEC was carried out using a Dionex Bio-LC system equipped with a Dionex CarboPac PA-100 (4 x 250 mm) column and a Dionex pulsed electrochemical detector in the pulsed amperometric detection (PAD) mode. A gradient of sodium acetate in 100 mM sodium hydroxide (1 ml/min) was used as follows: 0 to 50 min, 0 to 450 mM; 50 to 55 min, 450 to 1000 mM; 55.1 to 70 min, 0 mM. 3. R E S U L T S A N D D I S C U S S I O N
Preparation of a series of standards of RG oligomers Several types of RG oligomers, obtained by acid hydrolysis or enzymically, were prepared in order to have a series of standards available for the characterisation of the reaction products from linear RG oligomers by the action of RG-hydrolase and RG-lyase. These linear RG oligomers are of the type: ot-D-GalpA-(1---)2)-(ot-L-Rhap-(1-->4)-ot-D-GalpA-(1-->2))n-L-Rhap with n = 2 to 8, being oligomers with an even number of sugar residues (DP 6 to 18), abbreviated RGO's 6 to 18. They were purified from sugar beet pulp and characterised (Renard et al., 1995). The RGO's were treated with an RG-galacturonosidase from Aspergillus aculeatus, a new enzyme specific for RG fragments. This RG-galacturonohydrolase has a molecular mass of 66 kD and an isoelectric point of 5.1. The enzyme is able to remove the terminal nonreducing GalA from RG fragments and not from homogalacturonan fragments
266 (Mutter et al., in preparation). This enzyme was used to remove the nonreducing GalA from these RGO's to obtain oligomers of the type: (a-L-Rhap-(1-~4)-a-D-GalpA-(1--~2)).-L-Rhap with n = 2 to 8, defined as degalacturonosylated RGO's with an uneven number of sugar residues (DP 5 to 17). RGO 6 was sequentially treated with the RG-galacturonosidase and the RG-rhamnohydrolase (Mutter et al., 1994) to form oligomers ofDP 5 and 4. Branched RG oligomers of the type: a-D-us GalpA-(1 -->2)-(a-L-Rhap-(1 ---M)- a-D-GalpA-( 1--~2)).-L-Rhap
1"
1"
13-D-C~p-(1--M)
B-D-C~p-(1--)4)
with n = 1 to 4 (us = 4,5-unsaturated), were purified from saponified apple MHR by treatment with RG-lyase (Mutter et al., 1996 and unpublished results). These RG-lyase MHR oligomers were linearized using a 13-galactosidase from Aspergillus niger (Van de Vis, 1994). These linear RG-lyase MHR oligomers were of the type: a-D-us C~pA-(1---~2)-(a-L-Rhap-(1-+4)-a-D-GalpA-(1-~2))n -L-Rhap with n = 1 to 4, giving oligomers with a DP of 4 to 10. Oligomers with a DP of 4 to 8 were used as standards. A mixture of branched RG oligomers of the type: a-L-Rhap-(1--->4)-(a-D-GalpA-(1--~,2)-a-L-Rhap-(1---~4))n-D-GalpA
1' (I3-D- Galp-( 1--94))q
1' (I3-D- Galp-( 1--~4))~
with n = 1 to 3 and q/r = 0 or 1, was obtained from saponified apple MHR by treatment with RG-hydrolase (Schols et al., 1990a). These RG-hydrolase MHR oligomers were linearized by treatment with a 13-galactosidase from Aspergillus niger. These linear RG-hydrolase M]-IR oligomers were of the type: (a-L-Rhap- ( 1-~4)-a-D- GalpA-( 1---~2))n-a-L-Rhap- ( 1--~4)-D- GalpA with n = 1 to 3 (Schols et al., 1994; Mutter et al., 1994). These oligomers were derhamnosylated with the RG-rhamnohydrolase (Mutter et al., 1994) to form oligomers of the type:
267
((x-D-GalpA-(1-~2)-tx-L-Rhap-(1-->4)), -D-GalpA with n = 1 to 3, giving final DP's of 3, 5 and 7. The order of elution of the 24 different standard RG oligomers is shown in table I. The Rha residues are indicated by E! and the GalA residues by O, whereas the us-GalA units, which are introduced by RG-lyase action, are indicated by | The symbols are explained below the table, and will be used throughout the paper, although the shades of the symbols are different in the figures.
Table I Eluaon behaviour of different types of RG oligomers on HPAEC Structures in ascending order of elution on HPAEC DP of RGO or degalacturonosylated RGO n O-O-O-O-El DP 5 n-o O-I"l-O-El DP 4 n-o-n-o-n-o-ci DP 7 I"1-O-i"1-O
Ret. time (min) 3.0 12.2 12.5 13.3 19.8 20.8
O-I-I-O-I-I-O-l"!
21.2
DP 6
O-13-O 22.5 I-I-O-I-I-O-I"1-O-I"1-O-I"! DP 9 25.2 1"1-O-1"1-O-!"1-O 26.3 O-I-I-O-I"l-O-I"l-O-I"! DP 8 27.1 o-r'l-O-I"l-O 28.4 l-'i-O-I-I-O.r'l-O-I-'l-O-I--i-O-l-'l DP l 1 29.3 O-I"l-O-I"l-O-I-l- O-I"l-O-r'! DP 10 31.0 O-El-O-El 31.0 O-O-O-I"1-O-O-O 32.6 O-o-n-o-n-o-El-O-El-O-El-O-El DP 13 32.8 O-El-O-El-O-El-o-n-o-El-O-El DP 12 34.4 O-I"l-O-n-o-o 35.4 El-o-n-o-n-o-n-o-o-o-o-o-o-O-l-I DP 15 35 6 o-n-o-n-o-I"l-O-O-o-n-o-El-O-El DP 14 36 8 El-o-n-o-n-o-o-o-n-o-r'l-O-CI-O-CI-O-Cl DP 17 37 6 | O-l"l- O-I"i 38.4 O-El-o-n-o-n-o-El-O-O-O-O-O-Cl-O-El I DP 16 39.1 O-El-o-n-o-El-o-r'l-O-El-O-El-O-I"l-O-o-o-rl i DP 18 40.8 O, (z-D-CralpA (1-->2) linked to Rha, or D-GalpA at the reducing end; El, (z-L-Rhap (1-->4) linked to GalpA, or L-Rhap at the reducing end; | c~-D-us GalpA (1-->2) linked to Rhap. L
268 Since all oligomers in Table I have an alternating RG sequence, the differences in structure are determined by the sugar units at the reducing and nonreducing end. From Table I it can be seen that the retention time of the oligomers increases with DP within a series of similar structure, e.g. a Rha at the reducing end and a GalA at the nonreducing end. Differences in the end sugars influence the retention times drastically. When the RGO's are compared with the degalacturonosylated RGO's it can be seen that an additional Rha at the nonreducing end reduces retention time by a minute (e.g. DP 5 compared with DP 4, up to DP 17 compared with DP 16), even though the DP is higher. Structure I"1-O-I"!-O compared with !"1-O-I"1-O17 (DP 5), and structure I"1-O-I"1-O-i"1-O compared with I"!-O-!"1-O-I"1-O-!"! (DP 7), shows that an additional Rha at the reducing end has an even larger effect: the retention times are 6 to 8 minutes shorter. On the other hand, the removal of a GalA from the reducing end of O-17O-!"1-O (28.4 min) reduces the retention time much more (to O-I"!-O-I"! at 13.3 min) compared with removal from the nonreducing end (to I-1-O-i"l-O at 20.8 min). In conclusion, the reducing sugar seems to be of more influence on the retention time than the nonreducing sugar. For different types of oligosaccharides both the nonreducing and the reducing end have been reported to have the largest influence on HPAEC retention (Lee, 1990). The us-GalA at the nonreducing end causes strong retention on the CarboPac column: the largest RGO that elutes earlier than an oligomer with a nonreducing us-GalA is six units larger.
Degradation of RGO's by RG-hydrolase RGO's 6 to 18 were incubated with RG-hydrolase, under conditions estimated to be sufficient to reach an end-point situation. RG-hydrolase was not active toward RGO 6 to 10, although minor amounts of reaction products were released from RGO 10. RG-hydrolase was very active toward RGO 12 and higher. From RGO 12 two major products were formed (Fig. 1). When compared with the available standards, the earliest eluting peak corresponded with I-l_O_l-'l-O_r-I-O-I"l. The later eluting product had the same retention time as O-I"l-O-r-l-O. Together they match the DP 12 from which they originate. The designation of the products was confirmed by subsequent incubation of the reaction mixture with either the RGrhamnohydrolase or the RG-galacturonosidase. Figure 1 shows that upon incubation of the RG-hydrolase digest with the RG-rhamnohydrolase, the peak of O-I-I-O-El-O remains, a new peak is formed while l--I_O.r-l-O_l-l-O-l"i has disappeared and Rha is released. The newly formed peak has the same retention time as RGO 6, which can be explained by removal of the nonreducing Rha from l-I_O-EI-O-r-I-O-r-! by the RG-rhamnohydrolase. In case of incubation with the RG-galacturonosidase, shown in Figure 1 as well, the peak of I-1-O-!-1-O-1"1-O-I-1 remains,, while the peak of o-r-l-o-r-l,O has disappeared and a new peak and GalA are formed. The newly formed peak has the same retention time as I-I-O-r-I-O. This can be explained b y removal of the nonreducing GalA unit from O-17-O-1"!-O by the RGgalacturonohydrolase. In this manner, using the two exo-enzymes, the original assignments were confirmed.
:.
269
Since the original oligomer contains GalA at the nonreducing and Rha at the reducing end, O - D - O - D - O should result from the nonreducing end and D - O - D - O - D - O - D should originate from the reducing end and can therefore be positioned as follows: O-D-O-D-O
D-O-D-O-D-O-D.
Rha
/
3
5
5
g P
~
3
~ v
m u
.h v
n m
. V
m
~
m
~ v
~ ~
~,
0
.m ~ . .m .y . u. .w .L v
m m
~,
m
~. v
m m
v
~
m
m
m
4
II.
..r
I
0
10
20
30
m
~
m
v
v
,.I
---r
m
m
m
y
,,
m
m
v
~,
~
m
m
v
~,
m
m
m
40
Retention time (min) Figure 1: HPAEC elution patterns of RGO 12 (bottom chromatogram); RGO 12 incubated with RGhydrolase (second chrom, from bottom); RGO 12 incubated with RG-hydrolase and subsequently with RG-rhamnohydrolase (third chrom, from bottom); and RGO 12 incubated with RG-hydrolase and subsequently with RG-galacturonohydrolase (top chrom.). Numbers in chromatograms on the left correspond with structures on the right: Explanation of symbols see table I.
The results confirm that RG-hydrolase is a true rhamnogalacturonase, as it splits an alternating RG chain, and furthermore that it cleaves between GalA and Rha in the main chain by a mechanism of hydrolysis (Schols et all, 1990a). Similarly, the products from the RGO's with higher DP's were characterised. Figure 2 shows the cleavage of the various RGO's by RG-hydrolase. Differently dotted arrows indicate different cleavage options. No bond-cleavage frequencies could be given since the response factors of the various products were not known. The enzyme cleaves the chains five units from the nonreducing end. When the oligomer is larger, also seven units from the nonreducing end are cleaved off. From RGO 16 and 18 products were released whose formation could only be explained by assuming that a secondary cleavage of the reaction products occurred. Since RG-hydrolase cleaves between GalA and Rha, all primary cleavage
270 products from the RGO's have to be of uneven DP. In case of RGO 16 and 18 also products were formed that had similar retention times as the oligomers D - O - D - O (released from RGO 16 and 18) and D - O - D - O - D - O (released from RGO 18 only). In Figure 2 the secondary cleavage is indicated by a short arrow. The cleavage sites shown as primary in case of RGO 16 and 18 were considered as such for the following reasons. The smaller RGO's were cleaved five or seven units from the nonreducing end, and therefore it seemed logical that this also was the case for RGO 16 and 18. Furthermore, the resulting products after cleaving five or seven units from the nonreducing end were large enough to be cleaved by the RG-hydrolase (in case of cleavage of RGO 16, the resulting DP 11 was shown in separate experiments to be indeed cleaved by the RG-hydrolase). The (unbranched) RG-hydrolase MHR tetramer and hexamer as known to be liberated by RG-hydrolase from apple MHR-S (Schols et al., 1994) were both formed from RGO 18.
100--
12~ E 1 4 ,W= -i- , l- , = = l
r
r162162
9 ~ ~Jt ~, ~~, ~ C, ~ l
16r162162162162
/
/
i
I
i
D
i
I
i
i
|
I
/
i
|
I
l
I
r162162
I
18
Figure 2: Locations of cleavage of RGO's by RG-hydrolase. Differently dotted arrows indicate different cleavage options. A short arrow indicates a secondary cleavage, see text. Explanation of symbols, see table 1. Numbers refer to degree of polymerisation.
Degradation o f RGO's by RG-lyase When RG-lyase was incubated with RGO's 6 to 18, activity was observed toward RGO 14 and higher. From RGO 14 two peaks were formed, eluting at approximately the same retention time (Fig. 3). This retention time corresponded with | and RGO 10,
271 together matching with RGO 14. The designation of RGO 10 was confirmed by subsequent incubation of the reaction mixture with the RG-galacturonosidase, which resulted in release of GalA and the formation of a new peak with the retention time of D-O-l"l-O-D-O-D-O-D (DP 9) (Fig. 3). No confirmation of the unsaturated product could be given, since no enzyme was available (nor known) able to remove the us-GalA from the nonreducing end of RG fragments. For all oligomers, the structures of the products were assigned in this manner.
I
Gala
4,,,,4/ ~ j 3 4
P
.
21.
I1.
-
r
r
r162
1~. r162 . . . . . . . . .
0
i
. . . . . . . . .
10
,
. . . . . . . . .
20
i
. . . . . . . . .
30
Retention time (min)
-
r
-
r
-- 3 Q - B - ~ I I
r
r
-- 3 Cg--B-O~-IB
r
r162162
,
40
Figure 3: HPAEC elutionpatterns of RGO 14 (bottom chromatogram); RGO 14 incubated with RG-lyase (second chrom, from bottom);); and RGO 14 incubated with RG-lyase and subsequently with RG-galacturonohydrolase (top chrom.). Numbers in chromatograms on the left correspond with numbers of structures on the right. Explanation of symbols see table I.
It is clear that the unsaturated nonreducing GalA unit must result from the cleavage by RG-lyase, since the original RGO contains a saturated GalA at the nonreducing end. The structures therefore can be positioned as follows: O-D-O-D-O-D-O-D-O-D | The results confirm that RG-lyase is an RGase, able to cleave alternating RG chains, by a mechanism of 13-elimination, as already described by Mutter et al. (1996). Figure 4 shows the cleavage pattern of RG-lyase toward the various RGO's. Again no bond-cleavage frequencies could be given since the response factors of the various products were not known. The RG-lyase cleaved the chain four units from the reducing end. When the
272 oligomer is larger, also six units from the reducing end are cleaved off. This is in contrast with RG-lyase, which cleaved closer to the nonreducing terminus. Secondary cleavage products were observed only in case of RGO 18. The cleavage indicated as primary was considered as such for the following reasons. Since the smaller RGO's were cleaved four or six units from the reducing end, this seemed likely also to be the case for RGO 18. Furthermore, the resulting oligomer after a first cleavage at four units from the reducing end was RGO 14, which could be cleaved by the RG-lyase.
10
A
w
m
mm
A
~
m
A
m
~
m
mm~
A
m
mm
A
~
m
m
120--m-~ ~ ~, ~ ~r ~ ~, ~ ~' ~ 14
16
t
18
Figure 4: Locations of cleavage of RGO's by RG-lyase. Differently dotted arrows indicate different cleavage options. A short arrow indicates a secondary cleavage, see text. The thin arrow indicates a less abundant cleavage option. Explanation of symbols, see table I. Numbers refer to degree of polymerisation.
The data collected in this study can provide more insight in the structure of the RG part of native pectic hairy regions. Apparently the stretches of RG need to be at least 16 units long for RG-hydrolase in order to produce the tetramer and at least 18 units long to produce the hexamer known to be liberated from apple MHR. The stretches of RG need to be even longer for RG-lyase, since from RGO 18 only the tetramer is released, while from apple MHR oligomers with a backbone of four to ten units are released. It is also interesting to see that RG-lyase is able to degrade linear fragments, while from apple MHR only oligomers branched with single unit Gal side chains are released (Mutter et al., 1996). Together with the fact that the linear RG-hydrolase MHR hexamer is usually only released in small amounts (Schols et al.,
273 1994 and Schols et al., 1995), the results suggest that in apple MHR no strictly linear RG regions are present larger than about 16 units. 4. ACKNOWLEDGMENT The authors wish to thank Marjo Searle-van Leeuwen for purification of the 13-galactosidase from Aspergillus niger and Novo Nordisk Ferment Ltd Dittingen (Switzerland) for assistance in the purification of the RG-hydrolase and Novo Nordisk A/S Bagsvaerd (Denmark) for the girl of the crude recombinant RG-lyase. 5. REFERENCES An J, Zhang L, O'Neill MA, Albersheim P (1994) Isolation and structural characterisation of endo-rhanmogalacturonase-generated fragments of the backbone of rhanmogalacturonan I. Carbohydr Res 264:83-96 Diisterh6tt E-M, Bonte AW, Venekamp JC, Voragen AGJ (1993) The role of fungal polysaccharidases in the hydrolysis of cell wall materials from sunflower and palm-kernel meals. World J Microbiol Biotechnol 9:544-554 Kofod LV, Kauppinen S, Christgau S, Andersen LN, Heldt-Hansen HP, D6rreich K, Dalboge (1994) Cloning and characterization of two structurally and functionally divergent rhanmogalacturonases from Aspergillus aculeatus. J Biol Chem 269:29182-29189 Lee, YC (1990) High-performance anion-exchange chromatography for carbohydrate analysis. Anal Biochem 189:151-162 Matsuhashi S, Inoue S-I, Hatanaka C (1992) Simultaneous measurement of the galacturonate and neutral sugar contents of pectic substances by an enzymic-HPLC method. Biosci Biotech Biochem 56:1053-1057 Mutter M, Beldman G, Schols HA, Voragen AGJ (1994) Rhamnogalacturonan a-Lrhamnopyranohydrolase. A novel enzyme specific for the terminal nonreducing rhamnosyl unit in rhamnogalacturonan regions of pectin. Plant Physiol 106:241-250 Mutter M, Colquhoun IJ, Schols HA, Beldman G, Voragen AGJ (1996) Rhamnogalacturonase B from Aspergillus aculeatus is a rhamnogalacturonan ct-L-rhamnopyranosyl-(1---~4)-ct-Dgalactopyranosyluronide lyase. Plant Physiol 110:73-77 Renard CMGC, Thibault J-F, Mutter M, Schols HA, Voragen AGJ (1996) Some preliminary results on the action of rhamnogalacturonase on rhanmogalacturonan oligosaccharides from beet pulp. Int J Biol Macromol in press Sakamoto M, Shirane Y, Naribayashi I, Kimura K, Morishita N, Sakamoto T, Sakai T (1994) Purification and characterization of a rhamnogalacturonase with protopectinase activity from Trametes sanguinea. Eur J Biochem 226:285-291 Schols HA, Geraeds CCJM, Searle-van Leeuwen MF, Kormelink FJM, Voragen AGJ (1990a) Rhanmogalacturonase: a novel enzyme that degrades the hairy regions of pectins. Carbohydr Res 206: 105-115 Schois HA, Posthumus MA, Voragen AGJ (1990b) Structural features of hairy regions of pectins isolated from apple juice produced by the liquefaction process. Carbohydr Res 206:117-129
274 Schols HA, Vierhuis E, Bakx EJ, Voragen AGJ (1995) Different populations of pectic hairy regions occur in apple cell walls. Carbohydr Res 275:343-360 Schols HA, Voragen AGJ, Colquhoun IJ (1994) Isolation and characterization of rhanmogalacturonan-oligomers, liberated during degradation of pectic hairy regions by rhamnogalacturonase. Carbohydr Res 256:97-111 Searle-van Leeuwen MJF, Van den Brock LAM, Schols HA, Beldman G, Voragen AGJ (1992) Rhamnogalacturonan acetylesterase: a novel enzyme from Aspergillus aculeatus, specific for the deacetylation of hairy (ramified) regions of pectins. Appl Microbiol Biotechnol 38:347-349 Van de Vis JW (1994) Characterization and mode of action of enzymes degrading galactan structures of arabinogalactans. Koninklijke Bibliotheek, The Hague, The Netherlands, pp 89-108
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V.All rights reserved.
275
The Effect of Glycosylation of Endopolygalacturonases and Polygalacturonase Inhibiting Proteins on the Production of Oligogalacturonides
C.W. Bergmannl, B. Cook1, A.G. Darvill 1, p. Albersheim 1, D. Bellincampi:, and C. Caprari 2
1 Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, The University of Georgia, Athens, Georgia 30602-4712, USA
2Dipartimento di Biologia Vegetale, Universita di Roma "La Sapienza", Piazzale Aldo Moro, 00185 Rome, Italy
Abstract Fungal endopolygalacturonases (EPC~) are considered to be major pathogenicity factors, facilitating the breakdown of the plant cell wall and releasing biologically active oligogalacturonide elicitors which are also substrates for EPG. The inhibition of EPGs by plant polygalacturonase inhibiting proteins (PGIPs) has led to the hypothesis of PGIPs as general defense factors as a consequence of their proposed role in increasing the lifetime of elicitor active oligogalacturonides. Plant PGIPs exist in a variety ofglycoforms in which the peptide backbone is conserved with differing degrees of glycosylation. The population of the glycoforms change depending on the species, cultivar, and tissue investigated. Fungal EPGs also exist in a variety of glycoforms, which resulted in us studying the possible role that the glycosylation of EPG and PGIP may play in affecting the production of biologically active oligogalacturonides via their action on pectic substrates. Using HPAEC-PAD, we studied the oligogalacturonide products formed by the interaction of a variety of glycoforms of EPGs and PGIPs on polygalacturonic acid, allowing us to determine the effect of glycosylation on the sizes or lifetimes of the oligogalacturonides produced.
Results and Discussion Endopolygalacturonases (EPGs) are important pathogenicity factors of fungi and are among the first enzymes secreted when fungi are grown on isolated cell walls as their carbon source [1]. EPGs fragment homogalacmronan and solubilizc the other cell wall pectic polysaccharides, rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II), and appear to make cell walls more susceptible to the action of other endoglycanases [2,3]. Homogalacturonan fragments (i.e., a-l,4-1inked oligogalacturonides) with DPs 12-14 are oligosaccharins that, depending on the plant being studied, exhibit a wide range of signal functions [4]. EPGs m vitro rapidly degrade de-esterified homogalacturonan to mono-, di-, and
276 trigalacturonides, resulting in little opportunity for bioactive oligogalacturonides to accumulate [5,6]. All dicotyledonous plants have proteins in their cell walls that are able to inhibit fungal EPGs by forming one-on-one (stoichiometric) complexes [7-11 ]. We have demonstrated that in beans the concentration ofpolygalacturonase inhibitor proteins (PGIPs) rapidly increases in the cell walls of those cells immediately surrounding the site at which a fungus penetrates the plant, and that the epidermal cells accumulate the most PGIP [ 12]. The bean PGIPs, which we have studied extensively [ 12-15], are able in vitro to inhibit greater than 99% of the activity of a variety of fungal EPGs. The low amount of EPG activity remaining in the EPG-PGIP complex is sufficient to slowly depolymerize homogalacturonan and allow for the accumulation of bioactive oligogalacturonides [ 13,16]. Purified P. vulgaris PGIPs exist as a series of isoforms, and although the protein contains four potential N-linked glyeosylation sites, it has been speculated that these isoforms may be the result of multiple gene products [ 17]. The four PGIP isoforms of P. vulgaris have now been shown to be glycoforms, in agreement with work on tomato PGIP isoforms by Stotz et al. who estimate the presence of up to seven glycoforms [ 10]. P. vulgaris PGIPs from a variety of cultivars and tissues, including PGIP isolated from cv. cannelino cells grown in liquid culture, were compared and shown to have identical N-terminal sequences. These PGIPs also all yielded the same MW band, following chemical deglycosylation with TFMS, on SDS-PAGE silverstained gels as well as on Western blots probed with antibodies raised against a peptide from the N-terminus of P. vulgaris PGIP (Fig. 1).
1
2
3
4
5
Figure 1. Lane 1) cv "pinto" pod PGIP (native), Lane 2) deglycosylated cv "cannelino" cultured cell PGIP, Lane 3) deglycosylated cv "cannelino" pod PGIP, Lane 4) deglycosylated cv "blue lake" pod PGIP, Lane 5) deglycosylated cv "pinto" pod PGIP.
Comparative Western analysis of samples directly extracted from tissue without intermediate purification demonstrate that the relative amounts of the four P. vulgaris glycoforms vary from cultivar to cultivar, but their MWs and presence are invariant (Fig.2).
277
=~..,,,, ....
-' .~ ~( ":":'%~.;,~),~,(:~")~:~';'~...:<~;i:'=~':k~'.;:
a
' ~;::""' ""
.
Figure 2. Lanel 1) cv "cannelino", Lane 2) cv "pinto", Lane 3) cv "blue lake", Lanes a) epidermal tissue, Lanes b) extract of whole, immature pod.
The sole exception is for PGIP from cultured cells, which appear to have a higher percentage of carbohydrate than PGIP from whole tissues. The EPG from F. moniliforme has been cloned and shown to contain four potential N-linked glycosylation sites [18]. This protein has also been shown to exist as a series of glycoforms. The four glycoforms all yield the same MW band following enzymatic deglycosylation with Nglycanase or chemical deglycosylation with HF, demonstrating that the majority if not all the glycosylation is N-linked [19]. Further evidence for a single protein backbone comes from Southern analysis of fragments generated by three different restriction enzymes, all of which yielded a single fragment hybridizing to a 1272 bp probe corresponding to a cloned eDNA ofF. moniliforme EPG [ 18]. Northern blot analysis using the same probe revealed a single mRNA encoding the EPG. We are currently in the process of determining the structure and exact location of the carbohydrate side chains present in each of the glycoforms using MALDI-TOF and electrospray mass spectrometry. We have been interested in determining if the variations in glycosylation of the EPGs or PGIPs has any effect on their activities in vitro by monitoring their ability to produce defined patterns ofoligogalacturonide products when the glycoproteins are incubated in the presence of polygalacturonic acid. We first compared the profiles obtained by HPAEC-PAD of the oligogalacturonides produced by equal activities (in terms of reducing group units/rain or RGU) of the glycoforms of F. moniliforme EPG and found essentially no difference (Fig. 3).
I
m O
20
minum
40
60
!l O
m ~
minum
40
60
0
20
mmm
40
60
Figure 3. Dionex HPAEC profiles following 45 minute digestion of 1.25% PGA by 0.1 RGU each of 3 different glycoforms of F. Moniliforme EPGs.
278 An over-glycosylated form of the F. moniliforme EPG produced in Saccharomyces cerevicae was also tested. These heterogeneously expressed EPG glycoforms have molecular weights that are approximately 6-10 kD higher than the native enzymes, as the glycosylation sites are occupied by high-mannose chains. This mixture of EPG glycoforms also produced essentially the same oligogalacturonide profile as the native glycoforms for a given RGU activity (Fig. 4). Further, these over-glycosylated EPGs, because they contain only high-mannose chains, could be deglycosylated with endo-H, leaving a single GIcNAc residue present at each of the occupied asparagine residues. These S. cerevicae-expressed EPGs maintain their activity after deglycosylation and produce an essentially identical oligogalacturonide profile for a given activity as the wild type (Fig. 4).
tt
,l
n,,
10{
min~
O
i
i
''
''fill
il
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20 40 mblulm
,~:l
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Figure 4. Dionex HPAEC profiles following 45 minute digestion of 1.25% PGA by 0.1 RGU of F. moniliforme EPG expressed in Saccharomyces cerevicae (YPG) or the deglycosylated form (dYPG). This convinced us that, at least for Fusarium EPG, the glycosylation played no role in changing its ability to hydrolyze PGA.
We also tried to determine whether glycosylation of both EPG and PGIP affects the inhibition of EPG by PGIP, possibly providing a key to race-cultivar specificity. Consequently, PGIPs from various cultivars were each mixed with one of the F. moniliforme EPG glycoforms. The over-glycosylated PGIP from cultured cannelino cells was also mixed with the overglycosylated yeast-expressed form of the EPG. For these experiments a standard incubation was chosen based on the oligogalacturonide pattern obtained with the EPG m the presence or absence of pinto pod PGIP. There were little or no differences in oligogalacturonide product formation for all these EPG-PGIP combinations, except for the yeast-expressed EPG-cultured cannelino cell PGIP combination in which the most highly glycosylated forms of the proteins were used (Figs. 5 and 6).
279
,. 1 0
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Figure 5. Dionex HPAEC profiles following 48 hour digestion of 1.25% PGA by 0.1 RGU each of 3 different glycoforms of F. Moniliforme EPG inhibited by cv "pinto" pod PGIP.
m
~
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20
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o
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Figure 6. Dionex HPAEC profiles following 48 hour digestion of 1.25% PGA by 0.1 RGU each of 4 different EPGs inhibited by cv "cannelino" cultured cell PGIP. A and B) F. moniliforme EPG glycoforms, C) deglycosylated S. cerevicae expressed EPG, D) S. cerevicae expressed EPG.
For the latter case, an approximately 10-fold increase in the amount of PGIP was needed to obtain the desired level of inhibition. There are a number of reports that suggest there is significant variation in the abilities of the PGIPs from different plants to inhibit the EPC~ of different fungi. The PGIP from bean inhibits equally well the EPGs obtained from each of ten fungi [7,20-22]. In a comparative study done
280 in conjunction with John Labavitch's group (University of California, Davis), we found that when pear, tomato, and bean PGIPs were tested against F. moniliforme, A. niger, and B. cinema EPGs, only the bean PGIP inhibited all the EPGs. The pear PGIP was only able to inhibit B. cinema PGIP, and the tomato PGIP was unable to inhibit F. momliforme EPG and had only 5% of the inhibition against A. niger that was shown by the bean PGIPs ([22], manuscript in preparation). These PGIPs exhibit significant differences in homology, the bean being 50% homologous to both tomato and pear, which are 68% homologous to each other. Thus, it appears that the abilities of different species of plants to inhibit the EPGs of attacking fungi vary dramatically. We have been interested in determining what role the carbohydrate side chain of the various PGIPs may play in determining the specificity of inhibition. The PGIPs from all of the plant species investigated exist as a series of glycoforms [10,11 ]. A correlation appears to exist between the extent to which a PGIP is glycosylated and the increase in specificity of its inhibition, such that the greater the glycosylation the greater the specificity. The mature polypeptides of bean, tomato and pear all have calculated molecular masses of 34 +_0.3 kD [10]. When bean, pear, and tomato PGIPs are chemically deglycosylated, their polypeptides migrate as a single, sharp band at 34 kD, i.e. the calculated molecular weight [ 10,11 ]. Native pear PGIP migrates in SDS-PAGE as a diffuse band with a considerably larger molecular weight than bean PGIP, i.e. -~43 kD (Fig. 7).
. . . .
.........!j
48,500
1
2
3
4
Figure 7. SDS-PAGE of Lane 1) cultured cv "cannelino" cultured cell PGIP, Lane 2) cv "pinto" pod PGIP, Lane 3) tomato PGIP, Lane 4) pear PGIP.
Tomato PGIP with respect to its molecular weight falls in between pear PGIP and bean PGIP [22]. The presence of O-linked glycosyl side chains cannot be ruled out, but initial MALDI-TOF and lectin analysis experiments indicate that for the bean PGIP, all carbohydrate is N-linked. There are two conserved N-linked sites (based on the Asn-X-Ser/Thr consensus sequence) in all of the PGIPs that have been studied [ 10], but while there are four sites of potential N-linked glycosylation in bean PGIP, there are seven such sites in the pear and tomato PGIPs (Fig. 8).
281
T~
F T~I .... I ill /~iii!i/ I
Pear Been
ITI~: ii ;/
HIT, i i i
I/
~i~i'~i!t
l i
/i '~i.:ilri~"~i!
Figure 8. Sequence comparison of PGIPs (adapted from Stotz etal., [10]). Black = Glycosylation site, Gray = greater than 70% sequence homology.
Initial MALDI-TOF MS indicates that only two of the four bean sites have carbohydrate chains attached (data not shown). Bean PGIP expressed in tomato results in a PGIP with a decreased ability to inhibit some fungal EPGs (personal communication with F. Cervone). In a similar situation, pear PGIP expressed in tomatoes results in a 100-fold increase in the amount of PGIP but in only a 10-fold increase in EPG-inhibiting activity (J. Labavitch and A. Bennett, personal communication). Thus, over-expressing the bean PGIP in other plants will not necessarily result in an increased ability to inhibit fungal EPGs. Glycosylation differences may be one explanation when tmnsgenic plants often do not obtain the expected properties of the transgene [23]. For example, the bean PGIP may be glycosylated to a greater extent when it is expressed in tomatoes than in bean, resulting in an increased specificity of the PGIP. It is also important to remember that the experiments performed here were done on PGA, while the natural substrate of EPGs in cell walls is pectin. We cannot exclude the possibility that more profound effects could be observed if pectin were used as the substrate. Finally, the role(s) of EPG and PGIP in plant-fungal interactions are still being elucidated, and the carbohydrate moieties on either or both of these glycoproteins may be of consequence in binding or recognition.
References
English, P.D., A. Maglothin, K. Keegstra, and P. Albersheim. 1972. Plant Physiol. 49: 293-297. Cooper, R.M. 1983. In: JA Callow (ed) Biochemical Plant Pathology (John Wiley and Sons Ltd. New York, London) pp 101-135. Marfa, V., D.J. Gollin, S. Eberhard, D. Mohnen, A. Darvill, and P Albersheim. 1991. The Plant d. 1(2): 217-225. Darvill, A., C. Augur, C. Bergmann, R.W. Carlson, J.-J. Cheong, S. Eberhard, M.G. Hahn, V.-M. Lo, V. Marfa, B. Meyer, D. Mohnen, M.A. O~eill, M.D. Spiro, H. van Halbeek, W.S. York, and P. Albersheim. 1992. Glycobiol 2(3): 181-198. Goldberg, R., M. Pierrork L. Durand, and S. Mutat~hiev. 1992. J. Exp. Bot. 43:41-46.
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8. .
10. 11. 12. 13. 14. 15. 16.
17. 18. 19. 20. 21. 22.
23.
Riou, C., L. Fraissinet-Tachet, G. Freyssinet, and M. F~vre. 1992. FEMS Microbiol. Lett. 91: 231-238. Albersheim, P. and A.J. Anderson. 1971. Proc. Natl. Acad. Sci. USA 68: 1815-1819. Cervone, F., G. De Lorenzo, L. Degr/L, G. Salvi, and M. Bergami. 1987. Plant Physiol. 85:631-637. Johnston, D.J., V. Ramanathan, and B. Williamson. 1993. J. Exp. Bot. 44: 971-976. Stotz, H.U., J.J.A. Contos, A.L.T. Powell, A.B. Bennett, and J.M. Labavitch. 1994. Plant Mol. Biol. 25:607-617. Stotz, H.U., A.L.T. Powell, S.E. Damon, L.C. Greve, A.B. Bennett, and J.M. Labavitch. 1993. Plant Physiol. 102: 133-138. Bergmann, C.W., Y. Ito, D. Singer, P. Albersheim, A.G. Darvill, N. Benhamou, L. Nuss, G. Salvi, F. Cervone, and G. De Lorenzo. 1994. Plant J. 5: 625-634. Cervone, F., M.G. Hahn, G. De Lorenzo, A. Darvill, and P. Albersheim. 1989. Plant Physiol. 90: 542-548. Toubart, P., A. Desiderio, G. Salvi, F. Cervone, L. Daroda, G. De Lorenzo, C. Bergmann, A.G. Darvill, and P. Albersheim. 1992. The Plant J. 2: 367-373. De Lorenzo, G., Y. Ito, R. D'Ovidio, F. Cervone, P. Albersheim, and A.G. Darvill. 1990. Physiol. Mol. Plant Pathol. 36: 421-435. Cervone, F., G. De Lorenzo, G. Salvi, C. Bergmann, M.G. Hahn, Y. Ito, A. Darvill, and P. Albersheim. 1989. In: BJJ Lugtenberg (ed) Signal Molecules m Plants and PlantMicrobe Interactions. NATO ASI Series, Volume H36 (Springer Verlag, Heidelberg, FRG) pp 85-89. Frediani, M., R. Cremonini, G. Salvi, C. Caprari, A. Desiderio, R. D'Ovidio, F. Cervone, and G. De Lorenzo. 1993. Theor Appl Genet 87: 369-373. Caprari, C., A. Richter, C. Bergrnann, S. Lo Cicero, G. Salvi, F. Cervone, and G. DeLorenzo. 1993. Mycol. Res. 97(4): 497-505. Caprari, C., C. Bergmann, Q. Migheli, G. Salvi, P. Albersheim, A. Darvill, F. Cervone, and G. De Lorenzo. 1993. Physiol. Mol. Plant Pathol. 43: 453-462. Cervone, F., G. De Lorenzo, R. Pressey, A.G. Darvill, and P. Albersheim. 1990. Phytochemistry 29: 447-449. Clay, R.P., C.W. Bergmann, and M.S. Fuller. 1995. Plant Physiol. Submitted. Stotz, H.U., C.W. Bergmann, A.L.T. Powell, J.J. Contos, P. Albersheim, A.G. Darvill, and J.M. Labavitch. 1994. 7th International Symposium on Molecular Plant-Microbe Interactions, Edinburgh, UK. Stitt, M. and U. Sonnewald. 1995. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46:341368.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases
9 1996 Elsevier Science B.V. All rights reserved.
283
Erwinia pectate lyase differences revealed by action pattern analyses Stefan Bartling a, Patrick Derkx a,b,Christina Wegener c and Ole Olsen a a
Carlsberg Laboratory, Departmentof Physiology, Gamle CarlsbergVej 10, 2500 Copenhagen-Valby, Denmark
bWageningen Agricultural University, Section MolecularGenetics of Industrial Microorganisms, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands c Institute for Stress Physiologyand Qualityof Raw Materials, Institutsplatz, 18190 Gross L0sewitz, Germany Abstract
Erwinia bacteria cause soft-rot diseases of host plants by secretion of cell wall degrading enzymes, including pectate lyase (PL) isoenzymes. Sequencing of DNA fragments from Erwinia carotovora subsp, atroseptica established the genomic organization of three tandemly arranged pel genes encoding PLs. Expression of the individual genes in Escherichia coil followed by purification of the corresponding recombinant enzymes yielded 7-33 mg PLs (I culture) 1 suitable for kinetic analyses. Apart from their natural substrate pectate, the isoenzymes also degraded 31% and 68% esterified pectins. Although individual PL activities towards 68% esterified pectin were low, the presence of PL3 in enzyme combinations enhanced the activity by up to 64%. Analyses employing high performance anion exchange chromatography confirmed these findings and revealed that particularly pentamers, or heptamers, produced by the actions of PL1 and PL2, were further depolymerized by PL3. In line with this, individual PL isoenzymes were shown to exhibit distinctive action patterns. 1. INTRODUCTION
The cell walls of higher plants consist of cellulose fibrils embedded in a matrix of pectic substances, hemicelluloses and proteins. Pectins also constitute major functions in the middle lamellae by cementing adjacent primary cell walls (1). Accordingly, depolymerization of pectic components causes cell separation, release of cell wall bound proteins and leakage of the cell contents, resulting in complete lysis. Erwinia carotovora subsp, atroseptica (Eca) is a bacterial phytopathogen that invades plant tissues, predominantly potatoes, by pectolytic, cellulolytic and proteolytic degradation of its host causing blackleg and soft-rot diseases (2). Given the heterogeneous structure of pectin, its enzymatic degradation requires the synergistic action of pectin methyl esterase (PME), pectin lyase (PNL),
284
polygalacturonase (PG) and pectate lyase (PL). Bacterial synthesis of enzyme isoforms may additionally help to ensure the degradation of pectin in a variety of tissues or plants at various developmental stages. PLs, which are major determinants for the virulence of erwinias, have been distinguished from PNLs by their specificity for unesterified homopolygalacturonan regions in pectate (3). Both enzymes degrade the substrates by 13-elimination resulting in an unsaturated C-4--C-5 bond at the nonreducing end of the o~-1,4 linked products (4). Despite the differences, sequence comparisons imply similarities between the three-dimensional protein structures (5). Several homologous and recombinant PL enzymes have been purified for use in tissue maceration analyses, but there are limited data available regarding their mode of action. With the aim of studying the'biochemical properties of different isoforms of Eca PLs and elucidating their role in pathogenesis, we have initiated a systematic analysis of the PLs that are expressed during the infection by the bacteria. Eca genomic DNA spanning a cluster of three tandemly organized, individually regulated pel genes was examined. The properties of the corresponding proteins, PL isoenzymes 1, 2 and 3, were characterized after heterologous expression in Escherichia coli (6). Notably, the results revealed that the highly similar PLs depolymerize both pectate and pectin. Combinations of the enzymes were accordingly examined for synergism towards various pectins. Compared to incubations with the single enzymes, enzyme mixtures comprising PL3 were found up to 64% more active towards pectin of 68% esterification. In this report we describe the kinetics of pectate and pectin degradation by Eca PL isoenzymes. Moreover, action pattern analyses using various pectic substances provide further insights into specific differences among the isoenzymes. 2. MATERIALS AND METHODS 2.1. Cloning and heterologous expression of Eca pectate lyase genes Isolation and cloning of partially Sau3AI digested Eca genomic fragments, DNA sequencing, cloning of the coding regions of pell, pel2 and pel3 into the E. coil highexpression vector pT7-7 as well as production, purification and analyses of the recombinant PLs were as described before (6). 2.2. Studies on enzyme kinetics, substrate specificity and synergism Product formation by the action of PL was followed in a Model 8452A (HewlettPackard) diode array spectrophotometer equipped with a thermostat. Unless otherwise indicated, the experiments were carried out at 25~ during time courses of 2-5 min by monitoring the increase in absorbance at 236 nm caused by the formation of unsaturated products by PL. Pectic substances used were polygalacturonic acid (PGA), 31%, 68% or 93% esterified citrus pectin (all from Sigma). Enzyme assays were performed using a 100-mM Tris-HCI buffer, pH 8.5 (pH 8.0 for PL2) containing 0.1 mM CaCI2. To avoid breakdown of the pectic compounds at alkaline pH, the substrate solutions were prepared immediately prior to the measurements. An increase of 5.2 A23s units per min corresponds to the
285
formation of 1 l~mol unsaturated products per min or one unit (U) (7). Enzyme samples of 15 !~1 containing 0.04 U were added to solutions containing 985 1~1 substrate. For determination of KM and Vrnax,the PL isoenzymes were incubated with 0.02-0.10% (w/v) PGA or 31% esterified pectin. Subsequent calculations employed the direct linear plot (8). For analyses of substrate specificity and synergism, 0.1% (w/v) substrate was used. 2.3. High performance anion exchange chromatography (HPAEC) HPAEC analyses were carried out to determine the oligomeric products released from various pectic substrates after depolymerization by the PL isoenzymes. Action pattern analyses for the concerted action of PL isoenzymes utilized 68% esterified pectin as substrate. One-ml reaction mixtures in a buffer system as detailed in section 2.2. comprising 0.5% (w/v) substrate and 5 U of enzyme were incubated for 30 s to 18 h, and then thermoinactivated. Samples of 750 pl were applied to a Carbopac PA-1 (Dionex) column before the carbohydrates were eluted over a period of 70 rain using a gradient of 0.2 M KOH, 0.05 M K-acetate to 0.2 M KOH, 0.7 M K-acetate. Detection employed a Pulsed Electrochemical Detector (PED, Dionex) in the integrated amperometry mode according to the manufacturer's recommendations. 3. RESULTS AND DISCUSSION 3.1. Structure and organization of the pelgenes Screening of E. coli cells transformed with Eca genomic DNA fragments demonstrated the presence of several genes encoding PL. Alignment of overlapping DNA fragments yielded the genomic organization for a contiguous sequence of 7.5 kb encoding three PL isoenzymes (Fig. 1); alignment of the open reading frames revealed a similarity of ~80%. Unlike Erwinia chrysanthemi (Ech) in which one of its pel gene clusters encodes an acidic PL (PL A) and two alkaline forms (PL D and PL E), the triple gene cluster unveiled in Eca encodes three alkaline forms (see below). Sma I
I
i
Cla I Xho I
I
Bam HI
Eco RI
Cla I
,
Eco O 1091
Sma I
I
n~re 1 Structural organization of three pectate lyase genes in a 7.5-kbp region of Eca genomic DNA. Open reading frames encoding three PL isoenzymes are shown as open boxes with arrows indicating the direction of transcription. The locations of restriction sites used for cloning of four individual gene fragments are indicated.
286
The sequence 5' to the ATG translation initiation codon of each pel gene contains a typical ribosomal binding sequence, while segments sharing sequence similarity with binding sequences for prokaryotic -35 and -10 transcription factors (9) are located further upstream. Accordingly, each gene comprises a single independent transcription unit, further supported by the presence of sequences with high similarity to the identified consensus sequence for the binding of the Ech KdgR transcription repressor (10) which regulates genes encoding enzymes involved in pectin degradation (11). The presence of common regulatory sequences substantiates the argument that factors controlling transcription of pel genes are shared by the Eca and Ech bacteria. 3.2. Protein similarities and structural implications
Alignment of the deduced protein sequences revealed similarities of 88%, 86% and 82% for PLI:PL2, PLI:PL3 and PL2:PL3, respectively. Compared with Ech PL C - which has been crystallized and found to fold into a structure designated the parallel p-helix ( 5 ) - each Eca PL isoenzyme exhibits around 75% identity, including conserved residues important for enzyme integrity. While Cys-93, -176, -350, -373 indicate the presence of two disulfide bonds, linear stacks of amino acids in the aligned Eca PL isoenzymes have the potential of forming an asparagine ladder similar to that of Ech PL C. Moreover, Asp-131, Glu-166 and Asp-170, suggested to be involved in Ca§ and required for PL activity (5), correspond to Eca PL residues Asp-152, Glu-187 and Asp-191, respectively. Major sequence differences between Ech PL C and the investigated Eca enzymes are confined to regions shown to form loop structures protruding from the central core of the former enzyme. Accordingly, PLs synthesized and secreted by Eca are expected to fold into a conformation with close resemblance to the Ech PL C counterpart (6, 12). 3.3.
Purification of recombinant PLs
In order to analyze the biochemical properties of the Eca PLs, the protein coding regions of the pel genes were amplified by PCR and cloned in the vector pT7-7 for T7 RNA polymerase-directed expression (13). After growth of E. coil BL21(DE3)pLysS transformed with the plasmids, PL1, PL2 and PL3 were recovered from concentrated 10-1 cultures. Protein purification included ion-exchange chromatography followed by gel-filtration chromatography, yielding 159 mg PL1, 78 mg PL2 and 330 mg PL3. Analysis of the 15 N-terminal residues of the purified enzymes resulted in sequences corresponding to residues 23-37 deduced from the DNA sequences. These findings imply that Eca synthesizes PL1, PL2 and PL3 as pre-proteins. The 22-residue sequences preceding the mature N-terminus resemble typical signal sequences with a charged N-terminus, a hydrophobic core and a processing site consistent with the (-3,-1) rule for signal peptidase cleavage (14). Separation of the purified PL1, PL2 and PL3 isoenzymes by SDS-PAGE yielded single protein bands that corresponded to a molecular mass of 42 kDa (6), 4 kDa higher than calculated from the deduced amino acid sequences. Isoelectric focusing revealed a pl of >10 for each isoenzyme similar to that of the basic Ech PLs (15).
287
3.4. Substrate specificities
Plant cell walls, particularly the middle lamellae and primary cell walls, contain numerous chemically diverse pectic substances (1). Detailed analyses of potato tuber cell walls revealed that these account for approximately 50% of the total cell wall material, with around 20% of the pectins being 43% esterified (16). The unesterified form of pectin, pectate, is considered the natural substrate for PL. Highest enzymatic activity towards this compound was measured at 50~ pH 8.0 for PL2, while PL1 and PL3 exhibited optima at 40~ pH 8.5. The KM and Vmaxvalues for the PLs were analysed using various pectic substrates. Towards PGA, the kinetic d a t a - measured at 4 5 ~ hint at a significantly higher activity of PL1 compared to PL2 and PL3 (Table 1). Differences were also observed by analysing time courses for the depolymerization of pectate following addition of PL (results not shown). PL1 displayed the highest initial velocity which, however, leveled off rapidly. While PL3 degraded less substrate in the first 80 s, the total degradation after 160 s of incubation was higher. The time course for degradation of pectate by PL2 was between those of PL1 and PL3. Additional examinations of depolymerization using various esterified pectins supported this assumption. Unexpectedly, application of identical enzyme units to either PGA or esterified pectins revealed a significantly higher efficiency in degradation of 31% esterified pectin. Despite its low Vmaxvalue towards PGA, PL3 was more active in depolymerizing pectin with 31% esterification than PL1 and PL2 (Table 1). Pectin with 68% esterification was also attacked by the three enzymes, however, less efficiently, PL3 again being more active than the other two enzymes (data not shown). No degradation of 93% esterified pectin could be detected. Table 1
Kinetics of Eca PL isoenzymes using pectate and pectin 31% esterified pectin
PGA
PL1 PL2 PL3
KM (mg ml 1)
Vmax (#mol min 1 mg -~)
KM (mg ml 1)
Vmax (#mol min 1 mg ~)
0.27 0.30 0.27
846 641 634
0.20 0.20 0.10
1228 1041 2115
Intrigued by the finding that Eca PLs exhibit notable differences in their kinetics, HPAEC analyses were carried out to examine the products from the depolymerization of PGA and 31% esterified pectin. After 18 h of incubation with PGA, PL1 and PL2 had produced mainly di- and trimers. Similarly, main products of PL3 action were trimers, followed by dimers. Moreover, it was the only enzyme found to produce monomers from unesterified substrates with a degree of polymerization ___3.Using 31% esterified pectin as a substrate, similar end products were released by the PLs as from PGA. In addition to the products described, traces of tetra- up to octamers were detectable. While PL1 and PL2 released di- and trimers at almost
288
equal amounts from both PGA and 31% esterified pectin, quantities of trimers were again most substantial after PL3 action. Further evidence for the novel finding that pectate lyases depolymerize pectin more actively than pectate was provided by examination of scanning electron micrographs (6). Separate application of the three Eca PLs to potato tuber tissue not only degraded the middle lamellae, but also effected disintegration of pectic components localized in the primary cell wall. Moreover, the isoenzymes revealed activity differences in the degradation of potato tuber pectic material depending on the plant variety used. While the action of PL3 resulted in a breakdown of the middle lamellae, incubation of tuber tissue with PL1 or PL2 gave rise to disintegration of single cells and a subsequent liberation of the cell contents. These results support the notion that Eca PLs exhibit major roles for the depolymerization of pectins of intermediate esterification in vivo, thus being specially adapted to the chemical composition of potato tuber cell walls. Possibly, various individual isoenzymes degrade pectin subdomains of diverse cell wall types. Since the sequence motif (S/A/T)XXhWVDHXXh (h representing I, L or V, and X any amino acid), suggested to be part of the catalytic site or the substrate binding in several PL or PNL enzymes (17), is highly conserved in the pectin and pectate degrading Eca PL1, PL2 and PL3 at the positions 159-169, other subtle amino acid substitutions in substrate binding or catalytic residues may explain the differences in their substrate specificities. This is supported by the hypothesis that the substrate binding site of Ech PL C is located in the structure formed by loops 1 to 6 (18). Provided that similar loops are also involved in substrate binding of the Eca PLs, differential degradation of pectins by individual isoenzymes might be delineated to amino acid differences in these domains. The degradation of pectin is initiated by the action of PME, an enzyme removing methylester groups from highly methoxylated pectins. Production of incompletely deesterified pectin, probably due to end-product inhibition of PME (19, 20), may explain that an affinity of PL for both pectate and pectin is required for full pathogenicity of the bacteria. An intriguing environmental feature of an Eca invasion in potatoes is the change from pH 5.0 in fresh tissue to pH 8.5 in the infected tissue after --72 h. We propose that the involvement of PLs in the degradation of pectin is an evolutionary consequence of the alkalinization which inactivates PG [optimal activity for Eca PG is at pH 6.0 (unpublished results)]. Moreover, secretion of PL isoenzymes may ensure successful biological activity of Eca in diverse types of host cell walls. 3.5. Synergistic action between Eca PLs
Synergism is evident not only between enzymes utilizing identical substrates [e.g. cellulases (21, 22)], but also among enzymes with different specificities. For degradation of cell walls, strong synergism was found between pectolytic and cellulolytic enzymes (23, 24). Since naturally occuring pectins are structurally heterogeneous regarding side chains and degree of esterification, synergism among pectolytic enzymes displaying either identical or different modes of action might facilitate pectin depolymerization (19). To test this possibility the effect of exogenous PLs on the depolymerization of pectin was examined. All possible PL enzyme
289
combinations were added to solutions containing either PGA or pectin, followed by determination of product formation. Table 2 shows activity values of 70-90% of the sum of the single enzyme activities when using PGA or 31% esterified pectin as a substrate. Although the monoenzymic degradation of 68% esterified pectin was low compared to the less esterified compounds, combination of some PLs revealed significantly enhanced depolymerization of the substrate. Notably, synergism only occured in enzyme combinations comprising PL3, resulting in up to 64% increase in activity. Supplementation with PL1 and PL2 together showed no higher degradation of pectin compared to depolymerization by the individual enzymes (Table 2). Table 2
Synergistic depolymerization of pectic components by combined Eca PLs Synergism
PL1/PL2 PL1/PL3 PL2/PL3 PL1/PL2/PL3
PGA
31% esterified pectin
68% esterified pectin
0.89 0.99 0.83 0.92
0.78 0.72 0.73 0.67
0.96 1.37 1.32 1.64
Synergism is calculated by dividing the measured activity (enzyme combinations) by the expected activity (individual activities, data not shown). Values >1 indicate positive synergism. More detailed information was obtained from inspection of the time-course for product formation following degradation of pectin with enzyme combinations. Supplementation with PL1 and PL2 together caused high initial activities followed by a significant reduction after around 150 s. Further addition of PL1 or PL2 after 160 s effected no increase in product formation, probably due to exhaustion of available substrate. Alternatively, supplementation with PL3, either initially or after 160 s, stimulated a pronounced enhancement of pectinolysis. To obtain insight into the action pattern of pectin degradation by PL combinations, time-course experiments were carried out to analyse by HPAEC the products released from 68% esterified pectin. Supporting the findings described above, enzyme mixtures initially containing PL3 caused a notable degradation of 68% esterified pectin (Fig. 2a, b). Supplementation of the enzyme mixtures with PL1 or PL2 after 120 s of incubation showed no or only little enhancement in the depolymerization (Fig. 2a, b). Compared with mixtures comprising PL3, product formation by the combination of PL1 and 2 leveled off rapidly. Supplementation of PL3 after 120 s of incubation resulted in an increased formation of tri- and dimers (Fig. 2c). It is notable that the amount of oligomers, produced by the action of PL1 and 2 decreased upon addition of PL3, suggesting PL1 and PL2 to cleave pentamers that function as substrate for PL3 (Fig. 2c), off their polymeric substrate.
290
60 a
40-
i
i
I
i
I
I
l
i
""
I
1
L3
20
P
0
10" 0 "P r p + L1
' 2O0
'
300
60
g 402
PL1 + PL3
20
13_
+PL2
200
300
200
300
60, 40-
PL1 + PL2 I
_
_.--
20,
o
1;o
PL3
Time (s) Rgure 2 Action pattern analyses of pectin degradation. HPAEC data of oligomers released from 68% esterified pectin by combinations of Eca PLs are graphically represented. Arrows indicate addition of the third enzyme. Products with degrees of polymerization ranging from 2 to 9 were detected. The graphs illustrate the generation of dimers (A), trimers ( I ) and pentamers (~).
291
These data show that PL1 and PL2 generate products which can be further degraded by PL3, implying differences in substrate binding and action patterns among the isoenzymes, possibly based on structural differences in the loop regions of the enzymes. Thus, PL3 is likely to be less affected by structural features of the substrate, such as esterification and sidechain length. Because of the relatively low activity towards 68% esterified pectin, it remains to be established if these observations are of importance for the degradation of pectin in vivo. 3.6. Conclusion
To date, over 50 pel sequences have been determined. Functional analysis of the corresponding enzymes should help to further clarify the functional role of conserved residues within the pectate lyase/pectin lyase family. The results of the present study indicate not only that Eca PLs are good model enzymes in the elucidation of the structure-function relationship of pectolytic enzymes with special emphasis on protein-pectin interactions, but also that they have the potential for use in biotechnology. Aspects of possible application are provided in the accompanying contribution by C. Wegener et al. describing cell maceration and enhancement of resistance against pathogen attacks by Eca PL3 in transformed potato plants. The involvement in vivo of Eca PLs in degradation of esterified pectins in the presence of PME and PNL remains to be elucidated.
4. ACKNOWLEDGEMENTS
Diter von Wettstein is thanked for support and encouragement, Ib Svendsen for protein sequencing and Dr. Jacques Benen, Wageningen Agricultural University, for providing unsaturated oligogalacturonides. Eva Gertman is acknowledged for excellent technical assistance. This work was financially supported by grant 9315048 of the Danish Research Council to professor D. von Wettstein. 5. REFERENCES
1 Varner, J. E. and Lin, L.-S. (1989). Plant cell wall architecture. Cell 56, 231-239. 2 P6rombelon, M. C. M. and Kelman, A. (1980). Ecology of the soft rot Erwinias. Ann Rev Phytopathol 18, 361-387. 3 Collmer, A. and Keen, N. T. (1986). The role of pectic enzymes in plant pathogenesis. Ann Rev Phytopatho124, 383-409. 4 Rombouts, F. M. and Pilnik, W. (1980). Pectic enzymes. In Economic Microbiology. Vol. 5: Microbial enzymes and bioconversions, pp. 228-282. Edited by A. H. Rose. New York: Academic Press. 5 Yoder, M. D., Keen, N. T. and Jurnak, F. (1993). New domain motif: The structure of pectate lyase C, a secreted plant virulence factor. Science 260, 1503-1506.
292
6 Bartling, S., Wegener, C. and Olsen, O. (1995). Synergism between Erwinia pectate lyase isoenzymes that depolymerize both pectate and pectin. Microbiology 141,873-881. 7 Nasuno, S. and Starr, M. P. (1966). Polygalacturonase of Erwinia carotovora. J Biol Chem 241,5298-5306. 8 Cornish-Bowden, A. and Eisenthal, R. (1978). Estimation of Michaelis constant and maximum velocity from the direct linear plot. Biochim Biophys Acta 523, 268-272. 9 Studnicka, G. M. (1987). Nucleotide sequence homologies in control regions of prokaryotic genomes. Gene 58, 45-57. 10 Nasser, W., Reverchon, S., Condemine, G. and Robert-Baudouy, J. (1994). Specific interactions of Erwinia chrysanthemi KdgR repressor with different operators of genes involved in pectinolysis. J Mol Bio1236, 427-440. 11 Hugouvieux-Cotte-Pattat, N. and Robert-Baudouy, J. (1992). Analysis of the regulation of the pel BC genes in Erwinia chrysanthemi 3937. Mol Microbiol 6, 2363-2376. 12 Wegener, C., Bartling, S., Olsen, O., Thomsen, K.K., Bahlow, R. and von Wettstein, D. Differences in cell wall degradation patterns by Erwinia carotovora pectate lyase isoenzymes. Submitted to Protoplasma. 13 Studier, F. W., Rosenberg, A. H., Dunn, J. J. and Dubendorff, J. W. (1990). Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185, 60-89. 14 von Heijne, G. (1985). Signal sequences. The limits of variation. J Mol Biol 184, 99-105. 15 Ried, J. L. and Collmer, A. (1986). Comparison of pectic enzymes produced by Erwinia chrysanthemi, Erwinia carotovora subsp, carotovora, and Erwinia carotovora subsp, atroseptica. Appl Environ Microbio152, 305-310. 16 Weber, J. (1976). Untersuchungen 0ber Zellwandgehalt und -zusammensetzung der Kartoffelknollen. Biochem Physiol Pflanzen 169, 589-594. 17 Barras, F., van Gijsegem, F. and Chatterjee, A. K. (1994). Extracellular enzymes and pathogenesis of soft-rot Erwinia. Annu Rev Phytopatho132, 201-234. 18 Lietzke, S.E., Yoder, M.D., Keen, N.T. and Jurnak, F. (1994) The threedimensional structure of pectate lyase E, a plant virulence factor from Erwinia chrysanthemL Plant Physiol 106, 849-862. 19 RexovA-BenkovA, L. and Markovic, O. (1976). Pectic enzymes. In Advances in carbohydrate chemistry and biochemistry 33, pp. 323-385. Edited by R. S. Tipson and D. Horton. New York: Academic Press. 20 Pitk&nen, K., Heikinheimo, R. and Pakkanen, R. (1992) Purification and characterization of Erwinia chrysanthemi B374 pectin methylesterase produced by Bacillus subtilis. Enzyme Microb Technol 14, 832-836.
293
21 Irwin, D. C., Spezio, M., Walker, L. P. and Wilson, D. B. (1993). Activity studies of eight purified cellulases: specificity, synergism, and binding domain effects. Biotechnol Bioeng 42, 1002-1013. 22 Vincken, J.-P., Beldman, G. and Voragen, A. G. J. (1994). The effect of xyloglucans on the degradation of cell-wall-embedded cellulose by the combined action of cellobiohydrolase and endoglucanases from Trichoderma viride. Plant Physiol 104, 99-107. 23 Renard, C. M. G. C., Searle-van Leeuwen, M. J. F., Voragen, A. G. J., Thibault, J.-F. and Pilnik, W. (1991a). Studies on apple protopectin. I1. Apple cell wall degradation by pure polysaccharidases and their combinations. Carbohydr Polymers 14, 295-314. 24 Renard, C. M. G. C., Schols, H. A., Voragen, A. G. J., Thibault, J.-F. and Pilnik, W. (1991b). Studies on apple protopectin. II1. Characterization of the material extracted by pure polysaccharidases from apple cell walls. Carbohydr Polymers 15, 13-32.
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J. Visser and A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996Elsevier Science B.V.All fights reserved.
295
Functional Implications of the Three-Dimensional Structures of Pectate Lyases F. Jurnak a, N. Kita b, M. Garrett c, S.E. Heffron a, R. Scavetta a, C. Boyd d and N. Keen d
aDepartment of Biochemistry University of California Riverside, California 92521 bKanagawa Institute of Agricultural Science 1617 Kamikisawa Hiratsuka, Kanagawa 259-12, Japan c244 Manville Ave. Bowling Green, Ohio 43402 dDepartment of Plant Pathology University of California Riverside, California 92521
Abstract
The three-dimensional structures of two Erwinia chrysanthemi pectate lyases, PelC and PelE, have been refined to a resolution of 2.2 A. A superposition of the two structures has been used to correct the multiple sequence alignment of the extracellular pectate lyase superfamily. The corrected alignment has revealed two clusters of 'potentially catalytic' invariant amino acids, compatible with two active sites. Site-directed mutagenesis studies have confirmed that the pectinolytic active site includes the region around the Ca 2+ binding site. Furthermore, mutagenesis studies suggest catalytic roles for individual amino acids. Although PelC and PelE are structurally similar in the overall fold of the polypeptide backbone, there are significant differences in the size and conformation of the loops that comprise the pectinolytic active site. The differences in the surface charges between PelC and PelE in the groove extending from the Ca ~+ site suggest that the optimal oligosaccharide substrates are different for each enzyme. 1.
INTRODUCTION
Extracellular pectate lyases are virulence factors produced by plant pathogens that cause tissue maceration and cell death [1-4]. The enzymes are presumed to function by cleaving the a-l,4-polygalacturonic acid (PGA) component of plant cell walls. Among the most studied are the Erwinia chrysanthemi enzymes which are coded by multiple gene families that are independently regulated by complex mechanisms [5-7]. The extracellular isozymes can be grouped into two major subfamilies, pelBC and pelADE, that are characterized by their pI values and number of disulfide bonds. Within each subfamily, the sequence homology is 60% or greater but, between the subfamilies, the sequence homology is as low as 20%. Although the enzymatic mechanisms for all pectate lyases are believed to be similar, subtle differences between the subfamilies have been reported [8]. The peIADE subfamily cleaves PGA to a dimer and the pelBC subfamily produces a trimer as the major product. In addition to the sequence homology among the extracellular pectate lyases, the enzymes share sequence similarity with fungal pectin lyases as well as with plant pollen and style proteins [9]. The pectin lyases are related in function, cleaving pectin, a neutral methylated form of PGA. The function of the plant pollen and style proteins has not been elucidated but it is presumed to be important in development. Although it is generally
296 assumed that the plant proteins have pectinolytic activity, given the sequence similarity with the pectate lyases, the assumption has been proven for only one plant pollen homologue to date [10]. In contrast to the extensive genetic information, there is relatively little known about the details of the enzymatic mechanism of the pectate lyases. The presumed substrate is PGA, a plant cell wall component that is also commercially available. Since the late 1950's, it has been postulated that pectate lyases utilize a #-elimination mechanism to cleave PGA by exo- and endolytic mechanisms, resulting in an unsaturated bond between Ca and Cs at the nonreducing end of the polysaccharide [11]. Ca 2 § is required for in vitro pec'tinolytl'6 activity, but ils role is unknown. Prior to the structural studies, it had been commonly assumed that Ca z § bound only to PGA, inducing a substrate conformation that could be recognized by the enzyme [12]. The pH optimum of in vitro pectinolytic activity for all pectate lyases is rather high, ranging from pH 8 to pH 10. No amino acids had been identified that participated in catalysis or in saccharide recognition. Furthermore, there have been no reports of inhibitors which could be used to probe details of the enzymatic mechanism. Given the paucity of biochemical information, it is not surprising that the pectinolytic active site could not be delineated unambiguously from the three-dimensional structures of pectate lyases.
The E. chrysanthemi pel ~enes have been cloned and inserted into plasmid constructs with convenient properties for protein overproduction and purification [13-14]. The purity and quantity of the recombinant forms have facilitated structural studies. Although the recombinant enzymes are isolated from the periplasm of Escherichia coli, they have in vitro properties that are the same as the natural forms secreted from E. chrysanthemi. Two representative pectate lyases from different subfamilies, PelC and PelE, have been the focus of structural studies. PelC is composed of 353 amino acids with a molecular mass of 37,676 daltons and two disulfide bonds, Cys72-Cys155 and Cys329Cys352 [14]. PelE is composed of 355 amino acids with a molecular mass of 38,118 daltons and one disulfide bond, Cys291-Cys320 [7]. Although the enzymes crystallize readily, both require the presence of sulfate anions for large, ordered crystals suitable for X-ray diffraction studies [15-16]. 2.
METHODS
PelC was purified from the periplasm of E. coli cells containing the high-expression construct pPEL410 of the E. chrysanthemi EC16 pelC gene [7] and crystallized from ammonium sulfate [16]. PelE was purified in a similar manner using the high expression construct pPEL748 of the EC16 pelE gene [14] and crystallized from polyethylene glycol in the presence of lithium sulfate [15]. The PelC mutants were prepared by site-directed olig.onucleotide mutagenesis using PCR by overlapping extension methods [17-18]. The various PelC mutant proteins were prepared by overexpression of the respective genes cloned in pINK1 plasmid constructs expressed in Escherichia coli HB101 or in pRSET5A plasmid constructs expressed in E. coli HMS174(DE3). The pectinolytic activity of PelC wildtype and mutants was determined by monitoring the absorbance increase at 232 nm of a 1 ml reaction mixture containing sodium polypectate at 22 oC as previously described [13]. For each mutant, the Ca 2+ concentration was optimized to gwe a maximum specific activity. Protein concentration was determined by the method of Bradford [19]. Plant tissue maceration assays were done using mesocarp_ tissue of cucumber fruit according to the method of Mussell and Morre [20]. The Ca~z+ affinity for the PelC wildtype and mutants was determined by monitoring the changes in the intrinsic tryptophan fluorescence of PelC in response to the addition of Ca 2§ using a SPEX 112 Fluorolog spectrofluorimeter with a 150 Watt Xenon Arc lamp as source.
297 The three-dimensional structures of PelC and PelE were solved using multiple isomorphous replacements methods as previously described [21-22]. The PelC and PelE structures were superimposed using the command, 'lsq_exphcit ' in the program O [23]. The PILEUP multiple sequence alignment program o-f the GCG package was used to create the alignment of the extracellular pectate lyase superfamily as described previously [9]. All structural figures were generated by MOLSCRIPT [24]. 3.
RESULTS
The first three-dimensional structure of a pectate lyase, that of E. chrysanthemi PelC, revealed a novel topology, termed a parallel ~ helix [21]. The polypeptide backbone folds into parallel # strands that are wound into a large right-handed coil. Each level of the coil is composed of three distinct # strands, each of which forms backbone hydrogen bonds with neighboring # strands of adjacent levels. The overall appearance of the secondary structure resembles that of three parallel # sheets as shown in Figure 1A. Loops of various size and conformation protrude from the core structure. Another striking feature of the PelC structure is the high degree of internal organization. Every side chain that is oriented towards the center of the core participates in a stacking interaction with its neighbor [25]. There are extended hydrophobic stacks composed of valine, alanine, isoleucine or leucine; polar stacks of asparagme or serine; and aromatic stacks resembling DNA base-pair Interactions. Prior to the PelC structure, there were no biochemical data which identified any amino acids that participate in pectinolytic activity. Therefore, the active site could not be deduced unambiguously from the structural results alone. However, the structure provides two clues as to which region of PelC might participate in catalysis. First, the environment surrounding each of two heavy atom derivatives is compatible with that of a Ca 2 § binding site. The uranyl and Lu 3+ derivatives are coordinated directly to the enzyme through two carboxyl oxygens from Aspl31 and one carboxyl oxygen from each of Glu166 and Aspl70. Secondly, the putative Ca 2§ site is located in a narrow, highly charged groove, approximating the shape and length of 9-12 saccharide units of galacturonic acid as shown in Figure lB. The groove represents the only localization of charged amino acids on the surface of PelC and as a consequence, has been proposed as the substrate binding site. The spatial distribution of the positively-charged amino acids on PelC is particularly suggestive of an oligogalacturonic acid binding site. The periodicity of negative-charges on oligogalacturonic acid is anticipated to necessitate a pectate lyase-substrate interaction that is primarily stabilized by regularly-spaced electrostatic interactions. In 1994, two additional pectate lyase structures were reported, Bacillus subtilis Pel [26] and E. chrysanthemi PelE as shown in Figure 2A [22]. Both fold into the novel # helix topology with extensive stacking interactions among the internal side chains. The structure of B.s. Pel is complexed directly to Ca 2+ which is located at the .putative Ca 2+ site proposed for PelC. There is a groove in all three structures in the region of the proposed pectinolytic active site. However, a comparison of the PelC and PelE structures has revealed that, unlike PelC, the charged amino acids are randomly distributed over the entire surface of PelE as shown in Figure 2B. The difference in the distribution of the surface charges suggests that either the proposed pectinolytic active site region is incorrect or the ootimal substrate is not oligogalacturonic acid for both PelC and PelE. In particular, the surf'ace charges surrounding the Ca 2§ site in PelE are compatible with a substrate length of 3-4 adjacent galacturonic acid units, not 9-12 negatively charged units compatible with the PelC structure.
298
,.:%"-,-.
.... _
-:k--
A
II~
".\
il
B
Figure 1. The three-dimensional structure of PelC. A. A schematic diagram illustrating the major secondary structural features of the PelC polypeptide backbone. The three parallel/~ sheets are represented by arrows in light, medium and dark gray. B. Space-filling models of PelC. Neutral amino acids on the surface are shown in gray, negatively-chargec~ residues in white and the positively-charged residues in black. The location of the Ca: + is illustrated by cross-hatching.
A
B
Figure 2. The three-dimensional structure of PelE using the same view as Figure 1. A. A schematic ribbons diagram illustrating the major secondary structural features of the PelE polypeptide backbone. The three parallel ~ sheets are represented by the arrows in light, medium and dark gray. B. Space-filling models of PelE. Neutral amino acids on the surface are shown in gray, negatively-char~ed residues in white and the positively-charged residues in black. The location of the Ca 2 u is illustrated by cross-hatching.
299 In order to uncover additional clues as to the location of the pectinolytic active site, the structures of PelC and PelE have been superimposed and compared in atomic detail [9]. As shown in Figure 3, those aCs which superimpose within a deviation of 1.5 A or less comprise the backbone of the parallel ~ helix. Those aCs which deviate by 1.5 A to 3.0 A are located on one side of the core and comprise three to four loops, including the N- and C-terminal branches. The structural regions which differ by more than 3.0 2k include the loops protruding from the parallel/3 helix in the vicinity of the Ca 2§ binding site and the proposed pectinolytic active site. The results were ominous because the active site regions of functionally-related proteins are generally the most conserved structurally. Yet for the three known pectate lyase structures, the proposed active site region is the most diverse! In addition to an atormc comparison, the superposition of the PelC and PelE structures highlighted errors in the multiple sequence analysis of the extracellular pectate lyase superfamily based primarily upon evolutionary relationships [4,6]. The superfamily includes the extracellular pelBC and pelADE subfamilies, the fungal pectin lyases and the plant pollen proteins [9]. Approximately 252 amino acids, or the regions ranging from Gly6 to Phe257 in PelC nomenclature, could be aligned with confidence based on highly probable structural similarities. In the corrected sequence alignment, 10 amino acids are mvariant among all superfamily members. Such results are significant because amino acids which remain unchanged during evolution usually have a critical functional role. In PelC nomenclature, the 10 invariant amino acids include Gly6, Glyl2, Glyl3, Aspl31, Trp142, Asp144, His145, Thr206, Arg218 and Pro220 [9]. In addition to the latter 10 invariant amino acids, another 14 are invariant within the extracellular p.ectate lyase family [27], but not within the pectin lyose or plant pollen/style protein families. These 14 amino acids include three in the Ca ~+ binding site, Aspl70, Lysl90 and Arg223, as well as 11 in the vWiDH region: Thrl0, Thr92, Gly95, Asnll7, Thr179, Serl81, His228, Asn232, Ala338, Gly339 andLys342.
.:-
:..
..
...
Figure 3. A comparison of the a C backbone of PelC with PelE. The a Cs which superimpose within a root-mean-square deviation of 1.5 A are shown in black and those, within 3.0 A are shown in dark gray. The remaining backbone regions are shown in light ra),. The largest structural differences occur in the loops capping one end of the parallel ehx as well as in those comprising the putative substrate binding groove.
300 The locations of the set of 10 invariant amino acids cluster into two distinct regions as shown in Figure 4A and 4B. The first cluster includes Asol31, Arg218 and Pro220, all located in the vicinity of the Ca 2§ binding site. The remaining 7 amino acids are located on the opposite side of the parallel ~ helix, centered about the region containing the vWiDH pattern. Both clusters contain subsets of invariant amino acids which have chemically reactive properties and thus are potential candidates for catalytic residues. The finding suggested that either the pectate lyase superfamily members have two catalytic functions or the pectinolytic active site comprises two distinct regions, one for properly aligning the substrate via an interaction with t2az § and the other for catalyzing the cleavage reaction. The latter possibility has been eliminated by considering the distances separating the two regions. The shortest distance between the two clusters is approximately 25 2k, a distance which could accommodate binding of one end of an oligomer of 5-6 saccharide units at the Ca 2+ binding site and the other end at the vWiDH region. However, to span the shortest distance, the substrate would need to pass through the center of the parallel helix, a very unlikely possibility as a consequence of the extensive internal stacking interactions and the extensive hydrogen bonding network stabilizing the parallel ~ helix core. If the substrate were to span from one region to the other, then it is more probable that the substrate would lie on the outside of the parallel # helix core, but then the distances would be 95 2k. Such a distance would require that pectate lyases cleave substrates of 18-22 saccharide units in length. This analysis has led to the conclusion that the pectate lyase superfamily members have two active sites, one for pectinolytic function and the other as yet unidentified.
...............
...
........
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:
...........
..
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A
.
B
Figure 4. Superposition of the invariant amino acids in the superfamily upon the c,C tracing of PelC. A. Side view, with Aspl31, Arg218 and Pro220 o~the Ca z+ binding site on the left and Gly6, Glyl2, Glyl3, Trp142, Asp144, His145 and Thr206 of the vWiDH r e . o n on the right. B. Top view, looking down the axis of the parallel ~ helix, with the Cffz+ binding site on the left and the vWiDH region on the right. The loops covering the opening to the core of the parallel ~ helix have been removed for clarity.
301
To determine which site is the pectinolytic active site and what the function of the second site might be, many site-specific mutants of PelC have been prepared and characterized. Table 1 lists the most interesting mutations made in the region of the Ca 2 § binding site. All mutants in Table 1 crystallized isomorphously with wildtype PelC, indicating that the mutation did not result m a loss of structural integrity. In all mutants, the maceration activity on plant tissue parallels the observed pectinolytic activity, supporting the widely-held hypothesis that pectate cleavage is responsible for the observed "soft2+rot" damage. Because C~tz+ is required for m" vitro" pectinolytic activity, the affinity of Ca for the mutant has been quantitated by an intrinsic tryptophan fluorescence assay. As expected, pectinolytic activity is significantly reduced or abohshed in those mutations
ai
i~
(Lys172, Lys190, Arg218).
Table 1 PelC mutants near Ca 2 + binding site Tissue Maceration
%
' Max. Specific Activity %
Wildtype 1oo :too Mutations in Amino Acids Directly Coordinated to Ca 2 +:
Ca 2. Affinity
%
:too
DI31E DI31N EI66D EI66Q DI70N
<
<
14 76 39 113 44
DI29N KI72R KI90A R218K R218L R223A
50 69 <
48 45
67 40 12 ii 7 ?
Mutations in Amino Acids Linked to Ca 2+ through a Water Network:
The results for two mutants, D131N and E166Q, are particularly insightful because ' ' ' pectino 1 ytic activity, but not. Ca, 2 + affimt~r ~s abolished..These results, suggest that. Asp.131 and Glu166 have properUes which are critical for catalys~s. Not only is Asp131 an mvanant amino acid in the pectate lyase superfamily, but the loss of pectinolyuc activity in two mutations at this position, D131N and D131E, implies that Asp131 has a direct catalytic function. In contrast, Glu166 is only a conserved residue in the pectate lyase superfamily, occasionally being replaced by an aspartic acid. Moreover, the E166D mutation retains pectinolytic activity while the E166Q mutation looses it, implying that the negative charge on the side chain is a critical property for catalysis. An inspection of the PelC structure reveals that, while one carboxyl oxygen of Glu166 is coordinated to Ca 2+, the second oxygen forms an ionic interaction with the side chain of Lys190. Notably, the K190A mutation also disrupts the ionic interaction and results in a loss of pectinolytic activity. The role of the ionic interaction in catalysis is not clear, but may function to stabilize a particular orientation of Lys190 or its neighbors during catalysis. Altogether, the results in
302 Table 1 provide definitive evidence that the region surrounding the Ca 2§ site is, indeed, the pectinolytic active site as first hypothesized from the localization of the surface charges on the PelC structure. Table 2 summarizes the major mutational results in the vWiDH region as compared 9 9 to wildtype. In contrast to the Ca 2 + bmdmg region, the results from mutations in the vWiDH region are clouded by possible artifacts relating to the overexpression systems. Using the expression vector pINK1 [14], three mutants, W142H, T206A and K343E, were exported to the periplasm and purified using procedures similar to wildtype. Two mutants, D28R and D144N, were exported, but at a greatly reduced level relative to wildtype and purified with surprising difficulty. These five mutants were shown to have near normal levels of pectinolytic activity, suggesting that the vWiDH region is not involved in catalytic cleavage of a PGA substrate. The remaining mutant proteins in Table 2, H145Q, N210S, H228Q and E253D, could not be detected in either the membrane or in the periplasmic fractions. To circumvent the possibility that proteases might be cleaving the latter mutants over an 18-hour cellular growth period using the expression vector pINK1, the plasmid construct containing the pelC or mutated gene was inserted into a more efficient T7 expression vector, pRSET5A [28]. The wildtype protein is produced in large quantities within four hours; however, only 50% of the wll-dtype protein is exported to the periplasm. The remaining 50% remains associated with the membrane fraction in the form of unprocessedpreprotein. Using the T7 expression system, high levels of H145Q, N210S, H228Q and E253D could be detected, but were localized only in the membrane fraction as both the unprocessed and mature protein forms. Both forms were extremely sensitive to trypsin, in comparison to the periplasmic wildtype form, suggestin~ that the mutants were not properly folded. Continuing efforts are being made to determine if the results are an artifact of the expression systems or if the results imply that invariant amino acids in the vWiDH region participate in a catalytic function related to the export process of the protein through the inner membrane.
Table 2 PelC mutants surrounding the vWiDH region Tissue Maceration %
Max. Specific Activity %
Wildtype loo loo MutationsinlnvariantAmino AcidsinvWiDH region:
WI42H DI44N HI45Q T206A
88 67 membrane-bound 81
97 62 in T7 expression 83
system
Mutations in Conse~ed Amino Acids in vWiDH region: D28R
N210S H228Q E253D K342E
78
membrane-bound membrane-bound membrane-bound 72
76
in T7 expression in T7 expression in T7 expression 72
system system system
303 4.
DISCUSSION
The mutagenesis results provide the strongest evidence to date that those amino acids surrounding the Ca 2+ bindlng site in PelC, but not the vWiDH region, are involved in pectinolytic activity. However there is much work to be done to elucidate the actual catalytic mechanism as well as the substrate bindin~ site. Both are targets of current investigation. With regards to pectinolytic activity, ~t is well established that the PGA substrate is cleaved by a #-elimination mechanism in which Ca 2 + is essential. As shown in Figure 5, it is expected that the enzyme contribute a minimum of three groups to the catalytic mechanism: one to neutralize the charge on the uronic acid moiety of the substrate, a second to initiate the abstraction of the proton from C~ of the primary saccharide unit and a third, to facilitate the proton transfer to the cleaved glycosidic bond. The pectate lyase structures have conclusively demonstrate0 that Ca 2 § binds directly to the enzyme, in contrast to a commonly held hypothesis that Ca z+ only binds to the substrate to induce a p~roper conformation for binding to the enzyme [12]. Nevertheless, the precise role of Ca 2+ remains to be clarified. Does Ca 2+ participate directly in catalysis, such as activating a key water molecule, or does Caz+ serve a structural role to properly align amino acids about the catalytic site? Does Ca 2+ bind only to the protein or is Ca 2+ shared by both the protein and the polysaccharide substrate in order to neutralize the negativelycharged uronic acid moiety?
coo-... P.T o
\,
H
A '
H
\,
~ O - ~ O H
OH I
I
coo-... P.+ ,1 o'
I
CO0-
~ H
H I
OH
COO0
/
/ 0
I OH
0
H
+
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I OH
Figure 5. Diagram of catalyzed cleavage of oligogalacturonic acid. The enzyme may contribute a minimum of three groups to the catalyzed cleavage reaction: (1) a positivelycharged group such as Lys190 or Ca"z+ to neutralize the charge on the uronic acid moiety adjacent to the scissile bond; (2) an amino acid, such as Asp131, to initiate the abstraction of the proton from C~; and (3) an amino acid to donate a proton to the glycQsidic oxygen of the scissle bond. Th~ extensive hydrogen bonding network around the c a z+ site suggests that several amino acids and possibly water molecules may be involved in the transfer of the proton from C 5 to the glycosidic oxygen.
304 In addition to defining the role of Ca 2§ the identification of the amino acids that participate directly in catalysis is underway. The structural-based multiple sequence alignment highlighted 5 invariant, 'potentially catalytic' amino acids in the Ca 2§ region of the extracellular pectate lyase family. To date, site-specific mutagenesis results have suggested that Aspl31 and the ionic interaction between Glu166 and Lysl90 are critical for catalysis in PelC.2+ .Mutati~ in other invariant amino acids, such as Arg218, slgnfficantlv" "" decrease Ca affimty and not surprisingly, cause a loss of pectinolytic activity. Whicfa amino acid serves as the proton acceptor from Cs and which as the proton donor to the glycosidic bond? At this point, there are still too many possibilities to answer these uestions. However, it must be kept in mind, that the pectate lyases probably share a milar enzymatic mechanism with the pectin lyases which cleave a methylated, neutral pectin substrate. In the latter enzymes, Ca e+ ~s not required for catalysis; nevertheless, Ca e+ stimulates cleavage and raises the pH optimum from less than 6 to greater than 8 [29]. Thus, any enzymatic mechanism proposedfor the pectate lyases must be valid for the pectin lyases as well. The structural-based multiple sequence all/~nment demonstrates that the pectate and pectin lyases share only two invariant amino acids in this region, Asp l31 and Arg218 in PelC nomenclature, placing a constraint on the possible catalytic amino acids. Because all known polysaccharide-cleaving enzymes utilize an aspartic or glutamic acid as a catalytic residue, it is tempting to speculate that Asp l31 is the amino acid which initiates proton abstraction, both in the pectate as well as the pectin lyases. A difficulty with the latter hypothesis is that both carbonyl oxygens of Asp l31 are coordinated to the Ca 2§ in PelC and thus, are not available or sufficiently negative for a proton abstraction function. There remains much work to be done to elucidate the atomic details of the enzymatic mechanism; as yet, there are too many possibilities. Nevertheless, as a working model .and one2+that is consistent, with the structures, we are proposing the following view" of catalysis. Ca and possibly Lysl90 serve to align and neutralize t~e negative c~arge on the uronic acid group of the saccharide unit adjacent to the glycosidic scissle bond. The neutralization function is Rot essential in the pectin lyases, so these groups are not invariant. Furthermore, Ca z+ is shared by the enzyme and the substrate, probably through the uronic acid moiety. Upon substrate binding to the Ca.2+, at least one carboxyl oxygen of Aspl31 is released from the coordination sphere, of Ca z+, freeing the oxygen to initiate proton abstraction at C5. In the absence of Ca z+ in the pectin lyases, the analogous asl?artic acid is already available for proton abstraction. Despite the key role of an aspartic acid group in catalvsis, the pH optimum of the reaction for both pectate and pectin lyases in the presence o~' Ca 2§ is quite basic possibly because Ca 2§ might alter the pK of a neighboring group, such as the C5 proton or a coordinated water molecule, from very basic to the observed 8-10 pH range. What is the probable role of Arg218? For both pectate and pectate lyase reactions, proton abstraction at C5 occurs, either with the concurrent protonation of the carbonyl group of the uronic acid moiety [30] or with thegeneration of a negative charge on an unstable enolate intermediate. Arg218 may serve to donate a proton in the former case or to neutralize the negatively-charged enolate intermediate in the latter case. Which amino acid is responsible for transferring the proton to the glycosidic bond? Given the extensive hydrogen bond network around the Ca 2+ in the pectate lyase structures, the proton transfer step may involve more than a single amino acid. The conserved hydrogen bond network consists of amino acids as well as bridging water molecules, all of which could participate in the proton transfer step. Another major question under active investigation is the elucidation of the atomic details of the polysaccharide binding site. In structural terms, it is anticipated that most of the energy derived from an interaction between pectate lyase and a negatively-charged PGA will originate from ionic bonds between the negatively-charged uronic acid moieties and the positively-charged side groups on the protein. The energy which is used to define
305 the specificity of the enzyme for the saccharide conformation will be negligible in comparison to the electrostatic interactions, but will originate primarily from hydrogen bonds between the enzyme and the hydroxyl groups on the saccharide units. Because the binding energy for a single saccharide unit is characteristically small, saccharide-binding enzymes generally require rather long substrates, six or more saccharide units in length, to gain sufficient energy for specificity. For pectate lyases, the optimal substrate length is, as yet, undetermined and will be a focus of future studies. Moreover, the structuralview of protein-saccharide interactions suggest that pectate lyases probably bind relatively strongly to, but don't cleave, other negatively-charged polysaccharides, an expectation which has been observed in preliminary studies. Because the differences in binding energies are expected to be relatively small between those polysaccharides which only bind to the pectate lyases and those saccharides which bind and are cleaved by pectate lyases, it is essential to have a quantitate measure of saccharide affinity. The differences in the pattern of surface charges between the PelC and PelE structures has led to the conclusion that the optimal substrate is not the same for both enzymes. Based on structural considerations alone, PGA appears to be an excellent substrate for PelC. Although PelE probably cleaves between two galacturonic acid residues, the PelE binding site appears to be more compatible with an oligosaccharide substrate that includes neutral saccharide units at some distance from the Ca 2§ binding site. It is not yet possible to deduce whether the neutral units might be esterified galacturonates or another type of saccharide. The significance of elucidating the optimal substrate for each isozyme is twofold. First, subtle differences in the optimal substrate may account for the existence of multiple pectate lyase isozymes within a single pathogen or for the pathogenic differences among the isozymes on different plant tissues. In order to effect the damage, the pectate lyases must first reach their target substrate in the plant cell wall. Given the irregular distribution and composition of the saccharide components in the plant cell wall, the pectate lyases may have evolved to cleave oligosaccharides of slightly different composition. For example, PelE is 10 times more effective than PelC in macerating plant tissue, possibly because PelE may be capable of binding to partially esterified segments of pectate. Such segments are likely to be present at hi.~her concentrations in the mature plant cell wall than the negatively-charged unesterified form. A second reason for elucidating the atomic details of a pectate lyase-oligosaccharide interaction is to rationally alter the substrate binding site by recombinant DNA techniques in order to produce an enzyme which cleaves any desired oligosaccharide. Like specific proteases or nucleases, specific saccharidases may be of commercial importance. The structural view of the Ca2+-dependent pectinolytic active site has raised many interesting questions, all of which appear to be solvable by standard biochemical and genetic techniques. In contrast, the mystery of the vWiDH region is proving to be more difficult to solve. Several curious structural findings include the apparent aromatic specificity pocket surrounding the conserved aromatic residue analogous to Tyr7 in PelC [27]; the structural conservation of unusual conformations in loops that fold together around the vWiDH region [27]; and the clustering of 7 invariant amino acids in the vWiDH region [9,22]. Particularly curious is the glycine-rich consensus sequence, G(Aro)a(37X)GG, in the N-terminal branch of the superfamily. A similar pattern is found in approximately 25% of all precursor proteins and may represent a type of signal for exported proteins. Although most investigations have focussed on the composition of the N-terminal signal peptide fragment, from an enzymatic and structural viewpoint, it is also logical to expect another type of signal on the mature protein side of the scissile bond. For example, during enzymatic cleavage of an internal bond, in this case the cleavage of the peptide bond between the signal sequence and the mature protein, there are generally ~roups on both sides of the sc~ssile bond that contribute to the recognition of the substrate by the enzyme. The conserved G(Aro)a(3-7X)GG sequence may represent such a signal.
306 Equally curious is the relatively large number of invariant glycines in the region. The conservation of glycines during evolution usually suggests crucial functional roles in avoiding steric clashes or in controlling conformational changes. Interestingly, a small rotation around the ~ bond of the invariant glycine preceding the conserved aromatic residue, Gly6 in PelC, brings the N terminus to the imidazole nitrogen of the invariant histidine in the vWiDH cluster. Moreover, the orientation of the invariant aspartic acid and histidine of the vWiDH sequence resemble that found at the active site of serine proteases. Together these findings suggest that the conserved aromatic residue may have some type of alignment function for a catalytic activity occurring at the vWiDH sequence. What the function of the vWiDH reigion might be is only speculative. One possibility is that the vWiDH sequence participates in cleavage of the N-terminal signal peptide, perhaps in association with another Sec-dependent enzyme. To test this hypothesis, structurally conserved mutations have been made in each of the three 'potentially catalytic' invariant amino acids in the region. T206A behaves normally but the export of D144N is significantly reduced, sugigesting that neither residue is absolutely essential for catalysis. Unfortunately, a mutation in the most likely catalytic candidate, His145 in PelC, results in a mutant that does not fold properly. Only in the efficient T7 expression system can the H145Q mutant be detected at all, localized in membrane fractions as a doublet corresponding to the molecular weights of the unprocessed and mature protein. Taken at face value, the results suggest that H145Q and other problem mutants listed in Table 2 are defective in release of the protein from the membrane or in proper folding, but not in signal peptide processing because some mature protein is found m the membrane. Because it is generally believed that the signal peptide is cleaved and the protein folded in the periplasm [31], an alternative explanation is that H145Q and other problem mutants have produced artifactual results as a consequence of the expression systems used. Another question, as yet unresolved, is the functional significance, if any, of the novel parallel # helix fold observed first in the pectate lyase family. Only two other structures are known to have this novel fold and both are oligosaccharide-binding proteins [32-33]. Preliminary analyses of amino acid sequence patterns as well as circular dichroism spectra also predict that the parallel # helix fold will be found in other protein families [34]. These famihes include pathogenic factors affecting mammalian tissue as well as the leucine-rich repeat regions of polygalacturonase inhibitors and plant disease resistance proteins [35,36]. Thus, the initial surprise of a novel protein fold in the pectate lyases may not be the last as this family of enzymes is studied in more detail. 5.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the support of the U.S. Department of Agriculture (Award 94-37303), the National Science Foundation (Award MCB94-08999) and the San Diego Supercomputer Center. REFERENCES A. Collmer and N.T. Keen, Annu. Rev. Phytopathol., 24 (1986) 383. Q
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A. Kotoujansky, Annu. Rev. Phytopathol., 25 (1987) 405. A.K. Chatterjee and A.K. Vidaver, Adv. Plant Pathol, 4 (1986) 1.
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F. Barras, F. van Gijsegem and A.K. Chatterjee, Annu. Rev. Phytopathol., 32 (1994) 201. S. Reverchon, W. Nasser and J. Robert-Baudouy, Molec. Microbiol., 5 (1991) 2203. J.C.D. Hinton, J.M. Sidebotham, D.R. Gill and G.P.C. Salmond, Molec. Microbiol., 3 (1989) 1785. S.J. Tamaki, S. Gold, M. Robeson, S. Manulis and N.T. Keen, J. Bacteriol., 170 (1988) 3468. J.F. Preston, J.D. Rice, L.O. Ingram and N.T. Keen, J. Bacteriol., 174 (1992) 2039. B. Henrissat, S.E. Heffron, M.D. Yoder, S.E. Lietzke and F. Jurnak, Plant Physiol., 107 (1995) 963.
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S.E. Heffron, B. Henrissat, M.D. Yoder, S.E. Lietzke and E Jurnak, Molecular Plant-Microbe Interactions, 8 (1995) 331.
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MOLECULAR GENETICS AND REGULATION OF PECTINASE BIOSYNTHESIS IN SAPROPHYTIC AND PHYTOPATHOGENIC MICROBIAL SYSTEMS
This Page Intentionally Left Blank
J. Visserand A.G.J. Voragen(Editors),Pectins and Pectinases
9 1996ElsevierScienceB.V.All rights reserved.
311
Regulation of pectinase biosynthesis in Erwinia
chrysanthemi N. Hugouvieux-Cotte-Pattat, S. Reverchon, W. Nasser, G. Condemine and J. Robert-Baudouy
Laboratoire de G6n6tique Mol6culaire des Microorganismes, URA-CNRS 1486, INSA Bat 406, 20 Avenue Albert Einstein, 69621 Villeurbanne Cedex, France
Abstract Erwinia chrysanthemi synthesizes and secretes a large number of pectinases. The major pectinases include a pectin methylesterase PemA and five isoenzymes of endo-pectate lyases PelA, PelB, PelC, PelD and PelE. In addition, secondary pectinases were identified: a pectin methylesterase PemB, two endopectate lyases PelL and PelZ, an exo-pectate lyase PelX and an e x o p o l y g a l a c t u r o n a s e , PehX. The regulation of pectinase synthesis is very complex and dependent on many environmental conditions. It is induced by pectin catabolic products and affected by growth phase, catabolite repression, osmolarity, iron or oxygen starvation... Three regulators were identified by genetic analysis. The main repressor, KdgR, controls the transcription of pectinase genes, the intracellular catabolic pathway and the secretion machinery. The PecS repressor controls the production of pectate lyases and cellulases, the secretion machinery and the biosynthesis of a blue pigment. PecT acts as a repressor of the production of some pectate lyases. Other proteins are involved in the regulation of pectinase synthesis but their role is not well characterized.
1. I N T R O D U C T I O N
Among the family of Enterobacteriaceae, two species of the genus Erwinia define the soft rot group: E. chrysanthemi and E. carotovora. The main characteristic of the soft rot bacteria is their ability to produce large quantities of plant cell wall degrading enzymes. The maceration of plant tissue resulting from
312
bacterial infection involves the depolymerization of pectin by a set of bacterial pectinases. In addition, E. chrysanthemi synthesises ceUulases, proteases, nucleases, a lipase...
" " "
LO-C H 3
~O-CH3
~ OOH
O-CH3 O " L
.OH
{:)H
.,OH
pH ~ P H
IOH
L OOH ~ ~r162 ""
OH
OH
pectin
polygalacturonate (PGA)
PemA
&,;
PelA, B, C,
nlA
/
D, E, L,X,Z
PehX
PemB
unsaturated and saturated oligogalacturonides "-
,
,,
:
,,
iii
II
,
,
,,
,
'
cligogalactu ronides
i!iiili
galacturonate~
5-keto-4-deoxyuronate (DKI)
~T i
,
galacturonate
uxac
tagaturonate 2,5-diketo-3-deoxygluconate ( D K I I ) ~ U x a B altronate
i!ii!ii: iiilii
ii::ill
i,i i ~:~
, xa,
2-keto-3-deoxygluconate (KDG)-"
.....
... :.!i!i!
i:i
6-phospho-2-keto-3-deoxygluconate
i!!ii
:;i'......:i:,: .
.
i~ii~ ~!ii:!
::!iiiil
KdgA pyruvate + 3-phosphogl.vcsraldehyde
iii!i!~. ! (cytoplasm)
.
.
~:",,.(,bacterialcellwall~. " ", ........... (external medium)
T .=~!~! ,,, :i:i:
KDG
;i:iii
PelA, PelB, PelA, PelB, C a::l PelC, PelD, I ~ i.......II~PelC, PelD, PelE, PelL, PelE, eelL, iii'~ PemA, CelZ PemA, CelZ :::!ili ......
: .ii: .ii .;.ii.:.:ii.i:iii i"iiii:i:i!.i:::: i.":ii "i:(:i.i::..:~
Figure 1. Catabolism of pectin in Erwinia chrysanthemi.
313 Pectinases are classified according to their preferential substrate, pectin or polygalacturonate (PGA), their reaction mechanism (~-elimination or hydrolysis) and the endo or exo mode of action. E. chrysanthemi produces multiple isoenzymes of pectinases : two pectin methylesterases, seven endo-pectate lyases, an exo-pectate lyase, a pectin lyase and an exo-polygalacturonase. Another enzyme, oligogalacturonate lyase (Ogl), specifically degrades short pectic oligomers. The action of these pectinases leads to complete degradation of the pectic polymers and gives rise to short oligogalacturonides that are catabolized through an intracellular pathway (Figure 1).
2. T H E E R W I N I A C H R Y S A N T H E M I
PECTINASES
2.1. The major endo-pectate lyases PelA, PelB, PelC, PelD and PelE Endo-pectate lyases appear to be the major pectinolytic enzymes produced by E. chrysanthemi. They cleave internal glycosidic bonds in PGA by ~-elim~nation to yield oligomers that are 4,5-unsaturated at the non-reducing end. Their activity is Ca 2+ dependent, with a basic opfi_m_um pH (8.5 to 9.2). Pectate lyases exhibit a reduced activity on pectin. Their molecular size varies from 35 to 45 kDa. The multiplicity of isoenzymes secreted by E. chrysanthemi made difficult the biochemical analysis of each of them. Separation of E. chrysanthemi pectate lyases by electrofocusing in thin polyacrylamide gels followed by an activity detection (1, 2 ) revealed, in most strMns, five major isoenzymes with pectate lyase activity: one showed an acidic isoelectric point (PelA, pI about 4.5), two slightly alkaline (PelB and PelC, pI about 7.5 to 8.5) and two very alkaline (PelD and PelE, pI about 9.5 to 10.5). Despite an overall similarity, the isoenzymes differed in some of their biochemical properties. The alkaline isoenzymes seem to attack pectin more randomly than the neutral do, as evidenced by viscosimetry and analysis of reaction products (3, 4). The major end-product of PelE is the dimer. PelB and PelC predominantly produce a majority of trimers as reaction end products, with some dimers and tetramers. PelA produces a set of oligomers, from dimers to dodecamers (4). Structural analysis of the two pectate lyases PelC and PelE (5, 6), demonstrated that these proteins fold in a large helix of parallel ~ strands. A stack of asparagine residues parallel to the helix probably plays a role in the stability of this structure. Identification of the structurally conserved omino acids lead to a realignment of the protein sequences (7). In addition to Erwinia extracellular pectate lyases, the multiple alignment includes the Bacillus subtilis pectate lyase, Aspergillus niger and E. carotovora pectin lyases and plant proteins.
314
This study proposes amino acids most likely involved in the Ca 2+ -binding site, two putative distinct active sites, residues that probably fold in parallel ~ helix or constitute the Ash ladder. The degree of pectin methylesterification or acetylation and the presence of other chemical groups vary with the cell wall compartment. Gold-labelling and microscope analysis demonstrated that PelB and PelC are preferentially located in some domains of the middle lamella and cell junctions (8) whereas PelD and PelE are found along the plasmalemma of the cell wall (Y. Bertheau, personal communication), confirming a possible specialisation of each isoenzymes toward different pectic polymers. The various isoenzymes, which present weak differences in their ability to recognise and cleave pectin, can act co-operatively for the breakdown of a variety of pectic polymers. For instance, a synergism in pectate lyase action toward pectin was recently observed in E. carotovora (9). The molecular biology approach confirmed that most of the pectate lyase activity in E. chrysanthemi results mainly from the cumulative action of five major endo-enzymes encoded by the genes pelA, pelB, pelC, pelD and pelE (10). The five pel genes have been cloned from a variety of E. chrysanthemi strains, using special media for the detection of pectinase activity. The five genes are organised in two clusters -pelB, pelC and pelA, pelF,, pelD- which are widely separated on the bacterial chromosome (Figure 2) (11). A high homology was found between the genes belonging to a given cluster, suggesting that genes of each cluster have appeared by duplication of two ancestral genes. Similarly the three secreted isoenzymes of E. carotovora are encoded by three adjacent genes presenting a high level of homology (12). leu thr ser cys pelB-pelC -pelZ l | I n" pro , sm ~ / hmpX-pelA-pelE-pelD -pecY- pemA lys ~ V ~ / met ~ ~lpur / ~ outS-outB-outT-outC-M-outO pehX / " ~ " cbs, fct, cbu ile ~ ~ . pecG- pecT arg ~17 ~.. pelL-celZ mtl, xyl, argG, pecS, pecM " 4 . . . . . . . ~(..-ura pelX ~ t:rwinla c n r y s a m n e m l ~.- kdgT celY kdgK~ . , . ~ .,.R --I . . . . :_ o ~ L o g l - k d R, h,mA,
.... m2;
'ade " ~ set " \ "
l
Z~ man ~ trp
\
exuT-uxaCBA " ~
]~"
~
/'-...._
dsbC-recJ
thyA
nal put
,/~ gal
pomB
Figure 2. Chromosomal map of Erwinia chrysanthemi
exuR
zwf- kdgA his
315 Despite their common transcriptional direction and their vicinity within the two clusters pelB-pelC and pelA-pelE-pelD, diverse arguments prove that the pel genes are independent transcriptional units. Each of the cloned genes can be transcribed independently and insertions in the first genes of each cluster do not inactivate transcription of the other genes (10). All the sequenced pectate lyase genes of E. chrysanthemi strains have a TAA stop codon followed by a sequence similar to Rho-independent terminators. In the case of the pelB gene of strain 3937, S1 mapping confirmed t h a t this sequence really functions as a transcriptional terminator (13). The transcripts initiated from the pelB-pelC region were identified by Northern blot analysis, indicating the presence of only one transcript per gene, the length of which corresponded with that of the corresponding gene (13). This independent organisation for genes of the same regulon is preferable to a large operon arrangement since it gives the potential for individual flexibility of expression.
2.2. The exoopectate lyase PelX An exo-pectate lyase activity was first reported in E. chrysanthemi CUCPB1237 (14). Exo-pectate lyase generates a sole product identified as unsaturated digalacturonate. The pelX gene was cloned from two E. chrysanthemi strains EC16 (14, 15) and 3937 (V Shevchik, unpublished results). PelX was purified from an E. coli recombinant clone and presents an apparent pI of 8.6 (14, 15). This enzyme could utilise PGA and also methylated pectins as substrates. The pH for optimal activity depends on the substrate. It is between pH 7 and 8 with PGA and more alkaline with pectin, around 8 and 8.5 with partially (67%) and highly (97%) methylated substrates, respectively. The enzyme activity on PGA was activated in presence of Ca 2+ and Na +. In contrast, activity on highly methylated pectin was stimulated in the presence of cation chelating agents such as EDTA (14, 15). The PelX protein contains 749 amino-acids including an ~mino-terminal signal sequence of 26 ~mi,o-acids. The cellular localisation of the enzyme has not been rigorously determined. In E. chrysanthemi CUCPB1237, the exo-pectate lyase activity was cell-bound (14). The presence of this type of enzyme in the bacterial periplasm appeared normal since it acts better on oligomers produced by endo-pectate lyases than on long polymeric substrates (15). 2.3. The secondary pectate lyases, PelL and PelZ The deletion ofpelX and of the five major pel genes from the E. chrysanthemi chromosome failed to totally eliminate the capacity for tissue maceration (16). Analysis of the macerated tissue by electrofocusing followed by an activity detection, revealed the presence of a new set of pectate lyases (up to 5 forms). Because of their low activity in synthetic medium, they were described as secondary pectinases. The gene of one secondary endo-pectate lyases, PelL, has been characterized in two E. chrysanthemi strains. The pelL gene of E.
316
chrysanthemi 3937, was isolated from a genomic library of a strain deleted of the five major pel genes (17). Insertion mutagenesis of the ApelA,E,B,C,X, pehX derivative of E. chrysanthemi EC16 with a transposon containing an inducible promoter allowed hyper-expression of the pelL gene (18). Digestion of PGA by the PelL enzyme yielded a mixture of unsaturated oligogalacturonides, giving evidence that PelL is an endo-cleaving lyase (17). An exo-enzyme, such as the EC16 PelX, would generate a single product (15). The PelL protein differs from the major E. chrysanthemi pectate lyases in its ability to cleave both PGA and methylated pectin (17). The PelL activity has a basic optimum pH and an absolute requirement for Ca 2+ ions. Analysis of culture supernatants demonstrated that PelL is an extraceUular enzyme, such as the other secondary pectate lyases (17). The pelL gene is adjacent to the major cellulase gene, celZ (Figure 2). It encodes a 425 amino acid protein, including a typical N-terminal signal sequence of 25 amino acids. The PelL enzyme contains a high proportion of asparagine residues (12%) but has an apparent basic isoelectric point. The amino-acid sequence of the PelL protein shows some homology, restricted to the C-terminal region, with the exo-pectate lyase PelX of E. chrysanthemi (15). This region contains some series of 4 to 7 identical residues that could be involved in the active site of these enzymes. PelL and PelX define a new class of pectic enzymes since these two proteins lack homology with other pectinases. Predictional analysis of the secondary structure of PelL or PelX indicates that many regions of these proteins can potentially adopt ~ sheet secondary structure. The PelL and PelX 3D-structures may resemble that of the major pectate lyases, despite the absence of homology in their primary sequences. Recently, a new pectate lyase gene pelZ, was identified at the vicinity of the pelB-pelC cluster (Pissavin et al, submitted). A pelZ homologue was also found in Erwinia carotovora. PelZ defines a new family of endo-pectate lyase since its amino acid sequence displays only very low homology with that of other pectinases. 2.4. The pectin m e t h y l e s t e r a s e s PemA and PemB Pectin methylesterases remove the methoxyl groups of pectin to yield PGA and methanol. Despite its pectin methylesterase activity, E. chrysanthemi is unable to growth on highly (98 %) methylesterified pectin as a carbon source (19). The gene pemA, encoding the major extracellular pectin methylesterase is linked to the pelADE cluster encoding major pectate lyases (Figure 2) (10). This gene was cloned and characterized from two E. chrysanthemi strains, B374 and 3937 (20, 21). It codes for a 366 amino acid protein, including a signal sequence of 24 amino adds. Recently, a gene coding for a novel pectin methylesterase, has been cloned (19). This gene, pemB, codes for a 433 _amino acid protein including a N-terminal sequence of 21 amino acids which presents the characteristics of lipoprotein
317 signal sequences. After cleavage of this signal sequence, palmitate, the most prevalent fatty acid in bacterial lipoproteins, is incorporated into the mature protein. PemB is localised in the periplasmic face of the outer membrane and does not seem to be released into the extracellular medium. PemB was overproduced and purified to homogeneity (19). PemB activity is strongly increased by non-ionic detergents, such as Triton X-100, a characteristic of many membrane enzymes. This observation could explain the relatively low specific activity of PemB in vitro since this activity may depend on a specific lipidic and hydrophobic background. PemB activity is about 100-fold higher on pectic oligomers than on natural pectins. The action of extracellular pectinases on pectin probably liberates small methylated oligogalacturonides that can enter by diffusion into the periplasm. The role of PemB might be to degrade such methylated oligomers. Homology between PemA and PemB is quite low (19). Thus, it seems unlikely that the presence in E. chrysanthemi of two peru genes results from a recent duplication of an ancestral gene as proposed for pel genes. The six regions conserved in bacterial or plant pectin methylesterases are present in PemA and PemB (19, 21). Since the central regions II, III, IV and V are more conserved than regions I and VI, they are more probable candidates to be involved in the catalytic site.
2.5. The endo-pectin lyase Pnl Pectin lyase is able to cleave n a t u r a l pectin and highly (98%) methylesterified-PGA by ~-elimination but it is not active on PGA. Most soft-rot Erwiniae produce an endo-pectin lyase activity whose synthesis is induced by DNA damaging agents (22). Pectin lyase activity is generally higher in E. carotovora than in E. chrysanthemi, and has been more closely analysed in the former species. The E. chrysanthemi pectin lyase has a molecular mass of 35 kDa and a pI of 9.5 (22, 23). The activity is Ca 2+ independent, with a pH optimum of 8.3. The E. carotovora pectin lyase encoding gene has been characterised (24, 25) but this is not the case for the E. chrysanthemi gene. The E. carotovora pectin lyase contains 290 amino-acids and is not processed by a signal peptidase. Comparison of the protein sequences of the E. carotovora pectin lyase with the pectin lyases of Aspergillus niger and secreted pectate lyases of E. chrysanthemi or E. carotovora revealed a low degree of homology, dispersed throughout the length of the proteins (24). Invariant potentially catalytic omino acids of pectate lyases are also conserved in pectin lyase sequences (7). 2.6. The exo-polygalacturonase PehX While E. carotovora presents endo-polygalacturonase activity (10), the only hydrolase activity found in E. chrysanthemi is an exo-cleaving polygalacturonase. Polygalacturonases hydrolyse a-1,4-glycosidic bonds; they are active at acidic pH (between 4 and 6.5) and do rot require divalent cations. An exo-polygalacturonase
318 was purified from culture s u p e r n a t a n t s of the E. chrysanthemi strain CUCPB 1237 (26). It has an apparent molecular mass of 67 kDa and an apparent pI of 8.3. The pH optimum was 6, Km for PGA was 0.05 mM and specific activity 5.9 mmol/min/mg of protein. This enzyme attacks the non-reducing end of PGA to release a single product : digalacturonate. However, this enzyme has a preference for chains bearing a 4,5-unsaturated residue at the non-reducing end (as generated by pectate-lyases). The pehX gene was cloned from E. chrysanthemi EC16 (27) and 3937 (V Shevchik, unpublished results). The PehX protein contains 604 amino acids including a N-terminal signal sequence of 27 Amino acids. The different bacterial endopolygalacturonases characterised thus far belong to an homogeneous family. They also present homology to fungus or plant enzymes, mainly in a highly conserved motif: GHGxSIGSx4-9VxNVTVx9NGLRIKS (28). This motif is partially conserved in PehX from residues 427 to 462 and could be involved in the active site of polygalacturonases. 2.7. The oligogalacturonate lyase Ogl Oligogalacturonate lyase, encoded by the ogl gene, converts the oligogalacturonides that result from the extracellular action of pectinases into monomeric sugars. This cytoplasmic enzyme cleaves the a 1-4 glycosidic bond by transe!imination. Unsaturated digalacturonate, the best substrate, is cleaved into two molecules of 5-ke~o-4-deoxyuronate (4-deoxy-L-threo-5-hexosulose uronic acid, DKI). Digalacturonate is degraded to yield equimolar concentrations of galacturonate and DKI. The oligogalacturonides with more than two residues are also attacked by oligogalacturonate lyase, but the rate of degradation decreases strongly when the length of the s u b s t r a t e increases (29). PGA and tetragalacturonides are attacked very slowly, at approximately 1/400 the rate of u n s a t u r a t e d dimers. The Km is 5.3 and 1.6 mM for digalacturonate and unsaturated digalacturonate, respectively. The optimum pH is appro~dmately 7.2 and the enzyme does not require calcium ions for its activity (29). The ogl gene of E. chrysanthemi 3937 was cloned by complementation of an ogl mutation using a RP4 derivative plasmid (30, 31). Recently, the ogl gene of E. carotovora was cloned and sequenced and it showed 88% of homology with the ogl gene of E. chrysanthemi (32).
2.8. The i n t r a c e U u l a r steps of pectin catabolism E. chrysanthemi is not only able to cleave the pectic polymers but also to use them as carbon and energy sources for growth. Breakdown of pectic polymers results in the formation of two kinds of monomers : D-galacturonate and DKI which are catabolized by two independent pathways converging to a common i n t e r m e d i a t e : 2-keto-3-deoxygluconate (KDG) (Figure 1). KDG is then phosphorylated and cleaved to give compounds of the general cellular metabolism.
319 The main monomer produced after the action of pectate lyases and oligogalacturonate lyase on pectin is DKI. Since polygalactttronase activity is very low in E. chrysanthemi, only small amounts of galacturonate are generated from pectin degradation. The galacturonate catabolism is then of secondary importance for the growth of E. chrysanthemi on pectic polymers. Mutations in genes of the DKI catabolism (ogl, kduD, kduI) prevented growth on pectin as sole carbon source (14, 31, 33, 34) while mutants altered in genes of the galacturonate catabolism (uxaC, uxaB, uxaA) are capable of normal growth on pectin or PGA (35). Cloning of large chromosomal fragments on R-prime plasmids and mutational analysis established that the genes involved in these pathways are organised in 5 clusters on the E. chrysanthemi chromosome (Figure 2). Since the first steps of pectin degradation are extracellular, the intracellular catabolism is dependent on the permeability of the membrane to the external substrates. Oligogalacturonides can enter the cells but the corresponding transport system(s) have not yet been identified (14). Two transport systems which mediates entry of monomers were characterised: ExuT for galacturonate uptake (35) and KdgT for DKI, DKII or KDG uptake (36) (Figure 1).
3. PHYSIOLOGICAL CONDITIONS A F F E C T I N G PECTINASE PRODUCTION 3.1. Individual t r a n s c r i p t i o n of the pectinase genes Regulation analysis was performed using transcriptional fusions in each gene of the pectinolytic pathway. Such fusions are of particular interest in the case of the pel genes since the total pectate lyase activity results from the cumulative action of at least seven isoenzymes (37). When followed individually, each pel gene responds to a different extent to a given signal and sometimes they show opposite behaviours. Comparison of the basal level of each pel fusion (37) revealed the major role played by pelF, in the production of pectate lyase in the absence of pectin. The pelA gene is weakly transcribed in synthetic medium whatever the conditions tested, whereas the pelB and pelC genes are expressed at intermediate levels. The pelD expression shows a low basal level but is strongly inducible. This high inducibility allows pelD to reach a level of expression higher than that of the other pel genes. Generally, pelD and pelE genes are more sensitive to variations in the environment than other pel genes. 3.2. Induction by intermediates of pectin catabolism In E. chrysanthemi, the pectate lyase, polygalacturonase and pectin methylesterase activities are induced in the presence of PGA (26, 37). The inducer is not the polymer itself but some breakdown products, initially generated by the
320 basal level of pectinases (38). Pectinase synthesis is also induced in the presence of galacturonate or of saturated or unsaturated digalacturonate (14). The analysis of mutants that are blocked in various steps of the pectinolytic pathway allowed the identification of the true intracellular inducers :KDG, DKI and DKII (Figure 1). In ogl mutants, digalacturonates no longer induce pectinase synthesis, demonstrating that the formation of DKI or DKII is necessary for induction in the presence of oligomers (14, 31). Similarly, in an uxaA mutant, pectinase synthesis is no longer inducible in the presence of galacturonate, demonstrating that KDG formation is necessary for induction in the presence of galacturonate (35). In a kdgK mutant, which accumulates KDG intracellularly, pectinase production is strongly induced in the presence of either PGA or galacturonate, confirming that KDG is a true inducer (35). In kduD and kduI mutants, which accumulate DKII and DKI respectively, pectinase synthesis is also strongly induced in the presence of PGA, indicating that these compounds are also true inducers (33, 34). Individual investigation of the expression of each gene of the pectinolytic pathway demonstrated that they are all induced in the presence of PGA and that they are all sensitive to the intracellular inducers DKI, DKII or KDG. The isolation of regulatory mutants overproducing pectate lyases and constitutively expressing all the genes of the catabolic pathway conducted to the identification of the kdgR regulatory gene which mediates induction by KDG, DKI and DKII. The characteristics of this regulation, which ensures a co-ordinate control of pectin catabolism, will be detailed below. However, the residual induction of pectate lyase synthesis observed in a double kdgR-kdgK mutant, in the presence of PGA or galacturonate, demonstrated the existence of other regulatory proteins responsive to KDG (35). 3.3. Induction dependent on bacterial growth phase The genes pemA, B, pelA, B, C, D, E, L are induced from 5 to 60-fold in late exponential growth phase, when the bacterial population has reached its maximum (17, 19, 37, 39). In a variety of gram-negative bacteria, a regulation similar to that mediated by the LuxI-LuxR system of Vibrio fisheri (40) is responsible for the growth phase-inducibility (41, 42). Members of the LuxR family are regulators that respond to small ~ s i b l e molecules related to homoserine lactone (I-ISL) with variable N-linked acyl side chains. In E. carotovora, production of all extracellular enzymes is induced by N-(3oxohexanoyl)-HSL, synthesised by the action of the ExpI protein (a LuxI homologue). Acyl-HSL binds to the ExpR regulator (a LuxR homologue), allowing it to activate the expression ofpel, peh, prt, cel and expI. At high cell density, acylHSL reaches a concentration sufficient to activate ExpR. Acyl-HSL is a widespread regulatory s~:gnal since it controls various mechanisms in Vibrio, Pseudomonas, Agrobacterium, Rhizobium, Yersinia and Erwinia (43). A homologue of the expI gene was receatly identified in E. chrysanthemi 3937, suggesting that acyl-HSL mediates the growth phase dependence of pectate lyase synthesis in
321 this species (W Nasser, unpublished data). Mutants of E. chrysanthemi B374, in which extracellular enzyme production became independent of the growth phase, were designated as gpi. According to the ExpI-ExpR model, it is possible that gpi mutants produce the acyl-HSL signal constitutively. 3.4. Effect of carbon, oxygen and iron availability Pectate lyase production in Erwinia is subjected to cyclic AMP-controlled catabolite repression (44). Catabolite repression can be observed during growth in the presence of glucose but also in the presence of galacturonate or unsaturated digalacturonate (45). Moreover, the spectrum of Erwinia pectinases produced by the bacteria strongly depends on the nature of the carbon source present in the growth medium, suggesting that individual genes are differentially sensitive to catabolite repression (46). The catabolite repression is conserved when pectate lyases are expressed in E. coli, suggesting that the catabolite activator proteins (CAP) are interchangeable between the two bacterial genera (V James, unpublished data). Moreover, the 5'-untranslated end of most pel genes contains sequences homologue to the E. coli CAP binding site (30, 47). Molecular cloning of the E. chrysanthemi crp gene, encoding CAP, was recently performed (W Nasser, unpublished data). The E. chrysanthemi CAP displays strong homology with the E. coli CAP. An E. chrysanthemi crp mutant loses its ability to grow on several carbohydrates, including PGA, and is deficient in pectate lyase production (S Reverchon, unpublished data). Similarly, a cya mutant of E. carotovora, unable to produce cyclic AMP, was defective in pectate lyase synthesis (48). Mutants of E. chrysanthemi B374, in which extracellular enzyme production became insensitive to the catabolite repression, were designated as cri (49). In E. coli, mutations allowing expression of catabolite-sensitive operons in the absence of CAP were localised in the rpoD gene, encoding the Sigma 70 factor (50). The cri mutations of E. chrysanthemi may also affect the transcription apparatus. In E. chrysanthemi pectate lyase production increases under semi-aerobic growth conditions but with a differential effect on the individual pel genes. Expression of pelA, pelD and pelE is stimulated in anaerobiosis, pelB and pelC transcription is not affected and pelL expression is reduced (17, 37). Mutants affecting the E. chrysanthemi hmpX gene, which is adjacent to the pelADE region, synthesised reduced amounts of pectate lyases under low oxygen content (51). The hmpX gene codes for a flavohemoglobin which possesses a N-terminal hemoglobin domain and a C-terminal reductase domain. The regulatory effects of the hmpX mutations could be explained by a defect in sensing the oxygen status, either by a direct role of HmpX or by a secondary effect of the hmpX mutations disturbing the anaerobic metabolism. In response to iron deprivation, E. chrysanthemi induces the synthesis of siderophores and also the transcription of pectate lyase genes (52). The pelB, pelC, pelE and pelL genes are induced under limited iron-deprivation whereas pelD is only induced under severe iron-deprivation (53). Iron sensing in E.
322
chrysanthemi seems to be partially mediated by a Fur-like protein since in a fur mutant of E. coli the siderophore biosynthesis is no longer iron regulated (54). 3.5. Pnl production is induced by DNA damaging-agents In E. chrysanthemi and E. carotovora pectin lyase synthesis is subjected to a form of control completely different from that observed for the other pectinases. The pnl expression is induced by DNA damaging agents but it is not sensitive to PGA induction (22). In E. carotovora, RecA is required for transcriptional activation of the pnlA gene (55). The transcription ofpnlA gene is not controlled directly by LexA, the SOS repressor, but depends upon two genes rdgA and rdgB (56, 57). RdgB could be a direct activator ofpnlA transcription; its production is dependent of RdgA. Under non-inducing conditions, RdgA represses RdgB synthesis and prevents pnlA expression. In the presence of DNA-damaging agents, the activated proteolytic form of RecA processes RdgA in a form that activates rdgB expression which, in turn, activates pnlA expression (57). 4. T H E T R A N S C R I P T I O N A L
REGULATORS
4.1. KdgR, the main repressor of pectinolysis The regulatory gene kdgR is responsible for induction of all the genes involved in pectinolysis and in pectate lyase secretion in the presence of PGA and galacturonate (Figure 3). kdgR mutants were obtained through three different selection procedures: as mutants synthesising an increased level of pectate lyases in the absence of inducer (49), as mutants able to grow on KDG as the sole carbon source because of a derepressed expression of kdgT (36), and as m u t a n t s expressing at a higher level a kduD-lacZ fusion (58). In fact, kdgR inactivation results in a derepressed expression of all the steps of the pectin catabolism (58). In a kdgR mutant, the expression of the major pectinase genes is derepressed but remains inducible in the presence of PGA. Although its expression is inducible by PGA, the pectate lyase gene pelL is not regulated by kdgR (17). Thus, KdgR is probably not the sole regulatory protein that responds to the presence of PGA or PGA degradation products. The kdgR gene has been localised in a region encoding several steps of pectinolysis, downstream of the ogl gene (59). KdgR is a 306 amino acid protein with a molecular mass of 35 kDa. The native KdgR repressor was purified in the fraction corresponding to a molecular mass of 68 kDa, indicating that KdgR is a dimer. KdgR presents homology with other regulatory proteins controlling catabolic pathways (59) : GylR, a regulator of the glycerol operon in Streptomyces coelicolor; IclR, a repressor of the acetate operon in Salmonella typhimurium and Escherichia coli; and PobR, which controls the metabolism ofp-hydroxybenzoate in Acinetobacter calcoaceticus. The N-terminal part of all these regulatory proteins contains a region that could fold into a helix-turn-helix motif, characteristic of
323 DNA binding proteins. Since these proteins have no homology with proteins of the other families of transcriptional regulators, they define a new family of regulatory proteins. Comparison of the regulatory regions of the KdgR-controlled genes revealed the existence of a conserved motif which was proposed as being the KdgR-binding site (KdgR-box) (13, 30, 60). Using gel retardation assays, it was demonstrated that the purified repressor binds to this site (61) and that KDG is able to dissociate the KdgR-operator complex. The Km of KdgR for KDG varied from 0.3 to 0.6 mM depending on the operator (62). Analysis of the inducing properties of KDG analogues allowed for the determination of the structure of the inducer recognised by KdgR: all the inducing molecules contain the motif COOH-CO-CH 2CHOH-C-C included in a pyranic cycle (63). This motif is also found in DKI and DKII but a direct interaction of these two compounds with KdgR has not been verified. Modifications of KDG on carbon 5 have no influence on the inducing properties but render the molecules non-metabolisable since the KDG-ldnase is unable to phosphorylate them. In consequence, C5-KDG derivatives are gratuitous inducers of the expression of the genes of the KdgR regulon (63). In vitro analysis of the interaction of KdgR with its operators was performed for eleven genes of pectinolysis (pemA, pelA, pelB, pelC, pelE, ogl, kduI, kduD, kdgT, kdgK and kdgA) and two involved in enzyme secretion (outT and outC) (62, 64). In vitro, KdgR is able to bind to only nine of these operators (pelA, pelB, pelC, pelE, ogl, kduI, kdgT, kdgK, and outT). The KdgR-boxes usually overlap or are close to the sequences corresponding to the -35 or -10 putative promoter regions, suggesting that KdgR and the RNA polymerase compete for adjacent binding sites on DNA. In consequence, KdgR binding prevents gene expression. Alignment of the different KdgR boxes showed that they correspond to imperfect inverted repeats of 19 nucleotides AATRAAAYR-YRTTTYATT (R = A or G, Y = C or T). The moderate variations in the sequence of individual sites allow some modulation in the strength of repressor binding. KdgR binds to its operators with different affinities but there is no strict correlation between the affinity of KdgR for an operator and the degree of de-repression of the corresponding gene in a kdgR mutant (62). The duplication of the KdgR-box upstream from several genes (ogl, pelE, kdgT, and pelC) and the relative position of this operator within the different regulatory regions could affect the degree of occupancy of these operators by KdgR and modulate the efficiency of repression. Duplication of operators allows a stronger repressor binding through a co-operative interaction and can provide a more flexible response to changing environmental conditions. Although gene fusion studies proved that the expression of kduD, pemA, kdgA, and outC genes is controlled by KdgR, no direct interaction could be detected in vitro between KdgR and the regulatory regions of these genes. However, the KdgR-box is well conserved in the operators which interact in vitro with KdgR but degenerated in the operators that cannot interact in vitro with KdgR. The KdgR mediated regulation probably involves two different
324 mechanisms. KdgR can directly control the genes with an in vitro active KdgRbox. More complex mechanisms could explain the regulation of the other genes : either KdgR binding requires a co-factor or the KdgR effect is mediated via a cascade of regulatory proteins. For example, the in vivo regulation of the outC operon by KdgR requires a functional outT gene product (65) while outT expression is submitted to direct control by KdgR (62). The proposed model is that the outC operon is activated by the OutT protein whose production is KdgRdependent (65). This apparent complexity may contribute to some specific characteristics of the regulation. The KdgR regulon includes at least 13 cistrons involved in pectinolysis or secretion but also several genes of unknown function. Isolation of lac fusions whose expression is induced in the presence of PGA allowed for the characterisation of a set of pgi genes (PGA inducible) (66). Most of the pgi genes belong to the KdgR regulon. Some pgi mutations affect genes of pectinolysis but several pgi genes do not play a role in PGA metabolism and their functions are not yet identified. Two additional genes of the KdgR regulon, kdgC and kdgF, were identified on the basis of the presence of a KdgR-box in their regulatory region (60). kdgC is located downstream of kduD and encodes a 47 kDa protein that displays homology with pectate lyases of the periplasmic family (12). kdgF is transcribed divergently from kduI with which it shares a common KdgR-box. The product of kdgF is a 12 kDa protein that has no homology with proteins of known function (60). No pectinolytic activity could be associated with either KdgC or KdgF.
pelA pelEpelD pecY pemA . . . .
"~
-
"-
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-
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-
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exuR kdgA pemB
~
outT . . . .
,v
~
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..---
exuT uxaCBA
Figure 3. The KdgR regulon
. . . .
pecT celZpelL ~
-
~
~
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---..IP,
~l..---
. . . . . .
~
kdgT -
- - - I ~ -
pecS pecM
ogl~d'g'~kdgC kduD kdul kdgF -
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325 4.2. PecS, a r e p r e s s o r of p e c t i n a s e and cellulase s y n t h e s i s Isolation of mutations increasing pectinase synthesis led to the characterisation of the pecS locus (67). Inactivation ofpecS results in derepressed synthesis of pectinases, of the cellulase CelZ, of the secretion machinery and of an extracellular insoluble blue pigment (Figure 4).
-
pelA pelE pelD pecY pemA
outT
out
pecT
celZpelL
kdgT
-
ogl kdgR kdgCkduD kdul kdgF ..~--.~r
~,...--;..--. exuR kdgA
pemB
exuT uxaCBA
kdgK
~
pecM
pelB pelC pelZ
Figure 4. Regulation of pectinase genes by PecS The pecS gene was cloned by selection of an R-prime plasmid carrying the adjacent genes xyl and argG. Insertion mutagenesis of this locus revealed the existence of two divergently transcribed genes, pecS and pecM. These two genes have the same spectrum of action. However, the level of de-repression of the controlled genes is higher in the pecS mutant than in the pecM mutant. While a 15-fold increase in the pectate lyase activity was observed in the pecS mutant, only a 4-fold increase was obtained in the pecM mutant. The pecS gene encodes a protein of 166 amino acids with a calculated molecular mass of 19 kDa (67). The PecS protein was purified in the fraction corresponding to a molecular mass of 40 kDa, indicating PecS is a dimer (68). PecS displays homology with a family of small size regulatory proteins such as EmrR, SlyA, MarR and HpcR (68). Gel retardation assays demonstrated that the PecS protein is able to interact with the regulatory regions of several controlled genes (pelA, pelE, pelL, celZ and outC operon) with relatively low affinities. Analysis of the PecS-binding sites revealed no well conserved sequences, suggesting that the PecS repressor may not recognise a precise motif but rather some structural features of the DNA targets (68). The pecM gene encodes a protein of 297 amino acids with a calculated molecular mass of 32 kDa. The predicted PecM protein displays the characteristics of an integral membrane protein since it is largely hydrophobic, with potential trans-membrane domains. Subcellular fractionation confirmed that PecM is anchored into the bacterial inner membrane whereas PecS is
326 located in the cytoplasm. PecM could be involved in the sensing and transduction of an external stimulus (67). The relatively low affinity of PecS for its operators suggests either the requirement of cofactors or the existence of a post-traductional modification in E. chrysanthemi which could increase the PecS 8f~nity. The PecM protein would be a good candidate for PecS modification. In the absence of an inducing signal, PecM could modify PecS so that its affinity for DNA increases, giving rise to a strong repression. In contrast, when the PecM protein senses the external signal it no longer modifies PecS. The phenotypes of pecS and pecM mutants are consistent with this model. In a pecS mutant, the absence of the PecS repressor results in a high level of de-repression whereas in a pecM mutant, the presence of the unmodified PecS form, with low DNA affinity, results in a weaker de-repression. 4.3. PecT, an a d d i t i o n a l r e g u l a t o r of s o m e p e c t i n a s e s
Another type of mutation, resulting in the elevated synthesis of pectate lyases in the absence of inducer, was identified after Tn5 mutagenesis. The corresponding regulatory gene was calledpecT (Surgey et al, submitted). The pecT mutation de-represses the expression of a restricted set of pectinase genes: pelC, pelD, pelE, pelL and kdgC (Figure 5). The aspect of pecT m u t a n t s is quite different from that of the wild type strain (Surgey et al, submitted). They appeared as mucoid clones when grown on solid minimal medium and the cells floculate when grown in liquid minimal medium. Mucoidy usually results from an elevated synthesis of the cell surface exopolysaccharides (EPS). Therefore, PecT probably also controls EPS biosynthesis. The pecT gene encodes a 35 kDa protein which belongs to the LysR family of transcriptional regulators (Surgey et al, submitted). PecT most probably acts as a repressor on pel expression, in response to a signal which has not yet been identified.
pelA pelE pelD pecY pemA ....
~
- --"-I~'-'-I~
exuR
outT
- ~,,..- -~,,- - ~,,..-'-1~ . . . .
--'-1~
kdgA
pemB
. . . .
out:
~.. . . . . .
~
exuT uxaCBA
. . . . .
~-
( ~
celZ pelL
~
--"iP,,~"-"
~
kdgK
kdgT
~
pec5 pecM
Figure 5. Regulation of pectinase genes by PecT
ogl kdgR
- -'-1~ - "'-'~'-"lP
kdgC kduD kdul kdgF -q-'-
;,..-
-
~
-,~.-
pelB pelC pelZ
-
-"
327
5. C O N C L U S I O N Studies presented in this article underline the exceptional complexity and subtlety of the pectinolysis regulation which has evolved in Erwinia species. The different characterized regulators define distinct -but overlapping- regulons. KdgR controls expression of the pectin catabolism and related functions, such as pectinase secretion. The ExpI-ExpR system co-ordinates the production of all extraceUular degradative enzymes. Other regulators affect, in addition to pectinase production, cell surface properties (PecT) or pigment biosynthesis (PecS). Finally, other systems correspond to classical global regulators, such as CAP which is involved in carbon utilisation. These mechanisms probably also contain regulatory cascades, in which one regulator controls the synthesis of a second regulator, such as in the case of the OutT KdgR-dependent regulation of the secretion operon outC. The nature of the signal inducing the cell response has been established in only a few cases. The catabolic intermediates DKI, DKII and KDG indicate pectin presence and directly modify the DNA-binding properties of the KdgR repressor. The small diffusible molecule acyl-HSL is the signal through which Erwinia perceives and responds to high cell populations. For other regulons (PecS, PecT), the nature of the corresponding environmental stimulus and the means by which cells sense this stimulus are not understood. Moreover, many environmental conditions affecting pectinase production are not yet associated with any regulatory protein.
6. A C K N O W L E D G E M E N T S Work on various aspects of pectinolysis regulation has been supported in our laboratory by grants from the CNRS and from the Minist~re de l'Education Nationale, de l'Enseignement Sup~rieur, de la Recherche et de l'Insertion Professionnelle. Characterization of the Erwinia pectinases is part of a project financed by the European Community AIR programme (AIR2-CT941345).
328
7. R E F E R E N C E S
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J. Visser and A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
331
Molecular genetic and biochemical aspects of pectin degradation in Aspergillus Jacques Benen, Lucie Parenicova, Margo Kusters-van Someren, Harry Kester and Jaap Visser. Section Molecular Genetics of Industrial Microorganisms Wageningen Agricultural University Dreyenlaan 2, 6703 HA Wageningen, The Netherlands.
Introduction Pectin as one of the major plant cell wall constituents has received much attention both from a scientific and a technological point of view. Although pectin has been known to be a very complex heteropolysaccharide for quite some time, most progress on the elucidation of its structure has been attained in the last decade as a result of refinement and development of new more powerful techniques like HPAEC, HPGPC, NMR and the application of purified enzymes able to degrade specific parts of the complex molecule. Despite that there is still some debate on certain issues, like for instance on the small RG-II molecule, there is a consensus on the general structure of pectin: smooth regions of homogalacturonan interspersed with highly branched regions containing both rhamnose and D-galacturonate in the backbone which are either called hairy regions [de Vries et al., 1982] or RG-I [Darvill et al., 1978]. It is within the RG-I, reviewed by O'Neil et al. (1990), or hairy regions [Schols, 1995] and RG-II [Whitcombe et al., 1995] that the pectin displays its most intricate structure of a multitude of different carbohydrates like galactose, arabinose, apiose, fucose, xylose etc. which are linked, sometimes via various linkages, in varying composition depending on the species and season, to the backbone. It is obvious that both for the synthesis of pectin by plants as well as for its modification and degradation during development and for breakdown by organisms feeding on plant material a vast array of concertedly and/or consecutively acting enzymes is required. While in the field of pectin synthesis knowledge is still scarce due to the limited access to plants both biochemically and genetically, knowledge about the breakdown of pectin by microorganisms is compiling at a steady pace. In Table I constituents of pectin are listed together with enzymes involved in the degradation. For many microbial enzymes involved in pectin degradation corresponding genes have been cloned and sequenced as evidenced by the huge number of these genes, especially those encoding pectin and pectate lyases and polygalacturonases, deposited in nucleotide sequence databases. It should however be noted that the biochemical characterization of the corresponding enzymes is not in pace with the rapid accumulation of sequence data.
Enzyme
Reference
Pectin constituents
endo-polygalacturonase exo-polygalacturonase pectin lyase endo-pectate lyase exo-pectate lyase a-L- arabinofuranosidase endo-arabinase xy logalacturonanhydrolase rharnnogalacturonan rharnnohydrofase rhamnogalacturonan galacturonohydrolase rhamnogalacturonan hydrolase rhamnogalacturonan lyase pectin methyl esterase pectin acetyl esterase rhamnogalacturonan acetylesterase feruloyi esterase coumaryl esterase oligogalacturonate lyase oligogalacturonase 0-galactosidase
Jansen and McDonnel, 1945 Mill, 1966 Albersheim et al., 1960 Nagel and Vaughn, 1961 MacMillan and Vaughn, 1964 Akinrefon, 1968 Kaji and Saheki, 1975 Beldman et al., in preparation Mutter et al., 1994 Mutter et al., in preparation Schols et al., 1990 Azadi et al. 1995; Mutter et al., 1996 Fish and Dustman, 1945 Williamson, 1991 Searle-van Leeuwen et al., 1992 MacKenzie et al., 1987 MacKenzie et al., 1987 Moran et al., 1968 Hasegawa and Nagel, 1968 Borglum and Sternberg, 1972
galacturonate 6-O-methy l-galacturonate
3-0-acetyl-galacturonate 2/3-0-feruloyl-galacturonate 2/3-O-coumaryl-galacturonate xylose 2-O-methyl-xy lose rhamnose arabinose galactose apiose fucose 2-0-methyl-fucose glucose glucuronate aceric acid 2-keto-3-deoxy-D-manno-octulosonate 3-deoxy-D-l yxo-2-heptulosanate
332
Table 1. List of enzymes acting on pectin and constituents of pectin identified thus far. References are given for first characterization of the purified enzyme
333 Therefore it is presently in many cases not possible to assign a particular gene to a particular specific enzymic activity. Another aspect of pectinolysis which deserves more attention is the detailed analysis of the regulation of expression of the individual genes. This is again especially relevant for gene families like the afore mentioned pectate and pectin lyases and polygalacturonases and of course this should be addressed in relation to other genes of the full pectinolytic spectrum. The induction may be an interplay of several enzymes at three levels: 1) (a) constitutively expressed enzyme(s) with either endo- or exolytic activity generating specific mono-, di- or oligomers from either the reducing or non-reducing end, 2) a physical contact of the polymeric substrate and a membrane bound receptor and 3) an enzyme specific for the side chains of the pectin molecule generating monomers differing from galacturonate. In our laboratory we study all the above mentioned aspects of pectin degradation by fungi of the genus AspergiUus with special attention to the food grade species Aspergillus niger and A. tubingensis. In this contribution an overview will be given of results obtained so far with reference to work done on (other) Aspergilli by other groups where appropriate.
Genes encoding pectinolytic enzymes in AspergiUus The first Aspergillus pectinolytic genes cloned in our lab were pelD [Gysler et al., 1990] and pgaI and pgalI [Bussink et al., 1990 and 1991a] encoding pectin lyase I (PLI) and endopolygalacturonase I (PGI) and II (PGII), respectively. The technique used in these cases was reverse genetics using enzymes purified from commercial A. niger preparations Ultrazyme and Rapidase for pelD and pgaI and pgalI, respectively. Using these genes as probes complete families of pectin lyases (six members) [Harmsen et al., 1990] and endopolygalacturonases (seven members) [Bussink et al., 1992a] were discovered under heterologous hybridisation conditions. The individual genes were subsequently cloned and sequenced. Using the same approach of reverse genetics the pgaX gene encoding exopolygalacturonase (PGX) from A. tubingensis was cloned and sequenced [Kester et al., 1996]. With this gene as a probe two A. niger pgaX genes were proposed based on Southern analysis. One of these A. niger pgaX genes has in the meantime been cloned [Benen and Visser, unpublished]. Using an A. aculeatus cDNA expression library in E. coli the rhgA gene encoding rhamnogalacturonase A was isolated and its sequence established [Suykerbuyk et al., 1995]. The A. niger genes rhgA and rhgB were isolated with the A. aculeatus gene as a probe [Suykerbuyk et al., unpublished]. In our continued efforts to identify pectinolytic genes we also have obtained the pme genes from A. niger and A. tubingensis encoding pectin methyl esterases [Visser et al., 1996]. The search for additional genes like for instance oligogalacturonate lyase is still in progress. The A. niger pectin lyase gene family As already mentioned before a complete family of pectin lyases is present in A. niger. With all the genes sequenced and individually expressed, sometimes via promoter gene fusions (see below), the way is open to characterize the full spectrum of activities as outlined in the intro-
334 20
12 9 I3 I4 ] 42 ] 32 [
96
18 ]
78
379 aa 111
PLA
] 359mp
20 ~ i ~ | 47 I~.'.e. ~!~..'..~:~~.~.~.'.:~.:~.~
I1 149
I2 9 42
I3 ]
,
I3
, I8 I
109
112
.........................
18 !:~:i~]i:~:~i:i~.'.':! [~~,~:."~
144
: ;,
19
I1
I 12,
I3
I4 [
379 aa [ PLB 359mp
I7 56
378aa 138
I
1
PLC
360 mp
I5
373 aa 184
PLD 354 mp
22 :~. . . . .
!~" "~~
~.,.~g,:
I7 [
226
* 139
i
379aa
1
PLE
357 mp 20
,
,
I6
I7
,
,
,
476aa PLF 456 mp
:g':"~ '~-~ "~.'-'_~d::'!_~)~:.
[~i]
........................
= Signal peptide 9 I = intron;
9 = N-glycosylation site
Fig. 1. Schematic overview of AspergiUus niger pectin lyase genes aa, amino acid; m.p. mature protein.
pelA to pelF
duction. The entire set of data, once completed, will give valuable information about the role each member of this family plays in the pectin degradation. Genetic organization
In Fig 1. a schematic overview of the pectin lyase genes pelA to pelf is presented. As is clear from this scheme the pectin lyase genes are very similarly structured. With the exception of PLF all enzymes are almost equal in length and they all (including PLF) contain a signal peptide that directs the proteins to the secretory pathway. For PLA [Kusters-van Someren et al.,1991a] and PLD [Gysler et al., 1990] the cleavage position has been verified by N-terminal sequencing of the mature protein. For the other pectin lyases the signal peptide is based on amino acid similarity and conformation to the general von Heyne rule for secretory proteins [von Heyne, 1985]. Also the positions of introns is more or less conserved
335
'
'
I
'
'
'
'
-1.0
't
I
'
I
kb
pelA
pelB
I
'+'strand
I
0
I
I
V
T
'
O0
0
~
'
II
0
JX CREA
'
-0.5
'CCCTGA'
0
I
pelC
0
O I PACC
~L
' ' strand
Fig. 2. Analysis of Aspergillus niger pectate lyase promoters for putative regulatory sequences throughout, especially for the more closely related PLA, PLB and PLD, with the largest deviation again in PLF [Kusters-van Someren and Visser, unpublished]. This latter enzyme is also the most abberant with respect to potential N-glycosylation sites; while the other enzymes contain only one or two of these sites PLF counts five of these sequons.
Promoter analysis It is known, that production of pectolytic enzymes such as polygalacturonases, pectin and pectate lyases, pectin methylesterases and others by A. niger is induced when the fungus is grown on a medium containing pectin or sugar beet pulp and almost completely repressed when sugars like glucose, sucrose or xylose are added to the medium [Kusters-van Someren et al., 1991a; Bussink et al., 1991b and 1992b]. On the other hand in case of A. parasiticus or the phytopathogenic fungus Sclerotinia sclerotiorum some of the pectinolytic genes are constitutively expressed regardless of the carbon source present [Cary et al., 1995; Fraissinet-Tachet and Fevre, 1996]. This observation might suggest the different roles of the individual phytopathogenic pectinolytic enzymes in the process of plant invasion compared to pectinases of saprophytic fungi. The regulation of expression of pelA and pelB genes occurs at the transcriptional level as demonstrated by the Northern blot analysis of the multicopy A. niger transformants grown on different carbon sources [Kusters-van Someren et al., 1991a and 1992]. In Fig. 2 the results are shown of a computer analysis of the promoter of three pectin lyase genes of A. niger searching for potential regulatory elements (for the other pectin lyase genes not sufficient promoter sequences are available). As expected from the expression studies putative CRE A binding sites were detected. They appear at a distance ranging from approximately - 1.5 kb
336 to the translation initiation site of the pelA and pelC gene. Only the context independent DNA-binding sites (5" SYGGGG 3") for this protein [Cubero and Scazzocchio, 1994] are depicted in Fig. 2. A search for wide domain regulatory factors revealed the presence of the 5" GCCARG 3" hexanucleotide sequence identified as the binding site of PACC - the pH mediating transcription factor which in the model of Tilburn et al. (1995) acts as an activator of alkaline targets and as a repressor of acid targets. When compared to the promoters of the pga genes (Fig. 4) a remarkably high number of PACC binding sites is found. Whether this observation indicates pH dependent regulation of the pel genes awaits further analysis. Another common sequence in the promoters of pectinolytic genes of A. niger was discovered after scanning of upstream regions for hexanucleotide sequences [Bussink et al., 1992b]. The 'CCCTGA' sequence is present at least once in each of the promoters in either orientation. Whether this sequence has any importance as the common element for regulation of the pectinolytic gene expression in A. niger needs to be investigated in future.
Sequence comparison Since all pectin lyase genes presented here were isolated with one and the same gene as a probe using various stringency conditions it will be obvious that all the genes share a high degree of sequence identity. Recently two reports were published [Henrissat et al. 1995 and Heffron et al., 1995] that deal with the sequence comparison within the superfamily of pectate and pectin lyases including some of those presented here. It appears that throughout the entire superfamily only few amino acid residues are strictly conserved. Whether these invariant amino acids are really involved in catalysis or that they serve to keep the structural integrity of the enzymes awaits further studies like elucidation of the three dimensional structure and site directed mutagenesis.
Table 2. Compilation of features and properties of AspergiUus niger pectin lyase genes and enzymes. WT, wild type; n.d., not determined. gene
nucleotide sequence
expression mRNA(WT) protein
pectin lyase activity
localization
pelA
+
+
pelB
+
pelC
remarks
PLA/PLII
+
medium
+
PLB
+
medium
+
-
PLC
-
medium
pelD
+
+
PLD/PLI
+
peIE
+
n.d.
PLE
-
cell wall associated medium
A.nidulans
pelF
+
n.d.
PLF
+
(partial) medium
promoter
pkipromoter
pki-
337
Expression of pectin lyase genes and localization of corresponding enzymes A first question to be answered when studying a family of genes is whether the genes are expressed, cryptic or pseudo genes. The functionality of the individual genes can be approached by identifying the corresponding mRNA and the active enzyme. In Table 2, data concerning this are compiled. The mRNA identification, using the corresponding gene as a probe under stringent hybridisation and washing conditions, was in some cases done by using A. nidulans as an expression host. In one of our early studies with peIA aimed at investigating the functionality of promoter elements of this A. niger gene in A. nidulans Kusters-van Someren et al. (1991a) demonstrated that the carbon catabolite repressing response as operating on pelA expression in A. niger was completely absent in A. nidulans transformed with the A. niger peIA gene under the direction of its own promoter. When grown on glucose no PLA is formed in A. niger whereas, upon induction (growth on pectin) A. nidulans transformants expressed peIA at a much higher level than A. niger, even in the multicopy situation. Thus transformation of A. nidulans with the individual pectin lyase genes greatly facilitated the identification of the corresponding mRNA and enzyme. It is obvious that this can be particularly helpful in case of pectin lyases that are expressed at very low levels in induced A. niger. Another approach to demonstrate the functionality of the structural part of such a low level expression gene is by directing the expression from an alternate stronger promoter [Kusters-van Someren et al., 1992]. The latter of course does not demonstrate the functionality of the complete gene including its own promoter but when it results in a functional enzyme it provides at least support that the gene has a role in vivo. As can be seen in Table 2, mRNA was readily identified for pelA, pelB and pelD, genes that are expressed at high levels when wild type A. niger is grown on pectin. Also the pectin lyase activity was easily demonstrated for PLA and PLB as was their respective localization. For PLD this appeared not as straight forward. When grown in shaking batch cultures no PLD could be detected. However, in stationary cultures active PLD was identified in the mycelial mat formed on top of the culture fluid as was demonstrated by release of PLD upon washing of the mycelial mat in protease free buffer. Further analysis of this phenomenon revealed that PLD is rather sensitive to proteolyis by extracellular acidic proteases formed in large amounts by A. niger which is a notorious acidifier of its culture fluid. No mRNA could be detected in wild type A. niger for pelC under the growth conditions applied [Kusters-van Someren, 1991b]. This may indicate that the right substrate and/or condition for induction has not been found yet as is circumstantiated by the fact that no activity for PLC could be demonstrated on the substrates used so far despite the observation that PLC can be obtained by driving transcription via the strong promoter of the glycolytic pkiA gene encoding pyruvate kinase. The very presence of PLC in the culture fluid demonstrates that the gene is most probably functional; a non-functional gene would rather have resulted in a truncated protein that would not have been routed to the secretory pathway. The presence of residues in PLC that are conserved throughout the complete superfamily provides further support that PLC is indeed a functional enzyme. For pelE and pelF mRNA was not studied in wild type A. niger. However, for peIE high levels of PLE were identified in the growth medium under direction of its own promoter upon expression in A. nidulans [Kusters-van Someren and Visser, unpublished]. Similarly as for PLC we have not been able to detect any pectin lyase activity with the substrates used solar. PLF was well expressed in A. niger using the strong glycolytic promoter. The enzyme
338 exhibited typical pectin lyase activity and appeared to be partly associated with the cell wall and was partly found in the growth medium.
Biochemical properties of pectin lyases The identification of the pectate lyase family started with the cloning of the pelD gene via reverse genetics [Gysler et al., 1990]. The enzyme used to generate peptide fragments was PLI isolated from the A. niger pectinolytic enzyme preparation Ultrazyme [Kester and Visser, 1990]. It is therefore obvious that PLD and PLI are the same enzymes. This was demonstrated by the determination of the turnover number (4660 min -1) and Km (10 mM, calculated on the basis of the average molecular weight of the substrate 92 %DE citrus pectin) for both enzymes which were identical within error limit. The PLA turned out to be identical to the PLII enzyme from the Ultrazym preparation based on the turnover number (1600 min -1) and Km (1.3 mM). Both PLI and PLII have been extensively characterized by van Houdenhoven (1975). Both enzymes cleave the substrate in an endolytic way. Using methylated oligomers of increasing DP (2-10) it was demonstrated that the turnover number increases with increasing DP until it reached a plateau value. This is characteristic for enzymes with multiple subsites. On the basis of a plot of the turnover numbers and the DP of the substrates the number of subsites was estimated to be 9 or 10 for PLI and 8 for PLII [van Houdenhoven, 1975]. Since A. niger pelA was very well expressed in A. nidulans when grown in glucose, and thus free of contaminating pectinolytic enzymes [Kusters-van Someren et al., 1991a], we used A. nidulans to produce PLA. It appeared however that PLA consisted of two populations of enzymes as demonstrated by SDS PAGE. Analysis of the two populations revealed that the difference is due to different degrees of N-glycosylation. Despite the difference in glycosylation, the two purified PLA populations showed the same specific activity. However, for crystallization purposes it is necessary to use only one of the glyco-forms. As found for PLD, PLB appears to be very susceptible to proteolysis at lower pH by acidic proteases. For good yields of PLB it is therefore necessary to buffer the growth medium with a stronger buffer to avoid rapid decrease of the pH with concomittant production of acidic proteases (see below too). Interestingly, the pH optimum of PLB is quite high at pH 8.5-9.0 comparable with the pH optima found for the Ca 2+ dependent bacterial pectate lyases and much higher than found for PLA and PLD which both have a pH optimum of pH 6.0. Because of the high pH optimum the calcium dependence and potential pectate lyase activity of PLB were studied which resulted in the firm conclusion that PLB is a real pectin lyase [Kester and Visser, 1994]. During the purification of PLB it appeared that two fractions could be separated, one having a high turnover and the other having a much lower turnover. Detailed investigation of the two fractions showed that either can be converted into the other by dialysis against low or high salt buffers. At low salt concentration the 'low' activity form is generated while at high salt concentration the highly active enzyme form is obtained [Kester and Visser, 1994]. The 'high' activity PLB has a turnover of 30000 min -1, which is considerably faster than PLA and PLD using the same substrate (92 %DE citrus pectin), while the Km is 8.5 mM. As already mentioned in the previous section thus far we have not been able to demonstrate any pectin lyase activity for PLC and PLE. We believe that this is not a result of PLC and PLE not being real pectin lyases but rather our failure to identify the right substrate(s) or
339 method of analysis. Both enzymes show molecular masses as expected from the nucleotide sequences. Also their glycosylation seems to be homogeneous as only one band was seen upon SDS PAGE. PLF is an exceptional pectin lyase in that sense that it is heavily glycosylated and both Nand O-glycosylation occur. It proves therefore hard to obtain a pure, homogeneous enzyme form. Furthermore, PLF is some 100 amino acids longer than the other pectin lyases. The function of this carboxy terminal extension is not clear yet. Database searches using this extension as a query have not resulted in any clues about its nature. The extension may be involved in anchoring of the protein to the cell wall as at least part of PLF is cell wall associated. Similarly as found for PLB the pH optimum for PLF (pH 7.8) is also high. However the turnover number is at least two orders of magnitude lower using the standard substrate 92 % DE citrus pectin. The mode of action and substrate specificity of all pectin lyases together with the endopolygalacturonases is currently one of the main topics of our research.
The A. niger polygalacturonase gene family As shown for the pectin lyases endopolygalacturonases are also present as a gene family in A.
niger. The individual endopolygalacturonases are also characterized both at the genetic and biochemical level.
Genetic organization A schematic overview of the endopolygalacturonase genes sequenced thus far [Bussink et al., 1990, 1991a and 1992a and Parinicova, unpublished] is presented in Fig. 3. As is clear from this scheme the endopolygalacturonase genes are also quite similarly structured. All enzymes are almost equal in molecular mass and they all contain a signal prepropeptide that directs the proteins to the secretory pathway. The endopolygalacturonases differ from the pectin lyases with respect to the signal peptide as in the pectin lyases only a pre-sequence is found. After cleavage of the pre-sequence by the signal peptidase the propeptide is normally cleaved at the dibasic (Lys-Arg) kex2 site [Achstetter and Wolf, 1985]. The dibasic site, though not strictly conserved Lys-Arg but also present as Lys-Lys, is found in all prepro-endopolygalacturonases except for PGII where only the Arg is present. This sole basic residue provides the cleavage position of the pro-peptide and correct processing of PGII will take place as was demonstrated by N-terminal amino acid sequence determination of PGII isolated from the culture fluid. This is also indicative that at least two processing enzymes are present in A. niger, one with a monobasic specificity and a kex2 homologue. Also for PGI and PGE the cleavage position has been verified by N-terminal sequencing of the mature protein. For the PGC the prepro-peptide is based on amino acid similarity, conformation to the general von Heyne rule for secretory proteins (von Heyne, 1985) and the presence of the basic amino acid pair.
340 21+6
IB
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1362~a
,
18+13
[.~I
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|~I KR
IA
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I
I
337 m.p.
IC 43aa
[
*
KR
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,
] 383 aa 343 m.p.
IC *
PGC ll0aa
I
PGE 339 m.p.
Fig. 3. Schematic overview of Aspergillus niger polygalacturonase genes pgaI, pgalI, pgaC and pgaE. Grey squares signify the presequences; black squares signify the propeptides. Intron positions are indicated by IA, IB and IC. aa, amino acid; m.p., mature protein; *, N-glycosylation site.
The maximal number of introns detected so far in the pga genes is three in pgaC and pgaE. In the other pga genes either one (pgalI) or two introns (pgaI) are found at positions that are comparable to those of pgaC and pgaE. One potential N-glycosylation site is present in all endopolygalacturonases. This Nglycosylation sequon is conserved throughout whereas PGI has two sequons, the second one at a more C-terminal location. Promoter analysis Studies of the pga gene expression by A. niger, including the multicopy transformants of the major polygalacturonase activity encoding genes - p g a I and pgalI - confirmed that expression of these genes is regulated at the transcription level [Bussink et al., 1991b and 1992b]. Increased levels of mRNA were found when the fungus was grown on medium containing 1% pectin and 1% sugar beet pulp. The promoter regions of pgaI, pgalI, pgaC and pgaE were analyzed for DNA binding sequences of eukaryotic transcription factors (Fig. 4). As in the case of the pectin lyases several CRE A binding sites were detected in the region upstream of the translation initiation site of the particular genes. Again, only the context independent DNA-binding sites are depicted in Fig. 4. Also the presence of PACC binding site 5" GCCARG 3" was observed as were numerous 'CCCTGA' sequences [Bussink et al, 1992b].
341
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.
.
.
.
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.
.
.
.
i
-0.5
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~
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'-' strand
Fig. 4. Analysis of Aspergillus niger polygalacturonase promoters for putative regulatory sequences. In order to closer identify the upstream regulatory sequence of the pga promoters which could be involved in the positive regulation of the gene expression, a series of plasmids encoding the pgalI gene with truncated 5' upstream regions were made and used to transform A. niger [Bussink et al., 1992b]. These results revealed the necessity of the -799 to -576 sequence of the pgalI promoter for the high expression level of this gene under inducing conditions. A search for yeast regulatory DNA sequences in this region showed a sequence closely resembling the UAS2 of the Saccharomyces cerevisiae CYC1 gene [Guarente et al., 1984], which binds the heteromeric activation complex HAP2/3/4. As shown in Fig. 4 a similar sequence was found in the upstream region of pgaI and pgaC in either orientation [Bussink et al., 1992b] but this was missing in the promoter of pgaE [Parenicova and Visser, unpublished]. Furthermore when the promoter sequence upstream from -799 bp and the region from -576 bp to -300 bp of the pgalI gene were deleted PGII was produced much earlier in fermentation compared to transformants containing the whole promoter [Bussink et al., 1992b]. One explanation for this observation might be that in the upstream region of the 'HAP2/3/4' sequence the CRE A context independent binding site is located which probably still binds the repressor even under the inducing conditions and therefore slows down th e induction.
342
I
-11o
.
-0.5
.
.
.
~
!
0
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A. nigerN400
A. niger RH5344
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~ 'CCCTGA'
A. tubingensisNW756
~ PAC C
'+' strand
,~
~ HAP2/3/4 '-' strand
Fig. 5. Comparison of putative promoter elements in pgalI promoters from different Aspergilli. The sequence of strain RH53344 was taken from Ruttkowski et al. (1990) In a comparison of promoter regions of pgaII - like genes (Fig. 5) from A. niger N400, A. tubingensis NW756 and A. niger RH5344 - the same regulatory DNA sequences were found which in all cases appeared to be located in the same distance from the translation initiation codon. From this it is possible to deduce on the one hand that regulation of expression of the similar genes of closely related Aspergilli follows common rules and on the other hand that there is certainly a difference in the regulation of individual members of the A. niger pga gene family which might correspond to the nutritional conditions.
Endopolygalacturonase sequence comparison Since the endopolygalacturonase genes in our laboratory were isolated with one and the same gene as a probe, which resulted in the cloning of seven genes of which four have been sequenced, it is not surprising that all the genes share a high sequence identity. Alignment of twenty five endopolygalacturonase sequences of plant and microbial origin retrieved from the various databases available reveals that only very few amino acid residues are strictly conserved among endopolygalacturonases as is depicted in Fig. 6. We have prepared site directed mutagenized enzymes with respect to these residues in PGII which has recently also been crystallized. The characterization of one of these mutated enzymes, altered at the active site histidine 223, is presented by Benen et al. elswhere in this volume.
343 MH S FAS LLAYGLVAGATFAS AS PIEARDS CTFTTAAAAKAGKAKCSTIT LNNIEVPAGTTLDLTGLTSGTKVIFEGTTTFQYEEWAGPLISMSGEHITV TGASGHLINCDGARWWDGKGTSGKKKPKFFYAHGLDSSSITGLNIKNT PLMAFSVQANDITFTDVTINNADGDTQGGHNTDAFDVGNSVGVNIIKP WVHNQDDCLAVNSGENIWFTGGTCIGGI-IGLSIGSVGDRSNNVVKNVT IEHSTVSNSENAVRIKTISGATGSVSEITYSNIVMSGISDYGVVIQQDY_ED GKPTGKPTNGVTIQDVKLESVTGSVDSGATEIYLLCGS GSCSDWTWDD VKVTGGKKSTACKNFPSVAS C
Fig. 6. Deduced amino acid sequence of polygalacturonaseII with strictly conserved amino acid residues among twenty five polygalacturonases shown in bold and underlined.
Expression of endopolygalaeturonase genes None of the pga genes appears to be a pseudo gene as for all genes the corresponding enzymes were identified [Bussink et al., 1991b and 1992b], in part via Western blot analysis and in part via purification (for pgaE see Parenicova et al. elsewhere in this volume). All were also able to degrade polygalacturonate. Since it appeared that the pectin lyase genes were readily expressed in A. nidulans when grown on glucose, bypassing the carbon catabolite repressing response, the same was tried for the pgalI gene [Bussink et al, 1992a]. When grown on glucose high levels of expression of the pgalI gene were observed indicating that for the pga genes the carbon catabolite repression reponse can also be bypassed in A. nidulans. However, the other pga genes have not been studied in this respect.
Biochemical properties of endopolygalacturonases In our laboratory five different endopolygalacturonases were isolated from the commercial A. 1990]. The enzymes were subsequently characterized with respect to mode of action on oligogalacturonates and for two enzymes, the most abundant PGI and PGII, kinetic parameters like pH and temperature optima as well as Km and Vmax values using polygalacturonate as a substrate were determined. The recombinant PGI and PGII isolated from strains transformed with multiple copies of the p/a" promoter gene fusions were identical in all respects tested to those isolated from the Rapidase enzyme preparation with the exception that the glycosylation pattern of the recombinant enzymes was more homogeneous for each enzyme i.e. the number of bands on IEF decreased significantly to one [Kester and Visser, unpublished]. A more in depth kinetic study of recombinant PGI and PGII which includes a partial subsite map for PGII is presented by Benen et al. elswhere in this volume. For PGE the initial biochemical characterization is presented by Parenicova et al. elsewhere in this volume. PGE appears to be less active on polygalacturonate than PGI and PGII (at least one order of magnitude) which strongly suggests that the preferred substrate for this enzyme is not polygalacturonate. At present we are working on the subsite maps for all available endopolygalacturonases. The individual subsite affinities will provide insight in the preferred substrates of the individual enzymes.
niger Rapidase enzyme preparation [Kester and Visser,
344
Optimizing pectinolytic enzyme expression Promoter gene fusions The use of a strong constitutive or inducible promoter that is active under conditions where the secretion of enzymes other than the desired enzyme is low should enhance the yield and simplify the purification procedure of the individual pectinases. Since the pectinolytic genes are subject to carbon catabolite repression a promoter that is active during growth on glucose ensures that no other pectinolytic genes are expressed. The promoter of our choice is the strong promoter of the glycolytic gene pyruvate kinase A (pkiA) [De Graaff, 1989]. Cloning vectors have been constructed that allow the in flame ligation at the start codon of the pectinolytic genes after they have been engineered to contain the proper restriction site at that position. Using the obtained fusion constructs for transformation of the proper A. niger strain (see below) results in good expression of the genes and allows in general purification of the enzymes in a single chromatography step. Growth conditions As already mentioned in previous sections the pectinolytic enzymes yield depends strongly on the proteolytic susceptibility of the individual enzymes and therefore on the protease activity in the medium. A very recent study on this matter by Bartling et al. (1996) addresses this problem in more depth. They used a heterologous enzyme, Erwinia carotovora ssp atroseptica pectate lyase 3, expressed in A. niger, A. awamori and A. nidulans via special expression cassettes, as a marker for the assessment of culture conditions that allow higher yields of pectate lyase 3. From these studies it is clear that lowest PL3 yield is observed when the protease activity is highest which for A. niger is when the culture fluid has reached a pH of 4.0. For expression of PL3 in A. niger a significant increase in PL3 yield was observed when the pH of the medium was maintained longer at its starting value via a higher buffer concentration, phosphate in this case. Culturing of A. niger transformants is therefore done in high phosphate buffered minimal medium.
Strain improvement From the previous section it may be clear that a significant increase in enzyme yield can be expected when the protease activity can be lowered via downregulation of the protease expression. Since growth of the fusion transformants takes place in glucose or fructose and ammonium containing media, the protease expression is low since these enzymes are also under carbon catabolite and ammonium repression. However, still considerable protease activity is present at pH 4. Therefore a programme was started in our laboratory by van den Homberg and coworkers (1995) aimed at reducing protease activity in a systematic way. UV radiation of A. niger and suitable selection resulted in several protease deficient strains which were assigned to seven prt loci. Remaining protease activity in the prt strains, assayed with highly protease susceptible PLB, ranged from as low as 2% to 80% of wild type activity. One such strain, A. niger prtF, having only 2 % of residual protease activity was chosen as a host for the expression of the promoter fusion constructs of the pectinolytic genes. The use of A. niger prtF for expression, the strong glycolytic pk/A promoter to drive transcription under carbon and nitrogen repressing conditions and the use of high phosphate buffered media ensures a sufficient yield of individual pectinolytic enzymes.
345
Acknowledgement Part of the work presented here was funded by the EC, grant AIR2-CT941345, and part by Ciba Geigy Ltd, Basle, Switzerland. References Achstetter, T and Wolf, D.H. (1985) EMBO J. 4, 173-177. Akinrefon, O.A. (1968) New Phytol. 67, 543-556. Alberheim, P., Neukom, H.and Deuel, H. (1960) Arch.Biochem. Biophys. 90, 46. Azadi, P., O'Neill, M.A., Bergmann, C, Darvill, A.G. and Alberheim, P. (1995) Glycobiol. 5,783-789. Bartling, S., van den Homberg, J.P.T.W., Olsen, O., von Wettstein, D. and Visser, J. (1996) Curr. Genet. 29, 474-481. Borglum, G.B. and Sternberg, M.Z. (1972) J. Food Sci. 37, 619-623. Bussink, H.J.D., Kester, H.C.M. and Visser, J. (1990) FEBS Lett. 273, 127-130. Bussink, H.J.D., Brouwer, K.B., De Graaff, L.H., Kester, H.C.M. and Visser, J. (1991a) Curr. Genet. 20, 301-307. Bussink, H.J.D., Buxton, F.P. and Visser, J. (1991b) Curr. Genet. 19, 467-474. Bussink, H.J.D., Buxton, F.P., Fraaye, B.A., de Graaff, L.H. and Visser, J. (1992a) Eur. J. Biochem. 208, 83-90. Bussink, H.J.D., van den Homberg, J.P.T.W., van den IJssel, P.R.L.A. and Visser, J (1992b) Appl. Microbiol. Biotechnol. 37, 324-329. Cary, J.W., Brown, R., Cleveland, T.E., Whitehead, M. and Dean, R.A. (1995) Gene 153, 129-133. Cubero, B. and Scazzocchio, C. (1994) EMBO J. 13,407-415. Darvill, A.G., McNeil, M. and Albersheim, P. (1978) Plant Physiol. 62, 418-422. De Graaff (1989) PhD Thesis, Agricultural University, Wageningen. Guarente, L, Lalonde, B., Gifford, P and Alani, E. (1984) Cell 36, 503-511. Fish, V.B. and Dustman, R.B. (1945) J. Am Chem. Soc. 67, 1155. Fraissinet-Tachet, L.M. and Fevre, M. (1996) Curr. Microbiol. 32, 1-7. Gysler, C., Harmsen, J.A.M., Kester, H.C.M., Visser, J. and Heim, J. (1990) Gene 89, 101-108. Harmsen, J.A.M., Kusters-van Someren, M.A. and Visser, J. (1990) Curr. Genet. 18, 161-166. Henrissat, B., Heffron, S.E., Yoder, M.D., Lietzke, S.E and Jurnak, F. (1995) Plant Physiol. 107, 963-976. Heffron, S.E., Henrissat, B., Yoder, M.D., Lietzke, S.E and Jurnak, F. (1995) Mol. Plant-Microbe Interact. 8, 331-334. Heyne von, G. (1985) J. Mol. Biol. 184, 99-105. Homberg van den, J.P.T.W. van der Vondervoort, P.J.I., van der Heijden, N.C.B.A. and Visser, J. (1995) Curr. Genet. 28,299-308. Hasegawa, S and Nagel, C.W. (1968) Arch. Biochem. Biophys. 124, 513-520. Houdenhoven van, F.E.A (1975) PhD Thesis, Agricultural University, Wageningen. Jansen, E.F. and McDonnel L.R. (1945) Arch. Biochem. Biophys. 8, 97-112. Kaji, A and Saheki, T (1975) Biochem. Biophys. Acta 410, 354-360. Kester, H.C.M. and Visser, J. (1990) Biotechn. Appl. Biochem. 12, 150-160.
346 Kester, H.C.M. and Visser, J. (1994) FEMS Microbiol. Lett. 120, 63-68. Kester, H.C.M., Kusters-van Someren, M.A., Muller, Y. and Visser, J. (1996) Submitted for publication. Kusters-van Someren, M.A., Harmsen, J.A.M., Kester, H.C.M. and Visser, J. (1991a) Curr. Genet. 20, 293-299. Kusters-van Someren (1991b) PhD Thesis, University of Utrecht. Kusters-van Someren, M.A., Flipphi, M.J.A., De Graaff, L.H., van den Broek, H.C., Kester, H.C.M., Hinnen, A. and Visser, J. (1992). Mol. Gen. Genet. 234, 113-120. MacKenzie, C.R., Bilous, D., Schneider, H. and Johnson, K.G. (1987) Appl. Environm. Microbiol. 53, 2835-2839. MacMillan, J.D. and Vaughn, R.H. (1964) Biochem. 3,564-572. Mill, P.J. (1966) Bioch. J. 99, 557-561. Moran, F., Nasuno, S and Starr, J.P. (1968) Arch. Biochem. Biophys. 125,734-741. Mutter, M., Beldman, G., Schols, H.A. and Voragen, A.G.J. (1994) Plant Physiol. 106, 241 Mutter, M., Colquhoun, I.J., Schols, H.A., Beldman, G. and Voragen, A.G.J. (1996) Plant Physiol. 110, 73-77. Nagel, C.W. and Vaughn, R.H. (1961) Arch. Biochem. Biophys. 93,344-351. O'Neill, M.A., Albersheim, P. and Darvill, A.G. (1990) in P.M. Dey (Ed.), Methods in Plant Biochemistry, vol 2, Carbohydrates, Academic Press, London, 415-441. Ruttkowski, E., Labitzke, R., Khanh, N.Q., Loftier, F., Gottschalk, M. and Jany, K.D. (1990) Biochem. Biophys. Acta 1087, 104-106. Schols, H.A., Gereads, C.C.J.M., Searle-van Leeuwen, M.F., Kormelink, F.J.M. and Voragen, A.G.J. (1990) Carbohydr. Res. 206, 105-115. Schols, H. (1995) PhD Thesis, Agricultural University, Wageningen Searle-van Leeuwen, M.J.F., van den Broek, L.A.M., Schols, H.A., Beldman, G. and Voragen, A.G.J. (1992) Appl. Microbiol. Biotechnol. 38, 347-349. Suykerbuyk, M.E.G., Schaap, P.J., Stam, H., Musters, W. and Visser, J. (1995) Appl. Microbiol. Biotechnol. 43, 861-870. Tilburn, J., Sarkar, S., Widdick, D.A., Espeso, E.A., Orejas, M., Mungroo, J., Penalva, M.A. and Arst, H.N.Jr. (1995) EMBO J. 14, 779-790. Visser, J., Suykerbuyk, M.E.G., Kusters-van Someren, M.A., Samson, R. and Schaap, P. (1996) in Rossen, L., Rubio, V., Dawson, M.T. and Frisvad, J. (eds) Fungal Identification Techniques, European Commission, EUR 16510 EN, 194-201. Vries de, J.A., Rombouts, F.M., Voragen, A.G.J. and Pilnik, W. (1982) Carbohydr. Polym. 2, 25-33. Whitcombe, A.J., O'Neil, M.A., Steffan, W., Albersheim, P. and Darvill, A.G. (1995) Carbohydr. Res. 271, 15-29. Williamson, G. (1991) Phytochemistry 30, 445-449.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All fights reserved.
347
Expression of polygalacturonase and pectinesterase in normal and transgenic tomatoes. G. Tucker and J.Zhang. Applied Biochemistry and Food Science, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics. LE12 5RD. U.K.
Abstract
Polygalacturonase and pectinesterase are two enzymes involved in the degradation of pectin. These enzymes are particularly active in the degradation of pectin which accompanies fiafit ripening. The molecular biology of these two enzymes has been studied extensively in both normal tomato fruit and in transgenic fruit manipulated to reduce enzyme expression. Both enzymes exist in multiple isoenzyme forms. In the case of polygalacturonase these isoforms appear to arise from the post translational modification of a single gene product. In contract pectinesterase isoform expression seems to be much more complicated and appears to involve the function of several nonhomologous genes and also small multi-gene families. This paper provides an overview of the expression of these enzymes in normal and transgenic tomato fiafit. 1. INTRODUCTION
Fruit softening is often accompanied by degradation of the pectin components of the cell wall. These changes include a decline in the degree ofesterification, a loss of neutral sugars such as galactose and arabinose and an increased solubility and depolymerisation (Tucker et al 1993). These changes in pectin structure are brought about by the action of cell wall associated enzymes during ripening. In particular polygalacturonase (E.C 3.2.1.15), which has the capacity to depolymerise the polyuronide chains in pectin, and pectinesterase (E.C 1.1.1.11), which can deesterify the galaeturonic acid residues, are thought to be important degradative enzymes. These enzymes are associated with most, but not all, fiafit and often undergo sitmificant changes in expression during ripening. The expression and molecular biology of both these enzymes have been most extensively studied in tomato fiafit and this is the subject of this review. Tomato fruit during ripening show all the typical changes in pectin structure outlined above. 2. POLYGALACTURONASE Polygalacturonase (PG) activity is almost non detectable in green tomato fixtit (Hobson 1964). However, the activity increases dramatically during ripening. This PG activity has been
348 resolved into at least two isoforms (PG 1 and PG2) using ion-exchange chromatography (Pressey and Avants 1973,Tucker et al 1980). During ripening PG1 is the first detectable isoform, however PG2 activity can be detected very soon afterwards and then rapidly becomes the dominant isoform in ripe fi'uit (Tucker et a/1980). The PG2 isoform can be converted in vitro to a form resembling PG1 by simple incubation with an extract from green fiafit tissue (Tucker et al 1981). This converter activity has been identified and partially characterised Pressey (1984). Both PG 1 and PG2 have been purified and characterised (Moshrefi and Luh 1984, Tucker et al 1980). The PG2 isoform has been shown to be composed of a single polypeptide chain with a molecular weight of arotmd 43kD, and this has been fully sequenced (Sheehy et al 1987). Closer examination of this isoform by gel electrophoresis reveals two sub forms (PG2A and PG2B), however, these have been shown to contain exactly the same polypeptide chain and differ only in the extent to which they are glycosylated (Mohd-Ali et al 1982). In contrast purified PG1 appears to be composed of two heterologous polypeptide chains with molecular weights of around 43kD and 38kD (Moshrefi and Luh 1884). The 43kD polypeptide cross reacts with antibodies raised against purified PG2 and tryptic digestion of the 43kD polypeptide from both PG1 and PG2 result in the same digest pattern of peptide fragments. These results suggest that the 43kD polypeptide in each isoform is identical and presumably represents the catalytic subunit. The 38kD protein has been termed the 13-subunit and a corresponding cDNA has been identified and charactefised (Zheng et al 1992). The function of the 13-subunit and the role of the converter is unclear (Knegt et al 1991), indeed there is some evidence that the formation of PG1 is an artifact of the extraction and that this isoform does not exist in vivo (Moore and Bennett 1994). Recently expression of the 13-subunit has been downregulated in transgenic tomatoes using antisense RNA technology (Watson et al 1994). This resulted in the marked reduction in the level of extractable PG1 isoform and also influenced wall degradation. In these transgenic fiafit polyuronide solubilisation and depolymerisation were both significantly greater than in corresponding normal fiafit. This suggests that whilst the 13-subunit is not required for PG action it may have a possible regulatory role in limiting pectin degradation. The 13-subunit has been immunolocalised to the cell wall in green fruit and as such a role in targeting the PG within the wall has been postulated (Pogson et al 1991). This targeting may represent the mechanism by which the 13-subunit could limit pectin degradation. Several groups have isolated clones corresponding to the 43kD catalytic subunit of PG from fruit cDNA libraries (Grierson et al 1986, Sheehy et al 1987). The clone isolated at Nottingham was t e m ~ pTOM6 (Grierson et al 1986). Expression of the mRNA corresponding to pTOM6 occurs in a fiafit and ripening specific manner. This clone has been used to isolate corresponding genomic clones and thus the gene for tomato fruit PG has been characterised. There appears to be only a single gene encoding the fruit specific PG protein (Bird et al 1988). This finding is consistent with the biochemical data and again suggests that all the isoforms of PG identified in ripe tomato fruit are the product of a single gene and arise simply fi'om differential post translational modification of a common polypeptide or in the case of PG1 may arise purely as an artifact of the extraction procedure. Several groups have down regulated fruit PG expression using either antisense RNA technology or by co-suppression following transformation of plants with a partial sense DNA construct (Sheehy et al 1988, Smith et al 1988). The group at Nottingham have achieved this down regulation by both mechanisms using the pTOM6 cDNA clone. In both instances PG expression in transgenic fruit has been reduced to less than 0.5% of the activity occtnring in normal fruit. As expected all the isoforms of PG were effected by this transformation since only a single gene is
349 corresponds to pB8, the second to pB 16 (Hall et al 1994) and the third, which has 86% and 88% homology to pB8 and pB16 respectively within the coding region so far analysed, has no apparent cDNA product. It would appear that the first gene in this series accounts for the vast majority of the PE2 mRNA found in fruit and that this message codes for the major PE2 isoform. It is not clear whether or not the other two genes result in any appreciable production of PE2 protein. The second gene in the series is transcriptionally active but as yet no corresponding protein has been isolated. There is some evidence that PE2 in tomato fi~it may exist in multiple forms. Gaffe et al (1994) identified five PE isoforms in tomato fi-uit using isoelectric focusing. Three of these isoforms cross reacted with an antibody raised against an eqttivalent of PE2 and all three isoforms disappeared in transgenic fi~it produced by antisense RNA technology targeted against the PE2. It is possible that these isoforms arise from post translational modification of a single PE2 gene product, as for PG, this being the protein corresponding to the pB8 clone described above. It is also possible that these three PE2 sub forms may arise from the transcription and translation of two or more of the established PE2 multi gene family. Several groups have down regulated the expression of the PE2 gene(s) using antisense RNA technology (Tieman et al 1992, Hall et al 1993). In all cases total PE activity in the fiafit was reduced to around 10% of normal. The activities of the PE1, PE2 and PE3 isoforms in transgenic fruit from plants containing an antisense gene targeted at PE 2 are shown in figure 2. It can be seen that whilst the activity of PE2 has been reduced to almost zero, that of PE 1 and PE3 is unaffected and may even have increased slightly over the levels found in normal fi~tit. Thus the residual PE activity in these transgenic fruit is almost entirely due to the fact that expression of the PE1 and PE3 isoforms are both unaffected by the antisense gene. A similar finding has been reported by Gaffe et al (1994) since in this case of the five PE isoforms identified in tomato fruit only those three related to PE2 disappeared in transgenic fruit. 2.5 .
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Figure 2 Pectinesterase activity in transgenic tomato fruit. Activity is expressed as (ueq~,nin\gfwt} The effect on cell wall degradation of the down regulation of PE2 activity is not entirely clear. Tucker et al (1992) reported that the solubility and depolymerisation ofpolyuronide was unaffected by this treatment whilst Tieman et al (1992) reported that both aspects of wall metabolism were markedly reduced by antisensing PE2. These discrepancies may be accounted for by the different
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Figure 1. Pectinesterase activity in normal tomato fruit. Enzyme activity is expressed as (ueq\min\gfwt) cDNA library resulted in the identification and characterisation of two further clones with homology to PE2, these were termed pB8 and pB 16 (Hall et al 1994). The clone pB 16 differed from that isolated by Ray et al (1988) in only 4 positions and as such may represent a longer version of the original clone. The pB8 had 93.7% homology to pB16 in the coding region and the predicted amino acid sequence from this clone had a much greater homology to the actual PE2 protein sequence. The pB8 clone was si~ificantly different from both the original clone and pB16 in the 3' untranslated region. The pB8 clone was 1961bp in length and contained an open reading frame encoding a protein of 546 amino acids. Based on the N-terminal amino acid sequence of the mature PE2 protein this implies that the protein encoded by pB8 had an N-terminal extension of 229 amino acids. The function, if any, of such a large N-terminal extension is unclear. It is interesting to note that pTOM6, which encodes the PG enzyme, also encodes for a protein with a large, in this case 77 amino acids, N-terminal extension. Both of these N-terminal sequences carry typical hydrophobic signals but these are contained within the first 40-45 amino acid residues. The remaining parts of the N-terminal region may play important roles in the targeting or control of activity of the ~ e once it has been deposited within the cell wall. Using the si~ificant differences in the 3' untranslated regions of pB8 and pB16 it was possible to determine the relative levels of mRNA corresponding to each clone (Hall et al 1994). In both cases the mRNA expression peaked at the mature green\ breaker stage of development. However, at all stages of development and ripening the mRNA corresponding to pB8 was expressed at around 40 times the level of that corresponding to pB 16. It thus appears that PE2 may be encoded for by a small multi gene family. The precise number of active genes is unclear. The work above suggests two gene products but a report by Harfiman et al (1991) described the isolation of a partial PE2 cDNA clone which does not show homology to either pB8 or pB16 and which demonstrates a similar expression pattern i.e. with peak levels of mRNA occurring at the mature green\breaker stage. The two groups used different tomato cultivars and this may complicate any sequence comparisons. Both groups however, have carried out analysis of genomic clones and in both cases have shown that there are 3 PE2 like genes organised in tandem within the genome. It would appear that in the case of Ailsa craig at least, the first gene in the series
351 present. During the ripening of these transgenic fruit pectin solubility and degree of esterification were both unaltered whilst the depolymerisation of pectin normally associated with ripening was markedly reduced (Smith et al 1990). The 5' and 3' flanking regions of the PG gene have been used to generate a DNA construct in which they drive the expression of a CAT (chloran~renicol acetyltransferase) reporter gene. This reporter gale construct has been used to generate transgenic tomato plants in which CAT is expressed in a fruit and ripening specific manner Grierson et al 1990). This expression corresponds to that of the endogenous PG gale in normal fruit. In addition CAT activity in the transgenic plants was also found to be associated with abscission zone tissue. It is known that tomato tissues other than fruit express PG activity and abscission zone tissue is one of these tissues (Taylor et al 1990). Low levels of PG activity are detectable in zone tissue prior to separation, however, in response to ethylene treatment this activity increases dramatically as the separation zone develops. This occurs in both the leaf abscission zone and also in the zone associated with flowerkfrtfit abscission. Antibodies raised against the fruit specific PG protein do not reco~ise the abscission zone PG. In addition transgenic plants in which the expression of the fruit specific PG has been down regulated fail to show any corresponding down regulation of PG activity in the abscission zones (Taylor et al 1990).Thus the abscission zone PG gene presumably has insufficient homology to the fi'uit PG gene to enable antisense RNA constructs directed at the latter to have any effect on abscission zone PG expression. These observations suggest that the fruit and abscission zone PG enzymes are the products of two non homologous genes and thus other PG remain to be identified in tomato plants. 3 PECTINESTERASE Pectinesterase (PE) activity occurs in tomato fruit throughout both development and ripening (Hobson 1963). However, this activity has been resolved into several isoforms. Pressey and Avants (1972) showed that PE isoforms could be separated by ion-exchange chromatography and that the profile was different in different tomato cultivars. Tucker et al (1982) demonstrated that the PE fi'om Ailsa craig tomatoes could be separated into at least two isoforms, which they called PE1 and PE2, and succeeded in the purification of the major fruit isoform namely PE2. More recently Warrilow et al (1994) have separated three PE isoforms from tomato fi'uit which they termed PEA, PEB and PEC. Further studies on the PE1 fraction from the work of Tucker et al (1982) have shown that this can itself be resolved into two isoforms which have been termed PE1 and PE3. From comparisons of the properties of these groups of PE isoforms it is apparent that PEA, PEB and PEC, in the nomenclature of Warrilow et al (1994), correspond to PE2, PE3 and PE1 respectively. For the purpose of this review the PE isoforms will be referred to as PE 1,PE2 and PE3. Thus tomato fruit would appear to contain at least three isoforms of PE throughout both development and ripening. The relative activities of PE1,PE2 and PE3 during the development and ripening of tomato fruit are shown in figure 1. It can be seen that PE1 and PE2 both have peaks of activity at around the mature green\breaker stage of development. In contrast PE3 activity remains relatively constant throughout both development and ripening. It is also clear that PE2 is by far the predominant isoform at all stages. The PE2 isoform has been purified and fully sequenced (Markovic and Joumval 1986). Using this sequence data Ray et al (1988) succeeded in isolating a clone from a tomato fruit cDNA library. The predicted amino acid sequence from this clone had high homology to the actual amino acid sequence determined for PE2 but was not identical. Subsequent screening of the tomato
352 tomato cultivars used, the different stages used for analysis and by the different pectin extraction methods employed. However, in both cases it was clear that reduction in PE2 isoform levels had resulted in a marked reduction in the deesterification of pectin at all stages of fiuit development and ripening. It is clear from the antisense work described above that the PE1 and PE3 isoforms are derived from the expression of gene(s) which are non homologous and thus completely different to the gene family identified for PE2. This conclusion is supported by the fact that both PE 1 and PE3 isoforms have been purified and their N-terminal amino acid sequences determined. These sequences are completely different for PE 1 and PE3 and both in turn differ from that for PE2. This suggests that both PE1 and PE3 are coded for by distinct gene(s). The identification and characterisation of these genes remains to be carried out. The study of these other two PE isoforms is particularly important since these appear to be the isoforms which are prevalent in vegetative tissue. The PE2 isoform appears to be fi'uit specific. Antibodies raised against the fi'uit PE2 fail to cross react with either PE 1 or PE3 or with the PE extractable from leaf or root tissue. Similarly antisense PE2 plants whilst showing a marked decline in fruit PE levels show no effect whatsoever on levels of PE activity in leaves or roots. The vegetative forms of PE appear, at least on the basis of isoelectric focusing and ion-exchange chromatography, to be similar to the PE1 and PE3 isoforms present in the fruit. This has led Gaffe et al (1994) to describe the presence of two groups of isoforms in plant tissue. Group I are fnfit specific isoforms, all of which appear to be related to PE2. Group II are isoforms present in both fruit and vegetative tissue and these presumably correspond to PE 1 and PE3. 4 CONCLUSIONS The PG activity in tomato fi'uit appears to arise from the expression of a single gene. The various PG isoforms which appear during ripening being either artefacts of the extraction process or products of post translational modification of a common polypeptide precursor. This gene, and its product appears to be expressed exclusively in ripening fi'uit. The PG activity which is apparent in other tomato tissue arises from the expression of other non homologous genes. In contrast PE activity in tomato fruit appears to arise from the expression of at least two, and probably three, separate classes of genes. The major PE isoform (PE2) is fruit specific and maybe coded for by a small multigene family containing three members. However, one of this PE2 gene family, that corresponding to pB8 (Hall et al 1994), is preferentially expressed and the polypeptide encoded by this gene preferentially accumulated in the fi~it. The role, if any, of the other two gene members of this PE2 gene family remains to be elucidated. The remaining two fruit PE isoforms do not appear to be fruit specific and may also be expressed in a wide range of tomato tissues. It is clear however, that both these isoforms are encoded by a gene, or genes, which are separate from and non homologous to those for PE2. The nature and identity of these other PE genes or gene families also remains to be elucidated. REFERENCES
Bird, C.R., Smith, C.J.S., Ray, J.A., Moreau, P., Bevan, M.W., Bird, A.S., Hughes, S., Morris, P.C., Grierson, D. & Schuch, W. (1988) The tomato polygalacturonase gene and ripening specific expression in transgenic plants. Plant Molecular. Biology. 11.651-662.
353 Gaffe,J., Tieman,D.M and Handa,A.V (1994) Pectinmethylesterase isoforms in tomato (lycopersicon esculentum) tissues. Effects of expression of a pectinmethylesterase antisense gene. Plant Physiology. 105.199-203. Grierson, D., Tucker, G.A., Keen, J., Ray, J., Bird, C.R. & Schuch, W. (1986) Sequencing and identification of a cDNA clone for tomato polygalacturonase. Nucleic Acid Research. 14 85958603. Grierson,D., Smith,C.J.S., Watson,C.F., Morris,P., Gray,J.E., Davies,K., Picton,S.J., Tucker,G.A., Seymour,G.B., Schuch,W., Bird,C.R and Ray,J (1990) Regulation of gene expression in transgenic tomato plants by antisense RNA and ripening-specific promoters, in Genetic Engineering of Crop Plants. Lycett,G.W and Grierson,D Eds. Butterworths.pp 115-125. Hall, L. H., Tucker, G. A., Smith, C. J., Watson, C. F., Seymour, G. B., Bundick, Y., Boniwell, J. M., Fletcher, J. D., Ray, J. A., Schuch, W., Bird, C. R and Grierson,D (1993) Antisense inhibition of pectin esterase gene expression in transgenic tomatoes. The Plant Journal. 3. 121-129. Hall, L. N., Bird, C. R., Picton,S. P., Tucker, G. A., Seymour, G. B and Grierson, D (1994) Molecular characterisation of cDNA clones representing pectin esterase isozymes from tomato. Plant Molecular Biology. 25.313-318. Harriman,R.W.,Tieman,D.M and Handa,A.K (1991) Molecular cloning of tomato pectinmethylesterase gene and its expression in Rutgers, ripening inhibitor, non-ripening and neverripe tomato fruit. Plant Physiology. 97. 80-87. Hobson, G.E. (1963) Pectinesterase in normal and abnormal tomato fruit. Biochemical Journal. 86. 358-365. Hobson, G.E. (1964) Polygalacturonase in normal and abnormal tomato fi'uit. Biochemical Journal. 92. 324-332. Knegt,E., Verrneer,E., Pak,C and Bruinsma,J (1991) Function of the polygalacturonase converter in ripening tomato fruit. Physiologia Plantarum. 82. 237-242. Markovic,O and Jornvall,H (1986) Pectinesterase: The primary structure of the tomato enzyme. European Journal of Biochemistry. 158.455-462. Mohd Ali,Z. and Brady,C.J.(1982) Purification and characterisation of the polygalacturonase of tomato fiafit. Australian Journal of Plant Physiology. 9. 155-159. Moore,T and Bennett,A.B (1994) Tomato fruit polygalacturonase isozyme-1. Characterisation of the beta-subunit and its state of assembly in vivo. Plant Physiology. 106. 14611469. Moshrefi, M. & Luh, B.S. (1984) Purification and characterisation of two tomato polygalacturonase isoenzymes. Journal of Food Biochemistry. 8. 39-54. Pogson,B.J.,Brady,C.J and Orr,G.R (1991) On the occurance and structure of subunits of endo-polygalacturonase isoforms in mature-green and ripening tomato fruit. Australian Journal of Plant Physiology. 18.65-79. Pressey,R (1984) Purification and characterisation of tomato polygalacturonase converter. European J Biochemistry. 144. 217-221. Pressey,R and Avants,J.K (1972) Multiple forms of pectinesterases in tomatoes. Phytochemistry. 11.3139-3142. Pressey, 1L & Avants, J.IC (1973) Two forms of polygalacturonase in tomatoes. Biochimica et Biophysica Acta. 309. 363-369.
354 Ray,J.,Knapp,J.,Grierson, D.,Bird, C and Schuch,W. (1988) Identification and sequence determination of a cDNA clone for tomato pectinesterase. European Journal of Biochemistry. 174. 119-124. Sheehy,R.E.,Pearson, J.,Brady, C.J. and Hiatt,W.R.(1987) Molecular characterisation of tomato fruit polygalacturonase. Molecular and General Genetics. 208, 30-36. Sheehy,R.E.,Kramer,M. and Hiatt,W.R. (1988) Reduction of polygalacturonase activity in tomato fruit by antisense RNA. Proceedings of the National Academy of Sciences,USA. 85,8805-8809. Smith, C.J.S., Watson, C.F., Ray, J., Bird, C.J., Morris, P.C., Schuch, W. & Grierson, D. (1988) Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes. Nature .334. 724-726. Smith, C.J.S., Watson, C.F., Morris, P.C., Bird, C.R., Seymour, G.B., Gray, J.E., Arnold, C., Tucker, G.A., Schuch, W., Harding, S.E. & Grierson, D. (1990) Inheritance and effects on ripening of antisence polygalacturonase genes in transgenic tomatoes. Plant Molectdar. Biology. 14 9369-379. Taylor,J.E., Tucker, G.A., Lasslett,Y., Smith,C.J.S., Amold, C.M., Watson, C.F., Schuch,W., Grierson, D and Roberts,J.A (1990) Polygalacturonase expression during leaf abscission of normal and transgenic tomato plants. Planta. 183. 133-138. Tienmn, D.M, Harriman,R.W, Ramamohan, G and Handa,A.K (1992) An antisense pectin methylesterase gene alters pectin chemistry and soluble solids in tomato fruit. The Plant Cell. 4. 667-679. " Tucker, G.A (1993) Fruit ripening, in The biochemistry of fruit ripening. Seymour, G.B., Taylor,J.E and Tucker, G.A Eds Chapman and Hall.pp 1-51. Tucker, G.A.,Robertson, N.G. and Grierson,D. (1980) Changes in polygalacturonase isoenzymes during the ripening of normal and mutant tomato fi'uit. European Journal of Biochemistry. 112, 119-124. Tucker, G.A., Robertson, N.G. & Grierson, D. (1981) The conversion of tomato fruit polygalacturonase isoenzyme 2 into isoenzyme I in vitro. European Journal of Biochemistry. 115, 87-90. Tucker, G.A.,Robertson,N.G. and Grierson,D. (1982) Purification and changes in activities of tomato pectinesterase isoertzymes. Journal of the Science of Food and Agriculture. 33.396-400. Tucker,G.A.,Seymour, G.B.,Bundick,Y.,Robertson, D.,Smith, C.J.S and Grierso~D (1992) Use of antisense RNA technology to manipulate pectin degradation in tomato fruit. New Zealand Journal of Horticultural Crop Botany. 20. 119-124 Warrilow, A. G. S., Turner, R. J and Jones (1994) A novel form of pectinesterase in tomato. Phytochemistry. 35. 863-868. Watson, C.F., Zheng,L.S and Dellapenna,D (1994) Reduction of polygalacturonase betasubunit expression affects pectin solubilisation and degradation during fi-uit ripening. Plant Cell. 6. 1623-1643. Zheng,L.S., Heupel,R.C and Dellapenna,D (1992) The beta-subunit of tomato fruit polygalacturonase isoenzyne-1. Isolation,characterisation and identification of unique structural features. Plant Cell. 4. 1147-1156.
J. Visserand A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996ElsevierScienceB.V.All rights reserved.
355
Role of pectin methylesterase in tomato fruit ripening and quality attributes of processed tomato juice A.K. Handa a, D.M. Tieman a, K.K. Mishra a, B.R. Thakur b and R.K. Singh b Departments of aHorticulture and bFood Science, Purdue University, W. Lafayette, IN 47907-1165, USA.
Abstract We have created transgenic tomato fruits with over a 10-fold reduction in pectin methylesterase (PME) activity by expressing a PME antisense gene. Reduction in PME activity does not interfere with the fruit ripening and vegetative growth of plants, but is associated with increases in soluble and total solids, tool. wt. and methyl esterification of pectins, and modifies accumulation and partitioning of cations between soluble and bound forms. The processed juice from transgenic fruits shows increases in serum and efflux viscosity and precipitate weight ratio. Ketchup prepared from transgenic fruits has a significant lower Botswick value, reduced serum separation and higher serum viscosity t h a n control. Collectively, a marked improvement in fruit processing attributes results from lower PME activity in fruits. 1. I N T R O D U C T I O N Pectin methylesterase (PME, EC 3.1.1.11), an ubiquitous enzyme in the plant kingdom and in several plant pathogenic bacteria and fungi, catalyzes the deesterification of pectin to form a carboxylated pectin while releasing methanol and a proton [1,2]. Pectins are composed primarily of polygalacturonic acid, a homopolymer of [1-~4]a-D-galactosyluronic acid units containing partially m e t h y l e s t e r i f i e d carboxyl groups. Rhamonogalacturonan I and rhamnogalacturonan II are other types of pectins present in plant tissues [3]. It has been suggested t h a t pectins are synthesized with a high degree of methylesterification, but the esters are later cleaved by PME in the cell wall [4]. PME activity and Ca 2+ have long been implicated in the formation of Ca 2+ crossbridges in pectin leading to stabilization of the middle lamella. Although PME has been suggested to be involved in various growth and developmental processes in plants, including cell wall growth and extensibility, cation binding capacity of cell walls, ion acquisition, formation of abscission zones, textural changes in ripening fruit, root border cell separation, and symbiotic association with pathogens and insets [5-11], its in planta function in these processes is as yet unknown. PME
356 activity has been reported to increase during maturation and ripening of several fruits including tomato [1]. Since demethoxylated pectins are better substrates for polygalacturonase, it has been speculated that PME plays a role in fruit softening by increasing the in vivo susceptibility of pectin. Fruit specific PME isoforms have been localized to the primary cell wall and middle lamella of mature green and ripening tomato pericarp [ 12]. Pectin deesterification has been reported to start in the middle lamella of mature green tomato pericarp and continues in the primary cell wall until most pectins are deesterified in the ripe fruit pericarp [13]. To understand the molecular role of PME in tomato fruit development and fruit quality and processing attributes, we have created transgenic tomato plants expressing an antisense gene for the fruit specific PME. We identified several independent transgenic plants with greatly reduced levels of PME in fruits. We report here that reduced PME activity has a marked effect on the processing attributes of tomato fruit.
2. R E S U L T S AND DISCUSSION
C h a n g e s in PME Gene E x p r e s s i o n D u r i n g Tomato Fruit Ripening: Increases in PME activity in ripening tomato fruits have been demonstrated by a number of research groups [1,14]. We have purified a PME isozyme from mature green tomato fruits to an apparent homogeneity, raised polyclonal antibodies to the purified PME and isolated a PME cDNA clone from an expression library constructed from mature green pericarp poly(A)+RNA [1]. We have shown that PME activity is first detected in 10-d-old fruit and increases until the turning stage of fruit ripening. The increase in PME protein parallels the increase in PME activity until the turning stage but continues to increase thereafter while PME activity begins to decline [ 1]. PME mRNA becomes detectable in 15-day-old fruit and increases until the mature green stage before declining thereafter [1]. The highest level of PME mRNA are found in exopericarp followed by endopericarp, collumella and radial walls [15]. Our results indicate that in the ripening impaired tomato mutants, the t i n mutation strongly affects expression of the PME gene during the fruit development, while the N r and nor mutation have little effect on overall PME gene expression during fruit maturation [1].
Creation and Characterization of T r a n s g e n i c Tomato P l a n t s E x p r e s s i n g a n Antisense Gene for the Fruit Specific PME: To gain insight into the role of PME in tomato fruit development, we have introduced a truncated PME gene in antisense orientation under the control of the CaMV 35S promoter into tomato [16]. Fruits from independent transgenic plants expressing the introduced PME antisense gene exhibited PME activity ranging between 7 to 40% of wild-type Rutgers fruits [16]. One of the transformant 3781 ^, which contained single copy of the PME antisense gene was further characterized. Analysis of segregating progeny from this transformant showed that the introduced gene is inherited in normal Mendelian fashion and the reduced PME activity is due to the presence of the introduced PME antisense gene. Figure 1 shows changes in PME gene
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3 7 8 1 "T 3
Figure 1: Changes in PME gene expression during development of wild-type and transgenic fruits. 3781AT 1 and 3781^T 3 represent the primary transformant 3781A and its segregant homozygous for the PME antisense gene. Levels of PME activity, protein, mRNA and antisense RNA and PG mRNA were determined in pericarp tissue as described by Tieman et al. [16]. Abbreviations for stages of fruit development are as follows: 25d and 35d, 25 and 35 days after flowering; Br, breaker, Tu, turning; RR, red ripe fruits. Reprinted from Tieman et al., Plant Cell, 4 (1992) 667 with permission from American Society of Plant Physiologists.
358 expression during fruit development and ripening in fruits from wild-type Rutgers, primary transformant 3781 ^ and a segregant of 3781 ^ homozygous for the introduced antisense PME gene. PME activity in wild-type fruits increased until the turning stage, but remained fairly constant in transgenic fruits during fruit development. At red ripe stage, the transgenic fruits contained about 10% of wildtype fruit PME activity and undetectable levels of PME protein and mRNA (Fig. 1). Similar reduction in PME activity by a PME antisense gene has been reported in Ailsa Craig [17]. The remaining 10% PME activity in transgenic fruits is due to other PME isozymes that are also found in root, stem and leaves of both wild-type and transgenic plants [18]. Accumulation of this group of PME isoforms is not impaired by the expression of the fruit specific PME antisense gene [18]. Effects of R e d u c e d PME Activity on R i p e n i n g P a r a m e t e r s and P e c t i n C h e m i s t r y : Reduction in the fruit PME activity did not interfere with the ripening process (Table 1). During the ripening process, patterns of ethylene production, rate of respiration, lycopene accumulation and chlorophyll degradation in transgenic fruits were nearly identical with those observed in wild-type fruits (Table 1) [11,16]. However, reduction of PME activity caused a significant increase in degree of methylesterification of pectins in transgenic fruits, especially during the fruit ripening [16]. The levels of EDTA-soluble uronic acids were lower in transgenic fruit cell walls as compared to wild-type fruit throughout the ripening stages, while significant differences in EDTA-insoluble pectins in pericarp cell walls were not observed between wild-type and transgenic fruits (Table 1). Size fractionation of EDTA-soluble polyuronides from red ripe fruit pericarp showed higher levels of intermediate size polyuronides in transgenic fruits with reduced PME activity than in wild-type Rutgers fruits [16]. These results suggest that PME plays a role in depolymerization of pectins presumably by modifying the action of polygalacturonase. Synergism between polygalacturonase and PME has been suggested to play a role in depolymerization of fruit pectins during the ripening process [19]. A 5 to 10% increase in the levels of soluble solids was observed in transgenic fruits compared to wild-type fruits [16]. The effects of the introduced gene on soluble solids were more pronounced in mature green and ripening tomato fruits than in immature tomato fruits. Several factors such as environment, age of plant, nutrition, yield per plant and stresses are known to influence levels of soluble solids in fruits. However, several independent evidence suggest t h a t the increased levels of soluble solids in transgenic fruits were associated with reduced PME activity, including segregation of the higher soluble solid trait with the PME antisense gene [16]. Effects of R e d u c e d PME Activity on Cation Binding Capacity of Cell Walls and Cation Levels in Pericarp: Since the primary polyanionic charges in cell walls are those of polygalacturonic acid, changes in PME activity can greatly influence the ionic composition of cell walls [20]. To evaluate the role of PME on cation binding in fruit tissue, we have determined the in situ binding of Ni 2+ and 45Ca 2+ to fruit tissues from transgenic fruits with low PME activity and wild-type fruits [11]. The pericarp, radial cell wall, and collurnella regions of tomato fruit
359 Table 1 Effects of reduced PME gene expression on chemistry of ripened tomato fruits Genotypr Variable Rutgers Transgenic Lycopene (~g]gfw) a
87.4 + 17.1
121.6 + 8.6
Chlorophyll (~lg/gfw)a
2.1 _+0.3
1.4 + 0.1
Maximum Rate of Ethylene a Production (nl/gfw/hr)
25.3 + 2.4
24.3 _+ 0.7
Degree of Pectin a Methyle sterification (%)
38.5 _+ 7.4
48.5 +_ 5.5
Total Uronic Acid a (pmoles/mg cell wall)
1.128 _+ 0.047
1.023 _+ 0.042
Chelator Soluble Uronic Acid a (~moles/mg cell wall)
0.427 _+ 0.035
0.318 +_ 0.016
Soluble Ca 2+ (~g/gdw) b
453 + 33
554 _+ 53
Bound Ca 2+ (~g/gdw) b
452 _+ 56
228 _+ 15
Soluble Mg 2+ (~g/gdw) b
1193 _+ 12
698 _+ 79
Bound Mg 2+ (~g/gdw)b
275 + 19
95 _+6
Soluble Na + (~g/gdw) b
275 +_6
110 _+4
Bound Na+(~g&dw) b aModified from Tieman et al. [16]. bModified from Tieman and Handa [11].
195 _+ 12
105 _+ 13
exhibited high Ni2+and Ca 2+ binding, whereas the jelly in the locular region showed little binding of these cations. The transgenic fruits exhibited significantly reduced binding of both Ni 2+ and Ca 2+ t h a n wild-type fruits at all w of fruit ripening examined [11]. Reduced PME activity in transgenic fruits also had a marked effect on accumulation of cations and partitioning of cations between soluble and bound forms [11]. Lowered PME activity selectively impaired accumulation of Mg 2+ over other cations (Table 1). A 30 to 70% decrease in bound Ca 2+ and Mg 2+ was observed in transgenic fruits with low PME activity, while the levels of soluble Ca 2+ increased 20 to 40% in transgenic fruits compared to wild-type fruits [11]. Levels of soluble Mg 2+ and Na + decreased 20 to 40% in transgenic fruits with lowered PME activity. PME activity do not seem to influence levels of K +, PO 3" or CI" in transgenic fruits. We saw few differences in ion levels in leaf tissue of wildtype and transgenic plants [11], which is likely due to the use of a fruit specific PME antisense gene t h a t does not impair expression of PME isozymes present in vegetative tissues [18]. Conceivably, the decrease in cation binding capacity of cell walls, and increase in soluble Ca 2+ and Mg 2+ levels in transgenic fruits is a
360
750
B
_•
00
Rutgers
80
Transgenic
500
60 40
tq tq
250 20
MG
BR Ripening
TtJ Stage
RR
1
2
3
4
5
6
7
8
Weeks After H a r v e ~
Figure 2: Firmness and shelf life of wild-type and transgenic fruits. (A) Firmness of whole fruits at mature green (MG), breaker (Br), turning (Tu) and red ripe (RR) stages were determined using an Instron Universal Testing Machine as described by Tieman and Handa [11]. (B) Shelf life of ripened fruits from both genotypes was determined by storing ripe fruits at 25 _+2~ Shown is the percent of total usable fruits after increasing periods of storage. Fig. 2A redrawn from Tieman and Handa, Plant Physiol., 106 (1994) 429 with permission from American Society of Plant Physiologists.
direct consequence of the lower frequency of anionic changes on pectins due to higher degree of methylesterification. Effects of Reduced PME Gene Expression on Tomato Fruit Firmness and S h e l f Life: Changes in firmness of frmts from transgenic and wild-type plants
were examined by generating force-deformation curves using an Instron Universal Testing Machine [11]. The pattern and degree of softening in fruits from both genotypes during ripening were similar (Fig. 2A) suggesting that reduced PME has little effect on ripening associated fruit softening. However, when fruits from these two genotypes were stored at room temperature for 7 weeks, the transgenic fruits exhibited loss of tissue integrity while the wild-type fruits held tissue cohesiveness [11]. Interestingly, despite an increase in size of polyuronides in ripened transgenic fruits compared to wild-type fruit, no increase in firmness of transgenic fruits during ripening is observed. It is likely that the loss of bound Ca 2+ and the ability to form Ca 2+ cross-bridges might have negated any effects of reduced pectin depolymerization of pectins on ripening associated softening of transgenic fruits. The loss of tissue integrity of transgenic fruits during extended storage is likely due to continuous action of several cell wall hydrolases, including polygalacturonase, on structurally altered cell walls in transgenic fruits [21]. Fig. 2B shows the shelf life of field grown, wild-type and transgenic fruits. Tomatoes were discarded when signs of desiccation and deterioration were observed [21]. Transgenic fruits with
361 Table 2 Field performance of transgenic tomato plants with reduced PME activity*. Gcnotype Rutgers Transgenic Variable 1930 + 199(40)
2098 _+ 66(40)
392 _+ 30(40)
401 _+ 20(40)
3575 _+ 220(40)
3980 _+ 160(40)
Avg Fruit Weight (g)
95 _+5(40)
90 _+ 3(40)
Fruit/plant (no)
33 + 2(40)
45 + 1(40)
Soluble Solids (~
5.95 + 0.03(479)
6.16 +_ 0.03(488)
Total Solid (%)
7.61 _+ 0.6(288)
7.95+ 0.05(286)
Plant Fresh wt (g) Plant Dry wt (g) Fruit Yield (g)
4.43 + 0.01(449) 4.47 _+0.01(450) Juice pH *Modified from Tieman et al. [21]. Numbers in parentheses represent the number of independent samples analyzed.
reduced PME activity had a slightly shortened shelf life than wild-type fruits. This result is consistent with our result that PME plays a role in determining tissue integrity during fruit senescence [11]. Collectively these results show that PME is involved in maintaining fruit tissue integrity during ripening and senescence. These results are contrary to an earlier speculation that PME is involved in fruit softening by increasing susceptibility of pectins to polygalacturonase. E f f e c t s o f R e d u c e d PME A c t i v i t y o n F i e l d P e r f o r m a n c e o f T r a n s g e n i c T o m a t o P l a n t s : Transgenic and wild-type plants were grown in a randomized complete block design with four replications for each genotype to determine the effect of introduced PME antisense gene on plant biomass and fruit yield [21]. No effect of introduced PME antisense gene on plant biomass accumulation, as determined by fresh and dry weights, was observed in field trials (Table 2). Fruit yield and number were significantly higher in transgenic plants than in wild-type plants in one experimental year, but no differences were observed in the second year. Transgenic fruits had higher levels of soluble and total solids (Table 2). No differences were seen in fruit quality at the time of harvest among the transgenic and wild-type genotypes, however, a slight reduction in shelf life was seen in transgenic fruits as compared to wild-type fruits (Fig. 2B). Our results showed that the effects of the introduced PME antisense gene on PME gene expression was stably inherited through several generations [21]. Collectively, data showed t h a t inhibition of fruit PME activity did not adversely affect fruit yield or vegetative growth of plants [21]. Effect o f R e d u c e d PME o n Q u a l i t y A t t r i b u t e s o f P r o c e s s e d J u i c e : Fruits from transgenic 3781 ^ and wild-type Rutgers were processed by cold break, hot
362 break and MW methods and quality attributes of processed juice determined [22]. Processed juice from transgenic fruits exhibited a significant improvement in various quality characteristics compared to that from wild-type Rutgers (Table 3). Transgenic fruit juice contained higher levels of both total and soluble solids compared to that from wild-type Rutgers fruits (Table 3). Although, the method of processing influenced solid levels in the processed juice from both cultivars, the juice from transgenic fruits always contained higher total and soluble solids compared to juice from wild-type fruits. Depending on processing conditions, overall increases in total solids in juice from transgenic fruits compared to that from wildtype Rutgers ranged from 5.1-5.3% and soluble solids increased by 3.8-6.1%. The mechanism for the increased levels of solids in transgenic fruit juice is not clear. Table 3 Quality characteristics of tomato juice from wild-type and transgenic Rutgers fruits using various processing methods*. Processing Methodsa Variable Genotype Cold Hot MW Total Solids Rutgers 6.83+0.09 7.36+0.13 6.91+=0.03 (% Fr. wt) Transgenic 7.18+0.03 c 7.74+0.055 7.28+0.04 c Soluble Solids (% Fr. wt)
Rutgers Transgenic
6.18+0.10 6.56+0.04 c
6 . 6 3 + = 0 . 1 5 6.33+0.04 6.96+0.065 6.57+0.04 d
Precipitate Weightratio
Rutgers Transgenic
9.25+0.13 13.56+0.18 c
10.67+=0.13 11.22+=0.22 15.51+0.13 c 16.66+0.26 c
Serum Viscosity (sec)
Rutger s Transgenic
73.33+0.23 219.66• d
93.33+2.4 77.33+=0.6 262.91+10.7 d 258.58_+14.9 d
Effiux Viscosity (sec)
Rutgers Transgenic
28.00+1.04 43.17+4.50 d
30.67+1.04 58.16+2.30 d
29.11+2.11 50.28+1.72 d
pH
Rutgers Transgenic
4.25+0.02 4.35+0.02 d
4.28+0.01 4.33+0.01 c
4.27+0.02 4.35+0.02 c
Acidity Rutgers 0.49+0.01 0.53+0.01 0.51+0.01 (% citric acid) Transgenic 0.50+0.01 0.53+0.01 0.50+0.01 *Modified from Thakur et al. [22]. a Cold, Hot, and MW represent cold-break, hot-break, and break after microwaving tomatoes respectively; Transgenic represent a segregant of 3781 ^ homozygous for the PME antisense gene. b Significantly different from Rutgers (P<0.05). c Significantly different from Rutgers (P<0.01). d Significantly different from Rutgers (P<0.001).
363 Some of the increase may, however, be due to the highly esterified pectins of transgenic fruits (discussed below) which did not bind to the cell wall. Juice from transgenic fruits contained significantly (p< 0.05) higher amounts of total uronic acids compared to juice from wild-type Rutgers; percentage increase ranging from 35-50% [23] (Table 4). Pectins from transgenic fruits also had a significantly higher (p<0.05) degree of methoxylation (DOM), especially in the cold break processed juice. Reduction in PME activity in transgenic fruits resulted in an increase of 250% in DOM in cold processed juice and about 25% in hot and MW processed juice compared to juice from wild-type Rutgers. Pectins in transgenic fruit juice also had higher molecular mass compared to parental Rutgers under all the processing conditions (Fig 3). The observed increase in degree of methoxylation of pectin from transgenic fruit juice is the result of reduced PME activity. PME demethoxylates pectins; and in doing so increases their susceptibility towards degradation by developmentally regulated polygalacturonase [24]. Higher DOM of pectin reduces depolymerization by PG; resulting in a higher amount of pectin with a higher molecular weight in transgenic fruit juice. Increased uronic acid in transgenic fruit juice is likely due to reduced binding of methoxylated pectins to cell walls. Table 4 Effect of reduced PME activity on total uronic acid and degree of methylesterification of pectin in processed tomato juice*. Uronic Acid (~mol/mg cell wall) Degree of Methylesterification (%) Gen0typ~ Genotype Processing Method Rutgers Transgenic Rutgers Transgenic Cold Break
0.57+0.01 Be 0.76+0.03 A~
14.1+1.0 c~
51.4+0.8 A~
Hot Break
0.64+0.01 c~ 0.96+0.02 ha
50.1+1.1 ca
63.4+0.9 ha
MWHeating 0.59+0.01Bb 0.83+0.02 hb 39.1+2.9 cb 55.9_+2.4hb *Modified from Thakur et al. [23]. Upper case letters represent difference between samples from different genotypes processed under the same conditions while lower case letters represent difference between samples from the same genotype but processed under different conditions. Values with same letters are not significantly different. Values with different letters are significantly different at 95% confidence. Method of processing influenced the amount and DOM of pectin present in the juice from wild-type Rutgers and transgenic fruits; more so in wild-type fruit juice. Hot processed juice from both genotypes contained higher amounts of pectin with a higher degree of methoxylation and higher molecular mass compared to the cold processed juice (Table 4 and Fig. 3). This is expected as pectin degrading enzymes are heat inactivated in hot processed juice. In cold processed juice, the enzyme activity is not inhibited leading to pectin degradation. Even the cold processed juice from transgenic fruits contained higher amounts of pectin with a higher DOM and
364 higher molecular mass compared to pectins from hot processed juice from wildtype Rutgers. This shows the effectiveness of introduced gene in improving the quality of pectin in transgenic tomato fruits. 15
I
Hot
~
10
15
Cold
Processed
Processed
Rutgers 10-
o
s
5
o
II1
0_ 30
40
50
60
70
80
FRACTION NUMBER
90
30
40
50
60
70
80
90
FRACTION NUMBER
Figure 3: Size fractionation of EDTA-soluble polyuronides from Rutgers and transgenic fruit juice processed by cold- and hot-break methods. Pectin from processed juice was extracted as ethanol-insoluble solids and size fractionated on a Sepharose CL4B column. Under the same chromatographic conditions, elution of the branched dextrans with average molecular mass 2000, 500, 252, 151, 40 and 17.7 kD-peaked in fraction number 46, 50, 54, 62, 67 and 72, respectively. Modified from Thakur et al. [23]. Reduced PME activity in transgenic fruits resulted in a large increase in effiux and serum viscosities of the processed juice from transgenic tomato fruits [22]. Percentage increase in efflux viscosity ranged between 70-80%, while in serum viscosity it ranged between 180-220% in the processed juice from transgenic fruits over that of juice from wild-type Rutgers (Table 3). Hot processed juice from both genotypes had the highest effiux and serum viscosity, followed by MW and cold break processed juice. For transgenic fruit juice, both effiux and serum viscosities were about 60% and 200% higher even after cold break compared to hot break juice from wild-type Rutger fruits (Table 3). Processed juice from transgenic fruits also showed about 50% increase in precipitate weight ratio (PPT), an indicator of better processing attributes of tomatoes [25], over that of juice from wild-type fruits (Table 3). The observed increase in serum and effiux viscosity of juice from transgenic tomato fruits was most likely due to changes in pectin chemistry as a result of reduced levels of PME activity. Since viscosity is affected by the volume occupied by the molecule or the extent of molecular association in solution, both molecular weight and DOM will enhance the viscosity of the juice [26]. Since juice from
365 transgenic fruits contained a higher amount of pectin with a higher DOM and higher molecular mass compared to wild-type Rutgers, it had higher consistency. Increase in the consistency of tomato products due to higher DOM and higher molecular mass has been reported [27-30]. McColloch and Kertesz [27] reported that tomato juice with high consistency had pectin with higher degrees of methoxylation while that with lower consistency contained pectin with lower degrees of methoxylation. Bhasin and Bains [28] reported that hot break juice had higher consistency due to high molecular weight pectin with high degree of methoxylation. Kertesz and Loconti [31] observed that heating raw tomato fruits prior to, or immediately after crushing, inactivated pectic enzymes which could adversely affect consistency of the product. Our data demonstrated that in vivo reduction of PME activity can provide very effective means to improve viscosity of juice without heat inactivation of pectolytic enzymes. Increase in juice consistency and serum viscosity from polygalacturonase antisense tomato fruits has been reported by Schuch et al. [29,30]. Takada and Nelson [25] reported a correlation between PPT value and Bostwick and effiux viscosity of tomato products. Thakur et al. [22] also showed a correlation between Bostwick and effiux viscosity and PPT value. They also reported a linear relationship between PPT value and serum viscosity (r2=0.979), described by the equation, Y = 33 X-255 (X, PPT value; Y, serum viscosity). As juice from transgenic fruits contained higher PPT value, its higher viscosity values are in line with the reported literature. The juice from transgenic fruit had higher pH compared to wild-type Rutgers, but the increase did not affect the juice processing adversely (Table 3). Methods of processing had no effect on the pH of the processed juice. A small increase in pH of juice may partly be due to increased DOM of pectin which would reduce the number of-COOH groups resulting in an increase in pH of the juice. However, other environmental factors influence pH and acidity of tomato fruits as well [32]. Although, titratable acidity of the processed juice was similar for both the cultivars for a particular processing method, a slight reduction in the titratable acidity was observed in cold processed compared to hot processed juice (Table 3). C h a r a c t e r i s t i c s o f K e t c h u p f r o m T r a n s g e n i c T o m a t o e s w i t h L o w PME: Hot break juice from transgenic and wild-type Rutgers fruits was concentrated and processed into ketchup [22]. Quality characteristics of ketchup from both the cultivars is given in Table 5. The ketchup from transgenic fruit juice exhibited 10 fold increase in serum viscosity and a significantly lower Bostwick value compared to that from wild-type Rutgers fruits. Ketchup from transgenic fruits also showed a significant decrease in serum separation and a large increase in Brookfield consistency index. Serum separation decreased over 2-fold in ketchup from transgenic fruits compared to that from wild-type fruits (Table 5). Low Bostwick values and higher serum viscosity of ketchup prepared from transgenic fruit juice could be due to the nature and amount of pectin present. Lower serum separation may also be due to higher pectin content which would bind more water in the ketchup reducing serum separation [33]. Detrimental changes in pH or titratable acidity were not observed in ketchup made from transgenic fruits (Table 5).
366 Table 5 Quality characteristics of tomato ketchup from wild-type and transgenic tomatoes*. Genotvue Variable wild-type Rutgers 3781 ^ Homozygous Total Solids (% Fr. wt)
35.37•
34.00•
Soluble Solids (% Fr. wt)
33.00•
33.00•
Serum Viscosity (sec)
548.7+27
5400+102
Bostwick Consistency (Distance in cm in 30 sec)
4.62+0.17
3.12+0.13
Serum Separation (mL/5 min)
2.75+0.13
0.92+0.05
1.93 0.96
3.47 1.56
Brooldield Consistency Index at 33 Brix ~ 25 Brix ~ *Modified from Thakur et al. [22]. CONCLUSIONS
We have shown t h a t reduction in fruit specific PME isozymes activity contributes to changes in pectin chemistry, cation levels and their partitioning between bound and soluble forms, and the ability of fruit tissue to bind cations without any adverse effect on plant growth and development and fruit ripening. The transgenic fruits with reduced PME activity contained higher levels of soluble and total solids and showed loss of tissue integrity during senescence. Further characterization of cell walls from transgenic fruits may provide enlightenment on the role of pectin demethoxylation on the overall stability of cell wall and tissue integrity. It is evident from the data presented that inhibition of PME activity in tomato fruits improves many desirable quality characteristics of products processed from them. The processed products contain higher levels of soluble and total solids and pectin with higher DOM and molecular mass. Desirable changes in pectin chemistry leads to increased consistency of the processed products. Soluble solid content and consistency are among the most i m p o r t a n t quality characteristics of processed tomato products. A large increase in these attributes would be of great interest and economic significance to the tomato processing industry.
367 ACKNOWLEDGMENT
This research was supported by grants from USDA/NRI 94-37304-1110, USDA/NCBI 94-34190-1204, USDA/MPBC 90-34190-5207 and Indiana Value Added Grant Program to AKH. We thank Kurt Kausch for critical review of the manuscript. REFERENCES
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B.R. Thakur, R.K. Singh, D.M. Tieman and A.K. Handa, J. Food Sci., 61 (1996) 245. B.R. Thakur, R.K. Singh and A.K. Handa, J. Food Agric. Chem., 44 (1996) (in press). B.S. Luh, W.H. Dempsey and S. Leonard, Food Technol., 8 (1954) 576. N. Takada and P.E. Nelson, J. Food Sci., 48 (1983) 1460. G. Leach, H. Scols, L. Pyle and K. Niranjan, Int. J. Food Sci. Technol., 28 (1993) 261. R.J. McColloch and Z.I. Kertesz, Food Technol., 3 (1949) 923. U.B. Bhasin and G.S. Bains, J. Fd. Sci. Technol., 24 (1987) 247. W. Schuch, J. Kanczler, H.G. Robertson, G.D. Tucker, S. Bright and C. Bird, HortScience, 26 (1991) 1517. M. Kramer, R. Sanders, H. Bolkman, C. Waters, R.E. Sheehy and W.R. Hiatt, Postharvest Biol. Technol., 1 (1992) 241. Z.I. Kertesz and J.D. Loconti, New York State Agric. Exp. Station Bull., (1944) 272:2. J.N. Davies and G.E. Hobson, CRC Crit. Rev. Food Sci. Nutr., 15 (1981) 205. B.R. Thakur, R.K. Singh and A.K. Handa, J. Food Quality, 18 (1995) 389.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V.All fights reserved.
369
MOLECULAR CHARACTERIZATION AND EXPRESSION OF COLLETOTRICHUM LINDEMUTHIANUM GENES ENCODING ENDOPOLYGALACTURONASE
Centis, S., Hugouvieux, V., Fournier, J., Lafitte, C., Esquerr6-Tugay6, M.T., Dumas, B.
Centre de Biologie et Physiologie V6g6tale, URA CNRS n~ Route de Narbonne, 31062 Toulouse Cedex, France
Universit6 Paul Sabatier, 118
Abstract
Colletotrichum lindemuthianum, a fungal pathogen causing anthracnose on bean seedlings, secretes an endopolygalacturonase (endoPG) when grown on liquid medium containing pectin. The endolytic nature of the enzyme was demonstrated both by chemical analysis of the products it releases from the substrate, and through its specific interaction with the Polygalacturonase Inhibitory Protein (PGIP) isolated from the host-plant. Oligodeoxyribonucleotide primers designed from the N-terminal amino acid sequence of the endoPG of C. lindemuthianum race 13 and from an internal sequence conserved among different fungal endoPG were used in a polymerase chain reaction (PCR) to amplify genomic related sequences of the fungus. A fragment of 542-bp was obtained and used as a probe to screen a partial genomic library of C. lindemuthianum. Among the positive clones, one was further analyzed. Nucleotide sequencing of this clone revealed an ORF encoding a 363 aminoacid polypeptide showing a high degree of homology to ten fungal endoPG sequences. This genomic clone was thereafter designated Clpgl. Southern analysis, performed with a Clpglspecific probe, showed that this gene is present as a single copy in the C. lindemuthianum genome.
I. INTRODUCTION
Pectin, a methylated heteropolymer containing ot 1-4 linked galacturonic acid, is one of the predominant polysaccharides of plant cell walls (Carpita et al., 1993). Since this material constitutes a mechanical barrier as well as a carbon source, many phytopathogenic and saprophytic microorganisms secrete pectoi~j~ enzymes such as polygalacturonase, pectin methyl esterase, pectate lyase or pectin lyase. In filamentous fungi, pectolytic enzymes are rapidly secreted when these microorganisms are grown in vitro on pectin. Their action causes tissue maceration in the host plant thus allowing the fungal parasite to spread inter-, and intracellularly. These enzymes are important in pathogenicity for not only do they cause cell wall degradation (Cooper and Wood, 1975; Collmer and Keen, 1986), but also they act as indirect
370 elicitors of plant defence responses through the oligosaccharides they release (Hahn et al., 1981 ; Boudart et al., 1995). Endopolygalacturonase (or endoPG, EC 3.2.1.15) catalyses the hydrolytic cleavage of glycosidic or-l,4 linkages in polygalacturonide chains of pectic substances. Colletotrichum lindemuthianum is a fungal pathogen causing anthracnose on bean seedlings. When grown on pectin-containing liquid medium, this fungus secretes an endoPG which has been thoroughly characterized. It randomly cleaves a-(O)-D-galacturonosyl bonds of homogalacturonan and pectic polymers producing mono-, di-, and tri-galacturonic acid residues as final hydrolysis products. EndoPG activity is partly inhibited during the parasitic stage of the fungus by the bean cell wall polygalacturonase inhibitory protein (PGIP), (Albersheim and Anderson, 1971; Lafitte et al., 1984; Wijesundera et al., 1989). The use of gold-labeled PGIP allowed endoPG and associated cell wall degradation to be visualised in planta in infected bean tissues (Benhamou et al., 1991). The enzyme is a glycoprotein of around 38 kDa whose sugar moiety accounts for 4 % of the molecule and is composed of galactose, glucosamine and mannose (Keon et al., 1990). We recently reported that the endoPG purified from C. lindemuthianum race 13 elicits the biosynthesis of PR (pathogenesis related) proteins in bean cuttings, notably 13-1-3-glucanase, in a cultivar specific manner (Lafitte et al., 1993), suggesting an important role for this fungal protein in triggering the expression of the host defence genes. A better understanding of this dual role requires further studies of endoPG at the gene level. PG-encoding genes have been isolated from a few fungi, notably Cochliobolus carbonum (Scott-Graig et al., 1990), Aspergillus niger (Bussink et al., 1992), Aspergillus orizae (Kitamoto et al., 1993), Fusarium moniliforme (Caprari et al., 1993) and Sclerotinia sclerotorium (Reymond et al., 1994). As a prerequisite to evaluate the role played by C. lindemuthianum endoPG gene expression, we have cloned and subsequently characterized an endoPG gene from this fungus.
2. RESULTS 2.1. Endopolygalacturonase activity of C. lindemuthianum grown on pectin The production of polygalacturonase was first studied by measuring the PG activity in the culture filtrate of C. lindemuthianum race 13grown on pectin. PG activity was detected as soon as 2 days after inoculation of the medium and increased until it reached a maximum at 6 days (Fig. 1A). After this time, it dropped rapidly and stayed at a constant level of 0.8 nkatal ml-1, although the fungus continued to grow. This decrease was coincident with intense pectin degradation and subsequent release of galacturonic acid (GalA), (Fig. 1B). Since the assay for polygalacturonase activity did not allow to discriminate between exoPG and endoPG activities, experiments were undertaken in order to further characterize the nature of PG activity in C. lindemuthianum. Previous studies had ~hown that the pure PGIP from bean specifically inhibits the endoPG, but not the exoPG of C. iindemuthianum (Lafitte et al., 1984 ; Benhamou et al., 1991). Preincubation of the culture filtrate of C. lindemuthianum with PGIP led to 93 % inhibition of enzyme activity when the assay was performed on a 6-day-old culture, and only to 55 % on a 13-day-old culture (Table I). This indicated that, at early stages of growth, the fungus secreted almost exclusively endoPG, whereas exoPG was produced at later stages.
371 _
A ~'
e~
~N
3-
/"
2-
/
1O_
~
9 1
100-
B
~I,,,,,I
<~
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1
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3
4
5
6
7
8
9
D GalA DP2 m DP3
50-
0
_
~
3
5
7
Days of culture
9
Figure 1. Time-course measurement of polygalacturonase (PG) activity in the culture filtrate of Colletotrichum lindemuthianum grown on pectin and HPLC-Dionex analysis of mono-, di- and tri-galacturonic acid residues simultaneously released in the culture medium. The data are the mean of three independent experiments or represent one typical experiment in the case of galacturonic acid (GalA) residues. *DP = Galacturonic acid degree of polymerization.
Table I Inibition of endoPG activity in the culture filtrate of C. lindemuthianum grown on pectin with bean cell wall PGIP
Polygalacturonase Activity (nkat)
Days of culture
6 days
13days
PGIP No PGIP Inhibition %
0.009 0.138 94
0.06 0.138 55
372 Further characterization by SDS-PAGE and Western blot analysis of the culture filtrate of a 6 day-old culture of C lindemuthianum showed that only one endoPG of the same molecular weight (42 kDa) as the pure endoPG (Hugouvieux et al., 1995) was induced on pectin. A kinetic study of endoPG gene expression was then performed on total RNA extracted from the fungus subcultured on pectin, or on glucose as a control by using a cDNA coding for an endoPG of C. carbonum as a probe (Scott-Craig et al., 1990 ; Hugouvieux et al. 1995). A high increase in PG transcript intensity was recorded as early as 6 h alter transfer on pectin. This increase extended throughout the 72 hour-duration of the experiment and preceeded a corresponding increase of endoPG activity, which was about twenty times higher than on glucose at 18 hours. Only a faint hybridization signal starting 48 h alter subculturing was detected when the fungus was grown on glucose alone. The data show that the induction of endoPG activity by pectin results mainly from an enhancement of the endoPG transcript level. Additional experiments are needed to precisely characterize the level at which pectin regulates C. lmdemuthianum endoPG.
2.2. Amplification of endoPG genomic DNA sequences by PCR and isolation of genomic clones An oligo primer (5'-GTYCTIAACAACATYCC) based on the N-terminal amino acid sequence of C. lindemuthianum race 13 endoPG (Keon et al., 1990), and an antisense primer (5'-GCRASRCAGTCRTCYTGGTT) designed from an internal amino acid sequence well conserved among fungal endoPGs, were synthesized (R=A/G; S=C/G; Y=C/T). These two primers were used to amplify by PCR a genomic DNA fragment encoding a portion of endoPG. An amplified 542-bp product, named pgA, was sequenced. Sequence analysis showed a high degree of homology to fungal endoPG-genes. This fragment was used to screen a partial genomic library of C. imdemuthianum. Among positive clones, some of them showed high hybridization to the pgA probe while others gave much weaker signals. Clones of both types were purified and submitted to in vivo excision. Restriction patterns lead us to the conclusion that we had isolated two different classes of clones, designated Clpgl and Clpg2. The clone Clpgl which hybridized strongly to the probe, was retained for further analysis. The structure of Clpg2 will be described elsewhere (Centis et al., manuscript in preparation).
2.3. Characterization of the genomic clone Clpgl From the cloned genomic DNA, 1700 nt overlapping the region corresponding to pgA were sequenced according to standard procedures using both universal and specific primers. Analysis of this sequence with the Blast sequence comparison program revealed the occurrence of an ORF of 1152 bp. The coding region is interrupted by one intron of 70-bp (Fig.2). The encoded 363-aa (36,685 Da) is in good agreement with the MW of 37 kDa obtained by SDSPAGE analysis (Lafitte et al., 1984), and of 38,5 kDa obtained by gel filtration (Keon et al., 1990). A potential N-glycosylation site (Asn-Gly-Ser) was found at Asn 294. The presence of this site is correlated with the glycoproteic nature of endoPG (Keon et al., 1990). The Nterminal amino acid sequence of C. lindemuthianum endoPG published by Keon et al., (1990) and confirmed in our laboratory, corresponded to aa 27 to 62, thereby indicating that the first 26-aa peptide, rich in hydrophobic residues, corresponded to the signal peptide of endoPG.
373 This signal peptide might have been eliminated by a trypsin-like protease cleavage (Benoist et al., 1987) since an Arg 26 occurs in the CLPG1 sequence, as in the case of PG of F. momliforme and ofPGII of A. niger (Bussink et al., 1991; Caprari et al., 1993). The 5'-noncoding region of the clone representing 160 bp did not contain the typical eukaryotic TATA box (TATAAAT), but a CCAAT sequence which is often observed in fungal promoters, was found 55 nt upstream the ATG start codon. This sequence has been shown to be important in setting both basal and derepressed expression of amdS gene in A. nidulans (Littlejohn and Hynes., 1992), and could be involved in the repression of the endoPG of C. lmdemuthiamlm by glucose (Hugouvieux et al., 1995). Furthermore, pyrimidine-rich sequences which are also considered as important fungal promoter elements, were identified. C+T-rich regions were detected at positions-140, -104 et-65. The sequence surrounding the ATG start codon, 5'-CCAAGATGGT, closely resembles the Kozak sequence (CAMMATGq~IC) identified in filamentous fungi and higher eukaryotes genes (Kozak, 1986" Ballance, 1990). The 3'-noncoding region (340 nt) did not contain the typical polyadenylation signal, AATAAA, as already reported in the case of other fungal genes (Ballance, 1986 ; Benoist,1980). However, similar sequences situated 177 nt and 228 nt downstream the stop codon, respectively AATTAA and AAATA, were found. The CAATC sequence wich is present 38 nt downstream the stop codon is in agreement with the consensus sequence CAWTS involved in the termination of translation (Berget, 1984) and is proposed as a site Of polyadenylation (Mullaney et al., 1985). TAGT and TTT motifs situated at nt 1434, have also been considered as important functional components of polyadenylation or transcription termination in yeasts (Zaret and Sherman, 1982). Analysis of the codon usage of the Clpgl sequence showed that 44 out of the 61 sense codons are used. Codons ending in C are preferred and represent 74.4 % of the codons used.
XhoI A T G l m m CAAT box
TAA 1 1 m Intron
[]
Exon
BamHI 1
100bp m Termination signals
Figure 2. Structural organization of Clpgl, a gene coding for an endoPG of (2
lindemuthianum.
2.4. Comparison of the deduced aa sequence of endoPG with differents fungal PGs The CLPG1 sequence was compared to ten fungal endoPG sequences : PGN1 from C. carbonum (Scott-Graig et al., 1990), SCEEPG, SCEEPGA and SCEEPGB from S. sclerotiorium (Reymond et al., 1994, GenBank accession n ~ L29040), PECA from A. flavus ( accession n~ U05015), ANPG, ANPGA1 and ANPGA2 from A. niger (Ruttkowski et al., 1991; Bussink et al., 1991a, b), PGAC from A. nidulans (Bussink et al., 1992), PGA from F. moniliforme (Caprari et al., 1993) and ATPGA2 from A. tubigensis (Bussink et al., 1991b).
374 The different protein sequences showed similar sizes with an overall similarity of 33.3 %. At the amino acid level, the highest degree of homology was observed between the C. lmdemuthianum endoPG CLPG1 and PGN1 of C. carbonum (63% ; Table II). Most of the PGs contain one or two putative glycosylation sites. The CLPG1 sequence shows only one glycosylation site, located in the same region as the glycosylation site of four of the above mentionned endoPGs. In addition, CLPG1 exhibits the 231CXGGHGXSIGSVG243 sequence which is specific of fungal pectinases and the sequence 256RIK258found in all fungal endoPGs and plant ExoPGs, which is considered to be a specific signature of the PGs (Reymond et al., 1994).
Table II Comparison of C. findemuthianum endoPG aa sequence with other fungal endoP,Gs.
EndoPGs of various fungi
('. carbonum S. sclerotiorium
A. mdulans
A. tubigensis A. niger b: momliforme
CCLPGN 1 SCEEPG SCEEPGA SCEEPGB ANPGA2 ANPGAI ANPG ATPGA2 ANPGAC FSOWPGA
% identity with CLPGI
63 66 58 57 54 53 53 53 42 37
2.5. Two single copy genes coding for endoPGs are present in the C. lindemuthianum genome The previous analysis of C. lindemuthianum genomic DNA, using the cDNA coding for the endoPG of C. carbonum as a probe, suggested that at least two genes are present in the genome of this fungus (Hugouvieux et al., 1995). Two major fragments of 9 kb and 5 kb, hybridizing with pgA, were generated by XhoI cleavage of fungal DNA. To determine which of these fragments corresponded to Clpgl, a specific probe was prepared, corresponding to an adjacent DNA sequence at the 3'-noncoding region of the gene. This probe hybridized with only one of the XhoI fragments, suggesting that the Clpgl gene is contained in the 9-kb fragment and is present as a single copy in the C. lindemuthianum genome. Northern-blot analysis with the pgA probe showed only one transcript size (data not shown). Southern blot analysis using a specific probe of Clpg2 showed that this gene is also present as single copy in the genome of C. lindemuthianum (Centis et al., manuscript in preparation). Further investigations on the regulation of transcription of the Clpgl gene will involve the use of a specific probe for northern experiments and promoter studies.
375 5. DISCUSSION
The endopolygalacturonase of (1 lindemuthianum is abundantly produced during fungal growth both in vitro and m planta. In the latter case, the enzyme has been visualized in infected tissues through its interaction with PGIP, a protein that we have isolated from the host plant, and which specifically inhibits the endoPG of C. lmdemuthianum (Benhamou et al., 1991). A low level of endoPG transcript and activity was observed on glucose. We propose a scheme of regulation, in which this basal level ofendoPG would exist on glucose and result in limited hydrolysis of pectin. Substrate induction by pectin-derived molecules would then take over and highly amplify the basal level of endoPG leading to abundant production of small oligouronides and finally of galacturonic acid in the medium. Appearance of galacturonic acid would be hastened by the presence of exopolygalacturonase whose increased activity coincides with endoPG decline. The possible contribution of these final products to the regulation of endoPG gene expression will be investigated in the future. In vitro, pectin induction occurs in the very first hour following contact of the fungus with the substrate. This effect reminds the previously reported induction of cutinase by cutin monomers in Fusarium (Podila et al., 1988). Oligonucleotides derived from endoPG amino acid sequences were used to amplify a DNA fragment from the plant pathogenic C. lindemuthianum fungus. A 542-bp fragment was obtained and used to probe a partial genomic library of this fungus. Two genomic clones were isolated and one of them, Clpgl, was sequenced. The Clpgl coding sequence is interrupted by one intron and codes for a 363-aa protein which has a putative 26-aa signal peptide. The deduced aa sequence shows homology to other fungal endoPGs and contains one Nglycosylation site. Regulatory sequences were detected such as a CCAAT motif found in the 5'-non-coding region which is likely to represent a binding site for a regulatory protein as it was shown in the case of the amdS gene of A. nidulans. Southern analysis using a Cipgl specific probe shows the presence of Clpgl gene as a single copy in the C. lindemuthianum genome. The N-terminus sequence of the endoPG purified from the extracellular fluid of (: lindemuthianum culture is identical to the amino acid 27 to 62 of the protein sequence deduced from Clpgl, identifying this gene as being the major endoPG gene induced during saprophytic growth of the fungus in presence of the inducer, pectin.. However, the protein encoded by Clpg2 has never been isolated, suggesting that Clpg2 is poorly induced during in vitro conditions in classical media. Northern blot analysis confirmed that Clpg2 is only weakly induced by pectin. Further work will be aimed at determining the conditions for the induction of Clpg2 and to compare the biochemical and biological activities of the two endoPGs of C. lindemuthianum.
4.
REFERENCES
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376 Benoist. C., O'Hare, K., Breathnach, R. and Chambon, P. (1987) Nucleic Acids Res. 8 127142. Berget, S. M. (1984) Nature 309 179-182. Boudart, G., Deschamps-Guillaume, G., Lafitte, C., Ricart, G., Barthe, J.P., Mazau, D. and Esquerr6-Tugay6, M.-T. (1995) Eur. J. Biochem. 232 449-457 Bussink, H. J. D., Brouwer, K., De Graaf, L. H., Kester, H. C. M. and Visser, J. (1991a) Curr. Genet. 20 301-307. - Bussink, H. J. D., Buxton; F. P. and Visser, J. (1991b) Curr. Genet. 19 467-474. Bussink, H. J. D., Buxton, F. P., Fraage, B. A., de Graaf, L. H. and Visser, J. (1992) Eur. J. Biochem. 208 83-90. Bussink, H. J. D., Kester, H. C. M. and Visser, J. (1990) FEBS Lett. 273127-130. Caprari, C., Richter, A., Bergmann, C., Lo Cicero, S., Salvi, G., Cervonne, F. and De Lorenzo, G. (1993) Mycol. Res. 97 497-505. Carpita, N. C. and Cfibeaut, D. M. (1993) Plant J. 31 1-30. - Collmer, A. and Keen, N. T. (1986) Annu. Rev. Phytopathol. 24 1389-1409. Cooper, R. M. and Wood, R. K. S. (1975) Physiol. Plant Pathol. 5 135-156. Hahn, M. G., Darvill, A. G. and Albersheim, P. (1981) Plant Physiol. 68 1161-1169. Hugouvieux, V., Centis, S., Lafitte, C. and Esquerr6-Tugay6, M. T. (1995) C. R. Acad. Sci. - Paris 318113-120. Keon, J. P. R., Waksman, G. and Bailey, J. A. (1990) Physiol. Mol. Plant Pathol. 37193206. - Kitamoto, N., Kimura, T., Kito, Y., Ohmiya, K. and Tsukagoshi, N. (1993) FEMS Microbiol. Lett. 11137-41. - Kozak, M. (1984) Nature 308241-247. Lafitte, C., Barthe, J. P.., Montillet, J. L. and Touz6, A. (1984) Physiol. Plant Pathol. 25 3953. Lafitte, C., Barthe, J. P., Gansel, X., Dechamp-Guillaume, G., Faucher, C., Mazau, D. and Esquerr6-Tugay~, M. T. (1993) Mol. Plant-Microbe Interact. 6 628-634. - Littlejohn, T. G. and Hynes, M. J. (1992) Mol. Gen. Genet. 235 81-88. Mullaney, E. J., Hamer, J. E., Yelton, M. M. and Timberlake W. H.. (1985) Mol. Gen. Genet. 199 37-45. - Polida G. K., Dickman M. B., Kolattukudy P. E. (1988) Science 242: 922-925. Ruttkowski, E., Khanh, N. Q., Wientjes, F. J. and Gottschalk, M. (1991) Mol. Microbiol. 5 1353-1361. Scott-Craig, J. S., Panaccione, D. G., Cervone, F. and Walton, D. J. (1990) Plant Cell 2 1191-1200. - Reymond, P., Deleage, G., Rascle, C. and Fevre, M. (1994) Gene 146 233-237. Wijesundera, R. L. C., Bailey, J. A., Byrde, R. J. W. and Fielding, A. H. (1989) Physiol. Mol. Plant Pathol. 34 403-413. Zaret, K. S. and Sherman, F. (1982) Cell 28 563-573. -
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J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
377
CLONING OF GENES ENCODING P E C T I N - D E G R A D I N G E N Z Y M E S IN AZOSt:IRILL ~/ M I R A K E N S E
My Ali Bekri, Jos Desair, Leentje Van Lommel, Jos Vanderleyden. F.A. Janssens Laboratory of Genetics, K.U.Leuven W-De Croyiaan 42 3001 Heverlee Belgium
Abstract Azospirillum irakeuse is a nitrogen-fixing bacterium, isolated from the roots and rhisosphere of rice (Khammas et al., 1989). Within the genus Azo~pirillum, breakdown of pectin (and polygalacturonic acid) is best documented for A. irakense. From a gene library of A. irakense KBCI, we have isolated several cosmid clones that express polygalacturonase and pectate lyase activity in E. coli and A. brasilense. Genes encoding these enzymes appear to be closely linked in the A. irakense genome.
Introduction Bacteria of the genus Azo,spirillum are Gram-negative, vibrioid-type cells, able to fix nitrogen under micro-aerobic conditions. The association between bacteria of the genus Azo~spirilhtm and the roots of grasses and other plants has been studied extensively since 1976, when D6bereiner and Day reisolated Azo3pirillum lipoferum (originally named Spirillum lipoferum) from the roots of tropical grasses. In 1989, a new group of bacteria with the overall properties of Azo,spirillum and containing seven strains was described. They were isolated from the roots and rhizosphere of rice in the region of Diwaniyah (Quadisya) in Iraq (Khammas et al., 1989). These new isolates within the genus Azo~pirillum constitute a single DNA homology group corresponding with a separate phenotypic cluster. Therefore a new species, Azospirillum irakense, has been proposed and described (Khammas et al., 1989). So far, five species have been characterised within the genus Azo~spirillum: A. brasilense, A. lipoferum, A. amazonense, A. halopraeferans and A. irakense. (Tarrand et al., 1978; Magalhaes et al., 1983; Reinhold et al., 1987; Khammas et al., 1989). Azo,spiriihtm strains are unable to degrade starch and cellulose. Some strains of A. brasilense and A. litu?ferum were shown to be able to grow on xylan (Halsall and Gibson, 1985). Degradation of polygalacturonic acid and pectin has been reported for A. brasilense and A. lipt?ferum (Tien et al., 1981; Plazinski and Rolfe., 1985), and some characteristics of a polygalacturonic acid transeliminase of A. brasilense have been studied (Tien et al 1981). However, these species cannot grow on pectin or polygalacturonic acid, and the reported activities are extremely low when compared with other pectinolytic bacteria.
378
Among the five different species of Azo~pirillum, only A. irakense shows clearly pectinolytic activity on solid and in liquid medium. Moreover, this species can grow under non-diazotrophic as well as diazotrophic conditions when pectin is the sole carbon source (Khammas and Kaiser, 1991). Khammas and Kaiser (1991) analysed the pectinolytic activity of seven A. #'akense isolates, and gave evidence for the presence of two types of pectinolytic enzymes. All strains tested have inducible Ca 2+ dependent pectate lyase activity. Six strains, showed also pectin methylesterase activity. So far, none of the corresponding enzymes have been purified. The plant cell wall is a polymeric mesh consisting of cellulose, hemicellulose, pectin and protein. Cellulose and hemicellulose are integral components of the cell wall, but pectic substances are located mainly in the outer wall regions within the middle lamella (McNeil et al., 1984). Pectic substances are more susceptible to enzymatic degradation, because they are more exposed than other cell wall components. Therefore, pectin-degrading enzymes may play a central role in the penetration of plant tissue by bacteria. Pectinolytic enzymes are usually associated with virulence of a number of phytopathogenic bacteria, notably El~vinia (Schell et al., 1988). However pectinolytic activity has also been reported in non-phytopathogenic species such as Rhizobium (Hubbell et al., 1978; Mateos et al., 1992). Therefore, it is of interest to compare the structure, organisation and regulation of these pectinolytic enzymes and their corresponding genes in phytopathogenic and non-phytopathogenic bacteria. As a first step in the study of the pectinolytic activity of Azo,spirillum irake,se (non-phytopathogen), we isolated and partially characterised A. irakense genes that confer pectinolytic activity to Escherichia coil and Azo,spirillum brasilense
M a t e r i a l s and M e t h o d s Bacterial strains and Piasmids
The bacterial strains and plasmids used in this work are given in table 1. Media and culture conditions
Escherichia coli strains were grown at 37~ in Luria-Bertani medium (LB) (Miller., 1972), and Escherichia co# transformants were grown in LB medium supplemented with tetracycline (10ug/ml) or ampicilin (100ug/ml). Azo~spirillum strains were grown at 30~ in MMAB medium (Vanstockem et al., 1987) with 0,5% malate as a carbon source or in LB* medium (LB medium containing 2.5mM CaCiz and 2.5mM MgSO4). For the assay of pectinolytic activity, Escherichia coli transformants and Azo~spirillum strains were grown in the same media supplemented with 1% (w/v) of pectin (Sigma).
379 Table 1. Bacterial strains and plasmids used in this study Relevant characteristics
Strains and plasmid
Reference or
source
Strains Azo~pirillum brasileuse Sp245 Wild type strain, isolated from surface sterilized weat roots (Brazil) Azo,spirilhml itzlkeuse LMGI0653 wild type strain, isolated from (KBC 1) roots and rhizosphere of rice (Iraq) Escherichia col HBI01 F hsdS20(rBmB)recA 13 ara- 14 proA2 ktcY1 galK2 tpsL20 xyi-5 supE44 Km r Sl7-1::Tn5 Plasmids pLAFR1 pUCI9 pFAJ601
pFAJ603
pFAJ604
pFAJ605
Tc", IncP broad host range cosmid contains unique EcoRl site Ap r, Lac Z Tc', pLAFR 1 derivative containing 5 EcoRI fragments of DNA of A.irakeuse Td, pLAFR 1 derivative containing a single EcoRI fragment (9.2 kbp) of DNA of A. irakeuse Ap ~, pUC 19 derivative containing a single EcoRI fragment (9.2 kbp) ofDNA ofA. irakeuse Ap r, pUC 19 derivative containing an HmdIII-EcoRI fragment (3.2 kbp) ofDNA ofA. irakense
Tc r = Tetracycline resistant Km r = Kanamycine resistant Ap r= Ampicilin resistant
Baldani et al., 1986
Khammas et al., 1989
Maniatis el a/., 1982
Mazodier, P., (Paris cedex 15) Friedmann et cd., 1982 Yannish-Perron el al., 1985 This study
This study
This study
This study
380
DNA manipulations. Isolation of plasmid DNA, transformation, transfection and cloning procedures were performed using standard techniques (Sambrook et al., 1989).
Crude enzymes preparation. Azo,~pirillum strains were grown on LB* medium containing 1% (w/v) pectin at 30~ for seven days, and Escherichia coli transformants for two days. Culture media were centrifuged at 10.000 g for 10 minutes. The supernatant constituted the enzyme preparation.
Enzyme assays. On solid medium, Azo,~piriHum and Escherichia coil strains were plated on LB agar with l%(w/v) pectin and after six days of incubation, the plates were overlayed with 2% (w/v) solution of hexadecyltrimetyl ammonium bromide (HTAB) (Plazinski and Rolfe, 1985). In liquid medium , the thiobarbuturic acid test was used to determine polygalacturonase and pectate lyase activity (Sherwood, 1965). 1 ml of the crude enzyme preparation was added to 2 ml of 0.5 N HCI in a test tube. 4 ml of 0.01 M thiobarbuturic acid, dissolved in distilled water, were added. The tubes were heated in a boiling water for l h and centrifuged. The absorption of the supernatant was determined in the spectrophotometer over the range 480-580 nm. Reaction mixtures without enzyme, which showed no reaction with thiobarbuturic acid, were used as controls.
Results Pectinolytic activity in Azospirillum irakense. The A. irakense strain is clearly able to hydrolyze pectin on solid medium. A clear halo was observed after incubation for seven days and overlayed with HTAB, indicating the degradation of pectin, whereas non-hydrolyzed polymer is precipitated. For the other Azo,spirillum species, we were not able to demonstrate pectinolytic activity in this way.
Cloning of Azospirillum irakense genes encoding pectinolytic activity. A gene bank ofAzo~spirillum #'akense strain LMG 10653 (KBC1)was constructed in cosmid pLAFR1. A. irakense total DNA was partially digested with EcoRI and ligated into the EcoRI site of pLAFRI. The ligation mixture was packaged and used to transfect Escherichia coli HB 101. Approximately 3000 recombinant E. co# clones were obtained and tested on plates with Luria-Bertani medium, containing 1% (w/v) pectin. After 6 days of incubation at 37~ plates were overlayed with HTAB. Clones with extracellular pectinolytic activity show a clear halo indicating degradation of pectin. Eight positive clones were selected in this way. For all the positive clones,
381 DNA restriction analysis of the corresponding cosmids revealed one common EcoRI fragment of 9.2 kbp. The restriction map of the DNA region covered by the overlapping clones is shown in Fig 1. In order to characterize the pectinolytic enzymes encoded by these clones, the culture supernatants of all these clones were tested for pectate lyase and polygalacturonase activity, using thiobarbutiric acid as described in materials and methods. Absorption at 550 nm indicates the activity of pectate lyase whereas absorption at 510 nm indicates the activity of polygalacturonase. Three cosmids all having the 9.2 kbp EcoRl fragment in common, were transferred by conjugation to A brasileuse strain Sp245. The A. brasilense transconjugants all showed a clear halo on plates containing pectin. Analysis of the culture supernatant also revealed pectate lyase and polygalacturonase activity. A surprising observation is the fact that the A. irakense genes are expressed in E. coli, given the taxonomic difference among the corresponding genera.
Tn5 mutagenesis. We have subcloned the EcoRI restriction fragment of 9.2 kbp from the clone PFAJ601 in pLAFR1, and as expected, this fragment is sufficient to express pectate lyase and polygalacturonase activity in E. coli tranconjugants. This clone named PFAJ603, was mutagenized with Tn5, using E. coli Sl7-1::Tn5 as the donor strain. Kanamycin resistant Tn5-mutants were screened for the pectinolytic activity using the thiobarbuturc acid test. A total of five mutants that show no longer pectinolytic activity were obtained. DNA restriction analysis of corresponding cosmids, revealed that the genes encoding for the pectate lyase and polygalacturonase are located on a HindIII-EcoRI fragment of 3.2 kbp.
Preliminary characterisation of the A. irakense pectate lyase. We have started to purify the pectate lyase, using E. coli (pFAJ603) cultures. Crude cell extracts, obtained by breaking the cells with glass beads (FastPrep FP120 Instrument, Bio 101, Inc and Savant instrument, Inc, La Jolla, USA) were loaded on a SUPERDEX75 HR 10/30 (Pharmacia Uppsala, Sweden) gelfiltration colum, and fractionated with saline phosphate buffer (0.05M NaH2PO4; 0.075M NaCI; pH 7.2) as the eluent. Pectate lyase activity was obtained in a single protein peak. Further steps in the purification, using ion-exchange colums, are in progress.
Conclusions In this study we have isolated and partially characterised the Azo~spirillum mtkeuse genes that encode for pectate lyase and polygalacturonase activity. The genes are located on a 9.2 kbp EcoRI restriction fragment, and possibly within a 3.2 kbp HmdlII-EcoRI subfragment. Whereas for A. irakense, pectin or polygalacturonate is needed for expression of pectinolytic enzymes, no such induction is observed in the E. coil (pFAJ603) transconjugant. This raises the question of the regulatory elements for pg and pl gene expression in A. irakense. In A. irakense most of the pectinolytic activity is found in the culture supernatant. In the E. coli transconjugants, the pectinolytic enzymes need to be released by cell
E
E
E
E
E
E
E
E E
E
E
pFAJ601
I
I
I
I
382
E = EcoRl
1-1
pFAJ603
Fig 1: Physical map of the A. irakense genome containing pectinolytic genes
1 Kb
383 disruption. Therefore, it is very likely that the pectinolytic activity observed on plates with the E. coil transconjugants results from cell lysis. In some cases pectinolytic enzymes have been associated with virulence and it is generally accepted that pectinolysis by these bacteria facilitates their entryand spread in plant tissue. In Rhizobium, these enzymes may play a role in the root infection process that precedes nodule formation (Hubbell et al., 1978). A. irakense has never been reported to be pathogenic on plants. It can therefore be speculated that moderate and strictly regulated pectinolysis of A. irakense facilitates entry in the outer cortex of plants roots, since A. iraken,~'e has been isolated from surface-sterilized roots. It is likely that breakdown of plant polysaccharides by root colonizing bacteria can provide them with extra carbon source. Moreover, it will be of interest to evaluate the effect of A. brasilense expressing pectinolytic activity on plants. Four of the five species of Azo,spirillum, except A. irakense, produce considerable amounts of IAA. It could well be that combination of IAA synthesis and pectinolytic activity in the same species will drastically alter the interaction between Azo,spirillum and plants.
References Baldani, V.L.D., Alvarez, M.A. de B., Baldani, J.l. and DObereiner, J. 1986. Establishment of inoculated Azo~pirillum spp. in the rhizosphere and in roots of field grown wheat and sorghum. Plant Soil 90: 35-46. Day, J.M. and D6bereiner, J. 1976. Physiological aspects of N2 fixation by a SpiriHum from Digitaria roots. Soil Biol. Biochem. 8:45-50 Friedman, A.M., Long, S.R., Brown, S.E., Buikema, S.E. and Ausubel, F.M. 1982. Construction of a broad host range cloning vector and its use in the genetic anlysis of Rhizobium mutants. Gene 18: 289-296. Halsall, D.M., Gibson, A.H., 1985. Cellulose decomposition and associated nitrogen fixation by mixed cultures of Cellulomonas gelida and Azo,~pirillum species or Bacilhts maceraus. Appl. Environ. Microbiol 50:1021-1026. Hubbell, D.H., Morales, V.M. and Umali-Garcia, M., 1978. Pectinolytic enzymes in Rhizobium. Appl. Environ. Microbiol. 35, 210-213. Khammas, K.M. and Kaiser, P., 1991. Characterization of a pectinolytic activity in Azo,spirillum irakeuse. Plant and soil 137, 75-79. Khammas, K. M., Ageron, E., Grimont, P.A.D. and Kaiser, P., 1989. Azo,wiriihtm irakeuse sp. nov., a nitrogen-fixing bacterium associated with rice roots and rhizosphere soil. Res. Microbiol. 140, 679-693. Magalhaes, F.M.M., Baldani, J.I., Souto, S.M., Kuykendall, J.R. and D6bereiner, J. 1983. A new acid tolerant Azo,spirillum species. An. Acad. Bras. Cienc. 55: 417-430. Maniatis, T., Fritsch, E.F., Sambrook, J. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Mateos, P.F., Jimener-Zurdo, J.I., Chen, J., Squartini, A.S., Haack, S.K., MartinezMolina, E., Hubbell, D.H. and Dazzo, F.B., 1992. Cell-associated pectinolytic enzymes in Rhizobium legumiuosarum biovar trifo#i. Appl. Environm. Microbiol. 58, 1816-1822
384 McNeil,M., Darvill, A.G., Fry, s.c. and Albersheim, P. 1984 Structure and function of the primary cell walls of plants. Annu. Rev. Biochem. 53:625-663 Miller, J.H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY:354-358. Plazinski, J. and Rolfe, B.G., 1985. Analysis of pectinolytic activity of Rhizobium and Azo,spirillum strains isolated from Trillium repens. J. Plant Physiol. 120, 181187. Reinhold, BI, Hurek, T., Fendrik, I., Pot, B., Gillis, M., Kersters, K., Thielemans, S. and De Ley, J. 1987. Azo,spirillum halopraeferans sp. nov., a nitrogen-fixing organism associated with roots of Kallar grass (Leptochloafusca (L.) Kunth.). Int. J. Syst. Bacteriol. 37:43-51. Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor, new york. Schell, M.A., Roberts, D.P. and Denny, T.P;, 1988. Analysis of the Pseudomouas. solanacearum polygalacturonase encoded by pglA and its involvement in phytophathogenicity. J. Bacteriol. 170, 815-823. Sherwood, R.T. 1965 Pectin lyase and polygalacturonase production by Rhizoctouia solaui and other fungi. Phytopathology. 56:279-286. Tien, T.M., Diem, H.G., Gaskins, M.H., Hubbell, D.H. 1981 Polygalacturonic acid transeliminase production by Azo,spiriHum species. Can. J. Microbiol. 27:426431 Tarrand, J.J, Krieg, N.R. and DObereiner, J. 1978. A taxonomic study of the ,~'pirilhtm lipq)rbrunl group, with description of a new genus, Azo,spirillum gen. nov., and two species Azo,spiriHum iip~?['erum(Beijerinck) sp. nov. and Azo.spitillum brasileuse sp. nov. Can. J. Microbiol. 24: 967-980. Vanstockem,M., Michiels, K., Vanderleyden, J. and Van Gool, A., 1987 Transposon mutagenesis of Azo,spirillum brasileuse and Azo~pirillum lipoferum, physical analysis of Tn5 and Tn5-mob insertion mutants. Appl. Environ. Microbiol. 53:410-415. Yanisch-Perron, C., Vieira, J. and Messing, J. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M 13mp 18 and pUC 19 vectors. Gene 33:103-119.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
385
Transgenic potatoes that express an Erwinia pectate lyase isoenzyme Christina Wegene J, Stefan Bartling2, JOrgen Weber 2, Susanne Hoffrnann-Benning 3and Ole Olsen 2 Institut ~ r StreBphysiologie und Rohstoffqualitat, Bundesanstalt ~ r Ztichtungsforschung an Kulturpflanzen, Gross Liasewitz, Germany. 2 Department of Physiology, Carlsberg Laboratory, Copenhagen, Denmark. 3 Institut ~ r Genbiologische Forschung GmbH, Berlin, Germany.
Abstract
Pectate lyase isoenzyme 3 (PL3) from Erwinia carotovora subsp, atroseptica depolymerizes pectins of plant middle lamellae, leaving cells with intact primary and secondary cell walls. The sequence encoding mature PL3 was fused to the promoter of the potato patatin B33 gene and the CaMV 35S promoter, respectively, and inserted into the binary vector pBinl9 for Agrobacterium-mediated transformation of potatoes, cultivar Desiree. While enzyme production in plant lines transformed with pB33-PL3 was confined to tuber tissue, those plants transformed with p35S-PL3 exhibited tissueindependent production. The transformants differed considerably with respect to accumulation of PL3. After wounding of tubers, liberated PL3 caused tissue maceration. Moreover, some transformants expressing PL3 were more resistant against maceration by externally applied Erwinia bacteria or enzymes thereof.
1. Introduction Erwinia carotovora (Ec) is among the plant pathogens that cause extensive maceration of host tissue, leading to soft rot symptoms. Although the bacteria attack a wide range of plants,
main interest has focused on potato blackleg and tuber soft rot diseases (1,2). Several cell wall degrading e n z y m e s - including pectate lyases (PL), polygalacturonases, cellulases and proteases - are secreted by the bacteria, to depolymerize the plant tissue. The major enzyme activities are provided by PLs which cleave by B-elimination the ct-l,4-galacturonic linkage in pectic polysaccharides, releasing a series of unsaturated oligogalacturonides (3). Accordingly, potato tuber cell walls, which consist of 50-55% pectic substances (4,5), appear to be an optimal carbon and energy source for infecting erwinias. From Ec subsp, atroseptica strain
386 C18, three PL isoenzymes (PL1, PL2 and PL3), encoded by contiguous genes (6), were recently analyzed for their capacity to macerate potato tuber tissue (7). The three enzymes liberated host cells 5-10 min aider infiltrating the enzyme solution into the intercellular space of tuber tissue, as shown in the scanning electron micrograph of Fig. 1. Subsequently, differences in action among the isoenzymes became apparent. Alter incubation for 20-30 min, PL1 and PL2 produced lesions in the cell wall concomitant with the release of starch grains, while PL3 exclusively depolymerized the middle lamellae of tuber tissue, leaving a suspension of separate cells surrounded by intact primary and secondary cell walls (Fig. 2 and 3). These remained intact for more than 24 h. Previously, the function in vivo of PLs on pectin has been considered exclusively as the action of a pathogen-derived enzyme causing destruction of plant tissue, with main interest being confined to the role of PLs in host-pathogen interactions (8,9,10,11). The ability of PL3 to alter the intercellular cohesion is similar to textural changes observed when plant-derived polygalacturonases cause a depolymerization of pectic galacturonan during processes of fruit ripening and storage (12,13,14,15). To a certain extent this resembles cell separations causedby thermal treatment of plant tissue as used industrially for processing of potatoes and vegetables, aiming at liberation of cell aggregates or single cells with intact membranes.
Figure 1. Liberation of a single cell from potato tuber tissue following incubation with PL enzymes. Scanning electron micrograph. The'tissue was dried using a critical point dryer.
387
Figure 2. Cells liberated from potato tuber tissue by the Erwmia isoenzyme PL3. Scanning electron micrograph taken after air-drying of the preparation on the support.
Figure 3. Close up of the intact cell wall after digestion of the middle lamella with PL3. Scanning electron micrograph.
388 Such observations led to the hypothesis that regulated expression of PL3 in transgenic potatoes could contribute to enhanced tissue disintegration during storage or thermal treatment. This report addresses the properties of several independent transgenic potato plant lines that express PL3, either constitutively or tuber-specific. We also describe the results obtained following application of Erwinia bacteria to wounded potato tubers.
2. Materials and Methods Plants. Potato (Solanum tuberosum) cultivar Desiree was obtained through Vereinigte Saatzuchten eG, Germany. Plants for transformation as well as transformed plants were grown on sterile MS medium (16). Leaves and tubers were harvested from plants propagated in the greenhouse.
Plasmid constructions, transformation procedures and identification of transfomants. A BamHI fragment specifying the sequence encoding mature PL3 of Ec subsp, atroseptica strain C18 (6), was amplified by PCR and inserted downstream of either the potato patatin B33 promoter (17,18) or the 35S promoter (19) of the cauliflower mosaic virus (CaMV). Transcription termination was provided by the ocs sequence (20,21). The expression cassettes were inserted into the binary vector pBinl9 (22). Agrobacterium tumefaciens, strain GV2260, transformed with the resulting plasmids was utilized for transformation of S. tuberosum by the potato leaf disc transformation method as described (18). Integration of the gene encoding PL3 was analyzed by PCR using genomic DNA isolated from leaf tissue of regenerated, kan R plantlets. Western immunoblots utilizing antibodies raised against PL3 were used to identify plants that expressed heterologous enzyme. PL activity was determined by monitoring A236 for the formation of unsaturated products released from 0.1% polygalacturonic acid (P-1879, Sigma), dissolved in 0.1 M Tris/HC1 buffer, pH 8,0 supplemented with 0.1 mM CaC12. One unit of PL is the enzyme activity liberating 1 ~tmol of unsaturated oligoglacturonides from pectate per min at 25~ Activities are given in mU / ml extract per min. Maceration of potato tuber discs. Twenty tissue discs, each with a diameter of 10 mm and a thickness of 2 mm, cut from the medulla of transgenic and control potato tubers were incubated for 16 h at 25~ between filter papers soaked in water in order to determine endogenous maceration of the tissue. A second series of experiments was designed to analyze the maceration caused by attacking Erwinia bacteria. Twenty tissue discs were inserted between filter paper that had been soaked in a suspension of Ec bacteria (1 x 108 cfu/ml) and incubated as described above. In a third series of experiments, the intercellular spaces of the tissue discs were infiltrated with a solution of Ec-derived enzymes containing 0.1 U of PL activity per ml by applying a vacuum of 200 mbar for 5 min and then incubated for 30 min at 25~ After incubation the tuber disks were shaken in 40 ml of water for 1 h at 25~ in order to generate osmotic stress, and finally stained for 90 min in a solution of 50 mg/ml Neutral Red
389 dissolved in 0.2 M phosphate buffer, pH 7.5, containing 0.8 M KNO3. Subsequently, the discs were rinsed in water, and the absorbed dye extracted twice for 10 min in 96% ethanol. The volume was adjusted to 50 ml with 0.01 M H2SO4 before OD535 was measured (23). Maceration was determined by measuring the retained dye relative to a control sample comprising tuber discs incubated with either water or thermally inactivated enzymes. For baseline measurement of maceration potentially caused by endogenous PL-like enzymes in the transgenic tubers, control samples were stained immediately after cutting the discs. For triplicate analyses the standard deviation was <2%. Maceration of tuber tissue cylinders by Ec bacteria. Five 4-cm long cylinders, each with a diameter of 0.8 cm, were cut from the medulla of one potato tuber, then weighed and placed in wells containing 200 lal of an Ec suspension at 1• 108 cfu/ml; incubation was for 16 h at 25~ Cell debris was removed by using a spatula before the remaining, intact tissue was rinsed with water, dried on a filter paper and weighed. Maceration is given in % and corresponds to the reduction in weight. A total of 29 transgenic plant lines producing PL were analysed (5-6 tubers per line). Using untransformed tubers, it was determined that the standard deviation of the test was 4.4% (n = 30). Control samples were from transformed, PL-inactive tissue. Electrolyte leakage. Tissue discs, prepared from potato tubers as described above, were incubated for 16 h at 25~ between wet filter papers. After incubation, the discs were shaken in 20 ml H20 for another 60 min. One ml of this extract was diluted 30-fold with water, and subjected to conductivity measurements using a HI 8788 apparatus (Hanna Instruments). An increase in conductivity indicates a leakage of electrolytes through lesions in the cell wall caused by enzyme action. Control samples were not incubated, they were shaken in water only.
3. Results and Discussion Transformation. In Ec subsp, atroseptica, three genes encoding PL isoenzymes have been identified on a 7.5-kbp DNA fragment (6). One of these genes, pel3, encodes PL3. By using PCR, the coding region for mature PL3 was amplified and fused to the promoter sequence of the potato patatin B33 gene, which is specifically expressed in tuber tissue. In another construct the gene encoding PL3 was under control of the CaMV 35S promoter that provides constitutive expression in plant cells. The expression cassettes were cloned into the binary vector pBinl9 yielding pB33-PL3 and p35S-PL3. Since vector pBinl9 contains the Ti fight and left borders for Ti-mediated integration of DNA sequences into plant genomes, Agrobacterium-mediated transfection of potato leaf discs was employed to obtain transgenic potato plants. Moreover, as the integrated DNA fragment of pBinl9 includes a neomycin phosphotransferase II gene from Tn5 under the control of the nopaline synthase (nos) promoter and terminator, transformed plant calli were selected on kanamycin-containing media. A total of 97 and 82 kan R plantlets were found to be transformed with pB33-PL3 and p35S-PL3, respectively. Eventually, 25 pB33-PL3 and 20 p35S-PL3 containing plants exhibiting stable expression of PL3 were selected for further propagation in the greenhouse.
390 Compared with control plants, no visible differences were observed on neither the plants nor the tubers. However, the periderm formed after wounding the surface was thicker on transgenic tubers synthesizing PL3. PL3 in plants transformed with pB33-PL3. PCR analyses revealed that about 60% of the plants displaying kanR phenotype also contained the gene encoding PL3. Most of these plants synthesized PL3 as deduced from Western blot analyses. As expected, pronounced synthesis of active PL3 was detected in tubers, with the highest quantities in parenchymatic tissue. To determine the localization of PL3 enzyme in the cells ultrathin sections of conventionally fixed tuber tissue treated with PL3 antibodies were labelled with protein A-gold. Transmission electron micrographs (Fig. 4) showed that the density of gold grains was greater on the surface of starch granules (4,8+0,9 gold grains per 300 nm2) than in the cell walls (1,0+0,3) and the cytoplasm (2,3_+0,6). No PL3 protein was detected using tissue from untransformed controls.
r
,
$
9~ , : ,
:...
Figure 4. Transmission electron micrograph showing tuber tissue labelled with protein Agold. Localization of PL3 enzyme is indicated by arrow-heads. S= Starch grain; CW = Cell wall; Bar = 500nm. x 20 000. Although low, some synthesis of PL3 was detected in the stems, whereas leaves totally lacked the enzyme. Data presented in Table 1 show that independent transformants exhibit more than 10-fold differences in the production of PL3.
391
plants transformed with p35S-PL3. Putative transgenic plants were analyzed as described before. Although the production of PL3 was not as high as in plants where the patatin promoter directed expression, there was again pronounced differences in the production of PL3 among individual transformants, results that were supported by Western blot analyses (Fig. 5). Although the 35S promoter of CaMV is supposed to confer constitutive expression in all types of tissue, leaves exhibited higher activities of PL3 than tubers (compare Table 1 and Table 2).
PL3 in
Table 1.
PL activity in extracts from tuber tissue of potatoes transformed with the plasmids pB33-PL3 and p35S-PL3. pB33-PL3 Transformant
PL-activity a mU/ml
p35 S-PL3 Transformant
PL-activity a mU/ml
D28 D36 D33 D18 D14
30,27 48,69 96,04 168,12 258,12
D29
344,22
C4 Cll C1 C14 C7 C6
2,50 24,58 33,33 34,79 38,96 48,54
a Data represent the mean from 4- 6 tubers per line (lg tissue per trial; 3 replicons). Western blot analyses were positive.
Table 2. PL activity in extracts from leaf tissue of potatoes transformed with the p35S-PL3 gene fusion. Transformant
PL-activity b mU/ml
C1 C15 C3
75,00 95,72 259,38
C6
282,15
b Data represent the mean from 3-4 plants using 1 g leaf tissue per trial; 3 replicons.
392
Patatin-pro PL3 C
D17
D25
D2
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D3
~,
D19
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9
o
.
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.
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Figure 5. Western blot analyses. The blot was probed using antibodies raised against PL3. The PL3 protein bands corresponded to a molecular mass of 42 kDa.
Enzymic maceration of transgenic tuber tissue. To investigate the effects of heterologous PL3, tuber discs from transgenic plants with or without expression of PL3, as well as untransformed tissue, were incubated overnight between wet filter papers, and then compared for potential maceration caused by endogenous enzymes (Table 3). The data show that the maceration was enhanced in tubers synthesizing PL3. It seems that the degree of maceration was dependent on the quantity of PL produced, e.g. tissue of transformant D6 exhibited both the highest activity of endogenous PL (Table 5) and the highest level of maceration indicated by the decrease of cell viability (Table 3). Although these data show that heterologous PL3 enhance the rate of maceration, it remains to be established whether the combined action with other endogenous pectinases provide some additional degradation of the cell walls. Enzymic cell wall degradation in tuber tissue was also reflected by the release of electrolytes as shown by the increase of conductivity in extracts from the incubated tuber tissue disks (Table 4).
393 Table 3. The effect of endogenous PL3 (A) and externally applied E. c a r o t o v o r a enzymes (B) on the cell viability of tuber tissue disks from PL-inactive (I) and PL-active (II) plant lines.
Decrease of cell viability (%) Transformant
I) PL-inactive Untransf. control C4 D3 D30 II)PL-active (0,1-0,3 U/ml) D4 D24 D19 D29 06
A) Incubation with water
B) Incubation with Ec enzymes
5,1 7,7 6,9 9,0
42,2 44,4 33,7 28,8
12,0 12,8 25,5 25,8 28,8
16,3 15,1 13,5 11,6 10,2
C = p35S-PL3; D= pB33-PL3
Table 4. Electrolyte leakage due to the action of endogenous enzymes. Extracts from tuber tissue incubated with water were analyzed.
pB33-PL3 Transformant D19 D34 D4 Untransf Control
PL-activity mU/ml
Increase of conductivity laS
104,06 243,44 144,00
512,17 205,50 109,50
0
0
394
Maceration of tuber tissue by Erwinia-bacteria. The next question addressed how the presence of Erwinia bacteria may influence maceration. Notably, tuber tissue discs from plants synthesizing heterologous PL3 were more resistant against bacterial maceration than control plants (Table 5). Although transformant D6 exhibited a high degree of endogenous maceration (cf. Table 3), its protection against bacterial maceration was the highest observed (Table 5). This supports our previous analysis showing that tuber tissue infiltrated and preincubated for 2 h with 0,05 U ofPL3 /ml exhibited a significantly reduced level of Ec maceration (Fig. 6). Possibly, the action of PL3 caused some alterations in the plant cell walls, e.g. a thicker periderm at the wound site would reduce the effects of a bacterial attack - and/or the synthesis of PL inhibor proteins. Support for this notion is provided by the data giveri in Table 3, showing that application of an Ec enzyme extract to tuber tissue without heterologous PL3 caused higher maceration than following application to tissue already synthesizing the recombinant enzyme. Further experiments were initiated to confirm the results described above. Tuber tissue cylinder of 29 transformed and PL active plant lines (15 transformed with pB33-PL3 and 10 with p35S-PL3) were incubated in a humid chamber containing a suspension of 1• 108 Ec cfu/ml and tissue maceration caused by Ec bacteria was compared with that of the PL inactive plant lines (n=14). Again, tissue maceration was significantly reduced (P=0,01) in plants harboring active PL3. Table 5. Maceration of tuber tissue from pB33-PL3 transformants with Ec-Bacteria (1 x 10s cfu/ml).
Transformant
PL-activity mU/ml
Decrease of cell viability (%)
D3 D30 D2 D22 D 19 D4 D6
n.a. n.a. 57,50 85,73 104, 06 144,00 188,54
43,9 51,7 31,0 31,2 31,2 24,9 22,7
n.a., no activity.
Conclusion. We have shown that PL3 induces plant responses against maceration caused by attacking Erwinia bacteria. It remains to be elucidated whether this is caused by an enforcement of host cell walls and/or the production of compounds directed against the invading bacteria and their enzymes.
395 Products released by the action of PL have previously been reported to act as elicitors of plant defense reactions (24,25,26,27). Accordingly, the transgenic plants described in this report provides an excellent mutant collection for the study of factors conferring resistance against Erwinia carotovora bacteria. In order to obtain a better understanding of the processes that regulate tissue disintegration, additional experiments will include the heterologous expression in potato tubers of a secreted PL3 isoform.
Figure 6. Maceration of potato tuber tissue pre-incubated for 2 h with 0,05 U/ml of PL3 enzyme activity. Con = Control incubated with the inactivated enzyme.
4. Acknowledgements We thank Diter von Wettstein and Lothar Willmitzer for support and encouragement. Ilona Schollenberg and Jessika Dietze are thanked for excellent technical assistance.
396 5. References 1. KelmanA (1980) Ecology of soft Erwinias. Ann Rev Phytopathol 18:361-387 2. PerombelonMCM (1992) Potato blackleg: Epidemiology, host-pathogen interaction and control. Neth J P1 Pathol 98:135-146 3. Preston JF, Rice JD, Ingram LO, Keen NT (1992) Differential depolymerization mechanism of pectate lyase secreted by Erwinia chrysanthemi EC 16. J Bacteriol 174: 2039-2042 4. HoffJE, Castro MD (1969) Chemical composition of potato cell wall. J agr Fd Chem 17: 1328-1331 5. Weber J (1976) Untersuchungen tiber Zellwandgehalt und -zusammensetzung der Kartoffelknollen. Biochem Physiol Pflanzen 169:589-594 6. Bartling S, Wegener C, Olsen O (1995) Synergism between Erwinia pectate lyase isoenzymes that depolymerize both pectate and pectin. Microbiol 141:873-881 Wegener C, Bartling S, Olsen O, Thomsen KK, Bahlow R, von Wettstein D (1995) Differences in cell wall degradation by Erwinia carotovora pectate lyase isoenzymes. Prep. for publication. Collmer A, Keen NT (1986) The role of pectic enzymes in plant pathogenesis. Ann Rev Phytopathol 24:883-409 Collmer A, Batemann DF (1982) Regulation of extracellular pectate lyase in Erwinia chrysanthemi: evidence that reaction products of pectate lyase and exo-poly-ot-Dgalacturonase mediate induction on galacturonan. Physiol Plant Pathol 21:127-139 10. Hugouvieux-Cotte-Pattat N, Robert-Baudouy J (1992) Analysis of the regulation of the pel BC genes in Erwinia chrysanthemi 3937. Mol Microbiol 6:2363-2376 11. Heikinheimo R, Flego D, Pirhonen M, Karlsson M-B, Eriksson A, M/ie A, Koiv V, Palva ET (1995) Characterization of a novel pectate lyase from Erwinia carotovora subsp. carotovora. Mol Plant Microb Interact 8:207-217 12. Tucker GA, Grierson D (1982) The synthesis of polygalacturonase during tomato fruit ripening. Planta 155:64-76 13. Bird CR, Smith CJS, Ray JA, Moureau P, Bevan MJ, Birds AS, Hughes S, Morris PC, Grierson D, Sehueh W (1988) The tomato polygalacturonase gene and ripening specific expression in trangenic plants. Plant Mol Biol 11:651-662 14. Smith CJS, Watson CF, Ray J, Bird CR, Morris PC, Schuch W, Grierson D (1988) Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes. Nature 334:724-726 15. Smith CJS, Watson CF, Morris PC, Bird CR, Seymor GB, Gray JE, Arnold C, Tucker GA, Schuch W, Harding S, Grierson D (1990) Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes. Plant Mol Biol 14:369-379 16. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tabacco tissue culture. Physiol Plant 15:473-497 17. Rosahl S, Schmidt R, Schell J, WiUmitzer L (1986) Isolation and characterization of a gene from Solanum tuberosum encoding patatin, the major storage protein in potato tubers. Mol Gen Genet 203:214-220
397 18. Rocha-Sosa M, Sonnewald U, Frommer W, Stratman M, Schell J, Willmitzer L (1989)
19. 20.
21. 22. 23. 24. 25.
26.
27.
Both developmental and metabolic signals activate the promoter of class I patatin gene. EMBO J 8:23-29 Odell JT, Nagy F, Chua NH (1985) Identification of DNA sequences required for activity of cauliflower mosaic virus 35 S promoter. Nature 313:810-812 Gielen J, De Beuckeleer M, Seurinck J, Deboeck F, De Greve H, Lemmers M, Van Montagu M, Schell J (1984) The complete nucleotide sequence of the TL-DNA of the Agrobacterium tumefaciens plasmid pTiACH5. EMBO J 3:835-846 Herrera-Estrella L, Depicker M, Van Montagu M, Schell J (1983) Expression of chimeric genes transferred into cells using a Ti-plasmid-derived vector. Nature 303:209-213 Bevan M (1984) Binary Agrobacterium vectors for plant transformation. Nucl Acid Res 12:8711-8721 Weber J, Wegener C (1986) Virulence and enzyme production ofErwinia carotovora ssp. atroseptica on potato tuber tissue. J Phytopathol 117: 97-106 Darvill AG, Albersheim P (1984) Phytoalexins and their elicitors - a defence against microbial infection in plants. Ann Rev Plant Physiol 35:243-275 Yang Z, Cramer CL, Lacy GH (1992) Erwinia carotovora subsp, carotovora pectic enzymes : In planta gene activation and roles in soft rot pathogenesis. Mol Plant-Microbe Interact 5:104-112 Palva TK, Holmstr6m K-O, Heino P, Palva ET (1992) Induction of plant defence responses by exoenzymes of Erwinia carotvora subsp, carotovora. Mol Plant-Microbe Interact 6:190-196 Weber J, Olsen O, Wegener C, von Wettstein D (1995) Digalacturonates mediate tissue responses against potato soft rot. In press: Physiol Mol Plant Pathol.
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399
Pectins and pectolytic enzymes in relation to development and processing of green beans (Phaseolus vulgaris L.). K. Recourt a, T. Stolle-Smits ~, J.M. Laats ~, J.G. Beekhuizen ~, C.E.M. Ebbelaa#, A.G.J. Voragen b, H.J. Wichers a and C. van DijkL
aAgrotechnological Research Institute (ATO-DLO), Department of Biochemistry and Food Processing, P.O. Box 17, 6700 AA Wageningen, The Netherlands. bWageningen Agricultural University, Department of Food Sciences, P.O. Box 8129, 6700 EV Wageningen, The Netherlands.
Abstract Processing of green beans involves major changes within the composition of cell wall pectins. About 20% of homogalacturonan is degraded while 65% of rhamnogalacturonan is solubilized. Since B-eliminative breakdown, which is dependant on the degree of pectin methylesterification, is probably the main mechanism explaining this phenomenon, a biochemical and molecular biological study was initiated on the cell wall enzyme pectin methylesterase [PE]. Two groups of isoenzymes with molecular weights of 33 kDa and 42 kDa and different thermostabilities were partly purified. In addition, it appeared that two PE genes, most likely encoding precursor proteins of 63 kDa, are expressed during pod development.
1. INTRODUCTION The texture of processed vegetables is an important quality attribute and is determined by (i) the composition of the cell walls from the fresh product and (ii) cell wall changes occurring during processing. Cell walls of fruit and vegetables consist of about 40% of pectic polymers which can be distinguished in linear homogalacturonan (smooth) and branched rhamnogalacturonan (hairy) regions [1]. Plant enzymes capable to modify cell wall pectins include the de-esterifying pectin methylesterase (PE, EC 3.1.1.11) and the depolymerizing polygalacturonase (PG), existing as either endo-acting (EC 3.2.1.15) or exo-acting (EC 3.2.1.67) enzymes. Both PE and endoPG are considered to play a role during fruit softening and have been studied in detail during tomato maturation [2]. Processing of vegetables at 100 ~ to 120 ~ results in a major decrease of firmness. Several studies suggest that the major cleavage reaction leading to softening is the B-eliminative depolymerization of intercellular pectin. Since this chemical breakdown of pectins only occurs at methylated smooth pectic regions, the
400 demethylating properties of PE might be a tool to control the processing quality of vegetables. Moreover, for potatoes it has been shown that PE is activated during moderate heating procedures ranging between 50~ and 80~ resulting in an increased firmness of the end product [3]. In this paper, we report on changes of cell wall pectins during processing of green beans. Furthermore, biochemical and molecular biological research is presented on the role of PE and PG during pod development.
2. C E L L WALL CHANGES D U R I N G P R O C E S S I N G OF G R E E N BEANS To gain insight in textural changes during processing, cell walls were analyzed from fresh, blanched (5 min 90 ~ and sterilized (30 min 118 ~ green beans. This was accomplished by preparing AIR (Alcohol Insoluble Residue), WIR (Water Insoluble Residue) and WSP (Water Soluble Polymers). Sugar analysis showed that due to the sterilization procedure about 20% of uronic acids was degraded and could not be retrieved from any of the cell wall fractions. Since the overall degree of pectin methylation decreased during processing, this fraction was most likely highly methylated and poorly branched and might originate from the middle lamellae [4]. In addition, sterilization resulted in a significant shift of uronic acid, galactose and arabinose from the WIR to the WSP fraction suggesting a solubilization of branched pectic regions. To study the cell wall alterations during processing in detail, total pectic fractions were separated by extraction of AIR from fresh, blanched and sterilized beans with acetate buffer, CDTA and Na2CO 3. Analysis showed that sterilization caused a large increase in the amount of buffer soluble pectins (Figure 1).
mg/g AIR in: I I Buffer 171CDTA EEl Carbonate 4 "C I;~ Carbonate 20 "C
Fresh
Blanched
Sterilized
Figure 1. Distribution of pectins over different fractions during industrial processing of green beans.
401 Additionally, FPLC analysis showed that sterilization caused a significant reduction of the molecular weight of the solubilized pectins. Interestingly, blanching (5 min 90 ~ also affected the distribution of cell wall pectins (Figure 1). Moreover, processing of green beans at reduced blanching temperatures of 60 ~ to 70 ~ yielded relatively firm products which was accompanied by less pectin degradation (not shown). 3. P E C T I N METHYLESTERASE (PE) AND P O L Y G A I ~ C T U R O N A S E (PG) D U R I N G POD D E V E L O P M E N T To investigate the role of pectic enzymes during green bean development, plants were grown in a green house and pods were harvested at different 'developmental stages. Analysis showed that PG activities were only detectable during early developmental stages and activities ranged between 50 to 100 p k a t / m g protein. Due to the low activities, the endo or exo nature of the enzyme could not be determined. PE activities were measurable during all developmental stages and ranged between 100 and 150 n k a t / m g protein. Seeds contained significantly higher levels of enzyme activity which ranged between 200 and 250 n k a t / m g protein. To purify PE from green beans, mature pods and seeds were extracted with water, NaC1 (1.25 M) and total protein was precipitated with ammoniumsulphate (35%-90%). For seeds, PE activities were only detectable in the hull fraction which was used for further purification studies. After dialysis, PE activities were further purified using weak cation exchange chromatography and heparin affinity chromatography. The active fractions were characterized using gelfiltration chromatography, SDS-PAGE and isoelectric focusing. The identity of the isoforms was confirmed with polyclonal antibodies directed towards a pectin methylesterase from tomato fruit [5]. The results show that both pods and seed hulls contain at least two PE isoenzymes of 42 kDa and 33 kDa respectively. Isoelectric focusing of purified fractions showed the occurrence of one or more isoforms with relative alkaline pI values (Table 1).
Table 1 Molecular weights (MW) and isoelectric points (pIs) of PE fractions from green beans
Seed (hull)
Pod
Fraction
MW (kDa)
pI
I
42
9.8
II
33
>11.5, 10.5
I
42
9.8, 8.4
II
33
>11.5, 10.5
402 Both pod fractions were analyzed for thermostability by incubation at different time-temperature combinations. At a temperature of 70 ~ 50 % of fraction 1 activity was lost after 1 minute of incubation. Fraction 2 was more stabile and contained 50% of PE activity at 11 minutes of incubation. Total pods contained 50% of PE activity after 10 minutes of incubation at this temperature.
4. CHARACTERIZATION OF G R E E N B E A N PE (C)DNA CLONES 4.1 I s o l a t i o n o f (c)DNA c l o n e s Based on the homology between previously published PE sequences [6], three oligonucleotides were constructed and used to isolate green bean-specific PE clones. By using genomic DNA and mRNA from young beans of cv. Verona as templates for the PCR reaction, one putative genomic clone of 210 bp (PE1V) and two cDNA clones of 660 bp (PE2V&PE3V) were isolated. The identities of the respective cv. Verona clones ranged between 50% and 60% at the deduced amino acid level. In addition, a cDNA library was constructed using poly(A)+ RNA from young developing pods of cv. Masai. By screening this library with the cv. Verona PCR clones, a cDNA clone (PE3M) of 1990 bp with 99 % identity as compared to PE3V was isolated [7]. Figure 2 shows an alignment of part of the deduced amino acid sequences. The identity of the partial and full length cDNA clones was confirmed by producing the corresponding polypeptides in E.coli using the pGEX4T expression system and Western blot analysis with antibodies directed towards tomato fruit PE [5]. I PE3M PE3V PE2V PEIV
FIAKDIGFVN ********** **GQ**W*Q*
PE3M PE3V PE2V PEIV
IDFXFGNAAV ********** V********* **********
PE3M PE3V PE2V PEIV
.... PTYLGR .... ****** AGSIK***** .... KS****
II
NAGASKHQA ********* T**PQ****
VALRSGSDRS ********** ********Q*
VFFRCRFDGFQ *********** ******V****
DTLYAHSNRQ ********** ******T***
FYRDCDITGT ********** ****SF**A* .........
420
VFQSCKIMP ********* ***K*YLVA *L*E*N**S
RQPLPNQFNT ********** *KP*S**K*M *K**HG*ATV
ITAQGKKDPNQ *********** V****RE**** ****S**DP**
NTGIIIQKST ********** S**TS**QCN *TGIV**GCN
ITPFGNNLTA ******~*** ***SLDLKPV *KASFD*SSV
480
PWKDFSTTV IMQSDIGALL NPVGWMSWVPN VEPPTTIFYA EYQNSGPGAD *** ...... ......................................... *** ............................................... *** ...............................................
536
III
IV
Figure 2. Comparison of Phaseolus vulgaris pectin methylesterase cDNA clones PE3M (cv. Masai), PE3V & PE2V (cv. Verona) and genomic clone PEIV (cv. Verona). Homology boxes are printed in bold [6]. Numbers refer to the aa sequence of the full length cDNA clone PE3M.
403 The 2 kb clone PE3M contains an open reading frame of 582 amino acids (nucleotides 51 to 1796) encoding a polypeptide of 63.5 kDa. Following the rule of von Heijne, a putative signal peptide of 39 amino acids with the cleavage site at position Ala 45 can be predicted [8]. In addition, the N-terminal segment of the protein contains a putative transmembrane segment of 23 residues within the first 55 amino acids. This region is also identified for other (putative) plant PEs and forms part of the more than 200 N-terminal residues which precede the mature protein [5,9].
4.2 PE gene expression during pod development Southern blot experiments showed that the respective PE clones crosshybridized less than 10% using stringent conditions. Northern blot analysis was performed to determine the pattern of expression of the different PE genes. By using the genomic DNA fragment PE1 as a probe, no significant transcripts could be detected in any of the tissues analyzed. PE2 and PE3 cDNAs hybridized with transcripts of about 1.8 kb in length (Figure 3). Interestingly, PE2 expression levels were high during early stages of pod development while PE3 expression increased during pod maturation. Both bean PEs were also expressed in other plant tissues while tomato PE1 mRNA was only detectable during tomato fruit development (Figure 3, [5]).
A
Y.pods Seeds ,---=1 :=;~ ~:~: ~ , ;
2
! ~ ,; ~i
: ::ii=~i~::i~!;~
PE2
'~'"
D.pods
3 4 5 3 45 "~ . . . .
~"~=~~': ~~~, ~'~176 ~"~ ; ~ : :~" ~i: : , , ; ~ o~=o~.......; :~:~::?~:~:=:
9
iii
(1.8 kB) PE3
11.8 kB)
MG
11.9 kB)
Figure 3. Developmental pattern of expression of PE2 and PE3 in pods and other tissues of green bean cultivar Verona. Numbered samples concur with the following developmental stages in Days Post Flowering (DPF). 1" 1-5 DPF, 2:6-7 DPF, 3: 811 DPF, 4:12-22 DPF, 5:23-35 DPF. Abbreviations: Y.pods = Young pods, D. pods = Deseeded pods. rt, If and fl represent root, leaf and flower tissue respectively. Mature green (MG) tomato RNA was probed with tomato pPE1 [5].
404 5. S U M M A R Y
Processing of green beans involves modifications of the cell wall composition. Based on the analysis of the cell wall fractions from fresh and processed beans, we propose that due to the sterilization procedure demethylated smooth pectic regions (homogalacturonans ?) are degraded by means of B-elimination [10]. Analysis of the pectic fractions showed that (i) uronic acids are released into the brine and (ii) hairy regions are solubilized (rhamnogalacturonans). At decreased blanching temperatures of 60 ~ to 70 ~ a significant reduction of the release from uronic acids was observed. Further studies will indicate to which extent this effect is due to a temperature-dependant activation of PE. During early stages of pod development, low levels of endo- or exoPG were detected. In contrast, PE activities were measurable during all developmental stages. Purification studies showed that both pods and seeds contained two groups of PE isoenzymes with molecular weights of 42 kDa and 33 kDa respectively. Most likely, the relative thermostable isoenzymes of 33 kDa account for the firming effect at reduced blanching temperatures. At a molecular level, three PE clones were isolated from which the two cDNA clones (PE2V and PE3V), corresponding with mRNAs of 1.8 kb, were expressed during pod development and other plant tissues. Based on the deduced amino acid sequence of the full length cDNA clone (PE3M) we postulate that, similar to other plant PEs [5,9], green bean PEs are synthesised as precursor proteins of about 63 kDa. It is not known yet to which extent the proteins encoded by the PE2 and PE3 cDNAs correspond with the biochemically characterized isoenzymes. To investigate the function of PE3M in detail, this cDNA clone will be expressed in potato tubers using constitutive and tuber-specific promoter constructs.
6. R E F E R E N C E S
1.
J.A. De Vries, F.M. Rombouts, A.G.J. Voragen and W. Pilnik. Carbohydr. Polym., 2 (1982), 25-33. 2. G.A. Tucker and J. Mitchell. In: D. Grierson (ed), Biosynthesis and manipulation of plant products, London (1993), 55-103. 3. L.G. Bartolome and J.E. Hoff. J. Agric. Food Chem. 20 (1972), 266-270. 4. P. Ryden and R.R. Selvendran. Carbohydr. Res. 195 (1989), 257-272. 5. L.N. Hall, C.R. Bird, S. Picton, G.A. Tucker, G.B. Seymour and D. Grierson. Plant Mol. Biol. 25 (1994), 313-318. 6. D. Albani, I. Altosaar, P.G. Arnison and S.F. Fabijanski. Plant Mol. Biol. 16 (1991), 501-513. 7. K. Recourt, J.M. Laats, T. Stolle-Smits, H.J. Wichers, C. van Dijk and C.E.M. Ebbelaar. GenBank Data Base (1995) Accession no. X85216. 8. G. von Heijne. J. Mol. Biol. 189 (1986), 239-242. 9. L. Richard, L-X Qin and R. Goldberg. FEBS Letters 355 (1994), 135-139. 10. T. Stolle-Smits, J.G. Beekhuizen, C. van Dijk, A.G.J. Voragen and K. Recourt. J. Agric. and Food Chem. 43 (1995), 2480-2486.
APPLICATIONS.
A) DEVELOPMENTS IN PECTIN MANUFACTURING AND
APPLICATIONS
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J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All fights reserved.
407
Rheological Methods to Characterize Pectins in Solutions and Gels
H.-U. Endress, Claudia D6schl-Volle, K. Dengler
Herbstreith & Fox KG, Pektin-Fabrik NeuenbOrg P.O. Box 12 61, D-75302 Neuenbtirg, Germany
Introduction The development of new rheological measuring devices has given us a wide range of possibilities to characterize pectins in solutions and gels. This may give us important data for 9 the layout of technological processes 9 the quality control of raw materials, and standardized products or 9 the determination of material specific data in research and development. This treatise wants to give a short but not necessarily complete overview of the standard rheological equipment of a modem laboratory in the food industry, starting off with purely empirical rheometers like the Ridgelimeter, the Pectinometer of Herbstreith & Fox, and penetrometers, passing over to more sophisticated rheometers, capable to give fundamental data.
The Ridgelimeter The Ridgelimeter is the most commonly used device to standardize high methoxyl pectins for commercial use (Cox and Higby, 1944). This empirical Sag-test is a one-point, nondestructive measurement. In spite of the excellent reproducibility and easy handling this method has the disadvantage, that it takes in only the elastic properties of the gels. But Mitchell (1980) pointed out, that the breaking strength of a gel may not be related to the gel's elastic modulus, and therefore measurements based on gel-strength will not rank a series of gels in the same order as the Sag test. The reason for this behaviour is due to the molecular weight of the pectins. The elastic modulus will remain constant when reaching a certain molecular weight, while the breaking strength will further increase. Sag measurements are therefore not a sufficient quality criterion.
408
The Herbstreith Pectinometer The original method was developed by Lochmtiller and Ltiers in 1927 using a beam balance. The results of this method correlated well with the firmness and the sensory impression of the gels. The development of the Herbstreith Pectinometer is based on this method. The breaking strength of the gel is now measured with a wire strain gauge. The breaking strength obtained is the maximum value derived from the force variation during the entire measurement, according to the tension needed to pull the standardized shear insert out of the gel filled into a standardized test cup. With a new software program it is possible to measure the ,,Texture Constant" of pectins. This Texture Constant K is calculated by the ratio of the maximum force during the time interval of the measurement and the measured area below the force-time curve. The resulting ,,constants" K correlate well with the dynamic Weissenberg number of oscillating measurements carried through with the same pectin gels. An elastic brittle gel will give a relatively high Texture Constant. Compared to elastic brittle gels, elastic viscous gels resist the gliding of the shear insert to a greater extend, the area beneath the force-time curve is therefore larger, and the "Texture Constant" small. The comparison of gels formed from pectins of different raw materials confirms the test results and the sensory examination. The sensory examination reveals, that gels made from apple pectin, with their small values for the "Texture Constant" K, need a lower breaking strength than gels made from citrus or citrus/apple pectin to be regarded as equally firm. (Kratz, 1994)
Penetrometers Penetrometers are easy to use giving good results and correlating well with the sensory assessment of gels etc. Nevertheless these instruments yield a purely empirical method working on the principal of linear compression, penetration or back extrusion. At the penetrometers the form and the weight of the probe are preselected depending on the test material. The penetration speed or the penetration depth can not be preselected. The penetration measurements are based on the gravitational force applied by the probe.
The Texture A n a l y s e r s The texture analysers have been specifically designed to examine the texture of foodstuffs. They are multifunctional, giving values for the gel strength and penetration as well as load and penetration curves. The probe is pressed on or into the tested sample with a preselected
409 speed and distance. The starting point of the measurement is reached, when the plunger touches the surface of the sample with a certain minimum load. The force-distance curves show the breaking strengths of gels. The evaluation of the curves surpassing the breaking strength can be difficult because of poor reproducibility.
Advanced Rheometers
The flow behaviour of a material is described by the relationship between the force acting on the sample of a material, and the effect of this force. The effect may be elastic deformation or viscous flow. For technological and historical reasons, the standard measuring technique has been to force the sample to undergo a predetermined shear rate and measure the force required. The measuring modes of rotation rheometers can be divided in the following manner: 1. Controlled shear rate flow mode The measurements are carried out at preselected shear rates. The resulting curves are plotted in form of flow-curves ~ (D) or viscosity-curves rl (D) and give information about the viscosity of a substance at certain shear rates and their rheological character dividing the substances in Newtonian and Non-Newtonian fluids. 2. Controlled stress mode This method applies a constant force to the sample and monitors the strain (deformation) as a function of time. This is called the creep test and gives the elastic and viscous properties of substances. 3. Controlled stress flow mode The applied force is linearly increased and the results are plotted in a flow-curve. This is a test to attain the yield point of pseudoplastic material.
T h e C o n t r o l l e d S h e a r R a t e M e t h o d - T e s t i n g on A q u e o u s P e c t i n Solutions
Pectins are longchain macromolecules. In aqueous solutions they form more or less stiff rods or coils, depending on their degree of branching and linking as well as their molecular weight. In addition interparticular or intermolecular physical-chemical interactions like Vander-Waals forces, ionic interactions or hydrogen bonds influence the active volume of the molecule, the stiffness and the viscosity. The Non-Newtonian behaviour, i.e. the decrease of the viscosity as a function of the shear rate, becomes increasingly important when the polymer concentration and molecular weight
410 increase. Except for low hydrocolloid concentrations the viscosity is shear rate dependent and decreases as the shear rate increases. Pectins in aqueous solutions show pseudoplastic non-thixotropic behaviour, independent of their degree of methoxylation. Figure 1 shows the viscosity curve of a 2,5 % pectin solution, sheared the preselected shear rate-time function. The viscosity curves for the increasing and decreasing shear rate are superimposed. The pseudoplasticity of pectin solutions decreases with decreasing concentration.
.~i,
Shear RaID Prolh. as a F ~
of Tm~
"
!
,j"
~k
..............
r,,,,.(,)
Flow - and Vlsco6tty Curw (given. 8hear Rate D)
O,N
12i1 0Q
21 ) O r
'
tmn,
Most pectin solutions behave like Newtonian liquids below a pectin concentration of about 1 % (w/w). Onogi (1966) derived the critical concentration of polymer solutions from plotting the double logarithmic curves of viscosity (11) against concentration at constant shear rates. Each curve consists of two straight lines intersecting at the critical concentration. At higher concentrations than given by this point, the solutions have NonNewtonian flow. This behaviour is thought to be due to the formation of a network structure caused by entanglement of the longchain molecules in solution. Plotting the data of viscosity measurements of pectin solutions of different concentrations reveals the same behaviour, confirming Onogi's observation, with a critical pectin concentration of about 1% (w/w).
Figure 1. Visocsity curve of a 2.5 % pectin solution
The value of critical concentration depends strongly on the pectin being used. Figure 2 gives the viscosity curves of two different pectins at the same concentration of 2.5 % w/w. The different production parameters, that have been used for these pectins, have strongly influenced their flow behaviour. The enzymatic reduction of the molecular weight down to
411
~
Pectins with Different Viscosity Belvwk)ur due to Dilfemnt Production Pm(:esses viso~s~ (Pm
9 ~ ~ t y
! 0
125
ap~ peain
250
375
500
Figure 2. Viscosity curves of pectins with different molecular weight (2.5 % sol.)
approx. 50.000 dalton 1) has produced a pectin of very low viscosity and ideal Newtonian flow even at this higher pectin concentration. In comparison to this pectin, the plot shows the viscosity curve of a pectin produced under regular parameters. Its unchanged high molecular weight of approx. 100.000 dalton 1) leads to a much higher viscosity and Non-Newtonian flow. Commonly, the application of rheological data from aqueous pectin solutions is limited to a narrow field in the food industry. The direct transfer to other properties is limited, since the rheological behaviour of pectins show very complex dependencies on other additives like sugars, acids, salts, and many more.
1) intrinsic viscosity measurement using the constants of Owens et al. (1946)
The Term Thixotropy In pseudoplastic substances shear thinning depends mainly on the particle or molecular orientation or alignement in the direction of flow, this orientation is lost or regained at the same speed. Additionally many dispersions show this potential for particle or molecule interactions, this leads to bonds creating a three-dimensional network structure . They are often build-up from relatively weak hydrogen or ionic bonds. When the network is disturbed,
412 they break easily and the viscosity drops asymptotically reaching a minimum viscosity. This minimum describes the so-called ,,sol"-status of a dispersion. A thixotropic substance is defined by its potential to regenerate its structure when the shear rate is decreased or removed. The viscosity time curve marks the two phases of transformation: a) from gel to sol b) from sol to gel Thixotropic behaviour of pectins can be observed with the drop of their degree of esterification, and with the onset of a distinct reactivity towards divalent cations. The common measuring principal is pictured in figure 3.
Fiil.; 3
Measuring Profile f o r D e t e r m i n a t i o n andThlxo~oDv
of the Yield
Point ....
~
i: (Pa)
(Pa)
: f 3~
s
~,"
4
41
s
2b.
~S
1 time (t)
Shear Rate
Figure 3. Determination of the thixotropy of fruit preparations The shear stress time profile is divided in three sections: 9 the up-curve, the increasing shear stress destroys the structure 9 the plateau 9 the down-curve, the decreasing shear stress leads to the partial regeneration of the structure of the material, like a fruit preparation. The area of the hysteresis loop is a measure of the thixotropy of the tested system. These measurements are easy to carry out and give a quick overview of the thixotropic properties of
413 different pectins in specific preparations. Yet, the results obtained are only a relative measure and depend strongly on the preliminary treatment of the sample.
Rheometry of Fruit Preparations Fruit preprations for yoghurt are a typical example, how the influence of pectins in these preparations can be characterized with the help of rheological methods (Kratz and Dengler, 1995), using 9 the yield point determination, 9 the thixotropic behaviour 9 and the viscosity. For best results, the measurements are carried out in the controlled stress mode. The samples are subjected to a preselected defined stress profile, leading to comparable results. Figure 3 gives an example of a typical force profile. The force is increased continuously and reaches the point - at the end of the first part of the force profile - where the pectin preparations start to flow. The so-called yield point is reached. The further increase leads to the continuous destruction of the internal structure and the proceeding shear thinning. The applied stress in part 3 of the stress profile destroys the structure of the fruit preparations completely. Now the stress is reduced linearly, see part 4 and 5, down to zero stress. The resulting flow curves 2, 3 and 4 and the enclosed calculated area from the hysteresis loop give important evidence about the time-dependent decrease of viscosity and a relative measure of its thixotropy. The rheologically expected complete regeneration of the viscosity after the shearing experiment is not common for these fruit preparations. The reason for this loss of viscosity must be searched for in the destruction of those parts of texture being caused from a gelation process. A yield point is necessary to guarantee a homogeneous distribution of fruits in the big containers were fruit preparations are transported. The shear thinning of the preparations is an important aspect for their pumping and mixing properties. The loss of viscosity should be large enough to render a product, that can be easily pumped with no negative effect for the pieces of fruit, and can be easily mixed with the yoghurt, but afterwards will regenerate enough to produce a pleasant creamy texture in the final product. The yield points measured at the beginning and at the end of the measurement can be thought of as a criterion for texture. A big difference of the two yield points is a sign of a higher degree of gelation and a higher tendency for syneresis.
414 The advantages
o f p e c t i n s in f r u i t p r e p a r a t i o n s
are quite obvious:
1. Plastic flow: pectins build up a yield point without giving the product a slimy characteristic. 2. The thixotropic texture has the advantage, that the viscosity of the pectin preparations will decrease with increasing shear stress and regenerate quickly to a great extend when shear stress decreases. 3. The yield point regenerates within short time. 4. Pectins show great stability towards shearing. The very special demands on the pectins used in yoghurt fruit preparations show the usefulness of theological measurements. Figure 4 and 5 explain the difference in theological behaviour of fruit preparations made from 9 two low methoxyl apple pectins (Classic AY 901 and Classic AY 905) 9 an amidated pectin (Amid AF 015-A) and 9 this amidated pectin also in combination with locust bean gum (LBG). The yield points before and after shearing the yoghurt fruit preparations were measured at 20~ and different contents of soluble solids of 35 and 50 % . The ratios of the two yield points depend on the content of soluble solids. The apple pectin Classic AY 905 has the highest yield point of all at a content of soluble solids of 35 %, but as one can see from the regenerated yield point, this high value must be mainly due to gelation (see figure 4). For the apple pectin Classic AY 901 this ratio is much smaller, and its textur has less gel characteristics. Fruit preparations with the amidated pectin, with or without locust bean gum, do not have yield points high enough to prevent floating.
Comparison of Y'mld Points before and after Sheer Stress of Different Yoghurt Fruit Preparations with 50% SS at 20~
of Oflferent Yoghurt Fruit Preparations with 35 %
t
3S 30
J~ v
,o"
~
9
....
'
........... ! .................. !.................. +..........
t $
7
...........L............
i
.............~..........
t0
s 0
~Avt~
Cl'dslirAY N 6
.1 (~ YIeld P ~ . h r she~ glrms
~Ue~
~r
Ill lhid P~
AF 01$J~4.~1% LBG I
Ghls~ AYI~I
Ol~sdr &u
Amid AF 0'lS~lk Amid AFOler~t § (kl % LBG
l tin ~ t ~ Point alter Shear tlnms
at ~
Point
Figure 4 and 5 show yield points at soluble solids contents of 35 % and 50 %. Figure 5 shows both yield points for a content of soluble solids of 50 %. Now the amidated pectin has yield points also after the shearing process, which are sufficient to prevent floating.
415 For more details about the sensory properties of the pectin preparations additional sizes such as thixotropy and viscosity have to be referred to (see figure 6). Fruit preparations with apple pectin Classic AY 901 have a relatively high yield point after shearing, a small thixotropic area, and a relatively high viscosity. This supports the statement, that their texture is weakly elastic and highly reversible. The apple pectin Classic AY 905 gives products with a sufficiently high yield point at 35 % soluble solids and a pronounced area of thixotropy. The elastic components of the texture are destroyed and only partially regenerated in the course of the measurement, as can be seen in the large difference of the yield points. The viscosity being reached is sufficient for the product. The texture of the amidated pectin preparations shows little elasticity as indicated by the yield point differences. Nevertheless the thixotropic area is rather large due to shear thinning and the low viscosities leave a watery mouthfeel. The locust bean gum increases the viscosities remarkably, but does not result in a yield point (after shearing) large enough to prevent floating. .
.
.
...
,.,,,...".......
-
.
..
.... .
'Compa'rison of Measured Values of C l a ~ i c AY 901, with 35 % SS .
.
.
.
.
.
.
.
.
.
.
.
.
.
9
. . . . . . .
'. . . .
i .........
. 9. . . . . .
t
COmparison of Measured Values of Classic AY 905, with 35 % SS
;,i,
! i T
~
.
...............-t ~
............................................................ i...........I
4s
......................
-
15 o~
i
~
".
..9........................................i...................
.,e~-~: ~~,
.
.
.
.
.
.
.
.
.
.
0 ~
~-
I ~ - - - ~ Classic AY 901 .
.
.
.
.
.
.
~.
................."..~.
l l~lYield Point I'-IThixotropy IBostwick .
Classic AY 905 ,t
IVisc~
.
,.,
.................................... ,L
'
"
.~:~ Amld
I
"!
I.
,--77~, "s0,7
~06
...............................
AF eiS"4k""-
.V.~o..y
Compar~on of'Measured Values of ' " " AmidAF015-A+01%LBG with35%SS
.E~i] ........................... i~ ~ ~,~~ ..................................... -I ~' : 9,~I ............................ I~~.~~b~ .................................... -~0'~ *
o ,
,.
.l,m~o. Po,n, mTh,xot','oPyI.O.~,,~'k'
.
Comparison of Measured Values ~ ofAmidAF015-A, with35%SS i 1. -~' ........
~
.................. " ~ ....... ...L..I._...I.......I....I..I...-
,--I
- ....
"
"
'
I'm~o'dPOJ.iPITl~i'x'otropY'i~stwick " iVi'cos~Y
Figure 6. Comparison of measured values
"
'9
..
~
"
"
~ * ~
'
i
.
oV
.
.
,, :.,
- .....
-
_: . . . . . . _ _ _ 1
...................
o,7
...................................... i!....................................
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..................................... ~........ ~ I I
.~.~ ......~
Amid AF 01t-A §
0,1%
[t.~Yield'Point OThixotropY IBos'twick
~'
................. 4-
,~o iVis~.osity I
416 The Oscillating Mode- a Further Rheometric Technique
Visco-elastic fluids like pectin gels, behave like elastic solids and viscous liquids, and can only be clearly characterized by means of an oscillation test. In these tests the substance of interest is subjected to a harmonically oscillating shear deformation. This deformation ~/is given by a sine function, [ y = Y0 sin (03t) ] by % the deformation amplitude, and 03 the angular velocity. The response of the system is an oscillating shear stress x with the same angular velocity 03.
Ft~-8
Phase Displacement
between Stress and Strain
Schematic Construction of an Oscl.llatlon.Rheometer ~
Vis~.o-e~stlc Substance (phase d l e ~ t b e t w e e n G" u n d 9 0 ~)
i
Elastic - :o.d ( i t r a l m a n d strain in p h a e e )
.
Liquid
E m m d
(mr.~,.. a , d . t n , i n m " o . t o r ~p ~ u e )
b: ~ b e e e ~ c: meesu~ symm
d: foe~ceor ~ dmck~ e: ~ dmckw
Figure 7 shows the schematic construction of an oscillating rheometer.
Figure 8. Phase displacement between stress and strain.
The mathematical dependence applies only in the linear visco-elastic region. In this region the sample can be deformed up to a maximum deformation (Y0, max) without destroying the structure of the sample.
417 A purely elastic substance is linearly connected with the deformation via Hooke's Law producing a phase displacement 8 of 0 ~ (see figure 8). The deformation of the spring is caused by energy, this energy is stored and gained back when the applied force is removed. Ideal viscous substances can be described with the shock absorber model of Newton. The energy applied to the viscous body is completely transformed in energy of deformation or dissipated in form of heat. The mathematical description of this problem shows that the shear stress and shear rate curves are in phase compared to the deformation curve [? (t)], this is equivalent to a phase displacement ~5of 90 ~ (see figure 8). Visco-elastic substances can be described with the spring/shock-absorber model of Kevin and Voigt, and have phase displacements of 0 ~ to 90 ~ In analogy to other time dependent processes in physics, the oscillation tests are evaluated with complex arithmetics. Obtained are the complex quantities: 9 the complex viscosity (?*) 9 the complex Young modulus (G*) 9 the storage modulus (G'), as a measure for the reversibly stored energy/this is the elastic share of the complex Young modulus 9 the loss modulus, representing the irreversibly stored energy/this is the viscous share of the complex Young modulus 9 the loss factor giving the ratio of the viscous and elastic share (G"/G')
Different Tests and their Application Strain amplitude sweep The deformation amplitude is varied [? (t) = ?o sin (cot)] at a constant angular velocity. The resulting storage modulus (G') is plotted versus the strain. The test can be used 9 to determine the stability of a system 9 to determine the linear visco-elastic range, important for further measurements. The linear visco-elastic range ends when the elastic modulus G' starts to fall off with the further increase of the strain amplitude. This value is called the critical amplitude ?~. This is the maximum amplitude that can be used for non-destructive dynamic oscillation measurements.
Figure 9 gives the visco-elastic ranges of three different pectins with the same degree of esterification and under equal conditions. The gels based on citrus pectin have the highest storage modulus of the three pectins and the shortest linear visco-elastic range. In general citrus pectins form highly elastic, brittle gels. The lowest storage modulus is shown by the
418 gels based on apple pectin. They have a wide linear visco-elastic range correlating well with the sensory evaluation of an excellent spreadability and low tendency to syneresis.
~::.::::.:;:..: :::.:.
m a d e of.. P e c t i n s f r o m O i ~ n t
~7 ~
Raw Materials
!:..-i,-~.~ r: ........... . "t.~:::: :!.:i~,.~.;'i.!i~:i!~,!i~
o.ool
O~Ol
o.1
1
Figure. 9 Visco-elastic range of pectin gels
Strain frequency sweep The strain frequency sweep measurement can give answeres on questions concerning the stability of the structure of visco-elastic material. The measurement is conducted at a preset strain amplitude, increasing the oscillation frequency in dependence of time. The resulting double-logarithmic plot of the storage modulus G' versus the angular velocity 0~ shows characteristic spectra depending on the measured substances. Figure 10 gives the four types of spectra:
~: 9.
,
Storage Modulus Curves
/ ~~.:.-.~p.~f.)~::::~....::.-.
, .~
i~.~:~.:.':~-.-.,.
-::.::-.;_.:!.. :: .
. .......
. . ...... - . . . - - . .
:.~:...!.:,-:.:.::.,.=~:~-.;:,~!;~:.~i~.,,?.i~ ~
-: . . . . . .:.
G, .~
.~,
..... . . . . . . . . . . . .
,:., ,~:~~
...........! .:..:... .... , ..: ~:-~.,:--~.:.~:-~::~~.:~:.i.~i~.~i~?~
Figure 10. Graphical illustration of frequency sweep meas-urements taken from four different substances.
419
curve 1: curve 2: curve 3: curve 4:
substance with low molecular weight, no network substance with wide molecular weight distribution also no network substance with partial network or yield point substance with complete network
Short chain linear molecules which can't form a network change directly from the flow zone to the glassy zone. A plateau zone always indicates a network structure, its width depends on the molecular weight. In the transition zones the mobility of the chain molecules decreases, the chain segments can no longer relax completely. At even higher oscillation frequencies the chain segments can move no longer, this is called the glassy zone (G1). The spectra of pectin gels recorded follow the general rule for polysaccharide gels, being characterized by a flat dependency of the storage modulus (G') over the wide range of frequencies studied. This behaviour reflects the existence of a three-dimensional network. The values for the storage modulus G' depend on the parameters in the gels. The exceptionally strong influence of calcium-ions on pectin solutions especially made with HM citrus pectins can be shown by a frequency sweep. The addition of calcium leads to an increase of the complex viscosity. Additionally we can observe a stable trapping of air bubbles in the solution. This effect can not be caused by the increase of viscosity. The frequency sweeps of the solutions give the answer. The storage modulus curves show the significant increase of the elastic shares caused by the addition of calcium-ions. The dynamic Weissenberg number W' can be calculated from data obtained by the strain frequency sweep measurement. It's the ratio of the elastic to the viscous shares in the measured gel and leads to an objective description of the sensoric properties, representing the basis for the standardization of pectins. The dynamic WeiBenberg number of pectin gels is influenced by the raw material used for pectin extraction and by the degree of esterification of the applied pectin. With a decreasing degree of esterification the dynamic WeiBenberg number also decreases. The ratio of elastic to viscous shares in a visco-elastic gel influences the texture as follows: Very large elastic and very small viscous shares resulting in a high dynamic WeiBenberg number give a gel which is very brittle and has a high breaking strength. The gel is sensible to mechanical treatment and shows a high tendency to syneresis. Gels with a low dynamic WeiBenberg number that means with large elastic and increasing viscous shares are soft and show better spreadability. They are more stable against mechanical treatment and have less syneresis. There is a good correlation to sensory properties: Gels made from apple pectin have a wide linear viscoelastic range. Therefore a big effort is necessary to puncture the gel. On spreading the gel it remains joined, does not break into small lumps and shows a smooth and shiny surface. In the mouth the gel feels clear and smooth when squashing it between palate and tongue, the sweet fruity taste remains for a long time. The gels have ,,body". Gels made from citrus pectin have a small linear visco-elastic range. To puncture the gels takes a big but only short time effort. The gel breaks into small lumps immediately. In the
420 mouth the gels feel rather rough and crumbly, the gels have little body. Gels with higher dynamic Weil3enberg number need a higher breaking strength values here expressed in Herbstreith-Pectinometer units (HPU) to be judged sensorically equally firm.
Strain Temperature Sweep The strain temperature sweep measurement is conducted with a preselected amplitude for the applied strain (7) and a constant frequency (f). The changing parameter is the temperature T, which is given in a temperature-time profile [T = T(t)]. This test method serves to illuminate the structural build-up, the softening, the melting and the gelation of pectins influenced when the temperature changes. The gelling temperature is an important factor for the characterization and application of pectins. The pectin consumer wants a pectin fulfilling his special requirements, this can mean either working with or without pregelation. Pregelation, the weakening of gel structure, occurs when pectin preparations are stressed below their gelation temperature so that the mechanical treatment leads to an irreversible destruction of the three-dimensional network. i ~ : 11
Graph. Setting Temperature -
.,,.t~,~,.~..~,:~*.~,.~2
:~,.r
~:,'- ,.:,:.'-:.~ ,: ".~,"-~v ~ :...,~'. " :
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~
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::
~:
..... '::~:~,.il!i~i60"
2o ............
10
20
X.
SO
.......
100
200
Time(s)
Figure 11. Strain temperature sweep measurement
The pectin sol is prepared and filled into the measuring system at elevated temperatures higher than their gelling temperature. The system is cooled at the given rate and the gelation threshold is defined, when the loss modulus G " and the storage modulus G' are equal and the phase displacement is 45 ~ (see figure 11). The gelling temperature is an important criterion in
421 jam production, and must be chosen suitably for the desired packaging size, or the production of jelly fruits. The Temperature Sweep Figure 12 demonstrates the different gelling behaviour of three samples of so-called SprayNappage, an intermediate used in bakeries (Dengler, 1995). The ready to use liquid is produced from pectin, water, sugar, acid and fruit concentrate or pulpe, and is preheated to about 85~ before spraying. The setting temperatures at the phase displacement of 45 ~ were compared with the sensory evaluation. The sample with the highest setting temperature (sample 1, about 72~ had the shortest setting time the layer with the biggest thickness, and a very brittle texture.
Fla. 12
I~mmination of ~m~ing Temperature
of Nappage
.
.,.". . . . . . . . . ::":
0
-
'7
..
'
SO
"
.
60
70
80
tom~
90
(~
Figure 12. Determination of setting temperature of nappage
The gelation processes are not solely temperature dependent but also time dependent. The determination of the setting temperature is therefore dependent on the temperature profile being used. Results can only be compared, when the pectin preparations are pretreated and measured with exactly the same parameters. The Strain Time Sweep This test is based on a preselected amplitude for the applied strain (y) at a constant frequency (f) and a constant temperature (T). The method can be applied to test the stability of substances with temporary physical changes of structure or the course of chemical reactions like gelling at a given temperature. Again the pectin sols are filled into the measuring system
422 at temperatures higher than their setting temperature and afterwards immediately cooled down to the wanted measuring temperature. The criterion for gelation is also a phase displacement of 45 ~
Determination of Setting Time. of Nappage
..-....,:..
...: ; . . : :: .:;..(:. - ,-..:.~. :...........-.:~....,. .~....
e 2oo §
....
. : : " :'... i:,.:
-..
9
O.2
0.4
0.6
0.8
1.0
1-2
Figure 13. Determination of setting time of nappage A typical graph is plotted in figure 13, showing the storage modulus and the phase displacements for the previously mentioned Nappage samples. Sample 1 with the highest setting temperature builds up its network structure in a relatively short time compared to Sample 3 (ca. 30 min). In manufacturing, exact gelation times have the advantage that the products reach their final gel structure at the predetermined setting time resulting in a constant production process.
Conclusion
Using these rhelogical methods laboratories for quality control and research and development have good tools to characterize pectins in gels and solutions. The most important points are the reproducable handling, pretreatment, and measurement of the samples and the knowledge which information can be derived from the measured data regarding the texture, the production parameters, and the sensory evaluation of the product.
423 References:
1 Cox R.E., Higby R. H.: Food Ind. 16 (1944) 441-442 and 505-507. 2. Dengler K.: Getreide, Mehl und Brot, 49 (1995) 398-402. 3. Kratz E. Gums and Stabilisers for the Food Ind., 7 (1994) 403-411. 4. Kratz R., Dengler K.,: DMZ Lebensmittelindustrie und Milchwirtschat~, 14 (1995) 640-651. 5 Li~ers H., Lochmi~ller K.: Kolloid Zeitschrit~, 42 (1927) 154-163. 6 Mitchell J. R.: Journal of Texture Studies, 11 (1980) 315-337. 7 0 n o g i S., Kimura S., Kato T., Masuda T., Miyanaga N." J. Polymer Soc., Part C, 15 (1966) 381. 8. Owens H.S., Lotzkar H., Merill R.C., Peterson M.: J. Am. Chem. Soc., 66 (1946) 1178-1182.
This Page Intentionally Left Blank
J. Visserand A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996Elsevier ScienceB.V.All rights reserved.
425
Effects of extrusion-cooking on pectin-rich materials J.-F. Thibault, M.-C. Ralet, M.A.V. Axelos and G. Della Valle
Institut National de la Recherche Agronomique Centre de Recherches Agro-Alimentaires B.P. 1627 44316 Nantes Cedex 03 France
Abstract Some by-products from the food industry contain high proportions of plant cell walls which can be used in human nutrition to produce "dietary fibre" or "functional fibre", i.e. compounds which can be used for their waterh o l d i n g / b i n d i n g properties, oil-binding capacity,.., or as a source of polysaccharides such as pectins which are suitable after extraction, as gelling or thickening agents. Extrusion-cooking of cell-wall rich products (e.g. wheat bran, apple pomace, citrus peels, sugar-beet pulp, pea hulls .... ) led to an important solubilisation of polysaccharides of various types without extensive degradation of the polymeric structure. The possibility of obtaining gelled systems directly with the extruded pectin-rich materials was demonstrated. This treatment has the advantage of avoiding the acid extraction of the pectins; it could be also used to obtain extruded products which could be used in various preparations traditionnally requiring high methoxyl pectins as gelling agents. The final products could be therefore enriched in dietary fibres and devoid of additives.
1. I N T R O D U C T I O N Many plant products are very rich in cell wall materials. Cereal brans, seed hulls, various pulps (including beet pulp), citrus peels, apple pomace.., are typical exemples of such by-products (1,2). They can be used after simple treatments as dietary fibres, functional fibres or bulking agents, depending on the nutritional claims (2). They can be used also as sources of some polysaccharides. Indeed, apple pomace, beet pulp or citrus peels contain pectins (3). Chemicals, enzymes, microorganisms, or physical treatments can be used for thextraction (4).
426 The use of chemicals, essentially acid, is the method of choice for the extraction of pectins, at least at an industrial level (3,4). Enzymes such as "protopectinases", polygalacturonases, rhamnogalacturonases, have been tentatively used at a laboratory scale but their industrial interest is still an open question (4,5,6). There are only some reports on the use physical treatment such as various heat treatments. The same methods (chemicals, enzymes, physical treatments) can be also applied on the cell wall materials not with the aim of extracting polysaccharides but with the aim of obtaining modified fibres. New properties concerning for exemple fermentability, ratio soluble/insoluble dietary fibre, hydration., can be obtained (1). Our aim was to use extrusion-cooking and to investigate its effect on some pectin-rich materials, especially citrus fibres.
2. O V E R A L L E F F E C T S O F E X T R U S I O N - C O O K I N G Extrusion-cooking is an alternative method (7) for texturization, mixing or cooking which is nowadays currently used by the food industry for the production of various type of snacks, ready-to-eat cereal products .....In a typical twin-screw extruder, the product is feeded into the extruder and is conveyed by the rotation of the screws. In this part, the product is submitted to heat (by heating process or by self-heating) and to shear. At the end of the extruder, the product is forced to pass through a die, and then water can be vaporized leading to expansion of the product, at least for starchy products. In our studies, we used a twin-screw extruder from Clextral (BC 45) with a 1 meter long barrel. The degree of mixing and shear of t h e p r o d u c t was regulated by a combination of screw design (for example, the presence of reverse pitch element before the die), the die design and operating conditions such as temperature (100-180~ screw speed (150-250 rpm), feed rate (14-40 k g / h ) , amount of water added (10-60% on a dry matter basis). The main parameter to be calculated is the specific mechanical energy (SME) because it measures the severity of the treatment (7). The main effect (8,9) of extrusion-cooking on the plant cell wall materials was to increase very significantly the water-solubility in cold water (20-25~ of the products. Three straight lines can be drawn depending of the cell wall materials (Figure 1); one for wheat bran which needed high energy input and up to 15 % of the bran can be solubilised by water, one for the pea hulls for which a solubility similar to that obtained for wheat bran was observed but for lower energy, and a single curve for all pectin-rich materials as the points for beet pulp, apple pomace or citrus fibres fall on the same straight line. The amount of watersoluble material was up to 40%. Analysis of the products which become water-soluble after extrusioncooking showed that feroylated heteroxylans and glucans were solubilised from wheat bran (10), arabinans, heteroxyalans and pectins from pea hulls (11,12), and pectins from the other sources (13,14).
427
40
o~
v
beet pulp apple pomace citrus peels
30
0,6
..Q .
m
0 u}
s s o
s
s
s
f
%
s
99
20
Oj s
,OD
o 0
pea hulls
.11
s
10
S
.~'&
ot I
wheat bran
S I
0
1O0
200
300
9
!
400
Specific mechanical energy (kWh/t) Figure 1" Influence of the specific mechanical energy on the water-solubility (corrected from the blank values) of some raw materials
The effects of extrusion-cooking mainly on citrus fibres will be now detailed on samples which have been, unless otherwise stated, extruded in the same average conditions (SME -- 250kWh/t).
3. C H A R A C T E R I S T I C S
OF THE CITRUS PECTINS
The characteristics (14) of the crude extracts after water extraction of extruded citrus fibres and after acid extraction of the same material are indicated in Table 1. The yield in crude pectins and the intrinsic viscosity were slightly higher for the pectins obtained by acid extraction. This result suggested that extrusioncooking degraded more the galacturonic backbone of the pectin than acid extraction. However, the aqueous extract has contents in galacturonic acids and in neutral sugar slightly lower and higher than the acid extract, respectively. An important feature concerned the degree of methylation (dm). A value of 8 1 % was obtained after acid extraction and a very high value of 9 1 % was obtained for the pectins from extruded fibres. This is an unusual value for a dm and this result shows that extrusion-cooking had a very limited effect on the methylation of the pectin.
428 Table 1 characteristics of the pectins extracted from citrus fibres
pectins acid extraction a yield (% fibres, w / w )
34.1
Intrinsic viscosity (ml/g)
588
Galacturonic acid Rhamnose Arabinose Xylose Mannose Galactose Glucose
51.6 1.1 13.4 0.3 0.5 3.0 4.3
Degree of methylation
water-soluble after extrusion b
81
29.4 450 49.1 1.7 15.9 1.1 1.5 3.7 5.1 92
a: extraction by 0.05M HC1, 3x30 min, 85~ b. extrusion at 100~ 240 rpm, 20% water (SME = 250 kWh/t)
These crude pectins were submitted to chromatography on ion-exchange column (Figure 2). Some neutral polysaccharides were not bound to the column; they were essentially arabinans and the amount of arabinose represented less than 40 % of the total arabinose for the pectins obtained by aqueous extraction of extruded fibres, and more than 70 % for the acid-extracted pectins. This result shows that extrusion-cooking had a less degradative effect on the arabinan side-chains than the acidic treatment. Pectins were eluted by a gradient of ionic strength. A very narrow and homogeneous peak was obtained for the pectins obtained by water extraction after extrusion-cooking with an early elution in agreement with their very high dm. In contrast, acid-extracted pectins were eluted as a wider peak for a higher ionic stength in agreement to their lower din. The peak is also much more heterogeneous. The bound materials were collected and the structure of these purified pectins was further studied.
429
1000
o
500
ii
._1
E r
tO v
tO c-
O L,_
-1
0
r~ c-"
3
o (D
:::L
400 o
v
o o
rO ..=,_
r
o
200 (
0
200
400
600
elution volume (mL) Figure 2: Ion-exchange chromatography on DEAE-Sepharose CL-6B (elution by acetate buffer pH 4.8) of (a) dialysed water-soluble pectins from extruded citrus fibres (SME = 250 kWh/t) and (b) dialysed acid extracted pectins from the same raw material. (empty symbols: neutral sugars; full symbols= galacturonic acids) It is already well-known (4) that in various pectins extracted by chemicals including water, the neutral sugars are not evenly distributed along the pectin molecule but rather grouped in regions ("hairy" regions) containing few galacturonic acids while the bulk of the galacturonic acid constituted the "smooth" regions. As the functionality of pectins primarily arises from the "smooth" regions, it would be essential to know if this model also applied to pectins which become water-soluble after extrusion-cooking. For this purpose, the purified pectins have been first deesterified by cold alkali and submitted to degradation by pure endopolygalacturonase (15) which degraded the "smooth" region, leaving rather intact the "hairy" region. By gel permeation chromatography (Figure 3), the products were separated in two distinct populations.
430
t-
._o t" 0
"0 0
b
0
Kav
1
Figure 3: Gel-permeation chromatography on Sephacryl S-200 of the products obtained after endopolygalacturonase degradation of the (a) water-soluble pectins from extruded citrus fibres (SME = 250 kWh/t) and (b) the acid-extracted pectins from the same raw material. (empty symbols: neutral sugars; full symbols= galacturonic acids)
One peak is eluted at the void volume of the column and contained the bulk of neutral sugars and few galacturonic acid residues while the peak at the total volume contained almost pure oligogalacturonides. Therefore the same model of "hairy" regions and "smooth" regions also applied to these pectins. When comparing this result with that obtained with the acid-extracted pectins, the same two populations were separated but the amount of "hairy" regions is lower for the acid-extracted pectins (less than 10 % of the pectins) than for the pectins (=15 %). This result confirmed that extrusion-cooking is less degradative towards the side-chains that the acid treatment; it also shows that both pectins have large "smooth" regions.
431 4. G E L L I N G P R O P E R T I E S OF T H E W A T E R - S O L U B L E P E C T I N S It was possible (16) to obtain gels from the crude pectins in presence of sucrose and at acidic pH. The phase diagrams of the pectin systems (Figure 4) have been first established. The gel state was simply observed by visual inspection of the products obtained after heating (100~ 30 min) and cooling (to room temperature) solutions containing different quantities of pectins and sucrose at pH3 (citric acid). The gel/sol transitions cannot be distinguished for pectins extracted by after acid treatment and water-soluble pectins after extrusion. A minimal pectin concentration of 0.2% is required for gelation and no gels can be obtained below a sucrose concentration of 45 %. Commercial pectin (Hercules) with a dm 73 % has a lower phase transition line with a minimal pectin concentration of 0.1% and sucrose concentration of 40 %. sucrose (%) 65.
55-
GEL
45. SOL 35 0
I
0.5
I
1
I
1.5
pectins (%) Figure 4: Phase diagrams of pectins extracted by water from extruded citrus fibre and by acid from the fibres (upper curve), and of commercial citrus pectins (dm 73%) (lower curve) Some rheological measurements have been made (16) on the different gels. The Figure 5 shows the mechanical spectra measured for two pectins concentrations obtained after extrusion cooking. The constancy of the storage modulus in the low frequency range and its value 10 times higher than the loss modulus indicated that true gels were obtained.
432
G'. G" (Pa)
G' ooooooooor176
102
0.6% _oooeO*~176
101
G' u l n n l l l p l l i l l J G"
10 0
G" nmmlnnlnmm~illnllnmnU
10
~'mlinnmip _mninnu i l i
0.2%
-1 10 -3
10 -2
10 -1
100
101
Frequency (Hz) Figure 5: Mechanical spectra (Carri-med CS-50) of the gels (pH 3, 20~ 60% sucrose) made with 0.2 and 0.6% pectins obtained after water extraction of extruded citrus fibre (SME = 250 kWh/t). The plateau values of G' have been plotted as a function of pectin concentration; as expected, the gel elasticity increased with the amount of pectin (Figure 6).
G' (Pa) commercial pectin
103
acid e~~~~~~,on
102
10
0
0.2
0.4 0.6 Pectins (%)
0.8
Figure 6: Evolution of the storage moduli of the gels (pH 3, 20~ with the pectin concentration.
60% sucrose)
433 Whatever the concentration, commercial pectins formed the strongest gels followed by the acid-extracted pectins and the pectins from the extruded fibres. More differences have be seen when the storage moduli were measured as a function of the sucrose concentration (Figure 7).
G' (Pa) 104 commercial pectin
103
~
acid extraction
extrusion 102 10
40
50
60
Sucrose (%) Figure 7: Evolution of the storage moduli of the gels (pH 3, 20~ with the sucrose concentration.
0.6% pectin)
For commercial pectins, G' increased rapidly up to 45 % of sucrose and remained constant. In contrast, the value of G' for the other pectins increased more slowly and reached only a plateau for a concentration about 55 %. It is therefore possible to obtain gels with the pectins in the same conditions as other HM pectins. When these pectins are compared to the commercial pectins, it can be observed that higher pectin concentrations were needed and that the gelation was more dependent on the sucrose concentration; this fact can be ascribed to a higher purity of the commercial pectins (17,18). However, the difference was more marked between the commercial pectins and the other pectins. The reasons for this discrepancy can be found either in differences in the values of the dm or in the origin of the raw materials. Indeed, a lower dm value can lead to firmer gels (19,20). However, the main reason is probably the fact that the citrus fibre used in this study was a commercial citrus (dietary) fibres of which the pectin quality may be lower than the citrus peels used by pectin industry.
434
5. GELLING PROPERTIES OF THE EXTRUDED FIBRES The results showed (16) that the extruded products contained a large amount of water soluble pectins of high molecular weight which were able to gel. It has been shown that the all the extruded citrus fibres were able to gel in presence of sucrose at acidic pH without prior extraction of the pectins. The phase diagram of the sucrose/fibres (pH 3) is represented in Figure 8.
sucrose (%) m
65-
GEL
55L i
m m
m
m
I
- -
- -
45SOL
5
I
0
0.5
I
1
l
I
1.5
2
fibres (%)
Figure 8: Phase diagrams of the extruded citrus fibres (SME = 250 kWh/t) It was very simply composed by two perpendicular straight lines. No gels can be obtained for fibre concentration lower than 0.5 % and sucrose concentration lower than 50 %. The mechanical spectra of the gels are shown in Figure 9 for fibres which have been extruded in different conditions (16).
435
G', G" (Pa) 1000"
G OoOOOOOOOOOOOOOOOOOOOOOOOOOOO0 mmmmmmmmmmmmmmmmmmmmmmmmmmmmmm II
100
-
ooOO0OOO0:
G
mmmmmmmmm
io m
OOOoO0OOOO0OOOOOOOO0 .m m.mmmmmmmm mmmmmmmm 10
-2
10
-I
10
0
m
10
~7
1
Frequency (Hz)
Figure 9: Mechanical spectra (Carri-med CS-50) of the gels (pH 3, 20~ 60% sucrose) made with 1.5% citrus fibre extruded at two different (SME = 170 and 250 kWh/t) The data confirmed that gels can be obtained for all the extruded fibres. However, a slight decrease in G' with the frequency can be observed indicating that they did not behave as true viscoelastic solids. Indeed they contain some insoluble fibres embedded in the network and it is a composite gel rather an ideal gel. Some compression tests for gel made with extruded fibres have also be carried out in various conditions (Table 2). The Young moduli were in the range of the values obtained for commercial pectins. There was no marked influence of the severity of the treatment. The large difference between the Young moduli and the G' value confirmed that these gels are far from ideal networks. The breaking strength has also been measured (Table 2) and a more marked influence of the severity of the treatment was observed as lower breaking strengths were measured for fibres which have been extruded in severe conditions. The breaking strength for commercial pectins were much higher than those observed here for these gels.
436 Table 2 Young moduli and breaking strength (measured by an Instron apparatus) of pectin gel and of extruded citrus fibre at different concentrations ( 60% sucrose, pH3) concentration
SME (kWh/t)
commercial 0.4 pectins (dm 73%) 0.6
Young moduli (Pa)
Breaking strength (N)
1382 2439
6.2 6.9
extruded fibre
2.0 3.0
170
2165 2444
1.0 '2.2
extruded fibre
2.0 3.0
182
2121 2496
0.9 2.2
extruded fibre
2.0 3.0
220
1886 2239
0.7 1.5
extruded fibre
2.0 3.0
236
1617 2483
0.3 1.2
6. C O N C L U S I O N Extrusion-cooking increased very significantly the water-solubility of plant cell wall rich-materials. High amounts of pectins can be solubilised from sugarbeet pulp, citrus peels or apple pomace. The pectins from citrus have a very high dm, long side-chains. These pectins can be extracted by water from extruded fibres and they can gel as the other HM pectins. The extruded fibres can also gel in these conditions and must be considered as ingredients and not additives. Gelled products enriched in dietary fibres can be obtained. The extruded fibres can be used in jams, or other preparations requiring HM pectins as gelling agents.. REFERENCES
J.-F. Thibault J.-F., M. Lahaye. and F. Guillon, in: Dietary Fibre- a component of food- nutritional function in health and disease, (Eds. T.F. Schweizer et C.A. Edwards), Springer-Verlag (1992) 21. N.-G. Asp, I. Bjorck and M. Nyman, In R. Amado and T. Schweizer (Eds.),
437
10 11 12 13 14 16 15 17 18 19 20
Nahrungsfasern Dietary Fibres, Academic Press London (1986) 177. C.D. May, Carbohydr. Polymers, 12 (1990) 79. A.G.J Voragen A.G.J., J.-F. Thibault., M.A.V. Axelos, C.MG.C. Renard and W. Pilnik in: Food Polysaccharides and their applications, (Eds. A.M. Stephen), Marcel Dekker, (1995) 287. T. Sakai and Y. Ozaki, Lebensm. Wiss. Technol., 52 (1988) 1090. H.A. Schols, M.A. Posthumus and A.G.J. Voragen, Carbohydr. Res., 206 (1990) 117. G. Della Valle, A. Kozlowski, P. Colonna and J. Tayeb, Lebens. Wiss. Technol., 22 (1989) 279. J.-F. Thibault, G. Della Valle and M.C. Ralet, Produits riches en parois v6g6tales a fraction hydrosoluble accrue, leur obtention, leur utilisation et compositions les contenant.Brevet fran~ais n ~ 88-11601 (1988). M.C. Ralet and J.-F. Thibault in: La cuisson-extrusion, (Eds. P. Colonna et G. Della Valle), Tec Doc Lavoisier, Paris, (1994) 129. M.C. Ralet, J.-F. Thibault. and G. Della Valle,J. Cereal Sci., 11 (1990) 249. M.C. Ralet., G. Della Valle. and J.-F. Thibault, Carbohydr. Polymers, 20 (1993) 17. M.C. Ralet, L. Saulnier. and J.-F. Thibault, Carbohydr. Polymers, 20 (1993) 25. M.C. Ralet, J.-F. Thibault. and G. Della Valle, Lebensm. Wiss. Technol., 24 (1991) 107. M.C. Ralet and J.-F.Thibault, Carbohydr. Res., 260 (1994) 283. M.C. Ralet, M.A.V. Axelos. and J.-F. Thibault, Carbohydr. Res., 260 (1994) 171. J.-F. Thibault and C. Mercier, J. Food Biochem., 2 (1979) 379. C. Rolin, in R.L. Whistler and J.N. BeMiller (Eds.), Industrial Gums. Polysaccharides and their derivatives, Third edition, Academic Press (1993) 257. J. Hwang, Y.R. Pyun. and J.I. Kokini, Food Hydrocolloids, 7 (1993) 39. D. G. Oakenfull and A. Scott, J. Food Sci., 49 (1984) 1093. D.G. Oakenfull, in R.H. Walter (Ed.), The chemistry and Technology of Pectins, Academic Press (1991) 87.
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J. Visser and A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V.All rights reserved.
439
Pectin degradation in UF-membrane reactors with commercial pectinases C. Dinnellaa, A. Stagni a, G. Lanzarinib, F. Alfanic, M. Cantarellac and A. GallifuocoC
aDepartment of Industrial Chemistry and Materials, University of Bologna, 4 Viale Risorgimento, 40136 Bologna, Italy bDepartment of Biology, Defence and Biotechnology of Agroforestry, University of Basilicata, 85 Via Nazario Sauro, 85100 Potenza, Italy CDepartment of Chemistry, Chemical Engineering and Materials, University of L'Aquila, 67040 Monteluco di Roio (AQ), Italy
Abstract
Enzymatic degradation of pectin catalysed by Pectolyase Y23 RM (Sheishin Co., Japan) was studied in ultrafiltration membrane reactors. The mixtures of degradation products were continuously ultrafiltrated in four cells connected in series and equipped with membrane of decreasing molecular weight cut-off. The different fractions recovered. The ratio of enzyme to substrate loading affected product distribution and particularly the yield of oligomers from 10 to 15 monomeric units. TLC analysis allowed to identify the composition of the samples at low molecular weight (below 0.5 KDa and 1 KDa). The rate of pectin degradation in batch reactors was not significantly affected by the presence of oligomers between 1 and 6 subunits.
1. INTRODUCTION By-products of the agrofood industry the represent a severe problem for their high ecological impact and the high cost of their disposal. Consequently, the agrofood industry deserves continuously increasing interest in by-product utilisation in order to convert wastes in semi-manifactured products and to fulfil the day by day more severe prescriptions of laws. Moreover, valuable components are often present in considerable amount in the agrofood wastes, and their recovery and upgrading would help the overall process economy. Chemical processes have been suggested but most of them have a negative impact on the environment, while mild processes, such as those based on bioconversions, are strongly recommended.
440 The most studied system for agricultural waste recycling is their bioconversion into fuels [1 ], such as ethanol production from sugarcane molasses, citrus peels [2] and whey [3]. Considerable interest was also deserved to the transformation of wastes from agriculture and agrofood industry in proteic biomass for use as animal feed. These by-products are a potentially important source of energy for polygastric animals, but their nutritional value is poor because of the low content of protein and digestible dry substance. The growth of selected microorganisms on poor substrates considerably improves their nutritional characteristics since dry substance content, fibre digestibility and particularly the protein content are increased. Different fungi such as Fusarium [4,5], Penicillium roqueforti, Penicillium camemberti [6] and yeasts such as Candida utilis, Saccharomyces cerevisiae, Rhodotorula glutinis, Debaryomyces hansenii [7] were widely studied for this purpose. On the other hand, agricultural wastes can be alternatively used as substrates for edible biomass production. Cotton plant stalks [8], maize residues [9], olive milling wastewater [10] have been tested for cultivation of Pleurotus sp. fruiting body. Manufacturing by-products of the citrus industry, , such as exhausted citrus peel, represent more than 60-70% of the total material weight. They are generally eliminated by disposal in waste site or by burning. Only few Italian citrus industries have adopted by-product upgrading systems, such as drying processes for obtaining feed integrators. Besides, drying processes are not very interesting in industry because of the high energetic cost and the low dietetic value of final products. On the other hand, citrus fruit peel should be used as microorganism substrate for the production of high value substances such as macerating enzymes [11], ethylene [12] and piruvic acid [13]. Furthermore, citrus peel contains large amount of chemicals, particularly pectins, which posses a high commercial value since they are already and largely utilised as gelling, thickening and natural hazing agents in the food industry [14]. Consequently, considerable interest concerns both pectolytic enzyme action, reaction pattern of pectin degradation and product distribution. The optimisation of pectin biodegradation can largely contribute to the formulation of high quality food and beverages either via in situ processes of raw materials or via addition of pectin derivatives [ 15]. Pectin derivatives have also been recently used as starting materials for the synthesis of high value compounds. New surfactants have been obtained from ot-D galacturonic acid [16]. Pectin oligomers at defined polymerisation degree seem useful tools for studying physiological mechanism involved in plant host defence [ 17 ]. The overall reaction network of enzymatic pectin degradation is complex, since the initial random attack of the enzyme to the polymeric chain generates intermediates which in turn are substrates for further degradation steps. Some evidences are also reported in the literature that activity of pectolytic enzymes is significantly depressed in the presence of reaction products at low molecular weight which accumulate in batch reactors during substrate degradation [ 18]. This observation was also confirmed in preliminary tests carried out with Pectolyase Y23 and indicates that in industrial applications more efficient process schemes, allow continuous removal of low-molecular weight degradation products, should be designed. Ultrafiltration (UF) membrane reactors proved to be helpful tools in studies of enzymatic degradation of natural polymeric substrates, particularly in the presence of product inhibited kinetics [ 19, 20, 21 ]. Polygalacturonic acid (PA) degradation was previously studied in batch reactors and the oligomeric mixtures obtained at different operational conditions were characterised using three hollow fibre cells in series [22]. Various operational parameters may affect the overall reaction rate and product distribution; among them the molecular cut-off of the membrane reactor, the mean retention time, the substrate and enzyme weight ratio are the more significant.
441 Aim of this work was to optimise enzymatic depolymerization of pectins to valuable oligomers using commercial mixtures of pectolytic enzymes. Results of experiments in continuous and batch reactor configurations are presented which give some preliminary indications helpful to process optimisation. The use of continuous reactors equipped with ultrafiltration membranes, which assure removal of the reaction products, allows to identify possible operation policy for the improvement of the reaction yield.
2. MATERIALS AND METHODS
2.1. Enzymatic activity assay Pectolyase Y23 from Aspergillus japomcus (Sheishin Corporation, Japan) was used without further purification. The enzymatic composite was characterised for the endopectinlyase (PL), endopolygalacturonase (PG) and pectinesterase (PE) activities. Apple pectin (molecular weight 0.3-1.105, esterification degree: 70-75% Fluka Chemic GER) and polygalacturonic acid (molecular weight 3.104 Da, Serva Feinbiochemica GER) were used as substrates. Enzyme activity was monitored during incubation tests performed at 25 ~ The activity of PL was measured [23] from the formation of unsatured oligomers, during pectin degradation (5 g/L) in phosphate-citrate buffer 50 mM, pH 5.6 following the absorbance at 235 nm. The enzymatic activity was expressed as enzymatic units (EU), i.e. amount of enzymel that produces 1. ~tmol of product per minute. The extinction coefficient (5,550 M "1cm" ) was used for calculating product concentration. The activity of PG was measured [24] from the increase of reducing groups during polygalacturonic acid hydrolysis (10 g/L) in phosphate-citrate buffer 50 mM, pH 4.5. The colorimetric method with DNSA reagent (0.25 g of 3,5-dinitrosalicylic acid and 75 g of sodium potassium tartrate in 250 mL of 0.4 M NaOH) was used for product determination. The calibration curve was obtained using ct-D galacturonic acid as a standard. The PG activity was expressed as enzymatic units (EU), i.e. the amount of enzyme that produces 1 ~tmol of reducing group per minute. The activity of PE was measured [25] from the increase of carboxylic groups during the pectin deesterification (5 g/L) in phosphate-citrate buffer 50 mM, pH 4.7. Continuous titration of the reaction mixture with NaOH 0.01 M was employed for product determination. 2.2. Continuous experiments Pectin degradation was studied in a UF-membrane system. Five stirred cells (60 cm3 volume) were connected in series and thermostated at 25 ~ Each unit was equipped with a flat membrane (Amicon YM, USA or DDS, Denmark) at different molecular weight cut-off, from 20 to 0.5 KDa. Experiments were performed according to the following procedure. The first cell was filled with a buffered pectin solution (5 g/L), while the following ones contained pure buffer. The enzyn~3ewas loaded only in the first cell and the buffer solution was allowed to flow at roughly 12 cm h-1 across the system by means of a peristaltic pump (Gilson Minipuls 2, France). The solution outflowing from the first cell, containing degradation products at molecular weight less than 20 KDa, was continuously fed to the second cell, equipped with a 10 KDa cut-off membrane. The other cells were fed in series with the effluent of the previous cell. In each cell a build-up of oligomers rejected by the particular membrane occurred. The
442
REACTOR t 20KDa II
10KDa J.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
II
a
.
II
Q
time
$
L,o.t lo:::o,t FRACTIOO NNt COLLECTI
KDa
I REACTION & ULTRAFILTRATION
I FRACTION ANALYSIS
FRACTION RECOVERY
> KINETIC EXPERIMENTS & PRODUCT DISTRIBUTION
Figure 1. Flow-sheet of experimental procedure for continuous pectin degradation. whole system allowed to continuously monitor product distribution obtained in the first cell, which behaves as a well mixed and isothermal reactor. The final outlet of the cell sequence was the permeate from the last one which was recovered with a fraction collector (Gilson Redirac, France). Volumes and optical densities at 235 nm of the collected fractions, mainly rich in chemicals from one to three monomeric units, were measured. At the end of 24 hours of continuous process the system was shut down. The knowledge of flowed buffer volumes and of the optical densities inside and downstream each ultrafiltration stage allowed to estimate product distribution (see appendix for mass-balance equations and the calculation procedure). The content of each cell was recovered and freeze-dried in order to be stored and used for subsequent kinetic experiments. A schematic flow-sheet of the whole procedure is illustrated in figure 1.
443 2.3. Batch experiments Pectolytic activity was also studied in batch reactors, following the reaction progress in thermostated quartz cuvettes. The reaction medium (3 cm3) was prepared with 1.5 g/L pectin in the standard buffer and 0.063 mg of enzyme. The absorbance of the reaction mixture against the substrate blank was continuously recorded at the spectrophotometer (Perkin Elmer Lambda 2, USA). Typical reaction time was 15 minutes, but initial reaction rates were estimated considering only the absorbances recorded during the first 200 seconds, range of totally linear response.
2.4. TLC analysis TLC analysis of oligomers was performed on Silica gel 60 aluminium sheet (Merk) using buthanol and formic acid (1:1.5) as solvent [26]. The dye reagent was prepared by dissolving 9.5 mg of 1,3 dihydroxynaphtalin (Aldrich) in 5 mL of ethanol/H2SO4 mixture (1:0.05, V/V). The migration front (Rf) of each spot was measured and expressed as Rm, were: Rm = log (1/Rf- 1) 2.5. Separation of r 1-4 Galacturonide oligomers The oligomer separation procedure was performed according to the protocol described elsewhere [27]. Polygalacturonic acid (0. lg) was dissolved in 10 mL of citric-citrate buffer pH 4.5. Pectolyase Y23 solution (0.013 mg/mL final concentration) was added to the substrate solution and let to react for 12 hours under stirring at 25~ A 43 mL (55 cm x 1 cm ) column of DEAE-Sephadex A25 was equilibrated with starting buffer (100 mM KC1, 10 mM imidazole, pH 7.0). The pH mixture of oligomers generated by digestion was adjusted to pH 7.0 and to an ionic strength slightly less than that of the starting buffer by dilution with water. The medium was then loaded to the column. After washing the column with the starting buffer, the oligomers were eluted with a concave salt gradient: 100 mM KCI, 10 mM imidazole (pH 7.0) to 300 mM KCI, 10 mM imidazole (pH 7.0). The fractions were collected at a flow rate of 0.42 mL/min. The reducing sugar fraction content was measured using the DNSA reagent. Peaks from the anion exchange column were pooled individually, freeze dried and desalted on a Sephadex G-10 column.
3. RESULTS AND DISCUSSION
In previous papers it was shown that the enzymatic pool of Pectolyase Y23 possesses high catalytic efficiency either as free or immobilized form in solution of pectins [28, 29] and of fresh vegetable tissues [30]. According to Baldwin and Pressey [31 ] the following enzymatic activities were detected in the preparation: PL, PG and PE. The amount of the different enzyme detected per mg of Pectolyase Y23 and the main enzyme characteristic are quoted in Table 1 Since in continuous degradation processes it is expected to reach a molecular weight distribution of the products, which is optimal for their further use, the investigation was devoted to test the effect of a key parameter such as the enzyme to substrate ratio (E/S). For a fixed mean retention time in the UF-membrane reactor, the following behaviour can be
444 Table 1 Main characteristics of the e ~ e s Enzyme UE/mg
present in Pectolyase Y23 optimum pH Isoelectric point
molecular weight Da
PL
1
6.0
7.7
32000
PG
27
4.5
-
35500
PE
7.7
4.7
foreseen:the lower the E/S, the higher the mean molecular weight of products. In fact, under enzyme scarcity condition, the biocatalyst can meanly interact with undegraded molecules, the number of catalytic events would be limited and mainly high molecular weight species should be present in the reaction medium. On the contrary, when the enzyme amount in the reaction medium increases, the initial polymer breakdown is faster and more medium and low molecular weight species are available for repeated catalytic events, thus increasing the mean number of cleavages. Figure 2 illustrates results obtained with two different E/S: 0.006 and 0.0006. The percentage of total products is reported v e r s u s the molecular cut-off of each cell membrane. By inspection of figure 2, two interesting features arise. The mean molecular weight resulted 7.8 KDa for the lower E/S and 7.0 KDa for the higher. The E/S ratio seems to affect more markedly the product distribution in the range from 3 to 10 KDa as indicated by the product build-up in the cells equipped with the membranes at these cut-offs. This is Important since the fraction ranging from 1 to 3 KDa contains the higher value added chemicals (oligomers between 10 and 15 subunits). Moreover, the total extent of degradation (calculated from the volume of each solution recovered from the system and the corresponding optical density) is almost independent of the E/S. This finding indicates that productivity optimisation is to be carefully studied in order to design process conditions which maximises the yield per unit mass of enzyme. Fractions rejected by 1.0 KDa (C>1) and permeated through 0.5 KDa (C0.5) membranes were also subjected to TLC analysis. In Figure 3 are reported the R m values relevant to the various spots detected in the two samples as a function of an arbitrary polymerisation degree (DP). The good linear correlation between these parameters allows to hypothesise a difference of one monomer units between the subsequent spots [32]. Consequently, C0. 5 would correspond to the monomer, C 1 to an homologous series from the monomer to the hexamer. Since product inhibition due to the build-up of some oligomers into the reactor could seriously limit the process rate, the identification of the molecular weight of species candidate to act as inhibitors was considered important in order to design process conditions which could minimise the extent of inhibition. Figure 4 reports results obtained in the batch reactor configuration. The reference line fitted the open circle symbols, which depict reaction time course when oligomers are not initially added into the reaction medium. The other symbols represent experiments in which various amounts of the fractions C>0.5 and C> 1 are added. Significant decreases in the derivative are not observable, therefore the conclusion can be drawn that these species do not inhibit reaction rate, in the investigated range of
445
100
-
80 I ~ l
E / S = 0 . 0 0 6 rag/rag
Iiiiii!!iiiiiiiiT!~i!iiiilE/S = 0.0006
rag/rag
o
e~0
60
o r o
40
20
0.5 1
3
10
20
Cut-off, KDa Figure 2. Molecular weight distribution of degradation products. 1,5
corr. = 0 . 9 9 5 8 I
0,5
J [] Standards
J 0 !
I
J
,
1
Zx C0.5 0C1 t
2
,
I
,
3
I
4 D.P.
Figure 3. Migration function R m v e r s u s polymerisation degree.
446 0.25 -
0.20
o
<>
0.15
0 ",~
0
0.10 0.9 mg/mL C>0.5 A 1.6mg/mLC>l I'-'] 0.2 mg/mL C>I O No fraction added
0.05
0.00 0
I
I
I
100
200
300
t, S Figure 4. Batch hydrolysis of pectin in the presence of oligomers. concentrations. Finally, these oligomers apparently are not substrates for the enzyme since the slopes of the corresponding straight lines (product formation rate) are not different from the reference one. Preliminary experiments of or-l,4 oligogalacturonide separation by anion exchange chromatography have been done aiming to prepare pure fractions of oligomers at a defined polymerisation degree. Polygalacturonic acid was enzymatically degraded for 12 hours at 25~ The reaction mixture was then separated on a DEAE Sephadex column. The pertinent chromatogram is reported in Figure 5. The peak contents were pooled individually, freeze dried, desalted and then analysed by TLC. This analysis showed that peak 1 corresponds to the digalacturonic acid while peak 2 to the trigalacturonic acid, which also resulted the main components in the sample loaded to the column.
4. CONCLUSIONS
Enzymatic degradation of pectin can be satisfactory performed in UF-membrane reactors which have been proved to be helpful tool for laboratory scale investigations. Reaction products can be continuously recovered in a sequence of filtration stages. The obtained product distribution depends on the enzyme to substrate ratio, which affects particularly the
447 1,2 _
peak
1
_
1_
_
0,8
_
,r, 0 , 6 t ' " ,
peak
2
-
0,4-
_
0,2_
-
0 -' ,"W'm,'-',~" , " ~ 0 50
-
100
-
~
150 200 fraction number
~
"
,
.....
250
.,=~=Mea,
,
300
Figure 5. Elution profile of PA degradation mixture. yield of oligomers between 1 and 3 KDa. Evidences were found that products ranging from 3 to 5 monomer subunits do not depress the initial rate of pectin degradation. Research is in progress aiming to clarify the influence of other operational parameters such as residence time and molecular weight cut-off the membrane reactor on conversion yield and product distribution.
5. APPENDIX: MASS-BALANCE EQUATIONS The vessels were indexed by the subscript "j" (j = 0 refers to the reactor and j from 1 to 4 to the UF cells) and oligomers were lumped in two categories: "P" (Permeated) and "R" (Rejected). Let label "in" species entering a cell and "out" those leaving it. Instantaneous massbalance in the stream leaving a cell and feeding the following one is: Pj-l,out = Pj,in + Rj,in
(1)
The transient mass balances in the four filtration stages for the rejected and the permeated chemicals can be respectively written: dR./dt = R.. /x j j,m
(2)
dPj,out/dt = Pj,in/X "5,out/1:
(3)
448 where ~ is the mean retention time, i.e. the ratio of cell volume to volumetric flow rate. The corresponding equations for the first stage (enzymatic reactor equipped with the membrane at the highest molecular weight cut-off) are: dR o/dt = rR
(4)
rp = P0/x
(5)
+
dP0/dt
where r.. and r_ represent the net rates of production of oligomers respectively at molecular weight l~gher and e lower than 20 KDa. The system of differential mass balance equations (2)- (5) should be solved, provided that the "in" time functions were known, with the initial conditions: t = 0, P. =R. = 0 [j = 1, ..4] J J
(6)
Even if more than five time constants are elapsed when the system is shut down, the transient should not be vanished in each vessel, because at any time, the "0" cell could still furnish inlet species to the following ones due to the terminal, low rate reaction. Therefore, the constitutive rate equations are need for the solution of the above model system. Nevertheless, the complete knowledge of permeated and rejected product concentration is not necessary. In order to quantify product distribution in the whole system, it is sufficient to make an integral analysis and measure all "P" values and the overall concentration inside the cells at the end of a fixed process time (24h). In fact, if one considers the working conditions for the last stage, on the basis the perfect mixing hypothesis, P, .(24h) represents an instantaneous concentration permeate species at molecular weight le~'~t~en 0.5 KDa equal to the one still present in the cell. Subtraction of this value from the concentration detected inside the cell gives the amount of rejected species. This procedure can be extended backwards to the other stages.
Acknowledgement Research supported by National Research Council of Italy, Special Project RAISA, subproject 4, Paper N~
6. REFERENCES
1. 2. 3. 4.
W.F. Raymond and P. Larvor (eds.), Alternative uses for agricultural surpluses, Elsevier applied Science, London, 1986. M.K. Dowd, S.L. Johansen, L. Cantarella and P.Y. Reilly, J. Agric. Food Chem., 42 (1994) 283. J.H. Janssens, A. Bernard and R.B. Bailey, Biotech. Bioeng., 26 (1984) 1. M. Moresi, F. Clementi, J. Rossi, R. Medici and G.L. Vinti, Appl. Microbiol. Biotech., 27 (1987) 37.
449 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19 20. 21 22. 23 24. 25. 26. 27. 28. 29. 30. 31. 32.
M. Moo-Young, H.W. Blanch, S. Drew, D.I.C. Wang (eds.), Comprehensive Biotechnology, vol.3, The practice of biotechnology: current commodity products, Pergamon Press, Oxford (1985). A. Caridi, Abstract Book Convegno congiunto ABCD, AGI, SIBBM, SIMGBM, 1995 Montesilvano Lido (PE), Italy, 319. N. Nishio and S. Nagai, European Appl. Microbioi. Biotechnol., 11 (1981) 156. Y. Hadar, Z. Keren and B. Gorodecki, J. Biotech., 30 (1993) 133. A. Ginterov~ and A. MaxianovL Folia Microbiol., 20 (1975) 1974. E. Sanjust, R. Pompei, A. Rescigno, A. Rinaldi and M. Ballero, Appl. Biochem. Biotech., 31 (1991) 223. N. Nishio and S. Nagai, European Appl. Microbiol. Biotechnol., 6 (1979) 371. E. Chalutz, E. Kapulnik and I. Che, European Appl. Microbiol. Biotechnol., 18 (1983) 293. M. Moriguchi, Agric. Biol. Chem. 46-4 (1982) 955. G.G. Birch, N. Blakebrough, K.J. Parker (eds.), Enzyme and Food Processing, Applied Science Publishers, London (1980). A. Manganelli, Emmegi Agro-Industriale srl, private communication (1995). J.N. Bertho and R. De Baynast, Abstract Book of 7th European Congress of Biotechnology, Nice, France, 2 (1995) 42. A. Collmer, Ann. Rev. Phytopatol., 24 (1986) 283. S. Tipson and D. Horton (eds.), Advances in Carbohydrate Chemistry and Biochemistry, Academic Press, New York, 33 (1976). F. Alfani, M. Cantarella, V. Scardi, J. Membr. Sci., 16 (1983) 407 F. Alfani, L. Cantarella, A. GaUifuoco and M. Cantarella, J. Membr. Sci., 52 (1990) 339. F. Alfani, A. Gallifuoco and M. Cantarella, Chem. Eng. J., 43 (1990) B43 S. Todisco, V. Calabr6 and G. Iorio, J. Molecular Catalysis, 92 (1994) 333 P. Albersheim, P.U. Killias, Arch. Biochem. Biophys., 97 (1962) 191. M.F. Chaplin and J.F. Kennedy (eds.) Carbohydrate analysis: a practical approach, I.R.L. Press, Oxford (1986). Z.I. Kertesz, J. Biological Chem. 121 (1937), 589 E. Sthal (ed.), Thin layer chromatography, Springer Verlag Berlin, Heidelberg New York (1969). D.F. Jin and C.A. West, Plant Physiol., 74 (1984) 989. C. Dinnella, G. Lanzarini, A. Stagni and C. Palleschi, J. Chem. Tech. Biotech., 59 (1994) 237. C. Dinnella, G. Lanzarini and P. Ercolessi, Process Biochem., 30-2 (1995) 151. C. Dinnella and G. Lanzarini, Int. J. Food Science and Technol., 30 (1995) 391. E.A. Baldwin and R. Pressey, Plant Physiol., 90 (1989) 191. E. Le.derer (ed.), Chromatographic en Chimie Organique and Biologique, vol.2, Masson and C~eEditeurs, Paris (1960).
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APPLICATIONS.
B) APPLICATION OF PECTINASES IN BEVERAGE, FOOD, FEED AND NOVEL TECHNOLOGIES
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J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
453
A p p l i c a t i o n of P e c t i n a s e s in B e v e r a g e s Catherine Grassin and Pierre Fauquembergue Gist-Brocades, Beverages Ingredients Group, 15 rue des Comtesses, P.O.Box 239, 59472 Seclin cedex, France
Abstract Pectinases are necessary processing aids in the production of clear and con(:entrated fruit juices. Improving pressing and clarification, they are biochemical tools of first importance. In combination with hemicellulases and cellulases, pectinases can lead to fruits liquefaction. The improvement of our knowledge on fruits cell wall has allowed us to describe new enzyme activities to specifically degrade these substrates to optimize the performance of industrial equipments and to allow to produce juices and concentrates with a higher quality in terms of oxidization, aromas level and stability.
1. ENZYMES IN FRUIT JUICE INDUSTRY Among the food industries, the fiuit juice industry has to deal with the most delicate problems. Its role consists in processing raw material whose composition varies widely, to process fruits as much as possible at the lowest cost while maintaining or even improving the organoleptic quality and stability of the finished product. This ambitious objective has been made possible for the past years through the use of enzymes at different stages of the production of juice. Because the pectin structure and composition have been extensively described by specialists during this symposium, we will directly show you the role of pectinases in fruits processing by taken examples of different technologies. Each fruit has specific quantities and ratio of pectin, hemicelluloses and cellulose. These polysaccharides are important concerning enzymes activities required to produce juices and concentrates. Moreover, even if molecular weight and methylation degree of the pectin are specific for each fruit, during the fruit maturation, endogenous pectinases depolymerases and esterase are changing the pectin characteristics. This broad variability of raw material makes difficult the standardisation of fruits processing. Commercial enzymes are tools now used all over the world and widespread to every industry transforming fruits since 1930s. Whatever would be the finished product, the producer can take advantage of a wide range enzymatic activities contained in the commercial preparations. Since a few years, new activities have been discovered in relation with fruit cell wall architecture, like new pectinases [l] (i.e. rhamnogalacturonase for the apple) new glycosidases [2](i.e. apiosidase for the grape). The improvement of the knowledge of fruit composition, cell wall chemistry, enzymology of endogenous and exogenous enzymes allow to supply the juice producers with preparations more and more purified and specific, blended in optimal combinations.
454 Enzymes most frequently proposed to fruit juices producers are pectinases coming from Aspergillus. Pectinases are exocellular enzymes and are the main activities produced among numerous side activities type hemicellulases, glycosidases. The Table 1 gives the spectrum of enzymatic activities contained in three commercial preparations A, B and C.
Table 1 Comparison between commercial enzymatic preparations Enzyme activity U/g PG PL PE exo B ARA endoGlucanase exoGlucanase beta-Glucosidase
A
B
C
400 115,000 1,000 10 6,000 3 40
800 30,000 300 10 4,500 4 2
500 115,000 95 28 11,000 25 400
It appears from these results than tile enzyme spectrum is very different depending of the starting strain of Aspergillus and ratio are very important since enzymes act in synergy. It is necessary to choose the right enzymes preparation in relation with the fruit composition and with the final product people want to obtain. In this presentation, we will take examples of different fi-uit juice processes and will try to relate enzyme activities, the role they play and tile transformation they occur in terms of finished products. These examples will concern apple juice, french cider, pineapple and wine.
2. APPLE JUICE PRODUCTION About 40 millions tons of apples are gathered per year and about 10% are processed mostly to produce clear concentrate but a little part as cloudy juice. The aim of pectinases application is to increase the plants productivity by processing apples quantity as high as possible along the season with high yields (see Table 2).
455 Table 2 Comparison of juice yields without or with enzymes maceration with Rapidase Press 50g/ton - ih - 20~ after one pressing
Fresh apples Stored apples
Juice yield %
Horizontal presse
Belt presse
no enzyme with pectinases no enzyme with pectinases
83 86 72 84
72 75 60 70
2.1. Classical process with maceration and depectinisation In the classical process, enzymes are used at two stages of" processing: maceration of the pulp and depectinisation of the juice. Because endogenous enzymes have too low activities and no quick technological effect, the application of exogenous enzymes allows to regularize the production and allows a better productivity of the plant. Along the maturation, the unsoluble protopectin is slowly transformed into soluble pectin through the endogenous pectinases of the apple, the molecular weight and the degree of methylation of the pectin are decreasing. Apples become very difficult to press since the soluble pectin content increases. The producer solves this problem by an increase of enzymes dosage. Pectinases are essential tools to produce clear concentrates (see Table 3).
456 Table 3 Technological role of enzymes Enzyme Pectinlyase endo exo B Arabanases Rhamnogalacturonase P ect in acetylest erase Pectinmethylesterase Polygalacturonases Pectinlyase endo exoB Arabanases Arabinogalactanases Rhamnogalacturonase P ecti nacetyl est erase id pressing + endoGlucanases exoGlucanases cellobiohydrolase 13-Glucosidase Xylanase .p ecti nm ethyl est erase endoPolygalacturonase Galactomannanase Arabinogalactanases Polygalacturonases Polygalacturonases P ecti nmet hyl est erase Pectinlyase Arabinogalactanases exo B Arabinosidase Apiosidase Glucosidase Rhamnosidase
Importance
Role
Application
Fruits pressing
Apple juice extraction
+++ + + + +++ +++ ++ + +
Depectinisation of juices
+
Clear concentratable juices Apple, Pear, Grape
+
++ +
Cell wall destruction Fruits liquefaction
Apple Pear
Ca pectate formation Pulp maceration
French Cider Purees Clear Pineapple juice
+ +
, ,
+++ +++ +++ ++ + +++ + + + +++ +++ +++ +++
Gum hydrolysis
Must depectinisation
Grape must clarification
Aromas release
Wine quality
For apple maceration, the main activity is the endopectinlyase (PL) which decreases the viscosity due to the colloidal pectin by decreasing the molecular weight of this substrate methylated at about 90%. In association to this PL, exo B and endoarabanase (ARAs), pectinacetylesterase (PAE), rhamnogalacturonase (RG) and other hemicellulases allow to hydrolyse side chains of the pectin, decrease the steric hindrance and make the access of pectinases to the rhamnogalacturonic backbone more easy.
457 Added to the mash, these enzyme activities decrease its viscosity and improve its pressability. The free run juice volume is larger. The yield and then the capacity of the press are increased. Pectinmethylesterase (PME) and polygalacturonases (PGs) are key activities for the juice clarification. PME produces pectic acid the substrate of the PG which starts to work below a methylation degree of 60%. The ratio PE/PG is important also because a too high PE activity would make a too fast demethylation inducing the formation of unsoluble calcium pectate before hydrolysis of the pectic acid by PGs. The following curve shows the effect of the different pectinases on the decrease of viscosity of apple juice along the time. In addition, ARAs and other pectinases make partially hydrolysed hairy regions and side chains of the pectin to settle down. In the juice, pectinases hydrolyze the residual pectin decreasing the viscosity, leading to the clarification of the juice through the neutralisation of electrostatic charges of pectin, proteins and phenolic compounds. The sedimentation necessary for the clarification of an apple juice only occurs after enzymatic degradation of the colloidal pectin and the starch. The cloud is composed of 30-40% proteins with positive charges at the pH of the juice (3.5-4.0). These proteins are associated to polyphenolics surrounded by the partially hydrolysed pectin which has negative charges. The formation of these macromolecules induce the floculation of the cloud, leading to the clarification of the juice at an optimal pH of 3.5. The clarification is perfected by fining agents and filtrations or by ultra-filtration. In conclusion, the use of exogenous pectinases allows to produce a clear and stable apple juice easily concentrated. 2.2 Total liquefaction process The enzymatic liquefaction of apple consists in the hydrolysis of cell wall polymers by exogenous enzymes into soluble components, mainly acid and neutral sugars, thus increasing the extraction level and the yield. The aim of enzymes application is to decrease within three hours the viscosity of the pulp to be able to use a decanter to separate tlle juice from the solids. Total liquefaction occurs during two to three hours at 50~ under stirring. Enzymes work close to their optimal temperature. The liquefied pulp which contains 20% solids (in volume) is centrifuged with a decanter giving a juice 1. The pomace is added with about its weight of water and pumped to a second decanter giving a juice 2. Both juices are mixed and contain less than 2 g per liter solids. After pasteurization and aromas recovery, active carbon and gelatin are sometimes added to decolourize the juice and are then removed by filtration. Tile juice is then ultra-filtered and concentrated. If we compare liquefaction to maceration, more activities are needed to liquefy the cell wall. Since 1991, new pectinases activities such as rhamnogalacturonase, pectin acetylesterase and xyloglucanases complex have been found to be important in the apple liquefaction by Henck Schols, Jean-Paul Vincken and Voragen [3]. The cellulose-xyloglucan complex accounts approximatively 57% of the apple cell-wall matrix. In a liquefaction process, an efficient enzymic degradation of this complex is crucial to increase the sugars extraction, to decrease the viscosity of the pulp then to be able to ultra-filtrate the juice without second depectinisation, at last to have negative alcohol tests required by some concentrate customers. However, if theoritically, the combination of pectinases to cellobiohydrolases plus endoglucanases should release more than 80% of all polysaccharides from the cell walls (according to Voragen and al. [4]), in industrial conditions, we arrive ahnost at this level of degradation but only for the pectin. Commercial enzymes preparations contain pectinases, hemicellulases and cellulases.
458 Enzymes like Rapidase LIQ + contain enough pectolytic activities to depectinize the juice during the liquefaction time, giving an average yield of 93-95% according to this type of process. The juice has a good quality and its composition fits with RSK values. In conclusion, the technology of total liquefaction of apple allows to work with a continuous process with less labour and faster than with a classical one, to get a high and constant yield during the whole processing season at a very high level (93- 95%), to get a pulp with a low content of solids (about 20% in volume) which can be centrifuged instead of pressed (lower investment in equipment), to decrease the quantity of waste pomace, to decrease the production costs. Liquefaction technology allows to process different fruits with the same process, at last to liquefy fruits for which no equipment had been developed to extract the juice or for which the use of pectinases did not allow to get juice such as tropical fruits.
2.3. Pomace liquefaction process This new technology is interesting because it allows to produce different juices and concentrate with classical equipment with very high yields. Pomace liquefaction is the application of liquefying enzymes on the pomace a~er the extraction of premiurn juice. Hot water is added to the pomace, and the second juice is extracted with a decanter after enzymatic treatment. The first juice is extracted without enzyme by pressing with an average yield of 50-75%. It can be processed as cloudy or clear juice and concentrate. The first extraction yield can be adjusted to get a pomace with a certain quantity of residual juice to avoid to add too much water. The pomace with water and enzymes is stirred during about three hours at 50~ The liquefied pomace is centrifuged with decanters. The juice obtained is acidic and the brix degree increases from about 6.0 to 8.5 mainly due to the degradation of pectin and cellulose. This juice is added to the first pressing juice to be depectinized in the case of clear juice production (this second acidic juice can be added to increase the acidity of juices with low acidity). At last, the quantity of waste pomace is reduced in this process. The final yield can reach 100 to 102%. Added to the pomace after a first juice extraction, enzymes preparations such as Pomaliq contain pectinases, hemicellulases and higher cellulases activities than for total liquefaction. After a first pressing and elimination of the first juice, enzymes allow to decrease the viscosity of the pomace and to extract a maximal quantity of sugars from a substrate which is still pressable. In conclusion, this process allows the producer to diversify the types of products and to give a better value to his raw material. The producer can perform pomace liquefaction with his existing presses. With a low investment in equipment (decanter) the efficiency of the plant can be greatly improved. This process is very flexible regarding the diversity of products that can be made: single strength juices with high organoleptic quality from the first extraction (clear or cloudy juices) and clear concentrates from the second extraction. The benefits brought by this process to the juice producer is the highest in comparison with other processes, if we calculate theoritically the additional weight of concentrate per ton of apples he can produce and sell, out of the cost of enzymes (see Table 4).
459 Table 4 Comparison of technologies to produce apple juice
Technologie
Enzymes
Dose
Juice quantity
(kg) Pressing Depectinization Maceration Depectinization Liquefaction
no enzyme Rapidase C80 Rapidase Press Rapidase C80 Rapidase Liq +
0 3 g/hi 50 g/T 3 g/hi 125 g/T
Liquefaction Depectinization
Pomaliq Rapidase C80
450 g/T 2 g/hl
Juice brix
(~
70~ concentrate
Increase
(kg)
750
12.5
134.0 kg
reference
850
12.5
151.8 kg
+17.8 kg
860 750 455
13.5 12.5 7.0
165.9 kg
+31.9 kg
179.5 kg
+45.5 kg
Even if these liquefaction processes are still not accepted worlwide (for instance in Europe), they should grow within the next few years. We do really believe that they are the processes of the future, and especially the process of pressing / pomace liquefaction because it is an easy process, it allows the production of quality juice combined with high yields within a great flexibility. Such high yields, low production cost and flexibility to process different fruits make that fruit juice producers are more and more choosing tlle pomace liquefaction. 2. 4. Production of traditional fl'ench cider The french cider is produced with apples from cider varieties type bitter-sweet, sour. Traditionally, the clarification of the cider was carried out by defecation: the natural demethylation of the pectin by the apple pectinmethylesterase was giving pectic acid which became insoluble after linkage with the calcium in the juice. However, the clarification was uncertain due to variable endogenous pectinmethylesterase (PME) activity of apples and variable calcium content. The process was very slow. After fermentations, tile cider was aromatic but with a low acidity and sometimes contaminations. Nowadays, this process is completely under control. It has been developped by INRA Le Rheu (Mr Drilleau) and Gist-Brocades. Rapidase CPE a purified pectinmethylesterase is added with calcium chloride allows to clarify in a shorter time and to produce french ciders of high constant quality [5]. After a few hours, there is a formation of calcium pectate after the pectin demethylation. The bubbles at the beginning of the fermentation induce a retraction of the gel which goes slowly up at the surface of the tank. This coagulum entraps micro-organisms, phenolics and main part of the natrium content. After discarding, fermentations take place at low temperature to get maximum aromas (a few weeks until 3 months). Then the cider is centrifuged, clarified by fining and filtrated, carbonated or with a prise de mousse in bottles. About 30% of french producers are using this process.
460 With the use of exogenous pectinmethylesterase, the methanol content is lower because the demethylation of the pectin is not complete, part of the methylated pectin remaining in the coagulum and because the contact time between the PME and the pectin is shorter than in the past with the natural enzyme.
3. CLEAR PINEAPPLE JUICE PROCESS The pineapple fruit is mainly processed for canning as slices or cubes. After cutting, the residual pulp is removed from the peel for cloudy juice production. Then, by-products are used to produce clear juice for slices cover or as clear concentrate. In the process of clear juice, by-products are crushed and pressed. The juice is pasteurized, cooled down and depectinized with enzymes at 50~ before ultra-filtration and concentration. This pineapple juice contains a small amount of pectin but a high hemicelluloses content type galactomannans, arabinogalactans and galactoglucomannans when insoluble parts are rich in arabinoxylans. The presence of a natural gum in pineapple juice was found. This is a neutral polysaccharide containing 70% sugars which are predominantly galactomannans (2.25 mannose: 1 galactose). Because of this gum, the ultra-filtration flux rate quickly drops down and becomes the bottle neck of the process. Gist-Brocades has developped Rapidase Pineapple which contains high galactomannanases and xylanases activities with low pectinases activities. Its application decreases the juice's viscosity, the ultra-filtration flux rate and allows to process a clear concentrate.
4. WINEMAKING The winemaker is always facing problems due to the weakness of grapes which composition is variable and different for each vintage. He tries to prevent oxidation and to work with soft conditions to preserve grapes components important for the wine's equilibrium. The sanitary state of the harvest is of first importance. Grapes composition depends on the variety, terroir, viticulture and climatic conditions. The main objective for the winemaker is to keep and valorize grape components like aromas which will determine the quality of the wine.
4.1. Enzymes in must clarification Grape's cell wall and pectin structure have been extensively described by Saulnier, Brillouet and Pellerin [7,8]. The pectin content depends on the grape variety, maturity and the technology used after the harvest. Pectin as protector colloid makes the settling of particles and the clarification very slow, related to endogenous pectinases activities of tile grape which are weak and variable. Exogenous enzymes are now widely used (i.e. 20 millions hectoliters out of 60 are treated with enzymes in France). Enzymes are used to obtain a better initial extraction of the must. They allow to increase the free run juice volume by about +20%. Pectinases are the enzymes with the most important technological effects. They enforce endogenous PME and PG of the grape, allow to extract more juice by decrease of its viscosity, to improve the clarification of the must and the wine filtration. Because the methylation percentage of the grape's pectin is lower than for the apple, polygalacturonases activities (exo and endo types) play the main role (when it is the PL for apple) and are responsible of the decrease of the must's viscosity
461 The ratio PG/PL in enzymatic preparations for enology must be higher for the grape juice than the classical preparations sold to depectinize the apple juice. In association with hemicellulases, pectinases speed up the natural process of winemaking, make the fullest use of facilities and equipment and improve the quality of the wine. The turbidity can be adjusted to 100-150 NTU to allow a good alcoholic fermentation and elimination of particles which could induce off-flavors. This shorter processing time and better clarification reduce also the oxidation of the must since the contact of the juice with grape tyrosinase bound to particles is limited. The wine well depectinised is easily filterated and has an intensive clean and fruity flavor. 4.2. Enhancement of Wine Aromas with Enzymes Aromas coming from tile grape, tile yeast or aging are essential for the final equilibrium of the wine. The wine typicity is partially due to grape aromas. This is why the winemaker tries to enhance their level, to improve the fruity and fresh character of the wine. Grape aromas a r e present in the skin and bound to sugars. Aromas bound to glycosides called "precursors of aromas" is the major part. Glycosidic precursors in tile grape berry have been described by Cordonnier, Bayonove and Gunata [8,9]. These precursors are odourless. Their content goes from 6.5 to 28 mg per liter must. However, tile ratio between different types of precursors varies greatly depending of grape varieties. Moreover, this potential of aromas under glycosidic form is very stable during the alcoholic fermentation and remains soluble and stable in wines. Other precursors of volatile compounds exist such as linalol oxides, terpenic diols and triols, linear or cyclic alcohols, norisoprenoids in C13 and acid or volatile phenols. It is possible to enhance the wine in aromatic cornpounds. Aromas release is sequential. The sequential enzymatic hydrolysis of these precursors releases free terpenols very odorant. There are mainly linalol, nerol and geraniol. In the grape, glycosides are numerous and abundant. The osidic part is formed by rutinose 6-0-ot-L-rhamnopyranosyl-13-D-glucopyranose for rutinosides, by 6-0- cz-L-arabinosyl-13-D-glucopyranose for arabinosylglucosides and by 6-0-[3-Dapiofuranosyl-13-D-glucopyranose tbr apiosylglucosides. In a first step, one ot-L-rhamnosidase, one c~-L-arabinosidase or one 13-D-apiosidase must cut the terminal sugar. In a second step, one [3D-glucosidase must release the aromatic aglycon. This can be achieved by exogenous commercial enzymes. The preparation AR2000 is tile only commercial preparation containing pectinases and these four glycosidases types. Present in the right proportions to release the major part of terpenols, these enzymes come fi-om Aspergillus niger. Wines which have been added with AR 2000 are always different from the control, contain more terpenols, terpenic diols, norisoprenoids in C13. The addition of exogenous glycosidases enhance greatly aromas in wines in relation with the aromatic potential of grape varieties. Tastings confirm that the improvement is obvious for red and for white wines. Wines are always judged more fruity and more intense. The future will be to manage tile extraction of precusors of aromas from the grape during the skin contact steps of white and red winemakings.
5. CONCLUSION Exogenous enzymes are used to produce fruit juices more easily during different stages of the process i.e. maceration, liquefaction or juice depectinisation. These biochemical tools induce specific degradations that the processor can integrate into his process line to manage and valorize the fruits transformation into juice.
462 Enzymes allow to improve the quality of the final product and the productivity in the same time. Associated to new technologies, industrial enzymes allow to give value to raw material in Food industry and to reduce the wastes quantity. They are specific tools as important as the equipment. Thanks to specialists to help us to better understand fruits composition and to associate biochemical structures to acurate enzyme activities able to answer to technical needs. Gist-Brocades as enzymes producer is associated to european fruit juice producers, INRA and Wageningen University Professor Voragen, in a Eureka project dealing with "Fruits liquefaction with specific enzymes". With the description of new enzyme activities, we will be able to create very soon new fruits derivates and new types &beverages.
6. R E F E R E N C E S
1 H. Schols, C. Geraeds, M. Searle Van Leeuwen, F. Kormelink and A. Voragen, Carbohydr. Res., 206 (1990) 105. 2 Z. Gunata, C. Bayonove, C. Tapiero and R. Cordonnier, J. Food Chem., 38 (1990) 1232. 3 J.P. Vincken, G. Beldman and A. Voragen, Plant Physiol., 104 (1994) 99. 4 A. Voragen, R. Heutink and W. Pilnik, J. Applied Biochem., (1980) 452. 5 J.F. Drilleau, IAA, (1985) 885. 6 L. Saulnier, J.M. Brillouet and M. Moutounet, Conn. Vigne Vin, 22 (1988) 135. 7 P. Pellerin, E. Waters and J.M. Brillouet, Carbohydr. Polym., 22 (1993) 187. 8 R. Cordonnier, C. Bayonove and R. Baumes, Rev.Fr.Oenol., 26 (1986) 29. 9 Z. Gunata, C. Bayonove, R. Baumes and R. Cordonnier, J. Chromatogr., 331 (1985) 83.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V.All rights reserved.
463
Application of Tailormade Pectinases. H.P.Heldt-Hansen a, L.V. Kofod a, G. Budolfsen a, P.M.Nielsen a, S. H0ttel a'b, and T. Bladt ~'c. aNovo Nordisk A/S, Novo All6, 2880 Bagsva~rd, Denmark
Abstract Heterologous expression of pectinolytic enzymes has made it possible to tailormake pectinolytic enzyme preparations. Composition of pectinase products for existing applications can be improved, and products for new applications can be made. Apple mash treatment with galactanase, rhamnogalacturonase and rhamnogalacturonan esterase improves the cloud stability of the juice. Pectin methyl esterase is useful to gelate fruits, and the gelling effect can be enhanced by a pectin hairy region degrading enzyme combination. Rhamnogalacturonase releases soluble high molecular rhamnogalacturonans from the soy cell wall, and the use of this enzyme to produce a fibre enriched soy protein product is demonstrated. Carrot mash treatment with combinations of polygalacturonase, pectin lyase, and rhamnogalacturonase gives a puree.
1. INTRODUCTION Pectinolytic enzyme products have been commercially produced for more than two decades. These enzyme preparations are typically produced by fermentation of black Aspergilli on complex media which ensures that the pectinolytic enzymes are well expressed. The compositions of the pectinolytic enzyme preparations are optimized for their applications by strain selection, breeding, and fermentation optimization. Such enzyme preparations contain several pectinolytic enzyme activities, often with the homogalacturonan depolymerizing enzyme activities, pectin lyase, polygalacturonase and pectin esterase, present in high amounts, but also other pectinolytic enzymes like arabinanase and galactanase are often present. Furthermore rhamnogalacturonases and rhamnogalacturonan acetyl esterase have been reported from a pectinolytic enzyme preparation produced by e.g.A, aculeatus [1,2,3]. Not only pectinolytic enzymes, but also hemicellulolytic and cellulolytic enzymes are often present in such commercial pectinolytic enzyme products.
bCurrent address: Royal Veterinary and Agricultural University, B01owsvej 13, 1870 Frederiksberg C, Denmark. r address: Danish Crown, Marsvej 43, 8900 Randers, Denmark
464 The pectinolytic enzymes degrade the pectic substances of the plant cell wall. The pectic substances are characterized by a high content of galacturonic acid, which is present in homogalacturonan as well as rhamnogalacturonan [4]. In homogalacturonan long stretches of galacturonic acid are only occasionally interrupted by rhamnose, whereas rhamnogalacturonan is a polymer of alternating rhamnose and galacturonic acid often with arabinan, galactan and arabinogalactan sidechains [4]. The rhamnogalacturonan rich regions and the homogalacturonan rich regions of the pectin substances are often designated "hairy regions" and "smooth regions" respectively [5]. Pectinolytic enzyme products have been developed for numerous applications [6], most well known are mash treatment of fruits like apples and pears for higher yield of juice, mash treatment of grapes for higher colour release, clarification and depectinization of juice and wine. Depolymerization of the homogalacturonan by pectin lyase and/or by polygalacturonase and pectin methyl esterase plays an important role in such applications. Furthermore the arabinanases are known to be important to prevent the formation of arabinan haze in apples and pear juice [7]. The rhamnogalacturonan degrading enzymes have not been described to be of applicational importance. During the recent years modern biotechnology has made it possible to clone the individual components of the pectinolytic enzymes and to express them in new hosts, like Aspergillus oryzae, by the use of promoters, which do not require pectic substances for their expression. It is thereby possible in commercial scale to produce single pectinolytic enzymes, which are essentially free from most other pectinolytic enzymes. By combining such monocomponent enzymes, one can design tailormade enzyme preparations with a composition optimized for the established applications of the present pectinolotytic enzyme products. Furthermore one can design combinations very different from the present commercial enzyme preparations, tailormade for new enzyme applications. Examplewise pectinolytic enzyme preparations without homogalacturonan depolymerization activity can be made for applications where the homogalacturonan needs to be intact. The present paper demonstrates applications of combinations of cloned monocomponent enzymes, including combinations with rhamnogalacturonases, for production of cloud stable apple juice, gelation of fruits, degradation of soy cell walls, production of dietetic soy and production of carrot puree are demonstrated.
2. MATERIAL AND METHODS
2.1 Enzyme preparations The pectinolytic enzymes have been cloned from A.aculeatus. P,hamnogalacturonase B (RGase B), arabinanase, galactanase, arabinofuranosidase, polygalacturonase (PG) I and II, pectin methyl esterase (PME), pectin lyase (PL), endo-glucanase III, and endo-protease II have been cloned by expression cloning as described [2,8], whereas rhamnogalacturonan acetyl esterase (RGAE), and rhamnogalacturonase A (RGase A) were cloned by probes as described [2,9]. The genes were isolated and transformed into A. niger (for the polygalacturonases) or A. oryzae (for all the other enzymes) as described [10,11]. The transformants were fermented as described [2]. Enzyme preparations were recovered from the culture broth, and were shown to be essentially free from most interfering activities (rhamnogalacturonase, rhamnogalacturonan acetyl esterase, polygalacturonase, pectin lyase, pectin methyl esterase,
465 and arabinanase) other than the heterologously expressed enzyme. The enzyme preparations were used in all experiments, except for the degradation of soy CWM, for which purified enzymes were used (purified by ion exchange chromatography). 2.2. HPLC analysis The molecular weight distribution of enzyme digests was determined by high pressure size exclusion chromatography (HPSEC) which implied separation on three TSK gel filtration columns (POW G3500, PW G3000 and PW G2000 obtained from TosoHaas) connected in series followed by refractive index detection (RID) on a RID6A (Shimadzu). The saccharides were eluted with 0.4M Sodium acetate buffer pH 3.0 at a flow rate of 0.8ml/min using a Dionex gradient pump (Dionex Corporation). The chromatograms were processed by Dionex software AI450, and Dextran standards (Serva) were used for estimation of the molecular weight (Mw) and degree of polymerization (DP). The amount of soluble saccharide in the sample could be estimated from the area of the chromatogram. Oligomers obtained from the different substrates after enzyme digestion were separated by High Pressure Anion Exchange Chromatography (HPAEC). Oligomers were eluted from a CarboPac PAl column (Dionex Corporation) with a gradient of sodium acetate in 0.1M NaOH. Gradient mixing was controlled by the Dionex gradient pump. 25~tl were injected and eluting saccharides were detected by Pulsed Aperometric Detection (PAD) [ 12]. Rhamnogalacturonan oligomers were eluted with an acetate gradient according to Schols et al., 1994 [13]. Stachyose and raffinose were analysed according the the recommedation of Dionex [141 For the determination of monosaccharide composition enzyme digests were hydrolysed in 2M tri-flouroacetic acid (TFA) for 1 hour at 121~ followed by evaporation. The hydrolysate was redissolved in water and 25 !~1 was injected into the CarboPac PAl column. The monosaccharides were eluted with a step gradient from 0-12 min 5mM NaOH, from 12-28 min water, from 28-35 min 0.1M NaOH, and a linear gradient from 35-54 min from 0-300mM sodium acetate in 0.1M NaOH. The column was rinsed from 54-64 min with 0.5M NaOH and equilibrated from 64-70 min in 5mM NaOH. The eluting saccharides were detected by Pulsed Amperometric Detection (PAD). For calibration of the detector response standard solutions of 0.25mM, 0.5mM and lmM rhamnose, fucose, arabinose, galactose, glucose, mannose, xylose, galacturonic acid and glucuronic acid (all obtained from Sigma) were hydrolysed in TFA and analysed as described. The content of the individual monosaccharides in the enzyme digests was calculated from linear regression.
2.3. Production of cloud stable apple juice Apples (Red Belle de Boskoop, Jonagold or Mutzu) were cut and milled (1.5 mm) and 5% (w/w) of a 2% ascorbic acid solution was added immediately. Enzyme preparations (25 mg enzyme protein / kg mash) were added and the mash was incubated for 2 hours at 20~ whereafter it was pressed. The resulting apple juice was pasteurised at 85~ to discontinue further enzyme degradation. The cloud was measured as turbidity in EF/F units [15]. The cloud stability was determined by a centrifugation test as the amount of turbidity remaining after centrifugation at 4,200 x g for 15 minutes [15].
466 2.4. Gelation of oranges The oranges were washed, chopped in a meat mincer and homogenised by a Fryma mill. Water (0.6 volumes) were added before the slurry was heat treated by steam injection at 100~ for 2 minutes. The enzyme treatment was carried out for 1 hour at 40~ with 10 IU/g slurry of PME and 25 gg enzyme protein/g slurry of the other enzymes for each of the enzymes. The gelated orange slurry were treated at 85~ for 3 minutes to inactivate the enzymes before the strength of the gel was measured by a SMS Texture Analyser TA-XT2 (Stable Micro Systems, XT. RA Dimensions, Operations Manual versions) by compression analysis using a flat cylinder (20 mm dia.) with a speed of 2 mm/s. The force to provide a 20% compression was recorded. 2.5. Enzyme degradation of Soy CWM Soy cell wall material (Soy CWM) was isolated by Alcalase| treatment and jet cooking (115~ 4 minutes) of soy meal followed by centrifugation and recovery of insolubles. Aliqots of 1% suspensions of soy CWM in 0.1M acetate buffer pH 5.0 were incubated with enzymes (40gg of each to 1.5 ml of substrate) at 30~ for 24 hours and the solubilized material was analyzed by HPSEC and HPAEC. 2.6 Production of Dietetic soy. Defatted soy flour was suspended in water to 8% (v/w) protein, pH was adjusted to 6 by HC1 before 640 m g ~ Novozyme 415 (o~-galactosidase), 10 mg/L Phytase L (Novo Nordisk) and 53 mg enzyme protein/L rhamnogalacturonase B were added. After 4 hours at 50~ the pH was adjusted to 8 by NaOH, and the slurry was centrifuged. The supernatant was pasteurized (85~ 5 minutes) and freezedried. Protein was measured as 6.25 x Kjeldahl N. Phytate was measured as described in [16], and dietary fibres were analysed as described in [ 17]. 2.7. Production of Carrot puree Carrots (Boleo) were peeled by 2% NaOH at 88-96~ for 4 minutes, minced by a meat mincer (2 mm) and homogenised for 2 minutes by an ULTRA-TURRAX T25 homogenizer (from Jahne & Kunkel). The carrot mash was preheated to 45~ (20 minutes) before the enzyme preparations, 25 mg enzyme protein/kg mash, were added. The enzymes were dissolved in water to give a dilution of 5% (v/v) of the carrot mash. The mash was incubated at 45~ under stirring (60 rpm) for 2 hours, before the enzymes were inactivated at 86~ for 5 minutes in a microwave oven. Finally the purees were homogenised for 1 minute by ULTRA TURRAX. The viscosity of the puree was measured by a BROOKFIELD viscosimeter Model DV-H + with spindle A from HELIPATH SPINDLE SET at 2.5 rpm thermostated at 50~ The stability of the puree was measured as the sediment (in %) after centrifugation in 10 ml tube a 1660 x g for 10 minutes.
3. RESULTS AND DISCUSSION 3.1 Cloud stable apple juice Several combinations of pectinolytic and cellulytic enzymes were tested for their ability to give a cloudy apple juice of Belle de Boskoop apples. Selected results are shown in table I.
467 Table I Production of cloud stable apple juice from Belle de Boskoop Enzyme
Turbidity before centrifugation
Increase in turbidity relative to untreated control %
Cloud stability %
Untreated
1061 + 112
100
56 + 3
Galactanase
1212 + 28
114
77 + 24
1333 + 102
125
86+ 10
RGase RGAE
A
+
Gal
+
RGase A + Gal + RGAE + PG-II + PME
783•
74
5+
5
Average of 3 measurements. Gal: galactanase. The galactanase gives an improvement of the cloudiness and cloud stability. The cloud stability is further improved when combined with rhamnogalacturonase A and rhamnogalacturonan acetyl esterase. RGase A alone does not give any significant effect. When the cloud stabilising enzyme combination is mixed with the homogalacturonan depolymerisation enzyme combination of polygalacturonase and pectin methyl esterase, a very poor cloud stability is observed. The size distribution of the high molecule substances in the serum of the juices was studied by HPSEC, as shown on Fig 1. The HPSEC chromatograms of the sera from untreated and galactanase treated mash show a peak (A) at an apparent molecular weight of 200.000 Mw. The retention time and size of this peak is unaffected of treatment of the serum with arabinanase, galactanase and a combination of RGase A and RGAE, but disappears after treatment with a combination of polygalacturonase and pectin methyl esterase (data not shown), peak A is consequently assumed to consist of pectin. A peak (B) with an apparent molecular weight of 15.000 Mw appears in the serum after mash treatment with the combination of galactanase, RGase A, and RGAE. The retention time of this peak is unaffected of treatment of the serum by all the tested enzymes, but the size of the peak is about 35% reduced by treatment with arabinanase (data not shown). This peak is assumed to consist of rhamnogalacturonase resistant pectic structures with arab• side branches. The viscosity of both the juice and the serum increases after the mash treatment with the combination of galactanase, RGase A and RGAE (data not shown). It is assumed that the enzyme combination of galactanase, RGase A, and RGAE releases high molecular substances to the serum including the substances seen on the chromatograms as the B-peak. The high molecular substances are then assumed to increase the viscosity of the juice resulting in a stabilisation of the cloud particles.
468
+RGase A + RGAE + Gal + PG + PME
AA ~vl
AB
Ill
i ~ V ~ /"'il ++RGaseA+RGAEGa, + Gal
Untreated Uw DP
>500,000 125,000 >3,200 800
8,000 50
500 3
Fig. 1: HPSEC of serum of juice of Belle de Boskoop mash treated with enzymes. Chromatograms of the serum after mash treatment with galactananse (Gal), RGaseA + RGAE + galactanase, RGaseA + RGAE + galactanase + PG II + PME and the untreated reference are shown. The chromatogram of the serum from juice with poor cloud stability obtained after mash treatment with the enzyme mixture including the pectin depolymerisation enzyme combination of polygalacturonase and pectin methyl esterase is seen to be lacking the A-peak. High molecular pectin is therefore considered to be a prerequisite for a cloud stable juice. The cloud stabilisation effect of the enzymes depends on the apple sort. The cloud stabilisation obtained with three apple sorts with galactanase and with the combination of galactanase, RGase A and RGAE respectively is shown in Table II.
469 Table II Cloud stabilisation of three apple sorts Apple sort
Enzyme combination
Cloud stabilisation relative to untreated control %
Belle de Boskoop
Gal Gal + RGaseA + RGAE
138 153
Jonagold
Gal Gal + RGaseA + RGAE
105 150
Mutzu
Gal Gal + RGaseA + RGAE
78 115
Average of 3 measurements. Gal: galactanase None of the tested enzyme preparations gave a significant stabilisation effect on Mutzu, and galactanase did not give significant cloud stabilisation effects on Jonagold and Mutzu. In a study on apple protopectin, RGase A alone was shown to solubilize some pectic material (homogalacturonan as well as rhamnogalacturonan) and no synergism was seen with galactanase [18]. The synergistic results with galactanase and rhamnogalacturonan degrading enzymes obtained with Belle de Boskoop does therefore not seem to be general, but rather dependent on the source of substrate. 3.2. Gelation of oranges.
For production of jams and similar types of products, pectin is usually added as a gelling agent. An alternative is to let pectin methyl esterase react on the pectin containing material. This principle has been used in traditional oriental cooking, where the fruits of Ficus awkeotsang are used to form a gel. This has been shown to be accomplished by the reaction of the pectin methyl esterase originating from the red tepals with the pectin from the fruit. The hereby obtained low methoxy pectin forms a gel by natural content of calcium [ 19,20,21]. The same gelling effect can be obtained when PME is added to fruits like strawberry and oranges. The gelling effect must depend on the availability of pectin from the fruits. A mixture of possible pectin releasing enzymes was therefore tested to see whether it could improve gelling properties of oranges. The pectin releasing enzymes were a mix of pectin hairy region degrading enzymes, an endo-glucanase and a protease intended to degrade other parts of the plant cell wall than the homogalacturonan, in order to improve the availability of this part of the pectin. Oranges were chopped, minced and pasteurised, before the PME and/or the pectin releasing enzyme mixture were added to react. The texture of the orange treated with PME had a jam like appearance, with gel strength of 2 N (Table III), the gel strength was significantly improved by the addition of the pectin releasing enzyme mix. The gelling properties of pectin methyl esterase is thereby demonstrated as well as the ability of other enzymes to improve the gel strengh. The ability of the RGase and galactanase containing enzyme mixture
470 to increase the availability of the pectin might be comparable to the cloud stabilising effect of these enzymes in apple mash treatment, where pectin is believed to improve the cloud stability. Table III Gel strength of enzyme treated orange mash Gel strength (N)
Enzyme treatment None (control)
0.1
PME
2.1
Pectin releasing enzyme mix
0.1
PME + Pectin releasing enzyme mix
5.6
Pectin releasing enzyme mix: RGase A + RGAE + galactanase + arabinanase + a-arabinofuronasidase + endo-glucanase III + protease II. 3.3. D e g r a d a t i o n of Soy C W M The solubilisation of soy cell wall material (CWM) by the two rhamnogalacturonases (RGase A and RGase B) in combination with other pectinolytic enzymes were compared in order to identify enzymes for new soy processes or products. The experiments were carried out at pH 5.0, where both rhamnogalacturonases have about 25% of their maximum activity, and with high enzyme dosages to ensure that the maximum effects are obtained.
Table IV. Amount and composition of the released material from soy CWM by pectic hairy region degrading enzymes. Enzyme
Monosaccharide composition. Ratio
Released amount %
Gal A
:
rha
:
gal
:
ara
0.2
9
2
9
1
Soy CWM untreated
0
1
9
RGase A + RGAE + Gal + Ara + Araf
7 17 44 46
1 1 1 1
: : : :
1 1 1 1
: : : :
7 10 15 14
: : : :
6 7 8 8
RGase B + RGAE + Gal + Ara + Araf
37 52 48 47
1 1 1 1
: : : :
1.3 1.3 0.8 1
: : : :
14 19 15 14
: : : :
9 11 8 8
Gal: galactanase, Ara: arabinanase, Araf: t~-arabinofuranosidase
471 As seen on Table IV RGase A is only able to solubilize 7% of the CWM, whereas addition of RGAE and galactanase increases the solubilisation to 17% and 44% respectively. The solubilisation of the CWM by RGase B is less dependent on the presence of the other pectinolytic enzymes, as 37% are solubilized with RGase B alone. The ratio of galacturonic acid to rhamnose, galactose and arabinose is 1:0.2:2:1 for the untreated soy CWM, whereas the ratio is about 1:1:14:8 for the enzymatically solubilised material for most of the used enzyme combinations. The high content of galactan in Soy CWM is well known [22,23,24]. The RGase (A or B) and RGAE released material has a relatively high molecular weight, corresponding to a DP of 300 (data not shown). This polymer is depolymerized by addition of galactanase, arabinanase and arabinofuranosidase. Thus, it must be anticipated that the RGase and RGAE solubilized material is almost entirely composed of fragments of rhamnogalacturonan backbone with an almost 1:1 ratio of rhamnose and galacturonic acid and with long side chains of arabinogalactans and arabinan. The lower solubilization of Soy CMW obtained by RGase A alone compared to RGase B alone is most likely explained by a steric hindrance by the acetylation of the rhamnogalacturonan backbone and of the arabinogalactan and arabinan side branches. The explanation is verified by the monosaccharide composition which shows a lower galactose and arabinose to galacturonic acid ratio after hydrolysis with RGase A alone, compared to the ratio obtained with RGase B. Thus, RGase A preferentially cleaves in rhamnogalacturonan which is not extensively substituted with side chains. 3.4. Dietetic soy protein The ability of RGase B to solubilise soy fibres is useful for the production of a fibre enriched dietetic soy product. Soy milk has the disadvantages that it has a low fibre content and a high content of phytate and of stachyose and raffinose. To overcome these disadvantages tx-galactosidase was used to reduce the amount of stachyose and raffinose, phytase to degrade the phytate, and RGase B to release fibre material.
Table V Composition of dietetic soy product Produced with enzymes
Reference produced without enzymes
Dry matter, %
95.2
98.2
Protein, %
59.2
60.2
Dietary fibre, %
5.7
2.5
Stachyose, %
0.4
5.3
Raffinose, %
0.2
1.4
Phytate, %
0.2
1.5
472 The composition of the resulting soy product is shown on Table V, where it is seen that the protein content was unchanged, whereas raffinose, stachyose and phytate were almost removed, and the amount of dietary fibres was improved. This demonstrates that the availability of relatively pure pectinolytic enzymes, like the used RGase B, opens up for the new types of soy processes and products. 3.5. Carrot puree Enzymatic maceration, which is a softening of plant tissue by the use of enzymes, has some potential quality advantages over mechanical-thermal disintegration as maceration is obtained with less damage to the cell walls. The major part of the plant cells remains intact by enzymatic maceration [25], as the enzymes attack only the space between the cells, and with only rare injury to the cell membrane [26]. The intact cells protect nutritional components within the cells which minimise flavour changes and deterioration on storage [27,28]. Several pectinolytic and cellulytic enzymes were tested in the treatment of carrot mash for their ability to give a puree with a low degree of syneresis. The enzymes were divided into two groups: a) Pectin backbone degrading enzymes: PG I, PG II, PL, PME, and RGase B + RGAE, and b) RGase B + RGAE, arabinanase, galactanase and endo-glucanase, and all the possible enzyme combinations of each of the two groups were tested in carrot mash treatment. The ability to form a puree was evaluated subjectively as well a by the viscosity. Enzyme treated carrot mash with a viscosity lower than 2xl 0 5 cP, was found to correlate to what was subjectively classified as a nice puree-like consistency (fine structured homogeneous mass). Such a puree was obtained with 17 of the 31 enzyme combinations from group a), whereas none of the 14 enzyme combinations from group b) were able to give a puree-like consistency. The purees were obtained with combinations including either PG + PL, PG-I + RGase B + RGAE, or PL + RGase B + RGAE, which implies that either two enzymes depolymerizing the smooth regions of pectin or one enzyme degrading the smooth region and an enzyme system degrading the backbone of the hairy regions of pectin are needed in order to form a puree. A puree should preferably have a low degree of syneresis. The stability against syneresis were tested in a forced syneresis assay, where the sediment was measured after centrifugation. The results obtained with the purees with a viscosity lower than 2xl 0 5 cP is given in table VI. The most stable puree was found with combinations of either PG-I + PL or PG-I + RGase B + RGAE, whereas combinations including PL + RGase B + RGAE were found to give less stable purees.
4. CONCLUSIONS Heterologous expression of pectinolytic enzymes has made it possible to tailormake pectinolytic enzyme preparations. Such tailormade combinations without homogalacturonan depolymerizing pectin lyase and polygalacturonase activity were shown to be useful for the apple mash treatment for cloud stable apple juice and for gelation of fruits. Rhamnogalacturonase activities were found useful for these applications and for the solubilization of soy cell wall, which can be used for production of a dietetic soy product. Carrot mash treatment with combination of 2-3 pectin backbone degrading enzymes was shown to give a puree, and also here rhamnogalacturonase was shown to play a role. It is concluded that cloned pectinases can
473
be used to combine enzyme products very different from the present commercial enzymes, and that such new products can be used in applications where the present commercial enzymes have a suboptimal or no performance, like for production of cloud stable apple juice and for gelation of fruits.
Table VI. Stability against syneresis Enzyme combination
Sediment after centrifugation (1660 x g) %
PG-I + PL + RGase B + RGAE
63
PL + RGase B + RGAE
68
PL + PME + RGase B + RGAE
68
PG-I + PME + RGase B + RGAE
70
PG-I + PG-II + PME + RGase B + RGAE
70
PG-I + PL + PME + RGase B + RGAE
71
PG-II + PL + PME + RGase B + RGAE
71
PG-I + PG-II + PL + PME + RGase B + RGAE
71
PG-I + PG-II + PL + RGase B + RGAE
75
PG-II + PL + PME
76
PG-I + PG-II + RGase B + RGAE
80
PG-I + PL
81
PG-I + RGase B + RGAE
85
PG-II + PL + PME
85
8PG-I + PG-II + PL
86
PG-I + PL + PME
87
PG-I + PG-II + PL + PME
87
Standard deviation s = 2.8 (determined by multiple test with one of the enzyme mixtures).
5. A C K N O W L E D G E M E N T Thanks are due to Hanne L. Larsen, Helle F. Petterson, Susanne G. Jacobsen for skillful technical assistance, and to Gerda Jensen for secretarial assistance with this manuscript.
474 6. REFERENCES
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J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
Cations
Increase
Activity
and
Enhance
Permeation
475
of
Pectinesterase
in
Ultrafiltration Louise Wicker Department of Food Science and Technology, Food Process Research and Development Laboratory, University of Georgia, Athens, GA 30602, USA 1. ABSTRACT The effect of pH and cation concentration on pectinesterase (PE) activation and permeation on 30 kD MWCO ultrafiltration (UF) membrane was evaluated. In order of increasing effectiveness, PE activity was stimulated by monovalent and divalent cations, polyamines and trivalent cations. A similar trend was observed for permeation on UF membranes. Cation addition and higher pH releases PE from an inactive complex, increases activity, and increases permeation. Higher cation concentration decreases activity and permeation. These results suggest a common mechanism is involved in PE activation and permeation. 2. INTRODUCTION The citrus industry is a multi-billion dollar industry in the U.S. Citrus juice is highly valued as a refreshing beverage with good nutritional value, high in Vitamin C, and excellent aroma and flavor. Citrus juice (100%) has a high consumer appeal because it is one of few, processed food products that has no food preservatives or other additives. Another quality attribute of citrus juice is "cloud", which contributes mouthfeel and is due in part to the colloidal suspension of pectin, protein, lipid and hesperidin (1). Cloud loss or clarif'lcation in single strength juices appears as precipitates of insoluble material and a clear serum. Clarification is typically attributed to de-esterification of pectin by pectinesterase (PE). Formation of insoluble calcium pectate destabilizes the colloidal suspension and clarification results. In citrus juice processing, disruption of the cell wall matrix releases PE, enhances deesterification of pectin, and leads to cloud loss in single strength citrus juice and gelation of concentrates (1). In juice, PE is firmly associated with the particulates in juice (11, 15, 19) and PE activity is proportional to pulp content (18, 20, 21). Thermostable pectinesterases (TSPE), operationally defined as activity that survives 5 min at 70~ contribute most to cloud loss in juices at low temperatures and juice pH (26). The percentage of total activity that is thermostable is highly variable and differs in kinetic properties, (22, 26), ease of solubilization (28, 29), stability to low pH (25) and stability to freeze-thaw cycles (23). Some of the variability in reported total PE and TSPE may be related to limitations of current methods to quantify activity. Any processing treatment or assay condition that increases cell wall breakdown or release PE from a pectin complex would enhance detection of total and TS-PE activity. Commercially, PE is inactivated by pasteurization in a plate heat exchanger or during concentration in the TASTE evaporator.
476 Other methods to inactivate or inhibit PE have been identified, but violate standards of identity for 100% citrus juice (1,6), are highly variable (1), and change flavor (2). As a size separation process in juice processing, ultrafiltration (UF) is versatile, has lower labor costs, energy costs, and has higher ease of clean-up and maintenance (12). UF membranes come in a wide variety of materials, pore sizes and configurations. Polysulfone membranes are widely used due to chemical and physical stability, including a pH tolerance from 2-12 (8). Ultrafiltration (UF) has been used to separate juice particulates and concentrate serum (12) and to make a more flavorful juice serum (10). In citrus juice, PE did not permeate 100 kd (12) or 500 kd (10) UF membranes. The molecular weights reported for PE range from 27,000 to 54,000 (9, 14, 22, 27). Retention of PE in high MWCO membranes was attributed to fouling of the membrane by pectin (10). In this study, we show that PE permeates an UF membrane and discuss some of the biochemical parameters for PEpectate interactions, activity and permeation. 3. MATERIALS AND METHODS Marsh grapefruit (MGF) pulp was homogenized in 5 volumes of extraction buffer at 4~ and maintained at pH 8.0 (28). The homogenate was stirred for one hour, centrifuged and the supernatant used as the PE extract. Activity was measured by titration with a Brinkman (Westbury, NY) pH stat titrator at pH 7.5 and 30~ in 25 mL of 1% high methoxyl pectin (Citrus Colloids Limited, Hereford, UK) with 0.1M NaC1. PE units are expressed as the microequivalents of ester hydrolysed per minute. Uronic acid analyses were conducted based on the m-phenyl phenol (4) as modified for microplate reading (30). Activation studies were conducted at pH 7.5 at 30~ in 20 mL of 0.5 % high methoxyl citrus pectin (Citrus Colloids, Hereford, U.K.). Final cation concentration in PE extracts used for activation studies was less than 2 mM as measured by potentiometry. Controls were conducted to correct for non-enzymic alkali consumption, with no polyamine/no PE and polyamine added/no PE. PE activity was normalized as a percentage of activity with no cation addition. A bench top polysulfone hollow fiber membrane (0.0325m 2) with molecular weight cutoff (MWCO) of 30K (A/G Technology Corp., Needham MA) was used (24). UF was run in a total recycle mode at a rate of 1.2 L/min (flow speed of 0.73 m/sec), cross membrane pressure of 25 PSIG and 10 + I~ PE permeability is expressed as the fraction of PEU/mL in the permeate to PEU/mL in the retentate. Data presented are representative of at least duplicate replications. 4. RESULTS AND DISCUSSION There is strong evidence that PE-pectate complexes exist within the cell wall as well as in plant extracts. The formation of these complexes and release by cations and/or cell wall potential is thought to influence cell wall growth and extension (16, 17). The data in Fig. 1 and Fig. 2 depicts the effect on PE activity of calcium or ferric chloride and the effect of the
477 triamine, spermidine or lead acetate, respectively. In all cases, there is an initial activation followed by inhibition of activity at higher cation concentration. Not only do the cations differ qualitatively in the level of activation, they also vary in the concentration required for maximal activation as well as the range of concentrations prior to inhibition. PE activity was increased approximately 125% over a wide range of concentration by calcium (10-50 mM). Ferric chloride stimulated the activity by over 300% at approximately 10 mM, but was inhibited with a slight increase in concentration. Polyamines are naturally occurring, ubiquitous cations which are involved in cell wall regulation and growth (7). The spermidine increased PE activity about the same percentage as calcium but over a narrow range (0.5-2 mM). Likewise, lead acetate stimulated PE activity by about 175%, although to a lesser extent than ferric chloride and PE activity was inhibited within a narrow range of lead acetate concentration.
350 300 -~..4
250 200
*"~ 150 100 50 J f
0
I t
50 100 Cation conc (mM)
150
Figure 1. Effect of calcium chloride (0) and ferric chloride ( II ) on PE activity in 0.5 % pectin at pH 7.5. Qualitatively, the results observed with ferric chloride and lead acetate were highly variable. It was first noted with ferric chloride that gels were forming within the reaction vessel We observed the formation of translucent gels in the reaction vessel with calcium chloride, spermidine and ferric chloride. In the case of lead acetate, a feathery type precipitate formed in the reaction vessel. Macdonald et al. (14) observed formation of a clear gel and flocculated precipitate in high ester pectin treated with lemon endocarp and peel PE isozymes, respectively. They hypothesized that the different gel structures were due to unique mechanism of deesterification by the PE isozymes. Our results with different cations and formation of different gel structure or precipitate seems to be similar to that reported by Macdonald et al. (14). If there is a different mechanism of de-esterification for plant PEs,
478 then de-esterification and subsequent interaction with pectate may also be influenced by the type of cation present.
200 175 150 ~
125 100 75
N
50 25 0 0
2
4
6
8
10
Cation conc (mM)
Figure 2. Effect of spermidine ([3) and lead acetate ( 0 ) on PE activity in 0.5 % pectin at pH 7.5. MacDonnell et al. (15) first suggested competitive displacement for the stimulation of PE activity. An increase in activity was ascribed to competitive displacement of PE from an inactive complex followed by a decline in activity at higher concentrations due to competition for carboxylic acid binding sites between PE and cations. Competitive displacement of PE from pectic acid by cations was conclusively shown with Lineweaver-Burke plots (7). The effect was moderated by pH (7, 15). We have shown that other polyamines, including putrescine, a diamine, and spermine, a tetramine, "activate" PE activity similarly to inorganic cations (13). Ultrafiltration of heterogenous colloidal suspensions such as citrus juice is complex and many factors other than molecular weight contribute to fouling and permeation. For example, low MW aroma compounds were unevenly distributed in the permeate and retentate in UF in 500 kd MWCO system (10). The authors observed that the 500 kd MWCO UF removed all suspended solids, including pectin and PE. If PE is complexed to pectate in an inactive complex, then it is conceivable that release of PE from pectin with cations will enhance permeation in UF. At optimum salt concentration, less PE activation was observed at lower pH values than at higher pH (15). In juice systems, it is difficult to separate the effect of juice particulates on PE activity. Model studies with PE extracts allows UF in the absence of large or insoluble particulates and control of composition of the ultrafilter. In
479 these extracts, pectin was not completely removed and approximately 0.18 + 0.03 mg uronic acid/ml was present before ultrafiltration. Even this amount of pectin prevented complete permeation of PE at typical juice pH. The results presented in Table 1 summarize the effect of cation and pH on permeation of extracts of PE. Permeability is expressed as the ratio of PE in the permeate to that in the retentate. The permeability after three hours (P18o) is reported as a point of comparison. Flux was measured by timing collection of a fixed volume. The flux, LMH, after three hours 018o) is reported. At pH 8.0, P18o increased and J18o decreased with an increase in NaCL concentration. At pH 3.8, no permeability was observed at NaC1 concentrations less than 0.3M, although J18o values were higher than J18o at pH 8.0. P18o observed at pH 3.8, 0.4M NaCL was about 80% of the P~8o at pH 8.0, 0.15M NaC1. Table 1. Cations and pH Affects PE Flux and Permeability ab Permeability and Flux with NaC1 at pH 8.0 Conc., M
J18o
P18o
0.0
41 ___ 4.1
0.25 ___ 0.03
0.15
29 + 0.9
0.45 + 0.03
0.30
28 +__ 1.2
0.37 ___ 0.04
Permeability and flux with NaC1 at pH 3.8. 0.0
39 + 3.1
0
0.15
36 ___ 8.0
0
0.30
43 + 4.8
0.08 ___ 0.02
0.40
23 ___ 3.7
0.37 ___ 0.05
Permeability and flux with CaC12 at pH 3.8. 0.06
65 ___ 5.0
0.18 ___ 0.01
0.08
40 ___ 2.5
0.26 ___ 0.02
0.10
29 ___ 0.7
0.33 + 0.02
0.20
25 ___ 2.0
0.21 + 0.05
Flux
(Jt)
is described by:
Jt --" J1 -
Permeability (Pt) is described by:
b log(t). Pt =
P1 -
S log(t).
Table adapted from Snir et al. (24), with permission
480 Table 2. Permeability, flux, and uronic acid content of PE extract permeated at pH 8.0 followed by permeation at pH 3.8 a pH 8.0 time
P/R, PE
Flux, LMH
UA, R
UA, P
0
1.37
127
0.18 ___0.03
0.09 __. 0.01
5
0.62
57
0.17 ___0.02
0.11 + 0.03
30
0.92
41
0.08 ___0.02
0.06 + 0.01
60
0.56
39
0.21 _ 0.02
0.07 __+0.02
120
0.88
37
0.19 + 0.02
0.09 + 0.02
0.58
168
0.06 _ 0.01
0.03 + 0.01
0.60
137
0.03 __+0.01
0.04 + 0.00
30
0.72
113
0.02 ___0.01
0.03 + 0.01
60
1.18
101
0.02 _ 0.00
0.04 + 0.01
120
1.10
114
0.04 + 0.01
0.06 __+0.01
pH 3.8 0
P/R, PE = ratio of PE permeability; UA, R = uronic acid content in mg/mL in retentate; UA, P = uronic acid content in mg/mL in permeate. a
Table adapted from Snir et al. (24), with permission PE permeability was enhanced by CaCI2 at lower concentrations than effective for NaC1. P18o in 0.1M CaC12 was approximately 0.33, and similar to the permeability in 0.4M NaC1 at pH 3.8. There seems to be a unique cation effect rather than ionic strength effect. At equivalent ionic strength (0.3M NaC1 and 0.1M CaC12, pH 3.8), P18o of PE with CaC12, was approximately four fold higher. The concentration of cations at discrete pH values which resulted in the highest observed PE permeability was obtained at pH 8.0 and 0.15M NaC1, pH 3.8 and 0.4M NaC1, or pH 3.8 and 0.1M CaC12. An increase of pH and/or cation concentration promoted an increase in PE permeability. Conversely, flux decreased with an increase in cation concentration or pH. At similar pH and cation concentrations that stimulate PE activity by competitive displacement, permeation of PE through a 30 kD MWCO UF membrane was observed. At low pH and no salt, the low level of pectin present in the PE extracts bound PE and prevented permeation. To confirm that there was not degradation of pectin, changes in the membrane or changes in PE, UF was conducted at pH 3.8 after initial permeation at pH 8.0 (24). The
481 results are shown in Table 2. At pH 8.0, uronic acids were concentrated in the retentate, permeation of PE was high and flux was low. The pH 8.0 permeate was collected and adjusted to pH 3.8, the membrane was cleaned, and the UF was continued at pH 3.8. The permeation remained high and the flux was typical of previous UF at pH 3.8. These results indicate there is no change in the membrane, PE or pectin as a result of pH, cation concentration or membrane conditions that affects PE permeation. Polyamines act similarly to inorganic cations in UF and allow PE permeation. The addition of 0.8 mM spermidine to pectinesterase (PE) extracts significantly enhanced permeation through UF membrane at pH 8.0 and pH 3.8 (13). Membrane fouling for pH 8.0 with and without spermidine was high during the early stages of UF and remained high throughout UF (Fig 3). At pH 3.8, flux was initially greater than at pH 8.0, With or without spermidine. The initial flux near 100 LMH for pH 3.8 with spermidine was considerably higher than initial observations of 60 to 75 LMH for pH 3.8 or 8.0 alone, or pH 8.0 with spermidine. PE permeability over the UF process time was 0.22 and 0.52 at pH 8.0, and 0.18 and 0.30 at pH 3.8, without and with spermidine, respectively (13). Permeabilities were increased by higher pH and/or spermidine addition. In commercial processes, high temperatures are necessary to inactivate TSPE, which accounts for a low percentage of the overall activity (27). At 0.8 mM, the levels of spermidine are near that present naturally in citrus (3). The ability of naturally present polyamines, such as spermidine, to enhance permeation of PE may allow development of a UF process to separate PE from particulates in juice and avoid high pasteurization temperature. 120 100 80 J 60 40 ~ 20
r------o..._._
---'2---_ _......~1
! 0
50
100 150 Time, rain
200
Figure 3. Change in flux durL,ag ultrafiltration (30 kd MWCO) of PE at pH 8.0 (1"1), pH 8.0 with 0.8 mM spermidine (11), pH 3.8 (A), pH 3.8 with 0.8 mM spermidine (A).
482 An initial, steep decline in flux followed by a plateau is indicative of fouling. The generation of a pectin-pulp deposit on the membrane surface during UF of Mediterranean orange and lemon juices, was proposed to act as a dynamic membrane, becoming a UF process itself (5). Snir et al. (24) suggested a gel layer on the membrane, contributed to resistance to filtration as well as the membrane. They proposed that added cations shield electrostatic repulsion, enhancing hydrophobic interaction and aggregation in a gel layer that lowers flux. Because there is less charge on pectin at pH 3.8, they suggested that with less electrostatic repulsion, less aggregation of pectin occurred, and flux values were higher. 5. CONCLUSIONS The formation of PE-pectate complexes clearly influence PE activity and permeation in UF membrane systems. Particulates, "rag", juice sacs are rich in pectin (21). PE, if not physically entrapped, is at least bound to pectin in juice particulates in a tight electrostatic complex. In these extracts, however, there were no particulates and the pectin level was relatively low. At pH 8.0 and 3.8, a decline in permeability was observed at higher salt concentrations, suggesting a similar mechanism is involved in permeation of PE as in activation. If competitive displacement of PE from a pectate complex is the only mechanism involved in enhancement of PE activity and permeation, then complete PE permeation would be expected at higher cation concentration or higher pH values. Since there was a limit to permeation as for activation suggests that another mechanism is involved. All the cations tested stimulated PE activity to different extents and at different concentrations. A similar effect of NaC1, CaC12, and spermidine at pH 8.0 and pH 3.8 on ultrafiltration was observed as for the activation studies. There was an initial increase, followed by a decline in permeation. These results would suggest that there is a mechanism other than competitive displacement of PE from pectate which influences permeation. If there is a different mechanism of de-esterification depending on the PE isozyme (14) or cation present, then the type of PE-pectate complex that is formed may vary and that in turn will influence permeation, flux and fouling. Acknowledgements. The author gratefully acknowledges the contributions of V. Leiting and R. Snir. This research was partially supported by BARD Project No. US-2222-92R. 6. REFERENCES
.
.
Baker, R.A. 1979. Clarifying properties of pectin fractions separated by ester content. J. Agric. Food Chem. 27: 1387-1389. Balaban, M.O., Arreola, A.G., Marshall, M., Peplow, A., Wei, C.I., and Cornell, J. 1991. Inactivation of pectinesterase in orange juice by supercritical carbon dioxide. J. Food Sci. 56: 743-746, 750. Bardocz, S.; Grant, G.; Brown, D.S.; Ralph, A.; Pusztai, A. 1993. Polyamines in food-Implications for growth and health. J. Nutr. Biochem. 4: 66-71. Blumenkrantz, N.; Asboe-Hansen G. 1973. New method for quantitative determination of uronic acids. Anal. Biochem. 54: 484-489.
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Capannelli, G.; Bottino, A.; Munari, S.; Ballarino, G.; Mirzaian, H.; Rispoli, G.; Lister, D.G.; Maschio, G. 1992. Ultrafiltration of fresh orange and lemon juices. Lebensm. Wiss. Technol. 25:518-522. Castaldo, D., Lovoi, A., Quagliuolo, L., Servillo, L., Balestrieri, C. and Giovane, A. 1991. Orange juices and concentrates stabilization by a proteic inhibitor of pectin methylesterase. J. Food Sci. 56: 1632-1634. Charnay, D.; Nari, J.; Noat, G. 1992. Regulation of plant cell-wall pectin methyl esterase by polyamines -Interactions with the effects of metal ions. Eur. J. Biochem. 205: 711-714. Cheryan, M. Ultrafiltration Handbook. 1986. Technomic Publishing, Lancaster, PA. Evans, R.; McHale, D. 1968. Multiple forms of pectinesterase in limes and oranges. Phytochemistry 17: 1073-1075. Hernandez, E.; Chen, C.H.; Shaw, P.E.; Carter, R.D.; Barros, S. 1992. Ultrafiltration of orange juice: Effect on soluble solids, suspended solids, and aroma. J. Agric. Food Chem. 40: 986-988. Jansen, E.F.; Jang, F.; Bonner, J. 1960. Orange pectinesterase binding and activity. Food Research 25: 64-72. K6seoglu, S.S.; Lawhon, J.T.; Lusas, E.W. 1990. Use of membranes in citrus juice processing. Food Technol. 44: 124-130. Leiting, V.; Wicker, L. 1994. Polyamine interaction with Marsh grapefruit pectinesterase. Abstract No.: 11-7. Institute of Food Technologists, 53th Annual Meeting, Biotechnology Division Graduate Oral Paper Competition, Atlanta, GA, June 23-27, 1994. Macdonald, H.M.; Evans, R.; Spenser, W.J. 1993. Purification and properties of the major pectinesterases in lemon fruits (Citrus limon). J. Sci. Food Agric. 62: 163-168. MacDonnell, L.R.; Jansen, E.F.; Lineweaver, H. 1945. The Properties of Orange Pectinesterase Arch. Biochem. 6: 389-401. Moustacas, A.-M.; Nari, J.; Borel, M.; Noat, G.; Ricard, J. 1991. Pectin methylesterase, metal ions and plant cell-wall extension. The role of metal ions in plant cell-wall extension. Eur. J. Biochem. 279: 351-354. Nari, J.; Noat, G.; Ricard, J. 1991. Pectin methylesterase, metal ions and plant cellwall extension. Hydrolysis of pectin by plant cell-wall pectin methylesterase. Eur. J. Biochem. 279: 343-350. Rouse, A.H. 1951. Effect of insoluble solids and particle size of pulp on the pectinesterase activity in orange juice. F1. State Hort. Soc. Proc. 64: 162-166. Rouse, A.H. 1953. Distribution of pectinesterase and pectin in component parts of citrus fruits. Food Technol. 7: 360-362. Rouse, A.H.; Atkins, C.D.; Huggart, R.L. 1954. Effect of pulp quantity on chemical and physical properties of citrus juices and concentrates. Food Technol. 8:431-435. Rouse, A.H.; Atkins, C.D. 1955. Pectinesterase and pectin in commercial orange juice as determined by methods used at the Citrus Experiment Station. Florida Agric. Exper. Station Bull. 570: 1-19. Seymour, T.A.; Preston, J.F.; Wicker, L.; Lindsay, J.A.; Marshall, M.R. 1991a. Purification and properties of pectinesterases of Marsh white grapefruit pulp. J. Agric. Food Chem. 39: 1080-1085.
484 23.
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27.
28.
29. 30.
Seymour, T.A.; Preston, J.F.; Wicker, L.; Lindsay, J.A.; Wei, C.; Marshall, M.R. 1991b. Stability of pectinesterases of Marsh white grapefruit pulp. J. Agric. Food Chem. 39: 1075-1079. Snir, R.; Koehler, P.E.; Sims, K.A.; Wicker, L. 1995. pH and Cations Influence Permeability of Marsh Grapefruit Pectinesterase on Polysulfone Ultrafiltration Membrane. J. Agric. Food Chem. 43: 1157-1162. Versteeg, C. 1979. Pectinesterases from the orange fruit - Their purification, general characteristic and juice cloud destabilizing properties. Ph.D. thesis. Agricultural Univ., The Netherlands. Versteeg, C.; Rombouts, F.M.; Spaansen, C.H.; Pilnik, W. 1980. Thermostability and orange juice cloud destabilizing properties of multiple pectinesterases from orange. J. Food Sci. 45: 969-973. Versteeg, C.; Rombouts, F.M.; Pilnik, W. 1 9 7 8 . Purification and some characteristics of two pectinesterase isoenzymes from orange. Lebensm. Wiss. Technol. 11: 267-274. Wicker, L.; Vassallo, M.; Echeverria, E. 1988. Solubilization of cell-wall-bound, thermostable pectinesterase from Valencia oranges. J. Food Sci. 53:1171-1174 and 1180. Wicker, L. 1992. Selective extraction of thermostable pectinesterase. J. Food Sci. 57: 534-535. Wicker, L.; Leiting, V.A. 1995. Microscale Galacturonic Acid Assay. Anal. Biochem. 157: L14-N.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All fights reserved.
485
Production, Clmmcted~tion and Application of RhanmoC~acturonase H. Hennink, H. Stam and M~G. van Oort. Quest International, P.O. Box 2, 1400 CA Bussurn, The Netherlands.
Abstract In this paper an overview is given of all the work that is performed on the enzyme RhamnoC~acturonase by different groups (Quest Naarden, URL-Vlaardingen and LUW). The project was focussed on different areas conceming molecular biology, fermentation technology and biochemical characterization of rhamnogalacturonase. Also the functionality of this enzyme in different application areas was studied.
1. INaRODUCIION The use of pectolytic and cellulolytic enzyme has become an indispensable part of fruit juice processing in the last two decades. In this application enzymes improve the extraction yield, resulting in higher yields of juice. These enzymes can also cause viscosity reduction which improves filtration and clarification of the fruit juices and prevent haze formation. Such commercial enzyme preparations comprise a mixture of mainly pectinases together with quantities of other hydrolytic enzymes such as arabinases, cellulases and xylanases. One of the main substrates for these er~mes is pectin, a complex heteropolysaccharide which is a major component of the cell wall of higher plants. It is proposed that pectin consists of highly carboxyl-methylated linear homogalacturonan regions which altemate with the so-called hairy regions consisting of a rhmnnosegalacturonic backbone(RG) with arabinose rich side chains (Voragen 1989) (figure 1). Pectin degradation requires the combined action of various ertzymatic activities. However, evaluation of the contribution of individual pectinases in fruit juice extraction and clarification is rather complicated. Most commercial pectinolytic enzyme preparations are produced by fermentation with filamentous fungi, mostly strains belonging to the genus Aspergillus. Application studies with mixtures of isolated er~mes obtained by fermentation or by means of fractionation of commercial et~me preparations can be used to assess the importance of the various individual enzymes. Subsequently, molecular biology and fermentation technology can be used to enhance specific desirable enzymatic activities.
486
p I=
Ara I Ara I Ara-- Ara--- Ara I EA ~Alra] N
r'-- //P--I~A A[a| IOMe/./ | OMe Gal
RGase~
'smooth region'
'hairy region'
r
Ara I Ara-- Ara
IA~a
IPL
cl/FAE 1 Ara OA Me Oi i
EA:endo-arabinase PE"pectine-esterase PG:polygalacturonase AF:exo- arabinofuranosidase RGase:rhamno-galacturonase PL:pectin-lyase
'smooth region'
AE: pectine-acetylesterase PA:pectate-lyase
Figure 1. Schematic representation of the structure of the smooth and hairy regions of pectin, including the various pectinases. 1.1 RHAMNOGALACH~ONAN AND RHAMNOGALACHSRONASE A cell-wall polysaccharide fraction remaining after treatment with a "classical" pectinase is termed rhamnogalacturonan (RG) or modified hairy regions (MHR). It is characterized by a highly branched rhamnogalacturonan-polymer with some arabinan side chains. In 1990 Schols et al reported the purification of an enzyme which was capable of degradation of the hairy regions prepared from apple juice : RhamnoGalacturonase(RGase). This enzyme can split glycosidic linkages in the rhamnogalacturonan backbone of (apple)pectins producing a range of oligomers composed of galacturonic acid, rhamnose and galactose. Rhamnogalacturonase may be useful in the prevention of haze formation in apple juice concentrates. In combination with other enzymes it might improve liquefaction, resulting in increase juice yield and clarification.
487 1.2. EVALUATION OF THE FUNCIIONAIIIY OF RHAMNOGALACIURONASE In order to test the application of rhamnogalacturonase(RGase), a commercial enzymepreparation was separated into several fractions by ionexchange-chromatography (see figure 2). Fractions were screened on rhamnogalacturonase-activity. The active fractions were tested in apple-application tests. From application trials it was clear that addition of RGase containing fractions when used in combination with other pectinases has a beneficial effect on the apple-juice process.
Separation of commercial preparation
Ion-Exchange Chromatography, OD 280 nm OD 543 nm 0.4 100 L
80 60 40
20 0
i /,\
0.3
i' /"/i / '/
0.2
Protein-pattern RGase-activity
'/ \
0.1
i\
/\
/ Fractions
\ ,,\,,\ 0
Figure 2 Fractionation by ion exchange chromatography of a commercial pectinase preparation.
488 2. MATERIALS AND METHODS
2.1. Detection RhamnoGalacturonase activity. RhamnoGalacturonase activity was determined by following procedures :
Westem blotting Products were analysed by SDS-PAGE followed by Westem blotting (Bumette 1981). RGase antibody production is described by van der Veen et al. 1991 This method can be used to determine the presence of RGase in different enzymepreparations and culture-filtrates. The disadvantage of this method is that only presence, but not activity of the enzyme is monitored.
RhamnoC~actumnase-activity on isolated rhamnogalacturomn. For the isolation of modified hairy regions (see figure 3) the method of Schols et al. (1990a, 1990b) was used. Golden Delicious apples were crushed and treated with a pectinase-preparation without detectable RGase activity. After centrifugation the supematant was ultrafiltered in a microfiltrationsystem (cut off 50.000D) and the residue was dialysed and lyophilized. To determine the presence of RGase in the various preparations isolated RG was treated with these products. After incubation, the formation of reducing end-groups was measured by the DNS-method.
Breakdown products characterization by HPI~-Dionex chmmatogml~. The Dionex system uses a Carbo Pac PA-1 anion exchange column and a CarboPac PA-1 Guard. The column was loaded with 25 pl of the RG solution and eluted with a linear gradient of 0 - 0.5 M NaOAc in 0.1 N NaOH during 50 minutes. The flow rate was 1.0 ml/min and the process was monitored using a PE detector.
Application-trials Apples were chopped and mashed to a free puree. Apple mash was treated with enzyme preparation and incubated for 2 hours at 55~ Viscosity of mash was measured several times using a Brookfield DC3 viscometer with Helipath stand attachment and TD spindle. After two hours of incubation sample was centrifuged for 20 minutes at 10.000 rpm. Volume, clarity, pH and brix of the juices were measured. The pectin level of the juices was assessed by a standard alcohol test.
489
RHAMNO-GALACTURONAN ISOLATION
................................... ("Hairy regions") Apples (Golden Delicious)
I !
Rasp Mill Mash Liquefaction
(Pectinase mix 0.05%, 4 i~r a~ 5C:r',.,_; Centrifugation ...........................,,~- waste putD
l
Ultra Filtration (50kD) ~
t
Dialysis
permeate
LMW
t 1 Lyophilization
Centrifugation
I
Rhamno-Galacturonan (RG)
Figure 3 Isolation-process of rhamnogalacturonan. 2.2. Identification of Rhamnogalactumnase producing Aspergillus straim and lhe development of a RGase production process by lhe wild type strain A.aeuleatus 115.80. Various Aspergillus strains were cultured on rich medium (sugar beet pulp) in shake flasks and examined for RGase production. Westem blots were analysed for crossreactivity with antibodies raised against purified RGase. Various strains of Aspergillus showed 1 distinct band after hybridisation with rhamnogalacturonase antibodies, with molecular weights ranging from 51.000 D - 63.000 D. The highest RGase production as judged by Westem analysis was obtained after cultivation of A. aculeatus and further research was focused on A. aculeatus CBS 115.80.
490 To improve production of rhamnogalacturonase by Aspergillus aculeatus CBS 115.80 shake flask experiments were performed on several substrates. Cross reactivity was found after transfer to rhamnose in combination with galacturonic acid and on apple pectin, citrus pectin, beet pectin and sugar beet pulp. No cross reactivity was found after transfer to media containing simple carbon sources such as sucrose, glucose, fi'uctose, rhamnose or galacturonic acid. The process developed on lab scale was transferred to pilot plant scale. The process is a fed-batch fermentation with a growth-phase (24h) on complex sugars and a production phase (48h). During production phase a linear feed is added containing various carbohydrates. Activity of Rhamnogalactumnase from A. aculeatus CBS 115.80.
Addition of fermentation broth from Aspergillus aculeatus CBS 115.80 to the isolated substrate resulted in an extinction increase measured by DNS-method.. Analysis of degradation products by Dionex (HPAEX) (see figure 4) shows a large peak of galacturonic acid and other free sugars. However the chromatogram described by Schols et a1.(1992) differs from the profile as described in figure 4 in this report. They found various oligomers after incubation of rhamnogalacturonase with RG suggesting a partly degradation of the substrate whereas in this work a predominant peak of galacturonic acid is found indicating a more complete breakdown of the substrate. This suggest that the fermentation broth obtained after cultivation of A. aculeatus on pectins contains beside the known RGase activity other activities capable of breaking down the oligomers formed after RGase treatment. In the following sections we describe the isolation of the rhamnogalacturonase (rhgA) gene from Aspergillus aculeatus and the construction of Aspergillus strains which are capable of producing RGase in larger quantities. 2.3. Isolation of the d g ~ gene from A.aculeatus.
A.aculeatus CBS 115.80 was grown on apple pectin in a fermenter and strong induction of Rgase production could be observed. Total RNA was isolated from such an RCrase producing culture and used for the construction of a eDNA library. In this eDNA library the eDNA is placed under the control of an inducible promoter. After induction of this promoter transcription of eDNA will occur, resulting in the synthesis of proteins. Since the eDNA was synthesised from mRNA isolated fi'om a culture induced for RGase synthesis, the library is "enriched" with eDNA coding for RGase. RGase producing colonies can be detected using the antibodies raised against RGase. In this way eventually 4 colonies that scored positive with RGase-antiseaun were characterized further at DNA level, eDNA inserts from positive colonies were sequenced completely in both directions and were found to comprise the entire coding region of the rhgA gene. The amino acid sequence of the protein was derived from the coding region of the rhgA gene.
491 Regions encoding amino acid sequences corresponding to amino acid sequences of peptide fragments of isolated RGase were encountered within the gene, positively identifying the cloned cDNA as corresponding to A. aculeatus rheA. This clone was used as a probe to screen a genomic library of A. aculeatus, resulting in the isolation of the entire genomic A. aculeattts rhgA gene including the rhgA promoter region (3). Comparison of the genomic DNA sequence of the rhgA gene with the cDNA sequence identified the position and size of three introns. The rhgA gene is encoding a protein comprised of 440 amino acids with a deduced molecular weight of 46kD.
2.4. Ovelpmduclion of RGase In order to construct an A. aculeatus strain which is capable of overproducing RGase, multiple copies of the A. aculeatus rhgA gene were introduced into a suitable acceptor strain by co-transformation with the A. aculeatus pyrA gene (auxotrophic selection marker). A pyrA negative mutant from A. aculeatus, obtained by classical mutagenesis was co-transformed with a DNA fragment consisting of the rhgA gene including the rhgA promoter and a DNA fragment containing the pyrA gene from A.__,. aculeatus. Transformed strains were selected by their ability to grow in the absence of uridine. A selected number of transformants was analysed for co-transformation. Transformants which had incorporated one or more additional copies of the A~ aculeatus rhgA gene were identified by quantitative Westem analysis of the culture supematant after shake flask experiments as described above. In this way overproduction of A.aculeattts RGase could be demonstrated in a number of transformants. Since transformation was performed with DNA fragments instead of entire plasmids, the transformed strains contain only DNA derived from A. aculeatus (no vector material). To further increase the production level of rhamnogalacturonase the rhgA gene from A. aculeatus was fused to the endoxylanase promoter e(e__xlA)from A. awamori which has been isolated during the cloning of the endoxylanase gene (see below). This fragment was used to transform A. awamori strains again making use of the pyrA system (in this case from A. awamori) as selection marker. Transformants, selected by their ability to grow in the absence of uridine, were grown in shake flasks in the presence of xylose. Since xylose acts as an inducer for the endoxylanase promoter, expression of the rhgA gene in strains containing the rhgA gene fused to the exlA promoter will occur. Very high levels of RCrase could be efficiently produced in A. awamori using the exlA promoter.
Activity of Rlmmnogalacturonase produced by mcomi~nant strains. Westem blotting of above mentioned recombinant strains shows a clear increase of rhamnogalacturonase production compared to the wild strain. To check the activity, fermentation broths of the recombinant strains were added to the isolated substrate resulting in a small increase of the extinction. This is due to the fact that rhamnogalacturonase splits the substrate in a range of oligomers which have less effect on the DNS-value compared to the products formed by the wild strain which breaks RG into very small components.
492 Therefore this method can not be used to compare rhamnogalacturonase activity present in the wild strain and the recombinant strains. Dionex analysis of hydrolysis products of RG by these recombinant strains (see figure 4 ) shows the formation of various oligomers as published by Schols et a1.(1992). This proves our observation that the wild strain contains side activities which are responsible for a complete breakdown of RG. From these results it was concluded that the DNS method can be used as a method to optimize and follow the production-process of RGase by the wild strain. Dionex analysis is a technique which provides us to control the production of the fight ertzyme but does not give any indication about activity. Therefore apple-application experiments were performed to detem~e the amount of rhamnogalacturonase which is necessary for an optimal perfonmnce. With these application-experiments an impression could be obtained about the differences in RGase activity of the various strains.
RG BREAKDOWN
ANALYSED
BY DIONEX.
RG BLANCO
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Figure 4 Dionex patterns of RG treated with different strains.
493 APPLICATION-EXPERIMENt.
Apple application experiments for producing apple juice show a clear benificial effect when rhamnogalacturonase in combination with a pectinase was added to the process. As can be seen t~om figure 5 rhamnogalacturonase has a clear positive influence on viscosity-reduction. Rhamnogalacturonase also prevents formation of haze in juices and therefore gives a stable juice. Other processes wherein rhamnogalacturonase can play an important role are * Other Fruits - Improve yield and clarity. * Beer - Improve clarity and filtration.
All these applications are subject of study at the moment.
APPLE MASH VISCOSITIES VISCOSITY CPS Thousands 40r"
[] pect.mix O pect.mix + RGase WT 9 pect.mix + RGase A.ac 9 pect.mix + RGase A . a w
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Figure 5 Influence of rhamnogalacturonase of different strains on viscosity-reduction in apple-application-experiments.
494
REFERENCT.S 1 A.G.J. Voragen,(1989) Food enzymes: Prospects and limitations In: Roozen J.P., Rombouts F.M., Voragen A.G.J.(eds): Food science: Basic research for Technological Progress, PUDOC, Wageningen, The Netherlands. 2. H.A. Schols et al., RhamnoGalacturonase: a novel enzyme that degrades the hairy regions of pectins. Carbohydrate Research (206) 105-115. 3. P.van der Veen et a1.,(1991), Induction, purification and characterization of arabinases produced by Aspergillus niger. Arch.Microbiol.(157)23-28. 4. WN. Burnette (1981) "Western Blotting": Electrophoretic transfer of proteins from Sodium Dodecyl Polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal.Biochem. (112): 195-203.
J. Visser and A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996Elsevier Science B.V.All rights reserved.
495
Effect of a new canning process on cell wall pectic substances, calcium retention and texture of canned carrots H. Siliha% W. J a h n b and K. Gierschner b aDepartment of Food Science, Faculty of Agriculture, Zagazig University, E g y p t bDepartment of Fruit and Vegetable Technology, Institute of Food Technology, H o h e n h e i m University, Stuttgart, Germany Abstract
A new process was developed for the canning of carrots. The main features of this process are: filling the cans with carrots and a minimum amount of brine (ca.8.0% of the canned carrots), vacuum sealing, preheating at a certain temperature and time (67~ for 30min) followed directly by sterilization. Quality attributes of the resultant canned carrots were supelior to that conventionally canned in respect to texture, colour, flavour and nutritional value. In this research the effect of preheating time (15, 30 and 60 min) followed by sterilization as well as the comparison between the new and the conventional canned carrots was studied with regard to the texture, Ca *+ retention and cell wall pectic substances. Texture of canned carrots was improved when the time of preheating was increased, while the amount of Ca+* bound in the cell wall was not significantly different from that of fresh carrots. Degree of methylation(DM; measured by HPLC) of total pectin reduced when the time of preheating was increased. This demethylation was reflected on the DM of the oxalate-soluble pectin, while t h a t of the water-soluble pectin remained unchanged. Conventional blanching did not alter the DM of total pectin which was lowered after sterilization. Texture of conventionally canned carrots was considerably lower than t h a t produced by the new process. Addition of CaC12 resulted in similar improvement in texture of carrots canned by both processes. However, the amount of Ca** retained in the cell wall material of conventionally canned carrots was twice as much as that found in carrots canned by the new process. Both canning processes resulted in the conversion of the major part of acid- and alkalisoluble pectin into water- and oxalate-soluble pectin. This conversion was accompanied by alteration in DM, degree of acetylation and in the a m o u n t of individual n e u t r a l sugars. The results suggest that the improvement in texture of canned carrots can be partly ascribed to the activation of the native pectinesterase during preheating, consequently the quantity of free carboxyl groups increased which were cross-hnked with Ca**. Additionally, the improvement in texture was correlated with higher amount of alkali soluble pectin fraction.
496 1. I N T R O D U C T I O N Cell wall and middle lamella polysaccharides determine to a large extent the texture of fresh, stored and processed vegetables and fruits. Their structural modification by cell wall modifying enzymes, either endogenous or exogenous, and by heat treatments during industrial processing have a great impact on the texture of the resultant products. In vegetables and fruits canning i n d u s t r y h e a t processing causes excessive degradation in cell wall and middle lamella polysacchrides which results in an extreme soft texture (Stolle-Smits et a1.,1995). Therefore, informations concerning the course of these modifications are of prime importance for the understanding of the mechanism of the softening reactions and thus for selecting the proper process for retarding them. Cell wall polysaccharides consist of pectins, hemicelluloses and cellulose (Keegstra et a1.,1973 ; Carpita and Gibeaut, 1993) while the middle lamella consists predominantly of pectic polysaccharides cross-linked with Ca ++ (McCann and Roberts, 1991). Due to their high water binding capacity and gel properties pectic polysaccharides have direct correlation with tissue firmness (Jarvis, 1984). Thermal softening of fruits and vegetables appears to occur through two different pectin degrading reactions depending on the pH of the tissue (Van Buren, 1986). As early as 1965, Doesburg proposed that softening below pH 4 is consistent with an acid catalyzed cleavage, while at pH levels over 5, the normal conditions for vegetables, softening is consistent with a pectin depolymerization reaction that has the characteristics of a 13-elimination reaction catalyzed by hydroxyl ions and inhibited by demethylation of the pectin. Several attempts have been made to improve the texture of canned carrots. These include adding calcium (Sterling, 1968), changing the pH during processing (Van Buren and Pitifer, 1992) and modifying the blanching treatment before sterilization (Lee et al., 1979 ; Gierschner and Philippos, 1992 ; QuinteroRamos et al., 1992). These authors found that preheating of carrots at low temperature for long time resulted in firmer texture. The firming effect was attributed to the activation of the native pectinesterase which increases the number of free carboxyl groups on the pectin molecules, thus enabling them to be cross-linked with Ca *+ naturally present in the tissue. Interactions between Ca ++ and cell wall pectin play a key role in stabilizing the wall structure (Demarty et al., 1984) The structural features of cell Wall polysaccharides of carrots have been studied by Stevens and Selvendran (1984) and Massiot et a1.(1988). Plat et a1.(1991), Ben Shalom et a1.(1992) and Massiot et a1.(1992) investigated the changes in pectic substances of carrots after blanching, dehydration and extended heat treatment. Data on the changes in cell wall polysaccharides of canned carrots are lacking. This study aims to investigate the effect of preheating time at low temperature and the addition of CaC12 on texture and on the composition of various pectin fractions of carrots canned by conventional and by a new process.
497
2. M a t e r i a l s a n d M e t h o d s 2.1. C a n n i n g of c a r r o t s : Carrots (Daucus carota, var. carotan) were obtained from Ritz farm (Linkenheim/Baden-Germany). After washing, trimming and peeling the carrots were cut into cubes of 1 cm 3 by means of a cutting machine(Master, Nr. 3695). They were then screened to remove the undersized cubes. The carrot cubes were soaked in distilled water at 40~ for 2 min and allowed to drain. Two h u n d r e d and fourty grams of the cubes and 19.5 ml of distilled water were filled into the can which was then vacuum sealed (0.3 bar). The cans were put in rotary retort (Stock Pilot-Rotor 900, Germany) which was programmed to carry out the preheating and sterilization steps sequentially. Preheating treatments were performed at 65~ for 15, 30 and 60 min. Sterilization was carried out at 12 I~ (Fo=3.5). For investigating the effect of CaC12, the carrot cubes were soaked in and filled with 0.2%CaCI~ .The cans were preheated at 67~ for 30 min and sterilized. As for the conventional canning the carrot cubes were blanched in water at 85~ for 2 min, then 240 g of the cubes and 170 ml distilled water with or without 0.2% CaC12 were filled in the cans and sterilized as described earlier. 2.2. T e x t u r e m e a s u r e m e n t s : Texture of canned carrots was measured using Instron Universal Testing Machine (Model 1011) fitted with Kramer shear cell. Thirty grams of drained carrot cubes were evenly placed in the Kramer shear cell and were compressed, sheared and extruded using a crosshead speed of 100 ram/rain. Each measurement was repeated 10 times and the mean was used to express the firmness of carrot cubes in Newton(N).
2.3. P r e p a r a t i o n of the a l c o h o l i n s o l u b l e solids (AIS): The content of the can was drained and the carrot cubes were immediately frozen in liquid nitrogen, freeze-dried and milled. Carrot powder (ca. 10 g) was mixed with 200 ml 80% ethanol previously heated to 60~ After filtration the residue was extracted with ethanol until the filtrate was colorless (5 times) and gave negative reaction with phenol-sulfuric acid test (Dubois et al., 1956). 2.4 F r a c t i o n a t i o n of cell w a l l p o l y s a c c h a r i d e s : One gram of AIS was suspended in 100 ml distilled water at 40~ for 45 rain. The slurry was centrifuged at 15000xg for 20 rain and s u p e r n a t a n t was decanted. This step was repeated one more time. Both s u p e r n a t a n t s were combined and represented the water- soluble pectin (WSP). The pellet was sequentially suspended in 0.5% ammonium oxalate(at 40~ 0.05N HC1 (at 70~ and 0.05N NaOH (1~ Each extractant(100 ml) was applied two times (each 45 rain) followed by two times washing with distilled water. The extracts and the washing water were combined and represented, respectively, oxalate-soluble pectin (OXSP), acid-soluble pectin (I-ISP) and alkali-soluble pectin (OHSP). The pectin solutions were dialysed, concentrated under reduced pressure at 40~ and freeze-dried. The pellet was extracted with 4M NaOH (containing 10 mg sodium borohydride/ml) for 8 hrs at room temperature, followed by centrifugation.Extraction was repeated two times
498 and the extract was acidified with acetic acid to pH 5.2, dialysed and freeze-dried (hemicellulose). The remaining pellet was dialysed and freeze dried (celluose). 2.5. A n a l y t i c a l m e t h o d s : Anhydrogalacturonic acid (AUA) content was determined by the metahydroxydiphenyl (mHDP) method (Blumenkrantz and Asboe-Hansen, 1973). Degree of methylation (DM%) and degree of acetylation (DAc%) of the pectin fractions were measured by HPLC analysis (Voragen et al., 1986) using a Gynkotek-HPLC system equiped with Aminex HPX 87P column (25cm x 4mm i.d., Bio-Rad) and with refractive index detector (Shodex RI SE61). For the determination of ash and minerals content the AIS was ashed at 550~ The minerals (Ca ++, Na § K § and Mg +§ analyses were performed with PerkinElmer 3030 atomic absorption spectrophotometer. Neutral sugars content of the alcohol insoluble solids and the pectin fractions were determined as their alditol acetates after hydrolysis with sulfuric acid (Seaman et al., 1963) and trifluoroacetic acid, respectively. Derivatization was performed according to Englyst and Cummings (1984). The alditol acetates were separated with GC-14 gas chromatograph (Shimatzu) equiped with DB-225 capillary column 30m x 0.25mm (J & F Scientific)and FID-detector. Molecular size distribution of the pectin fractions was determined by HPLC. The pectin fractions were dissolved in 0.4M acetic acid/sodium acetate buffer (pH 3) containing 0.025M sodium sulfate. The high pressure size exclusion chromatography system (HPSEC)included a Gynkotek M480G pump, GINA 160 injection system, column thermostat at 40~ and Shodex RI SE61 refractive index detector. TSK columns GMPWXL (30cm x 7.8mm) and G6000 PWXL (30cm x 7.8mm)were used in combination with a guard column PWXL. Columns were eluted with 0.4M acetic acid/sodium acetate buffer pH 3 containing 0.025M sodium sulfate at a flow rate of 0.6 ml/min.
3. R E S U L T S AND DISCUSSION 3.1. Effect o f p r e h e a t i n g t i m e on f i r m n e s s a n d p e c t i c p o l y s a c c h a r i d e s Firmness of canned carrots preheated at 65~ for 15, 30 and 60 rain is illustrated in Fig. 1. Preheating for 15 rain showed a firmness of 261.6N. When preheating time was increased higher tissue firmness was observed. Similar observations have been reported by Lee et al., (1979) and Quintero-Ramos et al., (1992) who found that firmness of carrot tissues was increased with increasing the time of blanching at 65~ ' Degree of methylation (DM%) of total pectin of carrot alcohol insoluble sohds (AIS) was decreased from 60.73% for fresh carrots to 48.70, 44.62 and 43.83% for canned carrots preheated at 65~ for 15, 30 and 60 rain, respectively (Fig. 2). Similar levels of demethylation were also reported in potato (Bartolome and Hoff, 1972), in carrots (Lee et al., 1979) and in snap beans (Adams and Robertson, 1987) when they were blanched at low temperature between 65~ and 70~
499 400 35O 300 250 200 150 iZ 100. 50, 0
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Figure 1. Effect of blanching time on texture of canned carrots
70 60 50 40 30 20 100 t-, Lt~
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Figure 2. Degree of methylation (DM) and degree of acetylation (DAc) of the total pectin of fresh and canned carrots
The AIS of fresh and canned carrots were sequentially fractionated into watersoluble pectin (WSP), oxalate-soluble pectin (OXSP), acid-soluble pectin (HSP), alkali-soluble pectin (OHSP), hemicellulose and cellulose. The distribution of uronic acid among various fractions is presented in Table 1. WSP of fresh carrots accounted for 19.0% of the total pectin, while OXSP constituted 29.6%. HSP represented the lowest (12.0%) pectin fraction, whereas OHSP was the highest (35.6%) In the hemicellulose and cellulose fractions significant amounts of uronic acid were found. Heat treatments during canning altered the proportion Table 1 Distribution of uronic acid in the fractions of the alcohol insoluble solids of canned carrots preheated at 65~ for different times (as mg AUA/100 g fresh weight) Fraction Fresh Preheating Time (min) 15
WSP OXSP HSP OHSP Hemicellulose Cellulose Total AUA
30
60
183.96(19.0) 433.66(41.4) 418.64(39,3) 382.24(36.0) 286.62(29.6) 431.14(41.2) 459.35(43,1) 466.21(44.0) 116.70(12.0) 31.18(3.0) 36.78(3,5) 39,59(3.7) 344.84(35.6) 96.59(9.2) 100.00(9.4) 123.97(11.7) 12.47(1.3) 22.68(2.2) 20.02(1.9) 19.59(1.8) 25.01(2.6) 31.17(3.0) 29.90(2.8) 29.08(2.7) 969.60(100) 1046.42(100) 1064.67(100) 1060.68(100)
of various cell wall fractions. WSP and OXSP were increased, while HSP and OHSP were decreased. Results obtained by Plat et al., (1991) and Massiot et al., coincides with those reported in this study. Table 1 also shows that by prolonging the time of preheating to 30 and 60 min the amount of WSP slightly decreased and that of OXSP, HSP and OHSP increased. Since firmness of canned carrots increased as the preheating time was increased (Fig. 1),it seems reasonable to conclude that firmness retention corresponds with less conversion of protopectin
500 into water soluble pectin. This confirms the results obtained by Ben Shalom et a1.(1992) who found that firmness of carrots blanched at different pH values positively correlated with OHSP content DM (54.11%) and DAc (9.15%)ofWSP of fresh carrots were compared with those of canned ones (Fig. 3). After canning the DM was slightly increased, suggesting that native PE was inactive on the WSP and that the solubilized fraction from the protopectin which was added had almost similar DM as that of WSP naturally present in fresh carrots. During ripening of cherry fruits Batisse et a1.(1994) also found that DM of WSP remained constant. DAc, on the other hand, was increased twice as much as that found in WSP of fresh carrots, indicating that the solublilized pectin fraction, due to heat treatments, was characterized by relatively high DAc. Fig. 3 also shows that DM of WSP did not significantly change as the time of preheating was increased, while DAc was slightly decreased. The OXSP recovered from fresh carrots had lower DM (47.58%) and DAc (2.78) than those found inWSP (Fig.4). Sajjaanantakul (1989) obtained similar DM (46.0%) for the chelator-soluble pectin of fresh carrots. Preheating and sterilization lowered the DM of OXSP to 29.78, 25.79 and 26.95% after 15, 30 and 60 min, respectively, suggesting that this fraction was a prefered substrate for PE action. The same observation was also reported for cherry fruits native PE. It preferentially acted on OXSP of cherry fruits during ripening (Batisse et al., 1994). They also showed that this pectin fraction was characterized by the lowest DM and DAc among the various pectin fractions isolated from cherry fruits. However, it cannot be excluded that the pectin fraction which was added to OXSP, as a result of heat treatment, was previously demethylated and selectively cross-linked with the native Ca ++of the carrot tissue. 60~
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Fresh 15 30 60 B l a n c h i n g Time(min)
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of m e t h y l a t i o n and degree of a c e t y l a t i o n (DAc%) of oxalate-soluble p e c t i n of fresh and c a n n e d carrots Figure
(DM%)
4. Degree
,8
501 Molecular size distribution of various pectin fractions was monitored by HPSEC (Fig.5). WSP of fresh carrots comprised a high molecular weight fraction that consists of two unresolved peaks and a low molecular weight fraction which.consists of three distinct peaks. Two main observations can be deduced from Fig 5a due to heat treatments. First, the high molecular weight fraction was degraded and shifted to lower molecular weight with distinct separation of two different populations. The second observation is the increase in peak area of fraction II, giving an additional evidence for the increase in the amount of uronoides in the WSP due to solubilization of protopectin. Similarly to the pattern of WSP, the heat treatment affected the elution pattern of OXSP (Fig. 5b). The peak area of the high molecular weight fraction was increased after heat treatment. In contrast, HSP exhibited a reduction in molecular weight and in peak area of the high molecular weight fraction (Fig. 5c). It shifted to lower molecular weight. Massiot et a1.,(1992) obtained similar molecular weight distribution for HSP of carrots heated at 980C. When the preheating time was prolonged a fraction with high molecular weight appeared (peak I), of which the area was increased as the time of preheating increased. Due to the low solubility of the OHSP only a small peak was observed (Fig.Sd). As a result of heat treatment, similarly to HSP, a high molecular weight fraction (peak I) appeared. The composition of this peak is unknown and is a subject of our current research. .3.2. E f f e c t o f c a l c i u m o n f i r m n e s s a n d p e c t i c s u b s t a n c e s Carrots canned by the conventional process showed an extremely soft texture (185N) compared with those canned by the new process (Table 2). Addition of 0.2% CaC12 improved the texture of carrots produced by both processes to almost the same level. The AIS prepared from conventionally canned carrots was lower than that of fresh carrots due to leaching of salts, sugars and other soluble components into the brine. In the presence of CaC12 the amount of AIS recovered from carrots canned by both processes was increased. Table 2 also shows that DM of total pectin decreased from 60.73% to 52.0% after conventional canning. When carrots were ccnventionally blanched (85~ fo 2 min) the DM was unaffected. This indicates that the PE was inactivated under this blanching conditions and that sterilization process is responsible for the demethylation, presumably through 13-elimination breakdown of highly methylated segment of the pectin molecules. In canned green beans Stolle-Smits et a1.(1995) reported that DM of the AIS of sterilized beans was 14% lower as compared with the same samples of blanched and fresh beans. Carrots canned by the new process showed lower DM due to the action of native PE. Ash arid minerals (Ca ++, Na § K § and Mg +§ content was determined in AIS of fresh as well as canned carrots (Table 3). The native Ca ++bound in the cell wall components of fresh carrots was found to be 10.9 mg/g AIS. In the presence of CaC12 the conventionally canned carrots showed a sharp increase in Ca +*
502
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,
Figure 5. HPSEC elution pattern of pectin fractions of canned carrots preheated at 65~ for different times. (a) WSP, (b) OXSP, (c) HSP, (d) OHSP.
503 Table 2 Effect of CaC12 on firmness, alcohol insoluble solids content and degree of methylation (DM%) of carrots canned by conventional and by a new process Fresh Conv. Conv.+ New+ CaC12 CaC12 3888 185.8 371.3 385.4 Firmness(N) 3.15 2.86 3.12 3.5 AIS(% fresh weight) 60.73 51.2 52.7 46.92 DM % retention compared with those canned without CaCI~. Carrots canned by the new process retained equal amount of Ca ++ as that of fresh carrots, while the addition of CaC12 brought about higher Ca ++ retention. It is interesting to note t h a t although the new and the conventional canning processes exhibited similar firmness when CaC12 was added, the carrots of the former process retained much less Ca ++ t h a n the latter. Since the DM of the of the total pectin was higher in conventionally canned carrots t h a n those canned by the new process (Table 2), it seems t h a t not all the Ca ++ t h a t was retained in the AIS of conventionally canned carrots are cross-linked with pectin. Table 3 also shows t h a t this high a m o u n t of Ca ++ displaced the major p a r t of Na § and K + and to a lower extent Mg ++ in their binding in the cell wall constituents. Such displacement did not take place in the carrots canned by the new process. It should be stressed that the total a m o u n t of Ca § added during canning was higher in the conventional (170 ml of 0.2% CaC12) t h a n in the new process(19.5 ml of 0.2% CaC12) Table 3 Effect of CaC12 on ash and minerals content of the alcohol insoluble solids of carrots canned by conventional and by a new process (as mg/g AIS) Component Fresh Conv. Conv.+ New New+ CaC12 CaC12 Ash 82.00 67.00 76.00 91.80 92.30 Ca ++ 10.91 9.93 23.51 10.51 14.29 Na § 2.94 2.08 0.50 2.30 2.71 K§ 19.66 16.88 6.44 26.19 22.65 Mg++ 1.89 1.41 1.07 2.10 1.98 A comparison of the cell wall fractions of carrots canned by both processes is given in Table 4. H e a t t r e a t m e n t during canning process resulted in the solubilization of the major part of H S P and OHSP. This was reflected on an increase in WSP content in carrots canned by conventional process, in OXP content in carrots canned by conventional process with CaC12 addition and in WSP and OXP in carrots canned by the new process. The total uronoides content recovered from conventionally canned carrots was 21% lower as compared with
504 fresh carrots, suggesting a solubilization of uronoides in the brine. On addition of CaC12 the total uronoides content was 10.3% lower than that of fresh carrots and 13% higher than that canned without CaC12. Comparison of the amount of WSP and OXSP in carrots canned by conventional process (Table 4) shows that when CaC12 was added the amount of WSP decreased with a concomitant increase in the OXSP. This indicates a selective binding with Ca ++ of the water soluble-low methylated pectin molecules, thus becoming less soluble in water but soluble in oxalate. Table 4 Effect of CaC12 on the distribution of uronic acid in the fractions of alcohol insoluble solids of carrots canned by conventionaland by a new process (as mg AUAJ100 g fresh weight) Fraction Fresh Conv. Conv.+ New+ CaC12 CaC12 183.96(19.0) 292.15(38.2) 209.00(24.8) 343.50(33.3) WSP 285.52(29.5) 291.39(38.1) 410.40(48.6) 458.55(44.4) OXSP HSP 116.70(12.0) 35.88(4.7) 54.95(5.5) 35.32(3.4) 344.84(35.6) 93.21(12.2) 91.05(10.8)128.01(12.4) OHSP Hemicellulose Cellulose Total AUA
12.47(1.3) 27.42(3.6) 29.11(3.5) 29.40(2.85) 25.01(2,6) 25.11(3.3) 49.54(5.9) 37.60(3.64) 969.60(100) 765.16(100) 844.06(100)1032.49(100)
DM and DAc of various pectin fractions except those of OHSP (due to deesterification during extraction) are summarized in Table 5. The DM of WSP of conventionally canned carrots did not significantly differ from that of fresh carrots,while in the presence of CaC12 a considerable increase was observed. This increment can be ascribed to cross-linking of water soluble-low methylated Table 5 Degree of methylation (DM%) and degree of acetylation (DAc%) of the pectin fractions of carrots canned by conventional and by a new process in the presence of CaC].2 Fraction Fresh Cony. Cony.+ New + CaCI2 CaCI2 WSP 64.11 (9.16) 65.42(11.28) 73.64(13.33) 62.18(11.57) OXSP 47.68 (2.78)32.04 (5.23)37.50 (6.43)28.26 (5.33) HSP 55.94(14.94) 26.58(18.04) 29.85(20.73) 33.78(23.95) Data in paraenthesis are the DAc. pectin molecules with Ca ++leaving highly methylated ones in soluble form. This is evident from the increase in the amount of OXSP as previously shown (Table 4). The DM of OXSP decreased in both canning processes compared with the fresh carrots, due to the selective binding with Ca ++ of the low methylated pectin
505
molecules. It is worthwhile mentioning that although the amounts of OXSP of fresh and conventionally canned carrots (without CaC12) were similar, the DM differed. This can be explained by B-elmination breakdown of highly methylated segments during sterilization. The DM of HSP was sharply decreased after canning process due to solubilization of the major part of this fraction. Sugar composition of AIS of fresh and canned carrots is demonstrated in Fig. 6. Conventional canning either with or without CaC12 drastically altered the sugar composition. Rhamnose, arabinose, galactose and uronic acid decreased while other sugars known to be linked with hemicellulose and cellulose fractions increased. The extent of these changes was greater when carrots were canned without CaC12. The sugar composition of various pectin fractions isolated from fresh carrots considerably varied (Fig 7). OXSP was characterized by the lowest neutral sugars content, while OHSP was the highest. The ratio of total neutral sugars to uronic acid found in various fractions was 0.37, 0.08, 0.48 and 0.42 for WSP, OXSP, HSP and OHSP, respectively. These fractions underwent different changes during canning. Neutral sugars content of WSP of carrots canned by conventional (without CaCle) and by the new process was increased, with the latter being highly enriched with neutral sugars. The WSP of conventionally canned carrots withCaC12 did not show such an increament but rather a reduction in xylose, mannose, galactose and glucose content. Canning process resulted in an increase in sugars content of the OXSP in comparison to fresh carrots. Addition of CaC12 to carrots canned by both processes showed similar sugar composition which was higher than that canned without CaC12. On the other hand, neutral sugars content of HSP and OHSP decreased after canning. HSP of conventionally canned carrots with CaC12 addition had the highest content of neutral sugars among canned carrots, while OHSP of carrots canned by the new process was charaterized by the highest neutral sugars content. 1200,00 .'~ 000,00 -
WaFresh
~: 800,00 & 600,00 ~400,00 200, O0 0,00 Cell Wall Sugar F i g u r e 6. S u g a r c o m p o s i t i o n o f a l c o h o l i n s o l u b l e s o l i d s o f c a r r o t s c a n n e d by c o n v e n t i o n a l a n d by a n e w p r o c e s s in t h e p r e s e n c e o f CaCh
506
,.~ 350 -,-, 3 0 0
WSP
_
~: 250 200 t~ ,.~ 150 100 50
~
o
t I
I ~ l
i
I~ ' - ,
I
I~ '
--I
500
oxsP
oP,,I
400300200 ~- 100 h~
I -'-'r~
,= 120 "~ tD 100 ~:
'I
I
HSP
80-
cv 6 0 ~ 40 ~ 20-
-~
o
i -
-
350
,.~ 300 9 p,,,,I
a~ 250 -
,.= 200 150 -
OHSP m Fresh m Conv. D Cony. + CaCl~ D N e w + CaC12
100 -
o
50-
'~
o
r-,-4
i, < < Cell W a l l S u g a r
Figure7. Sugar composition of pectin fraction of carrots canned by conventional a n d b y a n e w p r o c e s s i n t h e p r e s e n c e o f CaCl2.
507
Conclusions Firmness of fresh carrots is governed by the physical strength and organized structure of cell wall and middle lamella. In the primary cell wall pectin is believed to be linked by acid- and alkali-labile bonds forming the protopectin (Van Buren,1991 ; Massiot et a1.,1992). Selvendran (1985) suggested that waterand chelator-soluble pectin are derived from the middle lamella. Based on these assumptions the protopectin (HSP and OHSP) in fresh carrots accounted for 47.6% of the total pectin while the middle lamella pectin (WSP and OXSP) made up 48.6%. Sterilization during canning dramatically altered the organized structure of both the protopectin as wen as middle lamella pectin. The major part of the protopectin was solubilized and the residual fraction accounted for 16% of the total pectin while middle lamella pectin increased to ca.75%. This conversion was accompanied by the dramatic loss of tissue firmness. In conventional canning part of the solubilized pectin was leached into the brine and the rest was retained in the tissue due to its molecular configuration. A water-soluble fraction is remained entangled in the tissue presumably due to the branched structure (Stolle-Smits et a1.,1995) whereas the other part is cross-linked with Ca ++. Stel~lization also altered the structure of the native OXSP as shown from the changes in DM, DAc, neutral sugars composition and the HPSEC patterns. The results suggested that highly methylated segments present in the OXSP were degraded by 13-elimination (Ryden and Selvendran, 1990). This alteration in the middle lamella pectin probably contribute in the weakening of the intercellular adhesion. Modification in the canning process either by addition of Ca ++ or by the new process (preheating and minimum amount of brine) lowered the harsh effect of sterilization. Addition of Ca ++ in the conventional canning allowed the available free carboxyl groups to cross-link and to form calcium-pectate gel which retarded, partly, the pectin solubilization into the brine and contributed in the intercellular adhesion. This cross-link selectively took place with those molecules having the lowest DM, and DAc, and poorly branched. In the new process, preheating activated the native PE which augmented the amount of free carboxyl groups and promoted pectin gelation with Ca ++naturally present in the tissue. Here the Ca ++ ions specifically cross-linked with the carboxyl groups while in conventional canning they were also bound in other positions which were not involved in tissue firmness. Furthermore, in the new process the prolongation of preheating time and increasing the amount of Ca ++ (data not shown) resulted in higher fimness. This was accompanied by slightly higher retention of OHSP. Since this pectin is linked via side-chains to other cell wall constituents by ester and]or covalent linkages, it is assumed that demethylation also occurred in the main chain, consequently 13-elimination breakdown in the demethylated segments is prevented.
Acknowledgment We thank Mrs. C. Tricker for her assistance in some experiments
508 References
Adams, J.B. and Robertson A. (1987), Texture technical memorandum, Nr. 449,The Campden Food Preservation Research Association, England Bartolome, L.G. and Hoff J.E. (1972), J. Agric. Food Chem. 20, 266-270. Batisse, C. Fils-Lycaon, B. and Buret, M. (1994) J. Food Sci. 59, 389-392. Ben-Shalom, N.; Plat, D.; Levi,A. and Pinto,R. (1992), Food Chemistry 44, 251. Blumenkrantz, N. and Asboe-Hansen, G. (1973), Analytical Biochemistry 54,484. Carpita, N.C. and Gibeaut, D.M. (1993). Plant J. 3, 1-30. Demarty, M.; Morvan, C.and Thellier, M. (1984), Plant, Cell Environ 7, 441-448. Doesburg, J.J. (1965), IBVT-Commun. Nr. 25 Dubuis, M.; Gilles, K.A.; Hamilton, J.K.; Rebers, P.A. and Smith, F. (1956) Anayltical Chemistry 20,350-356. Englyst, H.N. and Cummings, J.H. (1984), Analyst 109, 937-942. Gierschner, K. and Philippos, S. (1992), European Patent Nr. 81092. Jarvis, M.C. (1984), Plant, Cell Envitron. 7, 153-164. Keegstra, K.; Talmadge, K.W.; Bauer, W.D. and Albersheim, P. (1973), Plant Physiol. 51, 188-197. Lee, C.Y.; Bourne, M.C. and Van Buren, J.P. (1979), J. Food Sci. 44,615-616. Massiot, P.; Rouau, X. and Thibault, J.F. (1988) Carbohych'. Res. 172, 217-227. Massiot, P.; Guiller, I.; Baron, A. and Drilleau, J.P. (1992), Lebensm. Wiss.uTechnol. 25, 559-563. McCann, M.C. and Roberts, K.(1991), in Lloyd, C.W. (Ed) The Cytoskeletal Basis of Plant Growth and Form, Academic Press, London, pp. 109-129 Plat, D.; Ben-Shalom, N. and Levi, A. (1991), Food Chemistry 39, 1-12. Quinero-Ramos, A,; Bourne, M.C. and Anzaldua-Morales, A. (1992), J. Food Sci. 57, 1127-1128. Ryden, P. and Selvendran, R.R. (1990), Carbohydrate Res. 195,257-272. Saeman, J.F.; Moore, W.E. and Millet, M.A. (1963), in Whistler, R.L. (Ed) Methods in Carbhydrate Chemistry, Academic Press, London, pp. 54-70. Sajjaanantakul, T.; Van Buren, J.P. and Downing, D.L. (1989) J. Food Sci. 54,1272. Selvendran, R.R. (1985), in Roberts, K.; Johnston, A.W.B.; Lloyd, C.W.; Shaw, P.; and Woolhouse, H.W. (Eds) The Cell Surface in Plant Growth and Development, Company of Biologists, Cambridge, England.. Sterling, C. (1968), J. Food Technol. 3, 367-371. Stevens, J.H. and Selvendran, R.R. (1984), Carbohydr. Res. 128,321-333. Stolle-Smits, T.; Beekhuizen, J.G.; van Dijk, C.; Voragen, A.G.J.and Recourt, K. (1995) J. Agric. Food Chem. 43, 2480-2486. Van Buren, J.P. (1986) in Fishman, M.L. and Jen, J.J. (Eds.) Chemistry and Function of Pectin, ACS Symp. Ser. 310, 190-199. Van Buren, J.P. (1991), in Walter, R.H. (Ed) The Chemistry and Technology of Pectin, Academic Press, London, pp.l-22. Van Buren, J.P. and Pitifer, L.A. (1992), J. Food Sci. 57,1022-1023. Voragen, A.G.J.; Schols, H.A. and Pilnik, W. (1986), Food Hydrocolloids 1, 65-70.
STRUCTURE, PHYSICAL AND CHEMICAL PROPERTIES OF PECTINS
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J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V.All rights reserved.
511
Isolation and sequential extraction of cell wall polysaccharides from soy meal M.M.H. Huisman, H.A. Schols and A.G.J. Voragen
Wageningen Agricultural University, Department of Food Science, Bomenweg 2, 6703 HI) Wageningen, The Netherlands
Abstract Cell wall material was isolated as Water Unextractable Solids (WUS) from soy bean meal. WUS contained 89.9% carbohydrates, representing 92% of the carbohydrates present in the soy bean meal. Galactose, glucose, arabinose and uronic acids were the major constituent sugars. WUS was sequentially extracted with chelating agent, dilute alkali, 1 M alkali and 4 M alkali, to leave a cellulose-rich residue. All extracts were characterised by their sugar composition. For further characterisation, ChSS was fractionated by anion-exchange chromatography.
INTRODUCTION
The objective of the project described is to obtain insight in the relation between the chemical fine-structure of polysaccharides from soy bean cell walls and their functional properties in industrial products and how they effect processing. Soy meal is of great importance in the feed industry. The application of the (modified) soy bean cell wall polysaccharides as a food additive will be investigated. The obtained knowledge of the polysaccharide structures will also be used in studies concerned with the improvement of the in vivo digestibility of these polysaccharides.
METHODS
Isolation of Water Unextractable Solids (WUS) The Water Unextractable Solids were isolated from dehulled, defatted, untoasted soy bean meal (particle size < 0.5 mm) by removal of cold water solubles, proteins and starch. The soy bean meal was extracted with cold water, a solution containing sodium dodecylsulphate and 1,4-dithiothreitol, and incubated with a-amylase, to yield the CWS, SDSS and HWS
512 fraction, respectively. The CWS fraction was ultra-filtrated, resulting in a filtrate (UFF) and a retentate (UFR).
Sequential extraction of WUS Sequential extraction was performed as described by Redgwell and Selvendran [ 1]. WUS was sequentially extracted with chelating agent (ChSS), dilute alkali (DASS), 1 M alkali (1 MASS) and 4 M alkali (4 MASS) and a cellulose-rich residue (RES) remained.
Anion-exchange chromatography ChSS was fractionated on a column (550 x 15 mm) ofDEAE Sepharose Fast Flow using a Hiload System (Pharmacia), which was initially equilibrated in 0.005 M NaAc-buffer pH 5.0. The sample was dissolved in water, the insoluble residue was removed by centrifugation and the supernatant was applied onto the column. After applying the gradient shown in Figure 1, the residual polysaccharides were washed from the column using 0.5 M NaOH. Fractions (23 ml) were collected and assayed by automated methods [2,3] for total neutral sugars and uronic acids.
Analytical methods Protein content was determined by a semi-automated micro-Kjeldahl method [4]. The conversion factor used was 6.25. Starch content was determined using the enzyme-kit of Boehringer/Mannheim. NSP content of soy bean meal and HWS was determined by the method of Englyst and Cummings [5]. The starch was enzymatically hydrolysed, the residue was dried and the sugar composition was determined. Neutral sugar composition was determined by gas chromatography according to Englyst and Cummings [5], using inositol as an internal standard. The samples were treated with 72% H/SO4 (lh, 30 ~ followed by hydrolysis with 1 M H2SO4 for 3h at 100 ~ and the constituent sugars released were analysed as their alditol acetates. Uronic acid content was determined as anhydro-uronic acid (AUA) by the automated colorimetric m-hydroxydiphenyl assay [2,3,6] using an auto-analyser (Skalar Analytical BV, Breda, The Netherlands). Corrections were made for interference by neutral sugars present in thh sample.
513 RESULTS
The yield and the composition of the fractions from soy bean meal obtained with isolating WUS is shown in Table 1. The removal of cold water solubles, proteins and starch from soy meal was successful. The larger part of the material appeared in CWS, 59.1%. UFF contained mainly oligosaccharides and some water soluble proteins and UFR contained mainly water soluble proteins. The solution of SDSS and DTT extracted the residual proteins from the soy meal and the extract consisted for over 80% of proteins. Since the yield of the HWS fraction is only 0.4%, the composition is not discussed here. The remaining WUS contained 90% of NSP and the yield was 15.7%, which indicates that from the polysaccharides present in soy meal 92% was recovered in the WUS. By isolating WUS a fraction is obtained in which almost all cell wall polysaccharides are recovered and which contained only little other components. Table 1.
Yield and composition of fractions from soy bean meal as percentage dry weight. soy bean meal UFF UFR SDSS HWS WUS Yield 100 19.5 39.6 18.5 0.4 15.7 Protein content 57.3 21.1 87.8 84.2 15.4 2.1 Starch content 1.0 0 0 0 8.5 0 NSP content 15.4 50.2 13.7 3.0 43.3 89.9 The determination of the sugar composition was performed with and without prehydrolysis to determine the cellulose content. Cellulose was present in soy meal and in WUS, the content was respectively 17.2 and 17.9 mol%. Both soy meal and WUS contained mainly galactose, glucose (cellulose), arabinose and uronic acids and their sugar compositions were very similar. This indicates that no sugar residues were specifically removed during the isolation procedure. Table 2. extract WUS ChSS DASS 1 MASS 4 MASS RES
Sugar composition of extracts from so), WUS as mol%. yield rha fuc ara xyl man ~al glc AUA 100 2 2 19 8 2 28 20 20 38 2 3 24 6 1 36 1 28 7 2 3 24 6 1 37 1 27 16 2 3 23 11 1 34 5 21 7 1 3 12 27 2 18 26 11 18 1 0 3 3 4 2 75 12
WUS was sequentially extracted, which resulted in two pectin-rich extracts (ChSS and DASS), two hemicellulose-rich extracts (1 MASS and 4 MASS) and a residue. The yield of these extracts on sugar basis and the sugar composition is shown in Table 2. ChSS and DASS were rich in arabinose, galactose and uronic acids. These were, as expected, pectin-rich extracts and had a very similar sugar composition. The 1 MASS fraction was also rich in arabinose, galactose and uronic acids, but next to that it is also enriched in
514 xylose. The 4 MASS fraction was rich in xylose and glucose, which may indicate the presence of xyloglucans in this extracts. The residue was, as expected, rich in glucose (cellulose). The polysaccharides present in the pectin-rich extract ChSS were fractionated, based on their charge density for further characterisation. The elution pattern of ChSS is shown in Figure 1, and it can be seen that the relative uronide content of the fractions increased with increasing salt concentration of the eluens.
2.5
5O0 450 400
2
350
I
I
300 ~9
250
~
200
E
II
III
I
IV
I
I
9
V
I
....-
.. "
g
1.5 *~ Z
,, .,
1
-
150 100
0.5
~
_,,~.:.-.~-.-" ,
I
I
I
500
1000
1500
2000
Elution
Figure 1.
Table 5.
(nil)
Elution profile of ChSS on DEAE Sepharose Fast Flow. Eluent molarity ( . . . ) , galacturonic acid ( ~ ) and neutral sugars ( __. ).
Yield and sugar composition of the residue and the pools obtained after anion-exchange chromatography of ChSS expressed in mol%. Yield (% sugars) rha fuc ara xyl man gal 81c AUA
ChSS residue pool I p6ol II pool III pool IV pool V alkali wash recover),
volume
0 3000
2500
8.2 24.8 14.7 13.2 11.2 19.0 7.4 98.4
total
1.8
2.8
24.0
6.1
0.7
35.8
0.6
28.1
54.5
1.7 1.8 2.1 2.2 1.9 1.8 1.5 104
1.8 2.7 2.8 3.5 3.1 2.3 2.1 97
21.1 28 8 23 8 22 4 22 7 20.6 213 98
7.9 6.3 6.1 8.1 8.1 6.9 7.1 114
t 0.5 0.4 0.6 0.6 1.1 1.7 100
33.7 40.6 45.4 38.7 34.8 29.3 32.4 103
8.5 0.9 0.9 1.1 1.6 1.4 2.1 263
24.4 18.7 18.6 23.6 27.3 36.6 31.9 88
15.4 93.1 88.2 79.6 84.2 71.3 37.0
515 The recovery of this fractionation was 98.4%. The pools were characterised by determining their sugar composition, Table 3. All pools and the residue were rich in arabinose, galactose and uronic acids and differ in the ratio neutral sugars:uronic acids as was already seen from Figure 1. At the same time a shift from galactose to arabinose took place, the ratio arabinose:galactose increased from 1:1.9 (pool II) to 1:1.4 (pool V). These CDTA-extractable pectins contain an arabinogalactan and significant quantities of xylose residues, probably present as xylogalacturonans.
CONCLUSIONS
The isolation of polysaccharides from soy meal was successful, WUS contained only 2.1% of protein and 92% of the polysaccharides present in soy meal were recovered in WUS. The sugar composition of both the soy meal and WUS are similar and allow the conclusion that during the isolation procedure no sugar residues were specifically removed. The pectin-rich extracts (ChSS and DASS) obtained after sequential extraction of the WUS were the most abundant. Further research is directed to the determination of the fine-structure of the pectins and hemicelluloses isolated from soy meal, using chromatography and degradation with specific enzymes. With these results a model of the polysaccharides present in the cell wall of soy will be formulated. Furthermore, application directed experiments will be performed to obtain information about structure-function relationships.
ACKNOWLEDGEMENTS
This research is supported by the Dutch Technology Foundation (STW), Gist Brocades and the Animal Feeds Commodity Board (PVV).
REFERENCES
1. 2. 3. 4. 5. 6.
Redgwell, R.J. and Selvendran, R.R. Carbohydr. Res., 157 (1986) 183-199. Thibault, J.-F. Lebensm. Wiss. Technol., 12 (1979) 247-251. Tollier, M. and Robin, J. Ann Technol. Agile., 28 (1979) 1-15. Roozen, J.P. and van Boxtel, L. De Ware(n) Chemicus, 9 (1979) 196-200. Englyst, H.N. and Cummings, J.H. Analyst, 109 (1984) 937-942. Blumenkrantz, N. and Asboe-Hansen, G. Anal. Biochem., 54 (1973) 484-489.
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J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All fights reserved.
Modelling a pentasaccharide fragment of rhamnogalacturonan I. Max Broadhurst ~, Soizic Cros:, Rainer Hoffmann3, William Mackie 1 & Serge P6rez 1.
Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom. z Ingenierie Mol6culaire, Institut National de la Recherche Agronomique, Nantes, F-44026, France. 3 Unilever Research Colworth Laboratory, Colworth House, Sharnbrook, Bedford MK44 1LQ, United Kingdom. *Author for correspondence at: CERMAV-CNRS, BP 53X, 38041 Grenoble C6dex, France.
Abstract
A pentasaccharide fiagment related to rnamnogalacturonan I has been subjected to an exhaustive modelling studies using the MM3(92) and CICADA protocols. The results suggest that five major low-energy families of conformers may be considered in evaluation of the conformations of the pentasaccharide. The relative populations and energies of the families were also calculated and members superimposed on to the minimized lowest-energy conformation to show how sugar residues in different families differ in flexibility. The analysis shows that the non reducing end of the pentasaccharide is the most flexible part of the molecule. Nevertheless, the two most populated families (covering about 75% of the overall population) correspond to only one structure for the rhamnogalacturonan backbone having a stereoregular arrangement with a fairly extended conformation. INTRODUCTION Pectins are major plant cell wall constituents commonly used in the food industry as < additives. In biology, they are multifunctional compounds whose functions include structural, physiological, developmental and defensive roles. In industry, the applications as stabilizers, thickeners, and gel formers depend largely on the physical properties of their aqueous solutions. Chemically, pectic polysaccharides are complex structures co!ataining several component structural motifs. The pectic backbone is considered to consist of << smooth >> homogalacturonan regions interspersed with <> rhamnogalacturonan regions. The latter are described as <>because of the attachment of side chains (mainly galactans, arabans and arabinogalactans) at the (1-2)-linked aq~-rhamnose residues[I-3]. Energetically stable conformations of the rhamnogalacturonan backbone with various attached side chains polysaccharides have already been constructed using a new molecular builder procedure (POLYS) [4].
517
518 In the present study the pentasaccharide fragment related to the backbone structure of rhamnogalacturonan I, together with its rudimentary, galactan side-chain, has been subjected to further exhaustive modelling study using the MM3(92) [5] and CICADA protocols [6]. a- L- Rh ap-( 1-->4)-a- D-Ga ipA-( 1-->2)-a- L-Rh ap-( 1-->4)-a-D- GalpA 4 l
[5-o-Gaip The present study complements a parallel chemical synthesis of the target pentasaccharide and its di- and tri-saccharide precursors. The availability of these model compounds will allow conformational analysis by NMR spectroscopy to proceed with the calculation of features such as inter-hydrogen and inter-oxygen distances and other parameters for comparison with the theoretical modelling data reported here. The results should provide further conformational insight into factors such as the structure and functionality of rhamnogalacturonan I, including the function of rhamnose units as sites for further enzymatic glycosylation.
METHODS
A schematic representation of the pentasaccharide fragment of rhamnogalacturonan I is shown in Fig. 1 along with the labelling of the residues (from A to E) and the atoms of interest. The torsional angles of the glycosidic linkages of a disaccharide I (1-+x) J are defined as = O5I - C II - OxI - CxJ and W = C II - OxI - CxJ - C(x+l)J. The signs of the torsion angles are in agreement with the IUPAC-IUB conventions [7].
OH
COOH 14
I O
HO
OH
0 4 ~ O \ HOOCH 3
Fig 1. The pentasaccharide fragment of RGI along with labelling.
519
The MM3(92) force field [8-10] was used to generate relaxed maps for each of the constituent disaccharide fragments of the pentasaccharide. The MM3 force-field has been demonstrated to be successful for the modelling of many carbohydrates and it has been shown to account for the exo-anomeric effect satisfactorily [ 11 ]. Prior to the calculation of the relaxed maps, all starting conformers were first optimized by MM3 using the block-diagonal minimization method, continuing until the energy changed less than 0.00008 x n kcal/mol per 5 iterations, where n is the number of atoms in the structures. The dielectric constant (~) was set to 78.5 to simulate diminished electrostatic effects in aqueous solutions. The optimized coordinates were used as starting points for the calculation of the corresponding relaxed residue maps. The energies were calculated on a 10~ by 10 ~ grid in ~ and W. The use of CICADA (Channels in Conformational space Analyzed by Driver Approach) interfaced with MM3(92) enabled modelling to be undertaken. The procedure is best described as a method using an optimized tree branch search with full memory. The analysis of the potential energy hypersurface (PES) was explored for the pentasaccharide fragment of rhamnogalacturonan I. The potential energy hypersurface (PES) of the pentasaccharide was explored. The optimum analysis requires <> maps of the constitutent disaccharide units to be computed first, in order to obtain the low energy minimum torsion angles 9 and W. From these, twelve starting structures, all having low energy conformations, were generated. These starting structures were first pre-optimized by 1,,,,.,., using the block-diagonal method. During the CICADA search all of the eight glycosidic torsion angles were driven. The remaining torsion angles of the side chains were monitored (Table 1). Table 1: Driven and monitored torsion angles during the CICADA search.
Torsional Angle
Driven Monitored
Torsional Angle
Driven Monitored
AO5-AC 1-AO 1-BC4 9 1 AC 1-AO1-BC4-BC5 qJ 1 BO5-BC 1-BO 1-CC2 9 2 BC1-BO1-CC2-CC3 qJ2 BO5-BC5-BC6-BO6 CO5-CC 1-CO 1-DC4 9 3 CC 1-CO 1-DC4-DC5 W3 DO5-DC5-DC6-DO6 EO5-EC 1-EO1-CC4 ~ 4 EC1-EO1-CC4-CC5 q/4 EO5-EC5-EC6-EO6 AO5-AC5-AC6-AH61 AC 1-AC2-AO2-AHO2 AC2-AC3-AO3-AHO3
Driven Driven Driven Driven g~civen Driven Driven Driven Driven Driven Driven Monitored Monitored Monitored
AC3-AC4-AO4-AH4 BC 1-BC2-B O2-BHO2 BC2-BC3-BO3-BHO3 CC2-CC3-CO3-CHO3 CO5-CC5-CC6-CH61 DO5-DC 1-DO 1-DC7 DC 1-DC2-DO2-DHO2 DC2-DC3-DO3-DHO3 EC 1-EC2-EO2-EHO2 EC2-EC3-EO3-EHO3 EC3-EC4-EO4-EHO4
Monitored Monitored Monitored Monitored Monitored Monitored Monitored Monitored Monitored Monitored Monitored
A search of 20 ~ was applied to all driven angles. Two conformations were considered different if at least one of the driven or monitored angles differed by 30 ~ A relative cut-off of 50 kcal/mol was applied for exploring the PES, whereas a relative energy cut-off of 5 kcal/mol was applied for considering a new geometry as new starting conformer. The search was stopped after 1850 minima on the PES were found. The total number of MM3 calculations was 29000. The conformations and transition states found by CICADA were analyzed by the PANIC program [12] which explores the paths along the PES. Conformations were clustered into families and their relative populations were calculated applying a Boltzmann distribution at a temperature of 25~
520
RESULTS AND DISCUSSION As a first step in the molecular mechanics analysis of the pentasaccharide, fully relaxed energy maps of the component disaccharide fragment were constructed by means of the molecular mechanics force-field MM3(92). In Fig. 2, the adiabatic energy maps as a function of 9 and q~ for each type of disaccharide investigated, i.e. for a-l.-Rhap-(1---)4)-a-r~-GalpA, a-r~-GalpA-(l~2)-a-l.-Rhap anti fS-r)-Galp-(l~4)-ct-l.-Pdaap are shown.. In every map, the global energy minimum, as well as the different local energy minima are indicated. Iso-energy contours have been plotted at regular intervals of I kcal/mol up to 8 kcal/mol above the global energy minimum in each map. For the a-].-Rhap-(l--)4)-ct-r~-GalpA disaccharide., two low energy regions are found on the relaxed map (Fig. 2a), in which three minima are found. Two belong to the more extended low energy region with (~, W) values of (290, 160) (I), (270, 80) (II). The third-III is quite high in energy and is located about (270, 290). The relaxed potential energy surface computed for the a-r)-GalpA-(1-~2)-a-T.-Rhap disaccharide is displayed in Fig. 2b. Three low energy conformations, I, II, and III, are found; they are respectively located at (~, W) values of (80, 200), (100, 220) and (90, 90). The potential energy surface computed for the [3:r)-Galp-(l~4)-a-l.-Rhap is depicted in Fig. 2c, where three low energy conformers are found. They are respectively located at (~, W) values of (280, 290) (I), (280, 150) (II) and (220, 11 O) (III). .
300-
.
.
.
.
[ A--B,,I
.
.
360
I
240-
240-
~18o
,I,
-
(a)
f 8-C
180-
20-
120-
(b)
.i
60-
0
" ...... ' 0
I 6O
'
"
I 120
'
'
I
'
"
I
180
'
"
I
'
';
0
'
~
"
0
360
24O
1
'
'
60
I
'
'
120
I 180
"
'
1
'
'
240
I 300
'
(P
' 360
! D--E, ! 300-
240-
~leo
-
120"
(c)
60-
0
' 0
'
"I 60
'
'
I 120
'
'
I 180
'
'
I 240
'
'
I 300
'
' 360
Fig. 2. Potential energy surface computed as a function of 9 and qJ for a-T.-Rhap-(l---)4)-a-r~GalpA, a-r~-GalpA-( 1--)2)-ct-l.-Rhap and [3-~-Galp-( 1----~4)-ct-l.-Rhap. Isoenergy contours are drawn with interpolation of I kcal/mol above the minimum, up to 8 kcal/mol. On each surface, the location of the low energy conformers is indicated.
521 The complete ensemble of conformations resulting from the CICADA analysis has been clustered into different conformational families, within an energy window of 3 kcal/mol above the global minimum. Five families were found to have relative Boltzmann populations of at least 1%. The percentage of occurrence of the five familes are 57.5%, 16.6%, 6.8%, 4.5% and 3.6%, respectively. Of each of these families, the lowest energy conformation is listed in Table 2, along with the corresponding torsion angles for all glycosidic linkages.
Table 2: Conformational Families Derived from CICADA ~1
qJl
(I)2
qJ2
(I)3
W3
(1)4
qJ4
Erel
%
266.5
149.9
78.2
203.3
268.7
152.8
282.3
142.5
0.00
57.49
0.168
16.65
1.00
6.85
1.42
4.47
1.56
3.59
Family 1 Best Conf Min. Values
263.5
145.1
75.0
200.1
266.2
147.4
279.3
137.6
Max. Values
279.4
152.6
86.7
211.1
282.9
153.5
283.1
149.0
Best Conf..
285.8
156.3
79.7
204.6
286.7
158.7
282.1
145.3
Min. Values
285.8
156.3
79.7
203.5
286.7
158.1
281.1
145.3
Max. Values
287.(i)
157.2
79.8
204.6
287.4
158.1
282.1
146.5
Best Co~ff.
274.0
145.8
95.2
271.3
271.2
148.7
283.2
141.7
Min. Values
270.8
144.4
93.4
269.1
270.4
147.5
280.4
139.8
Max. Values
276.4
148.3
101.5
273.3
288.3
158.6
283.5
145.6
266.1
90.1
77.4
204.2
272.1
153.5
281.3
144.3
Family 2
Family 3
Family 4 Best Conf. Min. Values
260.1
80.80
75.70
202.8
266.1
150.5
278.5
139.0
Max. Values
266.1
90.1
83.4
215.0
272.9
150.5
282.5
145.9
Best Conf.
265.9
149.5
80.4
203.0
265.8
79.9
283.0
140.8
Min. Values
265.9
148.2
78.5
202.6
259.1
79.9
280.5
139.0
Max. Values
281.3
153.5
81.2
205.7
266.4
86.9
283.2
142.7
Family 5
In Fig. 3 all conformers are plotted within 3 kcal/mol above the global minimum found by CICADA for each of the glycosidic torsions, superimposed on the corresponding disaccharide relaxed map. Fig. 4 clearly indicates that most of the local minima present in the corresponding disaccharide fragments are also explored along the PES of the pentasaccharide by CICADA.
522
=_ r]
360 ~-7',
3oo
. . . . . .
I A
(~
'""'
' "
B-!
9 "' '
360 q-
'
300
. . . . . . . . .
I-B--C
-I .+
i
-
F3
24o -4
240
-4
"
1201 180
'Z--]i 1
1
0
360
i
I
60
FI, F2, F4, F5
I J
120-I
4 J
60-I 4 4
I
'i
~
120
I'
'i
i
i
180 PHI
I
t
O ~
I
240
300
380
(~
. . . . . .
' --
'. . . . . . . . . .
300 [ C - - D l
0
-+
360--,
1.o" FS'
60
0
,
,
l'"
60
,
,
'i
120
'
'
,
,
!
'
120
," . . . . . .
,
'l
180 PHI
'
+'-'~-~
'
i
240
'
'
'
9
|
i
'
|
9
300
360
1 ~
El
'~
F.~
-
120
'
i
60
FI, F2,
240-
Ok.
4
,
- VD
+~ 1 8 0 120
,
300 -
240 FI, F2, F3, F4,
0
:
'""
.
FI, F2, F3, F5
a_
9. . . . . .
60-
I
180 PHI
'
'
I
240
'
'
~
300
'
"--
360
0-0
60
120
180 PHI
240
300
360
Fig. 3. Projections on the.(~, qJ) maps of the CICADA conformational search of the pentasaecharide. The dots indicate the values of all the optimized conformations determined by CICADA at each glycosidic linkange in 8 kcal/mol energy window. For comparison, the isocontours, drawn in 1 Kcal/mol steps with an outer limit of 8 kcal/mol, represent the energy level of each disaccharide and calculated with the relaxed grid search approach. Dashed regions represent the locations of the low energy conformation of the pentasaccharide plotted on the potential energy surfaces of the constituting disaccharide segments.
523 In Fig. 4, for each of the families F 1, F2, F3, F4, and F5 the global minimum has been plotted, together with a number of structures that were clustered in the same family, exhibiting conformational freedom within each of the individual families. It is interesting to note that families F1 and F2 which represent almost 75% of the overall population, generate very comparable structures since their conformations about the glycosidic linkages belong to the same conformational energy wells, i.e. the (I) well for ct-~,-Rhap-(1---~4)-a-r~-GalpA constituents, the (I) well for a-r~-GalpA-( 1-->2)-a-I.-Rhap and the (I) well for [3-r)-Galp(l~4)-a-I.-Rhap. Families F3 and F4 correspond to conformations where the (II) energy wells of the two non-reducing units are occupied. The analysis indicates clearly that most of the conformational freedom, albeit restricted, is located at the non reducing end of the pentasaccharide. Conversely, the branching of the 13-r)-Galp- residue linked (1--)4) to the ct-l.Rhap does not induce any further flexibility since only one low energy conformation is found. From the complete ensembles of conformations found by the CICADA analysis of the pentasaccharide, ~H-~H distances have been calculated.These distances (not shown here) can be compared to the interesidual ~H-~H distances estimated from the NOE build-up rates of the corresponding cross-peaks in the 2D NOESY spectra, to be made un future experiments.
Family 1 (57.5 %) (F1) ~ .
Family 4 (4.5 %) (F4)
Family 2 (16.6%) (F2) ~ .
Family 3 (6.8 %) (F3)
Family 5 (3.6 %) (F5)
Fi,, 4 The five major families and their percenta,,e populations at 25~
524 Calculations of inter-residue oxygens distances were also made and, suprisingly, these indicated that none of the low-energy confomeres possessed strong intra-molecular hydogen bonds of the type commonly found in other oligosaccharide and polysaccharide systems (Table
3). Table 3. Inter-oxygen distances giving arise to possible hydrogen bonds. Distances
(A)
AO1---BO3 AO1---BO5 BO1---CO3 BO 1---CO5 BO5---CO3 BO6---AO5 CO 1---DO3 COl---DO5 CO3---EO1
Family 1
Family 2
Family 3
Family 4
Family 5
2.85 2.76
2.91 2.83
2.84 2.81
2.74 2.79
2.85 2.75
2.73
2.74
2.88
2.73
2.73
2.91 2.87 2.76 2.89
2.87
2.85 2.67
2.87
2.92 2.81 2.91
2.85 2.77 2.90
2.91 2.91 2.88 2.81 2.90
2.72 2.80 2.90
The present results must be considered in the context of the structural description of the polysaccharide backbone. Extended to local helical structures, the two major conformations would generate a right-handed, near integral (or pseudo) three-fold helical structure with an advance per disaccharide h = 6.84 A. In such a stereoregular arangement the rhamnogalacuronan chain adopts a fairly extended conformation. These findings provide strong support to previously stated conclusions [ 13] i.e. 1/the presence of rhamonse units in a strictly alternating fashion only slightly reduces the extension of the polysaccharide chains, 2/ the extended overall conformation remains relatively unchanged as a consequence of the selfcancellation of the kinking effects of successive paired rhamnose units.
4. CONCLUSIONS A pentasaccharide fragment of rhamnogalacturonan I has been throroughly examined by means of molecular mechanics calculations. The relaxed energy maps of each of the different component disaccharides generated by MM3 have provided insight into the conformational possibilities and flexibilities of the different glycosidic linkages in the polysaccharide. A CICADA analysis of the pentasaccharide structure demonstrated a total of five conformational families. Within each family, members which included transition states that could be related to possible routes of interconversions, were discovered. The relative populations were also calculated and members superimposed onto the minimized lowestenergy conformation to show how sugar residues in different families differed in flexibility. This showed that the non reducing end of the pentasaccharide is the most flexible part of the molecule. Familes 1 and 2, which represent almost 75% of the overall population, correspond to only one structure for the rhamnogalacturonan backbone having a stereoegular arrangement with a fairly extended conformation.
525 Extrapolated to the pectic backbone, the present findings support previous descriptions concerning the stuctural role that may be ascribed to the presence of rhamnosyl units. When << smooth >) homogalacturonan regions are interspersed with <~ hairy )) rhamnogalacturonan regions there is no major effect on the extension and the orientation of the pectic polysaccharide. ACKNOWLEDGMENTS The authors are grateful to the BBSRC, INKA and Unilever Research plc for financial support (MRB), and the use of facilities. We also thank Dr. D. Perkins for his assistance with the mathematical calculations.
REFERENCES
1 2 3 4 5 6 7 8 9 10 11
12 13
N.C. Carpita and D. M. Gilbeaut, Plant Journal, 3 (1993) 1. H.A. Schols and A. G. J. Voragen, Carbohydrate Research, 256 (1994) 83. H.A. Schols, A.G.J. Voragen and I.J.Colquhoun, Carbohydrate Research, 256 (1994) 97. S.B. Engelsen, S. Cros, W. Mackie and S. P6rez, Biopolymers (in press). MM3(92), QCPE, Creative Arts Building 181, Indiana University, Bloomington, IN47405 J. Koca, J. Mol. Structr. (Theochem), 308 (1994) 13. IUPAC-IUB, Commission on Biochemical Nomenclature, Arch. Biochem. Biophys., 145 (1971)405. N.L. Allinger, Y. H. Yuh and J. -H. Lii, J. Am. Chem. Soc. 111 (1989); 8551. N.L. Allinger, Y. H. Yuh and J. -H. Lii, J. Am. Chem. Soc. 112 (1990); 8293. N.L. Allinger, Y. H. Yuh and K. Chen, J. Am. Chem. Soc. 114 (1992); 6120. A.D. French, R. S. Rowland and N. L. Allinger in Computer modelling of carbohydrates molecules, American Chemical Society, Washington, DC, French A. D. and Brady J. W. (Eds), (1990), pp 120. J.Koca and P.H.J. Carlsen, to be published (1996). S. Cros, C. Garnier; M.A.V. Axelos, A. Imberty & S. P~rez, Biopolymers (in press).
This Page Intentionally Left Blank
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All fights reserved.
527
Influence of s o m e cations on the reaction of apple pectin with a m m o n i a in homogeneous media P.Denev and Chr.Kratchanov Higher Institute of Food and Flavour Industries, 26 Maritza Blvd., 4002 Plovdiv, Bulgaria
Abstract The running of parallel reactions of hydrolysis, ammonolysis and depolymerization of apple pectin in aqueous solution of ammonia (1M) at 25~ were investigated. It was examined the effects of monovalent cations (Na§247 +) and divalent cations (Ca§ §247 when they were added as chloride salts. It was found that the relative rates of the above mentioned reactions, depend on the nature and concentration of the added salts as well. The chlorides of sodium,-potassium and calcium accelerate hydrolysis and depolymerization, while magnesium chloride delays these reactions. Ammonolysis was increased in cases of ammonium chloride addition.
Introduction The deesterification of pectin with ammonia has industrial application because of the following reasons: - relatively fast reaction in comparison with the acid deesterification, but the rate of the reaction may be controlled (in contrast to the alkaline deesterification with NaOH); - the obtained pectin has new technological characteristics which contribute its application in different branches of the food industry; - there is no need of any special resistant equipment to carry out the reaction, as it is in the case of acid deesterification ; - the main disadvantage of the alkaline deesterification-depolymerization due of 13-elimination - is avoided. There are not many publications in the specialised literature examining the deesterification in the presence of mineral salts. R.M.McCready et al.[1] demonstrate that the adding of O.1M NaC1 to 0.33 M ammonia hydroxide enhances the deesterification more than twice. The adding of other chlorides, such as KC1, MgC12 and CaC12 also enhances the reaction. The effectiveness of the ions in relation to the deesterification and rate constant is arranged in the following order: Ca>Mg>Na, K. The equal values of pH and ion strength prove that only the specific cation effect is responsible for the enhanced velocity. Reistma et al. [2] establish the positive influence of NaC1 on the deesterification of ester groups of amidated pectins with ammonium hydroxide, without discussing the mechanism of the catalytic action of the sodium chloride.
528 In the specialised literature is paid greater attention to the influence of alkali and alkali-earth cations on the stability of the pectin macromolecules in water solution. Lineweaver [3] reported that the stability of the pectin is increased in neutral or slightly acid media when the salts concentration in the solution is minimum. In our previous publication [4] we demonstrated that the ammonium salts of different organic and inorganic acids influence the reactions which are performed at the interaction of pectin with ammonia, as they accelerate the ammonolysis and retard the hydrolysis and degradation of the pectin macromolecules. In the present investigation we set before ourselves the task to check the influence of chlorids of different alkali and alkali-earth metals on the rate of the hydrolysis, ammonolysis and depolymerization of apple pectin in aqueous solution of ammonia.
MATERIALS AND METHODS
Preparation of Amidated Pectin. Industrial apple pectin was washed off ballast substances and metal ions by intensive stirring of the pectin in 60 % acid ethanol for 30 minutes; filtering through a glass filter G-3 and repeated washing with 60 % ethanol until a negative reaction of C1- towards AgNO3 is established; washing with 96 % ethanol, and after that the prepared pectin is dried. The characteristics of the prepared pectin are: degree of esterification - 69.6 %, anhydrouronic acid content - 72.5 %, ash - 0.14 %. In a 500 ml conical flask with a grinded plug are put 4.0 g purified pectin, 6 ml ethanol, and then dissolved with 300 ml distilled water. After keeping at 20 ~ for some time, 100 ml 4 M NH4OH containing the corresponding amount of salts, are added. The reaction continues for 60 minutes at constant temperature of 20+0.1 ~ The reaction is interrupted by coagulating of the pectin solution with a mixture of 500 ml ethanol and 50 ml concentrated HC1, cooled up to -5 ~ The pectin solution is poured in a thin flow into the HCl-alcohol mixture at continuous intensive stirring to smash the obtained microgels. After 30 minutes the coagulate is filtered and pressed, then washed with 60 % ethanol to wash off the C I , twice rinsed with ethanol and dried at 40 ~ The used chlorides were solved in ammonia solution. The amount of the added salts was counted in a way that their concentration in the reaction mixture was 0.01; 0.02; 0.04; 0.08 and 0.2 M. Analytical methods. The anhydrouronic acid content (AUAC), degree of esterification (DE), degree of amidation (DA) and ash content were determined according to the methods described in Food Chemical Codex II ed [5]. Calculations. For determination of the intrinsic viscosity [li] the prepared pectins were solved in an 0.1 M phosphate buffer with pH 6.0. The relative viscosity was determined by a glass. Ubbelhode viscometer at 25+0.1 ~ The flow time of solvent (to) was 81.8 seconds. At least six pectin solutions with different concentrations were measured in a way that their flow times (ts) comply the order 1.2to
(2). lisp/C = [li] + kh. [1"1]2.C In lirel/C=[li] - kk. [li]2.C where li =l = tJ to
(1) (2) q sp = 1"1ro~- 1
529 The degree of deesterification RD shows the percentage of the initial ester groups participating in the deesterification. RD = (DEo- DE). !00/DEo DEo initial degree of esterification; DE - final degree of esterification. The degree of ammonolysis RA shows the percentage of the reacted ester groups converted into amides ones. R A = DA/(DEo - DE) The degree of hydrolysis RH shows the percentage of the reacted ester groups converted into carboxyl ones RH = 1-RA -
RESULTS The influence of the various cations on the pectin deesterification as a summarized process is shown in Fig. 1. As it can be seen from the graphics the examined ions are divided into two groups. From one side, Na +, K + and Ca ++ enhance the deesterfication, as this trend is mostly expressed at Ca ++ ions. In the second group are NH4 + and Mg +§ ions which retard this process. It is surprising that the cations from the same group - alkali-earth - Mg +§ and Ca ++ influence the deesterification in a different way. Ca§ have higher expressed promoting effect than Na + and K § The highly expressed inhibiting effect of Mg++-ions may be explained by its complexforming action at ligand participation of the esterified carboxyl groups. The inhibiting effect ofNH4 + is considerably lighter expressed in comparison with the one of Mg § which impose the assumption for different mechanism of action. o
o
o
-1-
o o 0
t( o
0
0.0'2 0.04 0.06 0.06 0.1 0.12 0.14 0.16 0.18 0.2
o
,
0
,
,
c,~centration[M ~~4c~---!~
A
Ka
X
,
,
i
i
|
,
,
0.0~ 0.04 0.015 0.08 0.1 0.12 0.14 0.16 0.18 0.2
c~er~dti~ N ~
X
cac~
Figure 1. Effect of cations on the pectin deesterification
--i~4c
--tl-r~c
A- ~
.-~e
X caae
Figure 2. Effect of cations on the hydrolysis
530 As it is known the consumption of the esterified groups of the pectin under the influence of NH4OH leads to obtaining o f - C O O H and -CONH2 due to hydrolysis and ammonolysis. In Fig. 2 we present graphically the effect of the added chlorides on the hydrolysis of the ester groups, and in Fig. 3 the influence of the same salts on the ammonolysis of these groups. It is obvious from Fig. 2 that Na +, K + and Ca ++ ions have enhancing effect on the hydrolysis, expressed mostly at adding of CaC12. NH4+ and Mg ++ ions demonstrate again retarding effect with approximately same intensity as a function of their concentration. The data from Fig. 3 show that at all experiments with alkali and alkali-earth chlorides the amount of the formed amide groups is smaller than the content of the amide groups of the pectin from the control sample. Ca++ ions have the strongest retarding effect. Only at the experiments with NHaC1, higher values for the content of amide groups have been observed, but this increase is relatively small - about 10 %. co
i I
9 tg~
O TO
i
i
i
i
i
0~
ClOI
CI(]B
Cl0B
Qt
i
i
C1"12 Q'I4
i
i
i
Qt6
Q18
C~
armratn[M
0
Q~
Q~
Q~
Q~
Q1
Q~
QH
Q~
Q~
Q2
~maicn[M --~r+~c--I--r,bc ---/k-~ --%-~'~r ---~c~
Figure 3. Effect of cations on the ammonolysis
Frigure 4. Effect of cations on the pectin degradation
The pectin depolymerization was followed by viscosimetry. The effect of the added chlorides on the intrinsic viscosity of the obtained pectins strongly depends on the type and concentration of the cations. The ammonium and magnesium chlorides slightly enhance the viscosity in comparison with the control sample, witch is a sign of retarding of the reaction of 13-elimination. In samples with Na + and K + the depolymerization is slightly enhanced. It is interesting in the case of Ca++ ions that their influence enhance the degradation. DISCUSSION Reactions of hydrolysis (3), ammonolysis (4) and depolymerization (5) are carried out at the interaction of pectin with ammonia in aqueous media (Fig. 5). We consider the deesteri-
531 fication as a summarized process, resulting from the competitively performed hydrolysis (3) and ammonolysis (4), since the esterified carboxyl groups react either with O H or with NH3 up to obtaining correspondingly of-COOH or-CONH2.
e
OH/H2%
o__J. ~
~0oo0r
o__.~ o ..L-----~
/..z" coo ' ~4O H
o--Z ~
/../o,-
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/~OH "~
W~OH Lo~cT~/
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0
~
,
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~OH
,,.o,~)/,,,
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o"
%H H3C
H3COOC~ \
~
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'~"
(5)
Figure 5. Interaction of pectin with ammonia in aqueous media For the course of each reaction we can judge in two ways: 1. From the change in the degree of esterification and amidation (Fig. 1 - 3). 2. From the degree of transformation - expressed by the relative change of each type of carboxyl group: RD (degree of deesterification), RH (degree of hydrolysis) and RA (degree of ammonolysis). It may be noted from the data for RD (Table 1) that at the conditions chosen by us for performing of deesterification with ammonia a degree of transformation above 80 % is reached. Both competitive reactions of hydrolysis and ammonolysis are commensurable in regard of their rate. This fact is very favourable for investigating cations influence on the course of the deesterification and the rate of the investigated competitive reactions(3), (4) and (5). Influence of the Monovalent Ions. The addition of ammonium salts retard the course of the deesterification by changing the reaction rate of hydrolysis and ammonolysis. By increasing the concentration of the ions the conversion of the ester groups is reduced from 83.3 % for 0.01 M to 62.8 % for 0.2 M (without added salt this value is 84.05 %), and the ratio hydrolysis:ammonolysis is changed correspondingly from 53.8:76.2 to 37.3:62.7 (without added salt this ratio is 57.6:42.4). The interaction between water and ammonia leads to the following equilibrium: 9.
NH 3 + H 2 0
9
O
- - NH 4 + O H
(6
The addition of ammonium salts will change this equilibrium to the le~, i.e. the concentration of the ammonia molecules will be increased and the concentration of the hydroxyl anions will be reduced. Due to this reason the hydrolysis of the ester groups in pectin, expressed in reaction (3), will be retarded because of the reduced O H content in the solution, and reaction (4) will be favoured.
532 Table 1 Effect of somme soults on the hydrolysis and ammonolysis of ester groups express by RD, RH and RA Concentration of salts [M] 0,00 0,01 0,02 0,04 0,08 0,2 Degree of conversion of ester groups expressed by RD NHnC1 84,0 83,3 79,5 76,7 72,2 62,8 NaC1 84,0 84,2 84,8 87,0 87,4 90,8 KC1 84,0 84,1 84,3 85,9 86,6 89,2 MgC12 84,0 76,8 72,0 66,9 54,9 50,6 CaC12 84,0 84,5 88,4 91,1 92,1 97,2 Degree of conversion of carboxyl groups expressed by RH NH4C1 57,6 53,8 51,7 50,2 46,9 37,3 NaC1 57, 6 64,4 65,9 66,2 67,1 72,2 KC1 57,6 63,4 63,9 65,6 68,6 71,9 MgC12 57,6 56,3 54,9 51,7 50,0 54,8 CaC12 57,6 71,4 72,2 77,8 79,3 83,1 Degree of conversion of amide groups expressed by RA NH4C1 42,4 46,2 48,3 49,8 53,1 62,7 NaC1 42,4 35,6 34,1 33,8 32,9 27,8 KC1 42,4 36,6 36,1 34,4 31,4 28,1 MgC12 42,4 43,7 45,1 48,3 50, 0 45,2 CaC12 42,4 28,6 27,8 22,2 20,7 16,9 The behaviour of the added alkali cations is different. Na + and K § enhance the deesterification and the differences in the interaction of both cations at the separate reactions are inessential. The use of these ions in the deesterification of highly esterified pectin in either enzyme or chemical method shows that they only enhance the hydrolysis. Since in the investigated case they cannot influence the equilibrium (6), the enhancement of the hydrolysis leads to faster exhausting of the reactable -COOCH3 groups and thus is reduced the rate of the competitive ammonolysis. Influence'of the Divalent lons. It is well known that the divalent ions form bridges between the separate pectin macromolecules, as they react with the dissociated carboxyl groups. This interaction is especially strongly expressed when DE is less than 50 %, and the strength of these bridges depends on the type of the cation and pH of the media [6,7]. As a result of this may be formed either gel or precipitate, but in both cases there occurs spatial netting of the pectin macromolecules, accompanied by conformation change in the main pectin chain. We noticed in our investigations that in all cases when Ca++ ions were added to the pectin solution, either jellying or precipitation occurred atter a certain period of time, but this never happened when Mg +§ ions were added. In the experiments with MgC12 an opalescence of the ammonia solution was noticed, due to the insolubility of the obtained Mg(OH)2
533
Mg+~-2C1o + NH4+OH ~-----Mg(OH)2 +NH4+C1 1
(7)
Considering only this equation, it could be suggested that in the model systems when MgC12 is added, the deesterification is retarded because of the reduction of both concentrations - first o f O H , due to the formation of Mg(OH)2, and second, of NH3, due to the shiffing of the equilibrium (6) to the right. It seems that the retarding influence on the hydrolysis is stronger (Rn = 54.8 % for concentration 0.2 M, and RH = 57.6 % in the absence of salts) and the presence of relatively higher number of ester groups leads to an increase in the conversion of these groups into amide ones (RA = 45.2 % versus RA = 42.4 %). The total effect of both these reactions leads to a stronger retarding of the deesterfication. Calcium ions are known for their specific behaviour towards the uronic polysaccharide [8, 9]. The shape of the calcium ions corresponds exactly to the space enclosed by the links of the uronic residues in the pectin and alginic acids and this favours the formation of stable gel structure, so called "Egg-Box" model [10]. A lot of investigations, directed towards the interaction between Ca++ and pectin [ 11 - 13] show that this is not only an ion interaction with the carboxylic groups, but it is also a coordinative one with the hydroxyl groups. May be this interaction is in the grounds~of the enhancing effect which the calcium ions express towards the hydrolysis and the deesterification and the relatively high retarding of the ammonolysis. The ammonium salts retard the depolytmerization, which is expressed by an increasing of the ammonia concentration and reducing of the ammonium hydroxide. The addition of Na + and K + ions leads to enhancing of the process, but in this case the values are not so close as it is in the other processes. From the investigated divalent ions, we found out that Mg ++ retard the depolymerization, while Ca ++ enhance it. Comparing the influence of the ions on the performing reactions, we will notice that these which enhance the ammonolysis retard the depolymerization, and these which enhance the hydrolysis enhance the depolymerization as well. REFERENCES
1. R.M McCready, H.S.Owens, W.D.Maclay, Food Industries, 16 (1944) 794. 2. J.C.E. Reistma, J.F.Thibault, W.Pilnik, Food Hydrocolloids ,1 (1986) 121. 3. H. Lineweaver- J.Am.Chem.Soc. 67 (1945) 1292. 4. Chr. Kratchanov, P.Denev, M.Kratchanova, Int. J. Food Sci. & Technol. 24 (1989) 261. 5. Food Chemical Codex II (1972), Washington DC, National Academy Press. 6. M.A.V. Axelos, C.GamieR, J.F.Thibault, A/R Conf. Prec. 1991. 7. J.F. Thibault, M.Rinaudo, British Polymer Journal 172 (1985) 181. 8. E. Racape, J.F.Thibault, J.C.E.Reistma and W.Pilnik, Biopolymers 28 (1989) 1435. 9. R.L.Whistler and J.N.Be Miller, Advances of Carbohydrate Chemistry, 13 (1958) 289. 10. G.T.Grant, E.R.Moris, D.A.Rees, P.J.C.Smith & D.Thom, FEBS Lett. 32 (1973)195. 11. R Kohn., I.Furda, Collection Czechoslov. Chem.Commun 33 (1968) 2217. 12. R.G.Schweiger, J.Org.Chem. 29 (1964) 2973. 13. A Kawabata, S.Sawayama, H.Nakahara, T.Kamata, Agric.Biol.Chem. 45 (4)(1981) 965.
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J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V.All rights reserved.
535
Heavy metals binding by pectins" selectivity, quantification and characterisation V.M. Dronnet, C.M.G.C. Renard, M.A.V. Axelos and J.-F. Thibault INRA, centre de recherche agro-alimentaire, rue de la G6raudi6re BP 1627,44316 Nantes Cedex 03, France
Abstract Citrus and sugar-beet pectins with similar degrees of methylation were put in contact with divalent metal ions in presence of 0.1M NaNO3 or in pure water, pH studies and binding isotherms allowed to establish the same scale of selectivity for both pectins, decreasing as follows: Cu 2+ Pb2+ >> Zn2+ > Cd2+ ~ Ni2+ > Ca2+. Binding isotherms showed the same features for both pectins according to the ionic strength, the pectin concentration and the cation, but differences of behaviour between pectins were highlighted by characterizing the binding process. This was ascribed to the presence of acetylated hydroxyl functions on C2/C3 of the galacturonosyl units of sugar-beet pectins. " "
1. I N T R O D U C T I O N Standards imposed to the industrial waste streams charged in heavy metals are more and more drastic in accordance with the updated knowledges of the toxicity of mercury, cadmium, lead, chromium.., when they enter the human food chain after accumulating in plants and animals (F6rster & Wittmann, 1983). Nowadays, the use of biosorbents (Volesky, 1990) is more and more considered to complete conventional (physical and chemical) methods of removal that have shown their limits and/or are prohibitively expensive for metal concentrations typically below 100 mg.l-1. The possibility of using natural substrates from different origins has already been suggested: microorganisms (Brady & Duncan, 1994), humic substances (Kerndorff & Schnitzer, 1980), lichens (Richardson, 1995), rice milling by-products (Marshall et al., 1993) and so forth. Such cheap natural adsorbents would not require complex regeneration unlike commercial synthetic ion-exchange resins. Divalent metal ions binding by pectins, anionic polysaccharides exhibiting high binding capacities, was carried out as a model of metal binding by pectin-containing beet pulp.
2. MATERIALS AND METHODS 2.1. Pectin samples Pectins from sugar-beet (degrees of methylation (DM) 58 and acetylation (DAc) 14, 700 mg galacturonic acid (GalA) per g of dry matter) and citrus (DM=54, DAc=0, GalA content= 911 mg.g-1 dm) were provided by Copenhagen Pectin Factory Ltd (Denmark) and SBI (France), respectively. Experimental measurements of their Cationic Exchange Capacities (CEC), 1.65 and 2.38 mequiv.COO- per g for sugar-beet and citrus pectins respectively, were in excellent agreement with theoretical CECs calculated from the GalA content and the DM. Pectins were put in the acidic form by treatment with 0.1M HC1 in 70% ethanol at 4~ overnight.
536
2.2. Selectivity scale Acidic pectin samples were dissolved in 0.1 M NaNO3 with gentle stirring overnight. The pH was adjusted to -5.1 by adding 0.05 M NaOH. pH measurements were performed at 25.0 + 0.1~ after sequential additions of the metal ion, stirring (15 min) and rest (15 min). To determine the selectivity scale of metal ions binding by pectins by means of pH measurements, we assumed that the exchange of the protons carried by carboxyl functions by metal ions involved a pH-decreasing which is more pronounced when the affinity of pectins for a given metal ion is higher.
2.3. Quantification of the binding Pectins in the acidic form were dissolved either in pure water or in 0.1 M NaNO3 and put to pH -7.2 by adding 0.05 M NaOH. Various amounts of metal ions were added to pectins solutions at two different concentrations, for 2 h under stirring at 25.0 + 0.1 ~ Concentration of metal ions in solution at equilibrium was determined either by a potentiometric method using ion-selective electrodes for Cu2+ and Pb2+ or by a spectrophotometric method using tetramethylmurexide dye ( Kwak & Joshi, 1981) for Ni2+, Zn2+ and Ca2+.
2.4. Characterisation of the binding mode Experimental data obtained from binding quantification measurements were plotted according to the Scatchard representation (Scatchard, 1949). This allowed to characterize the mode of binding either in terms of presence of more than one class of binding sites or in terms of anticooperativity (concave curvature) and cooperativity (convex-shaped curves) i.e. the fixation of one ligand decreases or increases the affinity of the ligand for a neighbouring binding site of the macromolecule, respectively (Cantor & Schimmel, 1980). 3. R E S U L T S
3.1 Selectivity scale The experimental results are presented by plotting A[H30+], the loss of protons from the pectic ionic sites, against the ratio [Me2+]t/Cp where [Me2+]t and Cp are the total cation concentration (equiv.l-1) and the pectin concentration (equiv. COO-.1-1), respectively. A[H30+] 6 10-6 n
4 106
-
2 10-6- n o
n
0
O
[]
OO o
9
-
9
"
<>A
0
_n~
I 0
[]
-
0 ~
~
o%,e *
nOO
n
9
0 <>0
.0 < > A A A
l
1
0 [Me2+]t / C
P
1
2 [Me2+]t/C
P
Figure 1. Effect of metal ions binding on the release of protons into the medium for sugar-beet (A) and citrus (B) pectins at 2 mequiv. COO-.1-1, at pH~5.1 in 0.1 M NaNO3 at 25~ (0) Cu2§ (#) Pb2§ (0) Zn2§ (A) Cd2+, (v) Ni2+, (11) Ca2+.
537 As shown in figure 1, the same two groups of metal ions may be distinguished for both pectins. A first one was composed of Ca2+, Ni2+, Cd2+ and Zn2+, for which a small and quasilinear pH-variation was found. Cu2+ and Pb2+, composing the second group, displayed a much higher pH variation, reaching a plateau in the final stages. The same scale of selectivity was therefore obtained whatever the pectins, decreasing as follows: Cu2+ ~ Pb2+ >> Zn2+ > Cd2+-Ni2+ > Ca2+.
3.2 Quantification of the binding The experimental results obtained by measuring ions activity after equilibration with pectins are plotted as binding isotherms [Me2+]b/Cp vs [MeZ+]dCp where [MeZ+]b is the bound cation concentration at equilibrium (equiv.l-1) calculated from measured activity using previously calculated activity coefficients.
[pb2+]b / Cp 1.2 0.81-
/_~0
04L 0
0.4
0.8 1.2 [pb2+]t / Cp
Figure 2. Influence of the ionic strength and the polymer concentration on the binding isotherms of Pbz+ by sugar-beet pectins in water (empty symbols) and in 0.1 M NaNO3 (full symbols) at 25~ (11) 2 mequiv. COO-.1-1, (e) 8 mequiv. COO-.1-1; (--): total binding of added Pb2+. Binding isotherms presented the same features, as shown in figure 2 for Pb2+ and sugarbeet pectins, for the five metal ions and for both pectins. In water, binding isotherms were independent of the pectin concentration, giving rise to a single curve. They followed an ideal "stoechiometric isotherm" for Cu2+ and Pb2+ at least in the early stages. In presence of ionic strength, the binding of metal ions was lower but increased with the polymer concentration.
[Me2+]b / Cp
o o u
r
0.2 0
0
oo
9
f"
0.8 [Me2+]t / Cp 0
9
v
B 0.8 [Me2+]t / Cp 1.6
Figure 3. Influence the metal ion type on the binding isotherms of sugar-beet (A) and citrus (B) pectins at 2 mequiv. COO-.1-1 in 0.1 M NaNO3 and at 25 ~ Symbols as in figure 1.
538 Binding isotherms of the five metal ions, in presence of supporting salt, were compiled (figure 3) either for sugar-beet or citrus pectins. It became possible to differenciate different levels of binding depending on the metal ion, by simple comparison of the binding isotherms. The following order was found for both pectins: Cu2+ ~ Pb2+ >> Zn2+ -- Ni2+ > Ca2+.
3.3 Characterisation of the binding mode The mode of binding was characterised by replotting experimental data obtained from binding isotherms in terms of the Scatchard representation, [Me2+]b /(Cp.[Me2+]f ) VS [Me2+]b/Cp where [MeE+]f corresponds to the final ion concentration at equilibrium. Metal ion concentrations were here exp__ressedin molarity and Cp in number of chain.l-1 (using the weightaverage molecular weights Mw).
[Me2+]b / (Cp.[Me2+]f)
[Mea+]b / (Cp.[Mea+]f) 10 5
2 107
A
v
B V v
Vv~
0
•,Vv~V V~TV~yV~v, 0
9
10 4
1 10 7 v v
100
..
V w-
[Me2+]b / C p
200
0
0
,
I 25
,
[Me2+]b / C p
50
Figure 4. Scatchard representation of binding of Ni2+ to pectins in water (A) and in 0.1 M NaNO3 (B) at 25~ with pectins at 2 mequiv. COO-.1-1: (V) sugar-beet pectins, (V) citrus pectins. In water, Scatchard plots showed clear concave-shaped curves whatever the pectin origin (figure 4A). Nevertheless, differences between sugar-beet and citrus pectins appeared in presence of ionic strength. While citrus pectins exhibited convex-shaped curves whatever the metal ion, sugar-beet pectins display convexe curvature for Cu2+ and Pb2+ but concave-shaped curves for the other three cations (figure 4B, in the case of Ni2+). 4. D I S C U S S I O N
4.1. Selectivity scale In presence of supporting salt, both pectins exhibit the same behaviour towards the two groups of metals consisting of (1) CuE+ and Pba+, the more strongly bound cations, and (2) Zn2+, Ni2+ and Ca2+, which are much less bound. The scale of selectivity, found by a rapid and simple pH method, is in good agreement with the typical sequence found for carboxylates based on Irving-Williams concept (Cotton & Wilkinson, 1976). This order is also in accordance with previously results obtained either for polygalacturonic acid (Jellinek & Chen, 1972; Deiana et al., 1983) or for oligomeric extracts of citrus pectins (Kohn, 1987). The acetylated hydroxyl functions on the galacturonosyl units of sugar-beet pectins (DAc=14%) did not seem to play a significant r61e in the selectivity of pectins for divalent metal ions. 4.2. Quantification of the binding Binding isotherms presented the same characterisitics for sugar-beet and citrus pectins according to the pectin concentration and the conditions of ionic strength. The single case of
539 Pb2§ is shown here (figure 2). In water, where polyelectrolyte effects take place, binding isotherms (figure 2) followed almost a single curve indicating that the level of saturation of the binding sites stayed the same whatever the polymer concentration. Binding was almost stoechiometric in the case of Cu2§ and Pb2+ even in advanced stages of the fixation, as already noticed by Gamier et al. (1994) for low-methoxyl pectins/calcium interactions. In presence of ionic strength, the binding of metal ions was lower than in pure water but increased with the pectin concentration due to increased [Me2+]/[Na+] ratios and competition of both ions for the pectin ionic sites. By comparing the level of the binding isotherms (figure 3) for both metals and pectins, it became possible to set up an affinity order of pectins, whatever their origin, for the five metal ions: Cu2+ ~ Pb2+ >> Zn2§ ~ Ni2+ > Ca2+. This scale, already found by pH-measurements, confirmed that Cu2§ and Pb2§ were more strongly bound than the other three cations with no difference between pectins.
4.3. Characterisation of the binding mode An anticooperative mode of interactions was assumed in case of concave-shaped Scatchard plots, as already proposed by other authors (Mattai & Kwak, 1986; Gamier et al., 1994). A convexe curvature of the plots indicated a cooperative binding process (figure 4). In water, anticooperative interactions were found for both pectins in presence of the five metal ions, as already found by Lips et al. (1991) and Gamier et al. (1994) in the case of calcium. In presence of ionic strength, the type of interactions depended on the metal ion and the pectin origin. Cooperative interactions with both pectins occured with Cu2§ as shown by Schlemmer & Decker (1993), and Pb2+, Which were the two more strongly bound cations. Nevertheless, cooperative and anticooperative binding modes were found for the three weaker bound cations (Zn2+, Ni2+ and Ca2+) with citrus and sugar-beet pectins, respectively. This difference of behaviour, found with sugar-beet pectins, was ascribed to the presence of acetylated functions on the galacturonosyl units, a main chemical difference between the two pectins (Thibault et al., 1993). These functionnal groups could create some specific steric hindrance involving a decreasing of the affinity of ionic sites of the macromolecule towards the more weakly bound ligands. 5. C O N C L U S I O N Interactions studies between some divalents metal ions and pectins from citrus and sugarbeet revealed that the chemical structure of the latter, namely the presence of acetyl functions, induces differences of binding process whereas the scale of selectivity was not affected. Some further studies could be carried out on the correlation between the binding mode and the degree of acetylation. Lastly, pectins showed a clear scale of selectivity towards heavy metals with high capacities of binding which make them suitable to be used in waste-waters depollution.
6. REFERENCES Brady D., Stoll A.D. & Duncan J.R. (1994) Biosorption of heavy metals cations by non-viable yeast biomass. Environ. Technol. 15, 429-438. Cantor C.R. & Schimmel P.R. (1980) Ligand interactions at equilibrium. In Biophysical Chemistry. Part III: The behavior of Biological Macromolecules. W.H. Freeman and Co., San Francisco, chap. 15, 849-886. Cotton F.A. & Wilkinson G. (1976) Basic Inorganic Chemistry, Wiley, New-York, USA. Deiana S., Micera G., Muggiolu G., Gessa C. & Pusino A. (1983) Interaction of transitionmetal ions with polygalacturonic acid: a potentiometric study. Colloids Surf. 6, 17-25.
540 F6rstner U. & Wittmann G.T.W. (1983) Metal Pollution in the Environment, Springer Verlag, Berlin. Gamier C., Axelos M.A.V. & Thibault J.-F. (1994) Selectivity and cooperativity in the binding of calcium ions by pectins. Carbohydr. Res. 256, 71-81. Jellinek H.H.G. & Chen P.A. (1972) Poly(galacturonic acid)-bivalent metal complexes J. Polym. Sci. 10, 287-293. Kerndorff H. & Schnitzer M. (1980) Sorption of metals on humic acid. Geochim. Cosmochim. Acta 44, 1701-1708. Kohn R. (1987) Binding of divalent cations to oligomeric fragments of pectin. Carbohydr. Res. 160, 343-353. Kwak J.C.T. & Joshi Y.M. (1981) The binding of divalent metal ions to polyelectrolytes in mixed counterion systems. I. The Dye Spectrophotometric Method. Biophys. Chem. 13, 5564. Lips A., Clark A.H., Cutler N. & Durand D. (1991) Measurement of cooperativity of binding of calcium to neutral sodium pectate. Food Hydrocolloids 5, 87-99. Marshall W. E., Champagne E.T. & Evans W.J. (1993) Use of rice milling byproducts (Hulls & Bran) to remove metal ions from aqueous solution. J. Environ. Sci. Health A28, 19771992. Mattai J. & Kwak J.C.T. (1986) Divalent metal ion binding to polyelectrolytes with different polyions structure and functionnal groups. Macromolecules 19, 1663-1667. Richardson D.H.S. (1995) Metal uptake in lichens. Symbiosis 18, 119-127. Scatchard G. (1949) The attraction of proteins for small molecules and ions. Ann. N. Y. Acad. Sci. 51, 660-672. Schlemmer U. & Decker H. (1993) On the mechanism of the copper-pectin interaction. In proceedings of Bioavailability '93: Nutritional Chemical and Food Processing Implications of Nutrient Availability. U. Schlemmer (Ed.). Ettlingen, May 9-12, 494-500. Thibault J.-F., Renard C.M.G.C., Axelos M.A.V., Roger P. & Cr6peau M.-J. (1993) Studies of the length of homogalacturonic regions in pectins by acid hydrolysis. Carbohydr. Res. 238, 271-286. Volesky B. (1990) Removal and recovery of heavy metals by biosorption. In Biosorption of Heavy Metals. Volesky B. (Ed.), CRC Press, Boca Raton, chap. 1.2, 7-43.
J. Visserand A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996ElsevierScienceB.V.All rights reserved.
541
Quantitative Vibrational Spectroscopy on Pectins. Prediction of the Degree of Esterification by Chemometrics S. B. Engelsen and L. N~rgaard The Royal Veterinary and Agricultural University, Food Technology, Department of Dairy and Food Science, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark
Abstract
The importance of the degree of esterification (%DE) to the gelation properties of pectins makes it desirable to obtain a fast and robust method to determine (predict) the %DE in pectin powders. Vibrational spectroscopy is a good candidate for the development of such fast methods as spectrometers and quantitative software algorithms (chemometric methods) becomes more reliable and sophisticated. Present poster is a preliminary report on the quantitative performance of different instrumentations, spectral regions, sampling techniques and software algorithms developed within the area of chemometrics.
1. I N T R O D U C T I O N
Pectins are important sugar-based hydrocolloids used in the confectionery industry. In US the sugar confectionery industry is growing approximately 3% per year and the consumption has increased to approximately 11 lb. per capita [1]. Pectin's are widely used in jelly confections often produced using fruit flavors. Pectin gels are characterized by providing a very tender, short texture with excellent clarity and outstanding flavor release properties. For commercial applications pectins are usual extracted from citrus peel, apple pomace and sugar beet and they can be processed to yield two general types of pectin products - high and low methoxyl pectins. Low methoxyl (<50 %DE) and high methoxyl pectins (>50 %DE) gel differently. High methoxyl pectins are capable of forming gel networks at acid pH's in the presence of highsoluble salts and the %DE controls their relative speed of gelation. Low ester pectin gelation properties are dependent on the presence of divalent ions such as
542 calcium and are much less pH dependent than that for the high methoxyl pectins. In low ester pectins the %DE controls their calcium reactivity. Finally the presence of amide groups (the degree of amidation, %DA) in low ester pectins provide a third parameter which strongly affects the calcium reactivity and the resulting gelation properties. Although the implications of structural details such as the degree of methoxylation, the distribution of rhamnopyranoses and the molecular weight on functional properties have been demonstrated, it is still not elucidated why such a biopolymer, unlike other similar structures, develops a jelly in the presence of water, sugars and acid [2]. At present our understanding of the 3D structures of pectins and interaction between pectin molecules is highly simplified. However, the still increasing knowledge about pectins and pectin systems combined with the arrival of new modeling tools specifically aimed towards complex carbohydrate structures [3] is likely to provide more profound insight in structural arrangements of pectins. For this spectroscopic investigation 98 amidated pectin samples were provided by Copenhagen Pectin A/S (Hercules Inc.). The samples are spanning a degree of esterification between 20 and 55 per cent and a degree of amidation between 4 and 24 per cent (see Fig. 1). The powder samples were all measured as is without any form of pre-treatment such as drying and dilution.
[
] --e
-'9
a-D-GalpA
-t
a-D-GalpA-6OMe
~o
~o
o~
a-D-GalpAm ~o
o~
o~
Figure 1. A schematic model of a short sequence of the pectin backbone including a-D-Galactopyranuronic Acid, Methyl a-D-Galactopyrnanosiduronate and a-DGalacto-pyranosiduronamide.
543 2. V I B R A T I O N A L S P E C T R A OF P E C T I N S
Five spectral ensembles were collected for the 98 amidated pectin powders (see Table 1 and Fig. 2). Two dispersive NIR reflectance ensembles were collected using a Tecator spectrometer. Two Fourier transform NIR spectral ensembles were collected using a Perkin Elmer System 2000 interferometer. One using a diffuse reflection cell and one using an integrating sphere cell. Finally two spectroscopic ensembles were collected in the region of the fundamental vibrations. One FT-IR ensemble using the diffuse reflection cell and one using the NIR FT-Raman technique (1064 nm).
NIR
Raman
10 00
90b0
80'00
70'00
60'00
50'00
40'00
30'00
20'00
10'00
2(}0
cm-1
Figure 2. The spectra of the amidated pectins (NIR, FT-IR and Raman shift).
Table 1 The spectroscopic data I n s t r u m e n t dispersive dispersive FT
FT
Sampling
integrating diffuse
reflectance reflectance diffuse
FT
FT 180 ~
method
(rot. cell)
reflection sphere
reflection scattering
X-variables 1050
1050
6001
3301
-1
6001 -1
-1
2771 -1
X-units
nm
nm
am
X-min
400
400
4000
4000
700
330
X-max
2500
2500
10000
10000
1750
3100
am
am
am
544 3. C H E M O M E T R I C S
Principal Component Analysis (PCA). Principal component analysis is an extremely important method within the area of chemometrics. By this type of mathematical treatment one finds the main variation in a multidimensional data set by creating new linear combinations of the raw data (e.g. spectral variables) [4]. The method is superior when dealing with highly collinear variables as is the case in most spectroscopic techniques: two neighbor wavelengths show almost the same variation. Partial least squares regression (PLS). Partial least squares regression applies to the simultaneous analysis of two sets of variables on the same objects. It allows for the modeling of inter- and intra-block relationships from an X-block and Y-block of variables in terms of a lower-dimensional table of latent variables [4]. The main purpose of regression is to build a predictive model enabling the prediction of wanted characteristics (y) from measured spectra (X). In matrix notation we have the linear model with regression coefficients b: y=Xb To investigate the variance structure in the raw physical/chemical data material a PCA was performed on the autoscaled Y-data. Figure 3 shows a loading plot of the Y-data as a function of the two first PC's describing together 57 % of the total variance. Loadings for PC-'# 1 versus PC-'# 2
0.8
,
Scores for PC# 1 versus PC# 2
,
2
,
,
,
1.5
ptl
0.6
,
21 22
Trans ~
0.5
~,
.a
0.2 Ca4 Ca3 Ca2
%DE
25 24
z3 24
2~5
24
21~2
20
2021"22
ZZ
-l
26 22
2!
%DFA
-1.5 - 25
22 22 2
-0.4
-2
SAG
.4).6 -0.6
2~176
%DA
Cal -0.2
r
25 2 1 ~ 2 2
, 20
20 2 2~ 2 ~ 12020 .
21
1
0.4
,
g.4
-0.2
F'C#1
o
oJ2
0.4
25
24
-2.5
4
PC#1
Figure 3. Loading (left) and score (right) plots from a PCA on chemical data measured on the pectins (%DE=degree of esterification, %DA=degree of amidation, %DFA=100-%DE-%DA, Transp=transparency, Cal-Ca4 and SAG are gel strength measures).
545 Different product sorts (20-22,24-26) are marked in the score plot of spectral NIR measurements (Fig. 4). Sorts 23 and 31 are in separate classes outside the range of this plot. A gradient is seen in the plot, indicating the chemical differences among the sorts: the %DE are increasing in the 20->25/26 direction while the opposite holds for the Ca-based gel strength measurements. xl0 -4 8
Scores for PC# 1 versus PC# 3 ,
,
xl0 -4
~
,
,
Scores for PL-'# 1 versus PC# 2 ,
,
,
22
4
21
3
t-q 2
1
2:~525 -1 2~ 23 23
3.5
-~
&
0
-2
20~2~ l
-3
2020
~26~
2121 ~.2 2122~2 21 22 21 21 22
-;
-;
-'1
o IK:#1
x 10-3
'1
25
25 2114 24 24 24
-5
015
1
25 2~5 26 ~25 26 2~
22 21 2: "'2122 21 2222
20
-4 2~2 2225
25
2/112~2 22 2,]1 12~
~o20 1.o
313131
,
25
2
25 22
,
22~
5
; xlO 4
Figure 4. Score plots from a PCA of NIR_R spectral data (2nd derivative). Left: PC#1 versus PC#3 shows a clear segregation of sorts 23 and 31. Right: PC#1 versus PC#2 (without sorts 23 and 31) shows a more distinct sort gradient/classification than the one produced by the chemical data. Two PLS-factors
.
.
.
(%DE) . .
/
4O
35 30
15 15
20
25
30
35
40
45
50
55
Measured
Figure 5. Predicted versus measured plot of %DE using the FTNIR_IS ensemble (C=cross validated samples and T= test set samples). The optimal PLS models obtained for the prediction of %DE using the five different spectral sources are listed in Table 2 (see also Fig. 5). The models
546 have been constructed using a conservative approach of systematic cross validation on app. 73 samples (objects) and subsequently tested on an independent test set consisting of 25 samples. Table 2 The optimal spectroscopic PLS models for the pectin powders.
degree of esterification of amidated
derivative
2
1
1
1
1
1
opt. PC's
2
3
3
2
4
4
RMSECV
1.19
1.17
1.54
1.54
1.46
1.90
R
0.98
0.98
0.97
0.97
0.97
0.95
The experimental errors on the %DE measurements are estimated to be between 1 and 2 %, taking into account a relative long time span and the involvement of different lab-workers. As indicated by Table 2 the best models converge to an RMSEP of 1.5 %; to refine the models further the experimental chemical errors have to be thoroughly investigated. Three PLS-factors (%DA)
C
_j T
/ ~
~
1~
1~
~o
~
30
Measured
Figure 6. PLS model for %DA using the NIR_R ensemble. Of the other physico-chemical data available to us we found good predictive correlations between the spectra and the degree of amidation (Fig. 6)
547 and for the pH (Fig. 7). Finally we obtained a somewhat weaker PLS model for the gel strength Ca2 (62 % explained using 3 PC's). Due to the large span in %DE/%DA for the samples it was necessary to use two levels of sugar contents in the Ca2 measurements which perhaps can explain the weak model. It may also be possible that the gelation potential is not "visible" in the spectroscopic ensembles but certainly the weak correlation is worth pursuing with further investigations. 5
,
,
,
Four PLS-factors(pH) ,
,
,
ThreePLS-factors(Ca2) ,
,
4.8
350 ~'42"42 . . . . . r C, ~ T
4.6
3O0
4.4
T
C j"Cc
C
12
4.2 4
200150
IZ 12
, : ~ C
~
C
CC
3.8 100
3.6 3.4 3.2 / 3.2
3.4
T 3.6
3.8
4 4.2 Measured
4.4
4.6
4.8
0
~
1~
1~
2t)0 ~ Measured
3()0
3~
400
Figure 7. PLS model for (left) pH and (right) Ca2 using the FTIR_DR ensemble.
4. CONCLUSIONS
From Table 2 it is observed that the dispersive NIR ensembles (NIR and NIR_R) result in the best cross validated models. The potential advantages of Fourier transform spectroscopy [5] are in practice outnumbered by a more reproducible setup and sampling procedures. When considering the two spectral models in the region of the fundamental vibrations (FTIR_DR and Raman) we observe that more PC's are needed to out-level interferences and describe the Y-variation. On the other hand these ensembles are needed to interpret the most important spectral elements in the models. The lower signal to noise ratio in the Raman spectra caused by the low energies of the scattered light and the poor reproducibility due to scattering effects are responsible for the relative poor performance of this spectral technique. However, the used sampling technique designed for micro analytical purposes (laser focused on a very tiny area, no control of depth focusing and pressure/density of the sample) could play a significant role. We are currently
548 making investigations to improve and control these factors to obtain a more fair picture of the potential of quantitative NIR FT-Raman spectroscopy. Another direction of future research is to investigate the relationships between IR and NIR spectra in the form of PLS2 modeling and 2D correlation spectroscopy [6]. A preliminary 2D correlation spectrum is shown in Fig. 8.
2D NIR/IR correlation spectrum "6 9
*
--~ l l h .
~
~...
~
.m
.,+ t
9
-..,.= P= ~.= = : I n
! m ....
9 l~a
NIR 7000 'gil ~. ~.~ o~N-'-9 " .,~ "'0 . -~=~- . ~ (cm-1) -.. =
6000
"
..'~'. ,=-'.."...m~_":,'-:.. 9~.;, . ~ , , . = , ; ~ = .~
1000
.
~
1500
.,~.,..,
2000
--.,=,, .d.. 2500
l~,ii
", " ~
~
~
~o 3000
3500
4000
IR (cm-1)
Figure 8. 2D NIR/IR correlation spectrum. Only contours for correlation coefficients with numerical values above 0.5 are shown.
7. REFERENCES
[1] J.M. Carr, K. Sufferling and J. Poppe, Food Technology, 7 (1995) 41-44. And references therein. [2] S. Cros, C. Herv~ du Penhoat, N. Bouchemal, H. Ohassan, A. Imberty and S. P~rez, Int. J. Biol. Macromol., 14 (1992) 313-320. And references therein. [3] S.B. Engelsen, S. Cros, W. Mackie and S. P~rez, "A Molecular Builder for Carbohydrates: Application to Polysaccharides and Complex Carbohydrates", submitted (1996). [4] H. Martens and T. Nees, Multivariate Calibration, Wiley, New York, 1993. [5] P.R. Griffiths and J.A. de Haseth, Fourier Transform Infrared Spectroscopy, Chemical Analysis Vol. 83, Wiley - Interscience, 1986. [6] F.E. Barton II, D.S. Himmelsbach, J.H. Duckworth and M.J. Smith, Applied Spectroscopy, 46 (1992) 420-429.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
549
POLYSACCHARIDES FROM Chorisia speciosa St. Hil.
E. B. BELESKI-CARNEIROa, b ; M. R. SIERAKOWSKI; J. L. M. S. GANTER; S. F. ZAWADZKI-BAGGIO; F. REICHERa aDepartament of Biochemistry, PO Box 19046. Universidade Federal do Paran~i, 81531-990. Curitiba - Paran~i - Brazil b Departament of Chemistry, Universidade Estadual de Ponta Grossa. Paranh- Brazil
ABSTRACT
The floss silk from Chonsia speciosa fitmished a polysaccharide with a main chain of (1 ~ 4) linked 13-Xylp substituted at O-2 by 5 % of uronic acid. The xylan structure also was interposed with ct-Rhap units in small amounts. The defatted seeds furnished on aqueous extraction a major fraction, (O-acetyl, 10 % and protein, 45 %) wich was hydrolysed and analysed by p.c. and GLC, showing Rha (20 %), Ara (16 %), Gal (64 %) and also uronic acids (45 %). Partial hydrolysis gave rise to a polysaccharide free of arabinose, with 46 % of uronic acids. Methylation analysis (GLC -MS) indicated a chain of (1 -~ 4) - linked Galp (42 % of 2,3,6-Mea-Gal ).
INTRODUCTION
Chorisia speciosa St Hil is a large tree of the family Bombacaceae, native to tropical South America, it grows in Brazil, being abundant in the States of Rio de Janeiro, Minas Gerais and S~o Paulo. Its is frequently found further South in Curitiba as an ornamental tree which flowers, appearing with ten new leaves having five pink, purple petals. The pear-shaped fruits contain an abundance of silk white floss, that is extremely elastic, light in weight and impervious to water, and is used in pillows, sleeping bags, upholstery and life preservers. The genus Chorisia has five species, the most important being C. speciosa, known as floss silk tree or "paineira".
550 Several aspects of this tree have been studied. One is a gum which exudes when the tnmk suffers injury, apparently to heal its wounds. It contains a complex polysaccharide with a backbone composed of glucosyluronic and mannose units [ 1]. Lufrano and Caffmi [2] compared four distinct species of Chorisia by phytochemical analysis, composition of the mucilages from the leaves and suggested a chemotaxonomic approach at genetic and specific levels. The seeds had a high protein content and furnished 22 % of oil. The triglyceride structures have been determined and showing fatty acids predominantely unsaturated [3]. This paper reports the structural features of the silk floss polysaccharide and the partial structure of a viscous acidic polymer obtained from the seeds of Chorisia speciosa. It was of interest to analyze these polysaccarides due to the relationship of Bombacaceae to Sterculiaceae, as well as for its possible commercial uses.
EXPERIMENTAL
General methods. Polysaccharides were hydrolyzed with M trifluoroacetic acid (5 h, 100 oC). Hydrolyzates were reduced with sodium borohydride, then acetylated in 1:1 pyridine-acetic anhydride (16 h, room temperature). The resulting alditol acetates were analyzed by GLC (gas-liquid chromatography), with a model 2440 Varian chromatograph operating at 180 oC, with columns packed with 3 % OV 225 or ECNSS (1.5 mm i.d. x 200 cm. Gas Chrom. Q support). The carrier gas was nitrogen (40 mL./min). Methylated polysaccharides were hydrolyzed with 72 % H2SO 4 (1 h. 0-4 oC) and thereafter water was added to a fmal acid concentration of 1 M (5 h. 100 oC). The solution was neutralized (BaCO3) and the products converted into O-methylalditol acetates that were analyzed by GLC-MS with a model 3980 Hewlett-Packard chromatograph equipped with and HP1 capillary column (0.2mm i.d. X 30 m) linked to an HP 5988 with mass spectrometer unit (electron impact, 70 eV). Injections were carried out at 150 oC and the column programmed to increase at 4 oC/min to 250 oC, then hold. Optical rotations were measured in water at 25 oC using an Acatec automatic polarimeter. I. r. spectra were determined using a Beckman Acculab TM-10 spectrophotometer. Uronic acid was estimated by the m-hydroxyphenyl method [4], O-Acetyl contents by the method of Hestrin [5], carbohydrate by the phenol-sulfuric acid method [6] and protein by the Hartree method [7]. Plant material The fruit of Chorisia speciosa at mature stage was collected from large trees in parks of Curitiba. Polysaccharide isolation. The silk floss (36 g) was cut and extracted with 2:1 benzene-EtOH in a Soxhlet apparatus for 16 h. The residue was extracted with 2 M
551 NaOH in the presence ofNaBH 4 for 6 h at room temperature. The alkaline extraction was repeated twice and the supematant combined the NaOH extract acidified to pH 5 with 50 % acetic acid, exhaustively dialyzed, polysaccharide precipitated with EtOH (2 vol.) and washed with EtOH and MeECO. The seeds (30 g) were ground in a Willey mill (60 mesh) and defatted as described above for 32 h and the residue submitted to sequential aqueous exctractions. The material was stirred in H20 (600 ml) at 5, 20 and 70 oC each for 12 h. Addition of EtOH (2 vol.) precipitated polysaccharide which was isolated via sucessive centrifugation, and washed with EtOH and Me2CO, yielding fractions FI, FII and FIII respectively (Figure I). Chromatoraphyc methods: For gel filtration of polysaccharide fraction PI, a Sephacryl S-300 chromatographyc column (1,1 X 46,7 cm) was calibrated with standard dextrans (molecular mass range 266, 72, 40, and 17 KDa; Sigma Chemicals), and the void volume determined with blue dextran. Polysaccharide sample (0.5 mL; 2 mg/mL) was applied and eluted with 50 mM NaOH, fractions 1 mL being collected and carbohydrate absorbance (phenol-H2SO4) being monitored. Analyses were performed by gel permeation chromatography (GPC) and by high performance liquid chromatography (HPLC). Gel filtration of oligosaccharides was effected in a thermostated (65 oC) column (210 X 1.5 i.d.) filled with polycrylamide gel (Bio-gel P2, 200-400 mesh; Bio Rad-USA), using distilled water as eluent (flow rate 30 ml.h-1). Smith degradation of PI. A sample of FI (50 mg) was dissolved in 0.5 M NaOH (5 mL) and acetic acid added to pH 7, followed by NaIO4 to a final concentration of 50 mM. At the conclusion of the oxidation (7 days) the product was reduced with NaBH 4 and dialyzed. An aliquot was removed and the remainder immediately treated with NaIO 4 and then reduced with NaBH 4. Samples of material subjected to one and two cycles of oxidation-reduction were hydrolyzed and reduced, and the products analyzed by GLC as alditol acetates. Methylation analysis of PI and Flbp. The fractions were methylated according to Ciucanu and Kerek [8]. The procedure was repeated until no absorbance was detected by I.r. at 2500-3500 cm-1 and the per-O-methylated polysaccharides hydrolyzed and analyzed by GLC and GLC-MS of the derived partly O-methylated alditol acetates. Oxidation of PI with chromium trioxide. Fraction P1 was twice acetylated as described above. The peracetylated polysaccharide (75 mg), together with 20 mg of mannitol hexacetate as internal standard was dissolved in 1.5 mL of HCC13, and treated with 1.89 mL of glacial acetic acid and 189 mg of chromium trioxide, at 50oC. Aliquots were removed at zero, 30, 60 and 120 min, water then added, and the material recovered by extraction with chloroform, hydrolyzed and analysed by GLC of derived alditol acetates.
552 F i g u r e 1. F l o w d i a g r a m
o f t h e i s o l a t i o n o f f r a c t i o n f r o m s e e d s o f C. s p e c i o s a
Milled seeds Benzene:EtOH
11 -I
E-I Lipid/pigment . . . . . . . . . . . . . . .
~
_
.~
MeOH
--
E-II Low Mw material
: water
- .. I w a t e r , 4 ~ _
!
.....
R-Ill
E-Ill FI
water, 25*C
R'!iv
centrifugate
water, 70~ E-Ilia Fla
E-Illb Fib NaOH
2 M
,[
PP. HAc 50% . . . . . . . .
. . . . . .
supernatant-I
precipitate Hem. A
EtOH
i
precipitate Hem. B
supernatant-i
i
553
Carboxyl redution. A sample of permethylated PI (5 mg) was carboxylreduced by a modification of the method described by Lindberg and LOnngren [9], as follows. The methylated fraction was solubilized and added a mixture of LiA1H4 (25 mg) in THF (5 mL) at 20 oC for 4 h. and refluxed during 1 h. The excess of reagent was destroyed with ethyl acetate (5-6 drops) and water (10 drops) and the pH of the mixture adjusted to neutrality with acetic acid. The insoluble residue was removed by centrifugation. The reduced fraction was precipitated with EtOH. The reaction was monitored by I.r. specroscopy. Hydrolysis products were analysed by GC-MS as methyl alditol acetates 13C NMR spectroscopy was performed with a Bruker AC-300 spectrometer at 75 MHz in the Fourier-transform mode, with proton decoupling at 30 ~ C, using 5 mm tubes and D20 as solvent. The spectral width was 200 ppm. Chemical shifts are expressed in ppm relative to the resonance of external DSS (sodium 4,4-dimethyl-4silapentane- 1-sulfonate). Partial acid hydrolysis ofFIa. A sample of FIa was treated with TFA at pH 2, 1 h at 100 oC.
RESULTS AND DISCUSSION
In terms of the average from 20 mature fi'uits, floss silk, seeds and fruit coat were isolated, the ratio being 14 %, 15 % and 7 1 % respectively. The filaments of floss silk on submission to microscopy, showed mainly unicellular trichomas, some of them being formed by two cells. The presence of lignin in the filaments was suggested by the characteristic reaction with phloroglucinol hydrochloride. The silk floss was defatted and hydrolyzed by Saeman's method [ 10] giving rise to xylose (73 %) and glucose (27 %), thus indicating the presence of xylan. When the time of hydrolysis was up to 24 h, the presence of rhamnose (5 %) was also evident. However, degradation of xylose was observed after 8 h at 100 oC. The defatted floss silk was submitted to extractions with aqueous NaOH giving rise to a polysaccharide (PI, yield 23 %) composed of xylose as the only neutral sugar. Uronic acid (10 %), was determined by a colorimetric method [4]. When products of acid hydrolysis of PI were fractionated by gel permeation chromatographic (GPC) on Bio-Gel P2, two fractions were obtanined, one being composed of Xyl and the other acid oligosaccharides. The acidic fraction was treated with MeOH-HCI and reduced with NaBH 4. After hydrolysis of the product, analysis by p.c. showed the presence of Xyl, Glc, Rha and 4-O-Me-Glc. These results were confmned by GLC-MS of derived alditol acetates indicating that the xylan contains a small amount of Rha, GlcA and 4-O-Me-GlcA as the acid components. Xylans from wood hemicellulose containing Rha (0.3 to 0.6 %) were related by Fengel and Wegener [11]. Aspinall and McGreth [12] obtained a xylan from
554 lucerne stems containg 11% of 4-O-methyl-D-glucuronic acid residues and 1 % of Rha. Geerdes and Smith [ 13] obtained a hemicellulose from flax straw which gave on hydrolysis, a mixture of aldobiuronic acid, Xyl and a small amount of Rha. Seed hair xylans are present in milkweed (Asclepias synaca) and kapoc (Ceibapentandra) and contain 4-O-Me-GlcA linked (1 ~ 2) to Xyl. The xylan from the silk floss (PI) was homogeneous on gel-column chromatography over Sephacryl S-300, having a molecular mass of z 52000. Methylation analysis yielded 2,3-Me2-Xyl, 3-Me-Xyl and 2,3,4-Me3-Xyl in a molar ratio of 93.0: 5.0: 2.0. When the permethylated product was carboxy reduced and remethylated, 2,3,4,6-Me4-Glc (3.1%) was also characterized (Table I).
Table I. Methylation analysis of products from original fraction PI carboxi reduced and remethylated material by g.l.c-m.s. (C. speciosa). Component
PI
2,3-Me2-Xyl 3-Me-Xyl 2,3,4-Me3-Xyl 2,3,4,6-Me 4 Glc
93.0 5.0 2.0
PI-CR mol % 91.2 4.9 0.8 3.1
Capillar column OV-225 and DB-210.
This results are consistent with a backbone of (1 --> 4) linked D-xylosyl units substituted at O-2 by glucuronic acid and its 4-O-methyl derivatives. Periodate oxidation followed by reduction and acid hydrolysis gave rise to glycerol and Xyl in a molar ratio of 88:12. However, when the polyol was reoxidized with periodate and reduced, acid hydrolysis gave rise to glycerol and Xyl in a molar ratio of 94:6, as expected. Interunit hemiacetal formation must have taken place during the initial periodate treatment inhibiting further oxidation. The anomeric configurations of the sugar residues were determined by chromium trioxide oxidation [ 14]. Oxidation of the fully acetylated polysaccharide and subsequent monosaccharide analysis by GLC indicated that the D-Xyl units are [3-1inked (oxidized more rapidly) and that the D-GlcA are a-linked (Table II).
555 Chromium trioxide oxidation of fraction PI.
Table II.
Monosacharide composition of product a
(mol%) Time (min)
Mannitol
Xylose
0
1.00
1.97
30
1.00
1.33
60
1.00
0.75
90
1.00
0.00
(a): GLC - ECNSS column.
The reaction was monitored also by p.c. which showed clearly on increase in the relative proportion of uronic acids related to xylose. After 90 minutes of oxidation, only uronic acids were observed indicating the a-configuration.
Table III. 13C NMR Data a for the Xylan (PI) from the floss silk of Chonsia
speciosa. Carbon
C-1 C-2 C-3 C-4 C-5 C-6
( 1 --~ 4)-13-D-Xylp
(1 -~ 4)-13-D-Xylp
unbranchedb
branchedb
103.7 74.8 76.1 78.4 65.0
102.8 81.2 78.5 75.9 64.7
a: shifts (5 in ppm); internal DDS reference. b: refers to branched and unbranched at 0-2.
(1 -~ 2)-~-DGlcA 99.4 74.2 75.9 84.2 73.4 178.3
Figura 2. aC NMR spectrum of the PI fraction from the floss silk of Chorkia speciosa, 75 MHz, D20,30"
C5
I
i
i/
ce
OL - D 6 t +
4-0-MI
f
556
I I
180
I
IM,
I
!to
f
f
s
120
1
IK)
1
loo
t
90
1
aa PPM
I
m
I
60
557 The 13C NMR spectrum of PI consisted of 5 large signals at 5 103.7 [C-l], 78.4 [C-4], 76.1 [C-3], 74.8 [C-2] and 65.0 [C-5], corresponding to (1 ---}4) linked 13 -D-Xylp. This polymer showed some residues of (1 ---}4) linked 13-D-Xylp substituted at 0-2 (5 81.2). The signals at 8 99.4 [C-l], 74.2 [C-2], 75.9 [C-3], 84.2 [C-4], 73.4 [C-5], 178.3 [C-6] corresponding to (1 ---} 2) linked o~-D-GIcA. The signal at 8 61.3 corresponding to 4-O-Me in glucuronic acid residues (Table III). The floss silk from Chorisia speciosa which envelops the seeds are important to their dispersion. Each fruit contains from 100 to 200 seeds. The seeds were crushed and defatted (see flow diagram, Fig. I) yielding 20 % of oil. This result is in agreement with Petronici et al [3]. The residual mass was refluxed w~th methanolwater in order to isolate low molecular compounds. Both treatments with organic solvents are efficient in enzyme inactivation [15]. Extractive-free seeds were subjected to aqueous extractions at progressively increasing temperatures (fractions FI to Fill, Table IV). The residue was treated with aqueous NaOH according to Whistler and Feather [ 16], yielding HA and HB (Table IV). All the aqueous extracts showed high viscosities. As FI was not completly soluble in water, it was centrifuged giving rise to FIa (supematant) and Fib (precipitate). The highest yield of polymers was obtained with water at 5 oC (FI) and at room temperature (FII).
Table IV. Yield and monosacharide composition of Fractions obtained by aqueous and alkaline extractions. Fraction
FIa Fib FII Fill Hem. A Hem.
Yield g%
Composition (mol %)a
3.3 15.2 21.6 1.1 3.0
Rha 20.0 13.5 48.0 48.0 13.0
Ara 16.0 16.5 19.0 29.0 4.0
Xyl --4.0 3.0 11.0 82.0
Gal 64.0 66.0 30.0 12.0 1.0
2.2
22.0
8.0
66.0
4.0
B
a: glc ECNSS column. b: Hem. = Hemicellulose
558 Rha, Ara and Gal are the neutral sugar components from all the fractions. Xyl is not present in FIa and is significantly present in the hemiceUulose fractions, indicating that this monosaccharide is component of hemicellulosic polymers. Chemical composition of the water fractions were determined (Table V). High protein contents and the presence of O-acetyl-groups were observed in four aqueous fractions. Neutral sugar and uronic acid composition points to inclusion of these polymers in the class of pectic polysaccharides.
Table V.
Determination of carbohydrates, proteins, uronic acids and O-acetyl of Fractions from aqueous extractions from seeds of C. speciosa.
Fraction
CarbohydrateProtein
FIa Fib FII FIII
33.0 51.0 37.0 24.0
uronic acids*
% content 41.0 48.0 34.0 31.0
45.0 25.0 33.3 23.0
O-Acetyl*
10.5 5.2 8.5 15.6
(*): in relation to carbohydrate.
Fraction FIa was chosen for structural purposes due to its better solubility in water and the absence of Xyl. In order to remove noncovalently associated protein, fraction FIap was submitted to sequential shaking cycles with a mixture of chloroform-buthanol, as indicated by Sevag and described by Staub [17]. The fraction was also treated with trichloroacetic acid. In both procedures, coprecipitation of carbohydrate and protein was observed, suggesting strong linkages and a more complex structure. FIa as was submitted to mild acid hydrolysis yielding FIas and FIap (Table VI).
559
Table VI.
Monosacharide composition of precipitate Flap and supematant Flas produced by partial hydrolysis of fraction Fla. (mol %)
Fraction
Ara*
Rha*
FIa Flap Flas
16 tr 64
20 24 35
Gal* 64 76 tr.
(*): determined by GLC. The removal of arabinose suggested the presence of peripheric ot-Larabinofuranosyl units. Flap was methylated by the method of Ciucanu and Kerek and products of derived O-methyl alditol acetates analysed by GLC-MS The resulting Flap contains uronic acid (46 %), protein (45 %) and was submitted to methylation analysis, GLC-MS of resulting methyl alditol acetates showed the presence of 2,3,6-Me3-Gal (42.6 %), 2,3,4,6-Me4-Gal (19 %), 2,3,4Me3-Rha (19 %), 3-Me-Rha (11.2 %), 3,4-Me2-Rha (4.9 %) and 2,3,4-Me3-Gal (3.3%). These results indicate a complex highly branched glycoprotein whose structure will be published in the future. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
J.L. Di Fabio and G. G. S. Dutton, Carbohydr. Res., 99 (1982) 41-50 N.S.P. Lufrano and N. O. Caffmi, Oiton, 40 (1981) 13-20 C. Petronici et al., La Rcvista Italiana delle Sostanze Grasse, 51 (1974) 11. N. Blumenkrantz and G. Asboe-Hansen, Anal. Biochem., 54 (1973) 484-489 F. Dowins and W. Pigman, Meth. Carbohydr. Chem., 7 (1978) 241-243 M. Dubois et al., Anal. Chem., 28 (1956) 350-356 9 E.F. Hartree, Anal. Biochem., 48 (1972) 422-427 I. Ciucanu and V. Kerek, Carbohydr. Res., 131 (1984) 209-217 B. Lindberg and J. L6nngren, Methods Enzymol., 50 (1978) 3-13 J. F Saeman et al., Tech. Assoc. Pulp Pap. Ind., 37 (1954) 336-343 D. Fengel and G. Wegener, Wood Chemistry, (1989) 108-127 G. O. Aspinall and D. McGrath, J. Chem. Soc. (C), (1966) 2133-2139 J. D. Geerds and F. Smith, J. Am. Chem. Soc., 77(13) (1955) 3569-3572 J. Hoffman and B. Lindberg, Meth. Carbohydr. Chem., 8 (1980) 117-122 F. Reicher et al., Appl. Biochem. Biotech. 34/35 (1992) 349-357 R.L. Whistler and M.S. Feather. Meth. Carbohydr. Chem, V (1965) 144-145 A. M. Staub, Methods Carbohydr. Chem., 5 (1965) 5
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J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All fights reserved.
561
Rigid and flexible pectic polymers in onion cell walls M.-A. Ha ~, B.W. Evans a, D.C. Apperley h and M.C.
Jarvis a
aChemistry Department, Glasgow University, Glasgow G 12 8QQ, Scotland hEPSRC Solid-state NMR Service, Durham University, Durham DH1 3LE, England.
Abstract The amount of thermal motion occurring in individual polymers of a composite material like a plant cell wall can be determined using NMR relaxation methods. This methodology can be used to indicate of the rigidity of each polymer and its contribution to the rigidity of the cell wall as a whole. We have applied this approach to onion cell walls. The proton T2 is used to identify individual polymer chains differing in rigidity whereas the proton T~p discriminates between spatial locations within which the average amount of motion differs. Through a Tfbased spectral editing procedure we reconstructed sub-spectra corresponding to a mobile, largely pectic fraction of the cell wall and a rigid microfibrillar fraction which also included a pectic component. There was a third extremely mobile pectic component consisting of a of 13(1,4)-linked galactan and highly esterified galacturonan. These highly mobile, hydrated polymers are not represented in a CP-MAS ~3C spectrum obtained under normal conditions. We found, however, that by a combination of a long-contact experiment and a delayed-contact experiment we could reconstruct a ~3C spectrum of the cell-wall components that are normally too mobile to be visible. Through a T~p-based spectral editing procedure we found some pectic material spatially located near cellulose. This included some 'eggbox' pectin. Pectic material was also located more than 2nm away from cellulose. These results show that, within a single cell wall, pectic polymers are very heterogeneous in rigidity as well as in composition, and are not distributed uniformly within the cell wall structure.
1. INTRODUCTION Recent models of the plant cell wall are simplified representations based mainly on what is seen by microscopy but also incorporating information from chemical techniques (1,2). However, electron microscopy is a moisture sensitive
562 technique and the dehydration it requires can affect moisture sensitive polymers such as pectin. Therefore techniques that do not require dehydration of the plant cell wall are needed to give us complementary information on the three dimensional architecture of pectin. Using solid state NMR, Jarvis and Apperley (3) have recently examined the conformation and aggregation of pectic galacturonans forming gels with calcium in vitro and proposed a 'cable' model for the structure of gels. Non-esterified galacturonan chains aggregated by calcium ions adopted both 2~ helical (eggbox) and 3~ helical conformations with some chain segments intermediate between these. Chains that were not aggregated, and were therefore more flexible, adopted a wide range of right-and lefthanded helical forms with the 31 helix as an approximate average conformation. In the cell wall, methyl esterification leads a larger number of chains to take up similar random conformations but there is evidence (4) that some methyl-esterified chain segments can also form aggregates in the 3~ helical form. Data on branched and acetylated chain segments are as yet, inconclusive. In addition to giving conformational information, solid state NMR relaxation experiments can be used to probe the thermal motion of polymers in the hydrated cell wall (5). The motion of the polymers can give us clues as to the environment of the polymer. When there are both rigid and mobile polymers within a composite material, NMR spin-diffusion experiments can be used to find out how far apart they are. We used modifications of the standard solid-state CP-MAS (cross-polarisation, magicangle spinning) experiment to allow the proton relaxation characteristics to be measured for each peak in the ~3C spectrum. It is known that highly mobile, hydrated polymers can not be seen using either usual CP-MAS ~3C spectrum or solution NMR (6). We found, however, that by a combination of a long-contact experiment and a delayed-contact experiment we could reconstruct a ~3C spectrum of the cell-wall components that are normally too mobile to be visible. With these techniques we were able to determine the mobility of pectins and their approximate spatial location in comparison to cellulose.
2. RESULTS AND DISCUSSION Our solid state NMR findings suggest that three distinct groups of pectins differing in mobility on the kHz frequency scale, coexist in at least two different areas of the onion cell wall.
2.1 Very Mobile Pectin The most mobile of the pectins were not visible in conventional solid state CP-MAS ~3C spectra, nor in solution 13C NMR. Because of the high mobility of this material, it was very slow to cross polarise (CP) (7,8). A spectrum of this slow-CP material was obtained by subtracting the signal intensities obtained in a delayed-contact experiment from those obtained in an experiment with long, variable contact times. The method is discussed in detail elsewhere (9). The resulting spectrum (figure 1) showed a highly methyl esterified o~(1,4')-D-galacturonan and a 13(1-4')-D-galactan. Both of these polymers are highly soluble in water. They are the most flexible polymers, identifiable by NMR, in the onion
563 cell wall. Foster et al (6) have arrived at similar conclusions by a quite different NMR approach.
Methyl Pectic C-4
galacturorlan C-6l
Crahctan C-6 Pectic Me
Pectic~C-1 ~ Galactan C- 1
I
180
'
I
'
I
160 "140
'
I
120
'
I
'
100
I
80
'
I
60
'
1
1
40
I
20
'
I
0
Figure 1. Spectrum of very mobile material in onion cell walls.
2.2
Pectins
of Low
and
Intermediate
Mobility
The conventional CP-MAS 13C spectra of hydrated onion cell walls can be seen in figure 2. This spectrum is derived from the low- and intermediate-mobility polymers present in the walls.
564
180
160
140
120
100
80
60
40
20
0
ppm
Figure 2" T2 CP-MAS ~3C spectrum of onion cell walls
Table 1 Solid state CP MAS ~3C signals Chemical Shift Carbon (ppm) . . . . Number 177 C-6 175 C-6 171 C-6 106.4,105.5, C-1 104.5 101 C-1 100-94 90.2, 89.4, 88.5 84.9,83.9 8O 79 77 75,72 69 66.2,65.4 62.5, 61.6 60.5 54 21 .
.
.
.
C-1 C-4 C-4 C-4 C-4 C-4 C-2,C-3 C-6 C-6 C-6 ,, ,,,
for plant cell walls (3,10) Assignment ... Calcium bound pectin Random coil pectic chains bound to monovalent cations Pectic carboxyl, methyl ester, free acid Cellulose Ia and Ib Pectic 3, and 2, helix conformations and other carbohydrates Intermediate pectic conformations Cellulose Io~ and 113 Cellulose Io~ and lib Pectic 3, helix 131-4 galactan Pectic 2, helix General carbohydrate Pectin Cellulose Io~ and 113 Cellulose Io~ and 113 1-4 galactan Pectic methoxyl Acetyl l~roup .....................
565 Peak assignments are given in table 1. A peak can be seen at 80ppm. This corresponds to the resonance from the C-4 of galacturonic acid occurring in a 31 helical conformation (3). The resonance of the C-4 of galacturonic acid in a 21 helical conformation occurring at 77ppm can not be distinguished here because of the large general carbohydrate peak seen at 75-76ppm. Intermediate conformations can be identified from the C-1 resonances (100-94ppm). By measuring the proton relaxation times, T2 and Tip, it is possible to estimate the mobility of polymer chains within the cell wall (11). Proton spin relaxation editing (PSRE) is a method of expressing these results. It separates the components seen in a conventional CP-MAS 13C spectra into low-mobility and intermediate-mobility components. If PSRE is applied to a T2 experiment (12) the mobility of the polysaccharide chains within the cell wall can be identified. Proton T2 increases with increasing mobility. In addition to the highly mobile pectin described in section 2.1, two further mobility classes of pectic galacturonans could be seen within the onion cell wall (figure 3).
Intermediate Mobility Low Mobility
r,
~/v-,.,~-uw,,r.--~/..~/~,,,,
~
I
-i " ~ l l I
180 160 140 120 100
80
I'~hlt_J'v~'vlm4N~'r
60
40
v \ ~ . t ~ "v-"
20
0
ppm
Figure 3: T2 PSRE spectrum of onion cell walls.
The pectic polymers of intermediate mobility included methyl esterified galacturonans and ~(1-4')- linked galactans, similar to those of the most mobile fraction. It is likely that differences in mobility of the galactan chains are a consequence of chain length, with the motion of short chains being more tightly constrained by the attachment to galacturonan at one end (6). The maximum intensity of the galacturonan C-4 resonance was about 79ppm. This may be assigned to a mixture of aggregated chains in the 31 helical
566
conformation and single chains as random coils with intermediate conformations between the 31 and 21 helical conformations. Considerable spectral intensity at around 78ppm suggested that some galacturonan chains of intermediate mobility were in the 21 helical eggbox conformation although overlapping signals from galactan complicate the interpretations. Small quantities of a low mobility galacturonan also appear to be present in both 21 and 31 helical conformations. 2.2 Location of Pectins
If PSRE is applied to the Tip experiment, the spatial location of the wall components can be identified. Within small regions of the cell wall, proton spin diffusion averages the Tip values. Thus the T~p measures the mean mobility within each of these small regions, not the mobility of individual polymer chains (13). It is therefore possible to deduce which polymers are located together in the same region. The proton Tip generally decreases with increasing mobility. Cellulose is the least mobile component of the cell wall. It exists in a crystalline form and there is very little movement of the chains (14). In the hydrated cell wall the Tip experiment averages the movement of the polymers over distances of about 2nm or less in the more mobile parts of the wall, and over 5nm in crystalline cellulose. Therefore any component that is within about 2nm of cellulose will appear in the PSRE spectrum with cellulose (15). It was not possible to work out the location of the highly mobile pectic components. However, there were indications that these pectic components did not exist close to cellulose.
Intermedi ate Mobility Low Mobility
I~1~11 ~ ' V l ~ ' U / ' l f v r g ' W . / - I I ' r ~ l v V r
I i~l~
180 160 140 120 100
I
80
60
40
20
ppm
Figure 4: Tip PSRE spectrum of onion cell walls.
0
567
Both the 21 helix and the 31 helix are represented in the low mobility spectrum of polymers close to cellulose (figure 4), although there was some interference from xyloglucan signals. Both conformational forms of the galacturonans could also be identified from their C4 signals spatially located at a distance from cellulose. Because of the nature of the experiment, we can not tell whether or not pectin from either location is covalently linked to cellulose. If cellulose exists in the cell wall as a network within a pectic matrix, the pectin that is within about 2nm of the cellulose network maybe on or near exposed surfaces of cellulose microfibrils. Both the gel and the eggbox pectins are represented in this low mobility spectrum. There are a number of possible locations within the cell wall for the pectin further away from cellulose. If there are covalent links between pectins and xyloglucans (16), then pectic chain segments close to these links would appear in the region sharing the same mean mobility characteristics as cellulose. The majority of the pectic molecule, diverging from the microfibrils would appear in the region with greater mean mobility. The intermediate-mobility pectin can exist in any space in the cell wall more than 2nm away from cellulose microfibrils. It could therefore be in the middle lamella, cell comers or between layers of microfibrils in addition to the above proposal. The pectin seen in this part of the spectrum are probably a heterogeneous mixture from a number of locations. It has been suggested that in cell walls other than those of onions, different types of pectic matrix are present in different parts of the wall (17). This work clearly demonstrates the existence of at least two spatially separate pectic matrices with polymers having at least two conformational forms and three distinct mobilities. It suggests pectins are more than just 'pore fillers' within the plant cell wall.
3. M E T H O D O L O G Y Onions, c v . Bobosa (100g) were homogenised in Triton-X-100 (2gl 1, 500ml), and cell walls collected on a sintered glass funnel. The walls were washed with water and the excess of liquid removed by suction. The walls were then stirred for 30 min in 15ml of phenol-saturated water. The walls were washed extensively with water, cryomilled in liquid nitrogen and dried to a 3.4:1 wall: water ratio prior to use. The NMR methodology has been previously described in detail (9). Briefly, the NMR experiments were carried out on a Varian VXR-300 spectrometer operating at 75.34 Mhz for '3C. MAS rates varied between 3.5 and 4.3 kHz. The proton decoupling field was nominally 35-36 kHz, and the Hartmann-Hahn match was optimised individually for each sample to allow for radiofrequency energy absorption by mobile water protons. The proton rotating-frame relaxation time Tip and cross-polarisation rate were measured in both variable-contact and delayed-contact experiments. In the variable-contact experiment 12 values of the contact time, in the range 25~ts-18ms, were used. Signal assignments were based on published data (3,10) and are given in table 1.
568 In the delayed-contact experiment a variable delay, during which proton spin-locking was maintained, was inserted prior to a fixed contact time of 0.5 ms. So that the results from the delayed-contact and variable-contact data could be compared, they were normalized to give equal signal intensities at a contact time of 0.5 ms (zero delay in the delayed-contact experiment) after the variable-contact data had been adjusted to allow for the fact that full equilibration of proton and 13C polarisation had not quite been reached after 0.5 ms. The difference in normalised signal intensity between the variable-contact and delayed-contact experiments, at a given time point r, is then a measure of the amount of 13C cross-polarising between 0.5 ms and r. The proton/'2 was estimated by subtracting the signal intensity with 0.5 ms contact after 9.5 ms delay from the signal intensity with 10 ms contact time, as above, but after the insertion of a variable delay (20#s to 2 ms) after the proton preparation pulse. The Tip based PSRE was carried out by taking linear combinations of the spectra at zero and 12 ms delay with the coefficients calculated to make the mean spectral intensity equal to zero between 83 and 85 ppm. The T2 -based PSRE was done in the same way using the spectra at zero and 8 #s delay.
4. R E F E R E N C E S
N.C. Carpita and D.M. Gibeaut, Plant J. 3 (1993) 1. 1 M.C. McCann, B. Wells and K. Roberts, J. Cell Sci, 96 (1990) 323. 2 M.C. Jarvis and D.C. Apperley, Carbohydr. Res., 275 (1995) 131. 3 R. Goldberg, C. Morvan, A. Jauneau and M.C. Jarvis, (1996) This volume. 4 K. Fenwick, M.C. Jarvis and D.C. Apperley, unpublished. 5 T.J. Foster, S. Ablett, M.C. McCann and M.J. Giddley, (1996) submitted. 6 x. Wu, S. Zhang and X. Wu, Phys. Rev. B 37 (1988) 9827. 7 L. Muller, A. Kumar, T. Baumann and R.R. Emst, Phys. Rev. Lett., 32 (1974) 1402. 8 M.-A. Ha, B.W. Evans, M.C. Jarvis, D.C. Apperley and A.M. Kenwright, unpublished. 9 10 M.C. Jarvis, Carbohydr. Res., 201 (1990) 327. 11 P.L. Irwin, M.D. Sevilla, W. Chamulitrat, A.E. Hoffman and J. Klein, J. Food Chem. 40 (1992) 2045. 12 P. Tekeley and M.R. Vignon, J. Wood Chem. Technol., 7 (1987) 215. 13 R.H. Newman, A.C.S. Sym. Ser. No. 489, (1992) 311. 14 R.H. Newman, M.-A.. Ha and L.D. Melton, J. Agric. Food Chem., 42 (1994) 1402. 15 R.H. Newman, Holzf0rschung, 46 (1992) 205. 16 C.M.G.C. Renard, A.G.J. Voragen, J.-F. Thibault and W. Pilnik, Carbohydr. Res. 16, (1991) 137. 17 M.-A. Ha, D.M. Gould, R.H. Newman, L.D. Melton and J.I. Mann, Proceedings of the 7th Cell Wall Meeting, (1995) 54.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
569
Changes in Pectic Polysaccharides during elaboration of table olives
A. Heredia; R. Guill6n; C. Sfinchez; A. Jim6nez; J. Fernfindez-Bolafios
Departamento de Biotecnologfa de Alimentos, Instituto de la Grasa, Consejo Superior de Investigaciones Cientfficas, P.O. Box 1078, 41012 SeviUa, Spain
Abstract A traditional system for the preparation of table olives, involves a treatment of the fresh fruit with a solution of NaOH to hydrolised the bitter glycoside oleuropein, followed by a lactic fermentation in brine. The modifications that take place on pectic polysaccharides of olives (Manzanilla variety) during this process was studied. Processing induced a net loss of polysaccharides soluble in sodium carbonate and a paralel accumulation of water and Imidazole/HC1 soluble polysaccharides. A general decrease of the apparent molecular weight of water and carbonate soluble polysaccharides was also detected.
1. INTRODUCTION Texture is one of the most important organoleptic characteristics of olives, on the other hand, the physical resistance of the fruit to some manipulations such as depiting is also related to it. Firmnes is, at least in part, related to the composition and structure of cell wall, therefore, from the point of view of controlling the process it is important to know the changes that take place on cell wall polysaccharides through it. On this work the main changes that take place on three pectic fractions, water, imidazole/HC1 and carbonate soluble polysaccharides, of olive cell wall are described and related with modifications of the fruits texture.
2. EXPERIMENTAL Table olives "spanish style" were processed as described by Jim6nez et al. (1). Cell wall were isolated (2) and the pectic polysaccharides were extracted according with their solubility on water (highly esterified polysaccharides), imidazolium (ionically linked polysaccharides) and sodium carbonate (covalent linked polysaccharides), as shows figure 1. Neutral sugars were quantified by trifluoroacetic acid hydrolysis (3) and gas chormatography of alditol acetates (4). Uronic acids were determined by Blumenkrantz method
570 (5). The main components of each fraction were purified by a combination of gel permeation and anionic interchange chromatography and their structure partially elucidated. CELL WALL MATERIAL
Water, 70~ (2X30 min.)
ImidazolelHCI buffer 0.SM 25~
v
WATER
v
IMIDAZOLE-HCI
y
CARBONATE
12 hours
Sodium Carbonate O.05M, 4~
16 hours
Sodium Carbonate O.05M, 25~
3 hours
Figure 1. Fractionation of cell wall
3. RESULTS
When the amounts of the three pectic fractions were determined through the different steps of the process, it was found that it is during the lye treatment and wash where the main changes took place. Neutral sugars increased in the water soluble fraction and d i d n t change on imidazole and carbonate ones. Uronic acids, on the other hand increased in the imidazole fraction and decreased in both water and carbonate ones. Water soluble polysaccharides: Table 1 shows that there was an important increased of neutral sugars associated with neutral polysaccharides on this group, and the glycosyl composition (table 2) reveals that they are basically arabinans (arabinose content about 90%). The acidic fraction in the fresh fruit is constitued by homogalacturonans (90% U.A) together with small amounts of ramnogalacturonans. Processing induced a decreased on the total amount of uronic acids and a parallel increased of neutral sugars, changing the ratio N.S./U.A. from 0.16/1 to
571 0.93/1. It can be concluded that during processing there is an incorporation of arabinans and a loss of homogalacturonans from this fraction. On the other hand a shift on the molecular weight distribution to lower values of the acidic fraction it was found. Table 1 Yield and composition of water soluble polysacchaddes (mg/fruit) NEUTRAL
ACIDIC
FF
PF
FF
PF
Yield (mg/fruit)
1,72
2,22
0,64
1,02
Neutral S.
0,68+0,01
Uronic A.
0
0
0,68+0,01
1,56+0,02
Total S. N.S./UA
1,564-0,02
. . . . . .
0,09a:0,02
0,28u
0,564-0,06
0,30-a:0,01
0,65+0,04
0,58+0,01
0,16/1
0,93/1
Table 2 Glycosyl composition of water soluble polysaccharides (mol%) NEUTRAL
ACIDIC
FF
PF
FF
PF
Rham
0,62
2,00
2,14(16,14)
11,04 (23,03)
Fuc
0,08
0,09
0,18 (1,43)
1,21 (2,51)
Ara
87,94
92,61
8,48 (63,76)
22,89 (47,70)
Xyl
1,15
0,56
0,05 (0,35)
3,54 (7,40)
Man
2,81
0,40
0,22 (1,64)
0,51 (1,10)
Gal
3,10
2,47
1,74(13,17)
7,83 (16,34)
Glc
4,20
1,86
0,46 (3,50)
0,93 (1,95)
Uronic A.
0,00
0,00
86,76
52,03
Ram/U. A.
-.
.
0,025/1
0,21/1
Rham/Ara
. . . . . .
0,25/1
0,48/1
.
.
.
572 Imidazole-HCl soluble polysaccharides: Most of the polysaccharides of this group didn't elute from the Q-Sepharose column with 10 mM buffer (Table 3). The glycosyl composition (Table 4) shows that these polysaccharides are homogalacturonans and ramnogalacturonans with Table 3 Yield and composition of lmidazole-HCl soluble polysaccharides (mg/fruit) i
NEUTRAL
ACIDIC
FF
FP
FF
FP
Yield (mg/fruit)
0,21
0,11
3,78
3,93
Neutral S.
0,04+0,00
0,04+0,00
0,24:L-0,01
0,64+0,09
Uronic A.
0,01
0
1,54+0,01
1,95+0,01
0,04+0,00
0,04+0,004
1,78+0,01
2,59-a:0,36
Total S. N.S/U.A.
-. . . . .
0,16/1
0,33/1
Table 4 Glycosyl composition of lmidazole-HCl soluble polysaccharides (mol%) NEUTRAL
ACIDIC
FF
FF
FF
PF
Rham
0,80
1,98
2,10 (15,50)
6,46 (26,30)
Fuc
0,15
0,60
0,20 (1,40)
0,55 (2,22)
Ara
57,83
66,02
8,84 (65,16)
11,58 (47,10)
Xyl
3,01
1,45
0,18(1,31)
0,91 (3,70)
Man
10,03
4,40
0,19 (1,42)
0,23 (0,92)
Gal
15,03
21,23
0,17 (1,25)
4,34 (17,65)
GIc
13,05
4,32
0,44 (3,28)
0,50(2,01)
Uronic A.
0,00
0,00
86,43
75,40
Rham/U.A.
. . . . . .
0,024/1
0,09/1
Rham/Ara
. . . . . .
0,24/1
0,56/1
573 side chains rich in arabinose, methylation analisis reveals that one of every three arabinose residues are substitued. The ratio Rha/U.A. is similar to that of water soluble polysaccharides meaning that the proportions of ramnogalacturonans are similar in both fractions. Processing induced a decreased on the percentage of uronic acids and an increase on those of galactose and ramnose, the ratio Rha/U.A. went from 0.02/1 to 0.09/1 while that of Rha/Ara from 0.24/1 to 0.56/1. It can be concluded that during processing there is an incorporation of pectic polysaccharides richer in ramnogalacturonans to this fraction. The molecular weight distribution of these polysaccharides d i d n t change as a consequence of processing.
Carbonate soluble polysaccharides: Unlike the other pectic fractions, tile total sugars of this one decreased during processing (from 1,13 to 0,41 mg/frui0, the most prominent decreased taking place on both neutral sugars an uronic acids of acidic polysaccharides (table 5). In the fresh fruit (table 6) this fraction contains high amounts of uronic acids (48 %) and arabinose (34 %), the ratio Rha/U.A. is much higher than that of water and imidazole soluble fractions, meaning that the hairy regions of the pectic polysaccharides are more abundant on this fraction. Methylation analysis shows that one of every four arabinoses is substituted. With processing it was found an increase in the molar ratio of uronic acids together with a decrease in that of arabinose, methylation analisis reveals that there was a decreased in terminal, 3- and 5-1inked arabinose and that the 3,5-1inked arabinose practically dissapeared. It was also found an important decrease in the molecular weight distribution of this group of polysaccharides. Table 5 Yield and composition of carbonate soluble polysaccharides (mg/fi'uit) NEUTRAL FF Yield (nag fruit) Neutral S.
Total S. N. S.FLI. A.
PF
0,35 0,05•
U.A.
0,2 0,02--1:0,01
0 0,05•
ACIDIC
0 0,02• ---
FF
PF
4,27
0,66
0,59•
0,16+0,01
0,54•
0,25•
1,13•
0,41•
1,10/1
0,64/1
574 Table 6 Glycosyl composition of carbonate soluble polysaccharides (mol%) NEUTRAL
ACIDIC
FF
FP
FF
FP
Rham
0,67
7,24
9,53 (18,20)
9,18 (23,00)
Fuc
0,20
0,83
0,72 (1,35)
0,61 (1,62)
Ara
82,55
50,50
34,10 (65,05)
lo8,96 (49,41)
Xyl
3,73
2,05
0,37 (0,70)
1,76 (4,60)
Man
4,04
8,05
0,04 (0,88)
0,44 (1,14)
Gal
5,16
16,20
6,36 (12,13)
6,41 (16,71)
Glc
3,63
15,33
0,87 (1,66)
0,91 (2,36)
Uronic A.
0,00
0,00
47,58
61,61
Rham/U. A.
-.
.
.
.
.
0,20/1
0,14/1
Rham/Ara
. . . . . .
0,28/1
0,48/1
Texture: In the figure 2 the values of firmness along the process for two consecutive seasons is shown, very similar results were found for both seasons. The most important decreases took place during the lye treatment and subsequent wash step, reaching values of 50% of the initial. When the fruits were placed in the brine solution the firmnes recovered to 80% of the initial and, finally, during fermentation there was a new decrease to 60% of the initial.
4. CONCLUSIONS
When the processing liquids were analyzed it wasnt found any important increased of uronic acids (data not shown), this together with the fact that the total amount of pectic polysaccharides quantified in the cell wall didn t change, bring us to the conclusion that there is no important solubilization Of pectic polysaccharides during olives processing and that the main process that take place, is an interchange of polysaccharides betwen different groups. Therefore processing induced a net loss of sodium carbonate soluble polysaccharides and a parallel
575 %
SEASON 1 90
-I- S E A S O N 2
u) uJ Z
:E 80 rr ,,m
u.. u.I >
.~70 60 501 UF
i
1/2LYE
t
LYE
I
WASH
I
EQ.BRINE
FE FIM
Figure 2. Changes in texture during elaboration
accumulation of water and imidazole/HC1 soluble ones. The accumulation of polysaccharides in the water soluble fraction is mostly a consequence of the incorporation of neutral arabinans, probably resulting from losses of pectic polysaccharides side chains. On the other hand, the uronic acid content of this fraction decreased, most likely due to deesterification of the galacturonic acid residues, increasing in this way their ability to linked ionically in the cell wall and becoming imidazole soluble. The fact that the molecular weight distribution decreased shows that together with the deesterification process it must also take place a limited degradation through B-elimination. In the carbonate soluble fraction there is an evident loss of arabinans side chains together with the breakdown of the linkages that held these polysaccharides with the hemiceUulosic matrix (probably phenolic linkages) yielding them imidazole soluble. This is supported by the fact that the imidazole soluble polysaccharides of the processed fruit are of high molecular weight, showing that there wasn Jt an important degradation of the backbone. It is clear that this changes on the cell wall must have important consequences on the firmness of the fruit. The breakdown of linkages between pectic polysaccharides and the matrix is likely to be an important factor on the irreversible loss of firmness. The deesterification of polysaccharides, on the other hand could have an effect of increasisng firmness. There is, however, another factor that should be taken into account, the alcali deesterification of the
576 galacturonic acid units increase the number of free carboxil groups on the pectic backbone this, together with the high pH during lye treatment and wash (between 12 and 10 respectively), creates a net negative charge on the molecule and repulsions between different pectic chains (the carboxil groups at these pHs are completed ionized), wich results in an important loss of texture. When the fruits are placed in brine the pH is corrected to about 7, this decreases the negative charge due to the deionization of carboxil groups and to the neutralization of some charges by sodium cations and deals to a recover of the firmness. The relative importance of the different factors is something that remains to be cleared.
5. REFERENCES
A. Jim6nez; J. Labavich; A. Heredia; J. Agric. Food Chem., 42 (1994) 1194. R.R. Selvendran. Phytochem. 14 (1975) 1011. J.M. Ruiter; J.C. Bums; J. Agric. Food Chem., 35 (1987) 308. H.N. Englyst; J.H. Cumming. Analyst, 109 (1984) 937. N. Blumenkrantz, G. Asboe-Hansen. Anal. Biochem. 54 (1973) 484.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All fights reserved.
577
Pectins from different tissue zones of apple: characterisation and
enzymatic hydrolysis P. Massiot, A. Baron and J.F. Drilleau
Institut National de la Recherche Agronomique, Station de Recherches Cidricoles, Biotransformation de Fruits et IMgumes, BP 29, 35650 Le Rheu, France
Abstract Pectins from different tissue zones, namely epidermis, the outer parenchyma, the parenchyma of the carpels zone, the carpels and the core line, were isolated from alcoholinsoluble solids (AIS. In both zones of parenchyma, the cell-waU material represented about 80% of the total cell-wall material from the whole fruit. The pectins from the outer parenchyma accounted for 70% of the total. However, there was no change in galacturonic acid concentration. The enzymatic solubilisation of tissues or AIS was higher in the parenchyma zones than in the others. Nevertheless, the depolymerisation of the soluble pectins from parenchyma zones with an endopolygacturonase required the action of pectin methylesterase. The depolymerisation of pectins from the other zones, however, did not.
1. INTRODUCTION
In apple processing, enzymatic treatment of the crushed fruit leads to a lower degree of degradation of the peel and the core than the rest of the fruit. Figure 1 shows the separate tissue zones in diagrammatic form. Their anatomic origins are different: the epidermis and outer parenchyma zones are tissues derived from the fusion of the calyx, corolla and stamens of the flower; the inner zones correspond to tissue derived from ovaries and carpels. The characterisation of the cell-wall material, especially pectins, from the different zones of the fruit may provide additional information on the possibility of finding uses for the discarded fractions.
578
A
Figure 1. Sections of mature apple; A: B: C: D:
epidermis zone, outer parenchyma, parenchyma of the carpels zone, carpels and core line.
2. DISTRIBUTION AND COMPOSITION OF C E L L - W A L L MATERIAL The outer parenchyma (B) is the major tissue zone of the fi'uit, corresponding to more than 80 % of dry matter and the edible zones (B and C) contained 80 % of the cellwall material (Fig.2).
Carpels and core line (D) 10% Parenchyma of the carpels reaion (C) 10%
Epidermis zone (A) 11%
Outer parenchyma (13) 70%
Figure 2. Distribution of alcohol-insoluble solid (AIS) in the different zones of Judeline apple. Total AIS: 12.6 g/100g of apple dry matter
579 The four AIS contained high and equivalent amounts of galacturonic acid (Table 1), indicating that the distribution of pectic substances was similar to the distribution of the AIS in the fruit.
Table 1 Composition of alcohol-insoluble solid (AIS) in different tissue zones of apple Tissue zones Yield (a)
Sugar composition (b)
Rha Ara
Xyl
Man Gal
Glc
Proteins Lipids
Gal.A
(b)
Co)
total
A
274
15
47
34
19
54
154 265
588
57
290
B
107
13
83
74
24
98
296 287
875
27
55
C
160
11
48
88
23
48
301 283
802
44
62
D
308
13
32
135
28
27
344 253
832
55
38
(a) mg/g of d.m. of tissue (b) mg/g of AIS
3. CHARACTERISATION OF PECTIC POLYSACCHARIDES (TABLE 2)
The composition of the pectic fractions confirmed the presence of highly esterified slightly branched rhamnogalacturonan in the CSP fractions and the presence of highly branched rhamnogalacturonan in the HSP fraction.
580 Table 2 Monosaccharides composition of pectic polysaccharides in different tissue zones of apple Tissue
Fraction
zones
Yield
Sugar composition (mole %)
~%AIS
CSP (a)
Rha Ara Xyl Man
Gal
Glc
GalA
DM
Total mg/g
C D
19.2 16.8 16.9 13.0
1.5 1.8 1.9 2.4
9.6 22.7 5.9 7.4
0.8 1.0 0.6 1.3
0.5 0.2 tr 1.0
6.3 11.1 5.4 5.8
1.3 0.4 0.2 1.3
80.0 62.8 86.0 80.8
70 71 62 65
582 722 516 428
A B C D
8.6 12.5 11.1 7.9
4.9 5.7 6.2 7.1
9.7 9.0 10.7 8.4
2.9 4.5 4.6 4.2
0.7 0.4 0.4 0.8
8.2 13.2 8.4 7.3
1.8 2.9 2.3 1.9
71.8 64.3 67.4 70.3
52 41 55 49
650 795 759 692
A B
HSP (b)
(a) CSP Cyclohexane-diamino-tetracetic acid Soluble Pectin. (b) HSP H C1 Soluble Pectin. DM: degree of methoxylation.
4. ENZYME HYDROLYSIS OF C E L L - W A L L POLYSACCHARIDES
When the apple tissues were treated with enzyme preparation for liquefaction (Fig. 3), the cell-wall materials were solubilised with different yields, 95, 86, 66 and 59 % for zones B, C, D and A, respectively. The sequence was the same with the maceration treatment (use of polygalacturonase [PG] only) but the yields were lower.
100
-
m
80-
Liquefaction
,-+.. Maceration
60-
4020-
t
A
C
I
D
A
B
C
Figure 3. SolubilisaUon of cell-wall material (as AIS) when apple tissues were treated with liquefying (SP249) or macerating (UM10) preparations
D
581
With PG [a], the kinetics of pectin solubilisation (Fig.4) were dose to each other, with limits around 40-50 %. A combined action of PG and pectin mcthylcstcrascs (PME) [1:)] or PG, PME and ccllulascs [c] increased these values, especially for the AIS from zone B. The sequence os the zones was D, A, C and B, in rising order of solubilisation.
(a)
100
1
80 60
+ jf
4O
'
20
~ =
A
i i
..---.--c~
B
:
~ : -
C
!
~ - - D
i,
0 0 ,
'qO .,m 0 0 .=. r"
1
2
3
4 ,,
,
,,
,,
100
..r
80
t
5 ,,,
,,,
,
....
OZ
-.--,Z.
_.___._..---~
~ 1 1 ~
A
~ r
C
0
!
60 40
~ t
,,,...,
ilm, 0
,,,
,
(b)
0L 0
,,,,,, ,
20
J
0 0
1
2
3
4
loo I 80 y.........o------c I i
5
~t
:_
(c)
I
-- I '---I"'- A t I
0
O 0
1
2
3
4
5
Time (h) Figure 4. Content of galacturonic acid in the soluble fraction after hydrolysis of AIS with polygalacturonases [a], with polygalacturonases and pectin methylesterase [b], with pectinases and cellulases [c].
582 The fact that the CSP pectic fractions from zones B and C were very little modified by the action of an endopolygalacturonase alone (Fig. 5) suggested that these fractions were structurally different than the others. In particular, the high degree of methoxylation of both fractions and the significant proportion of neutral sugars of the CSP fraction (zone 13) could limit the action of the endopolygalacturonase.
CSP(A)
r ,
~... [CSP(B)i
],': !
. I
& t_
N =
CSP (C~:'
, A
/t;.
I
i
0
-
Kay
i
1
Figure 5. High-Performance Size-Exclusion Chromatography of Cyclohexane-diamino-tetracetic acid-Soluble Pectin (CSP) from different tissue zones of apple (-----) or after hydrolysis with endopolygalacturonase ( - - -) or with endopolygalacturonase and pectin methylesterase (. . . . ).
5. CONCLUSION
The main part (80%) of pectins were in the edible apple zones but no change in galacturonic acid concentration was observed between the different tissue zones. So, carpels and epidermis zones are potentiel sources of highlymethylated pectins.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
I n v e s t i g a t i o n s of the influence of v a r i o u s p r o p e r t i e s of h i g h - e s t e r i f i e d pectin gels
583
cations
on
the
rheological
SybiUe Neidhart, Christa Hannak and Karlheinz Gierschner Institute of Food Technology, Department of Fruit and Vegetable Technology, Hohenheim University, Garbenstr. 25, D-70599 Stuttgart, Germany Abstract
The influence of sodium, potassium, calcium and magnesium ions on the gelling behaviour of apple pectins with degrees of methylation between 43% and 73% as well as the effect of these ions on viscoelastic properties and thermal behaviour of the final gels were investigated by means of small amplitude oscillatory shear experiments. The rheological data were obtained for each pectin according to mixture designs for the metal ions with least changes in pH and ionic strength and analyzed by multiple non-linear regression. With each pectin the various rheological properties were shown to be modulated in a complex way. The setting behaviour was always affected in a similar fashion by the ions as the thermal behaviour of the final gel, but not in the same way as the strength or the elasticity of the gels. The rheological results did not reveal any direct dependence on the charge or the size of the cations, suggesting various, more or less specific modes of action to be involved, depending on the nature of the cation and the amount of dissociated carboxylic groups in the sample. 1. INTRODUCTION High-methylated pectins (HMPs), classified as those with a degree of methylation (DMe) higher than about 50%, require high amounts of soluble solid substance, mainly sugar, and low pH for gelation. Besides type and concentration of pectin these parameters strongly determine gelation as well as resulting gel properties. The available results indicate that the junction zones of these gels are mainly stabilized by cooperative sequences of hydrogen bonds besides hydrophobic interactions between the ester methyl groups [1,2]. While low pH is needed to reduce dissociation of the carboxylic groups and hence to diminish repulsive forces between the pectin molecules, the sugars, reducing water activity, are discussed in terms of promoting hydrophobic interactions by their own interaction with water molecules, which depend on their stereochemistry [1]. As low-methylated pectins are able to form gels in the presence of various bivalent cations by chelating them, the influence of numerous metal ions on the physical behaviour of those pectins has been extensively studied [3-5]. As far as gelation of liMPs is concerned, there are somewhat contradictory indications for the influence of metal ions on the gelation behaviour and on the gel properties (e.g. [6-8]). It was felt that a more comprehensive study on this topic, considering various well characterized HMPs with different levels and patterns of methylation as well as different characteristics of the gelation process on basis of an experimental design, which allows for potential interactions between the cations, would be a useful contribution to the understanding of the complex mechanisms involved in HMP gelation.
584 Here data on apple pectins are presented. A comparison of corresponding data on apple and citrus pectins will be given in a future paper. Small amplitude oscillatory shear measurements have been widely used to follow the gelation process of biopolymers [9] because of their non-destroying character and the great variability of procedures, which allow simultaneous investigations of various features of a single sample. 2. EXPERIMENTAL
2.1. Pectin samples Three unstandardized, commercial pectins from apple (Herbstreith & Fox, Germany) were purified by washing six times with acidified ethanol (60%), afterwards with ethanol (60%), and, finally, in the absence of chloride in the filtrate, with ethanol (96%). The residue was dried for 2.5 hours at 105~ and extensively characterized (table 1) [10]. The percentage of anhydogalacturonic acid (AUA) was determined colorimetdcally with m-hydroxydiphenol. Degree of methylation (DMe) and acetylation (DAc) resulted from h.p.l.c, determination of methanol and acetate, while analysis of various neutral sugar contents was realized gas chromatographically. Cation analysis was done by atomic absorption spectrometry. For more details of analytical procedures and pectins, the reader is referred to [10]. The pattern of methylation of each pectin can be considered to be random The content of rhamnose was fairly constant. 2.2. Preparation of the gels The pectin/sucrose gels were characterized as follows (amounts per 100g gel): 0.3 g AUA, 65% soluble solid substance, 0.01 mol sodium acetate / lactic acid buffer, pH 3.0 (20~ The metal ions were added as combinations of chlorides according to a mixture design with constant amount of chloride ions (2.5 mmol / 100g gel). Thus the total amount of metal ions Table 1. Characteristics of the purified pectin samples. Pectin =
A73
A64
A43
AUA [g / 100g dry weight] DMe ~ [%] DAc ~ [%] Total neutral sugars~ [%] Calciumb [%] Magnesium ~ [%]
83.4 73.4 5.6 28.1 0.17 <0.001
83.9 64.1 3.2 24.9 0.02 <0.001
87.9 42.9 0.8 20.7 0.07 <0.001
0.03
0.03
0.03
Sodium~ [%]
Potassium~ [%] 0.13 0.09 0.25 Intrinsic viscosity 5.7 5.3 4.3 Weight average molecular weight c Mw (x 105) 12.7 8.56 35.6 Number average molecular weigh( Mn (x104) 10.6 9.04 13.1 Polydispersity~ D . . . . 11.9 9.4 27.1 a The terms for the purified apple pectins indicate the degree of methylation. b Values in mol / 100 mol AUA; total neutral sugars without the glucose content of starch. c in 0.4 M sodium acetate buffer containing 0.025 M sodium sulphate, pH 3.0; molecular weight distribution as determined by size exclusion chromatography referring to dextrans. 9
,
r
585 was similar to commercial jams with 45% fruit content. To simplify matters, the mixture designs for each pectin consisted of combinations ofNaC1, KC1 and CaC12 and were compared to corresponding experiments with mixtures of MgC12 instead of CaC12, not studying potential interactions between Ca ++ and Mg ++. Preparation of gels was performed by analogy to [ 11], carefully dispersing the dry pectin, premixed with a low portion of the sucrose, in the cold diluted solution of buffer and chloride solutions. Soluble solid substance was adjusted by cooking the whole mixture to a precalculated net weight of 170g. When this weight was reached, the sample was immediately poured into the hot measuring system of the rheometer. pH varied within minimal limits (pH 2.95 + 0.05). Lowering of pH was due to a higher ionic strength (A~t + 0.01), when the amounts of bivalent cations in the systems were increased. 2.3. Rheological experiments Dynamic theological measurements were performed using a Bohlin CS controlled-stress rheometer with improved angle resolution, equipped with a double gap device (diameter of the fixed cylinders 40 and 50 mm respectively), which had been modified to achieve more homogeneous temperature conditions within the sample. Thus the fixed inner cylinder, which is originally massive, was replaced by an equivalent hollow cylinder, forming the same inner one of the two (vertical) measuring gaps as before, together with the rotating cylinder. This hollow cylinder was screwed at the bottom to the outer fixed cylinder and showed on its top a flat basin with a deaeration screw. During the measurement this basin was filled with water, providing a humid atmosphere over the free sample surface by its combination with an adequate cover plate. The edge of the cover plate was additionally equipped with a wet sponge. In our previous studies the described system was shown to be suitable for the investigation of the gelling behaviour of pectin systems, even for low viscosity systems, which did not really gel. The gel formation was monitored by measuring the storage (G') and the loss (G") modulus as well as the related phase angle ~ = arc tan (G"/G') at fixed frequency (1 Hz) as a function of time on cooling first with constant rate (-1.041 _+0.007 K/min) from 93 to 20~ (deformation amplitude 7 = 0.015) and with further thermostatting at 20~ for 1 hour (7 = 0.005). In order to characterize the "final" gel, a mechanical spectrum, e.g. a frequency sweep, followed at the same temperature and deformation in the range of 0.005 to 10 Hz. This was completed by recording the thermal behaviour of the sample by reheating with constant rate (0.714 +_0.003) to 90~ (0.2 Hz, 7 = 0.005). Temperature control was achieved with an adjustable water bath, allowing the application of temperature gradients when cylindrical devices are employed as in this case. As the samples had to be reheated, the free sample surface was covered as good as possible with paraffin before starting the measurement. 3. RESULTS AND DISCUSSION 3.1. Influence of the metal ions on setting time and temperature By the applied method, the sol / gel transition can be recorded because of the accompanied, more or less sharp change from a viscous liquid to a viscoelastic solid-like character of the sample, leading to a drop of the phase angle during a relatively short time interval when G' rises sharply, generally exceeding the less rapidly increasing loss modulus G", due to increased network forming junction zone density. Thus, the exact gel point is directly accessible by this type of measurement. However, because of the superimposed frequency dependence of the dynamic moduli and related quantities, which vary with nature and concentration of
586 biopolymers [9], there is still some discussion on the proper detection criterion for this point, suggesting a multifrequency technique as the best method. For the present study, only measurements at one fixed f e q u e n c y were possible. Hence, the characteristic drop of the phase angle was used to estimate the gel point by considering the time t [s] (and overlaying temperature T [~ at 51 = 75 ~ and ~ = 45 ~ the latter indicating the point of intersection of both moduli. Both characteristic times were analyzed by multiple non-linear regression for each data set. The squares of the adjusted correlation coefficients lay between 0.99958 and 0.99994 for t~=75 o and between 0.94877 and 0.99985 for t,-_45o. The resulting simple models only contained parameters significant at levels with a < 0.15, describing direct influences of each of the three cations in one set, interactions between two cations and in only few cases quadratic influences of a single ion. With all three pectins the sol / gel transition was strongly influenced by the various cations within certain limits. Both characteristic times were affected in the same sense. However, when gelation proceeds less rapidly, the slowly decreasing phase angle is known to be more frequency dependent [9,12] and consequently the time interval between 51 = 75 ~ and ~ = 45 ~ will increase. With systems described here, in general, ~l = 75 ~ rather indicated the part of the ~5-t-curve with its highest change than ~2 = 45 ~ For this reason, only data based on ~1 = 75 ~ are shown in figure 1. In spite of this, it may be of some interest to note that the criterion ~2 = 45 ~
1'4
[Z-'] [mmol/100g gel] 2.5 2.1 1.7 1.3 0.9 0.5 ........................I........................!........................I........................!....................~ 2 0
~4
g3
40
2
~60
2
"-'1
_o ~
0.9 0.5 .................... 20
~3 O O
.2 p.
pectin A 73 i I
0.0 fig.la
[Z*] [mmol/100g gel] 2.5 2.1 1.7 1.3 ~-......~
0.2
I
I
I
0.4 0.6 0.8 [V++] [retool/100g gel]
1.0 ,
0.0 fig.lb
0.2
0.4 0.6 0.8 [V++] [mmol/lOOg gel]
1.0 ...
Figure la-c. Influence of CaC12 and MgC12 doses, replacing KC1 or NaC1, on the setting 2.5 2.1 1.7 1.3 0.9 0.5 behaviour of liMP / sucrose systems. .......................!.......................!........................I........................!.....................+20 Dependent variable t~75o, frequency 1 Hz, ~4 qdeformation 0.015. [V ++] = bivalent ion, [Z +] ..-, ", . , --40 ..... :'i ...... = monovalent ion, [CI'] = const. = 2.5 mmol / 100g gel, and 2[V++]+[Z+]=2.5. I Ca ++ . . . . . ~" " " "-" : : : : : : : :.. :...._. :~-60, + ++ + ++ + ~U' ~-+K , El Ca ++Na , r Mg ~-+K , O ? ', 86_~ Mg ++~-~Na+, --- a n d regression curves I pectin A43 I 8Q for Ca ++ and Mg ++ containing mixtures, I I I I / respectively. The thin dotted line at 20~ 0.0 0.2 0.4 0.6 0.8 1.0 marks the end of cooling. ,,?" indicates extrafig.lc [V++] [retool/100g gel] polated end of curve (pre-gelation). [Z T] [mmol/100g gel]
w
587 was not fulfilled under the given conditions for the pectin A43 when NaC1 was only added. This system thickened a little, as indicated by a slight drop of the phase angle, but remained liquid during the whole measurement. Statistical analysis did not reveal any si~ificant interactions between Na + and K+. Threedimensional plots for each design showed that the data of each mixture design could be easily described by the data belonging to the border lines of the defined fields, because minimum and maximum values were located there. Thus, without loss of information, figure 1 gives a representative idea of the influence, which the studied cations exert on the sol / gel transition of the three pectins. The curvature in the dotted curves reveal the interactions between Ca++ and a monovalent cation. In the presence of added salts, the onset of gelation was obviously favoured in the order Ca++ > K + > Mg ++ > Na + independent of the nature of the pectin (fig.l). In comparison to the other pectins, all systems of A64 (fig. lb) only gelled at extremely low temperatures despite the relatively high degree of methylation. This might be due to the great differences in molecular weight distribution among the pectins (table 1). 3.2. Influence of the metal ions on properties of the resulting gels In order to characterize the resulting gels, frequency sweeps were recorded alter a totally elapsed time of 135 rain. From the behaviour of the moduli during the preceding period at constant temperature (20~ and frequency, it could be concluded that the states of the gels at that time could already give a representative idea of the influence of the metal ions on the viscoelastic properties of the products, because only slight increases in the moduli, even at very low levels, were to be expected, which were not able to change the results on the whole. The majority of the mechanical spectra revealed the typical behaviour of gels [9]: The storage modulus only minimally increased when the frequency rised. However, the loss modulus was independent of the frequency in the limited range up to 0.1 Hz only. Within that range, the ratio between G' and G" was higher than 10:1. The mechanical spectra were similar to those of other HMP/sucrose gels [13]. In order to compare the gels, the values at 0.01 Hz were used because of the independency of frequency for both moduli. It might be interesting to point out to the few cases, where at least at higher frequencies, the storage modulus more strongly depended on frequency, as it was in general. But even those spectra could be characterized by their constant values in a low frequency range. The mechanical spectrum of the previously mentioned system of pectin A43, which remained fluid, containing only NaC1 as added salt, showed very low, roughly equal moduli about 1 Pa (at 0.01 Hz), which readily increased with frequency. The extent of the solid-like character, i.e. the strength of the samples, can be directly described by the storage modulus G'. As both moduli rised during gelation, it seemed to be more efficient to take the ratio of G' to G", describing the dominant elastic character of the viscoelastic sample, as a second characteristic quantity than to use the loss modulus G" itself. As shown previously (fig. 1), the complex influence of the metal ions even on viscoelastic properties of the gels might be safliciently demonstrated by considering the border lines of each defined field only (fig.2-4). The squares of the adjusted correlation coefficients lay between 0.9778 and 0.9965 for G' and between 0.9951 and 0.9996 for G'/G". The resulting simple models only contained parameters significant at levels with ~ _ 0.15, describing direct influences of each of the three cations in one set and interactions between two cations.
588 [27-] [mmoUl 0 ~ ~l] 2.1 1.7 1.3 I I I
2.5 1.2 ! ~1.0
~
.....
0.9 I
0.5
...............................................................................
-
~0.8
~-- [Z"] [mmol/100g gel] 2.5 2.1 1.7 1.3 1.2 I I I
0.9 I
0.5
~ 1 . 1 .......................................................................... t pectin A 73 1.0 ......--:.............................................................................................................
0o6
~_. 0.9 ........................v........:................a ......................................................... ~
0.4
~176
9176176176
"~
08
0.2 0.0 0.0
0.2
~.2a
0.4 0.6 0.8 [V++] [mmoYl 0 ~ ~1]
1.0
b 0.7 ....................................................................................................................... 0.6 I I I I 0.0 0.2 [ ~ 0.6 0.8 1.0 fig.2b [retool/100g gel] --~
Figure 2a/b. Pectin A73: Influence of CaCh and M g C h doses, replacing KC1 or NaC1, on (2a.) the strength and on (2b.) the elasticity of the resulting gel. Presentation in reference to the properties o f the gel, containing KC1 as added salt only (Go' = 177+8 Pa, Go" = 7.0_+0.2 Pa, (G'/G")o = 25.2_+0.5). Conditions: 20~ 0.01 ~ deformation 0.005, start o f frequency sweep 135 min. [V ++] = bivalent ion, [Z +] = monovalent ion, [CI'] = const. = 2.5 mmol / 100g gel, and 2[V++]+[Z+]=2.5. II Ca++~--~K+, r-! Ca++~__~Na+, , Mg++~__~K+, O Mg++~--~Na+, --- a n d - regression curves for Ca ++ and Mg ++ containing mixtures, respectively. ~-- [Z'-] [retool/100g gel] 2.5 2.1 1.7 1.3 1.2 I 9 I I .. 1.0 i
~
"
0.9 I
0.5
0.8 ......................~
............."-:-:-:-:--! .-II! ~
o
0.0 fig.3a
...
d 1.0 ~ , . , , .
0.5
~
b 0.9
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.................................................................. t oo.n I 0.2
0.9 I
......."~..........................--:::-..i
~. 0.6 ............................0.7- .....:: . . . :-~. ....... . . :--3-:---' . . . . -. . 0 0.4 . ...... 0.0
~-- [Z"] [mmol/100g gel] 2.5 2.1 1.7 1.3 1.2 I I I
.......
~0.8
64 t
I I I 0.4 0.6 0.8 [V++] [mmol/100g gel]
~'0.7
.......................................................................................................................
[D 0.6 1.0 fig.3b
0.0
I 0.2
I [~
I I 0.6 0.8 [mmol/100g gel]
1.0
Figure 3a/b. Pectin A64" Influence of CaC12 and MgC12 doses, replacing KC1 or NaC1, on (3a.) the strength and on (3b.) the elasticity of the resulting gel. Presentation in reference to the properties of the gel, containing KC1 as added salt only (Go' = 174_+9 Pa, Go" = 8.6+1.0 Pa, (G'/G")o = 20.3+1.3). Conditions and legend see fig. 2. In contrast to the setting behaviour, the pectins greatly differed as far as the relative effects of calcium and potassium ions on the strength of the gels were concerned (left sections (a.) of fig. 2-4). With the studied pectins, the importance of calcium ions to gel firmness rapidly decreased with increasing DMe in favour of the effect of potassium ions. But even in the case, when calcium extremely enhanced gel strength (pectin A43, fig. 4a.), potassium ions promoted gel elasticity, while increasing amounts of calcium ions, replacing potassium ions, were rather adverse with respect to the latter property (fight sections (b.) o f fig. 2-4).
589 [Z"] [mmol/lOOggel] 2.1 1.7 1.3 , , ,
2.5 8.0
........................................................
!
6.0 ~ 4.0
it
.............
0.9 ,
.-...-...'...'..2.2.2..7..'...'...-
0.5 ]
0.0 fig.4a
I
0.2
1
^
i
0.4 0.6 0.8 [V++] [mmol/100g gel]
.
. . . . . . .
-ii-
- . . . .
0.9 I
0.5
~
1.0 1 '"~-~............................ ~?:-......"'-:'~""?-'-:-::-..,~ o~
I
[Z'] [mmol/lOOggel] 2.1 1.7 1.3 I-I. I ~
.....
'"
~ 2.0 ...........Y: ...............;Y;;......."...................pectin A 43 " 0.0 4-
1.2
2.5
T
1.0
~
0.6
.
0.4-
~0.2 i (9 0.0 0.0 fig.4b
i 0.2
I [~
..... pectin A 43 I I 0.6 0.8 1.0 [mmol/lOOggel]
Figure 4a/b. Pectin A43: Influence of CaC12 and MgC12 doses, replacing KC1 or NaC1, on (4a.) the strength and on (4b.) the elasticity of the resulting gel. Presentation in reference to the properties of the gel, containing KC1 as added salt only (Go' = 34+_2 Pa, Go" = 3.4_+0.1 Pa, (G'/G")o = 9.8_+0.1). Conditions and legend see fig. 2. As it was shown for the setting behaviour, magnesium and especially sodium ions affected the viscoelastic properties of the final gels less favourably than potassium ions did (fig. 2-4). 3.3. Influence
of the metal ions on the thermal
behaviour
of the gels
As it was expected for HMPs, the gel state was stable when samples prepared of A 73 and A64 were reheated to 90~ In general, the storage modulus slightly decreased at first, but with further heating it strongly fised again, obviously passing a maximum, which, however, could not be measured with each sample because of slippage. The minima, which were reproducible, might indicate the onset of temperature dependending processes, which increase the junction zone density, probably due to forced hydrophobic interactions [ 1]. The gels made of pectin A43 collapsed, when the samples were reheated. Nevertheless, while the storage modulus was altogether decreasing during heating, for the majority of those samples, G' passed at least a local minimum and a local maximum. Referring again to the border lines of the examined mixture designs, table 2 shows the temperatures, where the absolute (A73, A64) or local (A43) minima were found. The metal ions seemed to influence the described behaviour in the same sense, as it was previously shown for the setting behaviour. The results may be summarized as follows: Na + seemed to inhibit the gelling ability of each pectin tested. Ca ++ always substantially increased the setting temperature in relation to other ions, but its influence on the strength of the gels greatly changed with the nature of the pectin. K + especially favoured the gel elasticity of all pectins. Replacing sodium ions, Mg ++ forced each property to a lesser extent than potassium ions did. By analogy to specific effects of various sugars [1], the obviously complex modes of action involved here may have to be discussed in terms of different effects on "solvent structure", which is caused by hydrophobic bonds between "water, soluted and dispersed molecules and induced, more or less strong hydrophobic effects depending on DMe. The metal ions are expected to interfere with this structure according to their charge density by forming different hydration shells in solution, while only the two bivalent ions, mainly Ca ++, are able to chelate with dissociated carboxylic groups [3,4]. The latter does not necessarily result in junction zone formation [3], but the
590 Table 2. Thermal behaviour of the gels. Temperatures [~ where minimal storage moduli were observed on reheating. Concentrations of ions in mmol / 100g gel, [V ++] = added bivalent ion, [Z +] = addedmonovalent ion, [CI'] = 2.5, 2[V++]+[Z+]=2.5 or [Na+]+[K+]=2.5. Exchange between monovalent ions [Na +] [K+] 2.5 0 1.5 1.0 0.5 2.0 0 2.5 Exchange between Na + and a bivalent ion [Na + ] [V ++ ] 2.5 0 1.5 0.5 0.5 1.0 Exchange between K + and a bivalent ion
pectin A73:
pectin TG,min [Ca ++ ] 20.0 29.1 29.2 pectin TG,min
To,rain [~ 20.0 24.2 26.2 27.3 A73: [~ [Mg ++ ] 20.0 23.4 26.6 A73: [~
pectin A64:
pectin T6,min [Ca ++ ] 20.0 20.0 25.4 pectin TG,min
To,min [~ 20.0 20.0 22.8 22.6 A64: [~ [Mg ++ ] 20.0 20.0 20.0 A64: [~
pectin A43:
pectin TG,min [Ca ++ ] no G'l,~m;, 35.8 49.1 pectin TG,min
Tc.,min [~ no G'~,~m~o no G'! .... i, 33.9 30.5 A43: [~ [Mg ++ ] no G'~,~m~, no G'I .... i, no G'ie~.min A43: [~
[K+]
[V ++ ]
[Ca++ ]
lMg ++]
[Ca++ ]
[Mg ++ ]
[Ca++ ]
lMg ++]
2.5 1.5 0.5
0 0.5 1.0
27.3 33.1 33.5
27.3 26.9 26.2
22.6 26.7 26.3
22.6 20.0 20.0
30.5 45.4 56.4
30.5 32.0 37.0
chelated metal ions might influence the system of hydrogen bonds in a more stabilifing sense than the corresponding ions in the surrounding solvent.
Acknowledgement The authors thank Herbstreith & Fox, Neuenbiirg, Germany, for the financial support as well as Prof. M. Kulm, Hohenheim University, who made the rheometer available for the present and other investigations.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Oakenfifll, D., Scott, A. (1984), J. Food Sci. 49:1093-1098. Walkinshaw, M.D., Amott, S. (1981), J. Mol. Biol. 153:1075-1085. Thibault, J.F., Rinaudo, M. (1986), Biopolymers25(3):455-468. Thom, D., Grant, G.T., Morris, E.I~, Rees, D.A. (1982): Carbohydr. Research 100:29-42. Kohn, R. (1987), Carbohydr. Research i60:343-353. Hinton, C.L. (1950), J. Sci. Food Agric. 1:300-307. Pilnik, W. (1964), Fruchtsafiindustrie 9:277-284. Endrel3, H.-U., Dilger, S., Gierschner, I~ (1987), Ver6ffentlichungen der Arbeitsgemeinschafi Getreideforschung, 214 (Ber. Tag. Lebensmittelrheologie, 2nd, 1987), p. 86-98. Doublier, J.L., Latmay, B., Cuvelier, G. (1992), In: Viscoelastic Properties of Foods, M.A. Rao, J.F. Steffe (eds.), Elsevier Science Publishers LTD, London, p. 371-434. Hannak, C. (1995), to be published. Zedler, C. (1983), Industrielle Obst- trod Gemiiseverwerttmg 68:523-527. Neidhart, S. (1995), to be published Lopes da Silva, J.A., Gon~alves, M.P. (1994), Carbohydr. Polymers24:235-245.
Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V.All rights reserved.
J.
591
C h a n g e s in m o l e c u l a r w e i g h t and c a r b o h y d r a t e c o m p o s i t i o n o f cell wall polyuronide and hemicellulose during ripening in strawberry fruit Yoichi Nogata, Koh-ichi Yoza, Ken-ichi Kusumoto and Hideaki Ohta Chugoku National Agricultural Experiment Station, Ministry of Agriculture, Forestry and Fisheries Fukuyama, Hiroshima 721 Japan
Abstract Polyuronides and hemicelluloses from the cell wall of developing strawberry fruit (Fragaria ananasa, Duch. cv. Toyonoka) were examined for molecular weight distribution, and carbohydrate composition prior to and following gel filtration chromatography. In nonfractionated polyuronides, the galacturonic acid and arabinose concentrations decreased during development. The concentration of rhamnose markedly increased, whereas that of arabinose declined in low-molecular-weight polyuronides (Mr < 50 kDa.). A significant reduction of hemicelluloses was detected in the high-molecular-weight region (Mr > 170 kDa.), whereas little change was observed in the sugar composition of these polymers. Polygalacturonase activity was consistently reduced during ripening.
1. INTRODUCTION Endo-polygalacturonase (PG) plays a crucial role in converting protopectin in cell walls to a soluble form during fruit ripening [1, 2]. The increase in pectin solubility is considered to result from the hydrolysis of large pectin polymers [3]. However, molecular weight distribution studies of polyuronides derived from strawberries [4] and apples [5], both of which have low PG activity [6, 7], showed little consistent change of these polymers during ripening. In addition, the work with a transgenic tomato suggested that degradation of polyuronides was not necessarily the cause of softening [8, 9]. Gel filtration chromatography analyses of tomato [10], strawberry [4] and muskmelon [11] hemicelluloses revealed marked changes in the molecular weight distribution of these polymers during ripening. Studies of the cell wall composition of ripening fruit showed that polymers rich in neutral sugars, particularly arabinose and galactose were lost from the cell wall during ripening [3, 11, 12]. Armed and Labavitch [13] showed that the loss of cell-wall arabinose in the ripening pear was a consequence of the hydrolysis of polyuronides rich in this sugar. The increase of pectin solubility may reflect the cleavage of linkages between side chains of pectin [14, 15] and hemicelluloses. Therefore, the modification of hemicelluloses must also be studied to elucidate the role of these polymers in fruit softening. Herein, we investigated the contents, molecular weight distribution and carbohydrate composition of polyuronides and hemicelluloses, and PG activity in the ripening strawberry.
592
2. MATERIALS AND METHODS 2.1. Plant material. Strawberries were grown in our experimental field and classified into five groups; small green (SG), large green (LG), reddish (W/R), red ripe (R) and over-ripe (OR) stage. 2.2. Preparation of polyuronide and hemicellulose. Fruit tissue was homogenized in four volumes of ethanol and refluxed for 30 min in a boiling water bath. The homogenate was passed through a glass filter and the residue washed sequentially with 500 ml of 80% of ethanol and 500 ml of ethanol. The powder (AIS) was dried over P205 in v a c u o . AIS was extracted at a concentration of 1 mg/ml in water with constant stirring overnight at room temperature and filtered through a glass filter. The residue was washed and filtered again. The filtrate was combined and made up to a known volume and used as a water-soluble fraction. The residue was resuspended with 0.05 M sodium acetate, 0.04 M EDTA, pH 4.5, at a concentration of 1 mg/ml with constant stirring for 4 hr at room temperature. The suspension was filtered and the residue washed with the same solution and filtered again. The filtrate was combined and made up to a known volume and used as an EDTA-soluble fraction. The residue was resuspended with 0.05 M HC1 at a concentration of 1 mg/ml and heated in a boiling water bath for 1 hr. The suspension was filtered and the residue washed with the same solution and filtered again. The filtrate was combined and made up to a known volume and used as a HC1soluble fraction. Polyuronides analyzed for molecular weight distribution was extracted directly from AIS with EDTA solution, freeze-dried and dialyzed against buffer solution for gel filtration. Hemicellulose fractions were prepared according to the method described by Huber [10]. Neutral sugar concentration was estimated by the anthrone method of Dische [ 16], and uronic acid concentrations were estimated by the m-hydroxydiphenyl method of Blumenkrantz and Asboe-Hansen [ 17]. 2.3. Sugar analysis and gel filtration chromatography. Sugar analysis was performed after methanolysis combined with TFA hydrolysis using a Dionex Bio-LC system (Sunny vale, CA) equipped with a CarboPac PAl column (4 x 250 mm). The experimental conditions were the same as those used by Ruiter et. al. [ 18]. The molecular weight distribution of cell wall polysaccharides was estimated by gel filtration with a TOSOH TSK gel G4000 PWXL (7.8 x 300 mm) column equilibrated and eluted with 0.05 M sodium acetate, 0.01 M EDTA, 0.05 M NaC1 (pH 5.0) in polyuronide and 0.05 M sodium citrate, 0.1 M NaC1 (pH 5.5) in the hemicellulose fraction. Samples (1 m g / m l ) o f 100 ml were injected. The eluate was monitored by a refractive index detector (Shimadzu RID-6A, Kyoto, Japan) and collected at the fraction size of 0.4 ml. 2.4. PG extraction and assay. One kg of tissue was homogenized with 2 1 of cold water and 100 g of polyamide. The suspension was adjusted to pH 6.0 with 1 M NaOH and centrifuged at 15, 000 g for 30 min. The insoluble fraction was washed with 1 1 of cold water and centrifuged again. The enzyme was extracted by suspending the pellet in 1 1 of 0.05 M sodium acetate, 1 M NaC1 (pH 5.5). After standing for 12 hr at 0-4 ~ with occasional stirring, the suspension was centrifuged and the supernatant was dialyzed against 0.05 M sodium acetate (pH 5.5). The reaction mixture consisted of 0.01 ml of 0.4 % purified polygalacturonic acid as substrate, 0.05 ml of 0.2 M sodium acetate (pH 5.5) and 0.05 ml of enzyme solution. After incubation at 37 ~ for 18 hr, the solution was analyzed for reducing groups by the cyanoacetamide reagent [19]. A unit of PG activity was defined as the amount of enzyme that catalyzes the formation of 1 mmole of reducing groups per hr.
593 3. RESULTS AND DISCUSSION 3.1. Changes in polyuronide and hemicellulose concentrations and PG activity. The concentrations of water-, EDTA- and HCl-soluble polyuronides steadily decreased during ripening (Figure 1). The decrease of these polymers was in agreement with the observation that the polysaccharide synthesis in the strawberry cell wall failed to keep pace with cell expansion that continued throughout the life of the fruit [20]. The reduction of EDTA- and HCl-soluble polyuronides was marked at an early stage of ripeness. This suggested that EDTAand HCl-soluble polyuronides were converted to water-soluble polyuronides during ripening. Conversely, the hemicellulose concentration consistently increased, which suggested that the synthesis of this polymer was more vigorous than that of polyuronides in the ripening strawberry.
,l,=a
F1 Water D EDTA I~ HC1 @ Hemicellulose -
"'~ 6
15 ~4 -= 2
r,.)
SG
LG W/P R Stage of ripeness
OR
Figure 1. Concentrations of polyuronides and hemicelluloses extracted from strawberry tissues at different stages of ripeness. Table 1 Changes in PG activities in strawberry fruit during ripening Stage SG LG W/P R OR
Weight (g) 4.11 6.62 10.61 12.46 12.73
Polygalacturonase (U/g) 0.313 0.143 0.092 0.079 0.061
The synthesis of endo-PG occurs in the ripening stage after an increase of ethylene production [21] and its appearance has been correlated with an increase in soluble pectin and softening [22]. Exo-PG is suggested to participate in the initiation of climacteric ethylene production [23]. Strawberry fruit has been accepted to be a non-climacteric fruit and ethylene
594 production declines steadily during fruit development approaching a basal level at maturity [20]. PG activity decreased consistently to be one-fifth of the initial level at the OR stage (Table 1). There may be a correlation between PG activity and ethylene production level, whereas the role of PG in the strawberry remains unclear.
3.2. Changes in sugar composition of polyuronides and hemicelluloses. In the polyuronides from the SG to OR stage, the galacturonic acid and arabinose concentrations decreased by 22% and 46%, respectively, and the rhamnose concentration increased by 2.5-fold on a mol% basis (Figure 2-I). The loss of arabinose was related to ripening; the loss of this sugar was more prominent at the ripening stage (W/P-R) than at any other stage. Glucose appeared at the LG stage and xylose at the W/P stage and accounted for 5% and 10% of the sugars in the polyuronides, respectively. The appearance of xylose was considered to be the result of cleavage of linkages connecting polyuronide and other cell wall polymers. The galactose concentration was persistent during ripening. These findings suggested that the side chains of the polyuronides are modified during the developmental process. Xylose and glucose were predominant neutral sugars in the hemicellulose at all stages of development (Figure 2-II). The concentration of arabinose decreased by 31% from the SG to OR stage, and the loss was not characteristic in the ripening stage. The proportions of other glycosyl residues remained constant.
100" 90--.-O 80. E 70--o 60--.,-~ 50--9 I:x,
II i:i:i:i:!: . . .
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. . . .......
40-
E ot o 30ca~
r~
2010-SG
LG
W/P
R
OR SG Stage of ripeness
LG
W/P
R
OR
Figure 2. Sugar composition of polyuronides (I) and hemicelluloses (II) extracted from strawberry tissues at different stages of ripeness. Galacturonic acid, Ill 9Rhamnose, [;~ 9 Arabinose, 1~ 9Galactose, [7]" Glucose, 1-'1 ; Xylose, ~ .
3.3. Gel filtration chromatography of polyuronides and hemicelluloses. Little change was observed in the molecular weight distribution of the polyuronides during ripening (Figure 3-I) as was reported for the apple [5]. However, from the SG to R stage the concentration of rhamnose markedly increased, while that of arabinose decreased in fractions less than 50 kDa. in molecular weight (Figure 3-II, III). It has been reported that insertions of rhamnose result in a marked kinking of the parent polymer and minimize the frequency of interaction of any two adjacent chains [24, 25]. The loss of arabinose in the polyuronides was due largely to that of low-molecular-weight polymers. The appearance of xylose was limited to
595 fractions less than 50 kDa. in molecular weight. The increase in pectin solubility may result from cleavage of linkages between branched polyuronides and other cell wall components containing xylose in strawberry ripening. A significant reduction of the high-molecular-weight polymers (Mr > 170 kDa.) was detected in hemicellulose fractions throughout development (Figure 3-IV). This change was more prominent in the developing stage than ripening stage. The low-molecular-weight polymers were newly synthesized from the result of increased content of these polymers. IV
I
SG so
J
j
J J
-,.R I
I
I
75 50 25 100. SG ......
~.
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I
I
5
1 II
.. . . .
9
f
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/
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~0 40-
o-
II 25
5
1
34
17
4.2
2
1
Molecular weight (xl0 kDa.)
Figure 3. Molecular weight distribution and sugar composition of polyuronides and hemicelluloses. I. Molecular weight distribution of polyuronides extracted from tissue at SG, W/P and R stages. II. Sugar composition of polyuronides extracted from SG stage tissue. III. Sugar composition of polyuronides extracted from R stage tissue. IV. Molecular weight distribution of hemicelluloses extracted from tissue at SG, W/P and R stages. V. Sugar composition of hemicellulose fractions extracted from SG stage tissue. VI. Sugar composition of hemicellulose fractions extracted from R stage tissue. Symbols as in Figure 2.
596 The concentrations of xylose and glucose increased and that of arabinose and galactose decreased in the fractions with a molecular weight ranging from 10 to 170 kDa. (Figure 3-V, vI). The present findings indicate that both polyuronides and hemicelluloses are modified during strawberry ripening. The increase in pectin solubility may not result from hydrolysis of the galacturonan backbone but by cleavage of linkages between polyuronides and hemicelluloses. 4. R E F E R E N C E S
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
S.J. Wallner and H. L. Bloom, Plant Physiol. 60 (1977) 207. S. Yamaki, Y. Machida and N. Kakiuchi, Plant Cell Physiol. 20 (1979) 311. K.C. Gross and S. J. Wallner, Plant Physiol. 63 (1979) 117. D.J. Huber, J. Food Sci. 49 (1984) 1310. H. Yoshioka, K. Aoba and Y. Kashimura, J. Amer. Soc. Hort. Sci. 117 (1992) 600. Y. Nogata, H. Ohta and A. G. J. Voragen, Phytochemistry. 34 (1993) 617. I.M. Bartley, Phytochemistry 17 (1978) 213. C . J . S . Smith, C. F. Watson, P. C. Morns, C. R. Bird, G. B. Seymour, J. E. Gray, C. Arnold and G. A. Tucker, Plant Mol. Biol. 14 (1990) 369. J.J. Giovannoni, D. DellaPenna, A. B. Bennet and R. L. Fischer, Plant Cell 1 (1989) 53. D.J. Huber, J. Amer. Soc. Hort. Sci. 108 (1983) 405. T . G . D . McCollum, D. J. Huber and D. J. Cantliffe, Physiol. Plant. 76 (1989) 303. K.C. Gross and C. E. Sams, Phytochemistry 23 (1984) 2457. A.E. Ahmed and J. M. Labavitch, Plant Physiol. 65 (1980) 1009. J.A. De Vries, F. M. Rombouts, A. G. J. Voragen and W. Pilnik, Carbohydrate Polymers 2 (1982) 25. H.A. Schols, M. A. Posthumus and A. G. J. Voragen, Carbohydrate Research 206 (1990) 117. R.L. Whistler and M. L. Wolfrom (eds.), Methods in Carbohydrate Chemistry, Vol 1. Academic Press, New York, (1962) 477. N.G. Blumenkrantz and Asboe-Hansen, Anal. Biochem. 54 (1973) 484. G.A. Ruiter, H. A. Schols, A. G. J. Voragen and F. M. Rombouts, Anal. Biochem. 207 (1992) 176. K. C. Gross, HortScience 17 (1982)933. M. Knee, J. A. Sargent and D. J. Osborne, J. Exp. Bot. 28 (1977) 377. D. Grierson, CRC Crit. Rev. Plant Sci. 3 (1985) 113. G. E. Hobson, Biochem. J. 92 (1964) 324. E. A. Baldwin and R. Pressy, Proc. Fla. State Hort. Soc. 101 (1988) 215. G. T. Grant, E. R. Morris, D. A. Rees, P. J. C. Smith and D. Thom, FEBS Lett. 32 (1973) 195. E. R. Morris, D. A. Rees, D. Thom and E. J. Welsch, J. Supramolecular Struct. 6 (1977) 259.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
597
Autoclave extraction of sugar beet pulp yields gel-forming pectic hairy regions A. Oosterveld a, G. Beldman a, J.M. de Bruijn b and A.G.J. Voragen ~ aDepartment of Food Science, Wageningen Agricultural University, Bomenweg 2, 6703 HD Wageningen, The Netherlands bCentral Laboratory, CSM Suiker bv, Valveeken 6, 4815 HL Breda, The Netherlands
Abstract
Arabinose and ferulic acid rich pectic polysaccharides were extracted from sugar beet pulp using an autoclave extraction. Three populations of pectic polysaccharides could be distinguished: two high molecular weight populations consisting of rhamnogalacturonans, highly branched with arabinose side chains ('hairy' regions), and a third, low molecular weight population consisting mainly of homogalacturonans ('smooth' regions). The extract contained considerable amounts of ferulic acid, which was located in the 'hairy' regions. Oxidative cross-linking of the extracted polysaccharides with hydrogenperoxide and peroxidase resulted in the formation of a high molecular weight population, which contained the 'hairy' regions and consisted mainly of arabinose and galactose. Almost all ferulic acid could be found in this population. Next to this high molecular weight population the low molecular weight material, containing the 'smooth' regions remained unchanged. These results were confirmed by enzymic degradation. Oxidative cross-linking at polysaccharide concentrations in the region of 0.5-1.5 % resulted in an increased relative viscosity. Above a concentration of 1.5 % a gel was formed.
I. INTRODUCTION Sugar beet pulp is a potential source for the extraction of pectins. However, the poor gelling capacity and low viscosity of beet pectin, which is mainly caused by a low molecular weight and the presence of acetyl groups [1 ], prevents it's use as gelling or thickening agent. An important characteristic of sugar beet pectin is the presence of ferulic acid in the 'hairy regions', which makes it possible to cross-link this material [2], thus increasing the molecular weight. Tradionally, pectins are being extracted under acidic [3,4] or alkaline [3] conditions. This results in the extensive degradation of the arabinan side chains, containing most of the ferulic acid, or saponification of the feruloyl esters, respectively. Therefore an autoclave extraction method was used to extract ferulic acid and arabinose rich pectic polysaccharides [5]. Additionally, the present study deals with the oxidative cross-linking of the autoclave extracted material.
598 2. M E T H O D S
Wet beet pulp (campaign 1991) was obtained, after sugar extraction, from CSM Suiker bv (Breda, the Netherlands). After two consecutive H20 washings of the beet pulp (40~ 30 min) pectic polysaccharides were obtained by an autoclave treatment (two times, 121~ 40 min) [5]. The sugar composition of both fractions has been determined after dialysis and freeze drying [6]. The degrees of acetylation, methylation and feruloylation were determined as described before [5]. The molecular weight distribution was determined by high-performance size-exclusion chromatography (HPSEC) with refractive index and UV detection at 335 nm [5] or by SEC on Sephacryl S-500 [7] as described previously. Hydrogenperoxide and peroxidase were used for oxidative cross-linking [3]. Enzyme degradability of the cross-linked material was studied by incubation (24 h, 30~ with rhamnogalacturonase + rhamnogalacturonanacetylesterase (RG+AE), Endoarabinanase + arabinofuranosidase B (EA+AF) or polygalacturonase + methylesterase (PG+PE). Shifts in molecular weight were determined by HPSEC. Relative viscosities were measured in 0.1 M phosphate buffer pH 6.0 using an Ubbelohde viscometer.
3. RESULTS AND DISCUSSION The autoclave extraction of sugar beet pulp yielded two extracts of which the second is being discussed below. Table 1 shows the sugar composition of this extract, in which arabinose was the predominant sugar. Galacturonic acid was present in relatively low amounts (27.7 %). Methylation analysis (data not shown) showed that arabinose was mainly found as terminally, 1,3-1inked and 1,3,5-1inked residues. Rhamnose was 1,2-1inked and 1,2,4-1inked. Galactose was mainly present as terminally linked and 1,4-1inked residues. Galacturonic acid was found to be mainly 1,4-1inked. These results indicate that the extract consisted mainly of highly branched rhamnogalacturonans. Relatively high amounts of methyl- and acetyl esters were found in the extract. Also ferulic acid was present in relatively high amounts. It is known that ferulic acid is linked to the galactose and arabinose residues in the 'hairy' regions [8]. From this it can be calculated that in the extract 1.3 % of all arabinose and galactose residues contained a ferulic acid group. Fig. 1 shows the molecular weight distribution of the extract and the ferulic acid content as a function of the molecular weight. Three populations could be distinguished: two high molecular weight populations containing most of the ferulic acid and a low molecular weight population with a low ferulic acid content. Probably the high molecular weight populations consisted of 'hairy' regions, based on the presence of ferulic acid [9]. Anion exchange chromatography also showed the presence of three populations (data not shown): a neutral population consisting mainly of arabinose, a anionic population consisting mainly of galacturonic acid and another anionic population containing most of the neutral sugars and ferulic acid, which strongly bound to the anion exchange column.
599 Table 1 Composition of the second extract obtained by consecutive autoclave treatment of beet pulp Yield ~ Total sugar content 2 rhamnose 3 arabinose xylose mannose galactose glucose anhydrogalacturonic acid DA 4
5.8 88.3 3.6 60.8 0.0 0.4 6.6 0.9 27.7 52.
DM 4
60.
DF s
1.3
!. Expressed as percentage dry weight of beet pulp, 2: Expressed as dry weight percentage of the extract, 3: Expressed as molar percentage, 4: Moles of acetyl or methyl groups/100 moles galacturonic acid residues, 5: Moles of feruloyl groups/100 moles arabinose + galactose residues.
tO
'
20
I
25
I
I
,
30 Time (min)
I
35
,
40
Figure 1. HPSEC results of the second autoclave extract before and after cross-linking. In literature it has been described that it is possible to cross-link ferulic acid containing pectins using hydrogenperoxide and peroxidase [3]. Accordingly, the autoclave extract could also be cross-linked using these reagents. After cross-linking two populations appeared to be present as could be demonstrated with HPSEC analysis (see Fig.1.). Compared to the original extract, almost all ferulic acid containing material shifted to the
600 high molecular weight region in the chromatograrn. The remaining low molecular weight population contained a low amount of ferulic acid. Separation of the cross-linked material on Sephacryl S-500 is shown in Fig. 2. The sugar composition of the high molecular weight population (Table 2) showed that arabinose and galactose prevailed in this population. Furthermore it contained most of the ferulic acid. Galacturonic acid was only present in small amounts. In the low molecular weight population a relatively high amount of galacturonic acid was present, indicating the presence of homogalacturonans, although also considerable amounts of arabinose wore present. The low amount of galacturonic acid in population I is an indication of the absence of large 'smooth' regions in the cross-linked material. Fig. 1 indicates that the two high molecular weight populations present in the original extract participated in the oxidative coupling, resulting in cross-linked material with the characteristics of 'hairy' regions (pool I, Fig. 2). This implies that these two high molecular weight populations also consisted of 'hairy' regions.
~..,,"
E
~ I
=,2.
II
\
=.... 0 0 L m
'XX
o0
j/
0
\ lOO
200 300 Elution volume (ml)
!--Neutral sugars---AUA
400
500
-- Ferulic acid
I
Figure 2. Molecular weight distribution on sephacryl S-500 of the second autoclave extract after cross-linking. Tabel 2 Sugar composition (mol %) of the S-500 pools of the second autoclave extract. Pool I Rha Ara Xyl Man Gal Glc AUA
2.7 76.2 0.5 0.7 10.8 2.4 7.0
Pool II 1.0 26.9 0.4 1.9 3.7 1.7 64.5
601 To obtain further information about its structure, the cross-linked material was treated with several combinations of purified enzymes: RG+AE, PG+PE or EA+AF. Fig. 3 shows the molecular weight distribution before and after enzyme treatment. RG+AE were able to degrade the cross-linked material only partially. EA+AF degraded the cross-linked material extensively and resulted in two populations containing ferulic acid. PG+PE could only degrade the low molecular weight, non cross-linked material. These results indicate that the arabinan side chains were mainly responsible for cross-linking. No homogalacturonan, degradable by PG+PE, was present in the high molecular weight population. Based on experiments with RG+AE using the original extract as substrate, it was expected that the cross-linked material would also be degraded by RG+AE. However, the cross-linked material was only partially degradable with this combination of enzymes. This could have been caused by steric hindrance in the network, inhibiting the action of the enzyme, or by the limited importance of the rhamnogalacturonan backbone for the integrity of the network.
PG+PE
x (1,) "o r.B
EA+AF
>
=o
(u 4= 4) rv'
RG+AE
Blank
20
25
30 Time (rain)
35
40
Figure 3. HPSEC results of the cross-linked extract after treatment with RG+AE, EA+AF or PG+PE. Viscosity measurements of the extracted polysaccharides cross-linked at increasing concentrations showed a clear increase in relative viscosity, when the cross-linking reaction took place at concentrations higher than 0.5 % (Fig. 4). At a concentration higher than 1.5 % a gel was formed.
602
~9
6
.~
4
Gel: 1.5%
| n,'
,
0
,
I
I
,
I
1 2 3 Polysaccharide concentration (%)
I--x-Blank
4
4
-~- Cross-linked I
Figure 4. Relative viscosity at increasing polysaccharide concentration in the second autoclave extract before (blank) and after cross-linking.
4. CONCLUSIONS Autoclave extraction of sugar beet pulp yielded pectic polysaccharides, which were rich in 'hairy' regions and ferulic acid. The 'hairy' regions and 'smooth' regions were present in separate populations. Most of the ferulic acid was found in the 'hairy' regions. Oxidative cross-linking Of the autoclave extracted polysaccharides lead to an increase in the molecular weight of particularly those polysaccharides containing the arabinan side chains ('hairy' regions). After cross-linking at higher polysaccharide concentrations the relative viscosity increased and eventually a gel was formed. Enzyme studies confirmed that only 'hairy' regions were responsible for cross-linking. The cross-linked material could be degraded by treatment with endoarabinanase and arabinofuranosidase B, and could only be partially degraded using rhamnogalacturonase and rhamnogalacturonanacetylesterase.
5. R E F E R E N C E S
E.L. Pippen, R.M. MeCready, and H.S. Owens, J. Am. Chem. Soc., 72 (1950) 813. J.F. Thibault and F.M. Rombouts, Carbohydr. Res. 154 (1986) 205. F.M. Rombouts and J.F. Thibault, in M.L. Fishman and J.J. Jen (Eds.), Chemistry and Function of Pectins, ACS Symp. Ser. 310, American Chemical Society, Washington, DC, 1986, pp. 49-60. C.C.H. Wang and K.C. Chang, J. Food Sei., 59 (1994) 1153. A. Oosterveld, G. Beldman, H.A. Schols and A.G.J. Voragen, submitted for publication in Carbohydr. Res. (1995). H.N. Englyst and J.H. Cummings, Analyst, 109 (1984) 103. H.A. Sehols, M.A. Posthumus, and A.G.J. Voragen, Carbohydr. Res., 206 (1990) 117. M.-C. Ralet, J.-F. Thibault, C.B. Faulds, and G. Williamson, Carbohydr. Res., 263 (1994) 227. F. Guillon and J.-F. Thibault, Carbohydr. Res., 190 (1989) 85.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
603
Pectins in m i l d alkaline conditions: [3-elimination and kinetics of d e m e t h y l a t i o n C.M.G.C. Renard and J.-F. Thibault Laboratoire de Biochimie et Technologie des Glucides, INRA, rue de la G6raudi~re, B.P. 1627, 44316 Nantes cedex 03.
Abstract Pectins were incubated in buffered medium in mild alkaline conditions (pH 8.5 to 11.2) at room temperature, leading to both demethylation and [~-elimination. At higher pHs 13elimination had increased initial speed but soon plateaued. Demethylation was slower but proceeded until completion. It followed a (pseudo)-first order kinetics with respect to concentration of methylesterified carboxyl groups. A rate constant of 27.2 + 9.0 mol ~ 1 min ~ was calculated after correction for the pH variation during the course of the reaction.
1. I N T R O D U C T I O N Loss of functional properties of pectins at high pHs has been recognised for more than 50 years (Kertesz, 1951, and refs cited therein), though the mechanisms in play have been recognised more recently. In alkaline conditions, pectins are degraded by two competitive reactions: 13-elimination, which creates double bonds next to a methoxylated galacturonic moiety, and demethylation by saponification (Neukom & Deuel, 1958; Albersheim et al., 1960). This competition is modulated by pH and temperature conditions: any increase of temperature increases the rate of [~-elimination more than that of demethylation, while an increase of pH increases demethylation more than [~-elimination (Kravtchenko et al., 1992). For exemple, amidated pectins with high molecular weight can be produced at low temperatures (McCready et al., 1944; Joseph et al., 1949), while heating at neutral to slightly acidic pHs (Albersheim, 1959; Kravtchenko et al., 1992) leads to extensive depolymerisation of pectins in solution. At room temperature, in the pH range 9-11, both reactions occur. To better understand these degradations and their competition, we have quantified rate constants of demethylation. 2. MATERIALS AND METHODS
Commercial Rapid Set citrus pectin (864 mg galacturonic acid per g, degree of methylation 73) was from SBI (Beaupte, France). The kinetics of 13-elimination were followed by the increase of the absorbance at 235 nm (Albersheim et al., 1960). The pH of the pectin solution (1 ml, already in the spectrometer) was adjusted at t = 0 by adding 1 ml of 0.2 M sodium hydrogenocarbonate sodium carbonate buffer. This buffer did not have a prohibitively high absorbance at 235 nm. The kinetics of demethylation were followed in hermetically closed vials in a water bath at 25~ Ethanolamine/HC1 buffer, at a final molarity of 0.2 M, was used to vary the pH. Tubes containing 1 ml of pectin solution in distilled water were prepared and at t = 0, 1 ml of ethanolamine buffer was added. One tube in each series was used to read the initial pH of the
604 pectin/ethanolamine buffer mixture, and determine the amount of acetic acid necessary to bring the pH to 4-4.5. In the other tubes, the reaction was stopped after a given time by bringing adding that predetermined amount of acetic acid. Free methanol was measured by gas-liquid chromatography. A sample solution (50 lxl) containing free methanol is mixed with 50 ~tl of a butanol solution (100 lxl in 1 1 of distilled water; internal standard) prior to injection (1 ~tl) on a DBwax column (30 m x 0.32 mm) at 50~ with hydrogen as a carrier gas at 0.85 bar. The injector and the flame ionisation detector were at 150~ After each third sample, the column was cleansed by heating to 150~ Ethanolamine was one of the few buffer systems usable in this pH range that did not give interfering peaks during glc. 3. RESULTS 3.1. B-elimination During 13-elimination of pectic substances, a double bond is created between C-4 and C-5 of the new non-reducing end, leading to absorbance at 235 nm (by conjugation with the carboxyl groups). This spectral property of I]-eliminated pectins was used to follow their degradation (Fig. 1).
0.4 0.3~
O. 1
. ..:..,. O:. ~ ~ O-. - .'. - ~ 2 - O .............
m~
~-
0 0
20
40
60 Time (min)
80
100
120
Fig. 1: Time-course of the 13-elimination reaction of a 2.5 mg/ml pectin solution at 25~ in 0.2 M sodium hydrogenocarbonate carbonate buffer. o : p H 11;O: pH 10.5; I : pH 10;ra: pH 9.5; II : p H 9 . The two most striking features of these time-courses are that, though the initial speed of the reaction increases markedly with the pH, the final level does not, as the reaction stops much earlier. This was not due to a drop of pH below the values suitable for ~-elimination, as the pH change were slight (-0.2 at pH 11, no measurable change at pH 9). In order to check wether this plateau was due to disparition of suitable reaction sites, i.e. methoxylated galacturonic acid residues, we have investigated in detail the deesterification reaction. 3.2. Effect of pH on deesterification The deesterification reaction, or more precisely the demethylation reaction, was followed by measuring the amount of methanol liberated (Fig. 2).
605
0.8
1
A
I
0.6-
I
J
o
f
.,>
0.40.2--
Ratio of residual/initial ester (log scale)
Ratio of free methanol/initial ester
i ,/" ,o v..-=..-
o'"
9 ,,-..D.
-
o-u -- -
"
~-e ~ ~ " o. _- - - -
-~
- ---
-
d,
,,,~.~~--.m" ...................... 0 u u u u u ...... 0 20 40 60 80 100 120 Time (min)
.1
I
100
2d)0 3d0 Time (rain)
Fig. 2: Time-course of demethylation of a pectin solution (-5 mg/ml) at 25~ ethanolamine/HC1 buffer. 9 : pH 11.25; O: pH 10.55; 0: pH 10.05; El : pH 9.53; I1: pH 9.02; O : pH 8.25.
4(~0
in 0.2 M
As for 13-elimination, the higher the pH, the faster the reaction rate: at pH 11.25, half of the methylesters are liberated within one hour, whereas at 8.25, the lowest pH we investigated, less than 20% are liberated after 24 h. The demethylation shows two major differences with 13elimination: the variation of the initial speed of the reaction is less drastic, and the reaction does not plateau so soon, particularly for high initial pHs. 3.3.
Determination
of
the
rate
constants
Classically, saponification reactions can be written as (Connors, 1990): RCOOR' + OH --) RCOO + R'OH, and follow second order kinetics with - d[RCOOR']/dt = d[R'OH]/dt = k [RCOOR'] x [OH]
(1)
with [RCOOR']: concentration of ester (here of methoxylated galacturonate residues); [R'OH]: concentration of free alcohol, here methanol; [OH]: concentration of OH ions; t" time and k: reaction constant. It should therefore be possible, by keeping the concentration of O H ions constant throughout the reaction, e.g. by buffeting the medium, to transform the reaction into a pseudo-first order reaction with respect to the ester. One of the typical features of a (pseudo)-first order reaction is that a plot of the logarithm of the advancement of the reaction versus time (Fig. 2B) should give straight lines. However we observed deviation from linearity before the first half-life, in spite of the fact that another characteristic features of (pseudo)-first order reactions, namely that plots of the extent of reaction vers.us time were independant of the initial concentration (Fig. 3), was verified. We therefore investigated whether variation occured in the reaction conditions as a function of time. We found a slight decrease of pH during reaction (0.1-0.2 pH units in the buffer zone of ethanolamine), which however translated as a decrease of about 20% of the concentration of OH ions. Above pH 10.5, the loss in OH ions reached about 40% of the initial concentration. This variation could be predicted by taking into account the need for replacement of the buffer ions: at any time t ~ 0 eletroneutrality implies that for every carboxylate liberated (i.e. every methoxylated galacturonate saponified), one molecule of ethanolamine is converted from the base form (EtNH2) to the salt form (EtNH3+). The concentration of the base and salt forms at
606 any time t, calculated from the intial pH and the amount of liberated methanol, gave a good prediction of the measured pH variation (Fig. 4). The deviation from the (pseudo)-first order behavior was thus explained by the impossibility to keep the OH ions concentration constant using a buffer. Ratio of residual/initial ester
p H = 10.05 016
-8
8
p H = 9.53 0
0.4 0"20 I
0
I
500 1000 Time (min)
I
0
I
500 1000 Time (min)
1500
Fig. 3" Demethylation of pectin at three different concentrations: 9 95 mg/ml; o: 2.5 mg/ml; ~x 91.25 mg/ml.
o -0.1
o*=.4
[]
;>
r
[]
-0.2
-0.3 0
100
200 300 Time (min)
400
500
Fig. 4: Variation of pH during demethylation of pectin (--5 mg/ml, in 0.2 M ethanolaminePrlC1 buffer). measured pH with initial pH of 10.48 (o) and 9.72 (tJ); pH calculated from the amount of liberated methanol for initial pH of 10.52 (-*-) and 9.75 (--~-). The kinetics of liberation of methanol were therefore recalculated taking into account the pH variations: equation (1) could be rewritten replacing the concentration of OH- ions by its expression as a function of the buffer. At each point the concentration of OH was calculated from the initial pH and the amount of reacted ester:
607 [EtNH3+], = [EtNH3+]0+ [MeOH]~; [EtNH2], = [EtNH2]0- [MeOH]~ with" [EtNH2] + [EtNH3§ = 0.2 M and pH, = pK, + log,0([EtNH2]] [EtNH3*],) (pK~ of ethanolamine/HC1 buffer: 9.5). The new expression of equation (1) is: d[MeOH]
dt
= k' ([RCOOMe]0_ [MeOH]t)
([EtNH2] ~ _
[MeOH],)
(0.2- ([EtNH2]~_ [MeOH]))
with k' = k x 10(PKa -
(2)
14).
Equation 2 can be integrated, giving an expression linear with time. Straight lines were indeed obtained when using this equation to plot the results of demethylation (Fig. 5), confirming that deviation from linearity was due to loss of OH ions during the course of the reaction.
1
Calculated with pH correction
0.8-
II II
0.6II 0.4-
II
Q o
0 0 0
, 1O0
I
I
200 300 Time (min)
I
400
500
Fig. 5: Kinetics of demethylation of pectin expressed from intergration of equation (2). O: pH 10.55; t : pH 10.05; El: pH 9.53; I1: pH 9.02. Twenty-three kinetics have been carried out at 25~ for pH values from 8.25 to 11.25. The rate constant, calculated as the average of all the ks, was of 27.2 + 9.0 mop 1 min ~ . The pH correction according to equation (2) was not perfect, as there was a tendency to obtain higher k values at lower pH values. However, this was specially true for extreme values of our pH range, where the buffer capacity of ethanolamine was limited (higher pHs) or the reaction proceeded very slowly (low pHs), impairing the precision of the data. Another factor that might explain the dispersion of the data is lack of precision of pH measurement (no better than + 0.02 pH units).
608 4. DISCUSSION In mild alkaline conditions, highly methylated pectin was demethylated following a (pseudo)-first order kinetics with respect to the concentration of methoxylated galacturonate moieties. Investigation in this pH range, where the initial concentration of methylesters was higher than the initial concentration of OH ions, was complicated by the necessity to use a buffer. This led to deviations from the theoretical behavior as the concentration of OH ions still varied in proportions which could not be neglected in the equations of the kinetics. However these deviations could be accounted for be the pH variation, and the pH variation itself predicted from the amount of liberated methanol. The constant we found was similar to previously reported data (Scamparini & Bobbio, 1982). 13-elimination had higher initial reaction rates at higher pHs, and the variation in reaction rates appeared more marked than for demethylation (for which the variation was linear with the concentration of OH- ions). Quantification of the demethylation reaction allowed to see that 13elimination reached a plateau long before total demethylation, which could be linked to topological requirements for the 13-elimination, e.g. necessity of two neighbouring methoxylated galacturonate residues. 5. REFERENCES
Albersheim P. (1959) Instability of pectins in neutral solutions. Biochemical and Biophysical Research Communications 1, 253-256. Albersheim P., Neukom H. & Deuel H. (1960) Splitting of pectin chain molecules in neutral solutions. Archives of Biochemistry and Biophysics 90, 46-51. Connors K.A. (1990) Chemical kinetics: The study of reaction rates in solution. VCH Publishers, NY. Joseph G.H., Kieser A.H. & Bryant E.F. (1949) High-polymer ammonia demethylated pectinates and their gelation. Food Technology 3, 85-92 Kertesz Z.I. (1951) The pectic substances, Interscience Publishers NY (specially p. 121-122). Kravtchenko T.P., Arnould I., Voragen A.G.J. & Pilnik W. (1992) Improvement of the selective depolymerization of pectic substances by chemical 13-elimination in aqueous solution. Carbohydrate Polymers 19, 237-242. McCready R.M., Owens H.S. & Maclay W.D. (1944) Alkali-hydrolyzed pectins are potential industrial products, part I. Food Industries Oct. 1944, 69-71, 139-140. Neukom H. & Deuel H. (1958) Alkaline degradation of pectins. Chemistry and Industry June 1958, 683. Scamparini A.R.P. & Bobbio F.O. (1982) Deesterification of citrus pectin. Industri Alimendari Feb.1982, 110-112, 116
J. Visserand A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996Elsevier Science B.V.All fights reserved.
609
P o t e n t i o m e t r i c t i t r a t i o n of p o l y ( a - D ) g a l a c t u r o n i c acid D.Rudan-Tasi5 and C. Klofutar
Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-61000 Ljubljana, SLOVENIA
Abstract A commercially available sample of poly(a-D)galacturonic acid, i.e. pectic acid, was characterized according to the size and shape of its molecule through the volumetric and transport properties of its aqueous solutions. Thus, the average molecular weight of polygalacturonic acid and its average degree of polymerization were estimated on the basis of viscosity measurements. The length-to-diameter ratio, calculated by means of S i m h a ' s equation, strengthened the assumption that pectic acid is a fairly rigid, rod-like molecule. By potentiometric titration of aqueous solutions of poly(a-D)galacturonic acid with several alkaline and tetraalkylammonium hydroxides, the effects of the size and nature of the counterion on the degree and extent of dissociation of the polymeric acid were estimated. In evaluation of the potentiometric curves the treatment proposed by M a n d e l for weak polyacids not exhibiting a conformational transition during titration was used. In addition, the nonelectrostatic character of polyion-counterion interactions was confirmed by the application of the cell m o d e l to the polyelectrolytic solute investigated.
1. INTRODUCTION
In the course of studies on the physicochemical properties of natural polymers in aqueous solution, attention has been drawn to pectic acid, i.e. poly (a-D)galacturonic acid as a potential model of a rigid polysaccharide. Extensive data are given in the literature for the potentiometric titration of polymer acids which may be used to study the behaviour of polyelectrolyte systems under different conditions. For poly(a-D) galacturonic acid there are few data of this kind, especially in connection with the occurrence of a conformational transition induced by pH variations, or with the effect brought about by the addition or the exchange of counterions. Since for a polyacid not exhibiting a conformational transition in the course of titration, p K a ( K a denoting the apparent dissociation constant) increases monotonously with degree
610 of dissociation,a, it is possible to represent this functional dependance by a converging series expansion of pK a in a [1]. The purpose of this study is to consider in more detail the influence of the size and n a t u r e of the counterion on the degree and extent of dissociation of poly(a-D)galacturonic acid already discussed in a previous paper [2], particularly to check the effect of screening the charges on the polyacid in the presence of different counterions.
2. E X P E R I M E N T A L
Solution preparation Commercially available poly(cz-D)galacturonic acid (PGA) was purchased from F l u k a Chemie. To obtain an aqueous solution of the polyacid, insoluble PGA was converted to its soluble sodium salt and then percolated through a cationexchange resin in the H-form [3].
Density measurements Density m e a s u r e m e n t s were carried out using an A. Paar digital densimeter (model DMA 100) at a temperature of 298 K over the mass concentration range 0.75- 6.00kgm-3 -The densimeter was calibrated with water [4] and dry air [5].
Viscosity measurements The viscosities of aqueous solutions of PGA were determined with an Ubbelhode capillary viscometer at 298 K in the same concentration range as the density measurements. The temperature of the water bath was maintained to + 0.05K.
Degree of esterification The degree of esteritication of the methyl ester of PGA was determined acidimetrically after hydrolysing the ester with sodium hydroxide [6].
Potentiometric titration Potentiometric titrations of aqueous solutions of PGd with some alkali hydroxides (LiOH, NaOH, KOH)and tetra-n-alkylammonium hydroxides, e.g.
(CH3)4NOH and (C4H9)4NOH,
were performed in three parallel determinations
in a titration vessel at 298 K using a Radiometer pH meter (type pH M4d) and a combined glass electrode (type GK 2501 C) [2] . The pH meter was standardized with six s t a n d a r d buffers (pH range 3 -10). The end point in the potentiometric titrations was determined using Gran's procedure [7].
611 3. R E S U L T S A N D D I S C U S S I O N
3.1.Transport
properties
of poly(~-D)galacturonic
acid
The mole fraction of polygalacturonate d e t e r m i n e d on the basis of potentiometric h y d r o g e n ion titrations was found to be 0.85. The degree of esterification of the m e t h y l ester of PGA was d e t e r m i n e d to be 5.34 per cent. The density of the investigated solutions is given by d - do - ac~ + bc~
where a a n d fl are empirical constants characteristic of the solute a n d the t e m p e r a t u r e , d e t e r m i n e d by the m e t h o d of least squares on the basis of the d a t a in Table 1. Their values a m o u n t to a = 0.427 andfl = -4.490. The e x p e r i m e n t a l viscosity data were analysed according to the relation 2
3
17-- 17o -- a l c 2 h - a 2 c 2 d - a 3 c 2
where ~ is the absolute viscosity of the solution ( k g m -1 s'l), 1]0 is the absolute viscosity of the solvent a n d a l , a 2 a n d a 3 are empirical coefficients ; their calculated values a m o u n t t o a I - 9.588x10 -5 , a 2 - -3.649x10 -6 and, a 3 - 6.929x10 -7 . The intrinsic viscosity was calculated as
[17] - a___~,= O.10769m3kg -1 r/o The viscosity average molecular weight of PGA was d e t e r m i n e d using the M a r k - H o w n i k - S a k u r a d e equation with the necessary constants from ref. [8] --b
[1"/]- a M v -
4.368xl
0- 7 ~1
Mv
8737
a n d was found to be M v - 30115. Thus, the average degree of polymerization was calculated to be 146. The a p p a r e n t specific volume was d e t e r m i n e d from the density m e a s u r e m e n t s via the relation [9]
1E
qgv = ~o 1
c2
1
where do is the density of the solvent a n d c2 is the m a s s concentration of the
612 solute (gcm 3) and the value of ~Ov*-0.574cm3g-1 was calculated at very low reference concentration, c2 = 3.75x10-4 gcm -3 For comparison, the value of the partial specific volume at infinite dilution for D-galacturonic acid is @o _ 0.554cm 3g-1. The viscosity increment was determined as v - B / v ~ - 172.7 ( ~ 2.5 for spheres) where B is the viscosity coefficient characteristic of a given solue-solvent --o
pair, and amounts to (9.91 + 0.24)x10 -zm3kg -~ for PGA in aqueous solution, v2 is the partial specific volume of the macromolecular component equal to (p: at v a n i s h i n g c2 . The length-to-diameter for P G A was then estimated via S i m h a ' s relation for elongated ellipsoids [ 10] and its value amounts to (a / b) = 49.6.
Table 1 Densities and viscosities (experimental and calculated) of aqueous solutions of P G A at 298 K in the concentration range studied.
C2 / kgm -3
d / kgdm -3
17xl 03 / kgm-ls -1
~L~l~xl 03 / kgm -~s -~
0.75
0.997390
0.9581 __+ 0.0012
0.9581
1.50
0.997700
1.0266 __+ 0.0012
1.0269
3.00
0.998287
1.1657 __+0.0014
1.1646
3.84
0.998629
1.2420 __+0.0015
1.2434
4.80
0.999020
1.3409 __+0.0014
1.3402
6.00
0.999462
1.4835 __+0.0017
1.4836
3.2. P o t e n t i o m e t r i c titration of poly(a-D)galacturonic acid
The degree of dissociation, a, was calculated from the electroneutrality condition
a
= Cp
613 where [BOH 1 is the number of moles of base added per dm a of solution, [H+]and
[OH-] are the molarities of free hydrogen and hydroxyl ions and c; is the concentration of polyacid in monomol dm 3. The apparent dissociation constant of the polyacid, pK a , was calculated by the relation
pica
- pH
+
log (1- a) a
3.2.1. For the systems investigated, the increase of pKawith expressed by a second degree polynomial according to Mandel [1]
pKa = pK ~ + qbla + r where pK~ and r and r
a
could be
2
~ - l i m p K a ) ~ ~ is the intrinsic dissociation constant of the polyacid are the regression coefficients. As an example the dependence of
the apparent dissociation constant pK a for the system PGA +(Call 9)4 NOH at 298 K is given. For an interpretation of the physical meaning of the regression coefficients, the Marcus titration equation for polyelectrolytes was combined with the Poisson-Boltzmann equation for the electrostatic mean potential, determining all charge interactions in a dilute polyelectrolyte solution. In this way it was found thatr depends on the distribution of macromolecular groups in V8 (the total electrically neutral sub-volume assigned to each polymeric ion) for the uncharged polyelectrolyte, and on the mean conformation of the uncharged macromolecule. The coefficients r are mainly determined by the expansion of the dimensions of the polyion in the course of the titration. The data in Table 2 show that 9 the values of pK: decrease with increasing size of the alkali counterion, which may be explained by the formation of contact ion-pairs between counterions and charged carboxylate groups on the chain. The extent of ionpairing depends primarly on the ionic potential, ~o, defined as ( ~ "--
charge ionic radius(A)
9 the positive and almost equal values of coefficients C1 for the investigated systems, C1 =1-616-+0.099, are related to the mean distribution of macromolecular groups for the uncharged polyelectrolyte, i.e. PGA, which is the same irrespective of the nature and size of the counterion. 9 the coefficients r are negative and decrease with increasing size of the alkali counterions, while in the case of tetraalkylammonium ions the sign of the coefficient changes from-0.151 for (CH3)4NOH to + 0.211 for(C4Hg)4NOg.
614 Table 2 Coefficients a n d s t a n d a r d deviation,s, of the l e a s t - s q u a r e s s e c o n d - d e g r e e p o l y n o m i a l r e p r e s e n t i n g t h e t i t r a t i o n curve of PGA with different s t r o n g b a s e s at 298 K.
Base
pK ~
r
r
s
LiOH
3.419 _+ 0.019
1.537 + 0.080
- 0.431 + 0.071
0.019
NaOH
3.413_+ 0.024
1.471_+ 0.099
- 0.439 + 0.091
0.018
KOH
3.327 + 0.034
1.776_+ 0.140
- 0.665 + 0.128
0.024
(CH3) 4 NOH
3.477 _+0.018
1.502 _+0.073
- 0.151 _+0.066
0.013
(C4Hg)4NOH
3.341+0.019
1.792 + 0.086
+ 0.211_+ 0.084
0.017
I
!
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
5:000
4.500
4.000
3.500
f
I
0.200
0.400 0.600 OK)O Of,
Fig. 1. C h a n g e of t h e a p p a r e n t dissociation c o n s t a n t with a for t h e s y s t e m PGA + (C4H9)4 NOH at 298 K. The curve w a s c a l c u l a t e d from t h e model given by Mendel.
615 3.2.2. The a p p a r e n t dissociation constant p K a is strongly dependent on the electrical potential on the surface of the macroion, v(a)according to the well known equation [ 11] p K a - p K ~ + ApK
where A p K - O . 4 3 4 s v ( a ) / k T . Following the cell model for rod-like ionized polyelectrolyte molecules, a generalized form of the equation which gives ApK in a polyelectrolyte solution of any composition may be written [ 12]
EvJna2h)]
ApK - log (qgpamm+ m~)
+log
1)22J} 2A
where v is the n u m b e r of charges t h a t the polyion carries, h is the effective total length of each molecular cylinder, a is the radius of the rod-like polyion stretched along the axis of a cylindrical cell with radius R, ~op is the osmotic coefficient of the salt-free polyelectrolyte solution, m,, the concentration (monomol cm ~) , m8 the n u m b e r of moles of added salt per cm 3 and 2 is a dimensionless p a r a m e t e r proportional to the n u m b e r of charges per unit length of the macromolecule 2
;~ .
VoC . .
aoc'
2
(h - Zb)
.
DhkT
DjbkT
if every jth monomer carries an ionizable group ( v - a Z / j ) , Z is the degree of polymerization, b the length of the monomeric unit, e the ionic charge, D the dielectric constant, and k T the B o l t z m a n n term. p is an integration constant [ 13] dependent on a and R. Relation 1-fl 2
D
1 + p coth(fly) connects fl with a and y w h e r e yis the concentration parameter, defined as 7 - In --R _ _1In _ _ 1 0 0 0 a
2
rla2bNA
1 In c 2
where c is the monomolar concentration (monomol dm 3) and NA the A v o g a d r o number. For the dimensional p a r a m e t e r s of PGA the values a = 7A and b = 4. 35A" from ref. [14] were used. The relation between 7" and concentration was expressed analytically in the form - 10g c - 0.8687" - 0.394
616 For a univalent counterion the charge density parameter A was found to be ~ = 1 . 5 5 1 a where a value of j = 1.06 was employed because of the degree of esterification of the methyl ester o f P G A ( 5.34 per cent). A comparison of calculated and experimental values of ( p K a - p K ~ at different degrees of dissociation is shown in Fig. 2. For p K ~ the values from Table 2 were used.
2.000
I
I
I
I 0
1.500
cL 1.000
0.500
-
o~176176
0.200 0.400 0.600 OE)O 1.000 et
Fig.2. A p K
of the potentiometric titration of PGA ( 0.011 monomol dm -3) with
strong base as a function of a" ( * ) theoretically calculated curve on the basis of the cell m o d e l , ( + ) LiOH , ( ~ ) (CH 3)4 N O H , and ( O ) (C4H9)4N O H .
4. C O N C L U S I O N S
On the basis of the experimental results, the following conclusions can be made: 9 The length-to diameter ratio strengthens the assumption that pectic acid is a fairly rigid, rod-like molecule and comparable to the structure of cellulose 9 The partial specific volume of the monomeric unit in PGA was found to be approximately 3 per cent higher than the value of D-galacturonic acid 9 For the investigated system the values of p K ~ can be explained by the formation of contact ion pairs between an alkaline ion and the carboxylate group
617 9 The differences in pK ~ values in the case of tetraalkylammonium ions as counterions can be explained by the size of the (CH3)4N§ ion in comparison with the (C4H9)4N+ ion and delocalization of the positive charge on the (CH3)4 N§ ion, which is probably the deciding factor for its stronger interaction with the polyion 9 The values of the coefficients r and r are directly influenced by the size and nature of the counterion 9 The theoretical potential calculated on the basis of the cell model shows that PGA is not suitable for testing a purely electrostatic theory since in this case significant specific binding of counterions to the polyion was detected. For an ion like (C4H9)4N§ this is offset to some extent by the four longer alkyl groups that protect the positive charge of the rigid sphere [ 15].
5. R E F E R E N C E S
.
2. 3. 4. .
.
7. 8. .
10. 11.
12. 13. 14. 15.
M. Mandel, Eur. Polym. J., 6 (1970) 807. D. Rudan-TasiS, C. Klofutar, Thermoch. Acta 246 (1994) 11. R. Kohn, B. Larsen, Acta Chem. Scand., 26 (1972) 2455. O.Kratky, H.Leopold, H.Stabinger, Digital Densimeter of Liquids and Gases (A. Paar K. G., A-8054, Graz, Austria). R. C. Weast (Ed.), Handbook of Chemistry and Physics, 62 Edn., CRC Press, Cleveland, 1981-1982. A. Mizote, H. Odagiri, K. T6ei, K. Tanaka, Analyst, 100 (1975) 822. G. Gran, Analyst, 77 (1952) 661. G .Y.M~dy, B. Lakatos, J. J. Kever, N.V.D.Yakonova, Acta Aliment., 21 (1992) 337. H. Durschlag, Specific Volumes of Biological Macromolecules and Some other Molecules of Biological Interest, in: Thermodynamic data for Biochemistry and Biotechnology, HansJurgen Hinz, Ed. (Springer-Verlag, Berlin, 1986) p. 45. R. Simha, J. Phys. Chem., 44 (1940) 25. A. Katchalsky, Z. Alexandrowicz, O. Kedem, Polyelectrolyte Solution, in: Chemical Physics of Ionic Solutions, Ed. 03. E. Conway, R. G. Barradas, J. Wiley, New York, i966), p.295. Z. Alexandrowicz, A. Katchalsky, J. Polym. Sci. Part A,1 (1963) 3231. S. Lifson, A. Katchalsky, J. Polym. Sci. 13 (1954) 43. J. C. T. Kwak, G. Murphy, E. J. Spiro, Biophs. Chem., 7 (1978) 379. J. Nagano, H. Mizuno, M. Sakiyama, J. Phys. Chem., 95 (1991) 2536.
This Page Intentionally Left Blank
J. Visserand A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996Elsevier Science B.V.All fights reserved.
619
Structural studies of a pectic polysaccharide from
Plantago major L. A.B. Samuelsen, Ellen Hanne Cohen, Berit Smestad Paulsen and Jens Kristian Wold. Institute of Pharmacy, Department of Pharmacognosy, University of Oslo, P.O.Box 1068 Blindern, N-0316 Oslo, Norway
Abstract
The leaves of large plantain (Plantago major L.) are used for wound healing in the traditional medicine. The effect might be due to biologically active polysaccharides. A pectin, PMII with anti-complementary activity has been isolated from the leaves by water extraction and ion exchange chromatography
(i).
Oligosaccharides were isolated from PMII by weak acid hydrolysis and separation by SEC and HPAEC-PAD. The isolated oligosaccharides were desalted, reduced and methylated. GC-MS analysis of the partially methylated alditol acetates has been used to reveal the structure of the oligosaccharides. Oligosaccharides consisting of galacturonic acid and rhamnose with dp 3-5 and a series of 1,4 linked galacturonic acid oligosaccharides of dp 4-10 were isolated.
1. M E T H O D S
1.1 W e a k a c i d h y d r o l y s i s PMII was dissolved in 0.5M TFA and incubated at 100oc under N2 for 2.5h. 1.2 SEC
The partially hydrolysed material was fractionated by size exclusion chromatography on a Bio Gel P10 (Bio Rad) column (2,6 x 90 cm) and eluted with 50 mM ammonium hydrogen carbonate at 20 ml/h and fractions of 3.2 ml each were collected. Fractions 45-70 were pooled and subjected to HPAEC-PAD for further separation.
620 1.3 HPAEC-PAD Oligosaccharides were isolated preparatively by high-pH anion exchange chromatography carried out on a LC-system (Dionex Corporation, Sunnyvale CA) equipped with a CarboPac PA-1 column (9 x 250 mm), coupled to a Spectra System AS 3500 auto sampler. The detection was carried out using Pulsed Amperometric Detection (PAD-II). Eluents: Gradient: Flow rate:
100mM NaOH (El) and 100mM NaOH/1000 mM NaOAc (E2). 0-5min 20mM NaOAc, 5-60 min 20-600 mM NaOAc. 1 ml/min.
100mM NaOH was prepared from a 50% solution NaOH (Baker) to minimise the carbonate content in the final eluent. 1000 mM NaOAc was prepared using NaOAc from Riedel-deHaan, Germany and the distilled water used was filtered in a Waters Milli-Q System. E1 and E2 were degassed by flushing with helium and pressurised continuously with the eluent degas module from Dionex.
1.4 Desalting Sodium ions were removed from the oligosaccharide fractions by treatment with Dowex H +. Acetate was evaporated by washing with methanol.
1.5 Reduction Galacturonic acid was methylesterified by shaking with 0.08M HC1 in methanol for 24h. Then water was added, methanol was evaporated and the remaining solution was neutralised. NaBD4 was added and the mixture was left over night. Sodium ions were removed by treatment with Dowex H + and borate was evaporated by washing with acetic acid in methanol followed by addition of methanol several times.
1.6 Methylation Oligosaccharides were methylated using NaOH in DMSO for sugar alkoxide formation(2).
621
2. RESULTS AND DISCUSSION 2.1 Separation of oligosaccharides by HPAEC-PAD Separation of oligosaccharides by HPAEC is shown in Figure 1. 16 fractions were collected (upper chromatogram). None of the oligosaccharides are identical with any of the standards available (lower chromatogram). Standard GalAI-~4 GalA was eluted before the oligosaccharides of the sample. Standard GalAI-,4GalAI-~4 GalA was eluted between fractions 3 and 4 of the sample. Fractions 1-4 and 6-12 were desalted, reduced and methylated for identification.
i,~le:olqqooolo~ol i r a . ~ 1 ~ .,~to 1200
100%
1000
7
000 PAD
"1
I
I
1.--z ....
...............
illtlYI ~
...........
t2
2100
'... .....
. . . .
i
!
:
10
'
;"'
'
~'~
20
....
"1"'
30
.
.
.
.
40
''
"
~
(
60
~
' ' " i
80
~
' '
~--'
70
"
9 9 1
80
"~
"
'
i
0%
N
Mlnutr
1800
~-
lOO%
-
eo~
1600 1400 1200 1000
PAD
60%
800 000
-
4O%
-
30%
4OO 200 0 0
'
' '"
I r ", t0
,',
I " ~'~ ~"1' ' 2O :10
'
"'
!'" 4O
;"~'1 ~' 80
'
'-] ;' 6O
""
~ ' 70
'
~ '
~ '"' 8O
' e0
M i r a
Figure 1. Separation of oligosaccharides from PMII by HPAEC-PAD.
622 2 . 2 0 l i g o s a c c h a r i d e structures The monosaccharide residues identified in fractions 1-4 are:
1-1inked GalA, 1,4-1inked GalA 1,2- linked Rha.
The amount of each monomer found in the different oligosaccharides could not be determined because of the small quantities that were isolated. The dp of the oligosaccharides are about 3-5 with 2-4 galacturonic acid residues and one or more rhamnose residues of each. The methyl glycoside formed at position 1 by methylation of reducing sugars will be hydrolysed off and appear as a l-linkage. Rhamnose is at the reducing end of each oligosaccharide and is also present between two galacturonic acid residues. Only small amounts of this type of sequences are found in the molecule since only minor amounts of each oligosaccharide were isolated. The monosaccharide residues identified in fractions 6-12 are:
1-1inked GalA 1,4-1inked GalA
Fraction 6 has the same retention time as a previously run sample of tetragalacturonic acid (not shown). The oligosaccharides consist of unbranched 1.4 linked galacturonic acid of dp 4-10. GalA 1~4 GalAI(-~4 GalA 1)n~4 GalA n=l-7
3. A C K N O W L E D G E M E N T S This work has been supported by the Norwegian Research Council, project no 100594/410. The authors are indebted to Finn Tonnesen for recording the GC-MS data.
4. R E F E R E N C E S 1. 2.
A.B. Samuelsen, B.S. Paulsen, J.K. Wold, H. Otsuka, H. Yamada and T.Espevik Phytother. Res. 9 (1995) 211 M.J. McConville, S.W. Homans, J.E. Thomas-Oates, A. Dell and A. Bacic J. Biol. Chem. 265 (1990) 7385
J. Visserand A.G.J. Voragen(Editors),Pectinsand Pectinases 9 1996ElsevierScienceB.V.All rightsreserved.
623
Structural characterization of a novel rhamnogalacturonan II with macrophage Fc receptor expression enhancing activity from the leaves of Panax ginseng C.A. Meyer K.-S. Shin, H. Kiyohara, T. Matsumoto and H. Yamada Oriental Medicine Research Center, the Kitasato Institute, Shirokane 5-9-1, Minato-ku, Tokyol08, Japan Abstract A complex pectic polysaccharide (GL-4IIb2') with macrophage Fc receptor expression enhancing activity has been isolated from the leaves of Panax ginseng C.A. Meyer. GL-4IIb2' consisted of at least 15 different component sugars which included the rarely-observed sugars such as 2-Me-Fuc, 2-Me-Xyl, apiose (Api), 3-C-carboxy-5-deoxy-L-xylose (Aceric acid, AceA), 3-deoxy-D-manno-2-octurosonic acid (Kdo) and 3-deoxy-D-lyxo-2-heptulosaric acid (Dha). Sequential degradation experiment provided evidence that GL-4IIb2' comprised of a highly branched a-(1-~4)-linked galacturono-oligosaccharide backbone with side chains such as aRhap-(1-~5)-Kdo, Ar~-(1-~5)-Dha, AceA-containing oligosaccharide and uronic acid rich-oligosaccharide. These results suggest that GL-4IIb2' resembles rhamnogalacturonan II (RG-II). However, structure of GL-4IIb2' possessed several differences from that of RG-II: rich in a-Rhap-(1 ~5)-Kdo side chain, some additional glycosyl linkages, and a relatively higher molecular weight. It was also found that GL-4IIb2' was directly purified from hot water exracts of ginseng leaves without treatment of endo-a-( 1 -+4)-polygalacturonase.
1. I N T R O D U C T I O N
The roots of Panax ginseng C.A. Meyer are a well known Chinese component herb widely used clinically for the treatment of gastrointestinal disorders as well as an erythropoietic and a tonicl Several pharmacologically active saponins and polysaccharides have been found in the roots of P. ginseng as active ingredients. The roots of P. ginseng is valuable because it takes 4 - 6 years for growing from the seed whereas the leaves of P. ginseng can be harvested every year. Therefore, if the leaves have a similar activity as the roots, the leaves will be available as well as the roots. As the results of investigation for the clinical value of the leaves of P. ginseng, we have previously reported that the leaves polysaccharides showed more potent anti-complementary [1] and anti-ulcer activities [2]. Recently, We have found that the crude polysaccharide fraction (GL-2) from the leaves and the purified polysaccharide (GL-4IIb2') from GL-2, showed potent
624 immune complex clearance enhancing activity of macrophages, and this activity was due to increment of de nove synthesis of Fc receptor (FcR) [3]. In the prsent sudy, GL-4IIb2' was found to be a complex pectic polysaccharide consisting of 2-Me-Fuc, 2-Me-Xyl, Api, AceA, Dha and Kdo which are characteristic in RG-II [4] of plant cell wall polysaccharides, therefore we describe structural characterization of a macrophage FcR expression enhancing polysaccharide (GL4IIb2') from the leaves of Panax ginseng C.A. Meyer.
2. Isolation of GL-4IIb2' Crude polysaccharide fraction (GL-2) was prepared from the leaves of P. ginseng by hot water extraction, ethanol precipitation and dialyw and GL-2 was fractionated by Cetavlon precipitation and weakly acidic polysaccharide fraction (GL-4) was obtained[3]. GL-4IIb2 was purified from GL-4 by DEAESepharose CL-6B as described previousely [3]. In order to remove the colormaterials, GL-4IIb2 was further purified by Q-Sepharose (CI form), and the major fraction, eluted with 0.3M NaC1, was repurified by gel filtration on Bio-gel P-30 column to obtain purified active polysaccharide, GL-4IIb2'.
3. Property of GL-4IIb2' GL-4IIb2' was eluted from Bio-Gel P-10 as a single peak and also gave a single peak in HPLC on Asahi-pak GS-510 + GS-320 and GS-320 + GS-220. It contained hexose (64.1%), uronic acid (33.7%) and acetyl (0.82%) and had a molecular weight of 8,000 - 10,000. Component sugar analysis indicated that GL-4IIb2' consisted of at least 15 kinds of sugars such as 2-Me-Fuc, 2-Me-Xyl, Api, AceA, Kdo and Dha which observed as rare sugars in RG-II [4] in addition to Fuc, Ara, Xyl, Rha, Man, Gal, Glc, GlcA and GalA. GL-4IIb2' did not react with ~-glucosylYariv antigen on single radial gel diffusion. Digestion with endo-a-(1-->4)polygalacturonase (endo-PGase) gave no oligogalacturonides from GL-4IIb2' and its molecular weight was not changed. Methylation analysis indicated that GL4IIb2' consisted of at least 32 kinds of sugar residues observed in RG-II. The results suggested that GL-4IIb2' has a similar structural feature as RG-II [4], but the proportion of Kdo and branching frequency were higher than those of RG-II.
4. Structural. analysis of GL-4IIb2' by sequential degradation 4.1. Characterization of fragments derived by procedure 1 In order to elucidate the differences between structures of GL-4IIb2' and RG-II, sequential degradation including partial acid hydrolysis was used for generation of characteristic oligosaccharides from GL-4IIb2' as shown in Scheme 1. After GL-4IIb2' was partially hydrolyzed with 0.1M TFA (60~ 30min), the products
625 GL-4IIb2' Procedure
1
I I
0.1 M TFA, 60~
30 rain
Bio-gel P-10 I
I
PA-1
PA-2 ]
0.1 M TFA, 40~
Procedure 2
PA-2'
24 hr
a-Rha-(l~5)-Kdo
Bio-gel P-6
I
I
PA-I-I
PA-I-III
PA-I-II AceA-containin~g oligosaccharide (Figure 2.)
Procedure 3 --
0.1 M TFA, 40~
--
Bio-gel P-6
PA-I-Ia
84 hr
i
I
PA-I-Ib
PA-I-Ic Rha-(~5)-Kdo
Endo-polygalacturonase
Procedure 4
Rha-(l~5)-Kdo Ara-(1-~5)-Dha
I
PA-I-Id Monosacc h andes
Bio-gel P-6
I PG-2
PG-1
Procedure 5
0.1 M TFA, 50~
I PG-3
48 hr
Bio-gel P-6
PG-1A
I
I
PG-1B •
PG-1C
Uronic acid r~ch oligosaccharide Scheme 1. Sequential degradation of GL-4IIb2'. were fractionated on Bio-gel P-10, and about 35% of TBA-positive materials (PA-2) was eluted in the small oligosaccharide fraction. PA-2 was further purified on QAE-Sephadex by using linear gradient of HCOONH 4, and it gave a single peak (PA-2') containing TBA-positive material. PA-2' consisted mainly of Rha (43.2%) and Kdo (40.8%). Methylation analysis indicated that PA-2' was composed mainly of terminal Rhap and 5-substituted Kdo (ratio 1:1). The methylated oligosaccharide-alditol derived from PA-2' gave only a single peak in the region of disaccharide-alditol in GC-MS. EI-MS of PA-2' contained prominant fragment
626 ions at m/z 189 (Rha) and m/z 308 (Kdo). The presence of a fragment ion at m/z 162 and the absence of m/z 177 showed that Kdo was substituted at C5 but not at C4. PMR of PA-2' exhibited a signal due to an anormeric proton (at 5.09 ppm), indicating that Rha is to be a-configuration. Therefore, it was indicated that PA-2' mainly contained a-Rha-( 1-~ 5)-Kdo.
4.2. Structural characterization of oligosaccharides obtained from PA-1 by procedure 2 PA-1 was partially hydrolyzed with 0.1M TFA (40~ 24 h) and the three fractions (PA-I-I, PA-I-II and PA-I-III) were obtained by Bio-gel P-6. Especially, about 50% of TBA-positive material (PA-1-III) was eluted in the fraction of small oligosaccharide. Permethylated oligosaccharide-alditols from PA-I-III were analyzed by GC-EIMS, and three disaccharide-alditols (1P, 2P and 3P) were detected. EI-MS and component sugar analysis suggested that the major peak, 1P was Rha-(l~5)-Kdo-ol and the minor peaks, 2P and 3P were two epimers of Araf-(1-~5)-Dha. The intermediate size fraction, PA-I-II consisted mainly of 2-Me-Fuc, Rha, Ara, Api, AceA and Gal in molar ratios of 19.7 935.1 913.6 97.6 94.1 914.7 and the glycosyl composition was similar to that of the AceA-containing oligosaccharide isolated from RG-II [5]. The glycosyl linkage composition of neutral glycosyl residues in PA-I-II was similar as that in AceA-containing oligosaccharide derived from RG-II, but PA-I-II contained the relatively large amounts of terminal Araf and 2-1inked Rha. It was also found that PA-I-II also consisted of 2,3-branched Arap which was not concluded the presence in AceA-containing oligosaccharide of RG-II. The presence of AceA-containing oligosaccharide in PA-I-II was confirmed by FAB-MS. Negative FAB-MS of PA-I-II gave pseudo-molecular ions [ (M-H) ] at m/z 1055, 1187 and 1201 which were suggested to be due to non-acetylated hepta- and octasaccharides, respectively. PA-I-II also gave (M-H) of monoacetylated heptasaccharide (m/z 1097) and octasaccharides (m/z 1229 and m/z 1243), and
1097
-132 205
-"
-(162,160) 337
~"
-160
'
... 659
/'
-146 " '' ; 8 1,9 :
....
965
,,,., ""]
.....
.J ~
I
rn/z
Figure 1. Negative FAB/CAD spectrum of the AceA containing oligosaccharide
627 diacetylated heptasaccharide (m/z 1137)and octasaccharides (m/z 1271 and 1285). CAD spectrum using B/E-linked scan of monoacetylated heptasaccharide (m/z 1097, as the highest ion peak) gave fragment ions at m/z 965, 819, 659, 337 and 205 due to fragments derived from the heptasaccharide by successive elimination of Api, Rha, AceA, (Gal + 2-Me-Fuc) and Ara, respectively (Figure 1). Recently, Whitcombe et al. have reported the presence of AceA-containing oligosaccharide having an octasaccharide unit in which non-reducing Araf was attached to position 2 of Rha of heptasaccharide unit [6]. Methylation analysis indicated that terminal Ara and 2-1inked Rha were present in PA-I-II, and it was assumed that the Ara was attached to 2-1inked Rha. These proposed that GL-4IIb2' comprised the same side chain of AceA-containing octasaccharide unit as RG-II. However it was also shown that GL-4IIb2' consisted of 2,3-branched Ara. Negative FAB-MS gave other ions at m/z 1333, 1375 and 1417. By comparison with (M-H) of hepta- and octasaccharides, it was suggested that (M-H) of hepta- and octasaccharides were produced by elimination of pentosyl or deoxyhexosyl units from the ions at m/z 1333, 1375 and 1417. From the results of methylation and FAB-MS analysis, it was proposed that GL-4IIb2' comprised AceA-containing oligosaccharide chains possessing nonasaccharide unit in which terminal Rha was attached to position 3 of 2-1inked Arap of octasaccharide chain (Figure 2). There was also a possibility that this structural unit is probably not unique for GL-4IIb2' because 2,3-branched Arap has been detected in RG-IIs from sycamore, and red wine in relatively large proportions [6,7]. From above results, it is inferred that the AceA containing oligosaccharide from GL-4-IIb2' has the following structure (see Figure 2.). Rha 1
$ 3 Araf-(l~2)-Rha-(l~2)-Arap-(1 ~4)-Galp-(1-~2)-AceA-(1-~3)-Rha-(l~3')-Api-(1-~ 2 1 2-O-Me-Fuc Figure 2. The possible structure of the AceA cotaining oligosaccharide obtained after procedure 2.
4.3. Characterization of fractions obtained by procedure 3 PA-I-I was further treated with 0.1 M TFA at 40~ for 84 h, and gave four subfractions (PA-I-Ia, PA-I-Ib, PA-I-Ic and PA-I-Id) by gel filtration on Bio-gel P-6. About 50% of remaining TBA-positive material in PA-I-I was eluted in the small oligosaccharide fractions (PA-I-Ic and PA-I-Id). Glycosyl sugar composition
628 analysis of PA-I-Ic revealed Rha and Kdo as the major constituents. GC-MS analysis of methylated oligosaccharide-alditols indicated that PA-I-Ic was consisted mainly of Rha-(l~5)-Kdo as same as PA-2'. The high-molecular-weight and major fraction, PA-I-Ia consisted mainly of Rha, Fuc, 2-Me-Xyl, Ara, Api, Gal, Dha, GalA and GlcA in molar ratio of 12.1 : 7.8 : 6.4 : 10.1 : 3.2 : 6.7 : 1.2 : 31.2 : 17.6. Methylation analysis showed that PA-I-Ia mainly contained terminal Ara, terminal 2-Me-Xyl, 3,4-branched Fuc, 2,3,4,-fully branched Rha, terminal Gal, 3'-linked Api, 2-1inked GlcA and terminal-, 4-liked, 2,4-branched and 3,4branched GalA. From these results, it was assumed that PA-I-Ia might be comprised uronic acid rich octasaccharide and Araf-(l~5)-Dha, which have been found in RG-II [4, 8], and branched galacturono-oligosaccharide.
4.4. Characterization of fractions obtained by procedures 4 and 5 After PA-1-Ia was digested with endo-PGase, and the product was fractionated by Bio-gel P-6. A large proportion (PG-1) of the products was eluted in the void volume and the product also gave an intermediate fraction (PG-2) and a considerable proportion of the lowest-molecular-weight fraction (PG-3). PG-2 contained a large proportion of GalA in addition to a small proportion of Rha, Fuc, 2-Me-Xyl, Ara, Gal, GalA and GlcA. Whereas PG-3 contained GalA only. These results suggested that the several side chains such as AceA-containing oligosaccharide, Araf-(1-~5)-Dha and Rha-(1-~5)-Kdo which were attached to the a - ( l ~ 4 ) galacturonan core in GL-4-IIb2'. In order to release uronic acid-rich oligosaccharide, PG-1 was further treated with 0.1 M TFA (in according to the procedure of Thomas et al. [9], Scheme 1, Procedure 5). The products gave PG-1A ~ PG-1C and PG-1A was shown to comprise similar component sugars as PG-1. Among the fractions, PG-1B was shown to contain Api, Rha, Fuc, 2-Me-Xyl, Gal, GalA and GlcA (5.4 : 15.1 : 12.0 : 10.0 : 9.3 : 26.4 : 14.7 in molar ratios), and the composition was almost consistent with ~he uronic acid-rich octasaccharide in RG-II [8]. Therefore it was assumed t h a t GL-4-IIb2' also comprised similar structural unit of uronic acid-rich octasaccharide side chains in RG-II. 5. CONCLUSION The primary structure of a complex pectic polysaccharide (GL-4IIb2') with macrophage Fc receptor expression enhancing activity, isolated from the leaves of P. ginseng C.A. Meyer, was elucidated (Figure 3.). The present results suggested that GL-4IIb2' resembles, in many respects, with a typical RG-II. However, RG-II from ginseng leaves presented discrete differences from previously reported RG-II [4] as shown in Table 1. Especially, GL-4-IIb2' consisted of 1) AceA containing oligosaccharide side chains possessing higher branched nonasaccharide unit than RG-II, 2) higher molecular weight than RG-II, and 3) greater richness of aRhap-(l~5)-Kdo side chains than RG-II. Because the pharmacological funtion of RG-II has not ever been reported up to now, present finding may be the first case for a pharmacological activity of RG-II. These postulated the possibility that the
629 aRhap-(1--S)-KDO-(2--2 or3) aRhap-(1--S)-KDO-(2--2 or3) aRhap-(1--S)-KDO-(2--2 or3) Uronlc acid-rich octasacchadde
I--
Uronlc acid-rich octasacchadde Ara-(1--S)-DHA-(2--2
or 3
Uronlc acid-rich octasacchadde Rha 1 i 3 Ara-(1---2)-Rha-(1---2)-Ara-(1-.4)-GaI-(I -,.2)-AceA-(1--3)-Rha-(1-.3')-Apl-(1--2 or 3)t 1 2-O-Methyl-Fire
aRhap-(1-*5)-KDO-(2---2 or3) aRhap-(1--5)-KDO-(2--2 or3).
Figure 3. The proposed partial structure of GL'4-IIb2' from the leaves of P.
ginseng C.A. Meyer.
Table 1. The decrete differences between GL-4-IIb2' and RG-II
GL-411b2 ~
RG-II
Preparation
Obtained w i t h o u t t r e a t m e n t of endo-PGase
dp
60
Pharmacological activity
Structural feature
Released f r o m p r i m a r y cell walls u p o n d i g e s t i o n with endo-PGase 60 - 30
Relatively s t r o n g m a c r o p h a g e FcR e x p r e s s i o n e n h a n c i n g activity [ Rha-(1-~5)-KDO
Not reported
Ara-(1-~5)-DHA 9 ]
5-1
1 -1
[ AceA containing oligosaccharide ]
:
R
i
,
3
,
Ara-(1 -, 2)-Rh a-(1 -:-)2)-Ara-(1 ~4)-Gal-(1 -)2)-AceA-(1 -~3)- Rha-(1 -)3')-A pl ,.. . . . . . . . . _, 1 2-O-MethyI-Fuc R = Rha
R = OH
630 phamacological activity may be related to some structural differences of GL-4IIb2' from RG-II. On the other hands, GL-4IIb2' was directly isolated from the hot water extracts of the leaves of P. ginseng without treatment of endo-PGase although RG-IIs have been purified from plant cell walls by endo-PGase digestion [10] or from crude preparation of the enzyme [11]. These facts suggest the possibility that GL-4IIb2' exists as a free form of RG-II. 6. R E F E R E N C E S
1. Q.-P. Gao, H. Kiyohara, J.-C. Cyong and H. Yamada, Plant Med., 57 (1991) 132. 2. X.-B. Sun, T. Matsumoto and H. Yamada, Plant Med., 58 (1992) 445. 3. X.-B. Sun, T. Matsumoto and H. Yamada, Phytomedicine, 1 (1995) 225. 4. V. Puvanesarayah, A.G. Darvill and P. Albersheim, Carbohydr. Res., 218 (1991) 211. 5. M.W. Spellman, M. McNeil, A.G. Darvill and P. Albersheim, Carbohydr. Res., 122 (1983) 131. 6. A.J. Whitcombe, M.A. O'Neill, W. Steffan, P. Albersheim and A.G. Darvill, Carbohydr. Res., 271 (1995) 15. 7. T. Doco and J.M. Brillouet, Carbohydr. Res., 243 (1993) 333. 8. L.D. Melton, M. McNeil, A.G. Darvill P. Albersheim and A. Dell, Carbohydr. Res., 146 (1986) 179. 9. J.R. Thomas, A.G. Darvill and P. Albersheim, Carbohydr. Res., 185 (1989) 261. 10. A.G. Darvill, M. McNeil and P. Albersheim, Plant Physiol., 62 (1978) 418. 11. Y.S. York, A.G. Darvill, M. McNeil and P. Albersheim, Carbohydr. Res., 138 (1985) 109.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V.All fights reserved.
631
Structural features of pectic polysaccharides of red beet (Beta
vulgaris conditiva) Georg R. Strasser, Daniel E. Wechsler, Renato Amadb Swiss Federal Institute of Technology, Institute of Food Science, ETH-Zentrum, CH-8092 Zurich, Switzerland
Abstract Cell-wall material from ripe red beet was extracted as alcohol-insoluble residue (AIR). The CDTA-soluble extract from AIR was fractionated by anion-exchange chromatography. Four fractions were isolated by a step-wise increase in the ionic strength of the elution buffer. The main fraction was further fractionated by gel filtration chromatography. This chromatogram showed one regular broad peak, which was divided into three parts and pooled. All fractions isolated from both chromatographic systems were freeze-dried and their neutral sugar compositions as well as uronic acid contents were determined. Furthermore methylation analysis of these fractions were performed prior and after reduction of the pectic polysacchaddes with NaBD4.
1. INTRODUCTION Pectins are a group of polysaccharides from the primary cell wall and the intercellular regions of higher plants [1]. They have been investigated for their structural features and their functions within the plant cell wall for many years, because changes in the texture of fruits and vegetables and in the properties of their products are related to changes in the pectic components [2]. From literature it is known, that the pectic backbone consists of ~t-(1---~)linked D-galacturonic acid units, interrupted by the insertion of o~-(1---)2)-linked L-rhamnosyl residues in adjacent or alternate positions [3]. Side chains consisting essentially of arabinans, galactans, arabinogalactans and single xylose residues are attached to this backbone. Other sugars have been found less frequently. In addition some non-sugar substituents, mainly methanol, acetic acid and phenolic acids are known to be present in pectins. Although the main structural elements of pectins are known, the complexity of these polymers has prevented a complete understanding of their fine structure so far. The aim of this project is to get additional information about the fine structure of pectic polysaccharides. Therefore pectins from red beet were isolated and fractionated by chromatographic methods. Some results obtained by methylation analysis of these pectin-rich fractions are presented.
632 2. MATERIALS AND METHODS
Extraction and fractionation of pectins (Figure 1): Red beets of the variety Red Ace F~ were purchased from a local store. Preparation of the AIR was done according to Selvendran and O'Neil [4]. Extraction with trans-l,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) was performed according to Selvendran et al. [5]. The CDTA-soluble extract was dialysed first against running tap water and subsequently against distilled water. Then it was fractionated by anion-exchange chromatography on DEAE Sepharose CL-6B using 0.04M phosphate buffer (pH 6.5) as eluent with a step-wise increase in the ionic strength (0, 0.1, 0.2, 1.0M NaC1). Related fractions were pooled. The main fraction (IE 0.2M) was further fractionated by gel filtration chromatography on two coupled columns, one packed with Sephacryl S-500 and the other one with Sephacryl S-200 using 0.04M phosphate buffer (pH 6.0) as eluent. The peak on the chromatogram was divided into three parts, which were separately pooled. All samples were dialysed and freeze-dried before further analysis. Analytical techniques: Fractions from anion-exchange chromatography were assayed for uronic acid and neutral sugar contents using an automated segmented flow analyser [6]. Neutral sugars (NS) of all samples were analysed by GC as alditol-acetates [7]. Uronic acids (UA) were determined by the m-hydroxy-diphenyl method [8]. Methylation analysis [9,10] was performed with polysaccharides prior and after carbodiimide-activated reduction with NaBD4. Peaks were identified by GC-MS, whereas the quantification was done by GC (FID) using calculated relative response factors on an effective carbon response (e.c.r.) basis [ 11]. 3. RESULTS AND DISCUSSION The results are summarised in Table 1. As expected, for the IE samples higher amounts of uronic acid are found with increasing ionic strength of the elution buffer. For the GF samples larger molecules (GF1) contain half as much NS compared to smaller molecules (GF3). The sum of differently linked NS residues determined by methylation analysis is calculated to 100%. 1,4-Galp is difficult to determine because of the high amount of 1,4GalAp present in most samples. Therefore 1,4-Galp was determined by methylation analysis without prior reduction with NaBD4. Whereas the percentage of some NS residues remain nearly unchanged in the different samples (e.g. T-Araf, 1,5-Araf and 1,6-Galp), others differ considerably. The relative amounts of 1,3-Araf, 1,3,5-Araf, 1,2,3,5-Araf, 1,4-Galp, 1,2-Rhap and 1,2,4-Rhap increase at higher ionic strength elution on anion exchange chromatography and decrease for smaller molecules on gel filtration chromatography. 1,3-Galp and 1,3,6-Galp show the opposite behaviour. This indicates that pectins eluted at high ionic strength and larger pectic molecules of the IE 0.2M fraction contain on average smaller side chains because of the relatively high amount of 1,2,4-Rhap compared to the total amount of NS residues (1,2,4-Rhap is the main branching residue in the pectic backbone). Furthermore it can be assumed that the side chains of these pectins mainly consist of structures similar to arabinans and arabinogalactans type I, because of the differently linked arabinose and galactose residues present. Pectins eluted at low ionic strength and smaller pectin molecules of the IE 0.2M fraction contain more galactose, which is mainly 1,3- and 1,3,6-1inked. These residues are believed to be part of structures, which are known to be present in arabinogalactans type II.
633 Red Beet Alcohol soluble extract
Extraction with ethanol ,,~ Alcohol insoluble residue (AIR)
CDTA insoluble residue
Extraction with CDTA =~ CDTA soluble material (CDTAS)
Anion exchange chromatography on DEAE-Sepharose CL-6B 1200 T --o- Uronic acids ~ --.- Neutral sugars ~ ~~ 800 + .... 0.04M phosphate buffer ~o~.= containing NaC1 a~ ~=
1 I/ .ll
400
............
q- 1.0 ] ] "=~=~ 0.5
,
0 i ,
I
20 ~ IEOM
i I '
I
40 i 60 I 1 ' 1 IE0.1M IE0.2M
i
=o
80 (I, IE1.0M
Fraction number
Gel filtration chromatography on Sephacryl S-500 and on Sephacryl S-200
~
Mw
(Pullulan)
6
]
i
L60
80
rlE 0.2M GF1 [IE 0.2M GF2] IE 0.2M GF3"[ Figure 1. Extraction and fractionation of pectins from ripe red beets
Fraction number
634
Table 1 Neutral sugar, uronic acid and methylation analysis (molS) of fractionated pectins from ripe red beets Chromatographic fractions
NS to UA ratio
NS residues
CDTAS
OM
0.1M
IE 0.2M
E 0.2M 1.OM
GFl
GF2
GF3
UA
25 75
99 1
92 8
17 83
18 82
10 90
16 84
20 80
Arabinose
T-Araf T-h a p 1,2-Araf 1,3-&af 1,5-Araf 1,3,5-Araf 1,2,3,5-Araf
22.9 0.9 1.2 2.1 17.4 12.0 2.0
26.2 1.1 1.3 3.3 22.4 1.2 0.5
22.6 0.4 0.8 0.9 19.0 0.6 0.0
23.4 1.0 1.6 2.1 17.4 14.1 2.2
21.8 0.8 1.3 2.9 17.3 14.9 3.1
22.1 1.3 0.8 2.6 16.9 17.9 2.9
23.7 0.8 1.6 1.9 16.3 13.2 2.7
22.3 1.2 0.9 1.6 16.0 10.1 1.8
Fucose
T-FUCP 1,3,4-Fucp
0.8 0.2
0.0 0.0
0.1
0.0
1.1 0.2
0.8 0.1
1.5 0.1
1.1 0.2
1.1 0.2
Galactose
T-G@ 1,3-G@ 1,4-Galp 1,6-Galp 1,2,4-Galp 1,3,4-Galp 1,3,6-Galp
2.8 6.8 n.d. 2.4 0.9 0.8 13.0
1.1 11.6 0.6 2.5 0.1 0.1 23.6
1.7 14.6 n.d. 2.3 0.0 0.2 33.8
2.4 5.1 n.d. 2.2 1.2 0.9 10.7
3.6 6.0 n.d. 3.0 0.9 0.7 5.6
3.7 3.7 n.d. 1.7 1.3 0.9 1.8
2.8 6.1 n.d. 2.1 0.9 0.7 11.7
2.0 7.8 n.d. 2.3 1.5 1.0 17.2
Glucose
T-GlCp 1,4-Glcp 1,6-Manp
0.9 3.3 0.1
0.2 3.1 0.1
0.0 1.5 0.1
0.5 2.8 0.0
0.0 4.4 0.0
2.8 5.2 0.3
0.0 3.7 0.4
0.0 2.2 0.4
NS
after reduction with
NaBD4
Mannose
Rhamnose
Xylose
T-Rhap 1,2-Rhap 1,3-Rhap 1,2,3-Rhap 1,2,4-Rh~
1.4 1.7 0.9 0.2 2.8
0.3 0.1 0.2 0.0 0.0
0.7 0.1 0.1 0.0 0.3
1.5 2.0 1.3 0.3 2.6
1.4 2.5 0.9 0.3 5.1
1.5 3.1 1.2 0.4 3.2
0.9 0.3 2.5
1.7 1.3 1.4 0.2 1.9
T-Xylp 1,CXylp
0.8 1.7
0.3 0.0
0.2 0.1
1.2 2.2
0.9 1.5
1.6 1.4
1.2 1.8
1.2 2.7
100
100
100
100
100
100
100
100
3.5
0.3
n.d.
3.6
6.6
4.7
4.2
2.2
2.5 0.3
100 0.0
49.4 8.6
2.1 0.0
2.8 0.0
0.7 0.0
2.7 0.3
2.8 0.4
0.9 93.3 0.6 1.4 1.0
0.0 0.0 0.0 0.0 0.0
0.0 41.7 0.0 0.3 0.0
1.0 93.9 0.7 1.2 1.2
0.6 92.8 0.6 2.0 1.2
0.4 95.9 0.6 1.1 1.3
0.8 93.2 0.7 1.2 1.1
1.2 92.7 0.7 1.4 0.8
100
100
100
100
100
100
100
100
Total NS
NS residues
Galactose without prior reduction
1,4-Galp
UA residues
Glucuronic acid T-GlcAp after reduction with 1,CGlcAp NaBD4 Galacturonic acid T-GalAp 1,4-GalAp 1,2,CGalAp 1,3,4-GalAp 1,4,6-GalAp
Total UA
1.9 1.7
Ratio branchinglterminal
after reduction with NaBD4 without prior reduction
1.1 1.5
0.9 0.7
1.2 ad.
1.1 1.1
1.1 1.1
1.1 1.1
1.1 1.1
1.1 1.1
Ratio Iinearhranchinp:
after reduction with NaBD4 without prior reduction
5.4 1.o
1.7 2.0
1.3
6.6 1.1
6.0 1.2
9.6 1.2
5.5
5.5
n.d.
1.2
1.0
n.d. not determined 635
636 The ratios between linear and branched residues are presented as well. All values obtained without prior reduction (NS residues only), are in the range between one and two. From this it can be assumed, that side chains contain only slightly more linear than branched residues. After prior reduction (NS and UA residues) much more linear than branched residues are found. This surplus of linear residues originates from the 1,4-1inked galacturonic acid backbone, which is also known as smooth region. The results presented allow the following assumptions to be made: CDTA-soluble material eluted at low ionic strength (IE 0M) contains hardly any uronic acids and consists mainly of neutral sugar residues, which indicate the presence of arabinans and arabinogalactans type II. Pectins eluted at high ionic strength (IE 1.0M) contain large amounts of uronic acids. The side chains consist mainly of arabinans, galactans and mixtures of them. High molecular weight pectins eluted at intermediate ionic strength (IE 0.2M GF1) contain large smooth regions of 1,4-1inked galacturonic acid and relatively small side chains. These side chains consist of sugar residues, which probably belong to arabinans, arabinogalactans type I and mixtures of them. Low molecular weight pectins of the same ionic strength fraction (IE 0.2M GF3) contain larger side chains, consisting of sugar residues, which indicate the presence of arabinogalactan type II similar structures. Additionally other sugar residues (e.g. T-Galp, T-Xylp, T-GlcAp) and non-sugar residues (e.g. methanol and acetic acid; results are not shown) are attached to these pectic polysaccharides, but further investigations are needed to clarify the fine structure in detail.
4. REFERENCES
8
9 10 11
Voragen, A.G.J., Pilnik, W., Thibault, J-F., Axelos, M.A.V., Renard, C.M.G.C. (1995). Food polysaccharides and their applications (Stephen A.M., ed.), Marcel Dekker, Inc., 287-339. Carpita, N.C., Gibeaut D.M. (1993). Plant J. 3, 1-30. McNeil, M., Darvill, A.G., Fry, S.C., Albersheim, P. (1984) Ann. Rev. Biochem. 53, 625-663. Selvendran, R.R., O'Neil, M.A. (1987). Meth. Biochem. Anal. 32, 25-153. Selvendran, R.R., Stevens, B.J.H., O'Neil M.A. (1985). Biochemistry of plant cell walls (Brett, C.T., Hillman, J.R., eds.) Cambridge University Press, Cambridge, 39-75. Thibault, J.F. (1979). Lebensnt-Wiss. u. Technol. 12, 247-251. Blakeney, A.B., Harris, P.J., Henry, R.J., Stone, B.A. (1983). Carbohydr. Res. 113, 291-299. Blumenkrantz, N., Asboe-Hansen, G. (1973). Anal Biochent 54, 484-489. Kvemheim, A.L. (1987). Acta Chent Scand. Ser. B41, 150-152. Harris, P.J., Henry, R.J., Blakeney, A.B., Stone, B.A. (1984). Carbohydr. Res. 127, 5973. Sweet, D.P., Shapiro, R.H., Albersheim, P. (1975). Carbohydr. Res. 40, 217-225.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V.All rights reserved.
637
Isolation and Physicochemical Characterisation of Xylose-rich Pectic Polysaccharide from Wheat Straw
Runcang Sun*, a, J. Mark Lawthera and W. B. Banksb aThe BioComposites Centre, bSchool of Agricultural & Forest Sciences University of Wales Bangor, Gwynedd LL57 2UW, United Kingdom
ABSTRACT Xylose-rich pectic polysaccharide (XRPP) was extracted from defatted and protein-free wheat straw with a solution adjusted to pH 1.6 using hydrochloric acid (HC1). The yield of XRPP obtained was 1.1% of the dried wheat straw. The isolated XRPP contained 44.8% galacturonic acid released by pectolyase treatment and 32.1% released by 2 N trifluoroacetic acid hydrolysis. The XRPP also contained 17.1% neutral sugars released by pectinase reaction and 28.4% released by the acid hydrolysis. A comparison of the FT-IR spectroscopic data of citrus pectin and XRPP showed that the extracted XRPP belongs to pectic substances and differs from hemicellulose with an intensive absorption band at 1740 cm -1.
Key words: xylose-rich pectic polysaccharide, pectin, wheat straw, extraction, sugars, lignin, FT-IR, phenolic acids and aldehydes.
INTRODUCTION There is growing interest in the use of cereal straws such as wheat straw for animal feed after increasing its digestibility by various methods, or as a raw material for paper and board production. This is particularly important in areas with limited forest resources (1). For all these purposes a good physicochemical characterisation of cereal straw is necessary. To date, the structural features of pectic polysaccharides and plant cell walls have been studied extensiv.ely using chemical analysis and enzymatic degradation. In addition, research on isolation and physicochemical characterisation of pectin from citrus peels, apple peels, sunflower head residues and sugar beet pulp has been reported (2). However, the pectic polysaccharides extracted from wheat straw have only previously been reported by Przeszlakowska (3). The author extracted 0.44% pectic substances from * Author to whom correspondence should be addressed.
638 wheat stem. In addition, an outline study of wheat straw pectin has been described by Harbers and co-workers (4) using scanning electron microscopy. The study indicated that wheat straw cell walls possess relatively small amounts of pectin. The objectives of the present study, which is part of an ongoing research program on the multiuse approach to cereal straw fractionation using thermomechanical pulping, were to isolate the pectic polysaccharides from wheat straw and to study the physicochemical properties of the polymers.
M A T E R I A L S AND M E T H O D S Materials Wheat straw was obtained from Silsoe Research Institute (Silsoe, Bedfordshire, UK), and was dried in a cabinet oven with air circulation at 60~ for 16 h. The dried wheat straw was then ground using a Christie Laboratory mill to pass a 60-mesh size screen and stored at 5~ until use. All chemicals were of analytical or regent grade. All experiments were performed in duplicate and yield is given on a dry wheat straw weight basis.
Isolation and analysis of wheat straw XRPP Xylose-rich pectic polysaccharide was extracted from defatted and protein-free cell wall preparation (5) using HC1 solution (pH 1.6) at 85~ for 4 h. The extract was adjusted to pH 5.0 with ammonia, concentrated on a rotary evaporator under reduced pressure at 40~ and precipitated with 5 volumes of 96% ethanol. After washing twice with 80% ethanol and drying in an air circulated oven at 40~ for 2 h, the pellet was redissolved with distilled water and then precipitated with 4 vols 96% ethanol. Before the pellet was gently ground, the precipitated pellet was washed twice with 70% ethanol and dried at 40 o in an air circulated oven for 16 h. The resultant white powder was labelled "xylose-rich pectic polysaccharide" and stored in a refrigerator. For measurement of the neutral sugars and galacturonic acid in extracted XRPP, both acid hydrolysis and pectinase digestion methods were used. During acid hydrolysis, XRPP (60 mg) was hydrolysed using 15 mL of 2 N trifluoroacetic acid (120~ for 2 h) in sealed pressure tubes. For pectinase digestion, 60 mg XRPP was dissolved in 15 mL of KH2PO4-NaOH buffer, pH 5.6 and 10 mg pectolyase (p-3026, 3.4 units/mg solid, Sigma) was then added. The mixture was incubated for 7 h at 35~ After filtration, the filtrate was evaporated to dryness at 40~ under reduced pressure. The released sugars arabinose, xylose, mannose, galactose and glucose were determined by gas chromatography after conversion to trimethylsilyl ether derivatives (5). myo-Inositol was used as an internal standard. The amounts of rhamnose released by acid hydrolysis and pectinase reaction were determined by the quantitative colorimetric procedure of Gibbons (6). The galacturonic acid released in both cases was assayed colorimetrically as anhydrogalacturonic acid using 3-phenylphenol colour reagent, according to the procedure
639 outlined by Blumenkrantz and Asboe-Hanson (7). Methyl ester content was determined using the method described by Wood and Siddiqui (8) whilst acetic acid was determined using the transesterification method described by Browing (9). The weight-average molecular weights of the XRPP were determined using gel permeation chromatography
(5). Viscosity was measured using a Brookfield Synchro-Lectric Viscometer (Model LV). A citrus pectin was used as a reference. XRPP samples (2%, w/v) were prepared in 0.1 M sodium phosphate buffer, pH 7.0, allowed to hydrate at 4~ for 16 h (10, 11). Viscosity was then estimated (cps) at 25~ Optical rotation was determined on a polarimeter (Perkin Elmer, type 108) according to the methods described by Phatak et al. (10) and McCready et al. (12). XRPP samples (1.0%, w/v) were prepared in double distilled water, and solutions were centrifuged before measurement. A citrus pectin was again used as a reference. The gelling properties of XRPP samples were tested according to the procedures of Phatak et al. (10), and Chang and Miyamoto (13). XRPP samples were prepared in distilled water at a concentration of 1.0% (w/v). IR spectra were obtained on an FTIR spectrophotometer (Mattson Cygnus 100), using KBr discs containing 1% finely ground samples. For the method of determination of lignin remaining attached to/associated with XRPP fraction see our previous report (5).
R E S U L T S AND D I S C U S S I O N
Composition of X R P P Extraction of wheat straw at 85~ for 4 h yielded an XRPP value of 1.1% for the given regime. The anhydrogalacturonic acid released by pectinase reaction and acid hydrolysis of XRPP were found to be 44.8% and 32.1%, respectively. This result indicated that pectolyase p-3026 treatment XRPP under the conditions chosen is more effective for release of galacturonic acid than 2 N trifluoroacetic acid hydrolysis (120 ~ for 2 h) which only released 71.7% of total galacturonic acid . Further hydrolysis with 2 N trifluoroacetic acid at 120~ or increases in trifluoroacetic acid concentration/hydrolysis temperature are necessary for release all of the galacturonic acid present in extracted XRPP. The methoxy content was low, 5.8%, indicating that wheat straw XRPP is a lowmethoxy XRPP. The data also shows that extracted XRPP possesses acetyl groups in its structure. The acetyl content of XRPP was 6.0%. Partial acid hydrolysis of the acetyl groupsrestored the gelation power of the pectin (14). The HC1 extracted XRPP contained a low amount of ash, 6.9%. This accords the study of Phatak and co-workers (10) on sugar-beet pulp pectin. Summarised in Table 1 is the neutral sugar compositions and anhydrogalacturonic acid content of XRPP released by pectinase reaction and acid hydrolysis, respectively. In both cases, XRPP were found to be rich in xylose and galactose content, but low in mannose content. The total neutral sugar content in XRPP released by acid hydrolysis was 28.4%,
640 which dropped to 17.1% for pectinase treatment. In contrast to the greater release of rhamnose during pectinase treatment, values obtained for the other sugars, arabinose, xylose, mannose, galactose and glucose, were higher in XRPP released by the acid hydrolysis. The greater release of rhamnose and galacturonic acid during pectinase treatment suggests that rhamnose coexists with galacturonic acid in the main chain of XRPP, and arabinose galactose and xylose are found in the side chains. Aspinall et al. (15), in discussions about pectin in soybean cotyledons, suggest the possibility that most of the xylose residues occur as xylosyl short side chains branched on the rhamnogalacturonan backbones. .Hence in wheat straw XRPP it is possible that a proportion of the xylose is present as an integral component of the acidic pectic fraction. In addition, as XRPP was readily degraded by pectinase, the material must contain homogalacturonan regions of the molecular chains. It is therefore concluded that XRPP belongs to a group of pectic substances. Although XRPP is a minor constituent of the polysaccharides in wheat straw, it probably has a distinct functional role in the cell walls.
Table 1. The composition of neutral sugars and content of anhydrogalacturonic acid (%) in XRPP extracted with pH 1.6 HC1 solution at 85~ for 4 h (1 g wheat straw/100 mL extractant) from defatted, protein-free wheat straw.
Sugars/anhydrogalacturonic acid
A(%)
B(%)
Rhamnose Arabinose Xylose Mannose Galactose Glucose Anhydrogalacturonic acid
1.2 0.9 8.2 0.1 5.8 0.9 44.8
1.1 3.5 14.0 0.2 6.8 2.8 32.1
Total
61.9
60.8
Areleased by pectinase reaction, Breleased by acid hydrolysis.
Matsuura and Hatanaka (16) observed that xylose-rich acidic polysaccharide having high mannose content was present in appreciable amounts in Japanese radish. This contained large amounts of neutral sugars, the galacturonic acid contents being only 11-25%. Xylose, arabinose and galactose were found to be the major constituents with xylose comprising more than 50% of the sugars in each sample, with the exception of one isolated from the leaves. Ray and co-workers (17) have also isolated a type of xylose-rich acidic polysaccharide, extracted with an aqueous 10% trichloroacetic acid, from the seeds of Acacia auriculaeformis. The composition of monosaccharides in this material were arabinose 13.5, xylose 18.0, galactose 23.0, glucose 10.5, and glucuronic acid 35.0%, respectively. Also, since pectin represents the material found in the primary cell wall of
641 plants, it is probable that the qualitative nature as well as quantity of various pectic polysaccharides found in pectin may vary with the degree of maturity/differentiation of the plant source (18).
Physicochemical characterisation of XRPP Due to degradation during acid (HC1, pH 1.6) extraction, the extract possessed a relatively low weight-average molecular weight: 8000. The viscosity (2%, w/v) was determined at 3.10 cps and was much lower than that normally observed for citrus pectin (93.50 cps). The pH, molecular size, degree of methylation, and temperature significantly affect the viscosity of wheat straw pectin. However, this low viscosity property of wheat straw pectin, which is similar to sugar beet pulp pectin, indicates a high potential for application in low-caloric, high fibre beverages (10). The optical rotation of XRPP (1.0%, w/v) was +60 ~ which was also low compared to that exhibited by citrus pectin (+ 162~ Because of the presence of acetyl groups, low viscosity and low molecular weight in the extracted wheat straw XRPP, no gel formation was observed at 1% levels of addition to water. Citrus pectin at 1.0% formed a firm gel. The FTIR spectra of citrus pectin and wheat straw XRPP (Figure 1) appeared to be similar. Both of the spectra have absorptions at 1740, 1608, 1430, 1360, 1244, 1080, 1060, 1035, 890 and 524 cm -1. The pectic substances belong to a class of carboxypolysaccharides which differ from neutral polysaccharides, with an intense band in the region 1740 cm -1 (for salts 1608 cm -1) related to vibrations of the carboxyl group (19). FTIR spectra of extracted wheat straw hemicellulose and cellulose do not exhibit this band (spectra not shown). From this point, the extracted wheat straw XRPP is also assigned to pectic substances. The intensity ratio of the bands uas(CO0-) at 1608 cm -1 and p(C:O) ester at 1740 cm -1 corresponds to fully deesterified pectin and Me pectate (20). Due to much stronger absorptions at 1608 cm -1 than that at 1740 cm -1 in wheat straw XRPP, it is also clear that wheat straw XRPP is a low-methoxy XRPP, which is in accordance with the results obtained by colorimetry. However, on closer examination of the spectrum of the citrus pectin, it can be seen that there is a specific feature in the 9501140 cm -1 region, where a group of six bands is observed at 950, 1008, 1035, 1060, 1080 and 1140 cm -1, whereas that of XRPP in this region has two weak absorption at 1008 and 1140 cm -1 and two very strong absorptions around 1060 and 1035 cm -1. This can be ascribed to the neutral polysaccharides present in the extracted wheat straw XRPP. The very weak absorption at 1510 cm -1 in extracted wheat straw XRPP is due to aromatic skeleton vibrations in wheat straw lignin. These data indicated that the extracted XRPP fraction contained small amounts of neutral polysaccharides and residual lignin. The total phenolic content in XRPP was 1.10%. The major components were found to be p-hydroxybenzoic acid (0.44%), vanillin (0.19%), syringic acid (0.13%), and syringaldehyde (0.13 %). The contents of p-hydroybenzaldehyde, vanillic acid and ferulic acid were 0.032, 0.015 and 0.020%, respectively. Gallic acid, protocatechuic acid and cinnamic acid were detected in trace amounts.
642
2.6 _
I
2.8 I
3.0 I
3.5 !
4.0 !
Microns 4.5 5.0 I I
6.0 I
7.0 I
8
!
tO I
t5 !
20 I
1 e,,
._o m
E c
IU
I-
000
''''
I ' ' ' ' 1 ' ' ' ' 1 ' ' ' ' 1 ' ' ' ' 1 , , , , I , , , , i
3500
3000
2500
2000
Wavenumber
1500
t000
500
(cm -1 )
Figure 1. FT-IR spectra of citrus pectin (a) and wheat straw xylose-rich pectic polysaccharide (b).
SUMMARY Our results have indicated that wheat straw XRPP contains 44.8% galacturonic acid and 28.4% neutral sugars and is particularly rich in xylose. The XRPP also contains 5.8% methoxyl ester content and 6.0% acetyl ester groups. The viscosity of XRPP was very low. The isolated wheat straw XRPP did not form gels under the experimental conditions. The XRPP extracted under acidic conditions, such as pH 1.6 HC1 solution, gave low molecular weight. In this work, wheat straw XRPP can be assigned to a group of polysaccharides termed as xylose-rich pectic polysaccharide. The fractional and structural characterisation of wheat straw XRPP is currently the subject of detailed further study in our laboratory.
643 ACKNOWLEDGEMENTS We acknowledge the financial support for the research from LINK Collaborative Programme in Crops for Industrial Use and Dr. James Bolton, Director of The BioComposites Centre. This study was supported by IlK. Ministry of Agriculture, Fisheries and Food for the LINK Collaborative Programme(Multi-use Approach to Cereal Straw Fractionation Using Thermomechanical Pulping) in Crops for Industrial Use under Agreement CSA 2054.
REFERENCES
1 0 . Theander and P. Aman, Swedish J. Agric. Res., 8 (1978) 189. 2 A. Miyamoto and K. C. Chang, J. Food Sci., 57 (1992) 1439. 3 M. Przeszlakowska, Acta Agrobot, 26 (1973) 115. 4 L. H. Harbers, G. L. Kreitner, G. V. Davis, M. A. Rasmussen and L. R. Corah, J. Ani. Sci., 54 (1982) 1309. 5 J. M. Lawther, R.-C. Sun and W. B. Banks, J. Agric. Food Chem., 43 (1995) 667. 6 M. N. Gibbons, Analyst, 80 (1955) 268. 7 N. Blumenkrantz and G. Asboe-Hanson, Anal. Biochem., 54 (1973) 484. 8 D. J. Wood and I. R. Siddiqui, Analytical Biochemistry, 39 (1971) 418. 9 B. L. Browing, (ed.) Methods of Wood Chemistry, New York, 1967. 10 L. Phatak, K. C. Chang and G. Brown, J. Food Sci., 53 (1988) 830. 11 S. A. Andon, Food Technol., 41 (1987) 74. 12 R. M. McCready, A. D. Shepherd, H. A. Swenson, R. F. Erlandsen and W. D. Maclay, Analytical Chemistry, 23 (1951)975. 13 K. C. Chang and A. Miyamoto, J. Food Sci., 57 (1992)1435. 14 E. L. Pippen, R. M. McCready and H. S. Owens, J. Am. Chem. Soc., 72 (1950) 813. 15 G. O. AspinaU, I. W. Cottrell, S. V. Egan, I. M. Morrison and J. N. C. Whyte, J. Chem. Soc., C (1967) 107. 16 Y. Matsuura and C. Hatanaka, Agric. Biol. Chem., 52 (1988) 2583. 17 B. Ray, P. K. Ghosal, S. Thakur and S. G. Maiumdar, Carbohydrate Research, 185 (1989) 105. 18 M. M. Baig, C. W. Burgin and J. J. Cerda, J. Agric. Food Chem., 30 (1982) 768. 19 M. P. Filippov, Food Hydrocolloids, 6 (1992) 115. 20 M. P. Filippov, G. A. Shkolenko and R. Kohn, Chem. Zvesti., 32 (1978) 218.
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J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
645
Chemical synthesis of oligosaccharides related to arabinogalactan-proteins (AGPs) J.F. Valdor and W.Mackie Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
Abstract
Synthetic routes to two terminal arabinose-containing oligosaccharide fragment.s related to arabinogalactan (AGP) polysaccharides have been devised using standard protecting group and coupling strategies. These new compounds represent possible carbohydrate epitopes of arabinogalactan polysaccharides that may be involved in developmental processes of plant cell walls. They may also be related to some of the observed pharmacological activities of AGPs.
INTRODUCTION Arabinogalactan-proteins (AGPs) are an important class of glycoproteins widely distributed in plant tissues and exudates ~. Their precise biological functions in plants remain unknown but, using a panel of monoclonal antibodies specific for carbohydrate structures, it has been demonstrated that the presence of certain AGP epitopes are closely related to cell development in plant morphogenetic processes 2'3. These monoclonal antibodies have been shown to cross-react with the type of AGP typified by Lolium multiforum (ryegrass) arabinogalactan-protein (Fig.l). This polysaccharide is characteristic of type II arabinogalactans and has a linear 13-(1-3) galactan backbonecarrying short 13-(1-6) galactan side branches substituted with arabinofuranose units4. More recently, oligosaccharide fragments from structurally similar arabinogalactans isolated from the Chinese herbs, Angelica acutiloba and Bupleurum falcatum have been shown to have various potential pharmacological activities5.
-~3.~.o.Galp.( 1-~3)-~-D.Galp-(1-~3)-~-o-Galp-( 1-~3)-~-o.Galp-(1-~3)-~-D-Galp-(1-~3)-I~-o-Galp-(1-~ 6 6 6 t t t 1 1 1 ~-t.-~a f-(1-~3)-o-Galp ~-t.-~a f-(1-~3)-o-Galp ~-L-Ara f-(1-~3)-9-Galp 6 6 6 t t t 1 1 1 ~-L-Araf-(l-~3)-o-Galp ~-L-PSaf-(1-~3)-o-Galp ~-t.-Ara f-(1-~3)-D-Galp 6 6 6 t t t 1 1 1 ~-L-~a f ~-I.-Araf ~-L-~V'af
Fig.l: Structure of ryegrass (Lolium multoflorum) endospermarabino-galactan-proteirf
646 At present there is little information concerning the detailed composition and number of sugar units that constitute the carbohydrate epitopes in the antibody interactions and in the pharmacological activities and very few well-defined arabinose-containing oligosaccharides are available for biological studies. Accordingly, to provide model examples of these putative bioactive oligosaccharides, we have undertaken the synthesis of some AGP fragments of welldefined composition. Initially these will be utilised to provide structural parameters that may be relevant to biological activity and will also be used in immunochemical studies as potential hapten inhibitors of anti-AGP monoclonal antibodies.
SYNTHETIC STRATEGY Possible oligosaccharide fragments considered to be potential bioactive epitopes or hapten inhibitors are trisacchafide A and tetrasaccharide B (Fig.2). To provide these target oligosaccharides, synthetic schemes using a stepwise approach have been devised. Routes to the oligosaccharides A and B and intermediate disaccharides are based upon standard procedures involving 1,2-transglycosidic linkage formation which requires the use of a donor glycosyl halide with participating-group assistance in the 2-position, a suitably protected glycosyl acceptor and the presence of silver salts as promoter. The (1-3) linked disaccharides 8 and 9 were prepared with high stereoselectivity and good yield from a single glycosyl acceptor, methyl-2,4,6-tri-O-benzyl-~-D-galactopyranoside 1 the synthesis of which involved selective 3-crotylation of methyl-13-D-galactopyranoside via an alkyl-stannylation reaction6. The optimum conditions to yield the desired products involved the coupling of I with either 2,3,5-tri-O-benzoyl-ct-L-arabinofuranosyl bromide 2 or 2,3,4,6-tetraO-acetyl-t~-D-galactopyranosyl bromide 3 in dichloromethane with silver triflate as promoter 7. As in Kochetkov's polycondensation which included glycosylation of trityl ethers with cyanoethylidene derivatives of sugars s, the preparation of the (1-6) linked disaccharide 10 required a triphenylmethylated acceptor, methyl-2,3,4-tri-O-acetyl-6-O-trityl-f3-Dgalactopyranoside 4. The glycosyl halide 3 was used without recourse to the usual corresponding orthoester and the coupling reaction was carried out rapidly under reflux in dichloromethane in the presence of a co-activator, silver cyanide, to trap the tritylium ion liberated during the reaction9. To form the internal galactosyl residues in the synthesis of the target oligosaccharides A and B, the selective and readily removable bromoacetyl group 1~was employed according to a previous study on the stereospecifity of the coupling reaction of galactosyl halides beating different substituents 1~. In the trisaccharide synthesis, this was introduced in the 3-position of the protected glycosyl donor 6, made available by pmethoxybenzylation of the dibutylstannylene complex of methyl-13-D-galactopyranoside, followed by selective oxidative removal with 2,3-dichloro-5,6-dicyano-benzoquinone (DDQ) in neutral conditions 12 after blocking the remaining hydroxyl groups. In the tetrasaccharide synthesis, the 6-position of the glycosyl donor 7 was bromoacetylated from the methyl-2,3,4tri-O-benzoyl-13-D-galactopyranoside 5 obtained after a temporary protection of the primary hydroxyl group as a trityl ether and followed by a perbenzoylation step and a selective detritylation. In both cases, bromoacetyl derivatives were converted to their corresponding txglycosyl chlorides, which were easier to purify than their bromo analogues, by using 1,1dichloromethylmethylether (DCMME) in the presence of zinc chloride as catalyst ~3. In this
647
-
Fig.2 (a): Synthetic routes to carbohydrate fragments from AGPs
\
mo
648
L..G OQ
Fig. 2 (b): Synthetic routes to carbohydrate fragments from AGPs
649 way, the monosaccharide glycosyl donors 6 and 7 provided the advantage of a substituent at 0-2 capable of neighbouring-group participation with the anomeric center and a protecting group at 0-3 or 0-6 that could be selectively removed. The coupling reactions were performed under base-deficient conditions 14 (taking care to minimize acid-catalyzed migration of acyl groups in the nucleophilic acceptor) in the presence of silver triflate and sym-collidine. Although good stereoselectivities were achieved yielding mainly the desired 1,2-trans products, there is scope for improvement and optimization of the coupling yields. It is possible that the reduced yields were due to competing formation of orthoesters as co-products in the formation of the required 1,2-trans compounds. Removal of the bromoacetyl group TM was achieved in the presence of other ester groups by treatment with thiourea in dichloromethane/methanol to afford a new glycosyl acceptor having a free hydroxyl group for further glycosylation. The above strategy has been applied successfully to obtain trisaccharide A of which the 13C NMR spectrum was fully consistent with the expected structure. Similar approaches leading to tetrasaccharide B are currently in progress. Although none of the synthetic oligosaccharides described above have yet shown any biological activity 15, it is intended in future work to use these oligosaccharides for the formation of synthetic antigens and the development of new monoclonal antibodies as molecular probes of plant cell wall development. In future syntheses of oligosaccharides, it is planned to utilise the application of triphenylmethylated glycosyl acceptors as used suceessfuUy in the synthesis of the (1-6) linked galactobioses. This should allow coupling and detritylation to proceed in one step avoiding the need for hydrolytic removal of trityl groups. In addition, since trisaccharide A and tetrasaccharide B have a common saccharidic unit, it is planned to utilise the synthesis of disaccharide glycosyl donors in block syntheses of these and higher oligosaccharides.
ACKNOWLEDGEMENTS The authors are grateful to the BBSRC for financial support (JFV).
REFERENCES
5 6 7 8 9 10
G.B. Fincher, B. Stone and A.E. Clarke, Ann. Rev. Plant Physiol., 34 (1983) 47. R.I. Pennel, J.P. Knox, G.N. Schofield and K. Roberts, J. Cell. Biol., 108 (1989) 1967. J.P. Knox, P.J. Linstead, J. Peart, C. Cooper and K. Roberts, The Plant Journal, 1 (1991) 317. R.L. Anderson, A.E. Clarke, M.A. Jermyn, R.B. Knox and B.A. Stone, Aust. J. Plant Physiol., 4 (1977) 143. H. Yamada, Carbohydr. Polymers, 25 (1994) 269. P. Kovac and C.P.J. Glaudemans, Carbohydr. Res., 138 (1985) C10. A. Rashid and W. Maekie, Carbohydr. Res., 223 (1992) 145. N.K. Kochetkov, Tetrahedron 43 (1987)2389. C. Bliard, G. Massiot and S. Nazabadioko, Tetrahedr. Lett., 34 (1993) 5083. P. Kovac, Carbohydr. Res., 153 (1986) 237.
650 11 12 13 14 15
T. Ziegler, B. Adams, P. Kovac and C.P.J. Glaudemans, J. Carbohydr. Chem., 9 (1990) 135. K. Horita, T. Yoshioka, T. Tanaka, Y. Oikawa and O. Yonemitsu, Tetrahedron, 42 (1986) 3021. P. Kovac, Carbohydr. Res., 144 (1985), C12. P. Kovac, C.P.J. Glaudemans, Carbohydr. Res., 142 (1985) 158. E.A. Yates, J.F. Valdor, S. Haslam, A. Dell, W. Mackie and J.P. Knox, Glycobiology, (1996) in press.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
651
Structural features of pectic substances during growth and ripening of apples Daniel E. Wechsler, Georg R. Strasser, Renato Amadb
Swiss Federal Institute of Technology Zurich, Institute of Food Science ETH-Zentrum, CH-8092 Zurich, Switzerland
Abstract
Pectic fractions, extracted with the chelating agent trans-l,2-diaminocyclohexaneN,N,N',N'-tetraacetic acid (CDTA) and with dilute sodium carbonate solutions from the alcohol-insoluble residues (AIR), were investigated by methylation analyses. To characterise structural changes related to growth and ripening, pectic fractions of unripe, mature and stored apples were analysed. Pectic fractions isolated from unripe and mature apples contained various amounts of starch. Therefore an amyloglucosidase treatment followed by a dialysis was carried out prior to further analyses. Linkage analyses were performed after a twofold reduction of uronic acids to the corresponding neutral sugar residues with NaBD4. Results of neutral sugar and uronic acid determinations revealed a loss of galactose during the first part of the ripening process. The linkage patterns of the investigated pectic fractions indicated that the loss of galactose is mainly due to a decrease in linear 1,4-1inked galactose residues.
1. INTRODUCTION Pectic substances make up about one third of the cell wall dry matter of dicotyledoneous plants, where they fulfill different functions. Pectins located in the middle lamella are important for the adhesion between adjacent cells, whereas pectins of the primary cell wall contribute to the water binding capacity by forming gels [8,11]. During plant development pectins undergo remarkable changes which lead to textural changes of plant tissues. As food additive with gelling, thickening and stabilising properties or as source of dietary fibre, pectins are also of technological and nutritional interest [9]. For a comprehensive understanding of the manifold functions of pectins detailed insights into the fine structure of this complex group of polysaccharides are needed. The ripening process has been subject of many studies [2,6,8]. Due to the large variety of plant tissues investigated, the results of these studies are quite heterogeneous. In general an increase in water-soluble pectins is observed, which is related to the combined action of
652 pectin methylesterases and polygalacturonases. In addition to the solubilisation of protopectin, losses of non-cellulosic neutral sugar residues also occur during the ripening of several fruits. The decrease in neutral sugar residues is not uniform for different species but is generally due to losses in galactose and/or arabinose residues [3]. L-arabinose and Dgalactose are known to be the major sugars in side chains (arabinans, galactans, arabinogalactans) of pectins. Losses of these residues during the softening process could therefore be related to the action of fruit endogeneous enzymes on pectic side chains. The degradation of these side chains might also affect the solubilisation of pectins by decreasing the entanglement with other cell wall constituents. The aim of the present investigation was to characterise the changes found in sugar composition of pectic fractions during growth, ripening and storage of apples. In order to determine structural changes, linkage analyses were performed on pectic fractions, extracted at different periods of development.
2. EXPERIMENTAL
Sampling: Golden delicious apples were sampled in intervals of three weeks over a hole season. Sampling started at the so-called "June-drop", which corresponds to the end of the cell division phase (= week 0). After harvest (week 14), the mature apples were stored over a period of 20 weeks at 4~ and 95% relative humidity. Preparation of AIR and extraction of pectic fractions: For the preparation of the alcoholinsoluble residue (AIR) the apples were peeled, cut into small pieces and boiled in 96% ethanol for 10min. After this enzyme inactivation step, the sample material was blended, homogenised and filtered through a G3 sintered glass filter funnel. The residue was washed with 96% ethanol, followed by acetone and diethyl-ether, dried overnight at 40~ under vacuum and stored at-20~ in the dark. Portions of about 10g of AIR were fractionated according to the method of Selvendran et ai. [ 10] as shown in figure 1. Removal of starch impurities: About 100mg of the extracted pectic fractions were dissolved in 30ml of NaOH 0.05M and stirred for 20h at 0~ The solutions were neutralised by the addition of 0.18ml glacial acetic acid and the pH was adjusted to 4.6. After the addition of 1 ml of an amyloglucosidase solution (60U/ml) without any detectable side activities, the samples were incubated at 60~ for 3h. Finally, the samples were cooled to room temperature, dialysed (cut-off: 12000D) against deionised water (4~ for 72h and freeze-dried. Reduction of uronic acids: Uronic acids (UA) were converted to the corresponding neutral sugars (NS) by carbodiimide activation of the carboxyl groups followed by a reduction with NaBD 4 according to the method of Kim and Carpita [5]. In order to achieve a complete reduction of the uronic acids the procedure was repeated once. Methylation analysis: Permethylation of the sample material was carded out according to Kvemheim [7]. After the first methylation excess of methyl iodide was evaporated with a stream of nitrogen and the methylation was repeated. Extraction, hydrolysis, reduction and acetylation was carded out according to Harris et al. [4]. The partially methylated alditol acetates (PMAA) were analysed by GLC-MS, using a Fisons GC 8065 gas chromatograph (Carlo Erba, Milano, Italy) coupled to a Finnigan MAT SSQ 710 mass spectrometer (Finnigan MAT, San Jose, CA, USA). For the gas chromatographic separation a DB-225
653 column (30m x 0.25mm id. 0.251am film thickness, J&W Scientific, Folsom, CA, USA) was used with helium (30cm s-1) as carder gas. The injector was hold at 220~ employing a splitless injection of 15s. The temperature programm was: 160~ for lmin, increasing by 2~ min -1 up to 220~ (holding time 19rain).
AIR
1
Stir with 1000ml 50mM CDTA at pH 6.5 for 6h at 20-22~ Filter on G3 glass filter, wash residue with water, centrifuge filtrate at 8000rpm for 20min, dialyse, concentrate. Reextract the residue under the same conditions and freeze-dry the combined filtrates CDTA-Fraction
Residue
Stir with 1000ml 50mM Na2CO 3 + 20raM NaBH4 (pH t0.8) for 16h at 1~ Filter on G3 glass filter, wash residue with water, bring f'tltrate to pH 5 with 2M acetic acid, dialyse, concentrate, freeze-dry Fraction N1
Residue
Stir with 1000ml 50mM Na2CO 3 + 20mM NaBH4 (pH 10.8) for 3h at 20-22~ Filter on G3 glass filter, wash residue with water, bring filtrate to pH 5 with 2M acetic acid, dialyse, concentrate, freeze-dry Fraction N2
l
Depectinated residue (DR)
Fig. 1: Extraction scheme for pectic fractions
3. RESULTS AND DISCUSSION The results of the linkage analyses indicated remarkable changes in the sugar composition as shown for fraction N1 in table 1. The decreasing ratio of neutral sugars to uronic acids is mainly due to a increase in galacturonic acid and to a loss of galactose residues during ripening. This trend was found in all the pectic fractions of golden delicious apples (data not shown) and is in good agreement with the results obtained by Gross and Sams [3] and Fischer et al. [2], respectively. Table 1 Ratio of PMAA derivatives of uronic acids and neutral sugars in fraction N1 Stage of development Weeks after "June drop" Neutral sugars Uronic acids
unripe 3
mature 15
stored 33
74.3 25.7
31.4 68.6
30.1 69.9
654 Methylation analyses of fractions N1 indicated that the linkage pattern of uronic acid residues (table 2) remains unchanged during ripening whereas the glycosidic linkage composition of neutral sugar residues showed different alterations. Table 2 Glycosidic linkage composition of the uronic acid residues in fraction N1 Stage of development Galacturonic acid 1,4-GalpA 1,2,4-GalpA 1,3,4-GalpA Glucuronic acid Total UA-PMAA
1,4-GlupA
unripe
mature
stored
94.9 1.2 3.4 99.6 0.4
97.0 1.0 1.9 99.9 0.1
96.4 0.8 2.7 99.9 0.1
100.0
100.0
100.0
The most important change in the glycosidic linkage pattern of the neutral sugars (table 3) was the decrease in linear 1,4-1inked galactose residues. Linear, (1--->4)-linked galactans have been isolated from pectic material of different sources [1,8]. Arabinogalactan type I has also a backbone of (1--->4)-linked I$-D-galactopyranosyl residues but contains short side chains of (1--->5)-linked a-L-arabinofuranosyl residues linked to position 0-3. The low amount of 1,3,4-branched galactose residues and the fact that the net amount of arabinose remains unaffected by the loss of (1--->4)-linked ~-D-galactopyranosyl residues indicate that the decreasein galactose might be related to a degradation of an unsubstituted galactan associated with pectins. The linkage pattern for arabinose residues showed an increasing relative amount of 1,51inked residues during ripening. Since the net amount of arabinose remains nearly unchanged during ripening, the observed changes indicate a slight linearisation of arabinan side chains. The detected rhamnose residues were typical for the backbone of pectin. Some of the (1--->2)-linked a-L-rhamnopyranosyl residues were branched, having side chains attached to 0-4 and to 0-3. Additionally terminal and double branched rhamnose residues were found in small amounts. Finally typical fucose and xylose residues were detected in all the analysed fractions. The starch content in apples reaches a maximum during growth and starts to decrease towards harvest. Therefore the AIR contained up to 40% starch for unripe apples and 10% for mature apples, respectively. During the extraction of pectic substances with CDTA and dilute sodium carbonate solutions starch was co-extracted. Although an amyloglucosidase treatment was carded out prior to methylation analysis, the fractions of all development stages still contained glucose residues. Assuming a complete starch degradation, the remaining glucose residues can not be considered as starch impurities but could be part of a xyloglucan. On the other hand most of the mannose residues must be considered as impurities, since they were found to originate from the commercial amyloglucosidase preparation.
655 Table 3 Glycosidic linkage composition of the neutral sugar residues in fraction N1 Stage of development Rhamnose T-Rhap 1,2-Rhap 1,3-Rhap 1,2,4-Rhap 1,2,3,4-Rhap Fucose
T-Fucp 1,3,4-Fucp
Arabinose
T-Araf T-Arap 1,2-Araf 1,3-Araf 1,5-Afar 1,2,5-Araf 1,3,5-Araf 1,2,3,5-Araf
Xylose
T-Xylp 1,4-Xylp
Galactose
T-Galp 1,3-Galp 1,4-Galp 1,6-Galp 1,2,4-Galp !,3,4-Galp 1,3,6-Galp 1,4,6-Galp
Glucose
T-Glup 1,4-Glup 1,3,4-Glup 1,4,6-Glup
Mannose
T-Manp 1,2-Manp 1,4-Manp 1,3,6-Manp
Total NS-PMAA
unripe 0.1 2.3 0.1 1.8 0.1
mature 0.3 2.8 0.3 2.8 0.3
stored 0.4 4.5 0.4 5.8 0.3
4.4
6.5
11.4
0.1 0.1
0.2 0.3
0.3 0.0
0.2
0.4
0.3
6.6 0.3 0.2 0.8 10.7 1.3 6.9 5.5
7.0 0.3 0.3 1.1 12.6 1.4 7.7 6.0
9.1 0.3 0.5 1.7 20.2 2.0 8.9 5.8
32.2
36.5
48.5
0.2 0.5
0.6 0.4
1.3 0.7
0.8
1.1
2.1
0.1 1.3 49.3 0.2 0.6 0.4 1.2 2.3
0.3 0.9 43.0 0.4 0.7 0.0 1.2 1.6
0.4 1.3 23.0 0.5 0.7 0.6 1.6 0.7
55.3
48.1
28.9
0.5 3.6 0.0 0.4
0.9 2.2 0.4 0.3
1.0 3.8 0.0 0.5
4.5
3.9
5.3
1.2 0.3 0.8 0.3
1.6 0.4 1.1 0.4
2.1 0.6 1.0 0.0
2.6 100.0
3.5 100.0
3.6 100.0
656 4. CONCLUSIONS -Losses of galactose residues occufing in pectic fractions during growth and ripening of apples are mainly due to a loss of (1--->4)-linked [~-D-galactopyranosyl residues. The low amount of branched galactose residues indicate the presence of a linear galactan associated with the pectic fraction of apple cell walls. -Branched arabinans with a backbone of (1--->5)-linked ot-L-arabinofuranosyl residues are present in pectic fractions of apples. During ripening a slow linearisation of the arabinans ocCurS.
5. REFERENCES [1] Eda, S., Kato, K. (1978). Galactan isolated from the midrib of the leaves of Nicotiana tabacum. Agric. Biol. Chem. 42, 2253-2257. [2] Fischer, M., Arrigoni, E., Amad6, R. (1994). Changes in the pectic substances of apples during development and postharvest ripening. 2. Analysis of the pectic fractions. Carbohyd. Polym. 25, 167-175. [3] Gross, K.C., Sams, C.E. (1984). Changes in cell wall neutral sugar composition during fruit ripening: a species survey. Phytochemistry 23, 2457-2461. [4] Harris, P.J., Henry, R.J., Blakeney, A.B., Stone, B.A. (1984). An improved procedure for the methylation analysis of oligosaccharides and polysaccharides. Carbohydr. Res. 127, 59-73. [5] Kim, J-B., Carpita, N.C. (1992). Changes in esterification of the uronic acid groups of cell wall polysaccharides during elongation of maize coleoptiles. Plant Physiol. 98, 646653. [6] Knee, M., Bartley, I.M. (1981). Composition and metabolism of cell wall polysaccharides in ripening fruits. In: Friend, J., Rhodes;, M.J.C. (eds.). Recent advances in the biochemistry of fruits and vegetables. Academic Press, New York, 133-148. [7] Kvernheim, A.L. (1987). Methylation analysis of polysaccharides with butyllithium in dimethyl sulfoxide. Acta Chem. Scand. Ser. B41,150-152. [8] Melford, A.J., Dey, P.M. (1986). Postharvest changes in fruit cell wall. Adv. Food Res. 30, 139-193. [9] Pilnik, W. (1990). Pectin - a many splendoured thing. In: Phillips, G.O., Wedlock, D.J., Williams, P.A. (eds.). Gums and stabilizers for the food industry. Elsevier, London, 209221. [ 10] Selvendran, R.R., Stevens, B.J.H., O'Neill, M.A. (1985). Developments in the isolation and analysis of cell walls from edible plants. In: Brett, C.T., Hillman, J.R. (eds.). Biochemistry of plant cell walls. Cambridge University Press, Cambridge, 39-78. [11]Van Buren, J.P. (1991). Function of pectin in plant tissue structure and firmness. In: Walter, R. (ed.). The chemistry and technology of pectin. Academic Press Inc., San Diego, 1-22.
PECTIN BIOSYNTHESIS AND BIOLOGICAL EFFECTS OF (DEGRADED) PECTIN
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J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
659
Metabolism of pectin in the gastrointestinal tract G. Dongowski and H. Anger
German Institute of Human Nutrition Potsdam-Rehbr[icke, Department of Food Chemistry and Preventive Nutrition, D-14 558 Bergholz-Rehbriicke, Germany
Abstract Pectin was depolymerized and de-esterified only to a small extent after treatment under conditions simulating the gastrointestinal lumen. The degree of esterification was slightly decreased in the upper part of gastrointestinal tract of germfree and conventional rats and additionally in caecum and colon of germfree rats. Pectin passes the small intestine as a macromolecule. The molecular weight distribution of pectins measured by gel chromatography with viscosity detection from faeces of germfree rats remained nearly unchanged. No or very low amounts of galacturonan were found in contents of caecum, colon and in faeces of most of the conventional rats. Oligogalacturonic acids were not detected in faeces of these animals. Diand trigalacturonic acid which could then be absorbed by the host were present in the colon of a few conventional rats. However during in vitro fermentation of pectin with human faecal flora unsaturated oligogalacturonic acids were found as intermediate products in variable concentration and composition within ca. 8 hours using thin-layer chromatography and HPLC (PAD, chiraliser and UV detection). Low-esterified pectin was fermented faster than highesterified pectins.
1. INTRODUCTION
The physiological importance of pectin as a soluble dietary fiber in the small intestine is closely related with its macromolecular properties (interactions with bile acids, lowering of serum cholesterol, effects on the postprandial lipemia etc.). Pectin is not depolymerized by intestinal enzymes. A partial degradation seems to be possible under the conditions of stomach and small intestine. Pectin is completely fermented in the (caecum and) colon by the microflora [1-4]. In order to be absorbed, pectin has to be degraded to mono- or oligomers having a low degree of polymerisation (DP). It is unclear whether oligogalacturonic acids (OligoGalA) as metabolites of pectin degradation are formed in detectable concentrations in the colon. OligoGalA intravenously applied or injected directly in the caecum were found in the urine of rats [5]. Pectin influences the absorption, incorporation and the renal excretion of lead [6].
660 This study reports on investigations of the metabolism of pectin by in vitro and in vivo experiments using chromatographic methods.
2. MATERIAL AND METHODS
2.1. Treatment of pectin under conditions of gastrointestinal tract (GI) Pectin solutions were treated 2 h at pH 1-2 and each then for 2 h at pH 6.0, 7.0 and 8.0.
2.2 Animal experiments 5 groups of 10 conventional rats and 4 groups of 6 germfree rats were fed ad iibitum over 21 days. The diet had the following composition: 6.5 % or 0 % pectin (galacturonan) with different degree of esterification (DE), 5.0 % cellulose, 63.0 % or ca. 54 % wheat starch, 20.0 % casein, 5.0 % sunflower oil, 5.0 % mineral mixture and 2.0 % vitamin mixture. The diets for the experiments with germfree rats were sterilized by T-irradiation (20 kGy). This resulted in a partial depolymerization of pectin (Table l). Faeces were collected in two periods (3 d during weeks 2 and 3). At the end of the experiments, the contents of ileum, caecum and colon were also investigated.
Table l Pectins used in experiments with rats Original pectins DE [ 11 ] (%) (mFg AG) 92.6 328 K 70.8 455 K'~; 34.4 294 K
7-irradiated pectins DE [ 11 ] (%) (ml/g AG) 92.6 208 c; 70.7 292 c 34.4 211G
K = conventional rats; G = germfree rats; AG - anhydrogalacturonic acid (galacturonan).
The lyophililized intestinal contents or faeces were treated for enzyme inactivation in 5 ml 96 % EtOH for 20 min at 75-80 ~ After addition of 5 ml water the mixture was stirred 30 rain and centrifuged at 6000*g also for 30 min. In the supernatant galacturonan was estimated by the m-hydroxydiphenyl (MHDP) reaction [7] and OligoGalA were determined using HPTLC. In the dried residues, the content of galacturonan and the DE were estimated after extraction with 0.5 % EDTA. Molecular weight distribution was analysed by gel chromatography (0.5 % AG) equipped with a Shodex Ohpak B 805 column (500*8 mm) with phosphate buffer (pH 6.5) using differential-refractometer/viscometer detection (Knauer). For calibration a pectin series from vibration milling was applied.
661 2.3. In vitro incubation 150 ml pectin media (0.5 % AG) were incubated with 4 g human faecal flora at 37 ~ without aeration. The contents of macr.omolecular pectin and OligoGalA were estimated in the culture after different periods. OligoGaiA were determined using the Camag HPTLC system on silica gel 60 developed with n-propanol-water (7+3.75) and (7+2.75). The spots were detected by measuring at 235 nm and after dipping in the MHDP reagent at 525 nm. Further a Kontron HPLC system equipped with UV (250 nm), chiralyser and PAD detectors was used for estimation of OligoGalA. The column was CarboPac PAl (250*9 mm) with a precolumn and the gradient of 40 to 100 % solvent B (0.15 M NaOH, 1 M Na-acetate) and of 0.15 M NaOH (A) was applied. The system was calibrated with a mixture of OligoGalA (DP 2-15) prepared from pectic acid using pectate lyase from Erwinia carotovora.
3. RESULTS AND DISCUSSION
Milieu conditions in gastrointestinal tract can influence the pectin structure and properties. Under the acid conditions of the stomach (pH 2-4) extraction of pectin from plant cell walls and hydrolysis of side chains can occur. In small intestine (pH 5-6) I]-elimination of main chains or de-esterification seems to be possible. In caecum and colon (pH 6-8) a strong fermentation of pectin takes place causing depolymerization to oligomers and leading to formation of short chain fatty acids and gases. The presence of OligoGalA is not yet clarified. After treatment under the conditions simulating the gastrointestinal lumen pectin was depolymerized only to a small extent. The DE of pectin is slightly decreased in the upper part of GI and as well as in caecum and colon of germfree rats (Table 2).
Table 2 DE of pectin Original pectin 92.6 70.8 34.4
(%) isolated from intestinal contents of conventional (K) and germfree rats (G) Ileum Ileum Caecum Colon K G G G 91.2 90.4 90.0-~.6 89.8_+0.3 70.6 70.4 69.9_+0.8 70.7_+0.6 34.1 34.5_+0.4 34.0-Z-O.5 34.6_+0.3
In intestinal contents and faeces from germfree rats quite high amounts of galacturonan were found, especially in the case of the pectin with the highest DE (Table 3). The isolated pectins were depolymerized to a small extent. The molecular weight distribution of pectins from intestinal contents and faeces remained relatively unchanged (Figures 1 and 2).
662 Table 3 Pectin in intestinal contents and faeces of germfree rats DE Ileum Caecum Colon (%) (mg) (mg) (mg) 92.6 87-+10 1599-+ 87 143-+59 70.8 42_+ 7* 655_+124'* 167_+28 34.4 55+ 0* 421+102"* 115+28 Soluble part in 50 % EtOH: 1.4-6.0 %; * P < 0.05; ** P <
Faeces I (mg/d) 317_+ 35 403_+ 39* 246_+146 0.01.
Faeces II (mg/d) 540-+67 514+95 362+56
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o
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ol
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R e t e n t i o n v o l u m o (ml)
Figure 1. Pectins isolated from contents of of ileum, caecum and colon as well as from from faeces of a germfree rat (DE 92.6%).
9
.
9 '
o. o
' R e t o n l l o n v o l u m e (ml)
Figure 2. Pectins isolated from faeces of germfree rats (collecting period I).
No or very low amounts of galacturonan were found in contents of caecum and colon and in faeces of most of the conventional rats (Table 4). Only in 1 or 2 animals of each group higher galacturonan concentrations were present in the lower parts of gastrointestinal tract as well as in faeces (Figure 3).
663 Table 4 Pectin in intestinal contents and in faeces of conventional rats DE lleum Caecum Colon Faeces I (%) (mg) (mg) (mg) (mg/d) 92.6 16-38 0-117 0- 5 15-212 70.8 26-38 0 - 64 0-62 3-217 34.4 28-38 0 0- 2 1- 18 Soluble part in 50 % EtOH: 0-7.0 %. .
.
.
.
.
.
Faeces II (mg/d) 4-108 0-145 0
.
OligoGalA could not be detected in faeces of conventional rats. Nevertheless unsaturated diand trigalacturonic acidsl were present in colon contents of a few rats (Figure 4).
r r-
4
T
....
Colen lr-Pl
1oo.
Yh~oe~
c h m m
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DE 92.11% DE 112.6%
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D e 70.8 %
~-2~
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De 7o.e%
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~
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Figure 3. High molecular and partially degraded pectins isolated from faeces of some conventional rats (collecting period II)
10 lO t.lel I enl Ilnlle: 255
90
40
So
6O
:PO
ee
~
J
Figure 4. Di- and TriGalA found in colon contents of conventional rats fed with a diet containing low-esterified pectin (above) and in a caecum extract with a standard mixture of OligoGalA (DP 2-5) (below)
664 During the in vitro fermention the amount of macromolecular pectin was diminished continuously. On the other hand the fraction of OligoGalA was increased at first and diminished later. The content of short chain fatty acids, which are typical end products of fermentation of dietary fibers rised permanently (Figure 5). Low-esterified pectins were fermented in vitro faster by human faecal flora than the high-esterified pectins.
5
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Figure 5. In vitro fermentation of low-esterified pectin (DE 34.4 %).
During this in vitro fermentation, a oligogalacturonic acids were found to be present in the culture as intermediate products in variable concentration and composition within ca. 8 hours depending on the fermentation conditions and the DE of the pectin. A typical experiment is shown in Figure 6. Generally OligoGalA with a higher degree of polymerization were mostly found in higher concentrations during fermentation of low-esterified pectins. The OligoGalA composition was estimated in the culture using HPTLC (Figure 7) with combined detections and using HPLC with different detectors (Figure 8). The double bounds of unsaturated OligoGalA can be detected at 235 nm. Interferences from acetate in gradient buffer may be suppressed by measuring at 250 nm. The pulsed amperometric detection is related to the reducing end groups of the oligomers. The sensitivity decreases with the chain length. An advantage of this method is its relative specificity for carbohydrates. The chirality detection is related to monomeric units in the chain. No significant decrease of response to the chain length was found. A slight effect of the signal from the chain length was detected at low DP. The combination of different detections improves the analyses of OligoGaIA by HPTLC and HPLC.
665 2,5 DE 0 % t._
DE 34.4 %
DE 66.0 %
DE 94.7 %
.......
.i-, O
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(h)
ImDiGalA m TdGalA n TetraGalA c-nPentaGalA INIHexaGalA m HeptaGalA I
Figure 6. Composition of OligoGalA during in vitro incubation of pectins with human faeces flora. !
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Figure 7. HPTLC determination of OligoGalA during in vitro action of human faeces flora on low-esterified pectin (detection at 23 5 nm [left] and alter treatment. with the MHDP reagent [fight])
m,m 50.0
Figure 8. Estimation of OligoGalA using HPLC with PAD, UV and chiralyser detection in culture a~er action of human faeces flora on low-esterified pectin (6 h)
666 4. CONCLUSIONS
Pectin passes the small intestine as a macromolecule. This is shown both by treatments of pectin under conditions of gastrointestinal lumen and in experiments with germfree rats. Because of its properties (e.g. viscosity, ion-exchange) pectin is able to interact with bile acids, neutral sterols or metal ions. By the action of microflora (experiments with conventional rats) pectin is intensively degraded. In general, pectin did not occur in the lower parts of intestine, but in some cases even macromolecular galacturonans were found in faeces. Di- and trigalacturonic acid were estimated in some colon contents. During in vitro fermentation, OligoGalA were present as intermediate metabolites of pectin degradation. This points out that OligoGalA could be absorbed by the host. Low-esterified pectins were fermented faster than highesterified pectins. Only unsaturated OligoGalA were detected as pectin metabolites in colon of conventional rats and in culture of in vitro experiments.
5. REFERENCES
1 J.H. Cummings, D.A.T. Southgate, W.J. Branch, H.S. Wiggins., H. Houston, D.J.A. Jenkins, T. Jivraj and M.J. Hill, Brit. J. Nutr., 41 (1979) 477. 2 W.D. Holloway, C. Tasman-Jones and K. Maher, Amer. J. Clin. Nutr. 37 (1983) 253. 3 B.J.H. Stevens, R.R. Selvendran, C. Bayliss and R. Turner, J. Sci. Food Agric. 44 (1988) 151. 4 C.J. Buchanan, S.C. Fry and M.A. Eastwood, J. Sci. Food Agric. 66 (1994) 163. 5 H. Anger, E. Waizel and B. Kahrmann, About the absorption of oligo-galacturonides from caecum of rats. FASEB Meeting (1994), Anaheim/USA; Abstr.: FASEB J. 8 (1994) A 152. 6 C. Stark, E. Walzel, G. Dongowski and B. Ozierenski, Influence of pectin on lead incorporation in germfree and conventialized rats. EUROTOX 95 (1995), Prague; Abstr. 7 N. Blumenkrantz and G. Asboe-Hansen, Anal. Biochem. 54 (1973) 484.
Acknowledgements: We thank Dr. Angelika Lorenz and Dr. Jiirgen Proll for conducting the animal experiments. This work was financially supported by the Federal Ministry for Education, Science, Research and Technology.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
Cell wall properties of transgenic tobacco plants that express acid invertase in their vacuole
667
a yeast derived
Susanne Hoffmann-Benning ~, Rudolf Ehwald b) Lothar Willmitzer c), Joachim Fisahn c) a)Institut for Genbiologische Forschung, Ihnestr. 63, 14193 Berlin, Germany, b) Humbold Universit~it, Sektion Biologie, Invalidenstr. 42, 1040 Berlin, Germany c~ Max Planck Institut ffir molekulare Pflanzenphysiologie, Karl Liebknecht Str. 25, 14476 Golm, Germany.
Abstract Transgenic tobacco plants that express a yeast derived acid invertase in their vacuole were used to analyse the effects of a modification in carbon allocation on cell wall properties and turgor pressure. Significant increases in turgor pressure, free and total acid content were detected in the transgenic lines. Cell wall neutral sugars were examined by gas chromatography and as a result the arabinose concentration was found to be significantly increased in leaf 6 of the transgenic plants. In contrast, fucose was reduced. However, the size exclusion limit of the pectin network was unaffected by the genetic manipulation.
Introduction The plant cell wall consists of cellulose microfibrils, hemicellulose, pectin, and proteins. The hemicellulose network and some cell wall proteins are discussed to play a major role in cell expansion. Pectins are synthezised in the ER and Golgi apparatus and then exported into the apoplastic space. A major component of the pectin network are chains of homopolygalacturonic acid that can either be esterified or cross-linked by calcium ions. Through the degree of crosslinking the strength of the cell wall may be modified and thus influence cell enlargement. Alternatively, it should lead to a varied pore size and may thus affect the accessability of the wall for cell wall enzymes. To analyse the effects of a modification in the carbon allocation pattern on the growth rate transgenic tobacco plants that express a yeast derived invertase in the vacuole were regenerated by genetic transformations. In particular, these plants exhibit a reduced internodal growth and accumulate hexoses in their vacuole (1, 2). Furthermore, these plants develop enlarged leaf cells in younger tissue. When the turgor pressure of these plants was investigated an increased value was measured that correlated with the rate of cell expansion (3). In the present study we used transgenic plants to analyse the amount of control exerted by an additional vacuolar invertase on the allocation of carbohydrates to the plant cell wall. Since physical parameters indicated a significant modification in the thermodynamic state of these invertase plants, the monosaccharide composition, the pore size and the amount of free and bound acids present in the cell wall were measured.
Methods The concentration of cell wall monosaccharides was analyzed by gas chromatography (4). Determination of total and free acids was performed according to Ehwald et al. (5). Cell wall size exclusion limits were measured as decribed by Ehwald et al. (6).
668
Table 1 Cell expansion rate, turgor, osmotic pressure, and water potential of leaf 6 and leaf 11 of wild type and invertase plants
Leaf 6
Leaf 11
WT
Inv
WT
Inv
669.8
3918.3
3242.2
567.1
Turgor [bar]
2.3 + 0.2
5.2 + 0.5
3.4 + 0.4
2.3 + 0.2
Osmotic pressure [bar]-
7.1 + 0 . 7
10.0+0.3
7.7 + 0.3 10.9 + 0.4
Cell expansion rate [l.tm3]
Water potential [bar] -5.2 + 0.3 -5.0 + 0.4
-4.3
-8.6
Turgor pressure of single leaf cells was measured by an improved version of the cell pressure probe according to Htisken et al. (7). Mikrocapillaries were pulled on a laser heated pulling device. Osmotic pressure. Leaf tissue was homogenized for 15-20 seconds, centrifuged at 13000 rpm for 1 min and the osmotic pressure of 50~1 of the supernatant was determined using a
40
35-
~ ~
wt, leaf 3 Inv, leaf 3 Wt, leaf Inv, leaf 6
0~ 3 0 o~ 2 5 II}
20=
15-
"6 e-
5-
N O" <
Rha
Fuc
Ara
Xyl
Man
Gal
Figure 1, Relative distribution of neutral cell wall saccharides. The bar indicates the means +/S.E. of 6 independent measurements.
669 microliterosmometer (Osmomat 030; Gonotech, Berlin, Germany) Cell dimensions were determined from micrografs and the expansion rates calculated from volume changes as a function of the time during which the leaf advanced from e.g. leaf 5 to 7. Results Since invertase cleaves sucrose into glucose and fructose it could be assumed that the turgor pressure within the leaves of the invertase plants was increased. A significant elevation in the turgor emerged in leaf 6 of the transgenic plants (Table 1). Parallel to the increase in turgor a large cell expansion rate was observed in leaf 6 (Table 1). Since the cell wall counteracts the turgor pressure the walls of the transgenic plants should be modified. Transmisssion electron microscopy revealed an increase in the cell wall thickness of 38% in the inner epidermal walls and a 68% increase in the mesophyll (Hoffmann-Benning et al. 1996). 0,8
A
0,7-
T
WT I-/7-/-~ Inv
~" 0 , 6 0,5o
E 0,4E v 0,3,<
i
u.. 0,2 1
0,1-~ r~.
~" 1 , 5 is}) o E 1,0-
E
,< I--
N~dt
N
0,5-
0~
_L
~g~74
~A
'
c
T
!
70,0 60,0 -, <
50,0 /
o"s 40,0-1 30,0 20,0 = 10,0 0,0
I 6
~ 11
Leaf age
Figure 2. Analysis of free (A) and total (B) acids in wild type (open bars) and transgenic (closed bars) plants. Fig. 2C shows the ratio of free to total acids (in %). The bars represent the mean +/- S.E. of 4-11 independent determinations.
670 Table 2 Size exclusion limits measured in 3 leaves of wild type and invertase plants.
Number
3
6
~i
WT n
3.00 + 0.10 4
2.96:1:0.09 12
3.00 + 0.07 7
Inv n
2.94 :t: 0.11 4
2.90 +_.0.09 10
2.72+ 0.04 8
To characterize modifications in the cell wall composition of transgenic invertase plants we determined the distribution of neutral saccharides (Fig. 1). Obviously, the relative amounts of fucose and mannose were slightly reduced in leaf 6 of transgenic plants, whereas arabinose was increased. The other saccharides remained unchanged. Additionally, the content of free and total acids as a measure of the degree of pectin esterification was investigated (Fig. 2). The amounts of both free and total acids were increased in the transgenic plants. However, their ratios are not significantly affected when both plant lines are compared (Fig. 2). The size exclusion limit of cell wall fragments is a measure of the density of the pectin network. When wild type and invertase plants were compared no significant differences in the pore size emerged (Table 2).
Discussion Several determinants of the cell walls within wild type and vacuolar invertase plants were investigated. Since an increased cell size and turgor pressure were described for the transgenic plants (Table 1; 3) we examined the amount of neutral sugars, free acids, bound acids, and the size exclusion limit of the cell walls (Fig. 1,2; Table 2). Among others, these parameters are known to be involved in the regulation of cell expansion. The growth of a plant cell results from an orchestrated interaction between turgor pressure and cell wall stress relaxation. These processes require metabolic and thermodynamic control. During cell expansion, polymers within the wall can be rearranged giving rise to controlled relaxation of the turgor pressure. Since cell wall tension balances the turgor, relaxation of this tension will lower cell turgor and therefore induce an influx of water into the cell (6). It is widely accepted that wall mechanical properties control whether, and at what rate, a plant cell can grow (6). However, the increase in turgor pressure associated with the transgenic invertase plants adds support to the hypothesis forwarded by Lockhard (5) that the turgor has a major impact on the rate of cell expansion. Although the transgenic plants exhibit dwarfism, the individual leaf cells are increased or are of the same size as in control plants. Cosgrove and Sovonick-Dunford (7) reported that chemically dwarfed pea seedlings showed lower wall extensibility than non-dwarfed controls when measured in a living cell. Measurements of wall extensibility in living plant tissue have revealed that the walls of rapidly growing cells are much more extensible than those of slowly or non growing tissue. This increase in extensibility may be correlated with an elevated amount of arabinose within the cell walls of the transgenic plants (Fig. 1). However, these changes do not result in a modified size exclusion limit or free-to-total acid ratio. Therefore, the cell wall thickness rather than the cell wall composition gives rise to an increase in turgor and the associated cell expansion.
671
Acknowledgements We thank Petra Lembke and Petra Heese for assistance during the cell wall analysis. We thank Dr. U. Sonnewald for the generous gift of the transgenic invertase plants. This work was supported by a DFG grant to SHB (HO 1605/1-2).
References D. Heineke, K. Wildenberger, U. Sonnewald, L. Willmitzer and H.W. Held, Planta, 194 (1994) 29-33. U. Sonnewald, M. Brauer, A. van Schaewen, M. Stitt and L. Willmitzer, Plant J., 1 (1991) 95. S. Hoffmann-Benning, L.Willmitzer and J. Fisahn, Plant Physiol., (1996) sub. R. R. Selvendran and P. Ryden, in: Methods in Plant Biochem. (P.M.Dey and J.B.Harborne, eds) (1990) 549. R. Ehwald, H. Woehlecke and C. Titel, Phytochem., 31 (1992) 3033. R. Ehwald, P. Heese and U. Klein, J. Chromatography, 542 (1991) 239. D.Htisken, E.Steudle and U. Zimmermann, Pant Physiol., 61 (1978) 158. J.A. Lockhart, J. Theor. Biol., 8 (1965) 264. S.J. McQuenn-Mason, J. Exp. Bot., 292 (1965) 1639. D.J. Cosgrove and S.A.Suvonick-Dunford, Plant Physiol. 89 (1989) 184.
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J. Visser and A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996Elsevier Science B.V.All fights reserved.
673
P e c t i c p o l y s a c c h a r i d e f r o m r o o t s of Glycyrrhiza uralensis: P o s s i b l e c o n t r i b u t i o n of n e u t r a l oligosaccharide in the g a l a c t u r o n a s e - r e s i s t a n t region to a n t i - c o m p l e m e n t a r y a n d mitogenic activities H. Kiyohara a, N. Takemoto b, J.-F. Zhao ", H. Kawamura b and H. Yamada" aOriental Medicine Research Center, the Kitasato Institute, Shirokane 5-9-1, Minato-ku, Tokyo 108, Japan. bTsumura Central Research Laboratories, 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300-11, Japan.
Abstract Digestion with endo-polygalacturonase liberated the enzyme resistant region ("ramified" region, PG-Ic) as an active site of the anti-complementary and mitogenic pectic polysaccharide (GR-2IIc) from Glycyrrhiza uralensis. Lithium degradation decreased the anti-complementary and mitogenic activities of PG-lc. Although the products from PG-lc were still active, the methylglycoside of r GalA-(1-->2)-~-L-Rha-(1--~4)-c~-D-GaiA did not show both activities. The lithium degradation of PG-lc gave various neutral oligosaccharide-alditols. The longest and short oligosaccharide-alditoi fractions had relatively potent anti-complementary activity, whereas all oligosaccharide-alditol fractions expressed weak but significant mitogenic activity. However, standard oligosaccharide-alditols consisting of GIc did not show any activity.
1.
INTRODUCTION
It has been found that pectic polysaccharides including pectins from medicinal herbs express various kinds of in vitro and in vivo pharmacological activities such as 1) potentiation of antibody response, 2) protection of adverse effects of anti-tumor drugs, 3) anti-ulcer activity, 4) complement activating activity, 5) mitogenic activity, 6) stimulation of IL-2 and IL-6 productions [1-3]. It is interesting to clarify which carbohydrate chains in pectic polysaccharides are responsible for expression of the pharmacological activities. We have compared anti-complementary and mitogenic activities of crude polysaccharide fractions from 10 medicinal herbs which are well used as component herbs in kampo (Japanese herbal) medicines, and found that crude polysaccharide fraction (GR-1) from roots of Glycyrrhiza uralensis Fisch et DC. shows both potent activities [4]. The roots of G. uralensis Fisch et DC. have been used as a component herb in many kinds of Kampo medicines, and clinically used for the treatments of inflammation,
674 allergy and gastric ulcer. Three acidic polysaccharides (GR-2IIa, GR-2IIb and GR-2IIc), isolated from the acidic polysaccharide fraction (GR-2) of G. uralensis, showed anti-complementary activity due to complement activation, however only GR-2IIc also had mitogenic activity [5]. GR-2IIa-IIc have been proposed to consist of an endoc~-(1-->4)-polygalacturonase-resistant region ("ramified" region) in addition to rhamnogalacturonan II-like regions and ~-(1-->4)-galacturonan regions with high heterogeneity, and the enzyme-resistant region (PG-lc) of GR-2IIc was shown to function as the active site for expression of the anti-complementary and mitogenic activities [6]. In the present paper we describe a contribution of the neutral carbohydrate chains in PG-lc for expression of its anti-complementary and mitogenic activities.
2.
PROPERTY OF GR-2IIc
The mitogenic and anti-complementary polysaccharide, GR-2IIc has been isolated from crude polysaccharide fraction (GR-1) by fractionation with cetyitrimethylammonium bromide and anion-exchange chromatography [5]. GR-2IIc consisted mainly of Glc, Gal, GalA and GIcA in addition to Rha, Fuc, Ara and Man. Fluorocytographic analysis indicated that surface IgD positive B cells in GR-2IIcstimulated spleen cells increased significantly in addition to surface IgM and IgG positive cells (Table 1). However, lipopolysaccharide (LPS) resulted increments of IgM and IgG positive cells but not IgD positive cells. These results indicated that GR-2IIc was novel and different mitogen from other known mitogens, and proposed that it proliferated immature B cells into mature resting B cells. Endo-~-(1-->4)-polygalacturonase digestion gave three fragments eluted in the void volume (PG-lc), intermediate fraction (PG-2c) and the lowest-molecular-weight fraction (PG-3c) from GR-2IIc [6]. Methylation analysis using base-catalyzed [~.elimination Table 1
Fluorocytographic analysis of GR-2IIc- or LPS-proliferated spleen cells in vitro
Percentages of positive cells Sample
control GR-2IIc LPS
Blast cells
Total Ig §
9.0 + 1.06 33.6 + 0.03 36.2 + 0.54
53.8 + 0.06 71.8 + 0.68 74.2 +0.29
Thy 1.2 + control GR-2IIc LPS
41.4 +_0.40 28.8 + 0.87 24.4 + 0.70
IgM § 51.7 + 0.55 70.9 + 0.08 76.7 + 0.16
IgG §
IgD §
46.2 + 4.26 67.9 + 0.90 76.5 + 0.49
51.5 + 1.01 69.8 + 0.46 17.0 + 0.43
675 and structural analysis of acidic oligosaccharides liberated by partial acid hydrolysis suggested that PG-lc comprised rhamnogalacturonan structure as acidic core, therefore PG-lc was considered to be "ramified" region. PG-3c was found to contain oligogalacturonide, and PG-2c consisted of 2-Me-Fuc, 2-Me-Xyl, Api as unusual component sugars in addition to Rha, Fuc, Ara, Man, Gal, Glc, GalA and GlcA. This result assumed that PG-2c comprised rhamnogalacturonan II structure. When PG-lc"--PG-3c were tested anti-complementary and mitogenic activities, PG-lc expressed more potent both activities, however PG-2c and 3c did not show any activity (Figure 1), therefore PG-lc was suggested to be the active site for expression of the activities of GR-2IIc.
0
CONTRIBUTION OF ACIDIC MOIETY IN "RAMIFIED" REGION (PG-lc) ON ITS ANTI-COMPLEMENTARY AND MITOGENIC ACTIVITIES
Methylglycoside of ~-L-Rha-(1-->4)-cx-D-GalA-(1-->2)-~-L-Rha-(1--)4)-cx-D-GalA had a similar partial structure as the rhamnogalacturonan core, however this tetrasaccharide did not show anti-complementary and mitogenic activities. When uronic acids in PG-Ic were degraded by lithium, the products (which contained neutral oligosaccharide-aiditols and degradation products from uronic acids) showed decreased anti-complementary and mitogenic activities compared to PG-lc (Figures 2A and 3). However, the products still showed weak but significant activities, and it was concluded that neutral carbohydrate chains in PG-lc might contribute to expression of these activities.
9..\\\\\\\'< control GR-2IIc PG-lc ("ramified region) PG-2c (RGII like region)
0%
PG-3c (oligoGalA) o~5
o.~5
Mitogenic activity (OD540)
o
0%
o
!
!
50
~oo
Anti-complementary activity (%)
Figure 1 Immunomodulating activity of fractions derived from GR-211c by endo-cx(1--~4)-polygalacturonase digestion
676
100-
PG-lc
A w
PG-Ic
J A w
I,
A v
v
.I
PG-Ic-4
t_
"~ 50 a_
s0 ,i
PG-Ic-2
Lithium-degraded PG-lc
i
o-
PG-Ic-5
~
<
0
9
0
160
200
3()0
4{)0
~
0
.
=.=
100
Concentration (It g/ml)
. 200
9 300
. 400
Concentration (~t g/ml)
Figure 2 (A) Effect of lithium-degradation on anti-complementary activity of PG-lc (B) Anti-complementary activity of oligosaccharide-alditois obtained from PG-lc by lithium degradation
control
PG-lc Li-deg-PG-lc PG-lc-1 PG-lc-2 PG-lc-3 PG-lc-4 PG-lc-5 9 0
~
*p<0.001 '
"
Incorporation of [ 3 H]-thymidine (X 10 s cpm) Figure 3 Mitogenic activity of products and oligosaccharide-alditols from PG-Ic by lithium degradation
677
0
ANTI-COMPLEMENTARY AND MITOGENIC ACTIVITIES OF NEUTRAL OLIGOSACCHARIDE CHAINS DERIVED FROM PG-lc
The lithium degradation products from PG-Ic were fractionated on Bio-gel P-10, and gave large amounts of short neutral oligosaccharide-alditol fractions (PG-Ic-4 and 5) in addition to fractions consisting of long (PG-Ic-1) and intermediate-size neutral oligosaccharide-aiditols (PG-Ic-2 and 3). Among these fractions, the longest (PG-lc-1) and shorter oligosaccharide-alditol fractions (PG-Ic-4) showed relatively potent anti-complementary activity (Figure 2B) whereas all the fractions had weak but significant mitogenic activity (Figure 3). In order to investigate whether the oligosaccharide-alditols from PG-Ic interact with spleen cells, PG-lc was incubated with spleen cells in the presence of equal amounts of the oligosaccharide-alditol fractions (PG-lc-I'---PG-lc-5). As shown in Figure 4, mitogenic activity of PG-lc was slightly but significantly reduced by PG-Ic-1, PG-lc-2, PG-lc-3 or PG-lc-5. These results suggested that the neutral carbohydrate chains in PG-lc might be able to interact with the cells through carbohydrate receptor-like molecule on the cell surface in order to express the activity.
control
PG-lc P G - l c + PG-lc/Li-deg P G - l c + PG-lc-1 P G - l c + PG-lc-2 P G - l c + PG-lc-3 P G - l c + PG-lc-4
!
P G - l c + PG-lc-5 -
i
-
|
0 1 2 3 I n c o r p o r a t i o n of [3H]-thymidine (X l0 s cpm) Figure 4
Effect of neutral oligosaccharide-aiditol fractions derived from PG-lc by lithium degradation on mitogenic activity of PG-lc
The oligosaccharide-alditols in PG-lc-4 were analyzed by GC-MS as permethylated oligosaccharide-alditols. The results indicated that PG-lc-4 contained di- to tetrasaccharide-alditols possessing glycosyl units such as Hex-(l--)4)-6-deoxyHex-ol, Pen--> Pen-~deoxyHex-oi, Pen-~Hex ~deoxyHex-oi, Hex ~Hex-~Hex-ol, Pen (~Pen)2-~Pen-ol, Hex (~Hex)2~deoxyHex-ol and Hex (--)Hex)~-~Hex-ol. Since PG-lc-4 consisted mainly of Ara as pentose, it was assumed that Pen in the
678 oligosaccharide-alditois might be Ara. PG-lc-4 also mainly comprised Man, Gal and Glc as hexose, however Hex units and glycosidic linkages in the oligosaccharidealditois could not be deduced in the present study. Since PG-lc-4 contained various kinds of neutral oligosaccharide-alditols, some standard oligosaccharide and oligosaccharide-alditols were measured for anticomplementary and/or mitogenic activities in order to investigate whether other oligosaccharide-alditols have such activities. However, all oligosaccharides and oligosaccharide-alditols tested, such as maltose, maltohexaose, isomatohexaose, laminarihexaose, maltitol, maltotriitol and maltoheptitol, did not show any activities. These results suggested that certain neutral oligosaccharide-alditols in PG-Ic-I and 4 might be responsible for expression of the activities.
5.
CONCLUSION
The present results proposed that the certain neutral carbohydrate chains in the "ramified" region of GR-2IIc plays important roles for expression of anticomplementary and mitogenic activities. However the activities of the neutral carbohydtae chains were weaker than those of the intact "ramified" region. It was assumed that substitution of the neutral carbohydrate chains to the rhamnogalacturonan core might be enhance the activities of the chains, and there might be a possibility to exist a minimum molecular seqeunce consisting of neutral carbohydrate chains and the acidic core for expression of the potent activities.
6.
ACKNOWLEDGEMENTS
We thank Dr. V. Pozsgay (NIH, U.S.A.) for his kind gift of methylglycoside of o~-L-Rha-(1-->4)-o~-D-GalA-(1--->2)-o~-L-Rha-(l-->4)-o~-D-GalA.
7. 1 2 3 4 5 6
REFERENCES H. Kiyohara, T. Matsumoto, N. Takemoto, H. Kawamura, Y. Komatsu, H. Yamada Planta Med., 61 (1995) 429. H. Kiyohara, T. Matsumoto, Y. Komatsu, H. Yamada, Planta Med., in press. H. Yamada, Asia Pacific J. Pharmacology, 9 (1994) 209. H. Yamada, H. Kiyohara, N. Takemoto, J.-F. Zhao, H. Kawamura, Y. Komatsu, J.-C. Cyong, M. Aburada, E. Hosoya, Planta Med., 58 (1992) 166. J.-F. Zhao, H. Kiyohara, X.-B. Sun, T. Matsumoto, J.-C. Cyong, H. Yamada, N. Takemoto, H. Kawamura, Phytotherapy Res., 5 (1991) 206. J.-F. Zhao, H. Kiyohara, H. Yamada, N. Takemoto, H. Kawamura, Carbohydr. Res., 219 (1991) 149.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All fights reserved.
Immunologically active
679
polysaccharides from cell suspension of H e l i a n t h u s a n n u u s
1805
M. Kratchanova a, M. Ilievab, E. Pavlova a, A. Pavlovb,N. Markova b
a Bulgarian Academy of Sciences, Institute of Organic Chemistry with Phytochemistry, 95 V. Aprilov Str., P. O. Box 27, LBAS - Plovdiv, Bulgaria 4002
b Bulgarian Academy of Sciences, Institute of Microbiology, Sofia, Bulgaria 1113
acad. G. Bontchev Str. 26,
Abstract Cell suspension cultures from H.annuus 1805 which release large amounts of extracellular polysaccharides were investigated. It was established that the main amount of polysaccharides was secreted during the logaritmic phase of the growth cycle. Three extracellular polysaccharides of pectic type were isolated from the spent culture medium of H.annuus 1805 alternately by 96 % ethanol precipitation and by freezing at -18~ The polysaccharide isolated by freezing manifested a higher immunostimulating activity. Three GelC fractions were obtained after purification on Sephadex G100. The fraction with the highest molecular weight and the highest content of D-galacturonic acid was the carrier of the biological activity.
Introduction
A considerable amount of extracellular polysaccharides is produced in the process of cultivation of certain plant suspension cultures and the spent culture medium has proved to be an accessible source for their production (1-3). The interest in investigating these extracellular polysaccharides has been quite strong over the past 10 - 15 years, motivated by their biological activity (4,5). Plants of the Asteraceae family, as well as their cell cultures, have been established to contain polysaccharides with immunostimulating activity (1-6). The object of our research was Helianthus annuus 1805 cell culture (Asteraceae), which according to the preliminary investigation produces a considerable amount of exopolysaccharides. The purpose of this research was to study the time course of growth of the cell culture, the production of extraceUular polysaccharides, and their characteristics.
680 Materials and methods
Cell culture and biosynthesis of polysaccharides. The Helianthus annuus 1805 cell culture was initiated according to a previously described method (7), using germs of Helianthus annuus as an explant. A medium of Linsmayer Skoog (8) supplemented with 0.2 m g ~ 2.4dichlorophenoxyacetic acid as a growth regulator was used for the growth of cell suspension. The cultivation was carried out in Erlenmayer flasks with 1/5 net volume on a shaker (11.6 rad/s) in the dark, at 26 - 28~ In compliance with the nature of the experiment, flasks of different size were used: 100 - 1000 cm 3, and the duration of the cultivation was 5 days for growing the inoculum and 12 days for studying the time course of growth. Experiments were carried out by varying the amount of inoculum (10, 15 and 20 % v/v) to determine the optimal quantity which ensures a steady growth. The time course of growth of the cell suspensions, inoculated with the corresponding amount of inoculum was traced by day-to-day determining the yield of dry cell biomass (7), while the time course of biosynthesis of extracellular polysaccharides was followed by their daily determination, using the carbazole method (9). Isolation ofpolysaccharide fractions: After a growth period of 8 days the cell biomass was separated by filtration and the spent culture medium was used for the isolation of polysaccharides. The following procedures were examined: Procedure 1:96 % ethanol was added to the culture medium (3:1), while continuously stirring. The precipitated polysaccharide was kept overnight at -4~ and then separated by filtration. It was washed with 70 % hydrochloric acid ethanol, then with 70 % ethanol to a neutral pH. In the end it was washed with 96 % ethanol and dried at 50~ (Sample 1). Procedure 2: The culture medium was frozen at -18~ for 24 hr's. The precipitated polysaccharide was filtered through a cloth, washed as in Procedure 1 and dried (Sample 2). The filtrate was concentrated 10 fold on a rotation vacuum- evaporator at 45~ Then 96 % ethanol was added to the concentrated filtrate and the sample was kept overnight at 4~ The precipitated polysaccharide was filtered, washed as in procedure 1, and dried (Sample 3). Gel chromatography on Sephadex G100 (2.8x50cm) of polysaccharide fraction I (Sample 2). The polysaccharide fraction was dissolved in a phosphate buffer at pH 6. After centrifugation, the supernatant was applied to the column at a flow rate of 0.8 ml/min. The elution was performed with a phosphate buffer and fractions of 10 ml each were collected. The refraction of each fraction was measured interferometrically. Fractions with coincident peaks were collected and analyzed for content of galacrturonic acid, neutral sugars and protein. Immunological tests were performed for studying the reactive of peritoneal-exudative cells, especially peritonial macrophages, which are the main effector cells involved in natural resistance (host defence system) against bacterial infection.
681 Tests:
1.Determination of number ofperitoneal-exudative cells after i.p. (intraperitoneal) application of polysaccharide fractions and during infection with Y. pseudotuberculosis in experimental animals. 2.Bioassay for "killing" ability (in vivo and in vitro against bacteria) of peritonial macrophages after treatment with polysaccharide fractions. 3.Bioassay for metabolic activity (glycolytic and acid phosphatase activity) of peritonealexudative cells after treatment with polysaccharide practions. Methods of analysis. The polyuronic acid content (PUAC) and the degree of esterification (DE) were determined according to Owens et al (10). The specific viscosity rl for different values of the concentration (C) of the analyzed polysaccharide solutions in 0.15 M NaC1 was determined by means of a capillary viscometer (Ubbelode) at 25~ The intrinsic viscosity [~1] and Huggins constant K H were calculated according to Huggins equation (11). The average molecular mass M M was determined by solving the equation, following the methods in (12). The content of neutral monosaccharides was determined after an acid hydrolysis, performed as follows: with 72 % sulphuric acid for 1 hr at 30~ and then, after dilution to 1M sulphuric acid, for another 3 hr's at 100~ The determination was performed by GC analysis of the prepared alditol acetates (13,14) The uronic acids in polysaccharide fraction I (Table 2) were determined colorimetrically with m-hydroxybiphenyl (15). The content of galacturonic acid in the fractions, obtained by gel chromatography, was determined by the carbazole method (9), and the content of neutral monosaccharides was determined by the anthron reaction (16). The protein content of the polysaccharide fractions was determined by the method of Lowry.
Results
Growth of the Helianthus annuus 1805 cell suspension and biosynthesis of extracellular polysaccharides. A particular characteristic of plant cell suspensions is the requirement for a high inoculation density in order to initiate growth. This is due to one of their special features: in order that their growth be initiated when transferred into the new medium, they need certain growth factors which are released and secreted into the medium by the cells themselves. Consequently, to ensure the growth of plant cell suspensions, a certain volume (in which plant cells have to be present at above certain densities) has to be used to import the necessary quantity of these substances (17). On the other hand, with cell suspensions synthesizing a considerable amount of polysaccharides, the excessive quantity of inoculum can lead to intensive aggregation before the maximum in the biomass synthesis is reached.
682 18
2.2
16-
2.0
~1~4 -
"~ .4
~O-
~9 .2
E 0
"~8-
4
ao.4
2
0.2
0 0
I
i
i
1
i
i
I
I
1
2
3
4
5
6
7
8
0.0 9
10
0
Time, days
Fig. 1. Growth of Helianthus annuus 1805 cell suspension inoculated with different amounts of inoculum
.
.
1
2
.
. 3
. 4
5
6
7
8
9 10
Time, days
Fig. 2. Time course of biosynthesis of polysaccharides from a cell suspension of Helianthus annuus 1805.
=
10% - i n o c u l u m
-"
15% - i n o c u l u m
=
15% - inoculum
"--
20% - i n o c u l u m
-"
20% -inoculum
=
1 0 % - inoculum
The time courses of growth of H.annnuus 1805 cell suspension for 10,15 and 20% (v/v) inoculum used, were followed (fig. 1). In all three cases the maximum amount of synthesised dry biomass was 15 - 15.5 g/dm 3, and it was attained on the 6th day, on the 8th and on the 10th day for a 20, 15 and 10 % inoculum used respectively. After the maximum was reached, the amount of biomass remained constant for 1 - 2 days, and then slight lysis was observed. The preliminary chromatographic analyses of the polysaccharides indicated that the Dgalacturonic acid is their major component. Thus the amounts the extracellular polysacchafides in the culture medium was examined by determining the quantity of D-galacturonic acid using the carbazole method. The time course of biosynthesis of extracellular polysaccharides from H.annuus 1805 cell suspension, when 10, 15 and 20 % v/v inocula were used, indicated that in all cases the maximum in the amount of extraceUular polysaccharide was achieved on the 8th day of cultivation. The use of a 10 % inoculum was inexpedient and inasmuch as the polysaccharide synthesis was considerably lower (0.95 mg/ml galacturonic acid), compared with the other two cases. The use of a 20 % (v/v) inoculum was most expedient. Maximum extracellular polysaccharides was the 8th day of cultivation. The increase of inoculum over 20% leads to problems, connected with the higher viscosity at the later stages of cultivation. Isolation and characteristics of the polysaccharides. It is known (1,3,4,6) that the polysaccharides from a culture medium can be precipitated by adding different volumes of ethanol (1:2, 1:3). Our experiments with precipitation with ethanol at a ratio of 1:3 led to the isolation of crude polysaccharides from the culture medium (Sample 1).
683 Table 1 Obtaining and characteristics of the polysaccharides from culture annuus 1805 cell suspensions Sam- Methodfor obtaining Polysaccharide Polyuronides pie yield g/L culture DE, PUAC, Nr medium, g % % Polysaccharide, isolated 1,6 56,0 61,0 by precipitation with ethanol (1:3) Fraction I 2. Polysaccharide,isolated 1,2 36,5 74,3 by freezing the culture medium at t=- 18~ for 24 hr's Fraction II 3. Polysaccharide,isolated 0,3 52;0 18,52 by precipitation of concentrated filtrate with ethanol (1:3) (after the removal of fraction I)
medium of the Helianthus Molecular Intrinsic Huggins mass viscosity constant dlxs- 1 KH 100 000
6,99
0,6
150 000
912,1
0,7
17 000
0.67
0.58
A new possibility for isolation of the exopolysaccharides in deep freezing of the culture medium (-18~ was arrived at in the course of our research (Sample 2, Polysaccharide fraction I). A second polysaccharide fraction (Sample 3) was isolated from the filtrates by concentration and following precipitation with ethanol (1:3). The yield and characteristics of the obtained polysaccharides (Samples 2 and 3) are given in Table 1. It is evident that the sum of the yields for Samples 2 and 3 is almost equal to the yield for Sample 1. The polyuronic content data are also well balanced. This fact indicates that the suggested method is suited for fractional isolation of the polysaccharides from the spent culture medium of H.annuus 1805 cell suspension. As can be seen from Table 1, the main part of the exopolysaccharide was in fraction I. The data on the molecular mass and on the intrinsic viscosity confirmed the expectation that the fraction, precipitated under freezing, had a higher molecular mass (Table 1). The values of Huggins constant were close and indicated that there was an increased tendency towards aggregation for the dissolved macromolecules. The results of the biological investigation showed that polysaccharide fraction I had a distinct immunostimulating activity. Characteristics of fraction 1. The carbohydrate content of Fraction I was 84.4 %, in which the main component is D-galacturonic acid (71%). Consequently, the polysaccharide is of a pectic type. The neutral sugars accounted for 13.4 % and according to their qualitative composition (Table 2) they correspond to the composition of pectin, isolated from sunflower heads (18). It is worth noting the high content of L-arabinose and D-galactose, compared with the other mon0saccharides. The protein content was 7.8 %.
684 Table 2 Composition of polysaccharide fraction I, isolated from the culture medium of H. annuus 1805
Nr 1
Compounds D-Galacturonic acid Neutral sugars: Rhamnose Fucose Arabinose
% n/w 71.005 13.4 0.5137 0 1177 6 258 0.3685 1 023 3.3231 1 7941 7.87 11
Xylose Mannose Galactose Glucose Protein by Lowry Ash
3 4
mol % 81.28 0.72 0.17 9.59 0.56 1.28
4.16 2.24
The investigated polysaccharide was further applied to gel chromatography. Three fractions were elued from a Sephadex G100 column (Table 3). The uronic acid content and the protein content were different for the three fractions. The uronic content was especially high in fraction I (4.5 times higher than both the level of neutral sugars and the level of protein). Fraction II had a higher protein content, and the ratio of the D-galacturonic acid to the neutral sugars was (1.1:1). In fraction III, whose molecular weight was the lowest, the content of neutral sugars was 4 times higher than that of D-galacturonic acid. Table 3 Composition of the fractions, obtained by gel chromatography of Fraction I on Sephadex G100 Eluate composition Composition of the fractions
Fractions
1
2 3
Total D-galactuvolume/eluate, ronicacid, ml mg
100 110 80
12,5 1,2 0,3
neutral sugars, mg D-glucose
2,8 1,1 1,1
protein, D-galactumg ronic acid %
2,8 0,7 -
69,1 40,0 21,4
neutral sugars %
protein content, %
15,5 36,7 78,6
15,5 23,3 -
Immunological tests indicated that fraction 1, obtaned by gel chromatography had an immunostimulating activity. It induced migration of peritoneal-exudative cells, respectively peritoneal macrophages into the peritoneal cavity of experimental animals. These cells are with high bactericidic metabolitic activity. The selective stimulation of these cells is of importance because they are the most active effector cells in host defense mechanisms against bacterial and viral infections.
685 The results suggest that polysaccharide fraction I may be thought as unspecific modulators of immune responsiveness.
Discussion
Immunoactive polysaccharides, containing galacturonic acid have been isolated from different plants and cell suspension culture liquids (2,4,19-22). Generalising the research into polysaccharides isolated from chinese herbs Yamada (22)concludes that most biological activities are observed in the case of pectic polysaccharides. Besides, results prove that the complementarily active power of these pectins is represented mainly through the branched regions and the activity is regulated by the polygalacturonic regions. Our studies showed that the exopolysaccharides from the H.annuus 1805 (Asteraceae) cell suspension culture were also of pectic type because the content of D-galacturonic acid varied from 22 to 69-70 %. Part of the crude polysaccharide (Sample 2, Table 1) was waterinsoluble, and the soluble part allowed the GelC isolation of three heteropolysaccharides. Polysaccharide fraction 1 had the highest molecular mass and the highest galacturonic acid content (69.1%). This fraction contained 15.5 % neutral sugars. It is worth noting that this polysaccharide of pectic type was different from the pectins isolated from sunflower plants (18) in that it contained a considerable amount of proteins. Consequently, this fraction is to be classified as a glycoprotein; it also has the highest biological activity compared to the other two fractions. Polysaccharide fraction 3 also manifested biological activity. It contained the highest percentage of neutral sugars (78.6%): mainly L-arabinose and D-galactose. Most probably, this is a polysaccharide of pectinoarabinogalactan type.
ACKNOWLEDGEMENT
The autors thank the National Research Foundation of Bulgaria for the financial support of this work. The valuable contribution of Prof. Dr. A. Voragen and his staff (Agricultural University, Wageningen, The Netherlands) on the Chromatographic analysis is gratefully acknowledged. Abbreviations: PUAC, polyuronic acid content; DE, concentration; I"1,specific viscosity; MM, molecular mass
degree of esterification; C,
686 References
1.Wagner H., Stuppner H., Schafer W and Zenk M. Phytochemistry 27: (1988) 119 2.Puhlmann J., Wagner H. Planta medica 55:(1989) 99 3.Uchiyama T., Numata M., Tereda S., Hosino T. Plant cell Tissue and Organ Culture 32: (1993) 153 4.Skvastava R., Kulshreshtha D. Phytochemistry 28: (1989) 2877 5.Labadie R. Immunomodulatory compounds Chapter in Bioactive Natural Products CRC Press London: 280 (1993) 6.Proksch A., Wanger H. Phytochemistry 26:(1987) 1989 7.Dixon K. Isolation of callus and suspension cultures in Plant cell culture a practical approach IRL Press (1985) 8.Linsmayer E., Skoog F. Physiol Plant. 18: (1965) 100 9.Bitter T., Murr H. Anal. Biochem. 4: (1962) 330 10.Owens H., Cready R., Shepheral A., Shultz T., Pippen E., Swenson N., Miers J., Erlander F., Maclay W AIC Report 340, Western Regional Research Laboratory, Albany, CA (1952) 11 .Moravettz H. Interscience Publishers, New York Macromolecules in solution: 254 (1967) 12.Anger H., Berth G. Carbohyrate Polymers 6:(1986) 193 13.Albersheim P., Nevins D., English P., Karr A. Carbohydr. 5:(1967) 340 14.Brakeney A., Harris P., Henry R., Stone B. Carbohydr. Res. 113: (1983) 291 15.Thiboult J. Lebensm. Wiss Technol. 12: (1979) 247 16.Briskorn C. Lebensm. Unters- Forsch. 108: (1958) 170 17.Kratchanov Hr. Helia 5:(1982) 49 18.Miyamoto A., Chang K J. Food Science 57: (1992) 1439 19.Kiyohara H., Cyong J., Yamada H Carbohydr. Res. 193: (1989) 201 20.Yamada H., Ra S., Kiyahara H., Cyahg C., Yang C., Otsuka Y. Phytochemistry 27: (1988) 3163 21.Yamada H., Ra S., Kiyahara H., Cyahg C., Otsuka Y. Carbohydr. Res. 189: (1988)209 22.Yamada H., Carbohydr. Polymers 25: (1994)269
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
687
Pectins and pectinases in stem rust-infected wheat M. Mierau a, B. Grael3ner a, A. J. Mort b and B. M. Moerschbacher c Institut for Biologie III, RWTH Aachen, Worringer Weg 1, D-52056 Aachen, Germany
a
b Department of Biochemistry and Molecular Biology, OSU Stillwater, 246B Noble Research Center, OK, USA c current address" Institut for Biochemie und Biotechnologie der Pflanzen, WWU MOnster, Hindenburgplatz 55, D-48143 MOnster, Germany
Abstract The dimer and trimer of galacturonic acid are able to suppress the hypersensitive defence reaction of wheat against the stem rust fungus. Our results suggest that the fungus produces an endopolygalacturonase during host tissue colonization and that thus, depending on the methylation patterns of the cell wall pectins which appear to be different in susceptible and resistant wheat plants, the suppressor active oligogalacturonides may or may not be formed. The rate of suppressor production during host cell wall penetration and the amount of suppressor accumulating around the growing fungal haustorium might then decide on compatibility or incompatibility of the host/pathogen-interaction.
1. I N T R O D U C T I O N The interaction between wheat plants (Triticum aestivum) and a biotrophic pathogen of wheat, the wheat stem rust fungus (Puccinia graminis f. sp. tritici), can range from full susceptibility to complete resistance. In highly resistant host plants, fungal growth is arrested by the rapid and active death of all host cells penetrated by a fungal haustorium, thus depriving the obligately biotropic parasite of its nutritional basis. The suicide of a few host cells only efficiently protects the plant from fungal colonization and the development of disease symptoms. The triggering of the hypersensitive response of penetrated host cells appears to involve a range of different molecular interactions. The defence reaction can be elicited by a range of fungal surface constituents, such as a glycoproteogalactan isolated from rust germ tube cell walls [Moerschbacher et al. 1986] or chitin-oligomers and chitosanpolymers [Vander & Moerschbacher, 1993]. Interestingly, these different elicitors
688 appear to induce differem aspects of the resistance reaction, and only their timely interplay may effectively trigger cell death. We assume that the host cell takes a well balanced decision based o n the recognition of several indicators of the presence of a pathogen before undergoing hypersensitive suicide. All of the isolated elicitors investigated so far were able to induce resistance reactions in both resistant and susceptible host plants. It has been speculated that the specific difference between resistance and susceptibility may be caused by the action of suppressor molecules in the susceptible plants only [Bushnell & Rowell, 1981]. We have shown that oligomers of galacturonic acid can suppress elicitor-induced resistance reactions [Moerschbacher et al., 1990]. The most active molecules turned out to be the dimer and trimer of galacturonic acid, known to be the main degradation products of pectins hydrolyzed by endopolygalacturonases [Collmer & Keen, 1986]. Our hypothesis is that the wheat stem rust fungus, like many other fungi [Cooper, 1983] including rusts [Deising et al., 1995], produces such an endopolygalacturonase to degrade the wheat cell wall locally when forming haustoria, thus producing these oligosaccharins which we call endogenous suppressors [Moerschbacher et al., 1990]. Though the pectins of grasses, in comrast to those of dicots, account for only around 5 % of their cell wall dry weight [Carpita & Gibeaut, 1993], a role of pectic fragments acting as oligosaccharins can still easily be imagined since any intercellularly growing fungus, upon penetration of a host cell wall, will invariably first meet the pectin-rich middle lamella which it will have to degrade. In this paper we present some evidence for the above proposed role of pectic fragments and of an endopolygalacturonase in the rust-infected wheat leaves. Firstly, we have chemically analyzed and compared the methylation patterns of the pectins isolated from cell walls of resistant and susceptible wheat plants. We have secondly chosen a molecular genetic approach in the search for an endopolygalacturonase of the stem rust fungus. In addition, we will summarize the effects of some pectic samples on the triggering of the hypersensitive response of wheat to the stem rust fungus or elicitors isolated from that pathogen.
2. MATERIALS AND METHODS 2.1 Biotests To test for suppressor activity, solutions of pectic substances in a concentration of usually 1 mg/ml were injected together with the glycoproteogalactan-elicitor isolated from germ tubes of the rust fungus (concentration 40 ~tg/ml glucose-equivalents) into the intercellular spaces of primary leaves of 7-day-old wheat plants, using a hypodermic syringe [Moerschbacher et al., 1989]. Control leaves were injected either with the
689 elicitor alone (positive control) or with distilled water (negative control). Further controls involved the injection of pectic fractions alone, to exclude possible elicitor activities. The leaves were harvested 24 h after injection, immediately frozen in liquid nitrogen. For spectrophotometric determinations of phenylalanine ammonium lyase (PAL) and peroxidases (POD), crude enzyme extracts were prepared [Moerschbacher et al., 1988], and the protein concentration of the extracts was estimated with the Biuret reagem [Gornall et al., 1949]. In additional tests, the suppressor activities of pectins in the intact host/pathogeninteraction were investigated by injecting genetically resistant plants with pectic substances prior to inoculation with the rust fungus. Infected leaves were harvested, cleared, and stained with Calcofluor one week after inoculation, and fungal growth was assessed under the UV-epifluorescence microscope.
2.2 Pectic samples Oligomers of galacturonic acid were produced chemically by autolyzing polygalacturonic acid in an autoclave for 20 min at 121~ followed by separation and fractionation using anion exchange chromatography [Robertsen, 1986]. Pectins were extracted from isolated cell walls of 5-week-old wheat plants using different methods. Enzymic digestions of the cell walls involved pectinases such as a commercial pectolayse or recombinant endopolygalacturonase [Maness & Mort, 1989]. Chemical extractions involved the chelating agent imidazole [Mort et al., 1991] or solvolysis with anhydrous HF at 0 ~ in a closed teflon line [Mort et al., 1989] followed by imidazole extraction.
2.3 Anion exchange chromatography Oligalacturonides were separated on a Dionex Bio LC with a CarboPac PA 1 column and pulsed amp~rometric detection using a linear Na-acetate-gradient in 0.1 M NaOH.
2.4 Analysis of methylation patterns [Mort et al., 1993] Pectins were isolated from cell walls of susceptible (Prelude-srx) and resistant nearisogenic wheat plants (Prelude-Sr5) by solvolysis in anhydrous HF at -23 ~ and subsequent imidazole extraction. The methyl-esterified galacturonic acid residues were quantitatively reduced to galactose by treatment with sodium borohydride [Maness et al., 1990]. The galactosyl bonds were then selectively cleaved by HF-solvolysis ( 1 % distilled water) at -15 ~ The oligomers formed were labelled by coupling of 2aminopyridine groups to their reducing ends [Maness & Mort, 1989], separated by anion exchange chromatography using a potassium oxalate gradient, and quantitated by fluorescence detection [Maness et al., 1991].
690 2.5 DNA-isolation and PCR DNA was isolated [Schillberg, 1994] from axenically grown mycelium of the wheat stem rust fungus [Fasters et al., 1993] and used as a template in PCR with degenerate primers, designed from highly conserved regions of known endopolygalacturonase genes from other fungi. PCR conditions were 55 ~ annealing temperature, 2.5 mM MgC12-concentration, and 40 cycles. The primers were kindly provided by H. Kusserow and W. Sch/ifer (AMP III, Hamburg, Germany).
3. R E S U L T S A N D D I S C U S S I O N Solutions of different pectic substances were injected into healthy wheat plants, with or without the glycoproteogalactan elicitor, and the activities of the enzymes PAL and POD were determined. These enzymes are involved in the hypersensitive reaction of wheat against the rust fungus, and increased activities can be expected after elicitation, whereas suppressor active substances will cause a reduction of the elicitor-induced enzyme activities. Oligomers of galacturonic acid with degrees of polymerisation ranging from 1-6 were produced chemically and tested for elicitor or suppressor activity. Injected alone, none of them were active as elicitors of PAL or POD activities, and only the dimer and trimer were active as a suppressor when co-injected with the elicitor. Using enzymic and chemical methods for the isolation of pectins from wheat cell walls, we produced fractions with different contents of galacturonic acid. We found that the higher the galA content in these fractions, the higher was their suppressor activity. This points to a possible role of the cell wall pectins, particularly the homogalacturonan, in the suppression of hypersensitive resistance in the wheat/stem rust-system. The suppressor activities of pectins isolated from wheat cell walls by HF-solvolysis and subsequent imidazole extraction increased from about 35 % to 74 % reduction of elicitor-induced PAL and POD activities, when the pectins were pretreated with a bacterial endopolygalacturonase. The production of small oligogalacturonides during this digestion was monitored by means of HPLC analysis. The suppressor active trimer of galacturonic acid was formed predominantly. This experiment further supports the influence of the length of the galacturonan oligomers on their suppressor activity towards the defence reaction. In additional tests, the suppressor activities of pectins in the intact host/pathogeninteraction were demonstrated. Injection of suppressors rendered genetically highly resistant plants more susceptible, i. e., we observed increased growth of the fungus, and in some cases sporulation occured. Obviously, oligogalacturonides do not only have an
691 effect on isolated elicitors in an in vitro-bioassay, but they can in fact play an important role in vivo in the intact wheat/rust-system. Analyzing the methylation patterns of wheat pectins, we found roughly equal degrees of methylation in resistant and susceptible plants (15 % and 19 %, resp.), but there were clearly different distributions of the methyl-esterified galacturonic acid residues along the linear pectin molecules. In the pectins of resistant plants, we found a presumed random distribution of methyl-esterifications, whereas in susceptible plants, these methylations appeared to be grouped blockwise. Consequently, an endopolygalacturonase secreted by the rust fungus would meet longer sections of nonmethyl-esterified galacturonic acid residues during the cell wall penetration in a susceptible plant than in a resistant plant. Accordingly, the enzyme might produce larger amounts of the suppressor-active molecules in susceptible plants, which in turn might be responsible for their susceptibility. Thus, the difference in the methylation patterns of cell wall pectins in susceptible and resistant wheat plants may result in a compatible or incompatible host/pathogen-interaction, respectively. It might be speculated that this difference may be involved in the expression of race/cultivarspecific resistance in the wheat/stem rust-interaction. In an attempt to investigate whether the stem rust fungus is in fact able to produce an endopolygalacturonase, we analyzed its DNA by PCR with degenerate primers designed from highly conserved regions of known endopolygalacturonase genes of other fungi. A ca. 650 bp long fragment was amplified. The size of this fragment tends to indicate that the fragment might in fact be part of an endopolygalacturonase gene, as a 630 bp fragment was amplified with these primers from PeniciUium olsonii DNA [Kusserow & Sch/ifer, personal communication]. Cloning and sequencing of this fragment are currently in progress. The results obtained so far suggest that the wheat stem rust fungus does possess a gene for an endopolygalacturonase. If the enzyme is synthesized during the infection of wheat plants, the production of suppressor-active oligogalacturonides will depend on the methyl-esterification of the host pectins, and will thus differ in resistant and susceptible plants. The amount of suppressor produced might then decide on compatibility or incompatibility of the host/pathogen-interaction.
692
5. Acknowledgements We like to thank Dr. H. Kusserow and Dr. W. Sch/ifer for providing the PCR-primers and Dr. M. Zimmermann for help with the molecular genetic experiments. Financial support from the German Research Council DFG, from the German Academic Exchange Service DAAD, and from the Land Nordrhein-Westfalen is gratefully acknowledged.
6. References Bushnell WR & Rowell JB (1981). Phytopathology 71:1012-1014 Carpita NC & Gibeaut DM (1993). Plant J 3" 1-30 Collmer A & Keen NT (1986). Annu Rev Phytopathol 24:383-409 Cooper RM (1983). in" Biochemical Plant Pathology, Callow JA ed; Wiley, New York: 101-135 Deising H, Frittrang AK, Kunz S, Mendgen K (1995) Microbiology 141"561-571 Fasters MK, Daniels U, Moerschbacher BM (1993). Physiol Molec Plant Pathol 42: 259-265 Gornall AG, Bardawill CJ, David MM (1949). J Biol Chem 177:751-766 Maness NO, Miranda ET, Mort AJ (1991). J Chromatogr 587" 177-183 Manees NO & Mort AJ (1989). Anal Biochem 178:248-254 Maness NO, Ryan JD, Mort AJ (1990). Anal Biochem 185:346-352 Moerschbacher BM, Flott BE, Noll U, Reisener HJ (1989). Plant Physiol Biochem 27: 305-314 Moerschbacher BM, Heck B, Kogel KH, Noll U, Reisener HJ (1986). Z Naturforsch 41c: 830-838 Moerschbacher BM, Noll UM, Flott BE, Reisener HJ (1988). Physiol Molec Plant Pathol 33:33-46 Moerschbacher BM, Schrenk F, Grael~ner B, Noll U, Reisener HJ (1990). J Plant Physiol 136:761-764 Mort AJ, Komalavilas P, Rorrer GL, Lamport DTA (1989). in: Modern Methods of Plant Analysis Vol 10 Plant fibers, Linskens HF, Jackson JF eds; Springer, Berlin Heidelberg New York: 37-69 Mort AJ, Moerschbacher BM, Pierce ML, Maness NO (1991). Carbohydr Res 215: 219-227 Mort AJ, Qui F, Maness NO (1993). Carbohydr Res 247:21-35 Robertsen, B (1986). Physiol Molec Plant Pathol 28:137-148 Schillberg S (1994). Ph-D-thesis, RWTH Aachen, Germany Vander P & Moerschbacher BM (1993). in: Chitin Enzymology, Muzzarelli RAA ed; EUCHIS, Ancona: 437-440
J. Visserand A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996ElsevierScienceB.V.All rights reserved.
693
Bioactive fragments from pea pectin O. Zabotina a, N. Ibragimova a, D. Ayupova a, O. Gurjanov a, V. Lozovaya a, G. Beldman b, A. Voragen b aInstitute of Biology, P.O.Box 30, 420503 Kazan, Russia bWageningen Agricultural University, The Netherlands.
Abstract Several bioactive fractions from pea stem cell wall pectin have been separated. The fractions contained mainly galacturonides inhibited the process of root formation in thin cell-layer explants, while the fractions contained only neutral sugars stimulated this process to different extend. Analysis of the last fractions showed that they mainly consisted of galactan and arabinogalactan fragments. INTRODUCTION The plant cell wall is a source of polysaccharide fragments named oligosaccharins which show several kinds of biological activity [1]. These activities have been demonstrated by some pectic and hemicellulose oligofragments produced by various means. Pectins are the major polysaccharides of the primary cell wall (especially in dicots) and of the middle lamella. The main structural components of the primary cell wall pectin of higher plants are homogalacturonan, composed of 1,4-1inked D-galactosyluronic acid residues and highly branched rhamnogalacturonans I and II, composed of DGalactose, L-Arabinose, L-Rhamnose, L-Fucose, Xylose, Apios, KDO, etc. Pectic oligosaccharides were the first oligosaccharins of higher plants to be detected [2]. Partial digestion of homogalacturonan generates oligogalacturonides that exhibit different regulatory effects in plants such as the elicitation of defense responses [2], the regulation of growth and development [3], the induction of rapid responses at the cell surface [4]. These oligomers can act as a trigger for hypersensitive responses [5] and as wound signals [6], can inhibit protein synthesis [7], induce lignification [8] and ethylene synthesis [9]. To date only homooligogalacturonides from plant pectin have been shown to exhibit the regulatory effects. Meanwhile pectin polysaccharides consist of neutral highly branched blocks, so it was of interest to search for possible biological activity of oligomers released from such structures.
694 This paper reports on the separation of some fragments obtained by acid hydrolyses of pectin from pea shoot cell walls, which had effect on thin cell-layer explant rhizogenesis. M A T E R I A L AND M E T H O D S
Plant material. Oligosaccharide fractions were isolated from 10 - 12 dayold pea shoots (Pisum sativum L.), cv. Bulat, grown at 25 C in light (12-h light period, 20 W/m 2 ). Plant material fractionation. The detailed steps of isolation and separation of oligosaccharide fractions were described earlier [10]. Pectin was separated by boiling the cell walls in 0.5 M ammonium-oxalate buffer, pH 5.2 at 100 o C for lh. The dialyzed solution of pectin was hydrolyzed with 0.15M HC1 for 3h at 100 C. Neutralized and desalted hydrolysate was loaded to the column (lx90cm) filled with biogel TSK HW-40 (Toyo Soda, Japan) equilibrated and eluted with 50mM sodium acetate (pH 5.2) at a rate of 0.3ml]min. In all fractions (lml) the sugars were determined by o-toluidine method (Resnikov et al., 1982) and fraction IP was collected as shown on Figure 1.
200
1P
-
-
I
I
100
0
0
10
20
.30
40
50
60
Fraction number
Figure 1. Size-exclusion chromatography on TSK HW-40 of pectin hydrolysate from pea shoot cell wall. The fractions (1 ml each) were pooled as indicated. o - Uronic acids, A- pentoses, x - hexoses.
695 This fraction was desalted and separated on a column (1X8cm) with DEAEcellulose (Serva, Germany) in 10raM phosphate buffer, pH 7.0. The separation was conducted using NaC1 gradient (0- 0.5M) at a rate of elution 0.3 ml/min, and 2 ml fractions were collected. In each fraction, the content of sugars was asseied by o-toluidine method. The fraction IPN was collected (see Figure 2), desalted and than separated on the column (2.5xl00cm) filled with Bio-Gel P-4 (Bio-Rad, USA).
300 1
1PN ,
1PK1 I
1PK2 I
1PK3 I
I
1PK4
~-0.6
I
0.5 "~ 200
0.4
o =
z
03
~} 1 0 0
F, i,
0.2
~
-0.1 O
i~i . . . . . .
0
~T--~;--T-~;TT i
10
u ; I;
20
"u" ~,-7 i
....
I i I i" i ' } ~ - i
30 Fraction number
i I i ~ i "31~. . . .
40
1 ~. . . . . . .
50
~ill~qlWiliU 0 . 0
60
Figure 2. Anion-exchange chromatography on DEAE Cellulose of fraction IP obtained after size-exclusion separation. Fractions (2 ml each) were pooled as indicated, o - Uronic acids, A- pentoses, x - hexoses.
Elution was conducted at 60 C with distilled water at a rate of 0.3ml/min and the fractions (2.5ml) were combined as shown on Figure 3. Analysis of pectic fractions. Qualitative analyses of pectic fractions obtained after separation on Bio-Gel P-4 was performed by HPAEC using a BioLC GPM-II q u a t e r n a r y gradient module equipped with a Dionex CarboPac PAl00 column (250x4mm, 20 C) (Dionex, Sunnyvale, CA). The eluate (lml/min) was monitored using a Dionex pulsed electrochemical detection detector in the PAD mode. The fractions studied and standard digests of arabinogalactan were analyzed by application of the following gradient: 0 to 30 min, linear gradient of 0 to 0.2M sodium acetate in 0.1M NaOH; 30 to 40 min, linear gradient of 0.2 to 0.5M NaOAc in 0.1M NaOH; 40 to 45 min, linear gradient of 0.5 to 1M NaOAc in 0.1M NaOH. After each analyses the column was rinsed for 5 rain with 1M NaOAc in 0.1M NaOH and equilibrated in 0.1M NaOH for 15min.
696
Sugar composition. Desalted fractions (IPN1-IPN14) were hydrolyzed using 2N TFA for 1.5h at 121 ~ C. The released neutral sugars were converted to their alditol acetates and analysed by GC as described [12]. 200
I !
II1~i 2
160~o
,.~
120-
.o ~
;/
3
4
I I
5
I
6
I
7
I
8
9 10 11 12
I II
13
I I
I
I
14
1
i/
vo I / 80-
0
o
40-
i
r~
,,
I
29
glucose
\ I
58
I
87
I
116
145
Fraction number
Figure 3. Size-exclusion chromatography on Biogel P4 of fraction IPN obtained after anion-exchange chromatography. Fractions (2.5 ml each) were pooled as i n d i c a t e d . - - neutral sugars, ---- uronic acid. Fractions 1, 2, 3 ... were named later as fractions IPN 1, IPN 2, IPN 3 ... r~spectively.
Enzymic hydrolysis. 2rag of each oligosaccharide fraction were incubated at 35 o C for 24h in lml 5raM sodium acetate buffer (pH 4.0) containing 0.01% (w/v) Na azide and 0.03 U/ml of endo-[3-galactanase type F (EC 3.2.1.89) [13] or a-Larabinofuranosidase type B (EC 3.2.1.55 ) [14]. Test for biological activity. The thin cell-layer explants approximately 5 mm long and 2-5 mm wide were cut from hypocotyls of buckwheat seedlings grown aseptically and cultured individually in 2 ml liquid RX medium in the absence of phytohormones as described earlier [15]. Oligosaccharide fractions were added to the medium at a concentration of 0.1 ~g/ml. Throughout the cultivation, the number of explants with roots was counted. After 20 days of cultivation, the number and weight of roots in each explant were asseied.
697
RESULTS AND DISCUSSION After multistep fractionation of cell wall pectin hydrolysate several bioactive fractions were obtained. They exhibited various influence on process of root development in buckwheat thin cell-layer (BTCL) explants (Table 1).
Table 1. Effect of oligosaccharide fractions on root formation in BTCL explants after 20 day culture period. Root fresh weight Number of Day when 50% of /explant, (mg) roots/explant explant form roots Control + lpg/ml IPN + l~g/ml IPA1 +l~g/ml IPA2 +l~g/ml IPA3 +lp~/ml IPA4
25_+6 45• 20• 11• 22• 18•
7+1 14• 5• 2• 4• 3•
9 6 15 19 16 18
Control +0. lpg/ml IPN1 +0. lpg/ml IPN2 +0. l~g/ml IPN3 +0. l~g/ml IPN4 +0.1pg/ml IPN5 +0.1~g/ml IPN6 +0. l~g/ml IPN7 +0. lpg/ml IPN8 +0. lpg/ml IPN9 +0. lpg/ml IPN10 +0. l~g/ml IPN11 +0. l~g/ml IPN12 +0. l~g/ml IPN13 +0.1p~/ml IPN14
20• 19• 17+3 21+3 35• 38+8 75+33 70• 61+14 61+15 58+9 66+20 59+25 59+10 25+10
5+1 6+1 4+1 5+1 7+2 7+1 16+3 15+3 14• 14• 15+2 15+4 13+3 10• 8•
9 10 11 11 7 7 6 6 6 6 6 6 7 7 10
Among them fractions IPA1, IPA2, IPA3, IPA4 contained mostly galacturonic acid (Table 2) and apparently consisted of oligogalacturonides. These fractions inhibited root development in BTCL explants, the same effect of such fragments has been observed in tobacco TCL explants [16]. Addition of other fractions IPN4-IPN14 to the culture medium resulted in activation of root development in BTCL explants to different extent (Table 1).
698 More r a p i d induction of roots on t h e e x p l a n t s in c o m p a r i s o n w i t h t h e control w a s observed. Oligosaccharides of t h e s e fractions i n d u c e d root f o r m a t i o n on t h e e x p l a n t s in n u m b e r s g r e a t e r t h a n in control v a r i a n t s , a n d the n u m b e r of roots f o r m e d on each e x p l a n t of such v a r i a n t s were h i g h e r t h a n on control. At the s a m e time t h e s e fractions p r o m o t e d g r o w t h of roots, w h i c h r e s u l t e d in h i g h e r for different e x t e n t dry w e i g h of t h e roots c o m p a r i n g w i t h the control. T h e y s t i m u l a t e d root i n i t i a t i o n as well (decreasing the time before t h e 50% of e x p l a n t s will form the roots). All t h e s e fractions c o n t a i n e d only n e u t r a l r e s i d u e s (Table 2).
Table 2. M o n o s a c c h a r i d e composition of bioactive fractions. Fucose R h a m n A r a b i n Galactose (mol%) ose ose (mol%) (mol%) (mol%) IPN IPA1 IPA2 IPA3 IPA4 IPN1 IPN2 IPN3 IPN4 IPN5 IPN6 IPN7 IPN8 IPN9 IPN10 IPNll IPN12 IPN13 IPN14
0.5 1.1 0.6 1.5 -
-
2.0 5.0 2.0 2.4 1.8 13.2 7.8 5.9 4.1 4.4 4.7 9.6 -
7.0 4.5 2.5 2.0 1.6 0.9 0.4 1.5 2.6 1.8 10.9 3.3 3.1 2.5 1.2 3.4 0.6 -
60.5 1.5 2.2 1.5 2.0 47.1 19.3 61.8 85.8 91.5 76.3 92.6 93.4 96.2 85.2 92.7 89.9 84.7 94.7
Glucose (mol%)
Xylose (mol%)
Uronic acids (mol%)
4.5 4.0 0.8 1.5 1.4 6.9 4.0 9.2 5.1 1.5 4.1 2.5 2.0 0.8 1.8 2.2 4.1 6.8 5.3
1.0 1.0 2.3 3.2 6.3 2.4 0.7 4.0 1.6 1.4 0.5 2.2 1.7 5.4 8.5 -
24.0 84.0 92.5 92.6 93.2 28.5 65.3 13.8
A n o t h e r fractions I P N I - I P N 3 did not exhibit a n y s t r o n g activity in t h e b i o a s s a y t e s t e d (Table 1). T h e s e fractions consisted both n e u t r a l a n d acidic r e s i d u e s (Table 2), so it m i g h t be t h a t the p r e s e n c e of oligomers consisted g a l a c t u r o n i c acid w i t h opposite effect on root f o r m a t i o n w a s the r e a s o n of inactivity of fractions I P N I - I P N 3 . F u r t h e r s e p a r a t i o n a n d i n v e s t i g a t i o n of t h e s e fractions will be done in the future. Thus, the hydrolysis of pectic p o l y s a c c h a r i d e s r e v e a l e d t h e f r a g m e n t s w i t h different effect on T C L e x p l a n t root formation. The n e u t r a l oligomers s t i m u l a t e d
699 this process were the subject of the next investigations. The fractions IPN4IPN14 were analyzed by HPAEC (Figure 4) and retention times of the products were compared with those of standard digests obtained after degradation of soybean arabinogalactan, apple xyloglucan and rhamnogalacturonan (data not shown). It seemed that elution patterns of all fractions represented the pools of the similar oligomers with decreasing molecular sizes. Monosaccharide analysis showed (Table 2), that all these fractions contained mainly galactose and some arabinose.
!
,
1 3
"
-
]:PN
12
]:PN
11
]:PI~T 1 0
i, :
L I
IPN~
i
I
0,00
,
5,00
,
10,00
,
15,00 Retention
,
,
20,00 time
25,00
,
30,00
35,00
(mizz)
Figure 4. Elution profile on HPAEC of pea shoot pectin fractions obtained after Biogel P4 separation.
The main peaks on their HPAEC elution profiles were corresponded to the peaks of unsubstituted galactan oligomers and some peaks to galactan oligomers
700 containing side chains of arabinose (data not shown). During the acid hydrolysis used, most of the arabinoses were cleaved from arabinogalactan fragments, so the result mixture could contain mostly different galactan oligomers and some arabinogalactan oligomers with the lower amount of side arabinoses. Incubation of fractions IPN4-IPN14 with galactanase resulted in disappearance of the main peaks corresponding to the peaks of the arabinogalactan digest. Meanwhile some new peaks with lower molecular sizes appeared and peaks of mono- and dimers increased (Figure 5: a,b). The elution profiles of HPAEC of the most active fraction (PN6) is shown on Figure 5 as the example. Very similar results have been obtained for all fractions analysed after their incubation with hydrolases. Some of the new products obtained after galactanase treatment disappeared after subsequent digestion of fractions by arabinofuranosidase (Figure 5: c).
rJl
o
0,00
5,00
10,00
15,00
20,00
25,00
30,00
35,00
Retention time (min) Fig.5 Elution profile on HPAEC of fraction IPN 6 before (a) and after treatment with galactonase (b) and arabinofuranosidase (c).
Results reported testify the possibility of galactan or arabinogalactan oligomers to be that active fragments which stimulate root formation on BTCL explants. The detailed structural analysis of the individual active oligosaccharide is currently in progress, meanwhile the data presented revealed new type of bioactive oligomers with new regulatory effect in plant tissues. Acknowledgment: This research was supported in part by NWO (The Netherlands, project N299.780 ) and ISF (USA, grant RH8000). We wish to thank Dr.Henk Schols for helpful discussion of HPAEC results.
701 REFERENCES
1.
2. 3. 4. 5. 6. 7. ~
9. 10.
11. 12. 13. 14. 15. 16.
P. Albersheim, A.G. Darvill, M. McNeil, B.S. Valent, J.K. Sharp, E.A. Nothnagel, K.R. Davis, N. Yamazaki. In "Structure and Function of Plant Genomes", O. Ciferri and L. Dure (eds), Plenum, New York 1983, 293312. M.G.Hahn, A.G. Darvill, P. Albersheim, Plant Physiol, 68 (1981) 11611169. S. Eberhard, N. Doubrava, V. Marfa, D. Mohnen, A. Southwick, A.G. Darvill, P. Albersheim, Plant Cell, 1 (1989) 747-755. C.A. Ryan, E.E. Farmer, Annu. Rev.Plant Physiol.Plant Mol.Biol., 42 (1991) 651-674. S.Aldington, S.C.Fry Oligosaccharins. Adv.Bot.Res., 19 (1993), 1-101 D. Bowles, Current Biology, 1 (1991) 165-167. N. Yamazaki, S.C. Fry, A.G. Darvill, P. Albersheim, Plant Physiol., 72 (1983) 864-869. R.J. Bruce, C.A. West, Plant Physiol., 91 (1989) 889-897. D. Roby, A. Toppan, M.T. Esquerre-Tugaye, Plant Physiol., 81 (1985) 228233. O.A. Zabotina, O.P. Gurjyanov, R.G. Malikhov, D.A. Ayupova, G. Beldman, A.J.G. Voragen, V.V. Lozovaya, Russian J. of Plant Physiol., 42 (1995) 366371. V.M. Reznikov, T.G. Matusevich, T.S. Selivestrova, Khimiya Drevesiny, 7 (1982) 109 H.N. Englyst, J.H. Cummings, Analyst, 109 (1984) 937. J.W. Van de Vis, M.J.F. Searle-Van Leeuwen, H.A.Siliha, F.J.M. Kormelink, A.G.J. Voragen, Carbohydr. Polym., 16 (1991) 167. F.M. Rombouts, A.G.J. Voragen, M.J.F. Searle-Van Leeuwen, C.C.J.M. Geraeds, H.A. Schols, W. Pilnik, Carbohydr. Polym., 9 (1988) 25. V.V. Lozovaya, O . A . Zabotina, N.I. Rumyantseva, R.G. Malihov, M.V. Zihareva, Plant Cell Rep., 12 (1993), 530-533. D. Bellincampi D, G. Salvi, G. De Lorenzo, F. Cervone, V. Marfa, S. Eberhard, A.G. Darvill, P. Albersheim, Plant J., 4 (1993), 207.
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IDENTIFICATION, MODE OF ACTION AND 3-D STRUCTURE OF PECTINASES
This Page Intentionally Left Blank
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V.All rights reserved.
705
Stereo chemistry of hydrolysis of glycosidic linkage by three AspergiUus polygalacturonases Biely, P.*, Benen, J.A.E.**, Kester, H.C.M.**, Heinrichova, K.* and Visser, J.** * Institute of Chemistry, Slovak Academy of Sciences, 84238 Bratislava, Slovakia. ** Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, Dreyenlaan 2, 6703 HA Wageningen, The Netherlands.
Abstract The stereochemistry of the hydrolytic action of endopolygalacturonases I and II (PGI and PGII, respectively) from Aspergillus niger and of an exopolygalacturonase (PGX) from A. tubingensis was investigated by ~H-NMR spectroscopy by following the configuration of the reducing ends in the products formed in D20 reaction mixtures. It has been shown that all three polygalacturonases are inverting enzymes; the newly formed reducing ends all showed the B-configuration.
Introduction Enzymic hydrolysis of glycosidic linkages either proceeds via a single chemical step, the so-called single displacement mechanism, or via a two step chemical process, the so-called double displacement [ 1]. Enzymes functioning according the single displacement mechanism generate newly formed reducing ends which show inversion of the anomeric configuration and are therefore called 'inverting'. Hydrolases using the two-step chemical process always generate reducing ends which have exactly the same anomeric configuration as found in the glycosidic linkage. Such enzymes are called retaining. It has been suggested that endoglycanases are retaining enzymes while exoglycanases are inverting enzymes [2]. This generalization is not completely valid however. Several exoglycanases have been shown to be retaining enzymes [3]. There is one generalization that is st~ll valid. Retaining enzymes are capable to catalyse glycosyl transfer reaction at high substrate concentrations while inverting glycanases have not been reported to be able to do this. For pectinolytic enzymes no glycosyl transfer reaction have been reported except for Dgalacturonan digalacturonohydrolase (EC 3.2.1.82) from Selenomonas ruminantium [4]
706 suggesting that this enzyme is a retaining hydrolase. For other pectinases the stereochemistry of the hydrolytic activity is unknown. In this report the stereochemistry of the catalytic reaction of two endo- and one exopolygalacturonase from fungal origin are described.
Materials and Methods Endopolygalacturonases PGI and PGII isolated from a recombinant Aspergillus. niger and exopolygalacturonase (PGX) isolated from A. tubingensis are described elsewhere in this volume (see Benen et al. and Kester et al., respectively). For NMR spectroscopy the enzymes were lyophilized three times from D20. Reduced galacturonic acid and oligogalacturonides (GalU-ol, diGalU-ol, triGalU-ol and pentaGalU-ol) were prepared by NaBH4 reduction of the corresponding alduronic acids. For NMR experiments the compounds were lyophilized three times from D20. PentaGalU-ol was used as a substrate for PGI and PGII and triGalU-ol was used for PGX. Substrates were used at 20 mM final concentration in 0.5 ml 100 mM D20 buffer pD 4.5. The amount of enzyme used was such that the rate of hydrolysis was much higher than the rate of mutarotation. Time courses of the reaction mixtures were recorded on a Brucker AM 400 spectrometer at 25 ~ The assignments of relevant resonances was based on data published by Hricovini et al. [5].
Results and Discussion PGI PentaGalU-ol is primarily hydrolyzed to diGalU-ol and triGalUA, by PGI with subsequent hydrolysis of triGalUA to diGalUA and GalUA. This is reflected in the 1H-NMR spectra of the reaction mixture recorded after time intervals. The signals of pentaGalU-ol (Fig. 1, Table 1) were replaced by the signals of the 13-anomer of triGalUA (H-113, 4.64 ppm, J1,2 7.8 Hz) (Table 2). Due to the further hydrolysis of triGalUA at a rate exceeding mutarotation, the ot-anomer of the trimer (H-lo~, 5.35 ppm, J1,2 3.8) was barely detectable. Instead the 13anomer of the diGalUA and GalUA appeared quite strongly at the expense of the 13-anomer of the primary product triGalUA. As a result of mutarotation the H-lot signals of diGalUA and GalUA appear later. PGII The cleavage mode of pentaGalU-ol by PGII is essentially the same as found for PGI. Only the secondary hydrolysis reaction of the primary product triGalUA proceeds much more slowly. Spectra are not shown. The time course of the relevant resonances depicted in Fig. 2 demonstrates that the 13-anomer of the triGalUA is initially formed. Thus, like PGI, PGII is an inverting enzyme.
707
PGX PGX releases GalUA from the nonreducing end of oligogalacturonides. TriGalU-ol is the smallest reduced substrate for PGX (See Kester et al., elsewhere in this volume). Therefore triGalU-ol which is hydrolyzed into GalUA and diGalU-ol by PGX was used in this study. The GalUA released immediately after hydrolysis appears to have the configuration of the 13anomer (H-113 4.60 ppm, J1,2 7.9 Hz, Table I) as is a proof for the inverting character of PGX (Fig. 3). Again, the ot-anomer appears much later as a result of mutarotation.
Table 1. ~H-NMR data (400 Hz, D20) for the anomeric and C-1 protons in galacturonic acid (GalUA), diGalUA, triGalUA, reduced diGalUA (diGalU-ol), triGalU-ol and pentaGalU-ol. 8 ppm values are centred for doublets and are relative to the D20 resonance (4.80 ppm)
Compound
Chemical shift 8 ppm
J1,2(Hz)
Coupling constant
Proton
GalUA
5.31 4.60
3.8 7.9
H- 1ot H-113
diGalUA
5.35 4.65 5.13
3.8 7.8 3.8
H-lot H-113 H'-I
diGalU-ol
5.16
4.0
H'-I
triGalUA
5.35 4.65 5.14 5.09
3.8 7.8 3.7 3.8
H-lot H-113 H'-I H"-I
triGalU-ol
5.18 5.10
4.0 3.9
H'-I H"-I
pentaGalU-ol
5.17 5.12 5.11 4.87
4.0 4.3 4.3 3.8
H'-I H"-I H'"-I H .... -1
708
A
---o
-
g
i" m
'~
",
9
9
,'.,
s'.,
,'.,
=g
,-,..,
9
,:-
,
.'r
t9 v _ =
~g
=-
;
c
~
.
.
.
.~
"
,:,
"
"
,:s
eg ,=
Ii~]I
e~
. 9 ~
t~2" -..-~ .-~,o :)
"
,-
,.,,
,~
"
','s
_~1= m . ,
B ,
,,
I
,'.s
m
,~
'
'
,'.s
.
.
.
.
,I,
H'-I?
A '~ P i l m
"
5.5
"
"
;
'
~,
[
5.0
4.5
4.0
315
Fig. 1. 1H-NMRspectra (400 MHz, D20 ) of pentaGalU-ol (A) and of its hydrolysis products with A. niger PGI for 3 rain (B), 11 min (C) and 3 h (D). The assignment of crucial signals (Several doublets and multiplet of H-213) is indicated.
709 Table 2. IH-NMRdata (400 Hz, D20) for the anomeric and C-1 protons in products formed in the initial stages of reaction by endopolygalacturonase I (PGI) from A. niger from reduced pentagalacturonic acid (pentaGalU-ol).
Enzyme
Chemical shift 5 ppm
Coupling constant Assignment J1,2(Hz)
PGI
4.64 5.09 5.14 5.15
7.8 3.9 4.2 4.9
>,, .,-, "~ t-
6
--
5
-
._~ o0 ._>9
4
-
3
-
N rr rr z -r-
2
H-113 in triGalUA H"-I in triGalUA H'- 1 in triGalUA H'- 1 in diGalU-ol
9
O
t-
!
1 0
I
I
I
I
I
I
5
10
15
20
25
30
T i m e (min.)
Fig. 2. Changes in relative intensity of anomeric proton resonances during hydrolysis of pentaGalU-ol by A. niger PGII: H- 113of triGalUA, O; H- 1ot of trigalUA, O.
710
_.=
8
~> 4
z,
o 0
10
20
30
40
50
60
Time (min.)
Fig. 3. Changes in relative intensity of anomeric proton resonances during hydrolysis of triGalU-ol by A. tubingensis PGX: H- 113of GalUA, O; H- 1ot of GalUA, O.
Conclusions The lack of glycosyl transfer reaction is the class of pectinolytic hydrolases is in agreement with the observed inversion of the anomeric configuration of the newly formed reducing ends of the products. All three polygalacturonases studied here utilize the single displacement mechanism of hydrolysis.
Acknowledgement This work was partly funded by the European Community grant no. AIR2-CT-941345.
References 1) Koshland, D.E. Jr. (1953) Biol. Rev. 28 416-436. 2) Reese, E.T. (1977) in Recent Advances in Phytochemistry (Loewus, F.A. and Runeckles V.C. eds.) Plenum Publishing Corporation, New York, vol. 11.311-367. 3) Svensson, B. (1994) Plant Mol. Biol. 25, 141-157. 4) Heinrichov~i, K., Dzurovh, M. and Rexov~-Benkov~, L. (1994) Carbohydr. Res. 235, 269280. 5) Hricovini, M., Bystrick~,, S. and Malovikovh, A. (1991) Carbohydr. Res. 220, 23-31.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All fights reserved.
711
PECTIN METHYLTRANSFERASES FROM SUSPENSION-CULTURED CELLS AND SEEDLINGS OF FLAX (Linum usitatissimum L. ).
Bruyant P., Bruyant-Vannier M.P., Bourlard T., Gaudinet-Schaumann A., Thoiron B., and Morvan C. Universitd de Rouen, SCUEOR, URA 203, F-76 821 Mont Saint Aignan cedex. Tel: (33) 35 14 67 50, Fax: (33) 35 70 55 20. E-mail: [email protected].
Abstract
Pectin methyltransferases solubilized from endomembranes of flax cells (Linum usitatissimun) (E.C. 2.1.1.1.18) consisted of several polypeptides, the molecular relative mass of which varied from very low (Mr < 5000) to very high (Mr ca 200,000) values. Several isoforms were detected from acidic to very basic pH with two main forms, 1) a basic form (pI 8-8.5) and 2) a neutral form (pI 6.5-7.5). The activity of pectin methykransferase measured in vitro was optimal at pH 7 in the presence of high-methylated pectins and at pH 5.5 when low-methylated pectins had been added. Besides, the activity increased significantly throughout the seedlings growth, but differently according to the organs and pH. Key Words: Plant cell-wall; Pectin biosynthesis; Methylester; Golgi apparatus. I. INTRODUCTION The methylation of pectin is an important process which limits ionic interactions between charged polymers in the wall, and hence, it might be involved in the control of cell-wall behaviour and cell elongation. Goldberg et al [ 1], while investigating the growth gradient exhibited by the mung bean hypocotyl, observed that highly esterified pectins were located in all the wall area of young cells and in the primary cell-walls of mature cells. For a better comprehension of the methylation of pectins, it is necessary to get information on the enzymes involved in the process. The methylation of pectins has been demonstrated to occur in endomembrane systems [2]. In Golgi-enriched preparation of flax, enzymes have been shown to catalyze the transfer of methylgroup onto endogenous pectic polysaccharides [3,4]. Pectin methyltransferase complex was easily solubilized from the endomembranes of flax cells (Linum usitatissimum L.), when using Triton X-100 detergent [5]. In this paper, first we will consider some properties of the complex. Then, we will report on the variations of these activities over the culture duration.
712 2. MATERIAL AND METHODS 2.1 Plant material Flax seeds were placed for germination on moist paper for three days at 22~ and in the dark; then, the plantlets were transferred under continuous white light on a liquid culture medium, as previously described [6]. Suspension-cultured cells of flax were obtained from hypocotyl-derived calli as described by Schaumann et al. [4] and cultured on a medium described by Murashige and Skoog [7] containing kinetin (0.75 mg 1-1)and 2-4 D (0.2 mg l-l). 2.2. Microsome preparation Flax cells or cut organs were plasmolysed for 15 min in 50 mM Tris-HC1 (pH 7.5) containing 12% (w/w) sucrose, 1 mM ethylen glycol-bis-(b aminoethylether)N,N,N',N' tetra acetic acid (EGTA), 1 mM dithiothreitol (DTT) and 0.1 mM MgC12 (Buffer E). The cells were ground in a tenbroeck glass Potter homogenizer in a minimum volume; the homogenate was strained through nylon cloth (30 ktm) and the filtrate was centrifuged at 11,000 g and supematant at 200,000. The pellet, resuspended in 4 ml of the buffer E, constituted the crude microsomal fraction (CM). All these operations were carried out at 4~ in less than 2 h. 2.3. Pectin methyltransferase assays PMT assays were performed as described by Vannier et al. [3] by adding an equal volume of an enzyme preparation to a 0.1 M Tris-HC1 buffer containing 3.36 ~tM of [14C]SAM (1.8 GBq mmol~, 740 kBq ml-~, NEN), 1% (W/V) BSA and 12% sucrose, with or without 0.2% pectic acceptor. The incubation was run at 28~ for 12 h. After precipitation of the reaction product in 70% ethanol, the methylated polymers were selectively extracted with 0.5% ammonium oxalate and radioactivity was measured in a Tricarb 2250 CA Packard scintillation counter.
3. RESULTS AND DISCUSSION 3.1. Characterization of pectin methyltransferases of flax cells. Figure 1 indicates that pectin methyltransferase (PMT) activity from freeze-thawed microsomes measured without exogenous substrate was maximal at neutral pH (6.5 to 7.5). When exogenous pectic substrates of various DE had been added, similar optimal neutral pH was observed, and the activity was slightly stimulated (1.2 to 1.8 times). A second optimal pH occured at pH 5.5, but in the presence of low methylated pectin (DE: 0.1). As suggested by Lineweaver and Ballou [8] to explain the behaviour of another pectic enzyme -i.e. pectin methylesterase (PME), the mobility and the activity of PMT might be influenced by the presence of polyanionic substrates. On the other hand, the existence of several forms of pectin methyltransferase in flax microsomes might be responsible for such variations of the activity.
Bruyant-Vannier et al [5] have shown that pectin methyltransferases solubilized from endomembranes of flax cells consisted of several polypeptides, the molecular relative mass of which varied from very low (Mr _<5,000) to very high (Mr ca 200,000) values.
713 3 "
5
0
0
~
3 0 0 0 ~ : : [ : ........iiiiiiiiiii~i~~ 2500 ~~iiii~ii iiiiiiiii~i!~ii_i!~iiiiiiii!i~~ ~" 2000 ~ ~ i i i ~ i iiiiiili!~!i:_iiiiiiiiiiiiii_~_~
D Crude microsomes
~,,'~ l1000~ii~_iiiiiiiii~iii!~ili!500 ~~iiii~iiiiiiii!ii~iii[iiiiiii_:.~:~
5000
m High-methylated pectins
i::i::::Low-methylated i pectins .
tr~ ~.~. ~ , ir~
":
~ ~ t-'L'-,-
pH
.
.
.
.
O0
Figure 1. Effect of pH and of the addition of exogeneous pectins of low and high degree of esterification on the pectin methykransferase activity from freeze-thawed microsomes of flax cells. Also, several isoforms were detected from acidic to very basic pH with two main forms, 1) a basic form (pI 8-8.5) and 2) a neutral form (pI 6.5-7.5). The purification of the PMT complexes is needed to determine their structure and catalytic behaviour. 3.2. PMT activity of flax seedlings over the culture duration. PMT activity measured without any exogeneous substrate from flax seedling microsomes was generally higher at pH 5 than at pH 7 (table 1) which was not the case in the suspensioncultured cells (see fig. 1). The activity was the most important in the cotyledons and particularly low at pH 7 in the hypocotyls. Whatever the pH, the activity increased over the culture duration.
Table 1" Pectin methyltransferase activity in dpm per organ of flax seedlings Days after transfer to light 0 Cotyl (pH 5.5) Cotyl (pH 7.0) nyp (pU 5.5) nyp (pH 7.0) Root (pH 5.5) Root (pH 7.0)
378 149 99 27 78 35
1
2
3
4
5
6
7
8
9
10
13
74 495 1976 1607 1054 2056 1750 1778 2486 2142 3528 39 335 585 832 709 501 401 693 1162 130 1207 127 106 443 429 312 558 538 584 523 441 438 138 49 34 48 88 34 94 178 64 474 241 123 126 516 423 524 509 527 589 374 807 696 26 106 122 235 234 372 373 474 333 420 497
Cotyl: cotyledons and developing up stem and leaves. Hyp: Hypocotyl In the part named <>which also contained the developing stem and leaves, the increase was linear, suggesting a relationship between PMT activity and growth and/or cell division. In the roots, the increase was slightly reduced from day 6, at both pH. Interestingly, the PMT activity in the hypocotyls raised up to a maximum but it occured earlier at pH 5.5 (between days 5 to 8, when the elongation was stopping) than at pH 7 (happening at the end of
714 the culture when the phloem fibres were differentiating). Moreover the increase of activity in the hypocotyl was related to light since Vannier et al [3] reported that alter 7 days of growth in the dark, the activky decreased to almost nil at pH 7. Figure 2 show that the specific activity from the cotyledons (mean value 38,200 and 13,900 dpm mg-~ protein at pH 5.5 and 7 repectively) was also larger than from hypocotyls (21,300 and 7,500) or roots (18,600 and 10,500) . Whatever the pH, two peaks occured in the cotyledons (days 3 and 9) while the specific activity was slightly increasing in the roots, during the culture. In the hypocotyls, the activity was rather constant at pH 5.5 and presented two peaks at neutral pH. In all cases, the specific activity was larger than that of suspensioncultured cells which had been estimated in the range of 250 and 2500 dpm mg1 protein.
110 = 105
"7
-
40
~"
C
,,1=
e-
9
0 0
2
4
6
8
10
12
0
2
4
6
8
10
1'2
0
Time after transfer to light in days
2
4
~
|
8
.
i
10
,
|
,
12
Figure 2: Specific activity of pectin methyltransferases from microsomes of flax seedlings. A: cotyledons; B: hypocotyls; C: roots. The activity was measured at pH 5.5 ( I ) and 7 (o) 4. REFERENCES 1 Goldberg R., Morvan C. and Roland J.C. (1986) Plant Cell Physiol., 27, 417-429. 2 Kauss H., Swanson A.L. and Hassid W.Z. (1967) Biochem. Biophys. Res. Commun.., 26, 234-239. 3 Vannier M.P., Thoiron B., Morvan C. and Demarty M. (1992) Biochem. J., 286, 863-868. 4 Schaumann A., Bruyant-Vannier M.P., Goubet F. and Morvan C. (1993) Plant Cell Physiol., 34, 891-897. 5 Bruyant-Vannier M.P., Gaudinet-Schaumann A., Bourlard T. and Morvan C. (1996) Plant Physiol. Biochem., (accepted). 6 Morvan C., Abdul Hafez A.M., Jauneau A., Thoiron B. and Demarty M. (1991) Plant Cell Physiol., 45,609-621. 7 Murashige T. and Skoog F. (1966) Physiol. Plant., 45, 473-497. 8 Lineweaver H. and Ballou G.A. (1945) Arch. BiocherrL, 6, 373-387.
J. Visserand A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996ElsevierScienceB.V.All rights reserved.
715
P e c t i n a s e s f r o m Rhizopus sp. E f f i c i e n t in E n h a n c i n g t h e Hydrolyzation of Raw Cassava Starch : Purification and C h a r a c t e riz a t i o n Lerluck Chitradon, Phuntip Poonpairoj, Polson Mahakhan a, Vichien Kitpreechavanich and Napha Lotong Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10903, Thailand. a Department. of Microbiology, Faculty of Science, Khon kaen Univ., Khon Kaen, Thailand. Abstract
Pectinase from Rhizopus sp. 26R, a fungal strain that capable of raw cassava starch hydrolyzation, showed high efficiency in enhancing the digestion of raw starch from whole cassava tuber when it was used as mixed crude enzymes with glucoamylase of Aspergillus niger J8. At the 1st hour of the reaction, the mixed enzymes gave the most efficient digestibility with hydrolyzation rate twice faster than that when glucoamylase was used alone and about 3 times faster than when pectinases were used alone. Rate of hydrolyzation, however, decreased upon further incubation that at the end of 8 hour the mixed enzymes gave 1.3 times faster rate than that by glucoamylase and twice of that by the pectinases. Moreover, it was shown that activity of the glucoamylase in digestion of raw ground cassava tuber that enhanced by the pectinase of Rhizopus sp. 26R was higher than that enhanced by the commercial pectinase. Purification of the pectinase was done with the enzyme produced in solid substrate composed of wheat bran, rice bran and rice husk in a ratio of 6:12:2 at 58% initial moisture content, pH 5.7. The crude enzyme was purified initially concentrated with ammonium sulfate 60-90% and further passed through anion and cation exchange chromatographs, respectively. DEAE- and CM-cellulose were the ion exchangers. The cation exchange chromatograph was an efficient step that could purified the enzyme to 107 fold which remained only 2 proteins of different sizes on SDS-PAGE. The 2 proteins was further purified with Hydroxyapatite column chromatograph. The enzyme was purified to 277 fold. On Toyopearl 55HW gel filtration chromatography, the pectinase was one peak of protein with molecular weight of 115,000 dalton. However, with SDS-PAGE, the molecular weight was 43,000 dalton. Optimum temperature and pH of the purified enzyme were 50~ and 5.25, respectively. The enzyme was stable at 2540~ and at pH 4.0-8.0. The purified pectinase of Rhizopus sp. 26R was proved
716 to have polygalacturonase and polymethylgalacturonase activities, that could enhance the hydrolyzation of raw starch from whole cassava tuber. INTRODUCTION Pectinases are the enzyme that widely used especially in industrial applications (1, 2, 3, 4). Types of the pectinases are different concerned on their properties and the actions on the pectic substances (5). Several genus of fungi could produce pectinases eg. Aspergillus, Penicillium, Sclerotium, Fusarium and Rhizopus (6,7). Rhizopus sp. 26R, isolated in Thailand was a strain that capable of raw cassava starch hydrolyzation. Its crude enzyme in solid substrates composed of agricultural products and wastes showed high efficiency in enhancing the digestion of raw starch from whole cassava tuber when it was used as mixed crude enzymes with glucoamylase of Aspergillus niger J8 (8,9). Moreover, the capability of the pectinases of Rhizopus sp. 26R was higher than that of the commercial one. The crude enzymes from solid substrates was purified and some of its properties are reported. MATERIALS & METHODS
Microorganism and cultivation conditions Rhizopus sp. 26R cultivated in 20 g solid substrates composed of wheat bran, rice bran and rice husk (6:12:2) in a plastic bags. Initial pH and moisture content were 5.7 and 58%, respectively. The fungus was incubated at 32~ for 6 days. Enzyme Assay The activity was assayed by the determination of substrate viscosity diminishing using Ostwald viscometer (10). The enzyme reaction was done at 37~ in 0.05 N acetate buffer pH 5.25. One unit of enzyme was defined as the amount of enzyme that could reduce the viscosity of 2% pectin by 50% in 10 rain. Pectin esterase activity was assayed by measuring the decreasing of pH in the reaction mixture which equal to methanol liberated by the method of Wood, et al. (11). Pectin lyase and pectate lyase activities were assayed by measuring the increasing of the absorbancy at 235 nm by the method of Albersheim and Kinias (12, 13) when pectin or sodium pectate was used as the substrate, repectively. Polygalacturonase activity was estimated by measuring the % viscosity diminishing of sodium polygalacturonate (14).
Protein determination Protein content was determined by Lowry"s method (15) with bovine serum albumin as standard. The protein content in the columneluates was measured by the absorbancy at 280 nm. E n z y m e e x t r a c t i o n a n d p u r i f i c a t i o n steps The crude enzyme was extracted from the solid state culture with 100 ml of 0.33% toluene at 4~ The enzyme was concentrated with 60-90% (NH4)2SO4, then the dialysed enzyme
717 solution was purified through 3 column chromatographs. First column was DEAE-cellulose chromatograph which was equilibrated with 0.05 N acetate buffer pH 5.25, then the enzyme was eluted with a linear gradient of 0-0.7 N KC1 in the same buffer. The second column was CM-cellulose chromatograph which was done under the same conditions except that the linear gradient of 0-0.7 N NaC1 in the equilibrate buffer was used for elution. The fractions from the second column that contained the enzyme activity was collected and dialyzed against 0.01 N phosphate buffer pH 5.8, then subjected through hydroxyapatite column chromatograph and the enzyme was eluted with 0.01-0.5 N of the same phosphate buffer. All experiments was done at 4~
Molecular weight determination The molecular weight of the purified enzyme was determined by both gel filtration chromatograph and SDS polyacrylamide gel electrophoresis (16). Toyopearl 55HW gel (Toyo SF 160K, Toyo, Co.,Ltd., Tokyo) was used for gel filtration and 0.05 N acetate buffer pH 5.25 was used as buffer. Effect of pH and temperature on the purified enzyme activity and stability The conditions of the enzyme activity and the stability was done followed Buranakarl, et al. (16). RESULTS
1. P u r i f i c a t i o n 1.1
of pectinase from
Rhizopus
sp. 2 6 R
DEAE-cellulose column chromatograph
The pectinase activity was found in void volume of the washing buffer while some contaminated protein was adsorbed to the ion exchanger (Figure 1. The activity was 100% recovered. Though, the enzyme was not adsorbed to the DEAE-cellulose, but it is useful since the some contaminated proteins could be separated. The enzyme was approx. 10 fold purified and its specific activity increased to 48.8 unit/mg protein (Table 1).
1.2
CM-cellulose column chromatograph
The enzyme activity was adsorbed to the cation exchange chromatography. Three peaks of activities were found (fraction no. 78-81, 83-85 and 86-89). The major peak (no.83-85) was collected (Figure 2. The enzyme was purified to approx. 100 fold with the higher specific activity, 534.7 unit/mg protein (Table 1).
1.3
Hydroxyapatite column chromatograph
The enzyme was purified to 277 fold with very high specSfic activity, 1387 unit/rag protein by hydroxyapatite column chromatography (Figure 3).
718 P~'n~ m~'ty (miq~ ~0-
OD28O / 14 NK(I
ul~t~ OD280NK~
~-~12 '0.7
Pealm~ act~vUy0nt~i) 5OO unit/rri OD280 NKCI 400 -4o • 30O
lml 120 i 8Oi 0
6
_"
~
.....
2!!
/
0.6I 0.7
20O
-0
_ A![! . . . . . ill21 . . . . .
OD~0 1 N KO 0.8
0
o
0.4
--.
lO 20 30 40 50 60 70 80 90100
0
Fl21~:ioIl nO.
Figure 1 The profile of pectinase on DEAE-ceUulose column chromatograph, size 3.5x40 cm, 0.05 N acetate buffer pH 5.25, flow rate 60 m l / h
Pec~m~ ac~'ty (umt~) O028O 1,200 6 u ~ t ~ (:I)280 m M ~ ~ a t e 1,030 5 o
Figure 2 The profile of pectinase on CM-cellulose column chromatograph, size 3x9 cm, 0.05 N acetate buffer pH 5.25, flow rate 60 ml/h
~
~
~
(D~ 0(]5 _,__
--9r
08
830
4
030
3m4
400
2
04
200
1
Q2
2-
0(~
o6
Q(s
-5OO
5
10
15 20 25
30 35
Frac~Oll ~
Figure 3. The profile h y d r o x y a p a t i t e column size lx60 mm, 10-500 buffer pH 5.8, flow rate
of pectinase on chromatograph mM phosphate 12 ml/h
001
5
9 ~ m
Figure 4. The profile of pectinase on Toyopearl 55HW gel filtration column size 1.1x55 cm. flow rate 15 ml/h , 50 mM acetate buffer pH 5.25
719 Table 1
Summary of the purification of pectinase from Rhizopus sp. 26R Total protein (mg)
Total activity (unit)
Specific activity (unit/mg)
4,000
56,600
283,160
5.0
567
12,049
283,500
3. DEAEcellulose
1,118
2,571
4. CMcellulose
168
5. OHapatite
100
Steps of purification
Volume (ml)
1. Crude enzyme 2. (NH4)2SO4
Yield (%)
Fold of purification
23.5
100.0
4.7
125,440
48.8
44.3
9.8
84
44,918
534.7
15.9
107.0
24
33,277
1,386.5
11.8
277.3
It was proved to be a single peak of protein and activity on gel filtration chromatography (figure 4) and single band on SDS-PAGE (figure 5).
1
2
3 4 5
6
kdal
1 2 3 4 5
6 7 a
205 116 97 66 45 29
Figure 5 SDS Polyacrylamide Gel Electrophoresis of pectinase from different steps of purification A: (1,6) standard protein (2) crude enzyme (3) ammonium sulfate precipitation (4) DEAE cellulose (5) CM-cellulose B: Electrophoretic pattern of the pectinase from hydroxyapatite (1,8) standard protein (2) fraction no.25 (3) no.26 (4) no.27 (5) no.28 (6) no.29 (7) no.30
720
2. P r o p e r t i e s o f t h e p u r i f i e d p e c t i n a s e 2.1
of
Rhizopus sp. 2 6 R
Molecular weight
The molecular weight determined by the gel filtration chromatography with Toyopearl 55 HW was 115 kdal while that estimated by SDS-PAGE was 43 kdal (Figure 4,5).
2.2 Effect of pH and temperature on the purified enzyme activity and stability The purified pectinase exhibited the maximum activity at pH 5.25 and 50~ The activity was stable up to pH 8.0 and at 25-40~ At 45~ the activity remained 80% and at 60~ the activity remained 50% (Figure 6).
2.3
Properties of the purified pectinase of
Rhizopus sp. 26R
The purified enzyme could hydrolyze pectin and sodium pectate. However, the absorbancy at 253 nm and pH of the reaction mixture were not changed. The purified enzyme showed the activity to be polymethylgalacturonase (PMG)and polygalacturonase (PG).
140
140
120
120
~100
~
optim
temp. temp
ility
o 100
60
20 3.5
4.5
5.25
6.0 pH
7.0
8.0
0
25
30
~ 37 40 45 50 T e m p e r a t u r e (~
55
60
Figure 6 Optimum pH and temperature of the purified pectinase and its stability to various pH and temperature.
2.4
D i g e s t i o n of raw starch from ground cassava tuber
Pectinase from Rhizopus sp. 26R showed high efficiency in enhancing the digestion of raw starch from whole cassava tuber when it was mixed with the glucoamylase. At the 1st hour of the reaction, the mixed enzymes gave the most efficient digestibility with hydrolyzation rate twice faster, than that when glucoamylase was used alone and about 3 times faster than when pectinases
721 were used alone. Rate of hydrolyzation, however, decreased upon further incubation that at the end of 8 hour the mixed enzymes gave 1.3 times faster rate than that by glucoamylase and twice of that by the pectinases. Moreover, it was shown that activity of the glucoamylase in digestion of raw ground cassava tuber that enhanced by the pectinase of Rhizopus sp. 26R was higher than that enhanced by the commercial pectinase. C O N C L U S I O N & DISCUSSION The crude enzyme extracted from the culture of Rhizopus sp. 26R cultivated in solid substrates composed of wheat bran, rice bran and rice husk in a ratio of 6:12:2 at 58% initial moisture content, pH 5.7 was used to purified. Concentration of the enzyme solution with 60-90% ammonium sulfate could increase its specific activity approx. 5 fold without any loss of the enzyme activity. Prior used of anion exchange chromatography help separate some contaminated proteins, though, the pectinase was not adsorbed to the DEAEcellulose. Ten fold purified enzyme could be collected by this step. The cation exchange chromatograph, CM-cellulose was an efficient step that could purified the enzyme to 107 fold which remained only 2 proteins of different sizes on SDSPAGE. The 2 proteins was further purified with Hydroxyapatite column chromatograph. The enzyme was purified to 277 fold. On Toyopearl 55HW gel filtration chromatography, the pectinase was one peak of protein with molecular weight of 115 kdal. However, with SDS-PAGE, the molecular weight was 43 kdal. Optimum temperature and pH of the purified enzyme were 50~ and 5.25, respectively. The enzyme was stable up to 40~ and at pH 4.0-8.0. The purified pectinase of Rhizopus sp. 26R showed the activity to be polygalacturonase (PG) and polymethylgalacturonase (PMG), that could enhance the hydrolyzation of raw starch from whole cassava tuber. In comparison to other pectic enzymes, PG of Rhizopus sp. 26R was not homogeneous protein, it might consist of more than one subunits. This was different from endo-PG of Rhizopus sp. strain LKN which was 38.5 kdal and homogeneous, optimum temperature of Rhizopus sp. strain LKN were 55-60~ which was higher than that of Rhizopus sp. 26R, optimum pH was 4.5-4.75 and stable up to 50~ pH 4.5-11.0 (17). However, molecular weight of the purified PG of Rhizopus sp. 26R was almost similar to a pectinesterase of Ficus awkeotsang which was 42 kdal (18). Moreover, Hara, et al. (19) reported 2 exoPG found in Aspergillus niger, but the characteristics were different. Molecular weight of the 2 exo-PG were 63 and 66 kdal which were higher than that of Rhizopus sp. 26R. Optimum pH of the 2 exo-PG were 3.8 and 4.5, pH stability were similar at 2.5-5.0 which were lower than PG of Rhizopus sp. 26R. On the otherhand, optimum temperature were 60~ thermal stability were up to 50~ which were higher than the purified PG of Rhizopus sp. 26R. According to the ability of the pectinase of Rhizopus sp. 26R that could efficiently enhance the hydrolyzation of raw starch from whole cassava tuber,
722 this enzyme was concerned to be very much useful in renewable utilization of raw cassava wastes which mostly contained cassava peel. The utilization of cassava wastes by enzyme reactions are further studied.
REFERENCES 1. A.D., Tressler and M.A. Joslyns, Fruits and Vegetable Processing Tech. 2nd ed., Avi Publishing Co. Inc., Connecticut, 1971. 2. K.R Sreekantiah, S. Jaleel and T.N.R. Rao, Indian Food Packer, 22 (1968) 1215. 3. K.R. Sreekantiah, S. Jaleel and T.N.R. Rao, J. Food Sci. Tech., 8 (1971) 201203. 4. M. Buenrostro and A. Lopes-Munguia, Biotechnol. lett., 8 (1986) 505-506. 5. W. Pilnik and F.M. Rombouts in G.G. Birch, et al. Enzymes and Food Processing. Appl. Sci. publishers, Ltd. London, (1980) 296. 6. O.B. Fawole and S.A. Odunfa, Lett. Appl. Microbiol., 15 (1992) 266-268. 7. R.H. Mflkhailova, L.I. Sapunova and A.G. Lobanok, World J. Microbiol.& Biotechnol., 10 (1994) 457-461. 8. P. Mahakhan., V. Kitpreechavanit, N. Lotong and L. Chitradon, Proc. in 30th Ann. Meet. of Kasetsart University, (1992) 627-638. 9. L. Chitradon, L., V. Kitpreechavanit, W. Yongmanitchai and N. Lotong, Thai J. ofAgric. Sci., 26 (1993) 109-121. 10. E. Roboz, R.W. Barratt and E.L. Tatum, J. Biol. Chem., 195 (1952) 459-471. 11. H, Deuel and E. Stutz, Adv. Enzymol., 1953. 12. P. Albersheim and U, Killias, Arch. Biochem. Biophy., 97 (1962) 107-115. 13. D.O. Silva, M.M. Attwood and D.W. Tempest, World J. Microbiol. & Biotechnol., 9 (1993) 574-578. 14. Yoshinari, Komae and Tanabe, J. Ferment. Technol., 63 (1985) 451-459. 15. O.H. Lowry, N.J. Rosebrough, A.F.Farr and R.I. Randall, J. Biol. Chem., 193 (1951) 265. 16. L. Buranakarl, K. Ito, K. Izaki and H. Takahashi, Enzyme Microb. Technol., 10 (1988) 173-179. 17. F.B. Elegado and Y. Fujio, World j. of Microbiol. Biotechnol., 10 (1994) 256259. 18. K. Komae, Y. Sone, M. Kakuta and A. Misaki, J. Agic. Biol. Chem., 54 (1990) 1469-1476. 19. T. Hara, J.Y. Lira, Y. Fijio and S. Ueda, Nippon Shokuhin Kogyo Gakkaisha, 31 (1984) 581-586.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
723
Isolation, characterization and immuno localization of orange fruit acetyl esterase. Tove M.I.E. Christensen, John E. Nielsen and Jorn D. Mikkelsen. Danisco Biotechnology, Langebrogade 1, DK- 1001 Copenhagen K.
ABSTRACT Acetyl esterase (AE) has been purified to homogeneity from orange peels. The purification steps included cation exchange chromatography and gel filtration. The enzyme has affinity for triacetin and sugar beet pectin with KM of 39 mM and KM of 26 mg/ml, respectively. AE has a MW of 42 kD and is a monomer. The isoelectric point is at pH > 9. Immuno localization using polyclonal antibodies raised against AE showed that AE was widely distributed in orange fruit but with more intensive immunological detection in the outer part of the peels e.g. albedo and flavedo and in the segments (juice vesicles). The results indicate that AE is located at the site where the major fraction of pectin is deposited.
INTRODUCTION Acetylated oligosaccharide polymers are present in - and have been characterized from - a number of plant species. Acetyl esterases which deacetylate polysaccharides have been isolated from numerous microorganisms and plants. In general, these enzymes are very specific for deacetylation of the cell wall components such as hemicellulose and pectin. Sugar beet pectin contains a high level of acetyl groups bound to C2 and/or C3 positions of the galacturonic acid residues. These acetyl groups influence the gelling properties. Acetyl esterases removing acetyl groups from pectin e.g. sugar beet pectin have been isolated from mung bean hypocotyl (1), from orange peels (2) and from Aspergillus (3). The purified enzymes are very specific for acetyl groups and are unable to remove the methylester groups linked to C6 of the galacturonic acid residues in pectin molecules. Orange peel has a high content of acetyl esterase compared to other plant sources. The present work reports the purification and characterization of acetyl esterase from orange fruit as well as the in situ localization of the enzyme by immuno histology.
MATERIALS AND METHODS
Plant material Spanish Navelina oranges were used for isolation of AE. The oranges were peeled manually and the peels were stored at-80~
724
Extraction of AE from orange peel 600 g frozen orange peels were thawed and cut into minor pieces. They were homogenized in a Waring blender for 2 min. in 1200 ml buffer (100 mM Na-succinate pH 6.2, 1 mM DTT). Solid NaC1 was added to the homogenate to reach an end-concentration of 3 % (w/v) in order to isolate ionically bound proteins (4). After two hours of incubation with gently stirring at 4~ the suspension was filtered through a nylon mesh and the filtrate was centrifuged at 15,000 x g for 20 min. to remove insoluble material. The supernatant was then fractionated using (NH4)2SO4 precipitation. The activity was precipitated between 30% and 60% (NH4)2SO 4 saturation. The precipitate was resuspended in 50 ml 50 mM MES pH 6.8, 1 mM DTT and dialysed against the same buffer over night. All operations were performed at 4~
Chromatography The dialysed sample was fractionated by cation exchange chromatography. A 40-50 ml sample was applied to a CM-Sepharose CL-6B column (1.5 x 15 cm). Unbound proteins were removed with 50 mM MES pH 6.8, 1 mM DTT, and the bound proteins were eluted with an increasing NaC1 gradient from 0 - 0.4 M NaC1 in a total volume of 500 ml. The flow was 25 ml/h and fractions of 8.33 ml were collected. The protein profile was measured at 280 nm. All fractions were analysed for AE activity and protein content. The protein content was measured spectrophotometrically according to Bradford using the BioRad protein assay kit with ~,-globulin as standard (5). The fractions containing AE activity were pooled and concentrated by ultrafiltration using Amicon filter system (YM 10). Desalting of the sample was performed by dialysis against 50 mM MES pH 6.8. The AE preparation (10 ml) was applied to a prepacked Mono S HR 10/10 FPLC column (Pharmacia). The column was equilibrated with 50 mM MES pH 6.8 with a flow of 4 ml/min. AE was eluted with an increasing NaC1 gradient (0 - 0.3 M). Fractions were collected manually according to the protein profile measured at 280 nm. Active fractions were concentrated as described above and buffer exchange to 50 mM Tris pH 7, 1 mM DTT, 0.1 M NaC1 was done on the same system as above. The concentrated AE sample (9 ml) was then applied to a Sephacryl S-200 (2.6 x 70 cm) gel filtration column. The column was equilibrated with the Tris buffer described above. The flow was 40 ml/h and fractions of 5.33 ml were collected. The fractions containing AE activity were pooled and concentrated.
Enzyme activity AE catalyses the cleavage of acetyl groups from different substrates. The enzyme activity was determined by measuring the release of acetic acid. The amount of acetic acid was measured spectrophotometrically using an acetic acid analysis kit (Boehringer, Mannheim). The activity of AE was measured in 0.6% sugar beet pectin solubilised in 25 mM Nasuccinate pH 6.2 and incubated with enzyme fraction in total 500 #l assay. The samples were incubated at 40~ and aliquots were examined after 0, 1, 2 and 3 hours of incubation. The enzyme reaction was stopped by incubating the samples at 100~ for 5 min. Precipitated
725 protein was removed by centrifugation and the amount of acetic acid in the supernatant was determined. AE was also detected by using the substrate triacetin. The enzyme fraction was incubated with 80 mM triacetin in 25 mM Na-succinate pH 6.2. The samples were incubated at 40~ for 30 min. After boiling for 5 min. the samples were analysed for released acetic acid. During purification triacetin was used as substrate.
SDS-Polyacrylamid gelelectrophoresis The purity of the AE fraction was investigated by SDS-PAGE using Pharmacia PhastSystem with 10 - 15 % SDS-gradient gels. Electrophoresis and silver staining of the proteins were performed as described by the manuals from Pharmacia. For determination of pI IEF 3-9 PhastSystem gels were used.
Amino acid analysis Amino acid composition was analysed as described by Barkholt and Jensen (6). Cysteine was determined after derivatisation with 3,3'-dithiopropionic acid. Tryptophan was not determined.
Immuno histology Fixation: tissue samples for immuno histochemistry were fixed in 2% paraformaldehyde, 0.25 % glutaraldehyde and 3 % sucrose buffered with 0.05M phosphate buffer pH 7. After incubation for 2 hours at 25 ~ and 63 hours at 5 ~ the specimens were washed 3 x 20 min. in phosphate buffer pH 7. Dehydration was carried out using series of ethanol washings 50, 70, 80, 96 % followed by 3 x in 99% (1A hr in each). After additional treatment with 2 x 2 hrs in petroleum ether (shellsol DT0k, Q7712) and 2 x 2 hrs in paraffin with 7 % beeswax, the samples were embedded in paraffin. Cross sections of 12.5 #m were made on a Supercut 2050 Reichart Jung pyramitome.
Immunology: Tissue sections were preincubated with 20% swine serum in TBS (0.5 M Tris/HC1 pH 7.6, 0.15 M NaC1, 0.1% Triton X-100) for 30 rain. before treatment for 1 hour with antibodies against acetyl esterase diluted in TBS/swine serum (1:1000). Excess antibody was removed by washing with TBS (5 x 5 min.) After washing the sections were incubated for 30 min. with secondary antibodies coupled with alkaline phosphatase (1:20) in TBS/swine serum. Excess of secondary antibody was removed by TBS washing as described above. Before staining the sections were treated with veronal acetate buffer pH 9.2 for 5 min., and then stained with Fast Red and Naphthol AS-BI phosphate (Sigma N4875) for 20 min. Excess reagent was removed by washing with water. Preimmune controls were run in parallel. RESULTS AND DISCUSSION Characterization of acetyl esterase AE was purified from orange peels. After homogenization, precipitation with 30 - 60% (NH4)2SO 4 followed by dialysis, the sample was applied to a cation exchange column (CMSepharose CL-6B). AE binds strongly to a cation exchange column material at pH 6.8
726 whereas most of the impurity does not bind to the column. With increasing NaC1 gradient AE eluted as one peak at 0.2 M NaC1. Pectin Methyl Esterase (PME) which also is present in high amounts in orange fruit eluted at 0.28 M NaC1. In order to remove the residual PME the AE fraction was further fractionated on Mono S cation exchange column. Unlike the DEAE-Sepharose column, where PME elutes after the AE activity, the order of elution was reversed on the Mono S column (Fig. 1). The last step in the purification was a gel filtration (Sephacryl S-200) column. SDS-PAGE revealed only one protein band in the purified AE fractions with a MW of 42,000 D (Fig.2). Isoelectric focusing of AE Showed that pI > 9. The amino acid composition of the purified AE is shown in table 1.
Table 1.
Amino acid composition of AE obtained after 24 hrs. hydrolysis. ND: not determined AE
Asp Thr Ser Glu Pro Gly Ala Cys Val Met Ile Leu Tyr Phe His Lys Arg Trp
43.8 20.9 29.3 27.2 18.3 33.8 36.5 16.0 24.0 9.9 16.4 26.3 12.1 23.1 11.3 21.5 14.6 ND
No. of residue MW (D)
385 42,000
Contrary to AE from orange peels described by Williamson (2) this AE has a higher molecular mass and pI > 9. AE isolated from mung bean, however, has similar MW and pI in agreement with the present findings (1). These differences could indicate the presence of several AE isoformes in different varieties of orange fruits.
727 The AE activity was strongly dependent of the pH. When measured with 1% sugar beet pectin and 80 mM triacetin an optimum was found at pH 5 - 5.5.
LU
AU 2.0
MW kD
O,.
~9 9 2 '~9 6 7 AE
1.5
30
1.0
21
14
0.5 0.0 ~ 0.0
~9 4 6
20.0
lie 40.0
60.0
Figure 1. Elution profile of PME and AE on the Mono S column.
min.
Figure 2. SDS-PAGE of acetyl esterase.
The affinity for sugar beet pectin was determined using a Lineweaver-Burk plot. The KM was calculated to be 26 mg/ml for sugar beet pectin whereas the KM for triacetin was 39 mM. This showed a very low affinity for sugar beet pectin and triacetin. Substrate specifity of purified AE is summarized in table 2.
Table 2. Substrate specificity of AE. In each experiment 16 #g enzyme was used. Substrate
Concentration
Sugar beet pectin Apple pectin Citrus pectin
10 mg/ml 10 mg/ml 10 mg/ml
Triacetin p-Nitrophenyl acetate ct-Naphthyl acetate
80 mM 2 mM 2 mM
Enzyme activity /~mol/hr/mg 2 0 0 816 472 0
728 AE hydrolyses acetyl groups from sugar beet pectin, triacetin and p-nitrophenyl acetate but no reaction was observed with et-naphthyl acetate. The specific activity of AE for sugar beet pectin is much lower compared to the activity with triacetin and p-nitrophenyl acetate. This could be due to the higher complexity of pectin. The enzyme showed no affinity with apple and lime pectin as substrates. In apple pectin the acetyl groups are mainly bound to galacturonic acid residues in the hairy region of pectin (7). It has to be further investigated whether AE can deacetylate isolated modified hairy regions. A rhamnogalacturonan acetyl esterase from A.aculeatus has been isolated which is specific for deacetylation of hairy regions of pectin, but this enzyme has no specificity for sugar beet pectin (8).
Immuno localization of acetyl esterase Immuno localizations of AE in sections of orange fruits are shown in Fig. 3. The most intensive depositions of acetyl esterase were found in the outermost parts of the peel (exocarp or outermost albedo and the flavedo) and in the segments (juice vesicles), although quite high levels of acetyl esterase were found in most other tissues as well. The acetyl esterase depositions were all intracellular. In the peel strong immunological depositions of acetyl esterase were found in epidermis, the small cells of the exocarp and in the oil cavities (Fig. 3 A,B,C). In the mesocarp and endocarp the immunological depositions were more moderate (Fig. 3 D), but strong immunological depositions were found in the vascular bundles, especially in xylem. The immunological depositions in the peel seem to be correlated with cell size or cell age. The small cytoplasma rich cells have a higher content of acetyl esterase. In the segments strong immunological deposition was found throughout the tissue. Again the results indicate a slight correlation of cell size and the amount of acetyl esterase. In the small cells in the periphery of the juice vesicles, acetyl esterase is clearly intracellular (Fig 3 D,E), whereas the acetyl esterase was found on the cell walls of the large inner juice cells. This
Figure 3" Immuno localization of acetyl esterase. Sections were incubated with antibodies raised against the acetyl esterase, followed by visualization with alkaline phosphatase conjugated secondary antibodies and staining with Fast Red. A: Overview of the acetyl esterase immuno localizations in the peel (40x) (Ex: exocarp, M: mesocarp, OC: oil cavity). B: Immuno localizations of acetyl esterase in the exocarp (Ex) and oil cavity (OC) (294x). The most intensive acetyl esterase depositions are found in the small sized exocarp cells and in the oil cavity. C: Immuno control with preimmune serum on the following section used in B (294x). D: Immuno localization of acetyl esterase in endocarp (En) and juice vesicle (JV) (94x). Acetyl esterase depositions in the juice vesicles are more intensive than those observed in the endocarp. No acetyl esterase was detected in the innermost cell layer of the endocarp (see arrows). E: Immuno localization of acetyl esterase in lamella (L) and juice vesicle (JV) (294x). Acetyl esterase depositions in the juice vesicles are more intensive than in lamella. Acetyl esterase was absent from the outermost cell layer of lamella (see arrows). F: Immuno localization of acetyl esterase in core, where intensive acetyl esterase deposition was found in the xylem (94x).
729
~
~
i
~.
~~i!~iii!!!~~,,~
~~,:~,~;~
.. ii!-'?q
~-
~-
~
~
~$~
- ~,-
-
_ .
730 is most likely a fixation artefact. Controls stained with PAS (Periodic Acid Schiff) and ABB (Aniline Blue Black) revealed that proteins form aggregates with the cell walls of the large inner juice cells (not shown). PAS (9) and ABB (10) stain carbohydrates red and proteins blue, respectively. In lamella and core the strongest immunological depositions were found in the vascular bundles (Fig. 3 F), whereas acetyl esterase was present in moderate amounts in all other cells. No acetyl esterase was found in the outermost parts of the tissues, cuticula of epidermis, innermost cell layer of endocarp, outer walls of juice vesicles and outer cell layer of lamella.
A high yield of acetyl esterase and the strong immunological reaction in situ indicate that AE playes an important role in the orange fruit. These findings are, however, in contrast to the low reactivity observed for AE when the substrate is acetylated pectin. This could be due to the steric hindrance exerted by the methyl groups linked to the galacturonic acid residues. For AE from mung bean (1) and orange peel (2) it has been found that demethylated sugar beet pectin increases the AE activity considerably. However, the distribution of AE in peel and lamella is consistent with the pectin distribution in orange fruits but significantly high levels of AE were found in the fruit segments. This could indicate that pectin deacetylation is not the main activity of AE and that a hitherto unidentified substrate is present in the orange fruit. To clarify this suggestion the principal activity of AE has to be found by testing the enzyme for different activities.
Acknowledgements The excellent assistance of Jytte Rasmussen, Bo Lindberg and Clive Phipps Walter is gratefully acknowledged.
REFERENCE (1) (2) (3) (4)
(5) (6) (7) (8)
(9) (10)
M. Bordenave, R. Goldberg, J.C. Huet and J.C. Pernollet, Phytochemistry 38 (1995) 315-319. G.Willliamson, Phytochemistry 30 (1991) 445-449. J.A. Matthew, S.J. Howson, M.H. Keenan and P.S. Belton, Carbohydr. Polymer 12 (1990) 295-306. C. Versteeg, F.M. Rombouts and W. Pilnik, Lebensmittel.-Wiss. u. Technol. 11 (1978) 267-274 M. M. Bradford (1976) Anal. Chem 72:248-254 V. Barkholt and A.L. Jensen, Anal. Biochem. 177 (1989) 318-322. H.A. Schols, M.A. Posthumus and A.G.J. Voragen, Carbohydr. Res. 206 (1990) 105-115. M.J.F. Searle-van Leeuwen, L.A.M. van der Broek, H.A. Schols, G. Beldman and A.G.J. Voragen, Appl. Microbiol. Biotechnol 38 (1992) 347-349. N. Feder and T.P. O'Brien, Amer. J. Bot. 55 (1968) 123-142. D.B. Fisher, Histochemie 16 (1968) 92-96.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
731
Enzyme-Mediated Substrate Immunolocalization of Polygalacturonic Acid Within Barley Epidermal Cell Walls Utilizing Endopolygalacturonase of Coddiobolus sativus and a Monoclonal Antibody Specific for the Enzyme
Clay R. P.~, Bergmann C. W. ~ and Fuller M. S. 2
Complex Carbohydrate Research Center, University of Georgia, 220 Riverbend Rd., Athens, GA 30602, USA.
2
Darling Marine Center, University of Maine, Walpole, ME 04573, USA.
Abstract The fungal pathogen Cochliobolus sativus is known to penetrate its barley host via the anticlinal cell wall junctions between leaf epidermal cells. Microscopic and cytochemical evidence that this area is rich in pectic polysaccharides led us to investigate the contribution of endopolygalacturonase to the penetration process. Accordingly, an indirect method was developed for localization of the substrate of Cochliobolus sativus Ito and Kuribay (strain SB85) endopolygalacturonase in barley epidermal cell walls at the electron microscope level. The localization of the polygalacturonic acid substrate was accomplished by exposing thin sections of the walls to purified Cochliobolus sativus endopolygalacturonase, followed by exposure to a murine monoclonal antibody specific to the enzyme and finally goat-anti-mouse conjugated to colloidal gold. The resulting substrate localization pattern was identical to that obtained directly with JIM 5, a monoclonal antibody specific for tmesterified pectin. We refer to this indirect localization method as EMSIL (Enzyme-Mediated Substrate Immunolocalization). Potential advantages of this indirect method relative to direct substrate localization methods are discussed.
1. INTRODUCTION
It is generally accepted that cell wall degrading enzymes (CWDEs) produced by plant pathogens during penetration and subsequent infection of their hosts, are crucial to pathogenesis (Misaghi, 1984; Cooper, 1983; and Hahn et al., 1989). Activities attributed to these CWDEs include not only the enzymatic cleavage of structural cell wall polysaccharides, thus compromising the integrity of the cell wall-barrier to pathogen entry, but also more
732 complex roles within the sphere of host-pathogen interactions such as the release of oligosaccharides that function as "elicitors" of plant defense responses (Hahn et al., 1989). In spite of current evidence and consensus as to the importance of CWDEs in pathogenesis, unequivocal evidence for the sole responsibility of any particular enzyme for virulence in a given system has yet to be presented. Rather, it appears that multiple enzymes act in concert or succession to achieve the state of successful pathogenesis (Bateman and Basham, 1976). In addition, much of the research concerned with the importance or requirement of specific enzymes during pathogenesis fails to address the specific roles of these enzymes in cell wall degradation. A better understanding of the roles of CWDEs during normal pathogenesis would likely render interpretation of these enzyme studies more meaningful. Such understanding would include a thorough knowledge of the composition and distribution of components within the host cell wall. Although the general .composition and distribution of plant cell wall polymers are known (Roberts, 1989), the specific distributions of cell wall components in areas traversed by penetrating pathogens are unknown for most systems studied. Without this information, meaningful conclusions concerning enzymatic penetration are limited. One method for determining the distribution of cell wall components is by application of cytochemical probes for the various wall components at the electron microscope level. Colloidal gold particles have been utilized as cytochemical markers at the electron microscope level since the early 1970's. During this time, molecules of several categories appropriate for investigating plant cell walls cytochemically have been conjugated to gold particles for probe production, including immunoglobulins, lectins, enzymes and various other proteins not included in these catagories (see Handley, 1989 for a historical reveiw). These probes exploit the affinity of the conjugated ligand for specific sites in the subject material. For most of the probe catagories mentioned above, the labeling mechanism is easily understood; antibodies bind respective antigens strongly, lectins bind specific polysaccharides with high affinity, etc. The mechanism by which enzyme-gold probes label their respective substrates in situ is less easily understood. The interaction between an enzyme and its respective substrate is typified by substrate recognition, substrate binding, catalysis and dissociation. Thus, the in situ recognition of substrate by an enzyme-gold probe is understandable but why such a probe would remain associated with its substrate in lieu of subsequent catalysis and dissociation is not fully understood. Not withstanding, the fact remains that numerous enzyme-gold probes have been applied successfully for many years (Bendayan, 1989). During a preliminary study of the early host/pathogen interaction between C. sativus (Ito and Kuribayashi) and H. vulgare L., we obtained microscopic and cytochemical evidence that pectin degrading enzymes are utilized by the fungus during the early penetration of the barley host (Clay, 1995). This evidence plus findings from the C. carbonum endopolygalacturonase (EPG) gene-disruption experiment by Scott-Craig et.al. (1990) led us to investigate the contribution of fungal EPG to the early infection process operative in the H. vulgare/C, sativus system by application of cytochemical techniques at the electron microscope level. A particular objective was to utilize the monoclonal antibody JIM 5 (Knox etal., 1990) specific for non-esterified pectin to visualize the progressive alteration or loss of EPG substrate from the host plant cell wall during penetration by the fungal pathogen. Although unesterified pectin (polygalacturonic acid) is the apparent substrate of the EPG, there was a need to confirm that the pectic polymer recognized by JIM 5 was indeed the substrate recognized by the EPG. Thus, we produced, purified and characterized an
733 extracellular EPG of C. sativus and subsequently generated both polyclonal and monoclonal antibodies to this EPG. The pectic substrate of the EPG was then localized at the typical penetration site of barley leaf epidermis by using a novel application of purified EPG and one of the anti-EPG monoclonal antibodies. The labeling pattern obtained with the purified EPG was identical to that obtained with JIM 5, thus supporting the singularity of the JIM 5 antigen and the EPG substrate. Subsequently, the loss of EPG substrate from the host plant cell walls during progressive stages of pathogen ingress was visualized cytochemically at the electron microscope level (Clay, 1995). The method by which the purified EPG was utilized to indirectly localize the substrate of the enzyme in situ constitutes a novel cytochemical method and is the subject of the present work.
2. M E T H O D S
Cytochemical localization of pectin in barley leaf epidermal cell walls was performed at the electron microscope level by both direct and indirect means. Direct immunocytochemical localization of pectin was accomplished using a monoclonal antibody (JIM 5) specific for non-esterified polygalacturonic acid (Knox et.al., 1990), followed by a secondary antibody (goat anti-rat IgG, whole molecule, Sigma Cat. # R-5130) coupled to colloidal gold. Antibody-gold conjugates were made and immunolabeling was performed as described in Freshour et.al. (1995). Tissue to which the direct immunolocalization was applied was processed for electron microscopy according to Knox et.al. (1990) using LR White resin as the embedding medium. Sections 100 nm thick were cut with a diamond knife on an RMC (Tucson, AZ) ultramicrotome, picked up on gilded slot-grids and placed on formvar bridges (Rowley and Moran, 1975) to dry. After immunolabeling, sections were post-stained with 4% (w/v) uranyl acetate and lead citrate (Reynolds, 1963) and observed with a Zeiss EM10 or EM902 electron microscope. Experimental controls for the specificity of the direct immunocytochemical localizations with JIM 5 included omission of the primary antibody, pre-incubation of primary antibody with polygalacturonic acid, and substitution of the primary antibody with an extraneous monoclonal antibody derived from rat. Indirect immunolocalization of pectin was accomplished using 70 nm thick sections of host tissue processed and embedded in Quetol 651 according to the resin manufacturer's recomendations. For labeling, sections were exposed to purified EPG as outlined in Bendayan (1989) for enzyme-gold probes, followed by exposure to a primary antibody (EPG-4) specific for the EPG and finally to a secondary antibody (goat anti-mouse IgG, whole molecule, Sigma Cat. # M-8642) coupled to gold. Antibodies were applied as outlined in Freshour et.al. (1995) and sections post-stained as described above. The authors have coined the term EMSIL (Enzyme-Mediated Substrate ImmunoLocalization) for this indirect localization technique. Controls applied to EMSILs included omission of the enzyme, omission of primary antibody, and substitution of the primary antibody with an extraneous murine monoclonal antibody of the same type-class. Cellulase-gold was made and applied according to (Berg et al., 1988) with sections from material embedded in Queto1651. Chromatographically purified cellulase complex from Trichoderma reesei was obtained from Worthington Enzymes (Cat. # CEL).
734
3. RESULTS
The EMSIL obtained with the purified EPG on transverse sections of barley leaf epidermal cells taken pependicular to the long axis of the cells and anticlinal to the leaf surface, revealed that EPG substrate is localized primarily in the cell comers and middle lamella of these cells (Fig. 1).
~ .-~~i-i.~i!ii~!~-.
..-..
: ?:-.. .":21
,,^
~,
..-,
,
..~
-..
Figure 1. Transverse section of barley leaf epidermal cells taken perpendicular to the long axis of the cells and anticlinal to the leaf surface. The section has been labeled by the EMSIL technique (see Methods) utilizing purified C. sativus endopolygalacturonase and monoclonal antibody EPG-4, which is specific for this enzyme, in order to localize the substrate of the enzyme at the typical site penetrated by the fungal pathogen. Bar = 1 lam. Inset: Comparable cell wall region as in Fig. 1 but labeled with monoclonal antibody JIM 5 to localize non-esterified pectin. Bar = 1 lam. Note the identical labeling patterns obtained with either method.
735 Immunogold labeling with JIM 5 exhibited an identical labeling distribution for polygalacturonic acid as was obtained indirectly with the EPG EMSIL (inset of Fig. 1). Control experiments for labeling specificities obtained by the direct or indirect methods resulted in total elimination of specific labeling. The cellulase-gold probe heavily labeled the epidermal cell walls (Fig. 2).
Figure 2.
Advanced stage of barley leaf penetration by C. sativus. The pathogen has penetrated the anticlinal cell wall junction between two host epidermal cells (e). The fungal appressorium (a) is visible above the cell comer. The host cell comer matrix has been displaced by an enlarged hyphal element (h) situated between the thin cell walls of the host epidermal cells. The host epidermal cell walls have been densely labeled with the cellulase-gold probe. An intercellullar hyphal element (ih) is present within the penetrated host cell. Bar = 1 laM.
Advanced stages of penetration by the fungus were characterized by displacement of the pectin-rich cell comer regions with concurrent stretching of the cellulose-rich primary cell walls (Fig. 2).
4. DISCUSSION
The EMSIL method may prove useful in cytochemical applications where lack of substrate immunogenicity precludes antibody generation, or for which enzymes specific to the
736 substrate cannot be labeled for use in enzyme-gold localizations due to insufficient size of the enzyme molecule, instability of the enzyme, inactivity of gold-bound enzyme, lack of enzyme purity, etc. EMSIL would also be appropriate when an enzyme is extremely difficult or costly to purify, rendering it unfeasible to obtain quantities of purified enzyme sufficient for coupling directly to gold. With the EMSIL technique, it may be possible to utilize a defined mixture of enzymes as a primary cytochemical reagent, followed by a complimentary mixture of enzyme-specific antibodies, each coupled to a different-sized gold colloid, thus labeling several different substrates in one step, on one thin-section. Accordingly, it may be be possible to simultaneously visualize the degradation or loss of multiple substrates at a given location within the host-pathogen arena.
5. REFERENCES
Bateman D. F. & Basham H. G. (1976). Degradation of plant cell walls and membranes by microbial enzymes. In: Heitefuss R. & Williams P. H., ed.; Encyclopedia of Plant Physiology, Vol. 4. New York: Springer-Verlag, 316-355. Bendayan M. 1989. The enzyme-gold approach: a review. In: M. A. Hayat, ed. Colloidal Gold: Pnnciples, Methods and A pplications. Vol. 2. Academic Press, Inc., New York, 118-145. Berg R. H., G. W. Erdos, M. Gritzali and R. D. Brown. (1988). Enzyme-gold affinity labeling of cellulose. Journal of Electron Microscopy Techniques 8:371-379. Clay R. P. (1995). Studies of the mechanism of host penetration during the infection of Hordeum vulgare by Cochliobolus sativus. Ph.D. Dissertation, University of Georgia, Athens, GA, USA. Cooper M. R. (1983). The mechanisms and significance of enzymatic degradation of host cell walls by parasites. In: Callow J. A., ed. Biochemical Plant Pathology. John Wiley & Sons Ltd., 101-135. Freshour G., R. P. Clay, M. S. Fuller, P. Albersheim, A. Darvill and M. G. Hahn. (1995). Developmental and tissue-specific structural alterations of the cell wall poysaccharides of A rabidopsis thaliana roots. Plant Physiology 110:1413-1429. Hahn M. G., Bueheli P., Cervone F., Doares S. H., O'Neill R. A., Darvill A. & Albersheim P. (1989). Roles of cell wall constituents in plant-pathogen interactions. In: Nester E. & Kosuge T., ed. Plant Microbe Interactions, Vol. 4. McGraw-Hill Publishing Co., 131-181.
737
Handley D. A. (1989). The development and application of colloidal gold as a microscopic probe. In: M. A. Hayat, ed. Colloidal Gold: Principles, Methods and Applications. Vol. 1. Academic Press, Inc., New York, 1-12. Knox J. P., Linstead P. J., King J., Cooper C. & Roberts K. (1990). Pectin esterification is spacially regulated both within cell walls and between developing tissues of root apices. Planta 181:512-521. Misaghi I. J. (1982). The role of pathogen-produced cell-wall-degrading enzymes in pathogenesis. In: Physiology and Biochemistry of Plant Pathogen Interactions. Plenum Press, 17-34. Reynolds E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. Journal of Cell B iology 17:208-212. Roberts, K. (1989). The plant extracellular matrix. Current Opinion in Cell Biology 1:10201027. Rowley J. C. III and D. T. Moran. (1975). A simple procedure for mounting wrinkle-free sections on formvar coated slot grids. Ultramicroscopy 1:151-155. Scott-Craig J. S., Panaccione D. G., Cervone F. & Walton J. D. (1990). Endopolygalacturonase is not required for pathogenicity of Cochliobolus carbonum on maize. The Plant Cell 2:1191-1200.
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J. Visserand A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996ElsevierScienceB.V.All rightsreserved.
739
I n f l u e n c e of glucose a n d p o l y g a l a c t u r o n i c acid on t h e s y n t h e s i s a n d a c t i v i t y of t h e p o l y g a l a c t u r o n a s e f r o m t h e y e a s t s t r a i n S C P P A. Gainvors and A. Belarbi Laboratoire de Microbiologie G~n~rale et Mol~culaire, Facult~ des Sciences, Europor Agro, B.P. 1039, 51687 Reims cedex 2, France. Abstract The yeast strain SCPP exhibits all of the panoply required for the degradation of pectins from various sources. This enzymatic machinery is secreted into the growth medium. Hence, we developped interest on the regulatory mechanisms controlling the synthesis of the main activity produced by the SCPP strain, i.e. the polygalacturonase. We observed that this activity is maximum five days after culture inoculation. Its biosynthesis is influenced by the glucose concentration. It is also stimulated by pectin concentrations similar to the ones seen in fruit juices.
1. INTRODUCTION Pectinases find industrial application in the food-industry during the extraction and stabilization ~f juices. Grape must contains pectins that are frequently removed by the ac:~ion of fungal pectinases. It would, however, be preferable to use Saccharomyces cerevisiae yeast strains that could produce pectinases. The catalytic capacity of several excreting pectolytic enzymes obtained from various yeast strains was examined using in vivo and biochemical techniques. Of the 33 yeast strains studied 30 were isolated from champagne wine during alcoholic fermentation. Only one yeast strain was found to excrete pectolytic enzymes and was identified as Saccharomyces cerevisiae designated SCPP. Three types of pectolytic enzymes were found to be excreted by SCPP : polygalacturonase (PG), pectin-lyase (PL) and pectin-esterase (PE) [1]. The polygalacturonase was studied and this enzyme was secreted constitutively both under anaerobic or aerobic conditions. A few physico-chemical properties of the secreted PG have been determined (PM, PI) [2]. The results obtained regarding its stability and pH-optimum are encouraging for its potential industrial usage. In effect, the SCPP strain is able to degrade pectins with varying esterification levels, thus participating in the clarification phenomena of fruit juices. The clarification potential of that strain was compared with the one of Several commercially available pectolytic mixes[3]. Recently, Gainvors and Belarbi [4] have set up a screening method for the selection of Saccharomyces cerevisiae yeast strains exhibiting pectinolytic
740 activities. This method is based on a particular physiological characteristic of this type of yeast. PG representing most of the pectinolytic power of the SCPP strain, we focussed on the role of glucose and polygalacturonic acid on the synthesis of this enzyme. 2. P O L Y G A I ~ C T U R O N A S E :ACTIVITY-SYNTHESIS R E L A T I O N S H I P Our study deals with the polygalacturonase activity in its globality and does not concern individual isoforms [2]. Gainvors et al. [1] have shown that the release of reducing groups in Pg glc growth culture medium (].% polygalacturonic acid, 1% glucose, 6.7 g/1YNB Base, 50 mM phosphate buffer pH 5.5) by the SCPP strain is significant after only 24 hours of culture. Even though it is very weak at first, it rises over the first four days of culture before reaching a plateau. Two distinct phases can be distinguished in the kinetic of release of reducing groups in order to study "~he regultion and synthesis of PG by the SCPP strain. Thus, we decided to estimate the effect of glucose on the PG activity at the start of the culture and define the cause of the stabilization phase in the release of the reducing groups Three hypothesis can be made in order to explain the existance of such a plateau: - The stable concentration of reducing groups may suggest that PG is absent from the culture medium on the fourth day. In the case PG is present, the plateau can be obtained by an inhibition of the enzymatic activity. - or by the enhanced breakdown of the reducing groups freed by the SCPP strain. -
In order to choose among these various hypothesis, we followed the kinetic of secretion of the polygalacturonase in the Pg glc medium. Hence, the proteins present in daily aliquots have been acetone precipitated [1]. The protein extracts were then either placed oil a reaction medium to assay for their PG activity [1] or studied by zymogram [5].
741 0,8
0,7
m
0,6 f~
0,5 SCPP 0,4 --O--- X2180-1A
~ 0,3 ~ 0,2 =L 0,I
2
3
4
5
6
Days
F i g u r e 1 9PG activity m e a s u r e m e n t of crude protein e x t r a c t of the S C P P s t r a i n grown on Pg glc m e d i u m C r u d e p r o t e i n e x t r a c t s a n d r e a c t i o n m e d i a w e r e m a d e as d e s c r i b e d by G a i n v o r s et al. [1].
Measurements were made on the reaction medium as described by Milner et Avigad [6].
W h e n studied on the PG glc m e d i u m , the PG e n z y m e of the S C P P s t r a i n is a l r e a d y p r e s e n t on the first day of culture and keeps on being secreted until the sixth day of culture (figure 1). On the other hand, the X2180-1A s t r a i n does not display and polygalacturonase activity during this period of time.
F i g u r e 2 9PG activity detection by zymogram[ 5] of the S C P P s t r a i n cultivated on Pg glc m e d i u m for two a n d five days Each deposited volume contains all ot' the proteins secreted by 5. 105 cells. 1 9Culture medium of SCPP cultivated on Pg glc medium for 5 days 2 9Culture medium of SCPP cultivat(.d on Pg glc medium for 2 days 3 9Culture medium of X2180-1A cultivated on Pg glc medium for 5 days
742 This results were confirmed by zymogram where a greater PG activity was observed on the fifth day over the second day (figure 2). Both techniques agree with each other and suggest a gradual enhancement of PG over time. At this point of our study, the stabilization in the release of reducing groups cannot be attributed to a disappearance of enzymatic activity beyong the fourth day. This phenomenom can thus only be explained on two ways : the first one is based on the idea t h a t galacturonic acid molecules are not metabolized by the SCPP strain. In the case, the reducing groups would accumulate to a given value which inhibits PG activity. Since the enzyme is still present in the medium, its inhibition would lead to a reduction in reducing group formation. the second one m a k e s the hypothesis t h a t the reducing groups are metabolized by the yeast cells, thus leading to an equilibrium between reducing groups biosynthesis by 1;he enzyme (still present in the medium) and their degradation by the SCPP strain. Earlier results obtained in our laboratory showed t h a t the concentrations of galacturonic acid found in the culture medium after four days of culture do not inhibit PG activity [2]. We, thus, favored the last hypothesis. This one is actually supported by an observation made during a search on PG activity of a dry active yeast. In the case of this strain, the quantity of reducing groups is m a x i m u m on the third day of culture and sharply decreases thereafter (figure 3), indeed suggesting that galacturonic acid molecules are utilized by this yeast strain and t h a t its basal PG activity is strong enough to lead to a fast enough renewal of the reducing groups and this maintain the equilibrium. Moreover, we have shown t h a t the SCPP s t r a i n is able to metabolize galacturonic acid [4]. -
-
0,8 0,6 O
--a-- SCPP .2 0,4
LSA X2180-1A
"6 0,2 -i
0,0
9
o
1
2
.
.
3
4
days
-
.
5
6
F i g u r e 3 9Comparison o:? the kinetics of release of reducing groups by various yeast strains grown on Pg glc medium Strains were grown at 30~ as non agitated cultures. Measurements were made on 300 ILl of the culture supernatants as described by Milner et Avigad [6].
743 This study has allowed us to demonstrate the existance of polygalacturonase after four days of culture, its activity being m a x i m u m on the fifth day. We have also observed t h a t the release of reducing groups stabilizes on the fourth day. The most plausible h y p o t h e s i s b e h i n d this p h e n o m e n o m is b a s e d on the existance of an equilibrium b e t w e e n the freeing of r e d u c i n g groups after polygalacturonic acid degradation and their utilization by the SCPP strain. 2. E F F E C T O F G L U C O S E O N T H E S Y N T H E S I S A N D E N Z Y M A T I C ACTMTY OF POLYGAI~CTURONASE
2.1. Effect of g l u c o s e on p o l y g a l a c t u r o n a s e b i o s y n t h e s i s In order to estimate the effect of glucose on the release of reducing groups in the culture medium, the SCPF' strain has been inoculated on Pg glc media with glucose concentrations ranging from 0.1% to 1%. The g r o w t h of the S C P P s t r a i n d e p a n d e d on t h e s e different glucose concentrations in the culture medium. It was raised by increasing glucose concentrations. Hence, in order to compare the release reducing groups in these media, we have expressed our results in terms of nmol of reducing groups freed per 0.5 unit of adsorbency at 600 nm which corresponds to 5.106 cells, i.e., the weakest cellular concentration obtained with 0.1% glucose. Figure 4 shows t h a t the highest release in reducing groups can be obtained with 0.25% glucose. This result r e m a i n s valid on the first and third day of culture.
120 m~ 110 100 ~ g 90
......
-
80
~N 70
"~a~~ 60 ~. 50
"iii~ii. . . . . ~ .............. .._.|
10 0
II !~1 11 P"A
1% glucose 0,5%glucose 0,25%glucose 0,1%glucose
~ 1
days
3
F i g u r e 4" Effect of glucose on reducing groups release by SCPP Measurements were achieved after growing the strain in the presence of 1% polygalacturonic acid and varying concentrations of glucose. They were done on the culture supernatants as described by Milner et Avigad [6].
744 It is difficult to interprete the result obtained when the SCPP strain was cultived in the presence of 0.1% glucose because its growth on such a medium was severely reduced. In the light of these results, it seems that PG activity is minimal at an initial glucose concentration of 1% in the presence of 1% Pg. It is maximal at an initial glucose c o n c e n t r a t i o n of 0.25% with the same polygalacturonic acid concentration. The best g r o w t h - P G activity ratio is t hus obtained with this l at er concentration. 2.2. Effect of g l u c o s e on PG activity A crude protein extract has been prepared by acetone precipitation on a three days old culture supernatant of the SCPP strain on Pg glc medium. In order to estimate the effect of glucose on PG activity, these protein extracts were deposited as dots on solid Pg glc medium. On this medium, the protein extracts of the SCPP strain exhibited PG activity similar to the one obtained on 1% Pg medium (data not shown). Hence, it seems t h at at this concentration (1%) glucose does not inhibit the PG activity of the SCPP strain. 3. E F F E C T OF P O L Y G A L A C T U R O N I C ACID C O N C E N T R A T I O N ON THE ACTIVITY AND SYNTHESIS OF PG The PG activity of the SCPP strain is expresssed on a constitutive basis. We have, however, observed that it rises in the presence of 1% Pg [1]. In order to determine the minimal substrate concentration capable of stimulating PG activity, we constructed Pg glc media containing 1% glucose and increasing concentration',5 of polygalacturonic acid (ranging from 0 to 10 g/l). These media were inoculated with 5.105 cells per ml and incubated at 30~ for three days in full tubes without stirring. The yeast cells were removed by centrifugation. Then, 500,~1 of the supernatants were acetone precipated, placed in reaction media and assayed for PG activity. Yeast growth was similar on all culture media whatever the polygalacturonic acid concentration used. Cell density was about 3.5.107 cells/ml. These results depicted on figure 5 are expressed in terms of nmol of reducing group per ml and min.
745
~ 0,5
-
,Phase 1
~ 0,4 ~o
E
Phase
3
-
S
0,3
. . 0,2 o
Phase 2
9
0
I
'
1
I
2
'
I
I
3
4
'
I
5
'
I
6
'
I
I
7
8
'
I
'
9
I
10
g/1 polygalacturonic acid
F i g u r e 5 : Detection of PG activity of crude protein extracts of the SCPP strain in the presence of 1% glucose and varying concentrations of polygalacturonic acid. Crude protein extracts and reaction media were made as described by Gainvors et al. [1]. Measurements were made on the reaction medium as described by Milner et Avigad [6].
These curve exhibits four distinct phases depending on the polygalacturonic acid concentration in the culture medium. Phase 1 (0 to 1.25 g/l) : PG activity corresponds to basal levels. The speed of substrate hydrolysis is 0.28 nmol/ml/min. Phase 2 (1.8 to 3.5 g/l) : rises in PG activity correspond to enhancements in polygalacturonic acid concentration in the culture medium. It is stimulated by a factor of 1.35 at a polygalacturonic acid concentration of 350 mg/1. Phase 3 (5 to 7.5 g/l) : At these concentrations, a drop in PG activity is observed. This phenomenom is reprocible and can be attributed to pectin-protein interactions. These interactions only occur between specific pectin-protein couples after a first depolymerization action of PG [7]. Depolymerized pectins associate with proteins to yield aggregates which can easily sediment. Their removal during medium centrifugation would explain the lowering in PG activity in the reaction medium over this polygalacturonic acid concentration range. Phase 4 (7.5 to 10 g/l) : A phenomenom similar to the one observed during phase 2 occurs, i.e., PG activity rises with the polygalacturonic acid concentration. Highest PG synthesis is actually obtained with 1% pectin, yielding a total stimulation factors of 1.7. PG induction levels are admittedly very weak. We would have been better off if we had taken the effect of glucose concentration on PG synthesis into account and had carried our experiment at a glucose concentration of 0.25%. 4. C O N C L U S I O N AND D I S C U S S I O N We display evidences showing t h a t glucose has inhibitory effects on PG
746 organisms [8-9]. Some of them are plant pathogens, PG enzymes playing a major role during cell penetration and break-down. It seems that the presence of large quantities of glucose in severals plants leads to the inhibition of the proliferation of the pathogenic micro-organisms [10-13]. Additionnally, we show that the PG activity of the SCPP strain is regulated. It is stimulated by polyglacturonic acid concentrations similar to the ones seen in various fruit juices. On an industrial point of view, this induction would be diminished by the elevated glucose concentrations found in these fruit juices when compared to the pectin concentrations. Basal activities would be sufficient to eliminate all pectins and obtain juice stabilization. It would also have been i n t e r e s t i n g to study the effect of glucose concentrations of 15% and 20% (concentrations found in fruit juices, beers and ciders) on the PG activity of the SCPP strain. The lack inhibition of enzymatic activity by glucose still leaves the PG enzymes produced on a constitutive basis free to hydrolyze their substrate. Glucose and polygalacturonic acid concentrations would have to be taken into account if an optimum production of polygalacturonases is to be obtained for its purification.
5. REFERENCES 1 A. Gainvors, V. Fr6zier, H. Lemaresquier, C. Lequart, M. Aigle and A. Belarbi. Yeast, 10 (1994a) 1311. 2 C. Lequart, A. Gainvors and A. Belarbi. Enzyme Microb. Technol., (Submitted) 3 A. Gainvors, N. Karam, C. Lequart and A. Belarbi. Biotechnol. Letters, 16 (1994b) 1329. 4 A. Gainvors and A. Belarbi. Yeast, 11 (1995)in press. 5 R.H. Cruickshank and G.C. Wade. Anal. Biochem, 107 (1980) 177. 6 Y. Milner and G. Avigad. Carbohyd. Res, 4 (1967) 359. 7 B. Perez. M6moire National d'(Enologue. Univ. Reims Champagne-Ardenne (1990). 8 F. Federici. Antonie van Leeuwennoek, 51(1985) 139. 9 V.E. Shevchik, A.N. Evtushenkov, H.V. Babitskaya and Y.K. Formichev. World J. Microbiol. Biotechnol, 8 (1992) 115. 10 J.C. Horton and N.T. Keen. Phytopathology, 56 (1966) 908. 11 S.S. Patil and A.E. Dimond. Phytopathology, 58 (1968) 676. 12 G. Holz and P.S. Knox-Davies: Physiol. Mol. Plant Pathol., 28 (1986a) 403. 13 G. Holz and P.S. Knox-Davies. Physiol. Mol. Plant Pathol., 28 (1986b) 411.
Acknowledgements We t h a n k L. Legendre for the translation of this manuscript. This work has been supported by Pascal Biotech sarl Paris, AEB Spindal and Europol'Agro.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
747
Pectin lyase from Fusarium oxysporum f. sp. radicis lycopersici" purification and characterization M.A. Guevara a, M.T. Gonz~ilez-Ja6n b and P. Est6vez a aDepartamento de Biologia Vegetal, Facultad de Biologia, Universidad Complutense, 28040 Madrid, Espafia. bDepartamento de Gen6tica, Facultad de Biologia, Universidad Complutense, 28040 Madrid, Espafia.
Abstract A pectin lyase has been purified from Fusanum oxysporum f. sp. radicislycopersici. Proteins from cultures of 4 days on pectin, were precipitated with ammonium sulphate and separated with a Superdex 75HR1030 column and by preparative isoelectric focusing (LKB column of 110 ml capacity). A single band, with isoelectric point of 9.20, was detected by silver staining on analytical isoelectric focusing. The molecular mass, calculated from its partition coefficient on the Superdex column, was 18 kDa. The highest activity of this enzyme was attained at pH 9.5 and 50 ~ C. The pectin lyase showed high specificity for pectin, an "endo" mode of action and calcium dependence.
1. INTRODUCTION Fusarium oxysporum f.sp. radicis-lycopersici Jarvis and Shoemaker (FORL) (Jarvis and Shoemaker, 1978) [1] is a pathogen of tomato which, with the arrival of intensive tomato culture under glass, has developed to serious proportions [2]. This forma specialis of F. oxysporum affects largely the root and crown tissues of tomato and the symptoms occur as foot and root rot. FORL isolates are pathogenic on tomato plants with genes for resistance to races 1 and 2 of Fusarium oxysporum Schlecht. f.sp. lycopersici (Sacc.) Snyd. & Hans (FOL), that cause the common Fusarium wilt of the tomato. However, although resistance to FORL has been found and incorporated into commercial cultivars, the disease is a severe problem in wide areas of the North Hemisphere [3-9]. Colonization of tomato root tissues by FORL is associated with striking modifications of host cell walls, as it has been shown by ultrastructural studies which have been carried out describing the penetration of the fungus through
748
infected root tissues [4, 10-19]. The pattern of penetration, with disruption or even loss of middle lamella matrices [16], implicates production of pectic enzymes by FORL [11]. In fact, pectic enzymes have been considered to play a critical role in parasitism involving pathogens of dicotyledons, in which rhamnogalacturonan has a key role in wall structure [20]. The r2 isolate of Fusanum oxysporum f. sp. radicis-lycopersici (FORL) produced several pectic enzymes that differ in substrate preference, reaction mechanism, and action pattern. We have detected three forms that have lyase activity, an absolute requirement for calcium, and pIs of 9.20, 9.00 and 8.65. The two most alkaline forms had a weak preference for pectin whereas the other was more active on pectate. The three lyases were produced when the fungus grew on pectin and on restricted galacturonic acid (data presented in the "XV Congreso National de Microbiologia" [21] and sent for publication). The objective of this work has been the purification of the pectin lyase with pI 9.20, which is the most abundant.
2. MATERIALS AND METHODS
Fungus The isolate of F. oxysporum f.sp. radicis-lycopersici used in this study was strain r2, supplied by Dr. J. Tello (Instituto Naeional de Investigaciones Agrarias, Madrid). It was isolated as a single-spore culture, from an infected tomato plant (Lycopersicon esculentum Mill.) and grown on potato sucrose agar (PSA) at 22 ~ C. The ability of the isolate to the infect tomato was periodically checked as described by S~nehez et al. [22]. Stock cultures were maintained in Petri plates on potato sucrose agar at 5~ (transferred every month) and in soil for long time preservation.
Enzyme production FORL cultures were grown in 250 ml Edenmeyer flasks containing a carbon source in 100 ml salts medium shaken (in)on a rotary incubator (150 rev.min 1) at 22~ The medium contained easamino acids 0.46, KHzPO 4 0.1, MgSO4.7H20 0.05g/100ml, FeSO4.7H20 0.2, ZnSO4.7H20 1.0, NazMoO4.2H20 0.02, CuSO4.5H20 0.02, MnC12.4H20 0.02 ~tg m1-1. Flasks were inoculated with 1 ml of distilled water containing 1 x 10 6 conidia obtained by flooding fungal colonies on potato sucrose agar. The carbon source (pectin, galacturonic acid or glucose) to shake cultures, was added at 0.5% (w/v) or supplied from diffusion capsules for restricted supply [23, 24]. The capsules, containing D-galacturonic acid or glucose (30g/100ml), were provided with membrane layers allowing
749 linear rates of release of sugar over 20 to 24h, so that capsules needed to be changed only once daily. Capsules with membrane layers were sterilized at 120~ for 10 min and filled with the carbon source (sterilized by filtration) before placing in cultures. The pH of cultures was adjusted at 5.5 with NaOH before sterilization. Cultures with restricted supply of the carbon source were first grown on 0.5% (w/v) glucose for three days on a rotary incubator (150 rev.min ~) at 22~ Then, cells were removed from culture fluid by centrifugation (1800 g, 30 min), and grown for one day on 100 ml fresh medium supplied with the capsule containing glucose. After one day with restricted supply of glucose, cultures were grown for three more days with restricted supply of galacturonic acid or with restricted supply of glucose. For cultures on pectin, 1 ml of grown cells for three days on glucose, were transfered to fresh inorganic salts medium with 0.5% (w/v) pectin (apple pectin, Fluka) and grown for six days. Cultures from different times of growth were collected. Culture fluids were cleared by passing through glass fibre filter. After dialysis for 16-18 h against distilled water at 5~ filtrates were assayed for enzyme activities and proteins. Assay Method Pectin lyase (PNL) activity was measured spectrophotometrically by the increase in absorbance at 235 nm of the 4,5-unsaturated reaction products. Reaction mixtures containing 0.25 ml of culture filtrate, 0.25 ml of distilled water and 2.0 ml of 0.24% pectin from apple (Fluka) in 0.05M tris-HC1 buffer (pH 8.0) with lmM CaC12, were incubated at 37~ for 10 minutes. One unit of enzyme is defined as the amount of enzyme which forms l~tmol of 4,5unsaturated product per minute under the conditions of the assay. The molar extinction coefficients of the unsaturated products is 5550 M-~cm-~ [25]. Also viscosity measurements were made using Cannon-Fenske viscometers or Ostwald micro-viscosimeter, at 37~ Reaction mixtures consisted of enzyme solution and 0.75% pectin in 0.05 M tris-HC1 buffer (pH 8.0) with 0.5 mM CaC12. One unit is defined as the amount of enzyme required to change the inverse specific viscosity by 0.001 min -1 under the conditions of reaction. Specific viscosity (n~p) is (t/t0)-l, where t is the flow time (sec) of the reaction mixture and t o is the flow time of the buffer. The inverse~specific viscosity (n~p-~) is proportional to the incubation time and the amOunt of enzyme used [26]. Units of enzyme activity were determined for 10 min of reaction. Protein determ ination. Protein was determined by Lowry's method [27], using bovine serum
750
albumin (Sigma) as a standar.
Purification of Pectin Lyase Preparation of enzyme. Culture fluids of three days on glucose 0.5% (w/v) and then four days on pectin 0.5% (w/v), cleared by passing through glass fibre filter, were used for the purification of PNL. A small quantity was remainder, dialyzed, and assayed for enzyme actitity and the remained was precipitated. Ammonium Sulfate Precipitation. The extract was made up to 40% saturation with the slow addition, with stimng, of ammonium sulfate at 4~ After several hours, the precipitate was removed by centrifugation at 30,000 g for 30 min and the supematant retained. It was brought to 100% saturation in similar conditions, the precipitate was collected by centrifugation, dissolved in the minimum of distilled water, dialyzed against water and then against 1% glycine, and lyophilized. Gel Filtration. The lyophilized protein was redissolved in 50 mM phosphate buffer, pH 7.4; 0.15 m NaC1; 0.013 % sodium azide and loaded on a Superdex 75HR1030 column equilibrated with the same buffer. Elution was downward flow (0.15 ml/min) and 0.25 ml fractions were collected. Fractions with pectin lyase activity were combined, dialyzed against distilled water and used in the next step. To estimate the molecular mass of PNL, the column was calibrated with standard proteins (Sigma MW-GF-70: Albumin, 66,000 Da; Carbonic Anhidrase, 29,00; Cytochrome, 12,400; and Aprotinin, 6,500). The proteins were eluted in the conditions described above and their volumes (Vo) were calculated from the peak maximum of the absorbance at 280 nm. The partition coefficient was obtained from the relationship K ~ - (Vo-Vo)-(V~-Vo) where Vt represents the bed volume of column and Vo the void volume (which was calculated using blue dextran, Sigma). The molecular mass was determined using a standard curve of K,v vs the logarithm of the molecular masses of the standards [28, 29] Preparative Isoelectnc Focusing. The PNL eluted from gel filtration was subjected to isoelectric focusing using a column of 110 ml capacity (LKB). The density gradient was formed with sorbitol [0-50% (w/v)]. Enzyme extract was distributed equally between the two gradient component solutions prior to the establishement of the gradient using a gradient former. The concentration of the career ampholytes, Servalyte 7-9 and 9-11 (Serva), was 1,2% (w/v) and the catode was placed at the botton of the column. The experiment was performed at 7~ with constant power (9.6 W) giving a maximum voltage of about 1600 V. After 48 h, fractions of 3 ml were removed from the bottom of the column. The pH values of the fraction were inmediately measured at 7~
751
Ultrathin-layer analytical isoelectric focusing Proteins were separated according their pI by isoelectric focusing (IEF) at 7~ on a LKB 2117 Multiphor II apparatus. Ultrathin layers (0.4 mm) of polyacrylamide gels with ampholytes pH 2-11 were cast for isoelectric focusing as recommended by the manufacturer. Polyacrylamide solutions containing 5.2 % acrylamide (Pharmacia), 0.17 % N,N'-methylenebisacrylamide (Pharmacia), 1.1 ml of Servalyte career ampholytes (Serva), 0.6 ml of 1 % ammonium persulphate (Pharmacia) and 20 lal TEMED (Pharmacia), per 12.72 ml of the total volume, were cast on a glass as support using an Ultro Thin Layer Casting Tray (Bio Rad). Electrode wicks for the anode and catode were soaked in 1 M H3PO4 and 0.5 M NaOH respectively. Gels were preelectrofocused for 30 minutes at a constant 5.0 W. Samples of 10 ~g of protein in 10 lal were applied onto the gel via a small tab of glass fibre paper. Subsequent electrofocusing was earned out for 60 min at a constant 15 W with a maximum of 1400 V; sample application tabs were removed 30 min after focusing began. Broad pI Calibration Kit standards (Pharmacia) were used for pI estimation. Agarose overlays (2 mm thickness) for enzymes detection, containing pectin, were cast by capillary action between two glass plates separated by spacers. On one of these glass plates, a GelBond support film (LKB) was affixed by a thin film of water. The agarose solution was heated to 95~ and the gel mold was heated to 50~ before casting. The agarose solutions contained 1 % agarose, 0 . 1 % of pectin in 0.05M tris-HC1 buffer (pH 8.0) with lmM CaC12 [25]. After focusing, gels were incubated in the appropriate buffer for 5 min. Then agarose overlays were placed on the surface of the isoelectricfocusing gels, incubated at 37 ~ for 20 min and the overlays stained with 0.05% ruthenium red. Afterwards, isoelectricfocusing gels were stained with silver (Bio-Rad kit) for protein detection.
Substrate specificity and mode of action Mode of action and substrate specificity of the purified enzyme were determined by following the decrease in viscosity and the increase in absorbance at 235 nm in reaction mixtures in the presence of 0.187 % substrate (pectin or pectate) at pH 8.0.
Optimum pH Optimum pH was determined by following the decrease in viscosity of the reaction mixture using 0.187 % pectin as a substrate in 0.05 M tris-HC1 buffer (pH 7.0-9.0) or glycine buffer (pH 9-10). Controls were run without enzyme preparation.
752
Effect of temperature Optimum temperature was determined at pH 8.0 by following the decrease in viscosity of reaction mixtures containing 0.187 % pectin, at temperatures between 30 ~ and 55~ Controls were run without enzyme preparation. Requirem ent of calcium The effect of C a 2+ w a s assayed by viscosimetry in reaction mixtures containing 0.187 % pectin, buffered at pH 8.0, and ethylene diaminetetraacetic acid (EDTA) (0.005 M) or GaG12 (0-0.02 M).
3. RESULTS
Production of pectin enzymes on restricted galacturonic acid and on pectin FORL was grown on restricted galacturonic acid and on pectin in order to ascertain the production of lyases by FORL and if different forms were produced. Figure 1 shows the time course of PNL activity during growth of the fungus in the two culture conditions experienced: pectin lyase was produced both (1)
4 1
E ctO ('O Cq
< (D o
200 o 3
0.02 restricted restricted galacturonic/ /' glucose acid ? // /
< .-- 0.01 t~
6
e-
1
2
3
4
5
'~ ~, '\\
6
100
7
Days of culture
,'7"
0.09 E t-
tO
O4
._= it) GI
(2)
40 ~-
0.06
""
p tin
0.03
7
r
i
1
2
3
20 '~
"""
,~
5
6
Days of culture
7
i
i
8
9
Figure 1. Time-course of pectin lyase activity in cultures of F. oxysporum f. sp. radici s - l y c o p e rsici. Fungus was first grown on unrestricted glucose for three days, then the biomass was: or shifted to restricted glucose and, after one day, to restricted galacturonic acid (1); or shifted to pectin (2). Enzyme activity was determined as increase in A235 nm (-*-) and by viscosimetry (-o-) and determined for 10 min.
753
on galacturonic acid as well as on pectin. Maximum of activity was obtained at 60 h on galacturonic acid (1) and 106 h on pectin (2).
Purification of Pectin liase Culture fluids of four days on pectin were cleared by passing through glass fibre filter and fractionated by ammonium sulfate, gel filtration and preparative isoelectric focusing: Table 1 summarizes the purification steps. A peak with PNL activity was eluted from the Superdex 75HR1030 column (figure 2) and subjected to preparative isoelectric focusing (figure 3). Table 1 Step
Protein
Total activity
Specific activity
Purification
Yield
mg
U
U/mg
fold
%
Extract
35.96
995.92
27.69
-
100
(NH4)2SO4
2.50
62.76
25.10
0.91
6.30
Gel Filtration
0.24
36.99
154.12
5.56
3.71
IEF
0.03
23.59
899.35
32.48
2.36
"13 0
!
~0
0.06
5"
:r,. .
E o~ o
ro
c~
m
o o3
5
0.03 ,-,
I
>
-
0
10
20
_
30
| | |
.
40
50
60
70
80
i
_
_
90
Fraction no.
Figure 2. Gel filtration. The dry residue obtained after ammonium sulfate precipitation was redissolved in 50 mM phosphate buffer, pH 7.4; 0.15 M NaC1; 0.013 % sodium azide, which was loaded on a Superdex 75HR1030 column equilibrated with the same buffer. Elution was downward flow (0.15 ml/min) and 0.25 ml fractions were collected. The fractions were assayed for protein content ( - - ) and PNL activity (-r
754
12
\
8"1" \
I
0
\\
r
A
0
5
10
_ _ _ I . . . .
i
20
25
15
. . . .
I
30
m
_
e
Fraction no.
Figure 3. Preparative isoelectric focusing. The PNL eluted from gel filtration was subjected to isoelectric focusing using a column of 110 ml capacity (LKB) with ampholytes pH 7-11. After 48 h (9.6 W constant power), fractions of 3 ml were removed and assayed for PNL activity ( § and pH (- -).
Molecular Weight The PNL, eluted from the Superdex column, showed a molecular weight of around 18 kDa (figure 4). lsoelectric Point Figure 5 shows the pattem of lyase isoenzymes along the purification process: at first, three bands with lyase activity (pls 9.20, 9.00 and 8.65) were detected in the ammonium sulfate precipitate (B 1); in the peak eluted from the Superdex 75HR1030 column, only one band with lyase activity was detected, that correspond to the PNL with pI 9.20 (B 2), but more proteins were detected by silver staining (A 2).
121 r
0
~
PNL
40
L.. m
~
1
80
2010-
~ -
4
4 1
0
0.1
I
I
I
0.2
0.3
0.4
K av
Figure 4. Estimation of molecular weight by calibration of Superdex 75HR1030. Standard proteins: 1, Albumin (66,000 Da); 2, Carbonic Anhidrase (29,000 Da); 3, Cytochrome c (12,400 Da); 4, Aprotinin, (6,500 Da). The line has been drawn using the equation lg m = 5.01930882 2.757789171 * kay; r = - 0.9965.
755
However, after the preparative isoelectric focusing column, the PNL was the only band detected both by lyase activity staining (B 3) and by protein staining (a 3).
A 9.30 8,65 8.45 8.15 7.35 6.85 6.55 --5.85
M 1
2
3
B 1
2
3
9 ~''~i
9.20 9.00 8.65
5.20 4.55 3.50
Figure 5. Analytical isoelectric focusing. Ultrathin layers (0.4 mm) of polyacrylamide with ampholytes pH 2-11 were used. Samples of 10 lag of protein in 10 lal of 1% glycine were applied. A.- Silver staining. B.- Stain for activity on overlays containing pectin in tris/HC1 buffer at pH 8.0 with CaC12. M.- Broad pI Calibration Kit protein (Pharmacia), samples of 5 lag of protein were applied. 1.Ammonium sulphate precipitated proteins from cultures on pectin. 2.- Fractions with PNL activity eluted from the Superdex 75HR1030 column. 3.- Purified PNL.
Properties of purified enzyme Substrate specificity and mode of action. Previous information, which we had obtained from FORL crude culture filtrates, showed that the pectin lyase (characterized by an isoelectric point of 9.2) had a predominantly "endo" way of action. This fact has been confirmed with the purified protein: it decreased the viscosity of reaction mixtures with pectin, but no increase in absorbance was detected in standard conditions. Moreover, the enzyme showed a great specificity for the substrate, as no activity was detected when the decrease in viscosity of pectate was tried. So, properties of the purified enzyme were studied by using pectin as substrate and following the decrease in viscosity of the reaction mixtures.
756
Effect of pH on the activity of PNL. The enzyme exhibited maximum activity at pH 9.5 (figure 6). Effect of temperature. The optimum temperature for the PNL activity was 50~ (figure 7). Effect of Ca2+. The addition of 0.005 M EDTA to the reaction mixtures, resulted in complete loss of activity, whereas the addition of CaC12 increased the activity (figure 8). Calcium concentrations of 0.001 M and lower were without effect on PNL activity, the optimum concentration being in the range of 5 to 15 M, and higher concentration resulted in a decrease in activity.
~
100
@9 80 ._> < 6o (!;)
.>-
40
"~ nr'
20 0
I
I
I
I
I
I
I
7
7.5
8
8.5
9
9.5
10
pH
Figure 6. Effect of pH on the activity. Reaction mixtures, buffered at different pH values: 7-9 (tris/HC1), 9-10 (glycine), were incubated under standard conditions. Both buffers were 0.05M of final concentration in the reaction mixture.
/ ~" 100 ~-~9 80
~> 40 ~.
20
o/
~I~
~1 ~
30
40
45
50
55
Temperature (~
Figure 7. Effect of temperature on the PNL. The optimum temperature was determined using temperatures between 30 ~ and 55~
under standard conditions.
757
"r
100 80
60
~: eo 0
EDTA 0
0.1
0.5
1
5
10
15
20
CaCI 2Concentration (mM)
Figure 8. Effect of CaC12 and EDTA on the PNL. The addition of EDTA (0.005 M) and CaC12 (0-0.02 M) to the reaction mixtures were assayed under standard conditions.
4. DISCUSSION It has been generally believed that, among plant pathogens promoting pectolysis, bacteria produce predominantly pectate lyase while fungi usually secrete pectin lyase (Phoma medicaginis var. pinodella synthesizes a pectin lyase [25 ). However, both types of lyase activity are frequently present in an organism: Fusarium solani f. sp. phaseoli produces a calcium-dependent lyase that degrades both pectin and pectic acid under alkaline conditions [30]. Fusanum solam f. sp. pisi produces an endopectate lyase that seems to be involved in pathogenesis [31 ]. It has been suggested that both types of enzyme should be considered as pectin lyase, and that they be distinguished according to their preference for highly esterified and low-esterified pectin [32]. As other pectolytic microorganisms the r2 isolate of Fusarium oxysporum f. sp. radicis-lycopersici produces a battery of pectic enzymes differing in substrate preference, reaction mechanism, and action pattern. When separated by isoelectric focusing and stained for activities, we have detected three forms that have lyase activity and pIs of 9.20, 9.00 and 8.65 (figure 5). Phoma medicaginis var. pinodella synthesizes a pectin lyase that has a pI of 7.9 [25], the pectate lyase from Fusarium solani f. sp. pisi has a pI of 8.3 [31], the multiple endopectate lyases from Hypomyces solani f. sp. cucurbitae obtained from culture and from infected tissue possess isoelectric points in the range of 10.2-10.3 and 10.510.6 respectively [33].
758
The more abundant lyase produced by FORL has been purified to homogeneity as it is shown by analytical isoelectric focusing (figure 5). The data in Table 1 show that a 32.48-fold increase in specific activity is achieved with a recovery of approximately 2.36%. The enzyme showed an "endo" type of action and a great specificity for pectin. The PNL exhibits an optimum pH of 9.5 (figure 6) and an optimum temperature of 55 ~ C (figure 7). Lyases catalyze the reaction in an alkaline or in a neutral medium at high temperatures [32]: pectin lyase from Phoma medicaginis var. pinodella showed an optimum pH of 7.5 [25], endopectate lyase from Fusarium solam f. sp. pisi showed an optimum pH of 9.4 [31 ], and pectate lyase from Rhizoctonia solani showed an optimum pH of 8.0 [34]. The molecular weight calculated by Superdex chromatography was 18 kDa (figure 2). Endopectate lyases from Hypomyces solani f. sp. cucurbitae from culture and infected tissue have molecular sizes between 32 and 42 kDa [33]. Fusarium solani f. sp. pisi possess an endopectate lyase of 26 kDa [31] and Erwinia aroideae possess one of 67 kDa [25]. Phoma medicaginis var. pinodella has two forms of the pectin lyase with molecular weight of 29.5 and 118 kDa which suggested the existence of monomeric and tetrameric components [25]. The enzyme had a requirement for calcium. The addition of EDTA to the reaction mixtures, resulted in complete loss of activity, whereas the addition of CaC12 increased the activity (figure 8). Presumably, sufficient contaminating calcium ions were present in the dialyzed enzyme and substrate mixture to permit the limited activity of the controls, but apparently these were removed by chelation with EDTA. The optimum concentration was in the range of 5 to 15 M, and higher concentration resulted in a decrease in activity. Phoma m edicaginis var. pinodella synthesizes a pectin lyase that lacked an absolute requirement for calcium ions but maximum enzyme activity required the presence of 1 mM C a 2+ [25]. The lyase from Fusarium solani f. sp. phaseoli, that is active on pectin and pectic acid, is calcium-dependent [30]. Most of the pectate lyases characterized are calcium-dependent: the pectate lyase from Rhizoctoma solam [34] and the endopectate lyase from Fusanum solam f. sp. pisi [31 ]. Two characteristics of the lyase that we have purified may be significant. First, the small molecular size of the protein may confer it a high mobility that could be helpful to its movement through host cell walls. In second place, it is an endo-type enzyme, fact that has been considered essential for maceration of plant tissues [35]. In this sense it is noteworthy that between the battery of pectic enzymes produced by FORL, this pectin lyase is the only protein that behaves as an endo-type.
759 REFERENCES
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W.R. Jarvis and R.A. Shoemaker, Phytopathology, 68 (1978) 1679. E.C. Tjamos & C.H. Beckman (ed.), Vascular wilt diseases of plants, Springer-Verlag, Berlin, 1989. C.H. Beckman, The Nature of Wilt Diseases of Plants. APS Press St. Paul, Minnesota (1987). R.A. Brammall, and V.J. Higgins, Canadian Journal of Botany, 66 (1988) 1547-1555. T. Katan, D. Zamir, M. Sarfatti and J. Katan, Phytopathology, 1 (3) (1991) 255. S. Kuninaga and R. Yokosawa, Annals of the Phytopathological Society of Japan, 57 (1991) 9. R.C. Rowe, J.D. Farley and D.L. Coplin, Phytopathology, 67 (1977) 1513. J.C. Tello and A. Lacasa, Boletin de Sanidada Vegetal. Plagas, 14 (1988) 307. D.J. Vakalounakis, Plant Pathology, 37 (1988) 71. N. Benhamou, H. Chamberland, G.B. Ouellette and F.J. Pauz6, Physiological and Molecular Plant Pathology, 32 (1988) 249. N. Benhamou, H. Chamberland and F.J. Pauz6, Plant Physiology, 92 (1990) 995. N. Benhamou, J. Grenier, A. Asselin and M. Legrand, The Plant Cell, 1 (1989) 1209-1221. N. Benhamou, M.H.A.J. Joosten and J.G.M. De Wit, Plant Physiology, 92 (1990) 1108-1120. N. Benhamou, D. Mazau, and M.-T. Esquerre-Tugaye, Molecular Plant Pathology, 80 (2) (1990) 163-173. N. Benhamou, D. Mazau, J. Grenier and M.-T Esquerre-Tugaye, Planta, 184 (1991) 196-208. R.A Brammall and V.J. Higgins, Canadian Journal of Botany, 66 (1988) 915. H. Chamberland, N. Benhamou, G.B. Ouellette and F.J. Pauz6, Physiological and Molecular Plant Pathology, 34 (1989) 131. H. Chamberland, P.M. Charest, G.B. Ouellette and F.J. Pauz6, Histochemical Journal, 17 (1985) 313. P.M. Charest, G.B. Ouellette and F.J. Pauz6, Canadian Journal of Botany, 62 (1984) 1232. J. Callow (ed.), Biochemical Plant Pathology, pp. 101-35. John Wiley & Sons, New York (1983). M.A. Guevara, M.T. Gonz~ilez-Ja6n and P. Est6vez, XV Congreso de la SEM. Madrid (1995), L.E. Sanchez, R.M. Endo and J.V. Leary, Phytopathology, 65 (1975) 726.
760 23 24 25 26 27 28 29 30 31 32 33 34 35
R.M. Cooper and R.K.S. Wood, Nature, 246 (1975) 309. R.M. Cooper and R.K.S. Wood, Physiological Plant Pathology, 5 (1975) 135. W.A. Wood and S.T. Kellogg, Methods in Enzymology, vol. 161, Academic Press Inc., London, 1988. W.A. Wood & S.T. Kellogg, Methods in Enzymology, vol. 160, Academic Press Inc., London, 1988. O.H. Lowry, N.J. Rosebrought, A.L. Farr, and R.J. Randall, Journal of Biological Chemistry, 193 (1951) 265. J. Bodenmann, U. Heininger and H.R. Hohl, Can. J. Microbiol., 31 (1985) 75. P. Prasertsan and H.W. Doelle, Appl. Microbiol. Biotechnol., 24 (1986) 326. D.F. Bateman, Phytopathology, 56 (1966) 238. M.S. Crawford and P.E. Kolattukudy, Archives of Biochemistry and Biophysics, 258 (1987) 196. L. Rexob~i-Benkov~i and O. Markovic, Adv. in Carb. Chem. And Biochem., 33 (1976) 323. J.G. Hancock, Phytopathology, 66 (1976) 40. W.A. Ayers, G.C. Papavizas and A.F. Diem, Phytopathology, 56 (1966) 1006. R.M. Cooper, B. Rankin and R.K.S. Wood, Physiological Plant Pathology, 13 (1978) 101.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
761
Enzymic release of ferulic acid from sugar beet pulp using a specific esterase from Aspergillus niger P.A. Kroon, C.B. Faulds, C. Br6zillon & G. Williamson Food Molecular Biochemistry Department, Institute of Food Research, Norwich Laboratory, Colney Lane, Norwich, NR4 7UA. U.K.
Abstract We have purified and characterised a novel esterase (CinnAE) from Aspergillus niger. The enzyme demonstrated activity towards various soluble feruloylated oligosaccharides derived from sugar beet pulp (SBP) but, when acting alone, the esterase released only 0.9% of the alkali-extractable ferulic acid from SBP. However, when incubated with a mixture of endo-arabinanase and o~-L-arabinofuranosidase, there was a 14-fold increase in ferulic acid release, demonstrating a strong synergy between these three enzymes. No increase in ferulic acid release was observed when SBP was incubated with CinnAE plus endo-(1,4)-13-Dgalactanase and /3-D-galactosidase. Hence, feruloylated arabinans in SBP are readily available for hydrolysis by arabinan-degrading enzymes, whereas feruloylated galactans ;are not available for hydrolysis by galactan-degrading enzymes.
INTRODUCTION Ferulic acid is a constituent of many plants and, in some, is accumulated to significant levels in the cell walls. Ferulic acid is found esterified to sugars in the cell wall polysaccharides [1-3] and/or etherified to components of the lignin [4]. Sugar beet pulp (SBP), a by-product of the sugar-refining industry, is a rich source of ferulic acid containing some 1% (w/w) [5]. Ferulic acid is associated almost exclusively with the pectic side chains in sugar beet [6]. Sugar beet pectins, which comprise some 25 % (w/w) of the whole pulp, are complex heteropolysaccharides containing galacturonic acid, rhamnose, arabinose and galactose as the major sugar constituents. The pectins are themselves composed of "smooth" regions comprising a backbone of (1-,4)-linked o~-D-galacturonic acid residues, and "hairy" regions where there is a 1:1 ratio of D-galacturonic acid and L-rhamnose residues in the backbone and a high degree of substitution of the rhamnogalacturonan. The pectic side chains in the hairy regions are comprised of highly branched (1--,5)-linked a-L-arabinans and linear (1--,4)-linked/3-D-galactans [6,7]. It has been shown that ferulic acid can be esterlinked to either C-2 of arabinofuranose residues or C-6 of galactopyranose residues in the pectic side chains [8]. Ferulic acid is distributed roughly equally between the arabinan and galactan components of the pectic side chains [9,10]. The European Economic Community have shown considerable interest in releasing ferulic acid from low value agricultural waste residues such as SBP, with subsequent bioconversion of the free acid to vanillin. Previous studies with commercial enzyme preparations have shown a high degree of solubilisation of ferulic acid from SBP is possible, giving rise to a mixture of free and esterified forms [9,10]. However, it is not known which
762 enzymes specifically are important in removing esterified ferulic acid from SBP. A major problem to date has been the isolation of a suitable esterase capable of cleaving the ferulic acid-sugar linkages present in sugar beet. Although a ferulic acid esterase (FAE-III; [11]) isolated from Aspergillus niger CBS 120.49 grown on oat spelts xylan (OSX) was able to release FA from wheat bran [11,12], it was not active on the FA-sugar ester linkages present in SBP [13]. The objectives of this study were (a) to isolate an esterase which is active on the ferulic acid-sugar ester linkages present in SBP, and (b) determine the enzymes required and the relationships between the various enzymes required, in the solubilisation of ferulic acid from SBP.
MATERIALS AND METHODS Source of Aspergillus niger strains, enzymes and substrates The source of A. niger strain CS 180 has been described [14]. The source and growth of A. niger CBS 120.49, and purification of FAE-III have been described previously [11]. Sugar beet pulp (0.02-0.8 mm particle size) was prepared as described previously [10]. Cinnamoyl esterase (CinnAE) was purified from culture filtrates of A. niger CS 180 grown with 1.5 % (w/v) SBP as carbon source [5]. Feruloylated oligosaccharides were purified from SBP as described elsewhere [9,10]. endo-Polygalacturonase, endo-arabinanase, o~-Larabinofuranosidase, endo-(1,4)-j3-D-galactanase and cellulase, were purchased from Megazyme Pty. (Australia). /3-D-galactosidase was purchased from Sigma. All the commercial enzyme preparations were of A. niger origin except for endo-cellulase which was from Trichoderma spp. Each enzyme was desalted (dialysis against water) and triplicate portions (20/xg) of the desalted enzyme assayed for esterase activity (see below). Enzyme assays Esterase activity was assayed by either a continuous photometric method [ 11] or using HPLC with detection at 310 nm [5]. Methyl esters of caffeic (MCA), p-coumaric (MpCA), ferulic (MFA) and sinapinic (MSA) acids, were used as substrates. The activity of purified CinnAE was determined on a range of SBP-derived feruloylated oligosaccharides as described previously [15]. Extracellular esterase and acetyl esterase activities were measured by a method similar to that described by Donnelly & Crawford [16] using p-nitrophenyl butyrate and p-nitrophenyl acetate, respectively (0.9 mM final concentration in 100 mM MOPS), at 30~ and pH 6.0. All p-nitrophenyl derivatives were purchased from Sigma. c~Glucosidase, ot-galactosidase, a-rhamnosidase, /3-xylosidase, o~-arabinosidase, xylanase, cellulase, polygalacturonase and arabinanase activities were assayed as described previously [5]. All enzyme assays were performed at least in duplicate, and concomitant with appropriate blanks to allow correction for any background reactions. Total protein was estimated using the Coomassie Protein Assay Reagent (Pierce). For all assays performed, one unit (U) of activity was defined as the amount of enzyme releasing 1 #mol of product min -~ under the assay conditions described. Electrophoretic methods SDS-PAGE was performed by the method of Laemmli [17]. The methods for native PAGE, isoelectric focussing, detection of esterase activity in electrophoresis gels, and assays for protein glycosylation have been described elsewhere [5].
763 Release of ferulic acid from SBP Portions (50 mU MCA-hydrolsing activity) of purified CinnAE were incubated at 37~ with SBP (10 mg), both in the presence and absence of other carbohydrases, in 100 mM MOPS (pH 6.0) in a final volume of 1 mL. Incubations containing boiled enzyme were performed as controls. Reactions were terminated by boiling (3 min) and the amount of free ferulic acid determined using a method described previously for de-starched wheat bran [18]. The total amount of alkali-extractable ferulic acid present in the SBP was 0.87 %
[51. RESULTS Induction of esterase activity Although A. niger CBS 120.49 produces high levels of ferulic acid esterase (FAE) activity when grown with oat spelts xylan as the major carbon source, the major esterase produced [11] is not active on the ferulic acid-sugar linkages present in SBP [13]. Another strain of A. niger (CS 180) is known to produce pectin-degrading enzymes and degrade sugar beet pectins [14,19]. We grew these two strains with 1% glucose, or with 0.1% glucose plus either 1% oat spelts xylan or 1% SBP as the carbon source. After 4 days, culture filtrates were assayed for FAE activity using methyl ferulate (MFA) as substrate. Both strains gave high activities when grown on OSX [180 and 80 U (L media) -1 for CBS 120.49 and CS 180, respectively]. Growth of CBS 120.49 on SBP yielded low levels of FAE activity [0.7 U (L media)-1], whereas a similar growth with CS 180 gave a ten-fold higher FAE activity. Hence, when grown on SBP, A. niger CS 180 gave highest FAE activity. Flasks containing basic media and either glucose (1% w/v) or glucose (0.1% w/v) plus SBP (1% w/v), were inoculated with A. niger CS 180 and incubated for 10 days, and portions removed after the first two days for measurement of esterase activity (Fig. 1). No esterase activity was detected in any of the flasks containing glucose as the sole carbon source. However, activity against all four of the simple phenolic methyl esters used as substrates was detected in SBP-grown cultures. The ratio of activites against the four substrates changed during the course of the experiment indicating that more than one esterase was produced. FAE-III was shown to be absent from SBP- and glucose-grown cultures using immunodetection with specific anti-FAE-III antibodies. Purification of the esterase A. niger CS 180 was cultured in shake flasks with 1.5 % SBP as carbon source for 108 h at 25~ Cultures were harvested by filtration through a single layer of muslin, clarified by centrifugation and concentrated prior to purification. Esterase activity was purified from the concentrated culture filtrate using (NH4)2SO4 precipitation, hydrophobic interaction chromatography and anion-exchange chromatography [5]. In total, 260 ~g of pure enzyme with a specific activity of 96.9 U (mg protein) -1 was purified form 22.5 L of crude culture filtrate. The purified protein gave a single, darkly-stained band on SDS-PAGE corresponding to a molecular weight of 75,800 Da (Fig. 2). Gel filtration chromatography (Superdex 200, Pharmacia) gave a native molecular weight of 145,000 Da, indicating the native enzyme is probably a dimer. A 1.1 ~g sample of partially pure enzyme gave a very darkly stained band corresponding to CinnAE when tested for glycosylation. IEF of the pure protein gave a single band after staining with a pI of 4.8, and this was shown to coincide with the esterase activity.
764 Figure 1 Changes in extracellular esterase activity with incubation time. Esterase activities were assayed using the following methyl esters; MCA (l--l), MpCA (m), M S A ( 9 and MFA (zx).
l
30
I
I
I
1
,-.25
I m~
3
E '~" 20 -
/
[]
/
-
/ >'15
o
o
uJ
5
0d
0
2
4 Time a f t e r
6 Inoculation
8
10
(days)
The pure enzyme was tested for activity against several methylated phenolic and cinnamic acids (Table 2). The enzyme was active on methyl esters of cinnamic acids : caffeic > p-coumaric > ferulic, and is therefore termed a cinnamoyl esterase (CinnAE). Assays using p-nitrophenol-acetate (pNPA) and butyrate (pNPB) confirmed the esterase activity of the purified enzyme was not due to the action of a "general" extracellular esterase since there was a decrease in specific activity between the crude culture filtrate and the pure enzyme, and the specific activity (for both pNPA and pNPB) of CinnAE is two orders of magnitude below that reported for general esterases, which possess activities in the region 220 U (mg protein) -1 [20]. The enzyme showed no detectable activity in assays for c~-glucosidase, c~-galactosidase, c~-rhamnosidase, /3-xylosidase, o~-arabinosidase, xylanase, cellulase, polygalacturonase and arabinanase activities.
Figure 2. SDS-PAGE of purified CinnAE (10% polyacrylamide gel). Samples in lanes as follows: Lane 1; 6/xg CinnAE after anion-exchange chromatography; lane 2, high molecular weight markers from Sigma.
LANE 1
2
MR ~205
116 ~---97.4
Imw
------------6s
~ 4 5
~
~ 2 9
765 Table 2 Properties of CinnAE purified from Aspergillus niger CS-180. (n.d. = not detected).
Substrate
Specific activity [U (mg protein) -1]
Methyl caffeate (MCA) Methyl p-coumarate (MpCA) Methyl ferulate (MFA) Methyl sinapinate (MSA) Methyl vanillate MVA) Methyl syringate (MSyA Methyl-3,5-dimethoxycinnamate (MDMCA)
96.9 84.2 22.4 n.d. n.d. n.d. n.d.
AEBSF, an irreversible inhibitor of serine proteases, was found to completely inhibit MCA-hydrolysing actMty in the concentrated crude culture filtrate at a concentration of 1 mM. We studied AEBSF inhibition of CinnAE at concentrations of 1 and 5 mM AEBSF and found activity was reduced to less than 1% of that found in the uninhibited sample within 18 h of treatment. These results indicate tlaat CinnAE has an active site serine residue. Activity of CinnAE on SBP and SBP-derived feruloylated oligosaccharides Several ferulolyated oligosaccharides have been isolated from SBP [9] and their structures determined by NMR [8]. CinnAE was able to release free ferulic from all the feruloylated oligosaccharides tested (Table 3). The enzyme therefore shows a different specificity compared to A. niger FAE-III [13] since, unlike FAE-III, it is able to release ferulic acid from feruloylated arabinose oligosaccharides [FA-(1--,2)-arabinose] and feruloylated galactose oligosaccharides [FA-(1-,6)-galactopyranose]. Further, the enzyme is active whether the primary arabinose is in the furanose or pyranose form. The enzyme was most active on the feruloylated arabinose trisaccharide (Ara3F) and disaccharide (Ara2F), and least active on the feruloylated arabinose monomer (Ara~F).
Table 3. Activity of CinnAE for a range of feruloylated oligosaccharides derived from SBP.
Feruloylated oligosaccharide
2-O-(trans- feru lo yl)-L-arap O-[2-O-(trans-ferulo yl)-a-L-araf]-(1-,5)-L-araf O-[6-O-(trans-feruloyl)-fl-D-galp]-(1--,4)-D-galp
Specific Activity
O-c~-L-araf-(1--,3)-[2-O-(trans-feruloyl)-~-L-araf]-(1--,5)-L-araf Feruloylated arabinose hexasaccharide
0.39 1.36 0.64 3.54 0.45
Feruloylated arabinose heptasaccharide Feruloylated arabinose octasaccharide
0.56 0.67
766 The sample of SBP contained 0.87% (w/w) of alkali-extractable FA and CinnAE was able to release only a fraction of this from the whole pulp when acting alone. In a 30 rain incubation, CinnAE released FA with a specific activity of 0.24 U (mg protein) -~. In a 24 h incubation, CinnAE was able to release 0.91% of the alkali-extractable FA. Synergy with other carbohydrases SBP was incubated in the presence of carbohydrases either individually, or in pairs, and in the absence or presence of esterase, and the soluble incubation products assayed for feruloyl groups by HPLC. None of the carbohydrases used contained FAE activity. Incubation of SBP with CinnAE alone gave a single peak of absorbance at 310 nm corresponding to FA (0.91% of the alkali-extractable ferulic acid was released). No feruloylated material was released when SBP was incubated with a mixture of endo-(1,4)-13D-galactanase and/3-D-galactosidase. There was no increase in FA release when CinnAE was supplemented with these two enzymes. A mixture of endo-arabinanase and c~-L-arabinofuranosidase gave three peaks of absorbance at 310 nm which corresponded to 4.78 (peak a), 0.84 (peak b) and 0.12 % (peak c) of the feruloyl groups in the pulp, while incubations containing these two enzymes and CinnAE gave no peak a, a reduced peak b, and a large increase in peak c (Fig. 3). Peak c corresponded to 12 % of the alkali-extractable ferulic acid in the SBP sample, indicating that in the presence of the endo-arabinanase and a-L-arabinofuranosidase, CinnAE was able to release 14-fold more ferulic acid than when acting alone. These results are consistent with a CinnAE-mediated hydrolysis of the (soluble) feruloylated oligosaccharide esters produced by the action of the endo-arabinanase and c~-L-arabinofuranosidase leading to release of free FA. Clearly, CinnAE is more active on some of the feruloylated arabinose oligosaccharide esters (e.g. peak a) than others (e.g. peak b), which was also seen in assays with isolated SBP-derived feruloylated oligosaccharides (Table 3). CinnAE requires ferulic acid to be in a readily accessible form to allow hydrolysis of the ester bond.
Figure 3. HPLC chromatogram illustrating the release of feruloylated material and free ferulic acid when SBP (10 mg) was incubated with a mixture of endo-arabinanase (2 U) and a-L-arabinofuranosidase (2 U), either in the presence ( ~ ) or absence ( ........) of CinnAE (0.5/xg). FA=ferulic acid. 1.0-
FA
r c
0.5a -
esterase
';'
~
+ esterase
i
0.0
o
!
1;
30 R e t e n t i o n t i m e (min)
767 DISCUSSION Growth of A. niger CS 180 with SBP as the major carbon source induces production of at least two extracellular esterase activities (Fig. 1), neither of which is due to a known A. niger esterase, FAE-III [11]. At least one of the novel esterases demonstrates activity towards methyl caffeate (MCA) which is not a substrate for FAE-III. We purified one of the induced esterases from culture supernatants of A. niger CS 180 grown on SBP using MCA as substrate, and obtained an electrophoretically homogeneous enzyme with a molecular weight of 145,000 (dimer), a pI of 4.80, and pH and temperature optima of 6.0 and 50~ respectively. The enzyme is one of several esterases produced by A. niger, but possesses both physical and catalytic properties that distinguish it from several others purified previously [21,11]. The enzyme demonstrated activity towards the methyl esters of several cinnamic acids : caffeic > p-coumaric > ferulic, and is therefore termed a cinnamoyl esterase (CinnAE). CinnAE demonstrated activity for all the SBP-derived feruloylated oligosaccharides tested (Table 3), releasing free ferulic acid from both feruloylated-arabinose and-galactose oligosaccharides. Thus, CinnAE is active on the ferulic acid-sugar linkages present in SBP, which distinguishes this enzyme from another A. niger esterase (FAE-III), which is not active on SBP-derived feruloylated oligosaccharides. However, when acting alone, CinnAE demonstrated only limited activity on SBP (0.91% of alkali-extractable ferulic acid released in 24 h). This indicated that physical rather than chemical factors were inhibiting the action of the enzyme on a complex cell wall substrate such as SBP. Incubation of SBP with individual carbohydrases including polygalacturonase, endoarabinanase and endo-(1-,4)-13-D-galactanase, failed to solubilise more than 1% of the feruloyl groups in the pulp. However, we observed a strong synergy between endoarabinanase and o~-L-arabinofuranosidase in solubilising feruloyl groups from SBP. These results indicate that th endo-arabinanase has only limited activity on (1--,5)-linked arabinan main chains in SBP due to a high degree of substitution with arabinofuranose residues or short (1->3)-linked arabinofuranose side chains [6,7]. The o~-L-arabinofuranosidase is able to cleave these arabinose substitutions, leaving the (1--,5)-linked arabinan main chains susceptible to hydrolysis by endo-arabinanase. The action of the endo-arabinanase on the debranched arabinan releases small, soluble feruloylated oligosaccharides which are good substrates for CinnAE. Ferulic acid is distributed roughly equally between the arabinan and galactan components of sugar beet pectins [9], and in the linear (1--,4)-/3-D-galactans, ferulic acid is linked to C6 of galactopyranose residues [8]. However, a mixture of endo(1--,4)-/3-D-galactanase and/3-D-galactosidase failed to solubilise feruloylated material from SBP, and did not increase the amount of ferulic acid released by CinnAE. Hence, although it has been demonstrated that treatment of SBP with more complex carbohydrase mixtures such as Driselase [9], or sequential treatment of isolated sugar beet pectins with endogalactanase and /3-D-galactosidase [22], leads to solubilisation of significant quantities of feruloylated galactose oligosaccharides, the results presented here show that the feruloylated galactans in SBP are not readily accessible for enzymic degredation by a simple mixture of endo-galactanase and/3-D-galactosidase.
The authors thank the Biological and Biotechnolgical Sciences Research Council and the European Commission (Grant No PL 920026)for funding. We would also like to thank David Archer for donation of the Asoergillus niger strain, lan Colquhoun for NMR analysis, and Marie-Christine Ralet for help in purifying the feruloylated oligosaccharides.
768 REFERENCES
10 11 12 13 14 15 16 17 18 19 20 21 22
M.M. Smith and R.D. Hartley, Carbohydr. Res., 118 (1983) 65. Y. Kato and D.J. Nevins, Carbohydr. Res., 137 (1985) 139. F.M. Rombouts and J.-F. Thibault, Carbohydr. Res., 154(1986) 189. A. Scalbert, B. Monties, J.-Y. Lallemand, E. Guittet and C. Rolondo, Phytochem., 24 (1985) 1359. P.A. Kroon, C.B. Faulds and G. Williamson, Biotechnol. Appl. Biochem. (In Press). F. Guillon and J.-F. Thibault, Carbohydr. Res., 190 (1989) 85. F. Guillon, J.-F. Thibault, F.M. Rombouts, A.G.J. Voragen and W. Pilnik, Carbohydr. Res., 190 (1989) 97. I.J. Colquhoun, M.-C. Ralet, J.-F. Thibault, C.B. Faulds and G. Williamson, Carbohydr. Res., 263 (1994) 243. M.-C. Ralet, J.-F. Thibault, C.B. Faulds and G. Williamson, Carbohydr. Res., 263 (1994) 227. V. Micard, C.M.C. Renard and J.-F. Thibault, Lebensm.-Wiss. U Technol., 27 (1994) 59. C.B. Faulds and G. Williamson G, Microbiol., 140 (1994) 779. C.B. Faulds and G. Williamson G, Appl. Microbiol. Biotechnol., (In Press). M.-C. Ralet, J.-F. Thibault, C.B. Faulds and G. Williamson, Carbohydr. Res., 263 (1994) 257. J.A. Matthew, S.J. Howson, M.H.J. Keenan and P.S. Belton, Carbohydr. Polym. 12 (1990) 295. P.A. Kroon and G. Williamson, Biotechnol. Appl. Biochem. (In Press). P.K. Donnelly and D.L. Crawford, Appl. Environ. Microbiol. 54 (1988) 2237. U.K. Laemmli, Nature 227 (1970) 680. C.B. Faulds and G. William son , J. Gen. Microbiol. 137 (1991) 2339. J.A. Matthew, G.A. Wyatt, D.A. Archer and M.R.A. Morgan, Carbohydr. Polym. 16 (1991) 381. M. Sundberg, K. Poutanen, P. Markkanen and M. Linko, Biotechnol. Appl. Biochem. 12 (1990) 670. C.B. Faulds G. Williamson, Biotechnol. Appl. Biochem. 17 (1993) 349. F. Guillon and J.-F. Thibault, Carbohydr. Polym. 12 (1990) 353.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
Characterization
of some e n d o - p o l y g a l a c t u r o n a s e s
769
f r o m Sclerotinia
sclerotiorum M. B. Martel, R. IAtoublon and M. FSvre Laboratoire de Biologie Cellulaire Fongique, CGMC, CNRS UMR 106, Universit6 Lyon I, 43 Blvd 11 Novembre 1918, 69622, Villeurbanne, France.
Abstract The isolation and characterisation of an endo-polygalacturonase from S. sclerotiorum is reported. The purified glycoprotein has a molecular mass of 42 KDa and a pI of 4.8 and shows the enzymatic characteristics an endo-polygalacturonase. Large amounts of the purified endopolygalacturonase have been prepared in order to raise antibodies which are found to be cross reactive with all isolated endo-polygalacturonases. The purification scheme shows at least fifteen chromatographic fractions enzymatically active. The four endo-polygalacturonases so far isolated and purified have the same molecular mass but differ by their charge. 1. I N T R O D U C T I O N Among the economically important group of plant pathogens, Sclerotinia sclerotiorum is an ubiquitous phytopathogenic fungus which attacks a wide range of plants. The fungus secretes a complete set of enzymes[ 1] that are able to degrade cell wall components, to macerate plant tissues and cause cell death. From all the pectinolytic enzymes secreted by S. sclerotiorum special attention is paid to the polygalacturonases frequently produced in several molecular forms, and considered as important in the pathogenesis and virulence [2, 3]. Previous works on S. sclerotiorum have shown the occurence of both exo and endo-polygalacturonases [4-6] but the number of isoenzymes is still in debate since seven endopolygalacturonase genes have been cloned [7]. The answer should be given by the isolation and characterization of a foremost endopolygalacturonase which preludes the overall knowledge of the secreted pectinolytic enzymes of S. sclerotiorum. 2. MATERIALS AND METHODS
2.1. Culture conditions Sclerotinia sclerotiorum (strain ssl3) was grown for 10 days under constant stirring at 22 ~ on a liquid minimal medium supplemented with 0.5 % of polygalacturonic acid (wt/vol) as carbone source. The minimal medium contained per liter NH4NO3 (2 g), KH2PO4 (0.1 g), MgSO4 (1 g), DL malic acid (3 g) and yeast extract (0.5 g) and the pH was ajusted to 6 with NaOH. Cultures were maintained on potato dextrose agar (PDA). For enzyme production, 2 liter cultures were inoculated with 4-days-old colonies removed from the growing edges of 2 Petri plates.
2.2. Enzyme purification The mycelium was harvested by centrifugation and the supernatant was dialyzed overnight at 4~ against distilled water and freeze-dried. The lyophilized filtrate containing the secreted
770 enzymes was solubilized in 50 ml of distilled water and brought to 50 % ammonium sulfate saturation. The precipitate was collected by centrifugation (30 min, 20,000 g) and the pellet discarded. The resulting supernatant was brought to 85 % ammonium sulfate saturation. The final pellet obtained after centrifugating the solution at 20,000 g for 30 min was the starting material for polygalacturonases purification.
2.3. Enzyme assays The polygalacturonase activity was determined by measuring the amount of reducing sugar released from polygalacturonic acid according to the 2-cyanoacetamide assay [8]. The standard reaction mixture (0.5 ml) was composed of 0.5 mg of polygalacturonic acid dissolved in 50 mM acetate buffer (pH 3-5). The reaction was initiated by addition of 1 to 20 Ixl of the enzymatic fraction and incubated at 45 ~ for 20 min. The reaction was stopped by addition of 1.2 ml of TBC reagent (100 mM sodium tetraborate, 100 mM boric acid and 0.1% 2-cyanoacetamide). After boiling for 10 min and cooling, the coloration was determined spectrophotometrically at 270 nm. A standard curve, 0-0.4 lamol of galacturonic acid, was prepared for each experiment. The unit of activity was defined as the amount of enzyme required to liberate 1 i~mol, of reducing group per minute at 45~ The exo-polygalacturonase activity was assayed in the same conditions using digalacturonic acid as the substrate. The endo-polygalacturonase activity was also assayed by measuring the decreasing viscosity of a 2% polygalacturonase solution according to [9]. 2.4. Chromatography Liquid chromatography was monitored by a Gilson HPLC system. Gel filtration was performed on a column (0.75 x 30 cm) of AcA 54 (IBF) equilibrated in 20 mM Tris-HC1 (pH 7.5), 25 mM NaC1. Ion exchange chromatographies were carded out first on a Macro-Prep High Q (IBF) column (10 ml) in Tris-HC120 mM (pH 8), then on a 5 ml Econo-Pac Q cartridge (BioRad) equilibrated in ammonium carbonate 20 mM buffer (pH 5) and on a 5 ml Econo-Pac S cartridge (BioRad) equilibrated in ammonium carbonate 20 mM buffer (pH 5). Hydroxy-apatite chromatography was carded out on a 1.7 ml HA-Ultrogel (IBF) column equilibrated in 10 mM phosphate buffer (pH 6). Elution was performed with a linear 10-300 mM phosphate buffer (pH 6) gradient. Chromatography on immobilized reactive dyes (1-4 ml) was performed in 10 mM ammonium acetate buffer (pH 4 or 5) and elution followed with a linear 0-1 M NaCI gradient in the same buffer. 2.5. Electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins was carded out according to Laemmli [10] in a 5% stacking and 10 % resolving gel. Analytical isoelectric focusing (IEF) gel electrophoresis was carried out on ready precoated gels (Serva) containing 5 % ampholine, pH range" 3 to 10. 2.6. Immunological detection of polygalacturonases on nitrocellulose Transfer of the proteins, from the polyacrylamide gel facing the anode, to the nitrocellulose sheet was performed in a Hoefer Semiphor semi-dry transfer unit, in Towbin buffer ( 25 mM Tris-HCl, 192 mM glycine buffer (pH 8.3), 1.3 mM SDS and methanol 15% ) for 1 hour at 0.8 mA/cm 2. The proteins from the IEF were blotted in the same apparatus, for 30 min at 0.8 mA/cm 2, with the following anode buffers : 30 and 300mM "Iris, 20% methanol and 25 mM "Iris, 40 mM 6-aminocaproic acid (pH 9), 20% methanol for the cathode buffer. The nitrocellulose sheets were incubated overnight in 5% non fat dry bovine milk in 0.15 M NaC1, 50 mM Tris-HC1 buffer ( TBS, pH 7.4) at 4~ then rinsed and incubated 1 hour at 20~ with a 1:1000 dilution of rabbit polyclonal antibodies raised against the acidic endopolygalacturonase (T) from S. sclerotiorum. The nitrocellulose sheet was washed three times
771 with the milk saline buffer and then incubated, for 1 hour at 20~ with a 1:1000 dilution of a HRP-conjugated secondary antibody (Pierce Goat anti-Rabbit IgG, (H+L) Horseradish peroxidase conjugated). After three washings in the former TBS saline buffer, the nitrocellulose sheet was soaked in a solution of 50 mg of D A B , 50 ml of 30% H202, in 100 ml TBS. The reaction was stopped by washing with distilled water and the blots were dried. 3. RESULTS AND DISCUSSION Treatment of the culture filtrate with increasing ammonium sulfate amounts showed that the bulk of polygalacturonase activity precipitates between 45 and 85 % ammonium sulfate saturation (Table 1). Table 1 Ammonium sulfate precipitation N ~ fraction (pH) P25 P45 P65 P85 P100 P100 S100
(5) (") (") (") (") (7) (")
% saturation S04(NH4)2
prot (mg)
vol (ml)
25 45 65 85 100 100 100
1.4 10 14.5 4.2 1.4 0.8 2
2.5 8.5 24 19.5 4.8 30 280
Polygalacturonase activity SA TA % (103U/mg) (106 U) 0 4.5 13 180 74 0 0
0 0.4 4.6 14.8 0.5 0 0
0 2 22 73 2.5 0 0
The filtrate supplied with a 2 liter culture, was treated with increasing amounts of ammonium sulfate. At each concentration step, the precipitate was checked for the polygalacturonase activity. SA : specific activity; TA : total activity. The purification of an endo-polygalacturonase secreted by S. sclerotiorum deals with the use of several chromatographic steps. The first one was an anion exchange chromatography at pH 8. The polygalacturonase acivity was essentially recovered in 2 fractions; the greatest specific activity was recovered in the fraction eluted with 500 mM NaC1. This last fraction referred to R1 was chromatographied on the same anion exchange medium at pH 5. The polygalacturonase activity was resolved in the four protein peaks separated on the column. The first fraction which possesses the highest specific activity was dialysed against 10 mM phosphate buffer pH 6 and then chromatographied on a HA Ultrogel column equilibrated in the same buffer. Two fractions were separated, one which did not bind to the column and a retained fraction eluted with 100 mM phosphate. The first fraction named NR2'A was lyophilized and subjected to gel permeation on AcA 54. Two active fractions were separated corresponding to proteins of >70 kDa and 45 kDa. The second peak with the highest specific activity was chromatographed on a Brown-10 column. A first fraction (S) was eluted with the equilibrium buffer and a second fraction, named T, was eluted with 700 mM NaCI. This last fraction was pure as judged by SDS-PAGE and IEF of Fig. 1. The purified polygalacturonase (T) is an acidic (pI : 4.8) glycoprotein with a molecular mass of about 42 kDa which is in the range of most fungal endo-polygalacturonases [2, 11-14]. The molecular mass is however slightly higher than those observed for the endo-polygalacturonases isolated from A. niger [ 15, 16], from G. candidum [ 17] and even from soybean hypocotyls
772
..~_ 97 9
..,,_.. 66
5.9
..~__45 ~__ 4.6
..~. 31
I .,~._ 4.2
4._.21 .,....14 1
2
3
MWkDa
1 2 3
4
pl
Figure 1. a - SDS-PAGE of fraction T (silver staining), lane 1 : fraction T, lane 2 : ovalbumine and lane 3 : molecular weight standards. b - IEF of fraction T (Coomassie blue staining), lane 1 : Soybean trypsin inhibitor, lane 2 : glucose oxidase, lane 3 : carbonic anhydrase (bovine) and lane 4 : fraction T. infected by S. sclerotiorum [ 18]. Acidic plfor polygalacturonases have already been described in saprophytic fungi like A. niger [19], in a mycorrhizal ericoid fungus [11] and in the same pathogenic strain [5, 6, 20]. The N-terminal sequence (A-T-X-X-T-F-S-G-X-X-G-A-A) is similar to the endo-polygalacturonases isolated from S. sclerotiorum by Waksmann et al [5] and is in good agrement with the sequence deduced from the genes pgl-3 [7]. This is not surprising since the deduced amino acid sequences of most of the fungal endo-polygalacturonases so far studied show a great similarity [21 ]. The enzymatic parameters of the purified polygalacturonase are 9an optimal temperature around 45 ~ and an optimal pH between 3.8 and 4.2. Under these conditions the kinetic is linear for one hour and the activity is linear with respect to the enzyme concentration up to 150 ng. All activity is abolished after boiling for 5 minutes and after proteolytic digestion with trypsin. From the differences observed during the release of reducing groups from polygalacturonate or from digalactm'onate ( less than 1% of the former activity) we conclude that the enzyme is an endo-polygalacturonase. This conclusion was corroborated by viscosimetry experiments. The isolated endo-polygalacturonase (T) is by its physical properties thus very similar to the enzymes named PG2 and PG3 by Waksmann [5]. However the enzyme which is a true endo-polygalacturonase has a Km of 0.4 mg/ml which is 2 times lower than the Km already allowed to S. sclerotiorum PG2 and PG3 and a Vm of 80 mmol/min/mg far much lower than the values attributed to the above mentioned enzymes [5]. These kinetic properties are much closer to those found for the polygalacturonase isozymes of Botrytis cinerea by 22]. The isolated endo-polygalacturonase (T) has been purified in great amount in order to raise polyclonal antibodies. The antibodies were used in assessing the purity and the enzymatic content of the various chromatographic fractions isolated according to the purification scheme (Fig. 2). The scheme is divided in two sections emerging from the first chromatographic step. The left part contains acidic, strongly charged polygalacturonases referred to as S to X. The fight part contains less acidic and less charged polygalacturonases named A to J. All these fractions are enzymatically active, some have a true endo-polygalacturonase activity and others like fractions E and I possess also an exo-polygalacturonase activity. The SDS-PAGE and IEF analyses (not shown) of the purified enzymes S, T, U and C show a similar mass but a different charge which indicates some variability among the charged aminoacid composition. This situation is corroborated by the analysis of the three genes already
773
Figure 2. Purification scheme of the polygalacturonases from Sclerotinia sclerotiorum Ammonium sulfate orecipitation
I
MacmPreD High 0 DH 8 I
I
Rl(100)
I
Econo Pac 0 D H ~ I1 I
NR2A(25)
I WB(30)
1
N R 2 q 10)
I
W30)
k(33)
I
AcA 54 + Green-19 DH
NR :not refined ,R : retained. , (number) : polygalacturonaseactivity in units, * : exo + endo-polygalacturonaseactivities.
774 sequenced (pgl, 2 and 3) which have more than 98% homology and slightly differ from their calculated pI[7]. We can assume that the herein purified enzymes are part of a secreted multienzymatic pattern composed of numerous endo-polygalacturonases which have almost the same molecular weight but differ slightly by their isoelectric point. This situation could be somehow similar to the four to nine closely grouped isoenzymes of the anaerobic fermentative yeast Kluyveromyces marxianus [23]. Anyhow this multiplicity must confer flexibility to the hydrolytic complex and increases its efficiency. Using different chromatography matrices, our results show that the endo-polygalacturonase activities could be resolved in 8 endo enzymes and 2 exo enzymes were also detected. This equipment appears much more complex than previously reported (5, 18). The reappraisal of the polygalacturonase equipment of S. sclerotiorum is in agrement with the molecular studies which reveals 7 homologous genes. One cannot exclude that this multiplicity is also due to posttraductional modifications of a limited number of gene products. However glycosylation is probably not involved in this process as separation of the enzymes was based on the charge of the proteins which is not affected by the extend and (or) the mode of glycosylation. 4. REFERENCES
1 C. Riou, G. Freyssinet and M. F~vre, Appl. Environ. Microbiol., 57 (1991)1478. 2 F. Cervone, G. De Lorenzo, G Salvi and L. Camardella, Ed.NATO ASI series. H vol 1. B Berlin: Springer-Verlag, (1986) 385. 3 P. Alghisi and F. Favaron, Eur. J. Plant Pathol., 101 (1995) 365. 4 C. Riou, G. Freyssinet and M. F~vre, Appl. Environ. Microbiol., 58 (1991) 578. 5 G. Waksman, J. P. Keon and G. Turner, Biochim. Biophys. Acta, 1073 (1991) 43. 6 F. Favaron, P. Alghisi, P. Marciano and P. Magro, Physiol. Molec. Plant Pathol., 33 (1988) 385. 7 P. Reymond, G. Deltage, C. Rascle and M. F~vre, Gene, 146 (1994) 233. 8 S. Honda, Y. Nishimura, M. Takahashi, H. Chiba and K. Kakehi, Anal. Biochem., 119 (1982) 194. 9 D. Bateman and H. Basham H., Physiol. Plant Pathol., Ed. R. Heitefuss & P. Williams Berlin : Springer-Verlag, (1976) 316. 10 U.K. Laemmli, Nature (London), 222 (1970) 680. 11 R. Peretto, V. Bettini and P. Bonfante, FEMS Microbiol.Lett., 114 (1993) 85. 12 M. Gupta, D. Guoqiang and B. Mattiasson, Biotechnol. Appl Biochem., 18 (1993) 321. 13 C. Caprari, C. Bergmann, Q. Micheli, C. Salvi, P. Albersheim, A. Darvill, F. Cervone and G. De Lorenzo, Physiol. Molec. Plant Pathol., 43 (1993) 453. 14 V. Hugouvieux, S. Centis, C. Lafitte and M. T. Esquerrt-Tugayt, C. R. Acad. Sci., 318 (1995) 113-. 15 E. Stratilova, O. Markovic, D. Strovinova, L. Rexova-Benkova and H. Jornvall, J. Chem., 12 (1993) 15. 16 C. Morvan, A. Jauneau, A. Flaman A, J. Millet and M. Demarty, Carbohyd. Polymers, 13 (1990) 149. 17 A. Golubev, A. Nuradieva, N. Rodionova and K. Neustroev, Biokhim., 57 (1992) 1855. 18 F. Favaron, P. Alghisi and P. Marciano, Plant Sci., 83 (1992) 7. 19 H. Kesler and J. Visser, Biotechnol. Appl. Biochem., 12 (1990) 150. 20 C. Riou, L. Fraissinet-Tachet, G. Freyssinet and M. F~vre, FEMS Microbiol. Lett., 91 (1992) 231. 21 H. Bussink, F. Buxton, B. Fraaye, L. de Graaff and J. Visser, Eur. J. Biochem., 208 (1992) 83. 22 R. Tobias, W. Conway, C. Sams, Molec. Biol. Intern., 30 (1993) 829. 23 S. Harsa, C. Zaror and D. Pyle, Enzyme Microbiol., 15 (1993) 906.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases
9 1996 Elsevier Science B.V. All rights reserved.
775
Analysis of the interaction between PGIP from Phaseolus vulgaris L. and fungal endopolygalacturonases using biosensor technology B.Mattei a , G. Salvib, C. Caprari b, G. De Lorenzo b, V. Crescenzia, and F. Cervoneb. a Dipartimento di Chimica, Universita' di Roma "La Sapienza", P.le A. Moro, 00185 Roma. b Dipartimento di Biologia Vegetale, Universita' di Roma "La Sapienza", P.le A. Moro, 00185 Roma.
Abstract.
The interaction between endopolygalacturonase t~om Fusarium moniliforme and PGIP from Phaseolus vulgaris L. was investigated using a biosensor technique based on surface plasmon resonance (BIAlite). This new analytical system provides information on the strength and the kinetics ofbiomolecular interactions. PGIP (the ligand) was covalently attached to the dextran matrix of a sensor chip while endopolygahcturonase (the analyte) was introduced in a flow passing over the surface. Results from real-time BIA are presented as a sensorgram, which is a plot of changes in the resonance signal as a function of time. The experimental data were evaluated with an appropriate software to derive the values of kinetic rate constants and to compare the affinities of PGIP for several variant endopolygalacturonases obtained by site-directed mutagenesis of the Fusarium
moniliforme enzyme. 1. INTRODUCTION Polygalacturonase-inhibiting protein (PGIP) is a cell wall protein that specifically binds to and inhibits the activity of fungal endopolygalacturonases (PG). It has been shown that formation of the P G - PGIP complex at pH 5.0 and at low salt concentrations results in nearly complete inhibition of PG activity; the activity is restored upon dissociation of the complex at high salt concentration and pH values lower than 4.5 and higher than 6.0 [1]. As a first step to study the structure-fimction relationship of fimgal PGs and PGIPs, we have employed a biosensor technology based on smface plasmon resonance (SPR) [2-3] in order to characterize the strength of the interaction between the two proteins in terms of the kinetic and equilibrium binding constants. This new powerful technique has shown a considerable potential for the characterization ofbiospecific interactions such as those between antigens and antibodies [4-5] biologically active ligands and receptors [6] and (oligo)saccharides and lectins [7]. As model proteins the endopolygalacturonase of the phytopathogenic fimgus Fusarium moniliforme and the PGIP of Phaseolus vulgaris L. were used. A systematic study of the relationship between the structural features of these proteins and their implications on interaction dynamics has been undertaken in our laboratory. The sequence encoding the endopolygalacturonase ofFusarium moniliforme [8] was cloned into the E. coli yeast shuttle vector Yepsecl for secretion in yeast [9]. The recombinant plasmid (pCC6) was used to transform Saccharomyces cerevisiae strain S150-2B. Three residues in a highly conserved
776 region were subjected to site-directed mutagenesis [10]" His 234 was mutated into Lys, Ser 237 and Ser 240, respectively into Gly. Both the wild type and the mutated enzymes were purified and characterized with respect to their enzymatic activity and the binding to PGIP.
2. EXPERIMENTAL PROCEDURES
Equipment and materials BIAlite* system, Sensor Chip CM5, HBS buffer (10mM Hepes, pH 7.4, 150 mM NaC1, 0.005% v/v surfactant p20 in distilled water), amine coupling kit were from Pharmacia Biosensor (Uppsala, Sweden).
Preparation of Sensor Surface PGIP, purified from P. vulgaris hypocotyls [11], was immobilized to the sensor chip via amine coupling. A continuous flow of HBS buffer (5 lxl/min) was mantained over the sensor surface. The carboxylated dextran matrix of the sensor sin-face was first activated by a 6-min injection of a mixture of N-hydroxy-succinimide and N-ethyl-W- (3-diethylaminopropyl) carbodiimide, followed by a 7-rain injection ofPGIP (10ng/lxl in 10 mM acetate, pH 5.0). The immobilization procedure was completed by a 7-min injection of 1 M ethanolamine hydrochloride to block the remaining ester groups.
Binding of endopolygalacturonases to immobilized PGIP Solutions of endopolygalacturonases in acetate buffer, pH 5.0, were injected into the flow cell and passed over the PGIP surface at the flow rate of 10 ~min. The interaction was followed in real time at different analyte concentrations. The binding was monitored as a mass change in the vicinity of the sensor surface, reflecting the progress of the interaction.
Surface plasmon resonance Surface plasmon resonance (SPR) is an optical phenomenon associated with total internal reflection that occurs at the boundary between substances of different ret~active index, e.g. glass and aqueous solutions. Normally, fight travelling through the material of higher refractive index (the glass) is totally reflected back when reaching an interface to the optically less dense medium (aqueous solution), provided that the angle of incidence is larger than the critical angle. Importantly, although the fight is totally reflected, a component of the electromagnetic field called the evanescent wave penetrates a short distance (of the order of one wavelength) into the solution. If the interface between the media is coated with a thin layer of metal, and the fight is monochromatic and p-polarized, the evanescent wave can interact with free oscillating electrons (plasmons) in the metal film surface, and the intensity of the reflected light is markedly reduced at a specific incident angle, producing a sharp "shadow". The angle at which the shadow is observed, called the SPR angle, is dependent on the refractive index in the solution close to the surface. Changes in the refractive index out to about 300 nm from the metal film surface can thus be followed by continuous monitoring of the resonance angle. All proteins, independent on their aminoacid composition, alter the refractive index of water by a similar amount per unit mass, and thus there is a linear correlation between the surface concentration of protein and the resonance angle shift. 1000 resonance units (RU) correspond to a 0.1 ~ shitt in the SPK angle and this is equivalent to a sm'face concentration change of about 1 ng mm-1.
777 Instrumentation
The instrument consists of a processing unit, reagents for ligand immobilization, exchangeable sensor chips and a personal computer for control and evah~tion. The sensor chip con~sts of a glass slide on to which a 50-nm thick gold film has been deposited. The gold film is then covered with a linker-layer to which a matrix of carboxylated dextran is attached. The dextran, which extends typically 100 nm out from the surface, provides a hydrophilic, activatable and flexible polymer to which biomolecules can be coupled through amine, sulphydryl, carboxyl and other groups. The sensor chip is held in contact with the prism of the optical system by a microttuidic cartridge that controls the delivery of sample plugs into a transport buffer that passes continuously over the sensor chip surface. By continuously monitoring the SPR response, expressed in resonance units (RU), in the detected vohnne and plotting this value against time a sensorgram is obtained. The sensorgram can be divided into three phases: association during sample injection, steady-state where the rate of analyte binding is balanced by dissociation from the complex and dissociation from the surface during buffer flow at the end of sample injection (Fig. 1).
E~ill'b~ Association
o
constants
Dissociation
?
2DO0
I I I I
0 1800-
1000-
Concentration
f
lzg~O-
I I
12D0-
$ 1000
800
0
'
I
100
"
I
200
'
I
300
'
I
400
'
I
600
'
I
600
Time (s) Fig. 1. Schematic sensorgram, showing association, equilibrium and dissociation phases.
Sensorgrams were analyzed by nonlinear least squares curve fitting using BIAevaluation 2.0 software (Pharmacia). A single-site binding model (A + B = AB) was used for the analysis of the interactions. The association rate and the dissociation rate can be expressed with the following equations, respectively:
778 dR/dt = -(konc + koff)R + koncRmax
(1)
dR/dt = - koff R
(2)
where Rmax is the maximum analyte binding capacity (in RU) of the PGIP surface and R is the SPR signal in RU at time t. The association phase was analyzed fitting the integrated form of equation (1)" R t = Req (1- exp( -ks (t-t o )))
(3)
where ~ = koncRmax/(konc + koff) was the amount of ligand bound in RU at equilibrium, to was the time the injection started and k s = konc + koff, where c was the concentration of the protein injected over the sensor surface. The association rate constant, kon, was determined from the slope of a plot ofk s versus c. The dissociation rate can be determined fitting the integrated form of equation (1)
gt = ~ exp(-kofr (t-to))
(4)
by non-linear least square analysis : R t is the amount of ligand (in RU) remaining bound at time t and to was the be~nning of the dissociation phase. The equilibrium association constant, KA,, was then calculated from kon/koff.
3. RESULTS AND DISCUSSION The study of the structure and function of fungal endopolygalacturonases and their plantderived interacting PGIPs is an essential starting point to understand some of the recognition phenomena occurring between plants and microorganisms [12]. We have undertaken a systematic study of the structure and function of PGIP from P. vulgaris and of endopolygalacturonase from F. moniliforme. The gene encoding this enzyme was introduced into S. cerevisiae via a shuttle vector and an active endopolygalacturonase was secreted into the medium by the transformed yeast cells. Point mutations were introduced in the amino acid sequence of the endopolygalacturonase ofF. moniliforme. Target sequence of these mutations was a region which is highly conserved in all endopolygalacturonases so far characterized [8]. Each mutated sequence was expressed in S. cerevisiae. His 234 was mutated into Lys, Set 237 and Ser 240 were mutated, respectively, into Gly. Both the wild type and the mutated enzymes were purified and characterized with respect to their enzymatic activity and the binding to PGIP. The replacement of His 234 with Lys abolished the enzymatic activity, confirming the biochemical evidence obtained by Cooke et al. [13] and by Rexovfi-Benkovfi et al. [14] that a histidine residue is critical for the activity of the enzyme. Replacement of either Set 237 or Ser 240 with Gly reduced the enzyme activity to 48% and 6% respectively, indicating that Set residues are also important for the activity. The interaction between PGIP of Phaseolus vulgaris and the different endopolygalacturonases was studied using a biosensor based on SPI~ PGIP was immobilized
779 as a ligand on the sensor surface, while endopolygalacturonase was passed in solution as an analyte on the surface. Sensorgrams for the interaction of PGIP with different amounts of F. moniliforme PG are shown in fig. 2. The increase in RU t~om the initial baseline represents the binding of the PG to the surface-bound PGIP. The plateau line represents the steady state phase of the PGPGIP interaction while the decrease in RU at the end of the injection represents the dissociation phase.
(RU) 9O3
i IO0
-lOa
,, -100
0
........
, IO0
,,
,
I
2DO
3OO
Time (=)
----; 4OO
.........
; ~00
........ : 6(7O
(,)
Fig. 2. Sensorgrams of Fusarium moniliforme endoPG injected over a PGIP surface at different concentrations.
The different interactions with either the wild type and the mutated polygalacturonases were analyzed kinetically as described in the experimental procedures. In fig. 3 , the slope of the plot ofk s versus c represent the association rate constant Icon for different endoPGs.
780 0.1 0.09 0.138 0.07 0.06 0.05 0.04
9 FmPG
0.03
D S240->G-.PG
0.02
y H234->K-PG 0.01
I Y,OG .,
O
0
8o-8
1.6e-7 Z4e-7
3.3s-7 4 e - 7 4.8e-7 5.6e..7 6.4e-7 7.2e-7 Concenb'atlo. (M)
8e-7
Fig. 3. Plots ofk s (or konc + koff) versus concentration for different endoPGs.
The kinetic parameters of each interaction are reported in Table 1. These data show that the values of the equilibrium association constant I ~ for the interaction of PGIP with the different yeast-expressed polygalacturonases do not differ si~ificantly, but in each case they are 2 + 5 times lower when compared to that of the F. moniliforme enzyme. This is probably due to the steric hindrance caused by hyperglycosylation of the yeast enzymes. Since the modification that causes loss of activity in the enzyme H - , K 234 does not interfere with the formation of the PG-PGIP complex, the site responsible for PGIP recognition may reside in a domain different from the active site. Studies are now under way to establish which site(s) and amino acid residues of the endopolygalacturonase are critical for interaction with PGIP.
4. ACKNOWI.J~DGEMENTS This work was supported in part by the National Research Council of Italy, Special Project RAISA, subproject N. 2 by the Ministero delle Risorse Agricole e Fores'taft (MIRAAF), and by the European Community Grant R 3 - C T 9 4 - 2 2 1 5 .
781 Table 1. Kinetic constants of the interaction between wild type and variant endoPGs with immobilized PGIP t~om P. vulgaris. PG cone
kon
koff
K A = kon/koff
(riM)
(105 M "1 s"1 )
(10.3 s"1 )
(107 M "1 )
Fusarium PG
25---400
1.17 + 0.11
1.2 + 0.2
9.7
S. cerevisiae PCC6 -PG
80---600
0.60 + 0.08
3.0 + 0.2
2.0
H 234 - , K PG
22--180
1.9 + 0.09
3.5 + 0.2
5.4
S 237-~ G PG (48 % activity)
10--70
1.1 __ 0.4
3.0 +_ 0.2
3.6
S 240--, G PG (6 % activity)
27--340
0.94 + 0.09
3.3 + 0.2
2.8
(0 % activity)
5. REFERENCES
1 Cervone, F., De Lorenzo, G., Degr~, L., Salvi, G. and Bergami, M., Plant PhysioL, 85 (1987) 631-637. 2
R. Granzow and R. Reed, Bio/Technology, 10 (1992) 390.
3 S.C. Schuster, 1LV. Swanson, L.A. Alex, ILB. Bourret and M.I. Simon, Nature, 365" (1993) 343. 4 G. Zeder-Lutz, D. Altschuh, I-I.M. Geysen, E. Trififiet~ G. Sommermeyer and M.H.V. Van Regenmortel, MoL lmmtmol. 30 (1993) 145. 5 D.J. O'Shannessy, M. Brigham-Burke, K.I~ Soneson, P. Hensley and I. Brooks, Anal. Biochem 212 (1993) 457.
782 6 L.D. Ward, G.J. Howlett, A. I-Iammacher, J. Weinstock, K. Yasukawa, 1LJ. Simpson and D.J. Winzor, Biochemistry, 34 (1995) 2901. 7 Y. Shinohara, H. Sota, F. Kim~ M. Shimizu, M. Gotoh, M. Tosu and Y. J. Hasegawa, Biochem (Tokyo) 117 (1995) 1076-1082. 8 C. Caprari, A. Richter, C. Bergmmm, S. Lo Cicero, G. Salvi, F. Cervone and G. De Lorenzo, Mycol. Res., 97 (1993) 497. 9 C. Baldari, J.A.H. Murray, P. Ghiara, G. Cesareni and C.L. Galeotti, EMBO J., 6 (1987) 229. 10 C. Caprad, B. Mattei, M.L. Basile, G. Satvi, V. Crescenzi, G. De Lorenzo and F. Cervone, submitted. 11 P. Toubart, A. Desiderio, G. Salvi, F. Cervone, L. Daroda, G. De Lorenzo, C. Ber~mann, A.G. Darvill, and P. Albersheim, Plant J. 2 (1992) 367. 12 G. De Lorenzo, F. Cervone, D. Bellincampi, C. Caprari, A.J. Clark, A. Desiderio, A. Devoto, 1L Forrest, F. Leekie, L. Nuss, and G. Salvi, Biochem_ Soc. Trans., 22 (1994) 396. 13 Cooke, 1LD., Ferber, C.E.M., and Kanagasabapathy, L., Biochim Biophys. Acta, 452 (1976) 440. 14
L. Rexov~-Benkov~, and M. Mrackovfi, Biochim Biophys. Aeta, 523 (1978) 162.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
783
Rhamnogalacturonan r pyranosyluronide iyase, a new enzyme able to cleave RG regions of pectin ~ M. Mutter', I.J. Colquhoun b, G. Beldman', H.A. Schols', A.G.J. Voragen" 'Department of Food Science, Biotechnion, Bomenweg 2, 6703 HD Wageningen, The Netherlands, fax +31 317 484893 blnstitute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA United Kingdom ~Financial support was from Novo Nordisk A/S (Copenhagen, Denmark)
Abstract The recently described rhamnogalacturonase B (RGase B), which is able to degrade hairy regions of pectin, was found to be a rhamnogalacturonan (RG) ot-L-rhamnopyranosyl(1-->4)-c~-D-galactopyranosyluronide lyase. The cleavage site and mechanism are different from that of the previously described rhamnogalacturonase A (RGase A), which is a hydrolase, and can now be termed RG a-D-galactopyranosyluronide-(1--->2)-ot-L-rhamnopyranosyl hydrolase.
1. INTRODUCTION The classical pectolytic enzymes, active toward the homogalacturonan or "smooth" regions of pectin, have been shown not to be active toward the RG or "hairy" regions of pectin. Schols et al. (1990a) were the first to discover an enzyme, rhamnogalacturonase (RGase), able to degrade the backbone of hairy regions. Since then a specific RG acetylesterase (Searle-van Leeuwen et al., 1992) and a rhamnohydrolase (Mutter et al., 1994), specific for the terminal nonreducing Rha unit in RG regions, have been found by our group. Recently, in our laboratory, another enzyme able to degrade the backbone of hairy regions was discovered (referred to by Kofod et al., 1994), named RGase B. Both RGase A (Schols et al., 1990a) and RGase B have been cloned and expressed in Aspergillus oryzae. In their study, Kofod et al. (1994) could not give evidence that RGase B was an RGase. In the present study we prove that RGase B is indeed an RGase. Furthermore we show that the two RGases are different, since RGase B was shown to be lyase (RG-lyase) while RGase A is a hydrolase (RG-hydrolase). A more specific nomenclature for the two enzymes is suggested (Mutter et al., 1996).
784
2. RESULTS AND DISCUSSION RG-lyase was purified from Pectinex Ultra SP-L, produced by Aspergillus aculeatus, using anion- and cation-exchange chromatography. The purified RG-lyase differed from RGhydrolase in pI and pH optimum and stability (Table I). Table I Characteristics o f RG-lyase compared with those o f RG-hydrolase (Schols et al., .19..9oq.).................................................................................................................................. .........................................
.............................
Mw 51 kD ~ pl 4.1-4.5 p H optimum 3-4 ~ p H stability below p H 6* T optimum 40-50 ~ Ts(qb(t!tY ..............UP tO5OOff* . . . . . . . . . . . * according to Schols et al, 1990a
R
e ........................
57 kD 5.1-5.3 6 p H 6 and higher 50-60 ~ up to 40 ~ ....
Saponified Modified Hairy Regions (MHR) of apple pectin (produced and saponified according to Schols et al., 1990b) could be degraded by RG-lyase, as observed using highperformance size-exclusion chromatography (HPSEC), producing a slightly different degradation pattern as compared with RG-hydrolase (Fig. 1). More markedly was the difference in elution behaviour of the oligomeric reaction products upon high-performance anionexchange chromatography (I-IPAEC) (see elsewhere in these proceedings, Beldman et al.). The oligomers as produced from saponified MHR were isolated using Sephadex G50 and preparative HPAEC. 1D and 2D NMR experiments (COSY and ROESY) were used to determine the structure of the smallest oligosaccharide, eluting at 23 min upon HPAEC. The chemical shifts of the assigned peaks in the ~H NMR spectrum are summarised in Table II. 1
f.................................................................................................................................................................................................. ~b/e lg ...................H Chem~c~t ~h!f:s f o r the smattest Rq-teq~e ot:ige~fehqrJde ............. ....v...~.!t.....................................................C...h..e.m...!.~t...~h~..(~)............................................................................................ H-1 H-2 H-3 H-4 H-5 H-6 Rha
GalA Rha
us-GalA Gaff'
A~ Ap B C D
5.22 4.94 5.08, 5.16 ~ 5.32 5.13 4.63
3.97 4.06 3.94, 3.98 ~ 4.32 3.80 3.50
4.09 n.d. 4.13, 4.15 ~ 4.08 4.34 3.66
3.71 n.d. 4.43
3.95 n.d. 4.63
1.34 n.d. -
3.62 5.81 3.90
3.85 n.d.
1.29 n.d.
n.d., not determined; a, Two values are for unit B linked to a- and fl-forms o f the reducing end unit, respectively; b, Two residues, 8 values differ by < 0.01 ppm.
The most important difference in the spectrum as compared with RGs released by RGhydrolase action (Colquhoun et al., 1990) was a doublet at 5 5.81 (J = 3.4 Hz). From the COSY experiments this doublet was found to belong to a four-proton spin-coupling network
785 that had chemical shifts and coupling constants characteristic of an a-linked A-4,5-unsaturated GalA residue at the nonreducing terminus (Tjan et al., 1974). For this residue the anomeric signal was at/5 5.13, and the doublet at 8 5.81 was assigned to the olefinic proton. Finally, the structure deduced for the oligosaccharide was: D
C
B
A
o~-D-us-GaleA-(1-->2)--tx-L-Rhap-(1-~4)-tx-D-GaleA-(1-~2)-L-Rhap. 4 4
1'
1'
1
1
l~-D-Galp
l~-D-Cralp.
The oligomer contained an alternating RG chain, similar to the previously published structure of the RG oligomers liberated by RG-hydrolase from saponified MHR (Colquhoun et al., 1990). However, the reducing and nonreducing ends had interchanged. Most surprising was the presence of the unsaturated bond in the nonreducing GalA unit. This showed that the new enzyme cleaved the backbone by 13-elimination (Mutter et al., 1996).
Absorbance
/~
~
RG-lyase RG-hydrolase ......~----~--'*~'~-~.---. Blank
16
20
24
28
32
Retention time (min)
36
16
L
20
24
28
32
Retentiontime(min)
36
Figure 1 HPSEC chromatograms of saponified A,fftR (bottom chromatograms); saponified MHR after degradation by RG-hydrolase (middle chromatograms); and saponified MHR after degradation by RG-lyase (top chromatograms). Left chrornatograms give the RI signal, right chromatograms the UV absorbance at 235 nm. After cleavage by [3-elimination, conjugation of the double bond with the carboxyl group at C5 of the 4,5-unsaturated GalA occurs. In homogalacturonan chains, the absorption maximum is at 235 nm. The absorption maximum for the 4,5-unsaturated GalA in RG chains was found to be the same. Figure 1 shows the HPSEC chromatograms of saponified MHR before and after degradation by RG-hydrolase and RG-lyase, using simultaneous detection by refractive index and absorption at 235 nm. In the region where oligosaccharides eluted from the column (32 min), upon UV-detection a large peak appeared only in case of the RG-lyase. Activity of RG-lyase toward various substrates was measured from the increase in the A:35 (Table III).
786 Table 111
Activity o f RG-lyase toward various substrates (0. 02-0.1%w/v) (U rag-l), determined
Substrate 6~ od' ............................................................................................ P_,H .............................. P_:H ...................... -Ca
MHR-S 8.8 Linear RG oligomers 3.9 pectin D M 92.3% 0 PGA 0 "~i%'iJO"~H::61:i'i:::20~p:H:8
+Ca c
-Ca
+Ca
9.8 n.d. 0 0
10.6 n.d. 0 0
11.8 n.d. 0 0 ; ~, 1 mM CaCI2;
RG-lyase was not active toward polygalacturonic acid or highly methoxylated pectin, also not with additional calcium ions. Considering the specificity of the enzyme for RGs, and given the type of linkage cleaved and the cleavage mechanism, the appropriate name is RG otL-rhamnopyranosyl-(1--->4)-a-D-galactopyranosyluronide lyase, abbreviated as RG-lyase. To our knowledge, the existence of an RG-lyase has not been reported before.
3. R E F E R E N C E S Colquhoun IJ, de Ruiter GA, Schols HA, Voragen AGJ (1990) Carbohydr Res 206:131-144 Kofod LV, Kauppinen S, Christgau S, Andersen LN, Heldt-Hansen HP, D6rreich K, Dalboge (1994) J Biol Chem 269:29182-29189 Muttter M, Beldman G, Schols HA, Voragen AGJ (1994) Plant Physiol 106:241-250
Mutter M, Colquhoun IJ, Schols HA, Beldman G, Voragen AGJ (1996) Plant Physiol 110: 7377 Schols HA, Geraeds CCJM, Searle-van Leeuwen MF, Kormelink FJM, Voragen AGJ (1990a) Carbohydr Res 206:105-115 Schols HA, Posthumus MA, Voragen AGJ (1990b) Carbohydr Res 206:117-129 Searle-van Leeuwen MJF, van den Brock LAM, Schols HA, Beldman G, Voragen AGJ (1992) Appl Microbiol Biotechnol 38:347-349 Tjan SB, Voragen AGJ, Pilnik W (1974) Carbohydr Res 34:15-32
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V.All rights reserved.
787
Purification and characterisation of galactose-induced pectinases from the exo-1 mutant strain of Neurospora crassa L.B. Crotti, J.A. Jorge, H.F. Terenzi and M.L.T.M. Polizeli Departamento de Biologia - Faculdade de Filosofia, CiEncias e Letras de Ribeir~o Preto, Universidade de S~o Paulo, Av. Bandeirantes 3900, 14040-901 - Ribeir~o Preto, S~o Paulo, Brasil Abstract Pectinases produced by the exo-1 mutant of N. crassa in galactose plus glucose supplemented medium, were separated by ion-exchange chromatography into two pools. Pool I contained pectate and pectin lyases, and variable polygalacturonase activity. Pool 2 contained polygalacturonase activity only. Gel filtration indicated a MWapp of 80 kDa (higher than those of separate enzymes) for all activities in the first pool, suggesting a complex. Polygalacturonase, pectin and pectate lyases were purified 39-fold, 22-fold and 33fold, respectively. Optima of temperature and pH were 45~ and 5.5 for polygalacturonase activity and 50~ and 9.5 for lyase activities. Km and Vmax values for polygalacturonase were 0.023 mg polypectate/ml and 2.08 ~moles (reducing sugar)/min/mg protein.
1. INTRODUCTION Previous studies from our laboratory [1,2] demonstrate that the filamentous fungus Neurospora crassa produces pectic enzymes as effectively as other hydrolases such as
cellulases [3], amylases [4], and xylanases [5]. The production of polygalacturonase was studied using the mutant strain exo-1. This interesting strain exhibits a rather exaggerated synthesis and secretion of several exoenzymes, among others amylase and invertase [6]. We demonstrated that this strain, when cultivated in the presence of pectin as the sole carbon source, secretes five to six times more than the wild type a glucose-repressible endopolygalacturonase [2]. Interestingly, the production of polygalacturonase was also induced by galactose, four times more efficiently than by pectin. The inducing effect of galactose, different of that of pectin, was not counteracted by glucose. Thus, we decided to investigate in more detail the effect of galactose as inducer of pectolytic activities and to biochemically characterise the pectolytic complex produced by the N. crassa exo-1 strain in the presence of galactose and glucose. 2. METHODS Culture conditions: The exo-1 strain was cultivated in two-stages: (I) pre-cultivation for 24 hours in Vogel's medium [7] supplemented with 2% glucose, and (II) transfer of the mycelial
788 mass to fresh medium supplemented with 2% glucose plus 2% galactose or other carbon sources, for 48 or 72 hours, according with the experiment, at 30oc, with agitation.
Enzymatic assays: Polygalacturonase was assayed: (a) by measuring the amount of reducing sugar released from sodium polypectate as a substrate. An enzyme unit is the amount which releases reducing sugar at an initial rate of l~mol/min at 30oc, using galacturonic acid as the standard [8]. Co) By the decrease in relative viscosity of a 0.2% pectin solution using an Ostwald viscometer. One activity unit was expressed as a percentage (50%) change in viscosity [9]. Lyase activities were measured by the increase in A232 nm of the unsaturated products of degradation of pectin or sodium polypectate. One activity unit was the amount of enzyme which released 1 lxmol of unsaturated product per minute [10]. Protein was determined by the Lowry method using bovine serum albumin as standard [11 ]. Separation of pectic enzymes: The crude filtrate was precipitated with 2 volumes of ethanol for 2 hours at -20oc and then centrifuged at 15,900g for 10 minutes. The precipitate was dissolved in 10 ml of Tris-HC1 buffer 10mM, pH 7,5 (buffer A) and applied to a DEAEcellulose column (1,6 x 20cm) equilibrated and eluted with buffer A. The flow-through protein was dialysed against 10raM sodium acetate buffer, pH 5,0 (buffer B) and applied to a CM-cellulose column (1,6 x 25cm). The column was eluted with a NaC1 gradient (0 500mM) in buffer B. Fractions (10ml) were collected at a flow rate of 33.5 ml/h. Determination of molecular mass of pectic enzymes: The molecular mass were determined by gel filtration in a Sepharose CL-6B column (1,8 x 88cm) equilibrated and eluted with TrisHC150 mM, pH 7,5 buffer, plus 100 mM KC1. Fractions (3,3 ml) were collected at a flow rate of 10 ml/h. Molecular mass markers were: tyroglobulin (660 kDa); apoferritin (440 kDa); 13amylase (200 kDa); alcohol dehydrogenase (150 kDa); bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa). Urea-SDS-PAGE (7%) was carried out according to Swank and Munkres [12]. Molecular mass markers were: myosin (205 kDa); [3-galactosidase (116 kDa); phosphorylase b (97 kDa); bovine serum albumin (66 kDa), ovalbumin (45 kDa) and carbonic anhydrase (29 kDa). Determination of neutral carbohydrate: Total neutral carbohydrate in protein samples was estimated by the phenol/sulphuric acid method of Dubois [13] using mannose as standard. Chromatographic characterisation of hydrolysis products: Hydrolysis products from sodium polypectate were analysed by thin-layer chromatography on silica gel G-60, using ethyl acetate / acetic acid / formic acid / water (9:3:1:4, by volume) as the mobile phase system. Sugars were detected with 0,2% orcinol in sulphuric acid-methanol (10:90ml) [14]. 3. RESULTS AND DISCUSSION. The effects of galactose and pectin as inducers of polygalacturonase activity in the exo-1 N. crassa strain is shown in figure 1. Both substances were efficient inducers, but in the presence of galactose the enzyme production was about four-fold higher than with pectin. A remarkable difference between induction with pectin or with galactose, was that the former was severely repressed by glucose, whereas galactose induction was not repressed by addition of glucose.
789
.~ > .i
1,0
ci.
t~
0,8
19 U) t-
0,6
/ / / / / /
/
/
/
/
. , . . , / / , ,
.• ,\/• ?
2
./
/
'
./
/
/
/
/
/
,/
/
/
/
I
,,
,,
/
/
/
/
/
/
/.,
/
~
i
i
0,4
i
I:~ i. 0
/
0,2
.
.
gal + glu
0,0
no carbon pect pect + glu
gal
Figure 1. Effect of 1% (w/v) pectin (pect), 1% galactose (gal), and of the simultaneous presence of 2% glucose (glu) on the production of extracellular polygalacturonase activity. Two-stages cultures were prepared as described under methods. Polygalacturonase was assayed in the culture filtrate as reducing sugar-releasing activity using sodium polypectate as a substrate. These results prompted us to examine the characteristics of the extracellular pectolytic enzymes secreted in medium supplemented with glucose and galactose. Figure 2 shows the profile of elution of pectolytic activities recovered from the flow-through of a DEAEcellulose column chromatographed on a CMC-cellulose column.
~~ NaCI0~'_ l:: 12
m
8
~"
0,25
v
0,20
t~ C
0,15
"~
2
I
im
6
0,10
19
0,05
0
E
0,30
19
10
t~ >,,
0,35
0
20
40
60
fractionnumber
9
80
I~ 0 Q.
0,00 100
Figure 2. CM-cellulose chromatography of pectolytic enzymes. The activity peaks of the flow-through of a DEAE-cellulose chromatography was applied to a CM-cellulose column. The column was eluted with a NaC1 (0-0.5M) continuous gradient at a flow rate of 34 ml/h. 10 ml fractions were collected and assayed for pectolytic activities Symbols: (O) pectate lyase; ($) polygalacturonase (reducing sugar-releasing activity); (x) protein. Other details in Methods.
790 Polygalacturonase activity eluted into two main fractions, the first coeluting with pectate lyase (and pectin lyase, not shown) activities, and the second free of other activities. When the first peak was rechromatographed under the same conditions, identical result was obtained. The distribution of polygalacturonase in the two peaks varied with the experiment. In other cases, lyases and polygalacturonase activities separated completely into two peaks, one containing the two lyases and the other containing polygalacturonase activity only. Interestingly, at this stage of separation all pectolytic activities had reached a considerable degree of purification: polygalacturonase was purified about 39-fold, while pectate and pectin lyases were purified 33-fold and 22-fold, respectively. Gel filtration of the peak showing associated lyases and polygalacturonase activity (Figure 3A) gave a single activity peak eluting with a MWapp of approximately 79.4 kDa, suggesting the existence of a multienzyme complex. On the other hand, the same peak run under denaturing urea-SDS-PAGE (Figure 3B) was resolved into two bands, one of pectate/pectin lyase activity (MWapp 56.2 kDa) and a second band with polygalacturonase activity (MWapp 44.7 kDa). 6,5 6,0 r
E _~
5,4 5,2
5,0 4,5
pectin lyases
4,8
pectate/pectin lyase
+ polygalacturonase
4,0
4,6
3,5 310
2 3
5,0
0
~
B
5,5
(D
O E
1
A 1 2
4,4 i
1,6
,
i
1,8
i
VeNo
i
2,0
,
i
2,2
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
Rf
Figure 3. (A) Determination of molecular mass of pectic enzymes by gel filtration in Sepharose 6B. Molecular mass markers:l- tyroglobulin, 2- apoferritin, 3- 13-amylase, 4alcohol dehydrogenase, 5- bovine serum albumin, 6- carbonic anhydrase. (B) SDS-PAGE of pectolytic activities. Molecular mass markers: 1- myosin, 2- 13-galactosidase, 3- phosphorylase b, 4- bovine serum albumin, 5- ovalbumin, 6- carbonic anhydrase.
Table i shows some biochemical properties of the pectolytic enzymes present in pool 1. The pectin lyase/pectate lyase activities (pool I) and polygalacturonase activity (pool II) were not significantly affected by NH4+, Na + and K + (0,25 - 2,5mM), while A13+, 13-mercaptoethanol, Hg 2+, EDTA, Ba 2+ and Zn +2 (2,5mM) inhibited 30-100% these activities. On the other hand, Ca2+, Mg 2+ and Mn 2+ at 2,5mM concentration activated 20-100% pectin/pectate lyases but Ca 2+ and Cu 2+ (2,5mM) inhibited polygalacturonase activity about 42 - 70%.
791 Viscosimetric assays and analysis of hydrolysis products by thin layer chromatography (TLC) were used to determine the mechanism of action of the polygalacturonase on sodium polypectate. The time required for 50% decrease in viscosity of a 2.0% (w/v) substrate solution at 45~ was approximately 105 min, at which time about 9% of total galacturonide bonds had been hydrolysed. The products of hydrolysis, analysed by TLC, demonstrated that oligogalacturonates accumulated initially, but the monomer was found after 24 h of reaction (not shown). These results suggested that the polygalacturonase of the mutant exo-1 of N.crassa induced by galactose in the medium exhibited a random mechanism of hydrolysis of sodium polypectate, suggesting that is was an endopolygacturonase.
Table 1 Kinetic constants and others intrinsic properties of pectolytic activities Parameters
Polygalacturonase
pectate/pectin lyases
reducing sugar
viscosity
pectin
pectate
KM (mg/ml)
0.023
n.d.
0.076
0.50
Vmax (U/min/mg protein)
2.08
n.d.
363.4
273.2
neutral carbohydrate
38.8%
38.0% (*)
optimal temperature
45~
45~
50~
500C
thermostability 60~ (T50 -min)
5
30
1.5
3
optimal pH
5.5
4.5
10
9
stability pH
5.0
4.0-5.5
9.5
10
(*) the sugar content of lyases was determined in a fraction containing both activities.
792
References
5 6 7 8 9 10 11 12 13 14
M.L.T.M. Polizeli, R.C.L.R. Pietro, J.A. Jorge and H.F. Terenzi, J. Gen. Microbiol., 136 (1990) 1463. M.L.T.M. Polizeli, J.A. Jorge and H.F. Terenzi, J. Gen. Microbiol., 137 (1991) 1815. B.M. Eberhart, R.S. Beck and K.M. Goolsby, J. Bacteriol., 130 (1977) 181. R.D. Sigmund, M.T. McNally, D.B. Lee and S.J. Free, Biochem. Genet., 23 (1975) 89. C. Mishra, S. Keskar and M. Rao, Appl. Environ. Microbiol., 48 (1984) 224. H.G. Gratzner and D.N. Sheehan, J. Bacteriol., 97 (1969) 544. H.J. Vogel, Am. Nat., 98 (1964) 435. G.L. Miller, Anal. Biochem., 31 (1959) 426. R. Tuttobello and P.J. Mill, Biochem. J., 79 (1961) 51. C.W. Nagel and M.M. Anderson, Arch. Biochem. Biophys., 112 (1965) 322. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. R.W. Swank and K.D. Munkres, Anal. Biochem., 39 (1971) 462. M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers and F. Smith, Anal. Chem., 28 (1956) 350. J.D. Fontana, M. Gebara, M. Blumel, H. Schneider, C.R. Mackenzie and K.G. Johnson, Methods Enzymol., 160 (1988) 560.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
Acetyl esterases of Aspergillus purification and mode of action
793
niger: on
pectins
M.J.F. Searle-van te, euwen, J.-P. Vincken, D. Schipper*, A.G.J. Voragen, G. Beldman. Dept. of Food Science, Agricultural University, Bomenweg 2, 6703 HD Wageningen, The Netherlands *Gist- brocades, P.O. Box 1, 2600 MA, Delft, The Netherlands
Abstract Acetyl esterases with different specificity occur in one AspergiUus niger preparation. Three acetyl esterases were purified and characterised: pectin acetyl esterase (PAE), feruloyl acetyl esterase (FAE) and rhamnogalacturonan acetyl esterase (RGAE). Only PAE, a novel acetyl esterase, could remove acetyl from beet pectin, to a maximum of 30%. This was shown to be one specific acetyl group in the,homogalacturonan chain of pectin (smooth region) by NMR spectroscopy. PAE activity was influenced by buffer salts and the addition of bivalent cations. PAE worked cooperatively with pectolytic enzymes. Contrary to PAE, RGAE removed at random the acetyl esters from apple pectic hairy ramified regions (MHR), to a maximum of 70 %. RGAE was essential for the activity of rhamnogalacturonase (RG), and as such comparable with A. aculeatus RGAE. FAE was specific for esterified xylan-oligomers, but did not show selectivity towards a specific ester. This enzyme could release ferulic acid as well as acetyl groups from esterified arabinoxylans in the presence of an endoxylanase.
1. INTRODUCTION Pectins are an important part of plant cell walls. Two structurally different regions can be distinguished in pectins: a "smooth" homogalacturonan region and a highly branched "hairy" or ramified region with side chains composed of arabinose and galactose [1]. The hairy regions contain a high amount of rhamnose in the main chain. The galacturonic acid of the two regions can be esterified with both methyl and/or acetyl groups. Also feruloyl esters occur in pectins, and these are found linked to the side chains. All the above mentioned substitutions influence the gelation properties of pectins [2], and consequently their use in the food industry as gelling agents.
794 Enzymes can be used to specifically modify the pectins. Pectin methyl esterase is already widely used to adjust the gelling properties of commercially available pectins. The acetyl esters also strongly affect the gelation [2,3] and removal is important for the upgrading of sugar beet pectin, extractable from a by-product of the sugar industry. This study deals with the purification and characterisation of acetyl esterases from A.niger with different specificity.
2. METHODS
2.1. Enzyme purification Rapidase C-80 (Gist-brocades) was used as enzyme source. The fractionation procedure of the crude preparation included chromatography on Bio-Gel P10 (100-200 mesh), DEAEBio-Gel A, and Bio-Gel HTP (Bio-Rad, Richmond, CA, USA). Other column materials used were cross-linked alginate (degree of cross-linking 2.34, prepared in our laboratory), Phenyl Superose HR 5/5 and Mono Q HR 5/5 (Pharmacia Biotech, Uppsala, Sweden).
2.2. Enzyme activities Acetyl esterase activities were determined toward either a solution of sugarbeet pectin (1% w/v), or one of several acetylated polysaccharides and synthetic substrates using equivalent acetyl concentrations. Incubations were carried out in piperazine buffer at pH 6.0 and 30 ~ for 1-24 hours. The reaction was stopped by heating for 5 min at 100~ Acetyl release was determined using HPLC [4] or using an enzymatic assay (BoehringerMannheim, Germany). Enzyme activities were expressed as units: one unit (U) corresponds to the release of one/~mol acetyl group per minute under the above standard conditions. KP pectin from sugar beet pulp was from Kobenhavns Pectinfabrik (Lille Skensved, Denmark). G-pectin was extracted from the whole sugar beet by Grindsted Products, Denmark. A preparation of modified hairy regions (MHR) was isolated from apple [5]. The non pectic acetylated substrates are described elsewhere [6,7,8]. Other enzyme activities, and protein content were determined as reported previously [8]. 600 MHz 1H NMR spectra were measured on solutions of ca 25 mg pectin/0.5 ml D20 on a Bruker AMX600 NMR spectrometer. The temperature was 350 K to diminish the viscosity of these solutions and 32 scans were measured. Solvent suppression was performed using presaturation during the recycle delay. 3. RESULTS AND DISCUSSION
3.1. Enzyme purification and characterisation Acetyl esterases were isolated from a Rapidase C-80 preparation according to the scheme shown in Figure 1. The purified acetyl esterases were devoid of relevant side activities, and showed great differences in their specificity towards the different acetylated substrates. The characteristics of the purified enzymes are summarised in Table 1. PAE was the only enzyme able to remove acetyl from beet pectin, FAE showed the highest activity towards
795 triacetin and xylo-oligomers, while RGAE was specific for MHR.The specificity of the enzymes will be discussed further for each enzyme separately
I crude A. niger prep]
11
i,, Bio..Gei P 10 i
]
.....
I,. D EAE Bio-GeI,A
lcrosslalginate I
IBio-Gel HTP
I FPLC
HTP-I
PAE
i ii
,l,
' Bio'-' Gel ,
iii
i
i
P 100
I
Mono Q i
AE I!1
,,,
HTP-un i
AE !1
IAEI
',''
ii
I
pectin acetyl eMerase
FAE
RGAE
feruloyl AE
rhamnogalacturonan AE
Figure 1. Purification scheme of acetyl esterases.
Table 1 Characterisation
of the acetyl esterases
activity in mU/mg protein G beet pectin Triacetin max acetyl release (% of total) KP beet pectin G beet pectin MHR ac xylan oligomers ac xylan polymers ac galacto gluco mannan Mol weight (Bio-Gel P-100, kDa) pI pH optimum temp optimum ( ~C) n.d., not determined
PAE
FAE
RGAE
6000 275
0 16358
0 0
28 30 10 9 0 0.5
0 0 0 31 4 0
0.6 0.4 70 0 0 0
60 4.1 5.5 50
>100 n.d. n.d. n.d.
42 4.5-6 5.5 50
796 3.2. PAE
This novel enzyme was the only esterase able to release acetyl from sugar beet pectin and removed about 30% of the total acetyl groups present. It also caused the release of acetyl groups from a range of other acetylated substrates, either synthetic or extracted from plants, in small amounts. PAE had an apparent molecular weight of 60 kDa and showed optimal activity at pH 5.5 and a temperature of 50 ~ The enzyme is sensitive to buffer composition and requires a bivalent cation for optimal activity and stability. In purified form this enzyme proved unstable, especially in phosphate buffers. Pectolytic enzymes appeared to have an influence on the initial rate of deacetylation of beetpectin by PAE, but not on the total amount of acetyl groups released. This remained a maximum of about 30 %. Pretreatment of the highly esterified G-pectin with pectin methyl esterase (PME) or pectin lyase (PL) resulted in an increase in the activity of PAE. As can be seen from Figure 2 removal of about 50 % of the original methyl esters resulted in a decrease of PL activity to almost zero, while PG and PAE activity increased at this point. At higher degrees of demethylation, the measurement of PG and PAE, with a strong requirement for bivalent ions is hampered by a tendency of the deesterified pectin to gelate. The actual increase of activity could therefore be higher than indicated. A decrease in the pectic molecular weight by pectin lyase resulted in an increase in the activity of PAE (Figure 2). This could be caused by easier access of the enzyme to the substrate, but PAE is not limited in its action by the substrate size as was found for FAE.
250 c Ot
250
PG
200
PAE
~= 200 E~ "E:
o 150
o 150
.=.. 0
0
z~
100
--- 100 z',
~
~ 5o
"~
50
........................................................................
PL 0
20
40 60 % demethylation
80
1oo
0
200 .. 400 600 800 1000 mint~es preincubation PL (200 mu/ml)
1200
Figure 2. The effect of enzymatic demethylation of G-pectin on the activity of PAE, PG and PL (left) The effect of pretreatment of G-pectin with PL on the activity of PAE (right) 'H NMR spectra analysis of KP-pectin and G-pectin revealed not only a substrate, but also a location specificity of PAE. The ~H NMR spectra of these pectines show two major types of acetyl groups; at 2.10 and 1.95 ppm. Upon treatment with PAE the resonance at 1.95 ppm disappears completely whereas the resonance at 2.10 ppm is only slightly reduced. The origin of these two acetyl groups is not completely clear. The signals might be related to substitutions on C-2 or C-3 of the galacturonic acid moieties, or even with single or double
797 substitutions on the same galacturonic acid. Due to the high molecular weight of these molecules and the high viscosity of the pectin solutions, even at high temperature, more sophisticated NMR techniques to locate the exact position of substitution failed as yet. 3.3. RGAE As this enzyme proved specific for the release of acetyl groups from MHR, to a maximum of 70 %, and is essential for the degradation of MHR by RG ( Figure 3), it was concluded to be comparable to that isolated previously from A. aculeatus[8,9]. The importance of this enzyme in the application of tailormade commercial pectinases is discussed elsewhere in these proceedings as well (H.P. Heldt-Hansen et al.).
(~ x (i)
,,
6
i
c-
,
I ~
.~..~,
,,
0
(I) '
o
;o
0 i
O ~
O
: ,,'/
','V O
O / O
',v
t.
,.'~:
:,,,q.'"
"~-,... _,.," ~".
d)
cc
:,/'
~-~, o o o
'qi
. ~-~-~
"8
20
I9
22
I-o- blank
I
24
I.
I
26
28
Time (min.) --,,,-- RGAE --~- RG
I
30
I
32
Comb. I
Figure 3. Degradation of MHR by RGAE, RG and the combination of these enzymes, as determined by HPSEC.
1H NMR spectra of MHR showed a more complex pattem of resonances in the acetyl group region. Although there appears to be two main groups of acetyl substituents, upon careful analysis after resolution enhancement both main signals consist of at least three different acetyl resonances. After treatment with RGAE (70% of acetyl groups removed) the overaU pattern remains unchanged, indicating that there is no preference of this enzyme for an acetyl group at a specific position. So RGAE differs from PAE in this aspect as well.
798 3.4. FAE This enzyme was shown to be specific for xylan oligomers and small acetylated synthetic substrates. Many characteristics have been published recently about this type of enzyme, purified from Trichoderma reesei, and A. oryzae [6], and a different A. niger preparation[7] FAE was identical to these enzymes in requiring an endoxylanase for activity on xylans. Under these circumstances ferulic acid could be released too. It has been observed that for the complete breakdown of different xylans, different enzyme combinations are required, as the substitutions on the xylan chain can vary widely [7]. No activity could be shown on pectins, neither in combination with other AE's and/or pectolytic enzymes.
4. CONCLUSIONS The A. niger preparation investigated in this study contains at least three different acetyl esterases, each with its own specificity. The activities of RGAE and FAE are comparable to those of similar enzymes isolated previously from A. aculeatus and a different A. niger preparation. PAE appears to be a new enzyme, with an activity specific towards one type of acetyl ester in the homogalacturonan chain of beet pectin.
5. LITERATURE J.A. De Vries, F.M. Rombouts, A.G.J. Voragen, and W. Pilnik, Carbohydr. Polym., 2 (1982) 25. G.L. Pippen, R.M. McCready, and H.S. Owens, J. Amer. Chem. Soc., 72 (1950) 813. J.A. Matthew, S.J. Howson, M.H.J. Keenan and P.S. Belton, Carbohydr. Pol., 12 (1990) 295. A.G.J. Voragen, H.A. Schols and W. Pilnik, Food Hydrocoll., 1 (1986) 65. H.A. Schols, M.A. Posthumus and A.G.J. Voragen, Carbohydr. Res., 206 (1990) 117. M. Tenkanen, PhD thesis Helsinki University of Technology (Espoo, Finland), VIT publications 242 (1995). F.J.M. Kormelink and A.G.J. Voragen, Appl. Microbiol. Biotechnol., 38 (1993) 688. M.J.F. Searle-van Leeuwen, L.A.M. van den Broek, H.A. Schols, G. Beldman and A.G.J. Voragen, Appl. Microbiol. Biotechnol., 38 (1992) 347. S. Kauppinen, S. Christgau, L.V. Kofod, T. Halkier, K. Dorreich and H. Dalboge, J. Biol. Chem., 270 (1995) 27172.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
799
Polygalacturonase inhibitor of Dieffenbachia maculata Archana Chitre and N.V. Shastri University Department of Biochemistry, Amravati Road, Nagpur, 440010, India.
ABSTRACT Stem juice of Dieffenbachia maculata contains an inhibitor of fungal polygalacturonase. The inhibitor is non dializable and heat stable. The double reciprocal plot indicates that the inhibitor causes a mixed type of inhibition. The paper also describes the distribution of the inhibitor in different varieties of Dieffenbachia, and some of its properties.
INTRODUCTION Naturally occuring enzyme inhibitors are often found as major components of the cytoplasm, secretions and intercellular fluids of many organs and tissues. Although a large number of reports are available on the presence of various enzyme inhibitors, both in animal and plant systems, comparatively very little work appears to have been done on inhibitors of pectolytic enzymes (Fielding, 1981; Resova et al., 1981; Akira and Murakawa, 1993; Mueller and Gersler, 1993). During the course of our investigations, a potent inhibitor of polygalacturonase (PG) was detected in the stem extract of Dieffenbachia, a plant known for its therapeutic effects (Barnes and Fox, 1955; Walter and Khanna, 1972; Ardditti and Rodriguez, 1982), which may, in part, explain the resistance of this plant to fungal attack. Polygalacturonase inhibitors have now gained increased importance in biotechnology, as the transfer of a PG inhibitor gene has yielded a transgenic tomato variety of much longer shelf life. This chapter describes investigations conducted on the PG inhibitor of Dieffenbachia maculata .
MATERIALS Polygalacturonase used in the present investigations was a A. niger pectinase (E.C. 3.2.1.15) purchased from Sigma Chemical Company (St. Louis, Missouri, USA). Polygalacturonic acid (PGA) sodium salt, polyvinylpolypyrolidone (PVPP), galacturonic acid (monohydrate) were also purchased from Sigma Chemical Company. 3,5-Dinitrosalicylic acid was the product of Loba Chemicals, Bombay, India. All other reagents were of analytical grade.
METHODS The A. niger PG preparation was suitably diluted with distilled water and was used as the enzyme source. Polygalacturonic acid was prepared as a one percent solution of in distilled water.
800
Preparation of inhibitor'. All the operations were carried out at 0-4~ unless otherwise mentioned. For the preparation of stem extract fresh stem was cut into small pieces and crushed mechanically. The pulp obtained was squeezed through two layers of muslin cloth. The juice was made 1% with PVPP to remove polyphenols, stirred gently for 10 minutes and cintrifuged at 1 ~ xg for 30 minutes 0-4~ The supernatant was dialysed against water. The precipitate formed was removed by centrifugation and the supernatent was used as the partially purified inhibitor. For the preparation of leaf and petiole extracts respective parts were homogenised with 1% PVPP using acid-washed sand and suspended in distilled water to get a 25 % homogenate, which was centrifuged at 10000 xg for 30 minutes. The supernatent obtained was dialysed and used as the inhibitor. Polygalacturonase assay: Polygalacturonase activity was assayed by measuring the liberated reducing groups by DNS reagent. The reaction mixture in a final volume of 2 ml contained 0.2 ml of a suitable aliquot of PG solution, 40 mM acetate buffer (pH 4.0) and 0.5% polygalacturonic acid. After 30 minutes incubation at 37~ the reaction was terminated by adding 1 ml of DNS reagent. In control tubes, the reaction was terminated prior to the addition of the substrate. The reaction mixture was then kept in a boiling water bath for 10 minutes, cooled and diluted to 13 ml with distilled water. The colour intensity was measured at 540 nm. A calibration curve established with galacturonic acid monohydrate was used to calculate the polygalacturonase activity. One unit of PG activity is defined as that amount of enzyme which liberates 1 mg galacturonic acid under the given assay conditions (30 min, 37~ Specific activity is expressed as units per mg of protein. Inhibitor assay: A suitable amount of inhibitor was preincubated with 0.2 ml of polygalacturonase and buffer in a total volume of 1 ml for 10 minutes at 37~ Control without inhibitor was run simultaneously. The enzyme reaction was initiated by the addition of 1 ml of substrate solution (1% polygalacturonic acid). The decrease in PG activity was a measure of the inhibitory activity. Proper controls containing only Dieffenbachia extract and no fungal PG in the assay mixture were also run to account for the inherent PG activity, if any, of Dieffenbachia extract. One unit of inhibitor activity is defined as the amount of inhibitor that reduces the polygalacturonase activity under the assay conditions by one unit. Specific activity of the inhibitor is expressed as units per mg protein. Protein estimation: Proteins were determined by the method of Lowry et al. (1951), using bovine serum albumin as standard.
RESULTS Each experiment was carried out several times and representative values are given.
PG inhibitor in different varieties of Dieffenbachia: Polygalacturonase inhibitor activity in whole stem was examined in different varieties of
Dieffenbachia. The data presented in Table 1 shows that the level of PG inhibitor in different species of Dieffenbachia is not significantly different. PG inhibitor activity in various parts of D. maculata: PG inhibitor was extracted from various parts of D. maculata as well as from different sections of young and mature stem by method described above. Table 2 shows that, although highest specific activity of PG inhibitor was observed in the petiole, stem tissue showed much higher concentration of inhibitor units per ml. Leaf showed the least levels of inhibitor activity.
801 Table 1 PG inhibitor in stem of different Dieffenbachia varieties.
Variety
]aahibitor units/ml
Dieffenbachia Dieffenbachia Dieffenbachia Dieffenbachia
maculata exotica amoena picta
2.00 2.87 2.62 2.17
Table 2 PG inhibitor in different parts of D. maculata.
Plant Part Units/ml
Leaf Petiole Stem (whole)
0.37 0.25 1.58
PG Inhibitor Activity Proteins/ml Specific activity (U/mg protein) 1.80 0.24 2.43
0.21 1.04 0.65
PG inhibitor in various parts of D. maculata stem: Various parts of the stem, in turn, showed variable inhibitor activity, the middle (1/3rd of the total stem length) section showing highest inhibition followed by the apical and the basal sections (Table 3). The age of the plant does not seem to have much significant effect on the level of PG inhibitor.
Table 3 PG inhibitor in different sections of D. maculata stem.
Stem Section Units/ml
9PG Inhibitor Activity Proteins/ml Specific activity (U/mg protein)
Young Stem Apical Portion Middle Portion Basal Portion
1.65 2.00 1.45
2.80 3.00 2.40
0.59 0.66 0.60
Mature Stem Apical Portion Middle Portion Basal Portion
1.62 1.87 1.25
2.56 2.64 2.08
0.63 0.71 0.60
802
Properties of D. maculata stem PG inhibitor: Effect of dialysis: Stem juice dialysed against distilled water for 16 hours. PG inhibitor activity was examined in the dialysate after 16 hours after removal of precipitate by centrifugation. Table 4 shows that the inhibitor is more or less non-dialysable although a part of its activity is lost during dialysis. Dialysis results in about 3 fold purification of the inhibitor. Dialysed inhibitor was used in subsquent studies.
TaMe 4 Effect of dialysis on PG inhibitor of D. maculata stem.
Fraction
Total Inhibitor Units/ml
Proteins mg/ml
Specific activity
Purification fold
Crude inhibitor
1.86
2.12
0.88
1.00
Dialysed inhibitor
1.38
0.56
2.46
2.78
Effect of heat treatment on PG inhibitor activity: Suitable aliquots of D. maculata stem inhibitor were kept in a boiling water bath for various periods of time, cooled quickly and assayed for residual PG inhibitory activities.
Table 5 Effect of heat treatment on PG inhibitor of D. maculata stem.
Time of exposure in boiling water bath (min) 0 10 20 30 40 50 60
Percent PG inhibitor activity remaining 100 100 93 93 93 83 83
As shown in Table 5, the PG inhibitor is relatively heat stable, retaining about 83 % of its activity after one hour at boiling water bath temperature.
Effect of preincubation of PG inhibitor with PG: A suitable aliquot of the inhibitor was incubated with 0.2 ml of polygalacturonase and buffer in a total volume of 1 ml for various periods of time at 37~ The reaction was initiated by addition of 1 ml of 1% polygalacturonic acid solution. It is apparent from Table 6 that the magnitude of inhibitory
803 activity was dependent on the preincubation of the inhibitor with polygalacturonase. Maximum inhibition was observed with preincubation for 10 minutes at 37~ Table 6 Effect of preincubation of PG inhibitor of D. maculata with PG.
Preincubation mixture Inhibitor + Polygalacturonase
Period (min)
Inhibitor activity (units/ml)
0 5 10 15
1.38 2.13 2.87 2.59
Effect of preincubation of PG inhibitor with polygalacturonic acid: One ml of 1% polygalacturonic acid was incubated with the inhibitor along with buffer in a total volume of 1.8 ml for various periods of time at 37~ The enzyme reaction was initiated by addition of 0.2 ml polygalacturonase. Results presented in Table 7 indicate that preincubation of the inhibitor with substrate for various time periods does not alter the inhibitor activity significantly. This probably indicates a minimal direct interaction involved between the substrate and the inhibitor during the course of reaction.
Table 7 Effect of preincubation of PG inhibitor of D. maculata stem with polygalacturonic acid.
Preincubation mixture Inhibitor + Polygalacturonic acid
Period (min)
Inhibitor activity (units/ml)
0 5 10 15
1.5 1.5 1.5 1.3
Effect of PG inhibitor concentration on PG activity: Various amounts of inhibitor were added in the assay mixture and the degree of inhibition of polygalacturonase activity was measured which showed a linear relationship between inhibitor concentration and percent inhibition (data not given). Effect of substrate concentration on PG inhibitor: Polygalacturonase activity was measured, with or without inhibitor, at different concentrations of the substrate in the reaction mixture to understand the mode of inhibition of polygalacturonase by D. maculata stem inhibitor. The data presented in Fig. 1 indicate that the nature of inhibition is mixed-type. The inhibitor affects both Km as well as Vmax of polygalacturonase.
804
DISCUSSION The results obtained in the present studies indicate for the first time that Dieffenbachia plants contain a potent inhibitor of polygalacturonase; the stem showing significantly higher activity than either leaf or petiole. Polygalacturonase inhibitor of D. maculata was observed to be comparatively heat-stable. Similar heat stable inhibitors of other enzymes such as amylases and proteinases have been reported by other workers also (Gudiseva et al., 1981; Shivaraj and Pattabiraman, 1980; Cinco et al., 1985; Brecher and Pugatch, 1969). Akira and Murakawa (1993) isolated a polygalacturonase inhibitor from a culture broth of Pezizales, which was completely heat stable at 100~ for 60 minutes. The magnitude of inhibition of polygalacturonase was found to be dependent on preincubation of inhibitor with the enzyme. Similar observations have been reported for other enzyme inhibitors (Shivaraj and Pattabiraman, 1980; Sharma and Pattabiraman, 1980; Padmanabhan and Shrasti, 1990). However, preincubation of the inhibitor with substrate did not show any effect on inhibitor activity. In contrast, Shivaraj and Pattabiraman (1980) and Buonocore et al. (1977), have observed inactivation of amylase inhibitor activity on pretreatment with starch. Lineweaver-Burke plot of polygalacturonase in the presence and in absence of inhibitor, suggests that the inhibition by Dieffenbachia PG inhibitor is of mixed type. Km as well as Vmax value of polygalacturonase changes in the presence of the inhibitor. Similar mixed inhibition has been reported by Mueller and Gersler (1993) for a PG inhibitor isolated from apple leaves. A heat stable, noncompetitive glycoprotein inhibitor (MW 1-2 x 106) of fungal polygalacturonase has been recently reported from Pezizales (Akira and Murakawa, 1993).
0.6
0.4 1/v 0.2
0.0
i
I
-2.0
-1.5
-1.0
-0.5
0.0
I
I
0.5
1.0
1/S Figure 1: Lineweaver - Burke plot of fungal polygalacturonase activity in the presence and in the absence of D. maculata stem PG inhibitor. S: Substrate concentration (mg/ml); V: Velocity of reaction (U/ml), o - o: Without inhibitor; o -o:With inhibitor.
805 REFERENCES
Akira, E. and Murakawa, S., Chem. Abstr., 118 (25) (1993). Abst. No. 250690n. Ardditti, J., and Rodriguez, E., J. Ethnopharmacol., 5 (1982) 293. Barnes, B.A., The Pharmacology and Toxicology of Certain Species of Dieffenbachia, Masters thesis, University of Hawaii, Gaines Ville 1 (1953). Brecher, A.S., and Pugatch, R.R., Experimentia (Basel), 25 (1969) 251. Buonocore, V., Petrucci, T. and Silano, V., Phytochem. 16 (1977) 811. Cino, F.J., Frels, J.M., Holt, D.L. and Rubnow, J.H., J. Food Sci, 50 (1985) 533. Fielding, A.H., J. Gen. Microbiol, 123 (2) (1981) 377. Gudiseva, C., Suryaprasad Raju, D. and Pattabiraman, T.N., J. Sci. Food Agr, 32 (1981) 9. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., J. Biol. Chem., 193 (1951) 265. Mueller, M. and Gersler, C., J. Dev. Plant. Pathol., 2 (1993) 68. Padmanabhan, S. and Shastri, N.V., J. Sci. Food Agr., 52 (1990) 68. Rexova, B.L., Heinrichova, K., Goebel, H. and Bock, W., Chem. Abstr., 95 (1981) Abstr. No. 92890c. Sharma, K.K. and Pattabiraman, T.N., J. Sc. Food. Agric., 31 (1980) 981. Shivaraj, B. and Pattabiraman, T.N., Ind. J. Biochem. Biophys., 17 (1980) 181. Walter, W.G. and Khanna, P.N., Eco. Bot., 26 (1972) 364.
This Page Intentionally Left Blank
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
807
Multiple forms of carrot exopolygalacturonase. Eva Stratilov~i, M~iria Dztirov~i, and Danica Mislovi6ov~i
Institute of Chemistry of SAS, Dtibravsk~i cesta 9, 842 38 Bratislava, Slovakia
Abstract
Four forms of exopolygalacturonase were found in carrot juice and one additive form more in carrot roots pulp. They differ in pH optima (3.6; 3.8; 4.7; 5.0 and 5.4), in molecular masses (about 50 000 for all excluding that with pH optimum 3.6, which has about 30 000), and in isoelectric points varying from 4.0 to 6.5. The action pattern on substrates with different degree of polymerization was different, too. The form with pH optimum 3.8 preferred oligogalactosiduronates, while the others the substrates with higher degree of polymerization. Exopolygalacturonase with pH optimum 3.8 was able to cleave the dyed D-galacturonan (DP 10). The D-galactopyranuronic acid inhibited the individual forms to various extend and with various types of inhibition. All forms showed an affinity to ConA-cellulose indicating the presence of Nglycosylation.
1. I N T R O D U C T I O N
Exopolygalacturonases [poly(1,4-ot-D-galacturonate)galacturonohydrolase (EC 3.2.1.67)] are exo-hydrolases catalyzing the hydrolytic cleavage of glycosidic ot-l,4-bonds of D-galactopyranuronic acid units situated at the nonreducing end of D-galacturonans and releasing D-galactopyranuronic acid as a sole reaction product. Substrates for these enzymes are polygalacturonic and oligogalacturonic acids and, in contrast to polygalacturonases, also digalacturonic acid [1]; the particular enzymes differ from each other by the range and rate of the effects on substrate in relation to the chain lenght [2]. Exopolygalacturonases are produced by microorganisms and by higher plants. The enzyme in plant tissue was supposed to be bound to cell wall materials via ionic interactions [3], what required hight salt concentrations for their solubilization. The most suitable substrates for exopolygalaeturonases of vegetal origin are D-galacturonan or partly degraded D-galacturonan of degree of polymerization 20 [4]. The common feature of all known exopolygalacturonases is that they do not fully degrade the substrate with
808 higher degree of polymerization [4]; the limit degradations being proportionally different with the content of neutral saccharides in the pectin molecule [2]. Therefore, it appears that these enzymes serve in cell wall metabolism, for instance in the partial degradation of pectic polysaccharides during cell growth and/or differentiation [3]. From this reason great interest is given to exopolygalacturonases from pollen in the last time [5 - 9] and to exopolygalacturonases at all [10]. Although the first study of exopolygalacturonases from carrots [11] indicated the presence of multiple forms of this enzyme based on the three present pH optima, the latter studies supported the idea of one form of exopolygalacturonase [2 - 4]. The present study deals with the whole spectrum of multiple forms of exopolygalacturonase, with forms described sooner and with forms found only now.
2. E X P E R I M E N T A L
Purification of exopolygalacturonaseforms. Juice from 10 kg of carrot roots (Daucus carota L. cv Zino) were extracted on juice extractor ES-3551 (Severin, Germany), submitted to two precipitation steps, with ammonium sulfate (std.) and ethanol (1:4), and desalted / equilibrated on Sephadex G-25 Medium (Pharmacia, Sweden) in 0.05 M acetate buffer pH 3.8. CM-Sephadex C-50 (Pharmacia, Sweden) stepwise elutions were utilized, with 0.05 M acetate buffer, pH 3.8, 0.10 M acetate buffer, pH 4.8, 0.15 M acetate buffer, pH 5.6, and finally this buffer containing 1.0 M NaC1. FPLC (Pharmacia, Sweden) utilized Superose 12 HR 10/30 in 0.05 M phosphate, pH 5.6, 0.15 M NaC1. Flow rate was 0.5 ml.min -1. Concanavalin A - cellulose chromatography [ 12] was performed in 0.1 M acetate buffer, p H 4.7 using 0.1 M m-methyl-Dmannopyranoside as an eluting agent. The enzyme forms strongly bound on cell walls were extracted by 1.0 M NaC1 solution from dry carrot roots pulp and handled as described for the multiple forms in carrot juice. Substrates. Sodium pectate (D-galacturonan content 89.8%, average M r determined viscosimetrically 27 000) was prepared by repeated alkaline deesterification of citrus pectin (Genu Pectin, Kobenhavns Pektinfabrik, Denmark) followed by precipitation with hydrochloric acid at pH 2.5 and neutralization with NaOH. Di(D-galactosiduronic) acid and penta(D-galactosiduronie ) acid were isolated from enzymatic hydrolysate [ 13] using gel filtration on Sephadex G-25 Fine in 0.05 M phosphate buffer pH 7.0 and desalting on Sephadex G-15 (both Pharmacia, Sweden) [14]. Enzymatic properties. Exopolygalacturonase activity was assayed at 30 ~ p H optimum, in 0.1 M acetate buffer, by measurement of the increase of reducing groups [15], using sodium pectate (0.5%) , di(D-galactosiduronic) acid or penta(D-galaetosiduronic) acid (1 mM solutions) as substrates and Dgalactopyranuronic acid as standard. Initial velocities were determined at five penta(D-galactosiduronic) acid concentrations, ranging from 0.1 - 1.0 mM and calculated by nonlinear regression. Products of the catalytic action on
809 oligogalactosiduronates were analyzed by thin-layer silica gel chromatography [16]. Relative molecular masses of individual exopolygalacturonase forms were approximately estimated by FPLC gel exclusion chromatography on Superose 12 utilizing low molecular weight protein markers (Pharmacia, Sweden). Ultrathin-layer isoelectric focusing in polyacrylamide gels on polyester films was performed as described [17]. Proteins were stained with Serva Violet 49 (Germany). Polygalacturonase activity was determined by the print technique with a dyed substrate (Ostazin Brilliant Red-D-galacturonan DP 10) [18] or by the print technique with colouress D-galacturonan DP 10 with following use of ruthenium red. The enzyme forms were washed out by water from freezed gel segments obtained by preparative izoelectric focusing. Activities of so separated enzyme forms were detected by pH optima values.
3. RESULTS AND DISCUSSION
Crude exopolygalaeturonases from carrot juice and carrot pulp were purified in two stages; to produce first an intermediate product and then partially separated exopolygalacturonases (Fractions A, B, C). The first stage utilized two precipitations and Sephadex step. Purification of resulting lyophilisate from juice by ion-exchange chromatography on CM-Sephadex C-50 gave a separation into two active fractions (Fig. 1).
0.8
~,b
fc
O r
~d
A
.
1.0 O
) 0.4
- 0.5 )
|
3O
9O 6O Fraction, No.
120
0
150
Fig. 1. Separation of two exopolygalacturonase groups (Fraction A, Fraction B) on CM-Sephadex C-50. Column size, 20x250 mm. Stepwise elution with 0.05 M acetate buffer, pH 3.8 (starting at arrow marked a), 0.10 M acetate buffer, pH 4.8 (at arrow marked b), 0.15 M acetate, pH 5.6 (at arrow marked c) and the latter buffer plus 1.0 M NaC1 (at arrow marked d). Fraction size 6 ml per half hr. Exopolygalacturonase activity determined with sodium pectate, pH 5.0 ( O - - O ) and expressed as As~o.
810 The course of separation of the lyophilisate from pulp by this method was very similar to separation of the juice lyophilisate but led to only one activity peak. The pH optima determination (Fig. 2) and gel chromatography on Superose 12 (Fig. 3 a,b,c) showed these fractions to be partially purified, complex mixture of more exopolygalacturonase forms with different molecular masses and pH optima. 100!
# :~_ 50 u
<
3.6
pH
Fig. 2. pH optima determination of Fraction A (O----O), Fraction B ( 4 ~ - - 1 ) , and Fraction C (0F-'~).
100
1.0
a
o
< 50
0.5
lb
|
30
50
30 s0 Fraction, No.
lb
30
50
Fig. 3. Molecular mass distribution of proteins on Superose 12 column (FPLC) in a - Fraction A, b - Fraction B, and e - Fraction C. Buffer - 0.05 M phosphate, pH 7.0, 0.15 M NaC1, fraction size 0.5ml/min. Exopolygalacturonase activity determined with penta(D-galactosiduronic) acid pH 5.0 (~----~) and 3.8 (O--'--O) as a substrate in a, pH 5.4 (II--"-O) and 3.6 (~----~) in b, and pH 4.7 (~----V) in c.
Fraction A was examinated to purify by affinity chromatography on ConA cellulose. Some impurities were removed but the separation of
811 exopolygalacturonase forms was not observed. On Fig. 4 is shown the elution profile of purified fraction on Superose 12 column.
100"
t
-1.o O
< 0.5
1'o
30
Fraction, No.
Fig. 4. Molecular mass distribution of Fraction A purified on Concanavalin A cellulose on Superose 12 column. Buffer - 0.05 M phosphate, pH 7.0, 0.15 M NaC1, fraction size 0.5 ml/min. Exopolygalacturonase activity determined with penta(D-galactosiduronic) acid pH 5.0 (k---A) and pH 3.8 (O---<)).
Partially purified Fraction A contained exopolygalacturonase forms with two pH optima - pH 3.8 and pH 5.0 (Fig. 2). The enzyme with pH optimum 5.0 is probably identical with one form of exopolygalacturonase present in carrot roots suggested by Hatanaka and Ozawa [11] and described by Heinrichov~i [2] with pH optimum 5.1 and action pattern characteristic for plant exopolygalacturonases which prefer D-galacturonan or partly degraded D-galacturonan (DP not lower than 20) [4]. The molecular mass of the enzyme in our preparation was about 50 000 and was identical with the molecular mass of enzyme with pH optimum 3.8 (Fig. 3a). Its action pattern was characterized by small difference in the initial ratio of degradation of pectate and pentasaccharide and markable decrease in the initial ratio of degradation of di(D-galactosiduronic) acid (Tab. 1).
Table 1 The inicial reaction rates of exopolygalacturonase forms distinguished on the base of their pH optima on substrates with various degree of polymerization [(GA)2- di(D-galactosiduronic) acid, (GA) 5 - penta(D-galactosiduronic) acid, MGA - D-galactopyranuronic acid] Activity on substrate (~tmol/min.mg) pH (GA) 2 (GA)s (GA)5 + 0. lmlvl sodium pectate MGA 3.8 0.502 1.837 1.722 0.549 5.0 0.300 1.820 0.976 1.919 3.6 0.022 0.030 nondet. 0.280 5.4 0.010 0.025 0.013 0.723
812 The presence of the enzyme with lower p H optimum was not described yet [2, 11]. It gives only negligible peak by this pH (Fig. 2) in comparison with pH optimum by 5.0 (only 28 % from it) when pectate was used as a substrate. Isoelectric focusing (ruthenium red staining) showed poor and unclear bands noncorresponding the real activity of both enzymes (Fig. 5) in the p H area 4.5 4.7.
i~!ii
B
A
B
A
Fig. 5. Isoelectric focusing (pH gradient 3-10) of Fraction A and Fraction B in ultrathin polyacrylamide layers. 5 l.tg of fractions were applied. Activity detection with ruthenium red (left) and with Ostazin Brilliant Red/Dgalaeturonan D P 10 agar print (fight).
The preparative isoelectric focusing helped us to attribute the lower isoelectric point (4.5) to the enzyme with pH optimum 3.8 and the isoelectric point 4.7 to the enzyme with pH optimum 5.0. The use of dyed substrate (DP 10) showed one marked band (Fig. 5) corresponding the value 4.5. The detection of exopolygalaeturonase with pH optimum 3.8 by this method was in discrepancy with the autors [18}, who use it as a diverse method for distinguishing the exopolygalacturonases and polygalacturonases. The affinity of these enzymes to such a dyed substrate is probably dependent on the arrangement of the active site of individual enzyme and cannot be generalized to exoenzymes or endoenzymes. Other difference between these two exopolygalacturonases was their action pattern expressed by various initial rates on substrates with various DP (Tab. 1), where exopolygalacturonase with pH optimum 5.0 preferred substrates with
813 higher DP and the enzyme with lower pH optimum preferred oligosubstrates. The affinity against penta(D-galactosiduronie) acid as a substrate was established by determination of K M and V for both pH. The K M value was 2.222 . 10 .4 mol.l "1 for pH 3.8 as well as for pH 5.0 while the V value was different: 4.348 ktmol.min-t for the pH optimum 5.0 and 2.121 ktmol.min-~ for the other one, respectively. Fraction B was more complex than Fraction A, containing enzymes from Fraction A and at least two other enzymes with pH optima 3.6 and 5.4 (Fig. 2). These forms can be considered to be minority exopolygalacturonases in carrot juice. The action pattern of enzyme with pH optimum 5.4 was characterized with an increase of initial rate of catalysis with an increase of polymerization degree of substrate (Tab. 1). Its molecular mass was identical with the molecular mass of enzyme with pH optimum 5.0 (Fig. 3b). The isoelectric points were less acidic as by enzymes in Fraction A - the main about pH 6.5, the other form about 5.7 (Fig. 5). Isoelectric focusing patterns showed more activity bands as corresponded the found four pH optima what was probably appropriated with the various degree of amidation of individual exopolygalacturonase structures. The K M and V value of this form were not established because of impurities from exopolygalacturonase with p H optimum 5.0. The results of Hatanaka and Ozawa [11] based on pH optima determination suggested the presence of such an enzyme in carrots, too. The courve of pH optima determination indicated a presence of acidic exopolygalacturonase form as it was in Fraction A but with slight shift to pH 3.6 (Fig. 2). It was impossible to commute this enzyme form with the acidic exopolygalacturonase from Fraction A because of its molecular mass about 30000 and action pattern identical with form with pH optimum 5.4. Further characterization of this form was not made because of its low content in lyophilizate. The third described enzyme form with pH optimum about 4.7 [11, 4], we found in Fraction C - the fraction from carrot roots pulp (Fig. 2 ) . W e supposed that this form of exopolygalacturonase is relatively strongly bound on carrot cell walls and so it can be released only by higher salt concentrations. The approximative molecular mass determination on Superose 12 (Fig. 3c) showed the molecular mass about 50 000 for this form and the second, with more acidic p H optimum, form present in the fraction. The further characterization of these enzymes showed the exopolygalacturonase with pH optimum 4.7 to be identical with enzyme described sooner by Pressey and Avants [4] and exopolygalacturonase with pH optimum 3.8 to be identical with the enzyme from Fraction A. In conclusion, the exopolygalacturonase form with p H optimum 3.8 can be considered to be the main enzyme form present in carrot roots. The affinity chromatography on ConA - cellulose indicated the presence of small N-glycosylation of all forms of exopolygalacturonases present in carrot roots (unpublished results). This method was usefull for purification of these enzymes from other protein inpurities but was completely uneffective by separation of individual forms (Fig. 4). In contrast with previous result [4] all exopolygalacturonase forms were inhibited by their product, D-galactopyranuronie acid [19] however the extent (Tab. 1) and the type of inhibition was various (competitive for enzyme with pH optimum 3.8 and mixed for the others).
814 This work should be considered as an introduction to plant exopolygalacturonase multiple forms structure studies.
4. R E F E R E N C E S
.
3. 4. 5. 6. 7.
.
10. 11. 12.
13. 14. 15. 16. 17. 18. 19.
IL. Rexov~i-Benkov~i and O. Markovi6, Adv. Carbohydr. CherrL, 33 (1976) 323. K. Heinrichov~i, Collect. Czech. Chem. Commun., 42 (1977) 3214. H. Konno, Methods in Enzymol., 161 (1988) 373. R. Pressey and J.K. Avants, Phytochemistry, 14 (1975) 957. S.M. Brown and M.L. Crouch, The Plant Cell, 2 (1990) 263. Y.H. Sheng and A. Collmer,. Bacteriol., 172 (1990) 4988. M.F. Niogret, M. Dubald, P. Mandaron and R. Mache, Plant Mol. Biol., 17 (1991) 1155. S.J. Tebbutt, H.J. Rogers and D.M. Lonsdale, Plant Mol. Biol., 25 (1994) 283. M.E. John and M.W. Petersen, Plant Mol. Biol., 26 (1994) 1989. K. Heinrichov~i, J. Heinrich and M. Dztirov~i, Collect. Czech. Chem. Commun., 60 (1995) 328. Ch. Hatanaka and J. Ozawa, Agr. Biol. Chem., 28 (1964) 627. D. Mislovi6ov~i, M. Chudinov~i, P. Gemeiner and P. Do6olomansk)~, J. Chromatogr., 664 (1995) 145. K. Heinrichov~i, Biologia (Bratislava), 38 (1983) 335. IL. Rexov~i-Benkov~i, Chem. zvesti, 24 (1970) 59. M. Somogyi, J. Biol. Chem., 195 (1952) 19. A. Koller and H. Neukom, Biochim. Biophys. Acta, 83 (1964) 366. B.J. Radola, Electrophoresis, 1 (1980) 43. O. Markovi6, D. Mislovi6ov~i, P. Biely and K. Heinrichov~i, J. Chromatogr., 603 (1992) 243. M. Dztirov~i, K. Linek and E. Stratilov~i, Biologia (Bratislava), 50 (1995) 1.
MOLECULAR GENETICS AND REGULATION OF PECTINASE BIOSYNTHESIS IN SAPROPHYTIC AND PHYTOPATHOGENIC MICROBIAL SYSTEMS
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J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
The exo-polygalacturonase from Aspergillus characterization and cloning of the gene
817
tubingensis:
H.C.M. Kester, M.A. Kusters-van Someren, Y. Mialler and J. Visser
Section of Molecular Genetics of Industrial Micro-organisms, Wageningen Agricultural University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands.
Abstract From the culture fluid of the hyphal fungus Aspergillus tubingensis an exopolygalacturonase (PGX) was isolated and purified. Besides the physico-chemical properties of the purified enzyme, the mode of action and the kinetics were studied on polymeric and oligomeric substrates. The enzyme hydrolyses galacturonic acid from the non-reducing end of polygalacturonic acid and from oligogalacturonates with different degree of polymerization (DP = 2-7). Kinetic analysis data on non-reduced and reduced oligogalacturonates were used for the calculation of the subsite affinities to obtain more insight in the substrate binding and catalyis on the molecular level. The PGX encoding gene, pgaX, was cloned by reverse genetics and the nucleotide sequence was determined. Restricted amino acid sequence identity is found with other polygalacturonases and a phylogenetic tree has been constructed with fungal, bacterial and plant polygalacturonases. The expression of PGX is induced by galacturonic acid and repressed by glucose at the level of transcription.
1. PURIFICATION
A. tubingensis strain NW756 was cultivated for 55 h at 30~ in minimal medium according to Pontecorvo et al [1] supplemented with yeast extract (2g/l) and 10 g/1 endo-PG II digested polygalacturonic acid (PGA). Complete hydrolysis of PGA by endo-PG II resulted in a mixture of mono, di and trigalacturonate [2]. Four subsequent column chromatographic steps were used for purification of the enzyme for which the data are summarized in Table 1.
818 Table 1 Purification of exo-polygalacturonase from A. tubingensis Step Culture fluid Crosslinked alginate fraction 1 DEAE-Sepharose 2 S-Sepharose 3 MONO Q 4
Volume (ml) 3000 non-bound 210 90 72 10.8
Specific activity (U/mg) 17.4 14.6
Yield (%) 100 59
8.6 21.4 39.0
16 3.6 2.3
The crosslinked alginate matrix was prepared by crosslinking sodium alginate (Kelgo Gel HV) with epichlorohydrin according to Rombouts et al [3]. All other column materials were purchased from Pharmacia Fine Chemicals. Polygalacturonase activity was determined by measuring the release of reducing sugars according to Stephens et al [4], in a reaction mix containing 0.25% PGA in 0.1 M Na-acetate buffer pH 4.2. Buffers used:
0.02 M Na-acetate pH 4.0; 2 0.01 M bisTris/HC1 pH 5.5,0-0.5 M NaC1 gradient; 3 0.02 M Na-acetate pH 4.0, 0-0.5 M NaC1 gradient; 4 0.02 M piperazine/HC1 pH 6.0, 0-0.2 M NaC1 gradient.
2. P H Y S I C O - C H E M I C A L PROPERTIES
An apparent molecular mass of 78 kDa was measured after SDS-polyacrylamide electrophoresis. Enzymatic N-deglycosylation resulted in two bands of 52 and 55 kDa respectively (Fig. 1). O-deglycosylation had no effect on the molecular mass. Charge microheterogeneity of the enzyme was demonstrated by isoelectric focusing. Five distinct bands could be detected in the pH range 3.7-4.4 all having polygalacturonase activity. The pH optimum for activity was 4.2 (Mcllvaine buffers).
819
1
2
3
Fig. 1. SDS-polyacrylamide gel electrophoresis of purified PGX before (lane 3) and after (lane 2) N-deglycosylation. In lane 1, molecular mass markers; 92.6, 67, 45 and 29 kDa, from top to bottom.
3. KINETIC ANALYSIS
The Michaelis constant (Km) and the maximum rate (Vmax) of PGX for PGA, reduced and non-reduced oligogalacturonates with increasing degree of polymerization (DP=2-7) were determined. Data are presented in Table 2. Table 2 Kinetic parameters of A. tubingensis PGX oligogalacturonates and polygalacturonic acid. Substrate
Km (mM) digalacturonate 1.44 trigalacturonate 0.94 tetragalacturonate 0.68 pentagalacturonate 0.53 hexagalacturonate 0.44 heptagalacturonate 0.32 polygalacturonate 3.2 (mg/ml) The turnover number was obtained concentration. 2 Values for the reduced oligomers.
Vmax (U/mg) 183 218 245 243 237 213 255
for
nonreduced
Turnover number (s- 1)l 159 188 212 211 205 185 221
and
Km 2 (mM) -1.31 0.89 0.65 0.49 0.29 n.d.
as V/e0, where e0 is the molar enzyme
reduced
Vmax 2 (U/mg) 150 248 249 237 243 n.d
820
For the oligomers Km decreases with increasing chain length whereas Vmax increases for the longer substrates and reaches a plateau value for tetragalacturonate and higher oligomers. The same was found for the reduced oligomers. PGX showed no activity on reduced dimer. Using polygalacturonate as substrate competitive inhibition was measured for D-galacturonate (Ki=0.3 mM) and reduced digalacturonate (Ki=0.4 mM). No inhibition was found for reduced D-galacturonate.
4. SUBSITE MAPPING
According to the method as described by Hiromi et al [5,6] the measured parameters Km and Vmax were used for the calculation of the subsite affinities (Ai) and the intrinsic rate constant. The calculated subsite affinities are summarized in Table 3. Table 3 Subsite affinities (Ai) and intrinsic rate (kint) for A. tubingensis PGX. Subsite I
1
2
3
4
5
6
7
Subsite affinity (Ai) (kJ/mol)
- 1.6
24.5
1.5
1.1
0.6
0.4
0.7
1The subsites are numbered from the non-reducing substrate end. The catalytic site is situated between subsite 1 and 2. The intrinsic rate constant (kint=716 S"1) is about three times as high as the highest values measured for the tumover number (221 s-1) of the enzyme which implies that there is always a large contribution of non-productive enzyme-substrate complex formation. The negative value of A 1 (-1.6 kJ/mol) and the high value for A 2 (§ kJ/mol) facilitates this nonproductive binding mode. The arrangement of the subsite binding affinities also explains the inhibition of the enzyme by galacturonate and reduced digalacturonate, preferentially occupying subsite 2 and the subsites 2 and 3 respectively. The fact that no activity was measured on reduced digalacturonate and that PGX is not inhibited by reduced Dgalacturonate leads to the conclusion that for binding at subsite 2 a closed ring structure is essential.
821
5. CLONING AND CHARACTERIZATION OF THE PGAX GENE.
Purified PGX was cleaved with CNBr. From three CNBr fragments the N-terminal amino acid sequence was determined. PCR with six oligonucleotides based on these amino acid sequences resulted in a 600 bp fragment. Sequencing of this fragment and alignment of the deduced amino acid sequence with known PG sequences showed homology, indicating that we had indeed cloned the pgaX gene. The PCR fragment was used to isolate the pgaX gene from an A. tubingensis gene library. The complete sequence of the pgaX gene has been determined. Seven introns are present in the gene which were all confirmed by eDNA sequencing. The introns are between 50 and 58 nt long, have fungal consensus splice sequences and all but one have in frame stop codons. The pgaX gene encodes a secreted protein of 47.1 kD with a predicted iso-electric point of 4.11. The calculated molecular weight is lower than the one determined by SDS-PAGE (78 kDa), even after deglycosylation (55 kDa). There are 12 possible N-glycosylation sites in PGX. A substantial discrepancy between calculated and determined molecular mass has also been found for the A. niger endo-PGI, 35 kDa and 55 kDa respectively [7], although this enzyme has a low degree of glycosylation.
6. INDUCTION OF THE PGAX GENE.
A. tubingensis mycelium was transferred to media with either 1% glucose or 1% galacturonic acid, and to these media with an extra 1% glucose after overnight pregrowth on medium with 1% sucrose. RNA was isolated 1 h after transfer and used for Northern analysis with the pgaX gene as a probe. It is clear that only after transfer to galacturonic acid pgaX mRNA is made (Fig. 2). Extra glucose completely inhibits transcription, showing that the pgaX gene is under carbon catabolite repression. 1
2
3
4 Fig. 2. Northem analysis of pgaX expression using a 2.0 kb pgaX PstI fragment as a probe. A. tubingensis was pregrown for 20 h on 1% sucrose and transferred to medium with different carbon sources. Lane 1: 1% glucose; lane 2: 2% glucose; lane 3: 1% galacturonic acid; lane 4:1% galacturonic acid, 1% glucose.
9
....
.
822
7. PGX PRODUCTION.
In order to obtain high level expression of the pgaX gene under conditions where no other pectinolytic enzymes are produced [8], a gene fusion with the A. niger pyruvate kinase (pk/) promoter [9] was constructed. A. tubingensis was transformed with this plasmmid and the A. niger pyrA gene as selection marker. One of the transformants has a high production level upon growth on medium with glucose as sole carbon source: 30-40 mg PGX per liter. Purification of PGX from the culture fluid was a one step procedure.
8. H O M O L O G Y WITH OTHER POLYGALACTURONASES.
Multiple sequence alignments were made between PGX and endo- and exo-PGs present in the SwissProt, PIR and GenBank databases using Clustal [10] and a phylogenetic tree [ 11 ] was constructed (not shown). Fungal, plant and bacterial PGs are known, which each can be placed into separate classes. The plant enzymes fall into two groups, the pollen-specific PGs and the fruit ripening related PGs. Their overall structures are probably similar, since almost all cysteine residues (12) are conserved in all these enzymes. One cysteine residue (C257 in PGI_BN) is present only in the pollen-specific PGs. Six of the twelve cysteines are also conserved in the fungal endo-PGs. A. tubingensis PGX is more closely related to plant PGs than to the fungal ones. The highest sequence identity (24%) is shared with the avocado PG (PGI_PA). Only four short stretches of amino acids are totally conserved between all PGs: 1) NXD (PGX:221-223), 2) DD (PGX: 244,245), 3) HG (PGX: 267,268) and 4) RXK (PGX: 302304), suggesting that at least some of these residues have a function in catalysis and/or substrate binding. Polygalacturonase hydrolyzes its substrate, polygalacturonic acid, by general-acid catalysis. Chemical modification of histidine residues by diethyl pyrocarbonate leading to inhibition of enzyme activity supported the hypothesis that a histidine is involved in catalysis. Chemical modification of carboxyl groups strongly suggests that at least one carboxyil acid residue is essential for PG activity. Since only one histidine residue and three aspartate residues are conserved in all enzymes, the histidine and one of the aspartates are likely to be active site residues. The positively charged residues arginine and lysine may be involved in substrate binding.
823
9. A C K N O W L E D G E M E N T S
Part of this work was funded by Gist-brocades NV (Delft, The Netherlands) and the Copenhagen Pectin Factory (Copenhagen, Denmark). J.V. also acknowledges a grant of the European Community, AIR2-CT-941345.
10. REFERENCES
9
10 11
G. Pontecorvo, J.A. Roper, L.J. Hemmons, K.D. MacDonald and A.W.J. Bufion, Adv. Genet., 5 (1953) 141. H.C.M. Kester and J. Visser, Biotechnol. Appl. Biochem., 12 (1990) 150. F.M. Rombouts, A.K. Wissenburg and W. Pilnik, J. Chromatogr., 168 (1979) 151. B.G. Stephens, H.J. Felkel Jr and W.M. Spinelli, Anal. Chem., 46 (1974) 692. K. Hiromi, Biochem. Biophys. Res. Commun., 40 (1970) 1. K. Hiromi, Y. Nitta, C.Numata and S. Ono, Biochem. Biophys. Acta (1973) 362 H.J.D. Bussink, K.B. Brouwer, L.H. de Graaff, H.C.M. Kester and J. Visser, Curr. Genet., 20 (1991) 301. M.A. Kusters-van Someren, M.J.A., Flipphi, L.H. de Graaff, H.C. van den Broeck, H.C.M. Kester, A. Hinnen and J. Visser, Mol. Gen. Genet., 234 (1992) 113. L. de Graaff, H. van den Broeck and J. Visser,. Curr. Genet., 22 (1992) 21. D.G. Higgins and P.M. Sharp, Gene 73, (1988) 237. J. Felsenstein,. Cladistics 5, (1989) 164.
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J. Visser and A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V.All rights reserved.
825
pgaE f r o m Aspergillus niger e n c o d e s a fourth p o l y g a l a c t u r o n a s e Molecular cloning and biochemical characterisation o f the gene product
Lucie Parenicov~i, Jacques A.E. Benen, Harry C.M. Kester and Jaap Visser
Section of Molecular Genetics of Industrial Microorganisms Wageningen Agricultural University Dreyenlaan 2, 6703 HA Wageningen, The Netherlands
Abstract: A gene encoding a fourth polygalacturonase from Aspergillus niger N400, designated as pga E, has been identified based on protein homologies found after database search. The entire coding region consists of 1296 bp and is interrupted by three small introns as revealed by cDNA sequencing. The deduced protein comprises 378 amino acid residues including 39 Nterminal residues of the pre-propeptide. The calculated molecular weight and pI are 35 584 and 3.6, respectively. For overproduction of PGE a pki-pgaE gene fusion was made using the strong glycolytic promoter from the A. niger pyruvate kinase gene. The protein was purified to homogeneity and used for further characterisation. The cleavage profile of PGE on polygalacturonate as a substrate showed a typical pattern as found for endopolygalacturonases. The pH optimum of PGE is 3.8.
I. Introduction
Aspergillus niger is a filamentous fungus which produces a whole range of enzymes involved in degradation of pectin. These enzymes include pectin lyases, polygalacturonases and pectinesterase. Because this fungus holds the GRAS status it is used in food and beverage industry in processes for preparation of juices, gels etc. [1 ]. Pectolytic enzymes with different substrates specificities can also serve as tools for preparation of pectic substances of defined qualities. Previously it was reported that gene families encoding pectin lyases [2] and polygalacturonases [3] are present in the A. niger N400. Two genes - p g a I and pgalI corresponding to the most abundant polygalacturonases [4] have been isolated using reverse genetics. Bussink et al. [3] used a probe derived from the A. niger N400 pgalI gene to identify five additional classes of recombinant phages based on restriction pattems. Transformants of
826
A. nidulans with selected phages of classes A, B, C, D and E resulted in increased polygalacturonase activity in comparison with the wild type strain. The culture media ofpga transformants were further analysed by Western blotting with polyclonal antibodies raised against PGI and in all samples examined cross-reactive bands with molecular masses similar to that found for PGI or PGII were detected. We set out to identify the polygalacturonase encoded by the class E phages.
2. Materials and Methods
The recombinant phages of classes A,B,C,D and E were isolated from the genomic library of
A. niger N400 by Bussink et al. [3]. Standard protocols for DNA hybridisation, cloning etc. were used [5]. cDNA was generated by a reverse transcriptase reaction and PCR [6]. For overproduction of PGE a pki-pgaE gene fusion was made by ligating a 0.7 kb promoter fragment of A. niger pyruvate kinase gene [7] and pgaE. This construct was used for cotransformation of A. niger strain 617.4 (prtF, leu, pyrA) using the pyrA gene of A. niger as a selection marker [8]. The pki-pgaE transformant 639.44 served for isolation of PGE. The minimal medium [9] used in 22h cultivation ofpki-pgaE transformant was suplemented with 2% fructose as a sole source of carbon. Under these conditions expression of other polygalacturonase-encoding genes is repressed. PGE was purified in a single step procedure using DEAE-Sepharose Fast Flow column chromatography. After polygalacturonate hydrolysis the products were analysed as described by Benen et al. [10]. The pH optimum of PGE was assayed in Mcllvaine buffers and the activity of the enzyme was determined by measuring the release of reducing sugars using the neocuproine method.
3. Results
3.1. Cloning and sequencing of the pgaE gene encoding a new polygalacturonase
The LE6 DNA was digested with several restriction enzymes and analysed via Southern blots using a probe derived from the pgaII gene. A restriction map of the LE6 clone revealed that the complete gene should be present on a 3.0 kb EcoRI fragment (see Fig. 1). fl::
Q::-~-~
~
~
-~
,,,
,,,:2Z=IZ
~
::E
co
-~ n
"~
o
_ _
co_
a::
_
cc
_
-
:
_--
-
._
~
=
Figure 1. The detailed restriction map of the 3.0 kb EcoRI fragment of the LE6 clone. The position of pgaE gene is shown by the black bar. This fragment was cloned into pBluescript SK + and further analysed.
o
827
The entire fragment was sequenced from both strands and after comparison of the sequence data with gene- and protein databases the presence of pgaE, encoding a new polygalacturonase, was confirmed. The 1296 bp long coding region is interrupted by three short introns ranging in size from 50 bp to 59 bp as was demonstrated by cDNA sequencing.
3.2. Deduced amino acids sequence of PGE and homology with other polygalacturonases from A. niger N400 The deduced PGE protein consists of 378 amino acid residues. Based on the known composition of N-termini of PGI and PGII proteins it is assumed that the 39 N-terminal amino acids of PGE form the pre-prosequence of the protein. The molecular weight (35 584) and isoelectric point (pI = 3.6) of mature PGE were calculated based on deduced amino acid sequence. 'Mature PGE' showed the highest homology - 79% (Fig. 2) - with PGC from A. niger based on a protein database search.
PGC PGE Consensus
mvrqliliSS llaavavRA .... padpAhP MVTeAPdvnl vEKRATtCTF ..... m v t S S s v i g l t l W A a i v s a s p v A d P L V T p A P k l e d I E K R A T s C T F . . . . . . . . SS . . . . . . . . A . . . . . . . . A - P - V T - A P . . . . . E K R A T - C T F
46 45
PGC PGE Consensus
SGSEGASkAS SGSEGASsAS SGSEGAS-AS
KSKTSCSTIy KSKTSCSTIv KSKTSCSTI-
LSDVAVPSGT LSDVAVPSGT LSDVAVPSGT
TLDLsDLNDG TLDLtDLNDG TLDL-DLNDG
THVIFqGETt THVIFeGETh THVIF-GET-
96 95
PGC PGE Consensus
FGYEEWeGPL FGYEEWsGPL FGYEEW-GPL
VrVSGTDITV VsVSGTDITV V-VSGTDITV
eGesdAvLNG tGadgAyLNG -G---A-LNG
DGSRWWDGEG DGSRWWDGEG DGSRWWDGEG
gNGGKTKPKF sNGGKTKPKF -NGGKTKPKF
146 145
PGC PGE Consensus
FYAHDLTSST FYAHDLTSST FYAHDLTSST
IksIYIeNSP IsgIYIqNSP I--IYI-NSP
VQVFSIDGST VQVFSIDGST VQVFSIDGST
dLTMtDITVD yLTMeDITID -LTM-DIT-D
NTDGDtDdlA NTDGD.DgeA NTDGD-D--A
196 194
PGC PGE Consensus
ANTDGFDIGE ANTDGFDIGD ANTDGFDIG.
STYITITGAe STYITITGAn STYITITGA-
IYNQDDCVAI VYNQDDCVAV -YNQDDCVA**
NSGENIYFSa NSGENIYFSg NSGENIYFS-
sVCSGGHGLS gVCSGGHGLS -VCSGGHGLS .
246 244
PGC PGE Consensus
IGSVGGRdDN IGSVGGRsDN IGSVGGR-DN
TVKNVTFYDv nVlkSQqaIR TVKNVTFYDs dIksSQngVR TVKNVTFYD ..... SQ---R
IKTIYGDTGS IKTIYGDTGS IKTIYGDTGS
VSEVTYhEIa VSEVTYkEIt VSEVTY-EI-
296 294
PGC PGE Consensus
FSDaTDYGIV LSDiTDYGIV -SD-TDYGIV
IEQNYDDTSk VEQNYDDTSe -EQNYDDTS-
tPTtGVpItD sPTdGItIeD -PT-G--I-D
FVLENIvGtc FVLDNVqGsv FVL-N--G--
EdddcTeVYI Essg.TnIYI E .... T - - Y I
346 343
PGC PGE Consensus
aCGdgSCsDW vCGsdSCtDW -CG--SC-DW
TWTgVsVTGG TWTdVdVTGG TWT-V-VTGG
svSdDCINVP ktSsDCeNVP --S-DC-NVP
sgISCdl* ddISC*.. --ISC---
383 378
Figure 2. Comparison of deduced amino acids sequences of PGC and PGE from A. niger N400. The conserved amino acids residues are in bold face. Conserved His and Asp residues among polygalacturonases from different origin [11] which are probably involved in catalysis [12] are marked (*) below the residue.
828 Fig. 3 represents the alignment of the four polygalacturonases from A. niger characterised so far. The amino acid sequences vary in number of residues especially in the pre-propeptides. Based on this alignment PGE seems to be intermediate between PGC and PGI. One to three introns have been identified in pga genes from A. niger, they are located in conserved positions as found also in polygalacturonase-encoding genes from A. oryzae, A. flavus and A. tubingensis but not in the phytopatogenic fungi Cochliobolus carbonum or Fusarium moniliforme which also show high homology with PGE. 21+6 .
IB
I
.
.
.
.
183 aa
I
R
18+ 13
[
IA
145aa]
IB 140aa
[
152aa
....
*
IB
IA 142aa
Xl0aa
I PGC 383 aa 343 m.p.
X07aa
] PGE 9 378 aa 339 m.p.
*
IB
IA
[ PGI 368 aa 337 m.p.
IC
~
KR 19+20
362 aa 335 m.p.
KK 16+24
il
152 aa *
IC ....
I......i l148aal KR
141aa
143aa ,
[
aa = amino acids m.p. = mature protein
Figure 3. Schematic representation of the PGII, PGI, PGC [13] and PGE proteins from A. niger, indicating the putative processing sites for the signal peptide ( m ) and the mono- and dibasic processing site for the propeptide ( 1 ) . The position of introns (IA, IB and IC) are indicated (I) and variation of amino acids number is shown in different parts of protein. The putative N-glycosylation sites are marked (*).
3.3. Partial biochemical characterisation of PGE
PGE was isolated as desribed in Material and Methods. SDS-PAGE electrophoresis of purified protein showed a single band migrating at approximately 60 kDa. This observation is not in the agreement with the calculated molecular weight of 35 584. However a similar effect has been observed previously in case of PGI and PGC. Apart of the N-glycosylation which plays role in all PGs (Fig. 3), O-glycosylation may also be present as indicated by the band size shift after a treatment of PGE with 0.1M NaOH (data not shown). PGE activity was demonstrated on polygalacturonate as the substrate. In the beginning of hydrolysis higher oligomers are released while as final products PGE generates mono-, diand trimers of galacturonic acid residues (Fig. 4). The specific activity is approx. 30 U/mg with polygalacturonate as a substrate. The pH optimum of PGE for polygalacturonate hydrolysis is pH 3.8 (Fig. 5).
829 G1
G1
zl( 3 :
IS
G2
24 h
i
IS
G2
'
G4
_ ,iL_J
4h 2h
. . . . . .
,
;
~
/I
G6
60 min
f .
.
.
.
~.._,~~
.....
I0 min 0 min
I
I
I
I
I
o
5
I0
15
20
Minutes
Figure 4. Time profile of polygalacturonate hydrolysis by PGE. 20 ktl of 10-times diluted samples were analysed by HPLC. PGE (2 mg/ml) was diluted 10-times and 20 ~tl was incubated in 1 ml of 1% (w/v) polygalacturonate, 50 mM Na-acetate buffer ( pH = 4.2) at 30~ IS = internal standard (0.1 ~tM glucuronic acid), G1 to G6 represent galacturonate to hexagalacturonate.
80 60 E
40 20
0
'"
2
i
i
3
4 pH
~! ' , ~ " - - " - ~ ,
5
, 6
Figure 5. pH activity profile of PGE on polygalacturonate. Mcllvaine buffers were used in the pH range from 2.5 to 5.5. Reducing sugars were detected by the neocuproine method. PGE (2 mg/ml) was 80-times diluted and incubated with 500 ~tl of a 0.25% (w/v) polygalacturonate solution, at 30~
830 4. Conclusions
The molecular analysis of the recombinant ~E6 phage confirmed the presence of a fourth endopolygalacturonase encoding gene in the genome of A. niger N400. The deduced amino acids compositon shows 79% of homology with PGC from A. niger. The schematic comparison of PGs from A. niger characterised so far showed a similar arrangement at the DNA level (position of introns) and also at the protein level (pre-prosequence, Nglycosylation site). The basic biochemical characterisation of PGE demonstrates that the enzyme has a random endolytic activity on polygalacturonate and that its pH optimum is similar to that of PGI and PGII [10]. The low specific activity with polygalacturonate as a substrate suggests that this is not the 'natural' substrate for PGE.
Acknowledgements:
This work was supported by a grant of the European Community, AIR2-CT-941345.
5. References:
[1 ] Voragen, A.G.J. (1989). Food enzymes: prospects and limitations. In: J.P. Roozen, F.M. Rombouts and A.G.J. Voragen (Eds) Food science: basic research for technological progress. Proceedings of the symposium in honour of Professor W. Pilnik. Pudoc Wageningen, pp 5981. [2] Harmsen, J.A.M., Kusters-van Someren, M.A. and Visser, J. (1990). Curr. Genet. 18, 161166. [3] Bussink, H.J.D., Buxton, F.P., Fraaye, B.A., de Graaff, L.H. and Visser, J. (1992). Eur. J. Biochem. 208, 83-90. [4] Kester, H.C.M. and Visser, J. (1990). Biotechnol. Appl. Biochem. 12, 150-160. [5] Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982). Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, New York. [6] Kawasaki, E.S. (1990). In: PCR protocols. A guide to methods and applications (Innis, M.A. et al., Ed.). Accademic Press Inc., San Diego, California, pp 21-27. [7] de Graaff, L.H., van den Broeck, H.C. and Visser, J. (1992). Curr. Genet. 22, 21-27. [8] Goosen, T., Bloemheuvel, G., Gysler, Ch., de Bie, D.A., van den Brock, H.W.J. and Swart, K. (1987). Curr. Genet. 11,499-503. [9] Pontecorvo, G., Roper, J.A., Hemmons, L.J., MacDonald, K.D. and Bufton, A.W.J. (1953). The genetics ofAspergillus nidulans. Adv. Genet. 5:141 -238. [10] Benen, J.A.E., Kester, H.C.M., Parenicova, L. and Visser, J. (1996). In: Proceedings of the International Symposium on Pectins and Pectinases. Kinetics and mode of action of Aspergillus niger polygalacturonases. [ 11 ] Kester, H.C.M., Kusters-van Someren, M.A., Mtiller, Y. and Visser, J., unpublished. [12] Rexov~t, L. and Mrackovd, M. (1978). Biochemica et Biophysica Acta 523, 162-169. [13] Visser, J., Bussink, H.J. and Witteveen, C. (1994). In: Gene expression in recombinant microorganisms (A. Smith, Ed.). Marcel Dekker Inc., New York, pp 241-308.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V.All rights reserved.
Identification of a seventh endo-pectate lyase phytopathogenic bacterium Erwinia chrysanthemi
831
of
the
C. Pissavin, J. Robert-Baudouy and N. Hugouvieux-Cotte-Pattat Laboratoire de G6n6tique Mol6culaire des Microorganismes, URA-CNRS 1486, INSA Bat 406, 20 Avenue Albert Einstein, 69621 ViUeurbanne Cedex, France
Abstract
The Erwinia genus includes soil bacteria with saprophytic or phytopathogenic ways of life. The species E. chrysanthemi is the causal agent of soft-rot disease. The E. chrysanthemi strain 3937 produces five major and several secondary endo-pectate lyases. The independent encoding genes are arranged in three clusters : peIA-pelE-pelD, pelB-pelC and pelL. Recently, molecular studies of the genomic region surrounding the pelB-pelC cluster have led to the identification of a new pectinase gene named pelZ. The PelZ protein is an enzyme with a weak endo-pectate lyase activity that generates mainly unsaturated trigalacturonate. Its activity requires the presence of Ca2§ or Mn 2§ a basic pH and the activity decreases when the degree of pectin methylation increases. The pelZ gene exists in different strains of Erwinia and its expression is subjected to many different regulations.
1. I N T R O D U C T I O N The major determinants of E. chrysanthemi virulence are extracellular depolymerising enzymes secreted by the bacterium to degrade the constituants of the plant cell wall. The production of pectate lyases is responsible for the maceration of parenchymous tissues [ 1]. These enzymes cleave the demethylated pectin, polygalacturonate, generated after the conjugated action of the pectin methylesterases PemA and PemB. The lyase action is characterized by a I~-elimination reaction resulting in unsaturated oligomers of various length. The five major pectate lyases are distinguishable by electrofocusing into one acidic (PelA), two neutral (PelB and PelC) and two alkaline (PelD and PelE) isoenzymes. E. chrysanthemi also has minor pectate lyases [2], among which the enzyme PelL has already been studied [3]. The major pectate lyase encoding genes are arranged in two clusters on the bacterial chromosome. One cluster contains the pelB and pelC genes which encode the neutral pectate lyases, the other contains the pelA, peID, peIE genes which encode the acidic and alkaline isoenzymes. Each gene represents one independent transcriptional unit. According to the degree of homology between their DNA sequences, the alkaline and neutral pel genes seem to result from the duplication of two ancestral genes [4]. In E. chrysanthemi, the synthesis of pectate lyases depends on the bacterial growth phase and is correlated with the variations of environmental parameters such as temperature, nitrogen starvation, oxygen pressure and the presence of pectic derivatives [5]. This induction results from the interaction of 2-keto-3-deoxygluconate (KDG) with the transcriptional repressor KdgR, which consequently cannot bind anymore on the operator region of the pel genes [6]. In a kdgR mutant, the residual induction of the pectate lyase synthesis suggests the
832 involvement of other(s) regulatory protein(s). The protein PecS was identified as a transcriptional negative regulator binding the regulatory region of the pelA, B, C, D, E, L genes [7]. No specific binding site has been found. The signal triggering the PecS regulation is still unknown. PecT, another regulatory protein of the LysR family, seems to act as a repressor on peIC, D, E (Surgey et al, submitted). We describe here the molecular identification of the pelZ gene and the genetic studies of the regulations affecting its expression. Following overproduction of the PelZ protein, biochemical studies were undertaken in order to define some enzymatic characteristics of PelZ activity.
2. G E N E T I C STUDIES OF PELZ 2.1. The pelZ gene The sequence of the 1 740 pb NheI - HpaI DNA fragment containing the 3' end of the peIC gene was determined. Use of uidA fusion showed that pelZ transcription was in the same orientation as that ofpelC. The pelZ ORF is made up of 1 260 pb (nt 228 to 1488). A putative ribosome binding site (GGAG) was observed 9 nucleotides upstream of the initiation codon ATG. pelZ is thought to be an independent transcriptional unit since only a monocystronic mRNA ofpelC was observed [8]. Separated by 15 nucleotides, the sequences GCGACA and "ITITAT (position 148 and 169) are putative -35 and -10 promoter boxes. The sequence observed from 188 to 207 presents some homology with a CRP binding site. The pelC and pelZ transcriptional terminators, from 136 to 174 and from 1520 to 1549 respectively, were characterised by the presence of GC-rich inverted repeats likely to form stem-loop structures, followed by a stretch of Ts. Comparison with the protein sequences of the EMBL bank reveals significant homology with the region adjacent to the locus containing the three genes pell, pel2 and pel3, encoding pectate lyases of E. carotovora ssp atroseptica [9] (Figure 1). The DNA coding sequence of pelZ is 70.3 % homologous with the E. carotovora DNA sequence of 1279 nucleotides, present downstream from the pel3 gene. Southern Blot experiments, using a specific pelZ probe, allowed the visualisation of pelZ homologs in different strains of E. chrysanthemi'EC16, B374, ENA 49. In consequence, pelZ may have a significant role in Erwinia pathogenicity since it exists in different strains of E. chrysanthemi and in E. carotovora ssp. atroseptica.
III peIB
I pelC
IIIII peIZ 1 kb
III
I
II IIIII r
pell
pel2
pel3
Figure 1. Organisation of the region surrounding pectate lyase encoding genes in E. chrysanthemi 3937 (A) and in E. carotovora ssp. atroseptica (B).
833
2. 2. Regulation of the pelZ transcription The construction of the chromosomal transcriptional fusions peIZ::uidA and pelZ::lacZ allowed for the study of the regulations affecting pelZ expression, pelZ is a weakly expressed gene with an optimum rate of transcription at the end of the exponential growth phase. It is induced in planta and also in synthetic media supplemented with plant extract, PGA or galacturonate. KDG formation is mainly responsible for this induction. Although pelZ transcription is modulated by the kdgR product, no well conserved putative "KdgR box" was identified in the 5' non-translated region upstream of the pelZ ORF. Based on the hypothesis that KdgR could bind on an unknown site of the pelZ regulatory region, retardation assays were performed using a DNA fragment containing the promoter region of pelZ and various concentrations of purified E. chrysanthemi KdgR protein. In vitro, KdgR does not bind with the regulatory region of peIZ, suggesting an indirect regulation by KdgR via an unidentified protein. In addition to this regulation, pelZ expression is also controlled by the pecT gene product. In contrast to the other pel genes, pelZ is not regulated by pecS. Moreover, the presence of glucose in the medium leads to a two-fold decrease of the pelZ expression. This catabolite repression could be correlated with the presence of a putative CRP-binding site upstream of pelZ.
3. CHARACTERISATION OF THE PELZ PROTEIN
3.1. Overproduction and localisation of PelZ in E. coli PelZ is a hydrophilic protein of 420 amino acids with a short hydrophobic sequence at its N-terminal end which has the characteristics of the signal sequences of exported proteins. The signal peptide may be 24 amino acids long, which would corroborate with the usual length encountered in prokaryotes. The molecular cloning of the pelZ gene in an expression vector pT7-6 allowed for the specific 35S-cysteine-methionine radio-labelling of PelZ in E. coli K38. We could detect, in crude extracts, the presence of a precursor and a mature form of PelZ. After cen fractionation, the mature form of PelZ could be localized in the periplasm of E. coll. So PelZ appears to be a protein exported by the Sec-dependent system of translocation. 1
2
3
kDa
6g -13
Figure 2. Overproduction of PelZ. Lane 1 9crude extract of the non-induced strain BL21/pTPZ. Lane 2" crude extract of the IPTG induced strain BL21/pTPZ. Lane 3" molecular weight marker. The arrow indicates PelZ.
834 The introduction of a multicopy plasmid carrying the pelZ gene into E. coli NM522 strain was not sufficient to detect any pectinase activity on a specific agar plate. In consequence, the pT7-6 derivative plasmid carrying the pelZ gene (pTPZ) was introduced into E. coli BL21 strain, allowing for the specific overexpression of the pelZ gene after addition of isopropyl-13-D-thiogalactoside (IPTG) (Figure 2). The overproduction of the PelZ protein allowed for the detection of pectate lyase activity after 24 hours of incubation at 30~ on pectate agar medium, supplemented with IPTG. The formation of unsaturated products from PGA could be spectrophotometrically assayed at 232 nm. No activity could be detected when the substrate was link pectin. 3.2. Characterisation of the enzymatic parameters of PelZ activity The pectate lyases are Ca2+dependent enzymes. On this basis, PelZ activity was studied both in the absence and in the presence of this cation. Without Ca z+, no activity was detected. The optimal Caz§ concentration was 0.225 mM in assay conditions with Tris-HC1 50 mM, pH 8.5, PGA 0.05% (Figure 3). Other cations, such as Mn 2§ Mg 2+ and Zn 2§ were also tested for their ability to contribute to the pectate lyase activity of PelZ. We observed that the presence of Mn 2+ led to a good activation of PelZ. The optimal concentration was about 0.2 mM. Mg z+ and Zn 2+ have weak effects on the pectate lyase activity of PelZ. A pH range of the reaction medium at 37~ allowed us to localise the optimal pH near 8.5 (Figure 3). The optimal concentration of PGA is 0.05% and we observed an inhibition with excess of the substrate. This inhibition is more evident with 0.1 mM of Ca z§ than with 0.2 mM of Ca 2+. The use of pectins with an increasing degree of methylation enabled us to see that PelZ is a pectate lyase which is very sensitive to the presence of methylated residues (Figure 3). With 40% methylated pectin as the substrate, the PelZ activity represented only 50% of the activity assayed in the presence of PGA. With 75% methylated pectin, only a residual activity of 10% was measured. These results could be correlated with the fact that PelZ is a weakly macerating enzyme.
Activity (%) lOO A
B
C
40
~ : - - - - ~ L___'___.~
o 7.5 8.0 8.5 9.0 9.5 10.0 0 pH
0.50
1.00 1.50 2.00 0 CaC12
7
22
45
60
75
Degree of pectin methylation (%)
Figure 3. Detemaination of the optimal parameters of PelZ activity. A : pectate lyase activity in a reaction medium containing PGA 0.05%, CaC12 0.2 mM and Tris-HC150 mM at various pH. B 9pectate lyase activity in a reaction medium containing PGA 0.05%, Tris-HC1 50 mM pH 8.5 and increasing concentrations of CaC12. C : pectate lyase activity in a reaction medium containing CaC12 0.2 mM, Tris-HC1 50 mM pH 8.5 and as substrate either PGA 0.05% or pectins 0.05%, with different degrees of methylation (from Copenhagen Pectin).
835 The products resulting from PelZ action were separated on thin layer chromatogaphy using silica gel. They were visualised after treatment of the dried chromatograms with a solution of phosphomolybdic acid (3%, v/v), sulfuric acid (10%, v/v) in ethanol, followed by brief heating. The PGA degradation by PelZ after 12 hours of incubation at 30~ leads to the formation of two sorts of product. They are, for the majority, trimers with a small proportion of dimers. In consequence, PelZ appears to be an endo-pectate lyase. The action of PelC generates both dimers and trimers while the action of PelL leads mainly to the formation of long oligomers.
3.3. Alignment of PelZ and PelC amino acid sequences No significant homology was found between PeLZ and the other pectate lyases, suggesting that PeLZ belongs to a new family of pectate lyases. According to the Pel realignment proposed on the basis of the structurally conserved amino acids [10], comparison between PelC and PelZ amino sequences was established (Figure 4). ATDTGGYAA-T-A--GGNVTGAVSKTATSMQ-D
I I
I::
I I
II
:
I: I
IVNI IDAARLDANGKKVKGGAYPLVITYT-GNEDSLINAAAANI
I
: :1
I I
II
I :1 :
I
CGQ
I
A P D L K G F G T E T __._._ VAG SGGKI I R-VT-T LDS--GGAGS
LREA-- L-A .... TKG---PRI
WSKDPR-GVE
. . . . . S S - D V V V O - -NMB.TA~Y L P G G A K . . . . D G D M - I R V D D S P
II
I
"
I
I-KEFTKGIT
"
I IGANGSSANFGIWIKK
"1 I'1
-EKDIRLA-E---PY---VT
9 I"
II
"1
'"
III
II
NVVVD
"
II
"111
I
II
9 I
125
o
II
"1
9
I
""
II
I
RCS-FA--W---GTDENLSVSGPRYDGPSGTAHNVT
I
I
I
.... VK-KVGLDGSSSSDTGRNITYHHNYY
9I
9
56 139
"
IAGQTAPSP--GITLVKGGMMITTHDVLVQHIRFRIGDN-GHAKKSGFEKDVSLYGPNAY
N.~.r~2~NELFAANHECDGTPDNDTTFESAVDIKGAS-NTVTVSYNYI-HG
II
II
I VFEVGG I --- IDLD .......
74
212
I'1
FSNN I IAEGLYDSSHSKGIHSMGTLVHD--NVTDAAI
IG
197
~DVN---ARL9L~RGG-L~HAYNNL-YTN-ITGSGLNyRQNG~ALIENNWFEKAINPVTsRYDGKNFGTW~-LKGNNITK 2 8 5 I I "1" III I I I " I I " I I I 9 I I I I NLYAHNNERN9WFKGATTGVVVNNLIYNPGIWGIRIGAIQs~WEGRSLPVNAKVAIAGNVMYHGANTKSGLSLVGSNTTG
277
o
PADFSTYSITWTADTKPYVNAD-S .
.
.
.
I"
"
.... WTSTGTFPT--VAYNYSPVSAQCV
9
I
"II
I
I I
I
....... KDKLPGYAGVG
II"
9
GDVWMSDNLAFDKAGKAVAQTSGTGINLLKSAPVWPTGLTAISSS•VANQVTQHA•ARPKDRDAVDKRIVSDFQKRSGTF ...........
TSTACK
VNSQSEVGGYP
TATATKRT
l ll'l
I
"I"
347 357
PelC
353 LTVP STNVDAWLQQMAKDLE
.... KNLATL
396
PelZ
Figure 4. Alignment of PelZ and PelC amino acid sequences. The vertical lines indicate identical amino acids and the two points indicate homologous amino acids. The bold letters correspond to the residues probably involved in Ca2+ binding or catalytic function(s). The two aspartate residues probably involved in Ca 2+ binding are indicated with an asterisk. The invariant residues, probably involved in PGA cleavage, are indicated with an open circle. The folding in I~-sheets is characterised by the underlined amino acids. Double underlining of PelZ residues is deduced from Chou Fasman and Robson Garnier folding predictions. Like the other pectate lyases, PelZ folding appears to be mainly based on 13-sheet structures. Moreover, PelZ possesses only one cysteine residue and thus can not present any intramolecular disulfide bond. The folding process of this protein may be different from that of the other pectate lyases. Invariant residues most probably involved in the Ca 2§ binding site are
836 also observed in the PelZ protein, confirming the requirement of this cation for activity. However, all the invariant residues of the Ca2+ binding site are not conserved. This may explain the fact that PelZ requires almost 0.2 mM of Ca 2+, instead of 0.1 mM for the other pectate lyases. The signatures of the two potential catalytic sites are also present. The catalytic site of pectate lyase activity is not well defined. The pectinolytic cleavage of PGA by a 13elimination mechanism probably requires an Asp and an Arg. These two invariant residues also exist in PelZ. However, certain of the putative residues implicated in the structure of the groove surrounding the pectate lyase catalytic site are lacking in PelZ. Consequently this could explain its weak pectate lyase activity. The signature of a second putative catalytic site, wViDH, is encountered in PelZ but in a degenerated form. This suggests the presence of some other enzymatic activity.
4. C O N C L U S I O N As previously described [2, 11], E. chrysanthemi possesses at least seven pectate lyases. Among them, PelZ seems to belong to a new family. However, its biochemical characteristics, basic optimal pH, calcium dependence and methylation sensitivity corroborate with those of the other pectate lyases of E. chrysanthemi. The weak expression of the pelZ gene and the weak activity of the PelZ protein seem to be correlated with specific environmental conditions. PelZ may act on pectin in synergy with the major pectate lyases.
5. A C K N O W L E D G E M E N T S This work has been supported by grants from the CNRS and from the Minist~re de l'Education Nationale, de rEnseignement Sup6rieur, de la Recherche et de rlnsertion Professionnelle. Characterisation of the Erwinia pectinases is part of a project financed by the European Community AIR programme (AIR2-Cq941345).
6. R E F E R E N C E S
1 2
A. Collmer and N. T. Keen, Annu. Rev. Phytopathol., 24 (1986) 383. G.P. McMillan, A. M. Barrett and M. C. M. Perombelon, J. Appl. Bacteriol., 77 (1994) 175. 3 E. Lojkowska, C. Masclaux, M. Boccara, J. Robert-Baudouy and N. Hugouvieux-CottePattat, Mol. Microbiol., 16 (1995) 1183. F. Barras, F. Van Gijsegem and A. K. Chatterjee, Annu. Rev. Phytopathol., 32 (1994) 201. N. Hugouvieux-Cotte-Pattat, H. Dominguez and J. Robert-Baudouy, J. Bacteriol., 174 (1992) 7807. W. Nasser, S. Reverchon and J. Robert-Baudouy, Mol. Microbiol., 6 (1992) 257. S. Reverchon, W. Nasser and J. Robert-Baudouy, Mol. Microbiol., 11 (1994) 1127. N. Hugouvieux-Cotte-Pattat and J. Robert-Baudouy, Mol. Microbiol., 6 (1992) 2363. S. Barring, C. Wegener and O. Olsen, Microbiology, 141 (1995) 873. 10 B. Henrissat, S. E. Heffron, M. D. Yoder, S. E. Lietzke and F. Jurnak, Plant Physiol., 107 (1995) 963. 11 S. Kelemu and A. Collmer, Appl. Environ. Microbiol., 59 (1993) 1756.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
837
Pectin methylesterase B of Erwinia chrysanthemi, the first pectinase characterised as a membrane lipoprotein. V. E. Shevchik, G. Condemine, N. Hugouvieux-Cotte-Pattat and J. Robert-Baudouy Laboratoire de GEn6tique Mol6culaire des Microorganismes, URA-CNRS 1486, INSA Bat 406, 20 Avenue Albert Einstein, 69621 Villeurbanne Cedex, France.
Abstract
The bacteria Erwinia chrysantherni secretes in the extracellular medium several pectinases of different types. A gene coding for a novel pectin methylesterase pemB has been cloned from an E. chrysanthemi strain 3937 gene library. The PemB sequence presents homology with other pectin methylesterases from bacteria and plants. PemB has been characterised as a bacterial lipoprotein. PemB is localised in the outer membrane and is not released into the extracellular medium. PemB was overproduced in E. coli and purified to homogeneity. It is activated by non-ionic detergents. PemB has a highest activity on methylated oligogalacturonides and this enzyme is necessary for the growth of the bacteria on this substrate as carbon source.
1. I N T R O D U C T I O N The phytopathogenic bacteria Erwinia chrysanthemi produce a number of extracellular pectinases, among which are at least seven isoenzymes of pectate lyases PelA, PelB, PelC, PelD, PelE, PelL and PelZ one pectin methylesterase PemA and one exo-polygalacturonase PehX (1, 2). The genes coding for these extracellular pectinolytic enzymes have been cloned and characterised. The pectinases have been overproduced, purified and their biochemical properties studied. Pectin methylesterase (PME) de-esterify pectin by liberating methoxyl groups to yield polygalacturonate (PGA), the preferential substrate of pectate lyases (PLs) (3, 4). The final end product of PGA digestion by PelA and by PelL is oligogalacturonates, from dimers up to dodecamers, while PelB and PelC produce tri and tetragalacturonates and PelD and PelE produce predominantly dimers (5, 6). The genes coding for PLs are gathered into three clusters. One contains pelA, pelD, pelE and pemA, the other one contains pelB, pelC and pelZ and the third one contains petL. Strains deleted for the pel genes have been constructed from different E. chrysanthemi strains (5, 7, 8). These strains are still able to macerate plant tissues, suggesting the existence of additional pectinolytic enzymes, so called "secondary pectinases". Similarly, a pemA mutant has a reduced virulence but still remains able to cause local necrosis on Saintpaulia (9). Thus, other enzymes with PME activity could exist in E. chrysanthemi. As an attempt to identify the genes of the secondary pectinases, we screened a genomic library of the E. chrysanthemi strain 3937 for pectinolytic activities and identified the gene of a novel PME. We propose to call this gene pemB and to rename pemA the gene coding for the already characterised PME which was previously called pem (3, 4).
838 2. RESULTS
2.1. Cloning and nucleotide sequence of pemB An E. chrysanthemi 3937 gene library was constructed and screened for new pectinolytic activities. We used a plate assay with pectin-agarose overlay, containing EDTA and Triton X-100. After staining with ruthenium red, two types of haloes could be observed over the pink background: clear haloes corresponding to degraded substrate surrounding PLproducing colonies, and purple haloes, characteristic of the formation of PGA from pectin, surrounding PME-producing colonies. Four colonies giving purple haloes were found among about 10 000 clones. Two of the colonies (class 1) produced haloes much larger than the two others (class 2). The plasmidic DNA of these clones was extracted and analysed by restriction endonuclease digestion (Figure 1). The inserts of the class 1 plasmids (pPME3) had an overlapping region and their deduced restriction map was identical to that of the already characterised pemA gene (4, 10). The plasmids of class 2 (pPME6) presented a totally different restriction map. When a DNA fragment containing the entire pemA gene was used as a probe for Southern blotting, hybridisation was found with the two class 1 plasmids but not with the two class 2 plasmids, confirming that they did not contain a deleted version of the pemA gene. pPME3
Pstl
I
I
Bgl II
BamHI
I
Notl
I
Nru I
I
i
Xbal i,,
pemA pPME6 Smal Stul Sacll EcoRI SnaBI I
,I
II
.........
I
I
Sacll ,
I
Maml EcoRI I
pemB r
Figure 1. Restriction nuclease maps of the pem carrying plasmids. The proteins, products of the pPME3 and pPME6 were analysed by IEF, followed by staining with ruthenium red. A band with PME activity at pI about 10, corresponding to PemA, was observed in E. chrysanthemi culture supernatant and in the extract of an E. coli strain carrying the pPME3 plasmid. A PME with pI of about 9.5 was detected in a lysate of an E. coli strain carrying the pPME6 plasmid. A very faint PME band with the same pI could be found in an E. chrysanthemi cell lysate. Therefore, a second gene coding for PME activity exists in E. chrysanthemi and was cloned in the class 2 plasmids. We called this gene pemB. A 2140 bp DNA fragment containing pemB was sequenced (The pemB sequence will appear in the EMBL/GenBank/DDBJ nucleotide sequence data library under accession number X84665). Only one large open reading frame that could correspond to pemB was found. This ORF had as a possible initiation codon, the GTG c0don at position 267, preceded by a good potential Shine-Dalgarno sequence (AAGGAG). The stop codon at position 1566 is followed by an inverted repeat (t~G= - 8.5 kJ mol- 1) and then a run of T residues that could function as a Rho-independent transcriptional terminator. This ORF codes for a 433 amino-acid protein with a deduced molecular weight of 46 767 Daltons. A sequence with homology (16/18 conserved nucleotides) with the KdgR binding consensus is present at position 135 to 156. A sequence with a good homology to the Sigma 70 promoter consensus sequence could be found overlapping the KdgR-box.
839 The amino-acid sequence of PemB was compared with that of other PMEs from bacteria (E. chrysanthemi and Pseudomonas solanacearum) or plants (Lycopersicon esculentum, Brassicus napus and Phaseolus vulgaris). In the central most conserved part of the proteins PemB has 36% identity with P. solanocearum PME, 27% identity with E. chrysanthemi PemA, 22% identity with L.esculentum and P. vulgaris PMEs, and 20% with B. napus PME. Construction of a phylogenetic tree of PMEs indicates that PemB is related to the PME of P. solanocearum, while E. chrysanthemi PemA is more related to plant PMEs. Although all these PMEs appear to be derived from a common ancestor, it seems unlikely that the presence in E. chrysanthemi of two pem genes results from a recent duplication of an ancestral gene. The six conserved regions present in PMEs proposed to be implicated in the catalytic site of these enzymes (11) were also found in PemB. 2.2. Characterisation of PemB as a lipoprotein and determination of its cellular localisation PemB has one hydrophobic region of 22 amino acids at the N-terminus of the protein. This sequence does not share the characteristics of typical signal sequences but has the features of lipoprotein signal sequences, cleaved by the lipoprotein signal peptidase: the hydrophobic region is terminated by the sequence L-X-A-C (12). To verify this hypothesis we tested whether PemB is synthesised as precursor processed later in mature protein by cleavage of its signal peptide, pemB was exclusively expressed and labelled with 35S methionine-cysteine using T7 RNA polymerase/T7promoter system of Tabor and Richardson (13) and then signal peptide processing was stopped by the addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) that dissipates the proton-motive force (14). Addition of 100 ~ CCCP blocked the formation of the 45 kDa mature protein, leading to accumulation of the 46 kDa species. Removal of CCCP allowed the processing of a part of the accumulated precursor, confirming the presence of a signal sequence. During processing of a lipoprotein signal sequence, N-acyl glyceride is added at its N-terminal cysteine (12). We used 3H-palmitic acid to label E. chrysanthemi lipoproteins. It was incorporated into several proteins among which was a 45 kDa protein that could correspond to PemB. The amount of the 45 kDa protein was strongly increased in an E. chrysanthemi strain carrying a high copy number plasmid bearing pemB. This 45 kDa protein was also extracted from E. chrysanthemi cells by Triton X-100, and immunoprecipitated with the PemB antiserum. These results indicated that PemB is a bacterial lipoprotein. The PemB cellular localisation was determined both in E. chrysanthemi and in an E. coli recombinant strain by Western blot of the cell fractions with a PemB-antiserum. No PemB was detected in the culture supernatant and only trace amounts were found in the soluble cell fractions - periplasm and cytoplasm (Figure 2). PemB was found mostly in the total membrane fraction from which it could be completely extracted by Triton X-100/Mg2+ and partially extracted by Sarkosyl (Figure 2). This behaviour is typical of inner membrane proteins, but since some exceptions have been noticed it does not positively indicate the PemB localisation (15). We performed cell membrane fractionation in sucrose density gradient centrifugation both by sedimentation and flotation, using several markers of inner and outer membrane vesicles. PemB was found in the outer membrane vesicles (data not shown). To check if PemB is surface exposed, E. chrysanthemi cells were subjected to proteolysis. Treatment of the cell suspension with trypsin, proteinase K or chimotrypsin at a concentration of 0.1 to 1 mg/ml for 1 h did not cause PemB proteolysis or its liberation into the medium. Cell pre-treatment with EDTA-lysozyme, which renders the periplasmic proteins accessible to proteases, gave no effect. PemB was also resistant to proteolytic digestion in extract of cells disrupted by sonication or in a French press. Only addition of Triton X- 100 (up to 0.1%) causing formation of the micelles with PemB lead to a quick proteolyis of this protein (data not shown). In another approach to analyse the PemB exposition, bacterial cells were labelled with sulfo-NHS-biotin. This compound is unable to cross membranes and biotinylation
840
A 1
2
3
B 4
5
1
2
3
4
5
Figure 2. PemB cellular localisation. (A) Fractionation of E. chrysanthemi cells by spheroplasting. Lane 1, culture supernatant; lane 2, total cell lysate; lane 3, periplasmic fraction; lane 4, crude membrane fraction; lane 5, cytoplasmic fraction. (B) Detergent extraction of PemB from E. chrysanthemi A837 cell envelopes. Lane 1: crude envelope fraction; lane 2: Triton-soluble fraction; lane 3: Triton-insoluble fraction; lane 4: Sarkosyl-soluble fraction; lane 5: Sarkosyl-insoluble fraction.
A 1 ..
o
.....?/~9:~:'!
B
2 - - - .
..........
- -
.
.........
3 :-
. . .
. . . . . . . . . . . . .
1 -----:..
............
,-~-:-::.:
2 .......
~---:-:-::.~
3 ...
--..-::
. . . . . .
::~:~-....
-
Figure 3. E. chrysanthemi cell surface labelling with sulfo-NHS-biotin. After labelling, the proteins were separated by SDS-PAGE, blotted onto nitrocellulose and revealed with PemBantibodies (A) or with streptavidin-peroxidase (B). Lane 1:A350 (wild type); lane 2:A837 (kdgR); lane 3: A350/pPME6. An arrowhead indicates the PemB position. is restricted to the cell surface (16). The experiment was performed with three strains producing different amounts of PemB, tested by immunoblotting (Figure 3). No variation in the amount of any of the labelled proteins was observed (Figure 3), indicating that PemB was not present among the cell surface biotinylated proteins. Thus, PemB does not seem to be a cell surface or
841 periplasm exposed protein accessible to proteolysis or biotinylation and it is embedded in the outer membrane. 2.3. Overproduction and purification of PemB PemB was overproduced in the recombinant E. coli strain using the T7 promoter/T7 polymerase system of Tabor and Richardson (13). Two hours after thermo-induction the cells were harvested, disrupted in a French press, unbroken cells were removed by centrifugation at 10 000 g for 10 min and the crude cell envelope fraction was sedimentated by centrffugation at 100 000 g for 1 h. The membranes were resuspended in 50 mM Tris-HC1 pH 8, 2% Triton X100, 5 mM MgC12, incubated with gentle agitation at 30~ and centrifuged at 100 000 g for 2 h. PemB represented about 90% of the proteins in this extract (Figure 4). It was further purified by preparative gel electrophoresis in a 10% polyacrylamide gel containing 8 M urea, 0.1% SDS. After electrophoresis, the gel was cooled to 10~ and the major opalescent band, that corresponds to PemB, was cut out and crushed. Proteins were extracted by three washes with 10 mM Tris-HC1 pH 8, 0.1% Triton X-100, dialysed against the same buffer without Triton and lyophilised. The enzyme obtained was electrophoretically homogeneous (Figure 4) and corresponded to the 45 kDa mature protein. Its N-terminal amino acid was resistant to Edman degradation, indicating that this lipoprotein is correctly processed in E. coli cells.
kDa
1
2
3
4
5
6
7
116 '-85"-"
I
==~
55--
39---
26-.-
,84184184 i
20-14--
Figure 4. Purification of PemB from E. coli K38 pGP1-2/pPME6-5 ceils. Proteins were separated by urea-SDS-PAGE. Lane 1, induced cell lysate; lane 2, soluble protein fraction from induced ceils; lane 3, membrane fraction from non-induced cells; lane 4, membrane fraction from induced cells; lane 5, membrane proteins not extracted by Triton X-100; lane 6, membrane proteins extracted by Triton X-100; lane 7, PemB purified by preparative electrophoresis. The molecular weight standard positions are indicated.
842 2.4. Biochemical characterisation of PemB; development of a double enzyme assay to measure PME activity The ability of purified PemB to demethylate pectin was confirmed using different techniques. Addition of PemB to a pectin solution (98% methylated) caused an acidification of the reaction medium tested by NaOH titration and by pH indicator colour change. Analysis of the reaction end products by gas chromotography indicated that methanol was formed. These results showed that PemB is able to demethylate pectin, liberating acidic groups and methanol. These assays currently used to measure PME activity are not easy and not suitable for the analysis of enzymatic properties. We developed a new double enzyme assay for PME activity using an approach similar to that described by McMillan et al. (17). E. chrysanthemi PelD is active on fully demethylated PGA. Its activity decreases when the level of methylation of the substrate increases. It is inactive on highly methylated pectin (18). Demethylation of pectin by PME leads to the formation of a substrate for PelD. Therefore, PME activity can be estimated by measuring PelD activity on pectin demethylated by PME. The reaction mixture (5 ml) containing 0.5% pectin (98% methylated), 0.1 M Tris-HC1 (pH 8.0) and 0.05% Triton X100 was incubated with 5 to 50 gl of sample at 30~ for 1-10 hours. At various times during the incubation, 0.3 ml aliquots were taken and used as the substrate for PelD: the aliquots were added to 2.7 ml of 0.1 M Tris-HC1 pH 8.5, 0.1 mM CaC12 and a standard amount of purified PelD (2 U). PL activity was measured spectrophotometrically at 230 nm at 30~ according to Moran et al. (19), against a control containing an identical reaction mixture without PME. One unit of PME activity was defined as the amount of enzyme that liberates one pmol of demethylated galacturonate residue per rain. PemB enzymatic characteristics were determined using this assay. The optimal pH for enzyme activity measured in Tris-HC1 buffer was 7.5. The enzyme showed highest activity at 40~ and was stable at this temperature for 5 h. The half time of PemB thermo-inactivation at 50~ was 10 min. PemB completely lost enzymatic activity after 5 min incubation at 60~ Addition of cations (K+, Na§ CaZ+, Mg 2+, Mn2+, and Zn2+) or EDTA had no influence on the enzyme activity. Urea up to 8 M did not change the enzyme activity. Sodium cholate had no influence up to 0.1%, while SDS at this concentration led to a complete loss of activity, that could be restored after SDS removal. Non-ionic detergents (Triton X-100, Tween 20, Tween 80, Brij 56) caused an effect on PemB activity characteristic for many membrane enzymes: stimulation at low concentrations (0.01-0.1%) and inhibition at higher concentrations. The strongest effect was obtained with 0.05-0.1% Triton X-100, which increased the PemB activity up to five-fold. The detergent concentration optimal for the enzyme activity was dependent on the PemB concentration, indicating that really Triton/PemB ratios should be taken into account. We tested the effect of substrate polymerisation level on the PemB specific activity. A mixture of methylated oligogalacturonides was obtained by digestion of 93% methylated pectin by the Aspergillus niger pectin lyase A (20). PemB was more than 100-fold more active on methylated oligogalacturonides than on polymeric pectin. 2.5. Presence of PemB homologues in other Erwinia species To identify PemB homologues in other Erwinia carotovora and Erwinia chrysanthemi strains, we used Western blot with PemB-antibodies. To avoid any cross-reaction with PemAlike proteins, the anti-PemB serum was pre-incubated with lysate of E. coli cells overproducing PemA. Erwinia total culture extracts and Triton X-100 soluble proteins were analysed by immunoblotting after separation by SDS-PAGE (Figure 5). PemB-antibodies interacted specifically with a 45 kDa protein in E. chrysanthemi strains 3937, B374, CU 1, and ENA49. In strain EC16, a 46 kDa protein showed a weak specific interaction with the anti-PemB serum. In contrast, no specific interaction was detected with several E. carotovora strains. We performed also Southern hybridisations using the labelled 0.8 kb SacII fragment included in the pemB open reading frame (Figure 1) as the probe and chromosomal DNAs of different Erwinia species. In the strain 3937 EcoRI-digested DNA, a single 1.6-kb EcoRI fragment hybridised with the pemB probe, as expected from the restriction map of this region
843 (Figure 1). Under stringent conditions, pemB homologues were detected in all the tested E. chrysanthemi strains giving hybridisation with a 9-kb PstI fragment in 3937 DNA, a 15-kb PstI fragment in B374 or ENA49 DNA, a 5.9-kb PstI fragment in CU1 DNA and a 7.6-kb PstI fragment in EC16 DNA. In contrast, no pemB hybridising signals could be detected in the two E. carotovora subsp, carotovora strains CC3-2 and SCRI193 or in the two E. carotovora subsp, atroseptica strains CA36A and SCRI31, even under low-stringency. Thus, PemB homologues have been detected in the four other E. chrysanthemi strains tested, suggesting that this enzyme is an essential element of the E. chrysanthemi pectin degradation machinery.
0 ur) e,')
<
kDa
r,,.. 03 oo
<
0"} ~ "~ Z w
~ r',.. eo
o3
t,..o ',0 w
":3
0
97--
68--
29
--It,
~--
"
.
18 _tt" .................................................
~ ......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Figure 5. Proteins extracted from different E. chrysanthemi strains by Triton X-100 were analysed by immunoblotting with PemB-antiserum.
3. C O N C L U S I O N We described here the characterisation of the pemB gene and its product the second PME of E. chrysanthemi. The biochemical analysis of the purified protein indicated that PemB is actually an enzyme that demethylates pectin, leading to formation of methanol and PGA. However, PemB is more active on methylated oligogalacturonides than on polymeric pectin. The activating effect of non-ionic detergents on PemB was never pointed out for other pectinases and it is a characteristic of many membrane enzymes (21). Up to now, the pectinolytic enzymes of E. chrysanthemi that have been detected were extracellular secreted enzymes (PelA, B, C, D, E, L, exo-Peh and PemA), periplasmic (exoPel), or cytoplasmic (OGL) proteins (1, 5). In contrast, PemB is an outer membrane pectinolytic enzyme. To our knowledge it is the first pectinase characterised as a membrane protein. We presented several lines of evidence showing that PemB is a lipoprotein: (i) Its Nterminal sequence has the characteristics of lipoprotein signal sequences. (ii) PemB is synthesised as a high molecular weight precursor processed into a lower molecular weight mature form. (iii) Palmitate, the most prevalent fatty acid in bacterial lipoproteins (12), is incorporated into PemB.
844
A pemB mutant showed a reduced growth on methylated oligogalacturonides as the sole carbon source, indicating the possible role of PemB in pectin catabolism. The action of extracellular pectinases on pectin may liberate small methylated oligogalacturonides that can enter by diffusion into the periplasm. PemB could demethylate these molecules in the periplasm or during their passage to periplasm.
A C KN O W L E D G EMENT S This work was supported by grants from the CNRS (URA 1486), from the Minist~re de la Recherche et de l'Enseignement Sup6rieur (DRED) and from the European Community (AIR 2CT94-1345 contract).
4. R E F E R E N C E S
8 9 10 11 12 13 14
15 16 17 18 19 20 21
F. Barras, F. Van Gijsegem and A. K. Chatterjee, Annu. Rev. Phytopathol., 32 (1994) 201. A. Collmer and N. T. Keen, Annu. Rev. Phytopathol., 24 (1986) 383. G. S. Plastow, Mol. Microbiol., 2 (1988) 247. F. Laurent, A. Kotoujansky, G. Labesse and Y. Bertheau, Gene, 131 (1993) 17. E. Lojkowska, C. Masclaux, M. Boccara, J. Robert-Baudouy and N. HugouvieuxCotte-Pattat, Mol. Microbiol., 16 (1995) 1183. J. Preston, J. Rice, L. Ingram and N. T. Keen, J. Bacteriol., 174 (1992) 2039. C. Beaulieu, M. Boccara and F. Van Gijsegem, Mol. Plant-Microbe Interact., 6 (1993) 197. S. Kelemu and A. Collmer, Appl. Environ. Microbiol., 59 (1993) 1756. M. Boccara and V. Chatain, J. Bacteriol., 171 (1989) 4085. N. Hugouvieux-Cotte-Pattat and J. Robert-Baudouy, Mol. Microbiol., 3 (1989) 1587. A. Sp6k, K. Schoengendorfer and H. Schwab, J. Gen. Microbiol., 137 (1991) 131. A. P. Pugsley, Microbiol. Rev., 57 (1993) 50. S. Tabor and C. Richardson, ProC Nail. Acad. Sci. USA, 82 (1985) 1074. E. T. Palva, T. R. Hirst, S. J. S. Hardy, J. Holmgren and L. Randall, J. Bacteriol., 146 (1981) 325. H. Nikaido, Meth. Enzymol., 235 (1994) 225. C. Kocks, E. Gouin, M. Tabouret, P. Berche, H. Ohayon and P. Cossart, Cell, 68 (1992) 521. G. P. McMillan, D. J. Johnston, J. B. Morel and M. C. M. P6rombelon, Anal. Biochem., 209 (1993) 377. V. E. Shevchik, A. N. Evtushenkov and Y. K. Fomichev, Biokhimiya (Russ), 53 (1988) 1628. F. Moran, S. Nasuno and M. P. Starr, Arch. Biochem. Biophys., 123 (1968) 298. J. A. M. Harmsen, M. A. Kusters-van Someren and J. Visser, Curr Genet, 18 (1990) 161. D. Bou6 and O. M. Viratelle, Biochem. Biophys. Acta, 1103 (1992) 120.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All fights reserved.
845
Synthesis of New Methyl Esters of 3-Deoxy.D.erythro. 2-hexulosonie acid (KDG) analogs, inducers of the Expression of Peetinase Genes in Bacteria Erwinia
chrysanr
Fr6d6ric Alessl I, Guy Condemine 2 , Alain Doutheau I, Janine R o b e r t - B a u d o u y 2 and Daniel Anker I*
1) Laboratoire de Chimie Organique. D6partement de Biochimie. Institut National des Sciences Appliqu6es. 20 avenue A. Einstein. 69621 Villeurbanne, FRANCE 2) Laboratoire de G6n6tique Mol6culaire des Microorganismes et des Interactions Cellulaires. URA-CNRS-1486. D6partement de Biochimie. Institut National des Sciences Appliqu6es. 20 avenue A. Einstein. 69621 ViUeurbanne, FRANCE
Abstract:
Four title compounds, 5-deoxy KDG Me ester, 5-epi KDG Me ester, 4-O-Me KDG Me ester and 4-deoxy KDG Me ester were prepared either from D-glucono-l,5-1actone or from 1,2:5,6 di-O-isopropylidene-D-mannitol. Biological tests .l~'formed on these molecules have shown that the compounds modified on the C-5 position (5-deoxy KDG Me ester and 5-epi KDG Me ester) are gratuitous inducers of the expression of pectinase genes in the phytopathogenie bacteria Erwinia Chrysanthemi when the C-4 modified molecules (4-O-Me KDG Me ester and 4-deoxy KDG Me ester) are not inducers.
INTRODUCTION Phytopatogenic bacteria of Erwinia type and, particularly, E. chrysanthemi have pectinolytic and cellulolytic properties which cause important damage in plants in the field and, after harvest, during storage. The enzymatic degradation of pectins by the lyases of this bacteria leads to the formation of 3-deoxy-D-erythro-2-hexulosonic acid or 2-keto-3-deoxy-D-gluconic acid (KDG) I which is then metabolized. This compound induces the expression of pectinase genes by binding to a specific site on the KdgR protein, a repressor of the expression of these genes (figure 1).
846
synthesis of pectinases
repression DNA KdgR ~
Inducers:
I
c
O OH
OH KDG 1
r---O OH
) coo. OH 5-O-Me KDG 2
Figure 1: Action of the inducers on the KdgR protein
As part of a study of the functionalities of inducers wich are necessary for recognizing the repressor protein we have previously shownl that" -only pyranose forms of KDG are r e c o # by the KdgR protein, -the 5-O-methyl derivative of KDG 2 (figure 1) is a gratuitous inducer i.e. is recognized by the protein but not metabolized. To complete this study we describe here the synthesis of four new analogs of KDG, methyl 3-deoxy-L-threo-2-hexulosonate (the methyl ester of 5-epi-KDG ) 13, methyl 3,5dideoxy-D-glycero-2-hexulosonate (the methyl ester of 5-deoxy-KDG) 20, methyl 3-deoxy-4O-methyl-D-erythro-2-hexulosonate (the methyl ester of 4-O-methyl-KDG) 24 and methyl 3,4dideoxy-D-glycero-2-hexulosonate (the methyl ester of 4-deoxy-KDG) 29.
RESULTS AND DISCUSSION Since KDG exists as a mixture of four species (a and [3 pyranose and ct and [3 furanose forms) in equilibrium (figure 2) it was obviously an unsuitable material for the preparation of C5 and C-4 modified analogs. We thus followed the same approach as that used for the preparation of KDG or for its 5-O-methyl methyl ester 2, starting from compounds 6 or 8 obtained in four or five steps respectively from commercially avalaible D-glucono-l,5-1actone (figure 3). 2
847 HO
/0.
OH
no [ .... ~C~OOH
~COOH
OH
OH
)) i(-Ok~oo.
); .O-~o .~oo.
OH
I OH
KD6 1 Figure 2
HO~6
o---
?o ~ , ~ ~ z ?o
CSA ~
Pyr
.o 7-[~ OH D-glucono- 1,5-1actone
-
\1\
5
1 OH
.~o.
............. /
U
o 3
o 4
.o
~ OBz
l
6
AcC1 MeOn
\OBz
Ro I
KDG I (R=H)
5-O-Me KDG SiO
>o
OBz
OH
I
"~
l
6
-'-, '~o AgN03
~
HO 3~~20~
7
8 Figure 3
2
(R=Me)
848 SYNTHESIS OF 5 - E P I - K D G METHYL ESTER 13 To obtain this compound the key step consisted in the epimerization of the C-5 in compound 6. This was acomplished by triflation 3 of the alcohol 6 and nucleophilic substitution of the triflate by a large excess of tetrabutylammonium acetate in diehloromethane. A controlled (4 ~ 3 h) basic methanolysis of the enol benzoate led to the keto-ester 114 whose hydroxyl functions at C-4 and C-6 were simultaneously deprotected under acidic conditions to furnish 12. Finally a Zemplen deprotection of the 5-acetoxy group led to 13 obtained in five steps and 11% overall yield from 6 (figure 4).
o-
AcO- Bu4N+
~ ~',~~COOMo
6
3
/ ~ 0
2"OBz
~
CO0 e
~10Ol~l
OBz M
'N/~~
O~ 9 x ~ ~ ~ C OOBz OMe
MeONa MeOH
O 11
AcOHO
PPTS /COOMe MeOH Ill
~
AcO O
O
OH
/
COOMe
~COOMe A_cO O 12
HO 6 4 I
O
OH
0
5
COOMe 3
1
I OH
.OH 2
HO 6 ~ 4 ~ C O O M e HO
OH
5-epi-KDG 13 Figure 4
CH2C12
MeONa MeOH
849 SYNTHESIS OF 5-DEOXY-KDG METHYL ESTER 20
As for the synthesis of 5-epi-KDG,compound 6 seemed to be a suitable precursor of the methyl ester of 5-deoxy-KDG 20 since only the C-5 hydroxyl was unprotected. In this case the key step was not the epimerization but the removal of that hydroxyl. Our attempts of radicalar deoxygenation of 6 were unsuccessful because the intermediate radical was intramolecularly trapped by the C-2,C-3 double bound. Therefore we first reduced the double bond and then converted the resulting diastereoisomeric alcohols 14 into the corresponding tdflates 15 which were submitted to the action of sodium iodide. Finally the iodides 16 thus obtained were hydrogenolyzed in the presence of diisopropylethylamine5 to give 17.
L /~
H2/Pd/C ~ ''O ~-~. \6 5.~OH = "0f ~ 4 ~~,,~,..,~COOMe / Et3N 6
3
= ~ O H (CF3SO2)20 O-- -,~.~COOMe tN,,~/x
2\O8z
14
NaI~ ~ ' ~ q 15
17
18
PPTS ~_ I ~ COOMe MeOH HO
/,
o 6
,--o
OH
5-deoxy-KDG
f
H2/Pd/C COOMe -~ "~OBz Et3N -
.COOMe
MeOH
"~OBz
0 ~ ~ 19
16
"a~om
.COOMe
~ O
-'OSz
20
Figure 5
"~OH
....~COOMe O
PDC AcOH
850 We checked that in the absence of hydrogen, compounds 16 did not undergo any elimination of the axial iodine. If such an elimination had occured (involving axial proton at C-4) the further hydrogenation of the enol ether thus formed would have led to a partially, or totally, racemised 17. The methanolysis of the benzoate ester in diastereomers 17 followed by an oxydation of the resulting alcohols 18 using Czemecki et aL conditions 5 led to the keto-ester 19 which was then deprotected to give the target comtxxmd 20.
SYNTHESIS OF 4-O-Me-KDG METHYL ESTER 23
The synthesis of this derivative of KDG was accomplished following the sequence depicted in figure 6. Methylation of the intermediate 8 with methyl triflate afforded compound 21. Subsequent removal of the enol benzoate group at C-2 and of the silyl ether at C-6 provided the target compound 23 in 11% overall yield based on 8. This low yield could be due to the unstability of compounds 8, 21 and 22 in a basic medium. However 24 was obtained in quantity sufficient for testing. I
-I--Si-O----!6
I
-I-si-o---1 ,
s,L.-o
CH3COOK MeOH
o
HO~ O B z
21
8 I
OBz
I
-I-si-
-I-~i-o-~
I
o
~
O
OMeCOOMe O~~tH
= 22
O
I
-I-~i-~
o
oH
Ho-~
//,,
' OMe
6 __
0
OH
1\4 / (COOMe HO I 3 1 OMe Figure 6
o
oH
" coo o
OMe
851
SYNTHESIS OF 4-DEOXY-KDG METHYL ESTER 29
In order to prepare this compound, since we anticipated that the intermediate 8 would not survive deoxygenation conditions, we decided to start from D-glyceraldehyde 26 having the required configuration (figure 7). The latter compound was prepared by oxydative cleavage of 1,2:5,6 di-O-isopropylidene-D-mannitol 257 and was reacted with phosphorane 248 to furnish the keto-ester 27. After hydrogenation of the C-3,C-4 double bond leading to 28 and, finally, methanolysis of the dioxolane group, the target compoud 29 was obtained in three steps and 42% overall yield based on 26.
COOEt
1 COOMe
0
2
0
3 H~PPh 3 Wittig
24
H
"
NalO~ OH
H20 ~
----
o,,,
Lo'/. 25 6
26 6
O O
0
coo.o
1143 /"'<1o"~ 27
H2/Pd/C=
2 COOMe
5~ 4
1
3
1
28 6
/OH PPTS
o
MeOH
/
COOMe
"I"
5 HO 4
HO
4 - d e o x y - K D G 29 Figure 7
0
OH 2
3
COOMe 1
852 CONCLUSION Compounds 13 and 20 were obtained in five and seven steps respectively from the intermediate alcohol 6. They, as well as the acetylated derivative 12, were tested in vivo 1 and proved to be, as their free acids 9, gratuitous inducers of pectinases in Erwinia chrysanthemi. These results provide evidence that the hydroxyl function in C-5 is not required for the recognition between inducers and the KdgR repressor protein. Therefore we can now envisage the preparation of affinity columns, by immobilizing inducers on supports using a spacer arm bound to the C-5 hydroxyl. Such columns could then be used for the purification of the repressor protein. Compounds 23 and 29 were synthesized in three steps from 8 and 25 respectively. These molecules showed no inducing effect, indicating that the hydroxyl in C-4 participates to the recognition process (or that the C-4 modified molecules could not enter the bacteria).
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the European Community (Contract AIR2-CT94-1345) for financial support. F. A. thanks the Minist&e de rEnseignement Sup6rieur et de la Recherche for a fellowship.
REFERENCES AND NOTES
Nasser, W.; Condemine, G.; Plantier-Royon, R.; Anker, D.; Robert-Baudouy, J. FEMS Microbiology Lett., 81 (1991) 73. Plantier-Royon, R.; Cardona, F.; Anker, D.; Condemine, G.; Nasser, W; RobertBaudouy, J. J. Carbohydr. Chem., 10 (1991) 787. Ambrose, M-G; Binkley, R. W. J. Org. Chem., 48 (1983) 674. Compounds 11 to 29 have been fully charactedsed (elemental analysis, 1H and 13C NMR). Augustine, R. L. Catalytic Hydrogenation; Dekker, M." New-York, 1965; pp. 125-126. Czemecki, S.; Georgoulis, C. ; Stevens, C. L. ; Vijayakumaran, K. Tetrahedron Lett., 26 (1985) 1699. Daumas, M.; Vo-Quang, Y.; Vo-Quang, L.; Le Goffic, F. Synthesis., (1989) 64. Shing, T. K. M. Tetrahedron., 48 (1992) 6777. The repressor protein recognized only free acids inducers. However, as already observed"1,the in vivo tests can be realized with the corresponding esters, since they are hydrolysed in situ by esterases.
J. Visser and A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996Elsevier Science B.V.All rights reserved.
Production Substrates
of Pectinases from
Lerluck Chitradon, Polson Mahakhan*, Kitpreechavanich and Napha Lotong
853
Rhizopus sp. in S o l i d Phuntip
Department of Microbiology, Faculty of Science, Bangkok 10903, Thailand.
Poonpairoj,
Kasetsart
Vichien
University,
* Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand. Abstract
Rhizopus sp. 26R that was isolated in Thailand and has the efficiency in hydrolyzing raw cassava starch was also found to have high pectinase activity. The pectinase from Rhizopus sp. 26R showed high potential in enhancing the digestion of raw starch from whole cassava tuber when used together with glucoamylase from an isolated Aspergillus niger JS. Production of the fungal enzymes was carried out by solid substrate fermentation composting of agricultural wastes in plastic bag in order to lower the cost of production. On the 20 gram of solid substrates composing of wheat bran and rice husk in a ratio of 18:2 Rhizopus sp. 26R gave high activity in 2 days and remained constant for 4 days. Addition of either 1 g raw cassava starch or 1 g pectin to a 20 g substrates increased the enzyme activity to 1.7 and 2.4 times, respectively. Addition of rice bran to the mixture of wheat bran and rice husk was the best substrates for the fungal pectinase production. Addition of pectin to the solid substrates that composed of rice bran could not increase the enzyme production but even inhibit the production. The highest activity obtained when the strain was grown on substrates containing a mixture of wheatbran, rice bran and rice husk in a ratio of 9:9:2 or 6"12:2 with 58% initial moisture content, pH adjusted to 5.7 and incubation temperature was at 32~ Under these conditions, Rhizopus sp. 26R produced ca. 700 units of enzyme activity per gram of solid substrates. Cost of the enzyme production in solid substrates was also estimated. INTRODUCTION Cassava is one of an important economic plants of Thailand. Thailand exported cassava products eg. cassava chip, pellet, flour and starch, etc. which are low value. The amount of the products was approx. 20 million metrictons a year in 1993. However, by the process, some carbohydrates in cassava tuber still waste and further cause pollution.
854 To obtain higher value products as well as improve the process for product recovery, efficient utilization of starch which is the main component in cassava tuber has been considered. Using mixed enzymes of Glucoamylase from Aspergillus niger J8 (1,2,3) together with commercial pectinase showed the increasing of the digestibility of raw cassava starch. Several strains of fungi capable of raw cassava starch hydrolyzation were assayed for their pectinase activitites. Rhizopus sp. 26R exhibited the highest activity of the enzyme and the activity remained high upto 6 days (2,4). Solid substrate fermentation using agricultural wastes was considered to be used for the production of both enzymes in order to reduce the production costs. Production of glucoamylase from Aspergillus niger J8 was reported (1,3). This report concerned on the production of pectinases from Rhizopus sp. 26R in solid substrates composting of agricultural wastes, optimization of the conditions for pectinases production in solid substrates and the estimation of the production cost. MATERIALS & METHODS
Microorganism Rhizopus sp. 26R, a fungal strain isolated in Thailand which capable of hydrolyzation of raw cassava starch (Figure 1). Cultivation conditions Spore of the fungus was inoculated into 20 g solid substrates composed of wheat bran, rice bran and rice husk in a 9x14 inch plastic bags (Figure 2). Different ratios of wheat bran, rice bran and rice husk were carried out as well as different initial pH, moisture content and incubation temperature were concerned. The fungus in the solid substrates was incubated for 6 days.
Rhizopus sp. 26R
Rhizopus sp. 26R grown in solid substrates of wheat bran, rice bran and rice husk (6:12:2) in a 9x14 inch. plastic bag for pectinase production.
855 Enzyme Assay The crude enzyme was extracted from the solid state culture with 100 ml of 0.33% toluene at 4~ The enzyme activity was assayed by the determination of substrate viscosity diminishing using Ostwald viscometer (5). The enzyme reaction was done at 37~ in 0.05 N acetate buffer pH 5.25. One unit of enzyme was defined as the amount of enzyme that could reduce the viscosity of 2% pectin by 50% in 10 rain. RESULTS and DISCUSSION
1. P e c t i n a s e p r o d u c t i o n in solid substrates of w h e a t bran, rice bran and rice h u s k at the ratio of 18:0:2 and 9:9:2 with the a d d i t i o n of inducers. When Rhizopus sp. 26R was cultivated in the solid substrates without addition of rice bran but composed of only wheat bran and rice husk at the ratio of 18:2. The pectinase activity from the culture was approx. 25-35 unit/ml within 2 days and the production remained constant for 4 days (Figure 3). One gram of raw starch from cassava tuber, 1 g of pectin or 0.5 g of yeast extract was added to the solid substrates in order to induce higher activity of the enzyme. The results showed that either 1 g raw cassava starch or 1 g pectin that was added to the 20 g solid substrates increased the enzyme activity to 1.7 and 2.4 times, respectively (Figure 3). The production of pectinase in solid substrates with wheat bran and rice husk could be enhanced with the addition of raw cassava starch and pectin. 160 -
.
120
Pe~in~
Activity (unit/ml)
100
160
addition
- - - ~ - - - n o addition
~wlth
staroh
- - - ~ - - - w i t h peotln
---~wlth
paotln
~wlth
yeast extraot
---4~-no
Pectinase Activity
so ,o
(unit/ml)
0
1
2
yeast extmot
---~--wlth
3
4
S
S
rsne (day)
Figure 3 Pectinase activity in 20 g of solid substrates composting of wheat bran, rice bran and rice husk (18:0:2) with the addition of 1 g raw starch from cassava tuber, 1 g pectin or 0.5 g yeast extract.
100
so
6o
0
1
2
$
4
5
6
"l-line (day)
Figure 4 Pectinase activity in 20 g of solid substrates composting of wheat bran, rice bran and rice husk (9:9:2) with the addition of I g pectin or 0.5 g yeast extract.
856 Addition of rice bran to the solid substrates to make the ratio of wheat bran, rice bran and rice husk to 9:9:2 helped increasing the activity of pectinases from Rhizopus sp. 26R as shown in Figure 4. The activity of the enzyme was approx. 4.3 times higher. Moreover, either 1 g of pectin or 0.5 g of yeast extract did not help increasing of the enzyme production. In contrary, the enzyme activity was decreased 2.6 times to that of the former one. Addition of raw cassava starch to the substrates did no effect to the enzyme production (data not shown).
2. P e c t i n a s e production in solid substrates of w h e a t bran, rice bran and rice husk at the different ratios. Different ratios of the solid substrates, wheat bran, rice bran and rice husk, were done and the activity of the pectinases was compared in Figure 5. The mixture of wheat bran, rice bran and rice husk in the ratios of 9:9:2 or 6:12:2 appeared to be two of the best composition ratios for growth of the fungus and the pectinase production.The ratio of 6:12:2 was selected for the enzyme production since rice bran was cheaper than wheat bran and locally obtained. 160 140
Figure 5 Pectinase activity in solid substrates of wheat bran, rice bran and rice husk at different ratios of substrates. Initial moisture content 66 % Incubation temperature 32~ Initial pH 5.7
120
Pectinase
Activity
(uniVml)
so 6O
0
1
2
3
4
6
6
7
Time (day)
3. P e c t i n a s e production in solid substrates of w h e a t bran, rice bran and rice husk (6:12:2) at the different ratios of initial m o i s t u r e content, initial pH and incubation temperature. Figure 6,7 and 8 showed the results of the pectinase activity when produced in the solid substrates containing wheat bran, rice bran and rice husk in the ratio of 6:12:2. The highest activity obtained when the strain was grown on the solid substrates with 58 % initial moisture content, pH adjusted to 5.7 and incubation temperature was at 32~ Under these conditions, the highest activity of the enzyme that could be obtained from Rhizopus sp. 26R was ca. 700 units of enzyme activity per gram of solid substrates.
857 160 140 120
Pectinase
Activity (unit/ml)
Figure 6 Pectinase activity in solid substrates of wheat bran, rice bran and rice husk (6:12:2) of different initial moisture contents. Incubation temperature 32~ Initial pH 5.7
100 80
3
4
Time (day)
160 140
Pectinase
Figure 7 Pectinase activity in solid substrates of wheat bran, rice bran and rice husk (6:12:2) at different initial pH. Initial moisture content 58% Incubation temperature 32~
lOO
Activity oo (unit/ml) 6O
3
4
Time (day)
160 140 120
Figure 8 Pectinase activity in solid substrates of wheat bran, rice bran and rice husk (6:12:2) at different incubation temperatures. Initial moisture content 58% Initial pH 5.7
IO0
Pectinaee Activity so
(un~ml)
60
0
1
2
3
4
Time (day)
858 4. The efficiency in r a w cassava s t a r c h h y d r o l y z a t i o n of p e c t i n a s e s from Rhizopus sp. 26R c o m p a r e d with a c o m m e r c i a l p e c t i n a s e w h e n m i x e d w i t h G l u c o a m y l a s e from Aspergillus niger JS.
Aspergillus niger J8, a strain of fungi isolated in Thailand, produced high activity of glucoamylase that could hydrolyze raw cassava starch (1,3). The crude pectinases from Rhizopus sp. 26R showed high potential in enhancing the digestion of raw starch from whole cassava tuber when used together with glucoamylase from Aspergillus niger J8 (Figure 9). For example, in the first half an hour, amounts of glucose liberated when the mixed enzymes were used, was 2.5 times of that liberated from the glucoamylase digestion. --ll-- nixed enzyrms 1:1, total I vol. l m l I pectinmm, total vol. 1 mlJ
s 4
0
O.S
1
2
3
~..(ho.r)
4
$
II
Figure 9 Pectinases from Rhizopus sp. 26R showed high potential in enhancing the digestion of raw starch from ground cassava tuber when the enzymes were mixed with the glucoamylase of Aspergillus niger J8
When compare the efficiency in enhancing the hydrolyzation of raw cassava starch between pectinases from Rhizopus sp. 26R and the commercial pectinase at periods of time (Figure 10).
i!i Time (hour)
Figure 10 The efficiency in raw cassava starch hydrolyzation of pectinases from Rhizopus sp. 26R compared with a commercial pectinase when mixed with glucoamylase from Aspergillus niger JS.
859 Figure 10 showed that using pectinases from Rhizopus sp. 26R with glucoamylase, in the first half hour, the efficiency of starch hydrolyzation could be increased approx. 2.6 times more than when using only glucoamylase. In the 2nd, 4th,6th and 8th hour, the hydrolyzation was 2, 1.6, 1.5and 1.4 times more efficient than using only glucoamylase, respectively. While using of commercial pectinase with glucoamylase showed less efficient in enhancing the hydrolyzation of starch. In the 2nd, 4th, 6th and 8th hours, the commercial one could enhance the hydrolyzation only 1.2, 1.4, 1 and 1 time, respectively. Therefore, pectinases of Rhizopus sp. 26R was more efficient in enhancing the digestion of raw cassava starch more than the commercial one when used with glucoamylase. 5. E s t i m a t i o n of t h e p r o d u c t i o n cost of p e c t i n a s e f r o m R hizopus sp. 26R. The production of the pectinases in the solid substrates composed of wheat bran, rice bran and rice husk (6:12:2) was considerably very low. The spore inoculum of Rhizopus sp. 26R was prepared on raw cassava starch agar which the cost estimation was US$ 1.0 per 1 litre. Wheat bran, rice bran and rice husk were approx. US$ 64 per 50 kg. The total cost of the production of pectinases from Rhizopus sp. 26R in the solid substrates, when considered only on the substrates was estimated to be only US$178 - 180 for 10 million units of crude pectinase. CONCLUSION Using agricultural wastes as solid substrates for the production of pectinase from
Rhizopus sp. 26R gave benefit, not only to the utilization of the wastes but also to the reduction of the cost of the enzyme production. The enzyme production in the solid substrates composed of wheat bran and rice husk (18:2) could be increased by the addition of either 1 g raw cassava starch or 1 g pectin to a 20 g substrates. The enzyme activity increased approx. 1.7 and 2.4 times, respectively. Addition of rice bran to the mixture of wheat bran and rice husk was the best substrates for the fungal pectinase production. The solid substrates that composed of wheat bran, rice bran and rice husk at the ratio of 6:12:2 was selected to be the best since rice bran are easily found in South-east Asian countries. Addition of either raw cassava starch or pectin as inducer is not needed. On the otherhand, pectin even inhibited the activity of the enzyme as well as that reported by Elegado and Fujio (6). The optimum conditions of the pectinase production that was carried out in a plastic bag contained 20 g solid substrates that composed of wheat bran, rice bran and rice husk (6:12:2) needs initial moisture content and pH of 58% and 5.7, respectively, and the fungus was incubated at 32~ for 6 days. Under these conditions, Rhizopus sp. 26R produced ca. 700 units of enzyme activity per gram of solid substrates.
860 The pectinases produced in solid substrates from Rhizopus sp. 26R showed the efficiency in enhancing the activity of the glucoamylase in digestion of rawground-cassava tuber higher than that of the commercial one. Cost of the enzyme production in solid substrate was estimated to be US$180 for 10 million units of crude pectinase. This price included the production of fungal spore inoculum. This production of pectinases from Rhizopus sp. 26R using agricultural wastes as solid substrates was one of the way to utilize agricultural wastes to value-added products and the cost of the enzyme production was very reductive. REFERENCES
1.
L. Chitradon, V. Kitpreechavanit, W. Yongmanitchai and N. Lotong, Proceedings on Workshop on AID/SCI Funded Research in Agricultural Biotechnology (1990) 20-27.
2.
L. Chitradon, P. Mahakhan, V. Kitpreechavanit and N. Lotong, Abstract in the 8 th NRCT NUS DOST-JSPS Joint Seminar on Biotechnology and the 4 th Annual Meeting of the Thai Society for Biotechnology (1992) 104.
3.
L. Chitradon, V. Kitpreechavanit, W. Yongmanitchai and N. Lotong, Thai Journal of Agricultural Science, 26 (1993) 109-121.
4.
P. Mahakhan, V. Kitpreechavanit, N. Lotong and L. Chitradon, Proceeding in the 30th Annual Meeting of Kasetsart University, (1992) 627-638.
5.
E. Roboz, R.W. Barratt and E.L. Tatum, J. Biol. Chem., 195 (1952) 459-471.
6.
F.B. Elegado and Y. Fujio, Journal of General and Applied Microbiology, 39 (1993) 409-418.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
861
Endo-polygalacturonase of the yeast Kluyveromyces marxianus is constitutive, highly active on native pectin and is the main extracellular protein R.F. Schwana,b, R.M. C o o p e r a and A.E. W h e a l s a aSchool of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, United Kingdom bCEPLAC/Cocoa Research Centre/SETEA, Cx. Postal 07, 45.600.000, Itabuna, Bahia, Brazil
Abstract Kluyveromyces marxianus is the most pectinolytic yeast found during early cocoa pulp fermentations. 85-90% of its total secreted protein comprises endo-polygalacturonase (PG). No pectic lyases (PL) or pectin methylesterases (PME) are produced. Regulation of PG is constitutive and not subject to carbon or nitrogen catabolite repression but galacturonic acid monomers and polymers are not utilized. Purified PG comprises four proteins of Mr 45, 42, 39 and 36kDa. Activity stained IEF gels revealed three major apparent isoforms (pls 5.9, 5.6 and 5.3) and six minor bands (pI ranging from 6.4 to 5.0). PG has a typical random mode of action and a very high macerating activity on plant tissues. 1. I N T R O D U C T I O N Yeasts tend to dominate the initial phase of the natural fermentation of cocoa pulp [ 1]. An isolate ofKluyveromyces marxianus (CCT 3172) was found to be the most pectinolytic of 12 yeast strains, resulting in degradation of pulp cell walls [ 1]. Pectinolytic enzymes secreted by the diploid K. marxianus have been partially purified and characterized [eg 2] but the results obtained were very variable, partially contradictory and paid little attention to the physiological aspects of PG secretion [3]. Commercial use and production of PG from K. marxianus has attracted considerable interest [4]. 2. M E T H O D S
AND MATERIALS
2.1. Strain Kluyveromyces marxianus CCT 3172 was isolated from cocoa fermentations in Bahia, Brazil and has been deposited in the culture collection of the Fundaqao Tropical de Pesquisas e Tecnologia "Andr6 Tosello" (CCT), Campinas, Sao Paulo, Brazil. 2.2. Screening for pectinase activity Screening of cells for pectinase activity was done on MP-5 medium [5] after precipitation of polygalacturonic acid with 1% Cetrimide (Sigma). Liquid culture experiments were done under the self-induced anaerobic conditions [6]. 2.3. Enzyme assays Culture medium supernatant was used as a source of extracellular enzymes. PG activity: Two methods were used to measure PG activity (versus polygalacturonic acid Na-
862 salt) in culture filtrates; release of reducing groups [7] or decrease in viscosity of substrate [8]. Macerating activity was determined by loss of coherence of tissue according to the method of [9]. Pectin Methylesterase (PME): Pectin methylesterase was assayed by continuous titrimetric determination of the carboxyl groups liberated from the methylester bonds [2]. Pectin Lyase (PL) and Pectate Lyase (PGL). Pectin lyase and pectate lyase activities were determined by increase in A240(PL) or A235 (PGL) of the unsaturated products from degradation of 0.25% (w/v) pectin or polygalacturonic acid Na-salt [ 10]. 2.4. Characterization of polygalacturonase i) Determination of relative molecular mass by gel filtration: A sephacryl S 100 HR column (90 cm x 1.6 cm) was equilibrated and eluted with 0.1M acetate buffer, pH 5.0 and fractions were collected and assayed for PG activity and A280. SDS-PAGE: Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) was performed by standard methods [ 11 ]. Gels were stained with Coomassie brilliant blue. ii) Isoelectric focusing (IEF): IEF was carried out using a fiat bed gel electrophoresis kit (LKB) and separated PG isozymes were visualized by a pectate-agarose overlay gel [ 10]. 2.5. Preparation and fractionation of sphaeroplasts: Sphaeroplasts were prepared by slight modifications to published methods [ 12,13 ]. Lysis of sphaeroplasts was effected by a combination of osmotic lysis and gentle mechanical disruption [ 14]. Discontinuous sucrose-density gradients were constructed and fractions were then assayed for protein, PG and marker enzymes for different organelles. 2.6. Mutagenesis Mutants were induced by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis [ 15]. Strains were selected on the basis of the size of halos on the basic medium (MP-5) [5].
3. R E S U L T S 3.1. Properties of polygalacturonase secreted by K. marxianus PG activity was assayed from cells grown in a medium containing 1% glucose in onelitre self-induced anaerobic fermentation for 5 days by increase of reducing sugars. Enzyme activity increased from pH 3.0 to pH 5.0 (citrate buffer) and decreased drastically above 5.0 (phosphate buffer), but activity was not affected differentially by the two buffers used (data not shown). PG activity increased almost linearly from 20~ to 40~ but above this optimum, activity was lost rapidly and the enzyme was completely inactivated at 60~ and 70~ after 10 and 6 min, respectively (data not shown). No PL, PGL or PME were detected. 3.2. Determination of relative molecular mass The culture supernatant ofK. marxianus was concentrated using PEG (20,000 Da) and the protein obtained was applied onto the column. Four peaks containing pectinolytic activities were resolved (Figure 1), and the molecular masses were calculated against markers as Mr of 47 kDa, 41 kDa, 35 kDa, and 33 kDa. SDS gel electrophoresis separation in total denaturing conditions was carried out on the protein of culture filtrates and proteins of known molecular mass. The four dark bands (Figure 2) which appear in the gel between 45 and 36 kDa of the standards were assumed to be PG based on the gel filtration results for PG activity and total protein. The relative molecular mass of the four protein bands were estimated as 45 kDa, 42 kDa, 39 kDa and 36 kDa. It was calculated that about 85% of total protein secreted into the culture medium by K. marxianus consisted of PG.
863 MrC=103)
66
45
29
24
-6
0.2
-5
~" >
0.15
o I
-4
,
II
E
cO
r(x)
<
-3
O. I -
c 0
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-2
~ >, o
0.05
-
-1
0 20
, 25
30
35
40
45
50
55
0 60
F r a c t i o n no.
Figure 1. Separation profile on Sephacryl S 100 column of extracellular PG. 2.7 ml fractions were analysed for protein ([3) and reducing sugars released (A). Peaks I, II, III and IV correspond to PG activity expressed as lamol galacturonic acid released rain-1.
3.3. Mechanism of enzyme action A viscometric assay and identification of hydrolysis products were used to determine the mechanism of action of PG. An endo-PG is characterized by a strong reduction in viscosity (e.g. 50%) with a concomitantly low (e.g. 1-3%) release of reducing groups [9]. The time required for 50% decrease in viscosity of a 3.0% (w/v) sodium polypectate solution at 25~ was approximately 10 min, at which time about 1.5% of the total galacturonide bonds had been hydrolysed (data not shown). These results reveal a random mechanism of hydrolysis of sodium polypectate and the enzyme was a poly oc(1,4)-D-galacturonide glycanohydrolase (EC 3.2.1.15) or endo-PG. 3.4. Hydrolysis of pectin from plant tissue by endo-polygalacturonase Endo-polygalacturonase was tested against pectin from cocoa seed pulp by measuring decrease of viscosity and against pectin present in potato and cucumber by loss of coherence of tissue (maceration activity). Cocoa pulp was removed from the seeds and centrifuged to remove solid material and the supematant was then used as a substrate for the viscometric assay. A sharp decrease in viscosity was observed after 10 min of incubation and 50% decrease of viscosity was achieved within 18 min (Figure 3). Endo-PG produced by K. marxianus had very strong maceration activity on potato and cucumber. Maceration of tissue from both species was extremely rapid with softening apparent even after only 5 min and complete cell separation occurring within one hour.
864
Mr
( X 10 3)
A
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_.= >
E 75
.--
o
To L
~.~
~.
se si
.
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4
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Figure 2. SDS-PAGE of culture supernatants. Freeze-dried samples were resuspended in distilled water, mixed with an equal volume of sampling buffer and heated to 100~ for 5 min. 101al aliquots were applied to the gel. The right lane contains standards of Mr 14 - 66 kDa. Lanes 2, 3, 4 and 1 are increasing dilutions of the supernatant respectively.
o 4., 6 0 E
o L
Q.
>25 " 0 0 > 0--
o
10
I
I
I
I
I
I
20
30
40
50
60
70
Time (rain)
Figure 3. Decrease in viscosity of cocoa pulp by PG secreted by K. marxianus.
3.5. Regulation of endo-polygalacturonase production PG production was constitutive and not subject to carbon catabolite repression. Highest yields were on glucose and fructose (upto 10% w/v) (Table 1). Inclusion of pectic compounds had no effect on growth or PG production (data not shown). 3.6. Distribution of endo-polygalacturonase in batch cultures The distribution ofPG (PG) as culture medium supernatant, cell-wall associated and cell-bound enzyme was observed in K. marxianus during the time course of growth in 5% glucose medium (Table 2). PG secretion started between 8 and 12 h alter inoculation and approximately 90% of total PG was secreted in early stationary phase. PG was not detected intracellularly alter 24 h of growth. 3.7. Intracellular location of endo-polygalacturonase The subcellular location of PG was studied in cells disrupted by osmotic lysis through formation and disruption of sphaeroplasts from self-induced anaerobically-grown cells. A discontinuous sucrose-density gradient produced four bands labelled I, II, III and IV. Band I included many vesicles and a peak of alkaline phosphatase activity (a vacuolar marker in yeasts), NADPH cytochrome c oxidoreductase activity, an endoplasmic reticulum marker, and
865
Table 1 Growth, enzyme production and enzyme yield Carbon Source
Growth (mg dry wt ml-~)
PG Activity (RVU m1-1 Jig protein -1)
PG Yield (RVU mg biomass -~ ml-~)
D-Glucose Sucrose D-Fructose D-Galactose Lactose D-Xylose
0.70+0.04 0.73+0.03 0.68+0.05 0.69+0.06 0.63+0.06 0.31+0.05
48 9 + 1 . 2 46 9+0.3 27 2+0.9 19 0+1.0 14 3+0.5 2.7+0.9
69.8 64.2 40.0 27.5 22.7 8.7
All cultures were grown with 10g 1-1 sugar under self-induced anaerobic conditions. Growth and PG activity were measured after 16 hrs. There was no growth on galacturonic acid, pectins, cellulose or rhamnose. • indicates standard deviation.
Table 2 Distribution of PG in sphaeroplasts and sub-cellular fractions. Cellular Fraction Time (hrs)** Sphaeroplasts Low-density vesicles Vacuoles * ~ ER*** p, . *** lasma-memorane
PG activity* 8
10
12
14
16
673 350 84 160 59
715 314 115 50 246
516 210 88 48 170
356 81 32 38 205
212 57 0 0 148
* Expressed as RVU. ** Hours after inoculation of self-induced anaerobic culture. *** Organelle identified by peak of marker enzyme activity. transmission electron microscopy revealed the presence of lipid-vesicles, identified by their affinity for osmic acid. Over 85% of the vanadate-sensitive Mg++ ATPase activity, marking yeast plasma membranes, was detected in bands II, III and IV and confirmed by TEM. Vesicles were also detected in these bands, but they appeared to be part of the plasma membrane and had not resulted from fraction contamination. Sphaeroplast lysates fractionated on sucrose-density gradients showed sequential movement of enzyme from the ER via low density vesicles to the plasma membrane (Table 2). PG activity was not found in fractions of cells harvested after 24 h. of growth.
866 3.8. Mutants
One hundred and thirty eight mutants produced clearing zones of repeatable and different diameter compared to the parent strain (diameter of 30 + 2 mm). PG activity of each mutant was then measured in the supernatant from liquid cultures. Only five mutants showed increased extracellular PG activity compared with the parental strain. The highest activity was 23.4 PG units, which is 25% above the wild type level. The great majority of the mutants (107) showed reduced PG activity ranging from 0 to 15 PG units. One hundred and thirty mutants showed similar or lower intracellular PG levels compared to the wild type; these comprised both over-and under-producers. Three mutants with higher intracellular activity secreted the enzyme later than wild type; five mutants with reduced levels of secretion had Very high intracellular PG activity, possibly indicating a defective secretory pathway. However, the total PG activity remained below wild type levels. Sub-cellular fractionation of five strains revealed the same numbers of bands. The distribution of PG activity in sub-cellular organelles was broadly similar in these five strains. PG activity was detected in low-density vesicles, vacuoles and ER fractions in samples harvested during the early exponential phase of growth. However, PG levels were always lower (at least 1.5 fold) than those found in wild type. Cells of the mutants harvested during stationary phase of growth showed that 84% of total intracellular PG activity was located in the vesicle fraction. No intracellular PG activity was found in stationary phase wild type cells.
pl 9.30
-
8.75
_
7.35
_
6.85_
5.85
-
5.20-
,d
3.50 -
~fl/t
W~[
4
3
6
7
27
30
31
32
35
Figure 4. Isoenzyme profiles of wild type and mutant strains. Detection of PG activity was by ruthenium red staining on pectate-agarose overlay gels after IEF. WT indicates wild type and the numbers refer to specific mutant isolates.
867
3.9 Isoenzyme profile of polygalacturonase in wild type and mutant strains Isoelectric focusing showed up to nine grouped apparent PG isoenzymes; three major bands (estimated pI's 5.9, 5.6 and 5.3) and six minor bands of apparent isoforms range from 6.4 to 5.0 (Figure 4). The time-course of growth showed that all nine isoforms were present in the culture filtrate aider only 24 h of incubation. Extracellular PG activities of 18 representative mutants showed a common pattern of at least three major isoenzymes, and five minor apparent isoforms. Profiles of PG from wild type and the very low under-producers (mutants secreting less than 1~ of the normal PG) were equivalent in band number (eight), but the intensity of all bands from the mutants was generally weaker. However, some mutants which are also under-producers (3 and 4) had almost identical profiles to the wild type, ie, they showed the three major and the five minor isoenzymes. 4. D I S C U S S I O N PG secreted by K. marxianus CCT 3172 showed activity from pH 4 to 6, with an optimum at pH 5 typical of PG secreted by yeasts. Unlike some pectinases, the activity of PG from K. marxianus CCT 3172 was not affected by buffers used across the pH range studied. The effect of temperature on the activity of PG from K. marxianus was similar to that reported for PGs from yeasts [eg 2]. The relative molecular masses of the four proteins revealed by gel filtration were in close agreement with previous estimates [ 16,17]. Activity-stained, IEF gels revealed nine multiple forms that could be divided into two acidic groups in relation to intensity of bands: the first one consisting of three major bands and the second with six minor isoforms showing weaker band intensities. The pI values in the present study are similar to those previously obtained [ 17]. Numbers of apparent isoenzymes are sometimes caused by variations in glycosylation or by degradation due to the use of old cultures. However, the presence of all nine PG isoforms in the growth medium from young cultures might suggest that they are not artefacts from IEF. None of the under-producer mutants had lost a single isoenzyme indicating that none of the mutations were in structural genes. Those under-producer mutants showing 8 isoenzymes with similar intensities of each band, seem to have had a mutation in the regulatory gene(s) affecting a subset of the isoenzymes. Differences were found in isoenzyme profile, growth rate and total excreted proteins when mutants showed about 20% of wild type extracellular PG activity, a phenotypically pleiotropic effect in whiizh various enzyme systems were modified (unpublished data). The endo-action of the K. marxianus PG was demonstrated by a extremely rapid attack on plant tissue. This activity appears to be at least equivalent to that of several commercial preparations used for separating plant cells for protoplast preparation (RMC, unpublished data). Most of the endo-PGs produced by plant pathogens and saprophytes have so far been reported to possess macerating activity. PG secreted by K. marxianus CCT 3172 also had a strong activity in reducing the viscosity of cocoa pulp. Cocoa pulp generally contains 1 - 1.5% (w/w) of pectin consisting of 68% esterification and 11.6% methoxyl content [ 18]. The distribution of PG activity, among supernatant, cell-wall and cell-bound fractions changed throughout the time-course of growth and it is likely that PG activity in cell-wall fractions was enzyme in the process of being released, rather than located in the periplasmic space. Studies on the subcellular location of PG using sucrose density gradient suggested that PG was synthesized and secreted by the classic yeast secretory pathway [ 19]. This conclusion is consistent with the results from the five mutants which synthesized PG but were unable to release it into the medium. 90% of intracellular PG activity was located at the vesicle fraction, indicating a specific transfer block at the stage from vesicles to plasma-membrane. This is the first study of subcellular location of PG in K. marxianus.
868 The screening method based on diameter of halos yielded only five strains with at least 20% more PG than wild type. Most attempts to enhance pectinase production have been made in filamentous fungi, in which enzyme production is regulated by induction and catabolite repression mechanisms [8]. By contrast, PG produced by K. marxianus is constitutive and not subject to catabolite repression [6]. Failure to find greatly enhanced secretion levels is consistent with the idea of a constitutive gene that is capable of only modest further induction. PG was found in this study to be already the most prolific secreted protein ofK. marxianus. This strain ofK. marxianus was found during the first 36 h of cocoa fermentation when the pulp is degraded which suggests that PG from K. marxianus may be utilized directly on cocoa beans to speed up the fermentation process and also to obtain higher quality fermented beans. No other pectinolytic enzymes, apart from PG, were secreted by K. marxianus into culture medium. This should facilitate PG purification. PG from K. marxianus may in future, be utilized directly on cocoa beans for the extraction of cocoa juice. The pulp obtained with the aid of pectinolytic enzymes has a lower viscosity and this aids processing of pulp for pasteurized juice and soft drinks from cacao. Another potential industrial application for PG produced by yeasts, is in the manufacture of fruit nectars or in softening of vegetables for preparation of baby foods. Advantages of this enzyme over PGs from filamentous fungi, mainly Aspergillus niger, appear manifold, and include no requirement for pectin inducers, absence of numerous contaminating enzymes, absence of PME which releases toxic methanol, and much faster growth rates of K. marxianus.
5. R E F E R E N C E S
6 7 8 9 10 11 12 13 14 15 16 17 18 19
Schwan, R.F., Rose, A.H. and Board, R.G. (1995) In: Fermentation: Foods, Feeds and Condiments. R.G. Board, D. Jones and B. Jarvis. (Eds) Supplement to J. Appl. Bacteriol. Symposium 79, pp 96S-107S. F.M. Barnby, F.F. Morpheth and D.L. Pyle (1990) Enzyme Microb. Technol. 12, 891. A.P. Espinoza, E. Barzana, M. Garcia-Garibay and L. Gomez-Ruiz, (1992) Biotech. Letts 14, 1053. S. Harsa, C.A. Zaror, and D.L. Pyle (1993) Process Biochem. 28, 187. L. Hankin and G.H. Lacy (1984) In Compendium of Methodsfor the Microbiological Examination of Foods, 2nd edition, ed. M.L. Speck. American Public Health Association, Washington, D.C. R.F. Schwan & A.H. Rose (1994) J. Appl. Bacteriol. 76, 62. G.L. Miller (1959) Anal. (7hem. 31,426. R.M. Cooper and R.K.S. Wood (1975)Physiol. Plant Path. 5, 135. R.M. Cooper, B. Rankin and R.K.S. Wood (1978) Physiol. Plant Path. 13, 101. P.K. Durrands and R.M. Cooper (1988) Physiol. Molec. Plant Path. 32, 343. U.K. Laemmli (1970) Nature 227, 680. F. Altherthum and A.H. Rose (1973) J. Gen. Microbiol. 77, 371. T.G. Cartledge, A.H. Rose, D. Belk and A.A. Goodall (1977) J. Bacteriol. 132, 426. P.A. Henschke, D.S. Thomas, A.H. Rose, and F.J. Veazey (1983) J. Gen. Microbiol. 129, 2927. C.W. Lawrence (1991) In Guide to Yeast Genetics and Molecular Biology, C. Guthrie and G. Fink. (eds)Methods in Enzymology 194. Academic Press, New York. S. Inoue, Y. Nagamatsu, and C. Hatanaka, C. (1984) Agric. Biol. Chem. 48, 633. A.B. Smith and D.L. Pyle (1990) J. Food Biochem. 14, 273. P.R.F. Berbet (1979) Revista Theobroma 9, 55. R. Schekman (1985) Ann. Rev. Cell. Biol. 1, 115.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
Polygalacturonase and pectinmethylesterase activities during growth of 1805 cell suspension
869
Helianthus annuus
M. Ilievaa, M. Kratchanova b, E. Pavlovab,T. Dimova a, A. Pavlov a
a Bulgarian Academy of Sciences, Institute of Microbiology, acad. G. Bontchev Str. 26, Sofia, Bulgaria 1113
b Bulgarian Academy of Sciences, Institute of Organic Chemistry with Phytochemistry, 95 V. Aprilov Str., P. O. Box 27, LBAS - Plovdiv, Bulgaria 4002
Abstract Time courses of growth of Helianthus annuus 1805, changes of the amount of cell walls, as well as changes in polygalacturonase and pectinmethylesterase and related changes in the degree of esterification and polyuronic content of the cell walls were investigated. Dependences between the cell enzyme activities and the cell wall changes, as well as between the cellular and extracellular enzyme activities, were established. Growth of the cell suspensions and the related remodelling of the cell walls were considered a process in which the enzymes under study were active both in the cell and in the culture medium.
Introduction
The growth of plant cell suspensions is connected with changes in the mechanical and structural elements of the cell wall (1). This process may involve changes in the amount and/or the structure of the pectic polysaccharides (2). The loss of the native strength of the wall and the secretion of structural fragments from the latter is facilitated by the activity of endogenous hydrolytic enzymes during growth (2-4). Recent studies have followed their changes and have related them to the process of development (5-8). The objective of this research was to follow the course of growth of H.annuus 1805 cell suspension in parallel with the changes in the amount of cell walls, as well as the course of biosynthesis and secretion of the enzymes polygalacturonase and pectinmethylesterase and the
870 related changes in the polyuronic content and the degree of esterification of the cell polysaccharides.
Materials and methods
Cell culture. The Helianthus annuus 1805 cell culture was grown in Linsmayer-Skoog medium (9), supplemented with 0.2 mg/L 2.4 - dichlorphenoxyacetic acid and 3% sucrose. The callus cultures were kept in an agar medium of the same composition. They were grown in a thermostat in the dark at 26-28 ~ for two weeks and could be stored up to two months in a refrigerator. H.annuus 1805 cell suspension was cultivated on a shaker (11.6 rad/s) at 26-28 ~ in the dark in 1/5 net volume flasks. Duration of growth was different: from 5 days for obtaining the inoculum to 10 days for studying the course of growth, the course of changes in the cell walls and the courses of biosynthesis and secretion of the enzymes polygalacturonase and pectinmethylesterase as well. For inoculation 20 % (v/v) five-day cell suspension, containing 9 g/L dry cell biomass, was used. By daily taking samples during the 10-day cultivation, changes in certain parameters were followed, using the relevant methods: Course of growth. The course of growth of H.annuus 1805 cell suspension was followed by measuring the amount of dry cell biomass/DB/(10). Polygalacturonase /PG/ and Pectinmethylesterase /PME/ A ctivities Cellular enzyme activities. The cell PG and PME were determined after their extraction from the cell biomass. For that purpose the cell biomasses were frozen for 12 hours, then, after defreezing, 0.2 M acetate buffer (pH 7.9) was added at a ratio of 1:2.5 and the mixture was homogenized for 10 minutes using Polytron homogenizer. The sample stayed in the refrigerator for 24 hours, after which it was centrifuged for 40 minutes at 6,000 xg. The supernatant was separated and ammonium sulphate was added to 40 % concentration and the precipitated interfering proteins were removed by centrifugation (30 min, 4500 xg). Once again, ammonium sulphate was added to the separated supematant until 70 % saturation was reached which led to precipitation of the proteins carriers of both enzyme activities. The precipitate was separated by centrifugation (30 min, 4500 xg) and diluted with 5 ml 0.1 M phosphate buffer (pH 7.0). The solution was dialyzed for 12 hours against distilled water and after dilution to the required volume with the same phosphate buffer, polygalacturonase and pectinmethylesterase were determined. Extracellular Enzyme Activities. The protein, carrier of the polygalacturonase and pectinmethylesterase activities, was salted out from the H.annuus 1805 culture medium by adding ammonium sulphate to 70 % saturation. The precipitate was separated by centrifugation (30 min, 4500 xg), diluted with 0.1 M phosphate buffer (pH 7.0) and dialyzed for 12 hours against distilled water. After dilution to the required volume with the same buffer, both enzyme activities under study were determined in the solution.
871 Polygalacturonase was determined by the viscosimetric method (11), and pectinmethylesterase - according to the titrametric method (12). Cell Walls (CW). After the extraction of the cellular enzymes from the biomass, the residue of homogenized cells was repeatedly washed with water and finally with 96 % ethanol, dried and the dry weight of cell walls was determined (10). The polyuronic content (PUC) and the degree of esterification (DE) of the dry cell walls were determined according to the method of Gee (13). The results for these two parameters are given in terms of per cent of the total amount of cell walls. The presented results are average values from three independent experiments.
Results and discussion
Course of growth of H. a n n u u s 1 8 0 5 cell suspension and of changes in the cell walls. The plant cell wall contains different types of polysaccharides, proteins (structural glycoproteins and enzymes), lignin and water, as well as some inorganic components (1, 14-16). The plant cell suspensions, however, grow as a population of cells with a primary cell wall(17). The main components of these walls are cellulose-free polysaccharides and pectic polysaccharides in particular, which constitute 1/3 of their dry weight. (18). Some fragments, e.g. methanol, acetic, ferulic and p-cumaric acids, are connected with the pectic polysaccharides by ester bonds with the carboxylic and hydroxylic groups. Besides, it is known that the culture medium acts as a common external sink like a lamella (15) or a vacuole (19), in which polysaccharides, enzymes and other metabolites are secreted during growth. Consequently, the growth of plant cell suspensions is a complex process, connected with structural and metabolite changes both in the cell wall and in the culture medium, involving a complex of hydrolytic enzymes. Our results, regarding the course of growth of H.annuus 1805 and the course of changes
in the cell walls show that the cell suspension underwent an intensive growth from the 3rd to the 7th day of cultivation (Fig 1). The changes in the amount of the cell walls followed the course of growth, i.e. by the 6th day the maximum was reached both in the amount of cell biomass and in the amount of cell walls. It is worth mentioning that after the 6th day of cultivation of H.annuus 1805 the synthesized biomass was preserved until the 8th/9th day, while the amount of cell walls decreased considerably. As it is known (2), at the beginning of the stationary phase of growth the cell mass does not change substantially, but the cell wall thickness, as well as the number of the cells decrease as a result of the processes of decomposition of the cell walls. The latter reflects more tangibly on the total amount of cell walls, than on the total amount of biomass.
872 60 55 5O
~s
16 14 12
4
_1
8 2
3O 1
25 2O
1
i
i
i
i
I
i
i
i
2
3
4
5
6
7
8
9
Time,
10
days
Fig. 1 Time course of growth ofH. a n n u u s 1 8 0 5 cell suspension, variations in the cell walls, their polyuronic content and degree of esterification. =
DB
"--
CW
-" "~
DE PUC
Course of biosynthesis and secretion of PME and PG, and related changes in the degree of esterification /DE/ and in the polyuronic content/PUC/of the cell walls. During cultivation the polyuronic content of the cell walls varied within narrow limits (Fig. 1). It is known that polygalacturonase is the enzyme, included in the decomposition of acid pectic polymers (3). As follows from the course of biosynthesis and secretion of polygalacturonase
(Fig 2), its amount at the end of the lag phase (4 th day) was considerable. With the beginning of the intensive growth of the cell suspension, PG gradually decreased. Part of it was secreted into the culture medium and the main peak in the extracellular enzyme activity was observed on the 8th day, preceded by a smaller one on the 5th day. It is known that the polygalacturonase activity is manifested along with other hydrolytic enzymes (3) and, as it was mentioned above, its chief role is to decompose the acid pectic polymers to polysaccharide chains of certain structure and molecular mass and also to ensure a certain polyuronic content of the cellular polysaccharides. Consequently,' its activity is probably regulated by relevant inhibitors. On the other hand, the polyuronic fragments, separated from the cell wall, are secreted into the culture medium. This is a potential substrate for the polygacturonase and when they are present in a considerable amount, then, by certain mechanism, part of the enzyme activity is secreted into the culture medium. It is probable that here it takes part in the remodelling of the secreted polysaccharide fragments to molecular sizes and structures with certain physiological functions for the cell. These functions are similar to those of the polysaccharides from the central lamella (15) and are related primarily to intercellular interactions and more precisely to information transfer regarding the development of the cells and their protection (20). It is also probable that the extracellular polygalacturonase takes part in carbon regeneration by decomposing the soluble acid polymers to compounds that can be
873 assimilated by the cell once again to provide for the growth and construction of new cell walls (3,19). Fig.1 follows the course of change of another important characteristic of cellular polysaccharides - their degree of esterification, which is regulated by the pectinmethylesterase (6). A sharp change in the degree of esterification of the cell walls of H . a n n u u s 1 8 0 5 was observed from the 3rd to the 6th day of cultivation (from 35 % to 58 %, Fig 1). The same period of growth marked an increase in the PME activity as well (Fig 3) and coincided with the period of intensive growth (3 rd - 7th day, Fig 1). The highly esterified polysaccharide fragments were more loosely connected and more water soluble, consequently they were more easily secreted into the culture medium, which is an important peculiarity of the process of suspension growth (6). After the 7th day of cultivation, as a result of secretion of a considerable amount of highly esterified watersoluble polysaccharide fragments, the degree of esterification in the cell walls was estimated to be 50 % by the 8th day. For the period between the 3 rd and the 7th day part of the cellular PME was secreted into the culture mediumand peaks of extracellular PME were observed on the 6th and
120
40
25
~oo
3o ~
20
25 ~
-~
25
35
-~
20 "5
-~6o
9
~
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is ~
40
.
20 -~
~ "5
-5
~0
5
10
5
5
0 20
0 2
3
4
5
6
7
8
9
10
2
3
"9
C eUular PG
-
Eztrac ellular PG
m
5
6
7
8
9
10
0
]]me, days
Time, days
Fig. 2 Time course of biosynthesis and secretion of polygalacturonase from H a n n u u s 1805.
4
Fig. 3 Time course of biosynthesis and secretion of pectinmethylesterase from H. a n n u u s 1805.
v~
Cellular PIvEE
"
Extrac ellular PIVIE
.
the 8th days of cultivation (Fig 3). They can be related to the sharp decrease (consequently secretion) of intracellular activity between the 6th and the 8th day.
874 Apart from PME, pectinmethyltransferase (6) and some other enzymes, connected with polysaccharide esterification, were involved in the process of regulating DE of the pectic polysaccharides. Therefore, a more detailed interpretation is difficult to make, but the data from this research confirm the fact that in the case of H. annuus 1805 cell suspension the processes of esterification and deesterification in the cells and in the culture medium are in a certain equilibrium and should be considered in correlation. Pectinmethylesterase, like polygalacturonase, is not limited in its involvement to remodelling the cell wall during suspension growth. When the highly esterified pectic fragments accumulate in the culture medium in considerable amounts, the enzyme is secreted and takes part in their deesterification, which allows the formation of acid regions in the polysaccharide fragments. These acid regions link the polysaccharide fragments with other components until polysaccharide structures with certain functions are established (17).
Acknowledgement The authors gratefully acknowledge the financial support for this work from the National Research Foundation of Bulgaria.
References 1. Sakuzai N., Bot. Mag. Tokyo 104 (1991) 235 2. Konno H., Y.Yamasaki, K. Katoh, Physiol. Plantarum, 69 (1987) 405. 3. Konno H., Y.Yamasaki, K. Katoh, Physiol. Plantarum 76 (1989) 514. 4. Konno H., Y.Yamasaki, K. Katoh, Physiol. Plantarum,68 (1986) 46. 5. Uchiyama T., M. Numata, S.Terada, T. Hosino, Plant Cell Tissue and Organ Culture 32 (1993) 153. 6.Schaumann A., M. P. Bruyant, V. F. Ogubet, C. Mowan, Plant cell Physiol. 34 (6) (1993) 891. 7. Amino Sh. Z., Naturforsch. 44C, (1989) 754. 8. Mc Camm, M.C., K. Roberts, Y. of Exp. Botany 45 (1994) 24. 9. Linsmayer E. M., F. Skoog, Physiol. Plantarum 18 (1965) 100. 10. Dixon R. A. (Ed.) Plant cell culture- a practical approach IRL Press (1985) 15. 11. Pherr D. M., P. B. Dickinson, Plant Physiol. 51 (1973) 577. 12. Kertesz Z. I. - Methods in Enzymology, New York, 1 (1955) 159. 13. Gee M., Mc Comb E. A., Mc Cready R. M., J. Food Sci. 23 (1958) 72. 14. Van Cutsen R., J. Messiaen, Acta Bot. Neerl 43 (1994) 231. 15. Ficher G. B., B. A. Stone, Ann Rev Plant Physiol. 34 (1983) 47. 16. Mutaftschiev S., A. Macaya, R. Prat, P. Devillers, R. Golberg, Plant Physiol. Biochem. 31 (4) (1993)459. 17. Heredia A., A. Jimenez, R. Guillen, Lebensm. Unters. Fosch., 200 (1995) 24. 18. Buwn J. B., S. C. Fry, Plant Physiol. 103 (1993) 993. 19.Wink M, Plant Cell Tissue and Organ Culture 38 (1994) 307. 20. Mohnen D., M. G. Hahn, Seminars 4 (1993) 93.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All fights reserved.
875
Differential expression of Erwinia chrysanthemi strain 3937 pectate lyases in pathogenesis of African violets: importance of low iron environmental conditions C. Masclaux, a N. Hugouvieux-Cotte-Pattat, b and D. Expert a aLaboratoire de Pathologie V6g6tale, INA P-G / INRA, 16 rue Claude Bernard, F-75 231 Paris, France bLaboratoire de G6n6tique Mol6culaire des Microorganismes, URA CNRS 1486, Biochimie 406, INSA de Lyon, 20 Avenue Albert Einstein, 69 621 ViUeurbanne, France
Abstract
Multiplication of Erwinia chrysanthemi pel mutants and pel gene expression (via the use of GUS fusions) were studied during the first hours following plant inoculation. The expression of pel::uidA mutants affected in iron assimilation was compared to the wild type background. The results showed that low iron condition encountered by bacterial cells during pathogenesis modulate the e• of pel genes. Moreover, the expression of peID::uidA was stimulated by the presence of iron chelators in the growth medium and the absence of functional chrysobactin mediated iron uptake.
1. Introduction The pathogenicity of Erwinia chrysanthemi 3937 on African violets involves at least five pectate lyases (PelA to PelE), one methyl pectin esterase (Pem), encoded by pelA to pelE, pem genes and an iron assimilation system mediated by chiTsobactin [ 1], (Fig. 1). Soft rot symptoms produced by E. chrysanthemi consist of a disorganisation of parenchymatous tissues following the release of bacterial pectinolytic enzymes. The diverse enzymes do not contribute equally to the virulence on a given host and their implication may vary according to the host considered. For instance, inactivation of pelE, pelD, pelA or pem in strain 3937 considerably reduces the virulence on African violets while mutations in pelB or pelC remain ineffective [2]. Pectinolysis is regulated by the transcriptional repressor KdgR, inactive in the presence of pectic inducers. Soft rot spreading depends on the efficiency of the iron uptake pathway mediated by the siderophore chrysobactin. Biosynthesis of the ferrichrysobactin outer membrane receptor (Fct) and of the chrysobactin precursor, i.e. the activated form of 2,3-dihydroxybenzoic acid, are encoded by an operon,fct cbsCEBA [3]. Furthermore, the occurence in strain 3937 of a second iron acquisition zystem, dependent of a siderophore designated achromobactin, has been recently demonstrated. The structure of achromobactin is still unknown and it is likely to be an iron ligand less competitive than chrysobactin. The ascA gene is involved in achromobactin synthesis. Ferriachromobactin uptake pathway utilises a specific ABC transporter referred to as the Cbr permease. Mutations in cbr locus, interrupting the transport of ferriachromobactin into the cytosolic compartment give rise to derepression of chrysobactin production [4-5].
876 KdgR is a well knownrepressor of pectate lyase genes. In kdgR mutants, pectate lyases are still inducible in the presence of pectin derivatives, suggesting the existence of other regulatory factors [6]. Pectinolyticenzymes ~'(~lant cellwalldegradation.) insaturated oligogalacturonides f
<~eatabolic_ inducers
pela._ petC~
~//~~~
~
peIA pelE pelD
activatpe%ruvate
pem
Iron and pel genes regulation?
m:;:::.:. ............:':':':::: ::i
I
acsA
~,,-.i . ~__~
I
cbrA B
C
D
~ v
I
~MV1
Cbrpermease
..... ............
F
i
i
I
f c t . cbsC E B A
I ilan~membr-L~ ! outermernbr~
1---, I Ferr,chr' !oba'ct,nI Chrysobactin ""~/ ~
Ac~omobactin] erriachromob~tin] low affinity
~
high affinity
Figure 1: Pathogenicfactorsof Erwinia chrysanthemi. Transcriptionnal activity of pel genes responds differentially to catabolic repression, growth phase, temperatureand nitrogen starvation. Iron limitationproved to be involved in the induction of severalof the pectinaseencoding genes [7].
877 (i) pelB, pelC and pelE are induced under iron limitation, i.e. when no iron is added to culture media [7]. (ii) in cbr mutants that produce chrysobactin independently of iron availability, pelB, pelC and pelE genes are no more sensitive to iron limitation [7]. (iii) cbr mutants produce delayed symptoms on African violets [7]. Plants represent an environment where iron is not directely available for bacteria: (i) a chrysobactin like compound was produced in diseased African violet leaves in a relative advanced stage of the soft rot symptom [8] (ii) strain 3937, harbouring lacZYA chromosomal fusions in the chrysobactin operon ~ct::lacZ) was inoculated on African violet plants. Expression of the fusion was detected in iron deplete medium and in planta 10 hours after plant inoculation [9] Does the regulatory effect of iron, observed under laboratory conditions, have a physiological significance in the soft rot disease caused by E. chrysanthemi strain 3937 on African violet ?
2. R e s u l t s 2.1. Activity of pel::uidA gene fusions in strain L37, after leaf inoculation To study the transcriptional activity ofpel and pem genes during pathogenesis, a culture of L37 cells harboring one of the peIA::, pelB::, peIC::, peID:: and pelE: uidA fusions was inoculated into the leaf parenchyma of African violet potted plants. GUS activity, produced by the bacteria present in extractable fluids, was assayed at 3 hour-intervals over a 72 h period after inoculation. The growth of pel mutants was compared with that of the wild-type strain.The growth rate of mutant and parental strains was similar during the first 48 h following inoculation. After this period of time, the growth of mutants was slightly reduced. The activity ofpel::uidA fusions fell roughly into threedasses of response (Tab. 1): (i) pelA::uidA activity was very limited throughout the growth period (ii) pelB::, pelC:: and pelE::uidA fusions were expressed at a moderate level. Induction of those genes was apparent after 9 h and maxima were reached 24 h after inoculation. (iii) high level of expression was observed with pelD::uidA. PelD::uidA appeared to be induced after only 6 h (Tab. 1) and reaching a maximum 60 h after plant inoculation. These results indicate that the differeni pectinase genes are not regulated in the same manner during the first stage of pathogenesis. 2.2. Activity of pelD::uidA and pelE::uidA gene fusions in mutants L37 cbrA21 and L37 kdgR, after leaf inoculation In-the light of the above data, we examined the incidence of the cbrA21 (chrysobactin siderophore produced constitutively) and kdgR mutations on the transcriptional activity of pel genes. We chose to study pelD: :uidA and peIE: :uidA that proved to be well expressed in planta and whose suceptibility to iron limitation is different: Table 2 shows the lack of any significant difference in pelE::uidA activity between L37 and L37 cbrA21. In contrast, peID::uidA failed to be induced in cbrA21 cells (Tab. 2). In L37 kdgR, the activity of peID::uidA remained unchanged over a period of 15 h following inoculation, after which a significant increase, with a maximum around 20 h, was observed. In addition, Table 2 indicates that the activity of a kdgR::lacZ fusion increased after 12 h post-inoculation, with a maximum after 15 h. This shows that between 6 h and 18 h after inoculation, pelD::uidA expression does not depend on KdgR control, but is sensitive to iron assimilation efficiency.
878 Table 1" Expression of pel::uidA fusions in L37 strain after inoculation onto African violets (noticed in hours). Specific GUS activity correspond to 109 CFU. Experiments were performed in triplicate and standard deviation is indicated between parenthesis. fusions
pelA::uidA pelB::uidA pelC::uidA pelD::uidA pelE::uidA
3 h
6 h
9 h
12 h
24 h
60 h
0.2 (0.0)
0.3 (0.1)
0.3 (0.1)
0.6 (0.4)
0.4 (0.0)
0.4 (0.1)
0.5 (0.4)
0.9 (0.6)
4.6 (3.4)
2.8 (1.s)
11.1 (2.2)
6.9 (2.3)
0.2 (0.2)
0.7 (0.7)
1.6 (1.1)
3.0 (2.3)
7.8 (1.7)
7.3 (2.9)
0.4 (0.5)
6.1 (4.9)
0.9 (0.3)
0.8 (0.5)
19,0 (15,0) 17.5 (7.2)
55.0 (20)
91 (10)
1.5 (1.0)
11.6 (4)
10.4 (5.0)
5.4 (4.0)
Table 2: Expression ofpeID and pelE::uidA fusions in strains L37, L37 cbrA21 and L37 kdgR after inoculation onto African violets. Specific GUS activity correspond to 109 CFU. Experiments were performed five times and standard deviation is indicated between parenthesis. genotypes
3 h
6 h
9 h
pelD::uidA 0.4 (0.5) 6.1 (4.9) 19.0 (15.0) pelD::uidA cbrA21 1.2 (1.2) 1.3 (0.9) 1.5 (0.2) pelD::uidA kdgR 4.1 (2.0) 6.9 (2.9) 8.9 (8.1) peIE::uidA 0.9 (0.3) 0.8 (0.5) 1.5 (1.0) peIE::uidA cbrA21 1.6 (1.0) 0.9 (0.3) 0.9 (0.4) kdgR::lacZ 26.0 (9.9) 38.5 (21.0) 30.0 (12.7)
12 h
18 h
24 h
17.5 (7.2) 47.0 (19.0) 55.0 (20) 2.4 (0.8)
5.9 (2.9)
5.9 (1.2)
24.1 (11)
113 (20)
72.4 (46)
5.4 (4.0)
11.1 (3.0)
10.4 (5)
1.5 (1.5)
4.6 (1.6)
8.5 (5)
67.3 (22.8) 130 (11)
79.6 (lo.3)
2.3. Control of pelD::uidA according to" the presence of Fe(III) chelators and to iron availability in intercellular fluids of African violets Iron inaccessibility in the medium was achieved by addition of EDDA (log FeL = 34) to Tris medium. The activity of pelD::uidA wasrecorded in wild-type and cbrA21 cells during their growth in Tris medium supplemented with FeC13 or with EDDA (Fig. 2A). The presence of EDDA appeared to stimulate pelD::uidA activity in both strains. Interestingly, deferration of Tris medium with Chelex, an iron-binding resin which can remove iron from relatively weak ligands (log FeL < 12), had no effect on expression of the fusion (data not shown). We concluded that the activity ofpelD::uidA increases only in severe conditions of iron starvation, i.e. when iron-scavenging agents are present. Leaf intercellular fluid harvested from healthy plants (IF) reflects the environmental conditions that may be encountered by bacterial cells during pathogenesis [9]. To examine whether the expression ofpeID:" relative to pelE::uidA was influenced by the iron status in IF, peID':uidA activity was recorded in bacterial cells grown under such conditions. Activity of pelD::uidA was analysed in wild-type and cbrE-1 (chrysobactin deficient mutant)backgrounds. In parental cells, pelD::uidA expression in IF decreased when previously supplemented with FeC13. In cbsE-1 cells, expression of pelD::uidA was higher than in parental cells and addition of FeC13 into IF was ineffective.
879 These data show that disruption of chrysobactin biosynthesis enhances the expression of
pelD::uidA. In addition, the lack of repression of the fusion by iron in chrysobactin-deficient cells indicates that the iron supplied cannot be internalized via the achromobactin dependent pathway. Hence, IF must contain strong iron-free ligands scavenging the Fe(III) supplied.
17 2 '
I
,
,,
i O 600nm
|
,,,,
,
!
--[-
1,0
8
iiiiii iiiiiii
Ii
0,8'
........................
0,6"
i-}:!:?i:i~i~::::?i-! .:_:__,_,__:_:_.~ ....
--y--
r~
it.ITI +Fe +EDDA Tris medium
l-Fe
iiiiii!ii!!ii! iiii!iiiiiiii
074"
~
i!iiii!il ii
-::-::-:-:-:-:-:-:-:-:-:........................
iiiii~iiiiii!!iiiii
|
iiii!i!i!ii!iiiiil!ii!!!!ii!!i!ii
Intercellular fluids of healthy plants
Figure 2: Expression of pelD::uidA fusion in strains L37 (open bars) and L37 cbsE-1 (filled bars) grown in: (A) Tris medium supplemented with FeC13 (20 I.tM) or EDDA and (B) intercellular fluids of healthy African violets, supplemented or not with FeC13 (20 l.tM). OD at 600 nm of the samples was 1.5. GUS activity is expressed per OD units.
3. Conclusion This work was initiated to elucidate whether the regulatory effect of iron on pel gene expression, observed under laboratory conditions, had a physiological significance in the soft rot disease caused by E. chrysanthemi strain 3937 on African violets. Regarding the prevalence of pectinolytic enzymes in the soft rot symptoms, it is noteworthy that the experimental model developed on African violets stresses the dynamic aspect of the disease and illustrates a number of points which have long been questioned. In wild type strain: globally, we found that all pel genes are induced after 9 hours following inoculation and concomitant with the expression of fct cbsCEBA, except for peID::uidA which is turned on earlier, i.e. after 6 h. Induction of chrysobactin operon, 10 hours after inoculation, and sensitivity of peID::uidA to several mutations affecting iron assimilation, converge to provide evidence that iron availability in the host plant controls pel gene expression. Furthermore, we noted that soft rot symptoms apparition is correlated with pel genes induction. The mutant L37 cbrA21 is affected as regards to its iron uptake pathway mediated by the siderophore achromobactin. Because this mutation results in derepression of the chrysobactin mediated iron transport pathway, the mutant is probably less susceptible to iron deprivation than wild-type cells are, when entering the host. This results in a delay in Pels production thus leading to delayed symptoms, as reported by Sauvage and Expert (1994).
880
4. Materials and Methods Two month potted plants of African violets were used [8]. The derivatives of E.
chrysanthemi strains L37 [10], L37 cbrA21 [4], L37 cbsE-1 [4] and L37 kdgR (harboring the kdgR mutation of strain A1077 [11]) used in this study were a set of mutants with a transcriptional fusion to one of the five pectate lyase genes with the reporter gene uidA [ 11]. Assays of 13-glucuronidase were performed as described by Sauvage and Expert [7]. Plant inoculation was performed as described by Masclaux and Expert [8]. Intercellular fluids of African violets were prepared as described by Neema et al. [9].
5. References 1 Bertheau, Y., Madgidi-Hervan, E., Kotoujansky, A., NguyenThe, C., Andro, T. and Coleno, A. 1984. Detection of depolymerase isoenzymes after electroporesis or electrofocusing 2 Boccara, M., Diolez, A., Rouve, M., and Kotoujansky, A. 1988. The role of individual pectate lyases of Erwinia chrysanthemi strain 3937 in pathogenicity on saintpaulia plants. Physiol. Mol. Plant Pathol. 33:95-104 3 Enard, C., Diolez, A., and Expert, D. 1988. Systemic virulence of Erwini chrysanthemi 3937 requires a functional iron assimilation system. J. Bacteriol. 170:2419-2426 4. Mah6, B., Masclaux, C., Rauscher, L., Enard, C., and Expert, D. 1995. Differential expression of two siderophore-dependent-iron acquisition pathways in Erwinia chrysanthemi 3937: characterization of a novel ferrisiderophore permease of the ABC transporter family. Mol. Microbiol. 18:33-43 6 Reverchon, S., and Robert- Baudouy, J. 1987. Regulation of the expression of pectate lyase genes pelA, pelD, and peIE in Erwinia chrysanthemi. J. Bacteriol. 169:2417-2423 7 Sauvage, C., and Expert, D. 1994. Differential regulation by iron of Erwinia chrysanthemi pectate lyases: pathogenicity of iron transport regulatory (cbr) mutants. Mol. Plant-Microbe Interact. 7:71-77 8. Masclaux, C., and Expert, D. 1995. Signalling potential of iron in plant-microbe interactions: the pathogenic switch of iron transport in Erwinia chrysanthemi. Plant J. 7:121128 9 Neema, C., Laulh~re, J.-P., and Expert, D. 1993. Iron deficiency induced by chrysobactin in Saintpaulia ionantha leaves inoculated with Erwinia chrysanthemi. Plant Physiol. 102:967-973 10 Hugouvieux-Cotte-Pattat, N and Robert-Baudouy, J. 1985. Lactose metabolism in Erwinia chrysanthemi. J. Bacteriol. 162:248-255 11 Hugouvieux-Cotte-Pattat, N., and Robert-Baudouy, J. 1992. Analysis of the regulation of the pelBC genes in Erwinia chrysanthemi 3937. Mol. Microbiol. 6:2363-2376
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
881
Regulation of Polygalacturonases in Two Isolates of Fusorium oxysDorum f. sp. radicis IvcoDersici (FORL). B. Patifio ~, M.L. Posada ~, M.T. Gonz~ez-JaEn 2, M.J. Martfnez 3 and C.
V~quez t . ~Dpt. of Microbiology III. UCM. 28040 Madrid. Spain. 2Dpt. of Genetics. UCM. 28040 Madrid. Spain. 3CIB. CSIC. Madrid. Spain.
Abstract In the present work we studied the regulation of polygalacturonases (PG) in two FORL isolates (r2 and r~3) grown on different carbon sources. Polygalacturonases were strongly induced by apple pectin, citrus pectin and polygalacturonic acid, apple pectin and citrus pectin being the best inducers. with galacturonic acid the enzyme activity started later. Although a band with similar mobility to purified PG was visible on SDS-PAGE, no detectable extraceUular polygalacturonase activity was found in a medium containing glucose as the carbon source. Specific antibodies were obtained against a purified polygalacturonase from FORL. To determinate the inmunological specificities to the antisera, the ability of the antibodies to crossreact with extaceUular proteins from apple pectin and glucose crude culture filtrates was examined by SDS-PAGE and inmunoblotting. The anti-PG detected one band corresponding to the molecular mass of the purified enzyme preserit in PG-inducing and non-inducing conditions. Simultaneously, we studied the polygalacturonase gene expression by the RNA analysis through reverse transcription and subsequent amplification with specific oligonucleotides, and hybridization with a PG probe generated by PCR were also done. The RNA was obtained from FORL r2 and r~3 grown in PG inducing and non-inducing conditions.
INTRODUCTION
Fusarium oxysporum Sehlecht. f. sp. radicis lycopersici Jarvis &
882
Shoemaker (1) (FORL) is a soilborne pathogen that colonizes the roots of tomato plants (Lycopersicon esculentum Mill.) and causes crown and root rot disease. This fungus enters the roots through wounds and colonizes the tissues through xylem vessels, where it grows abundantly (2). Root colonization is accompanied by a large amount of damage and irreversible cell wall alterations including the pronounced disruption and loss of inner layers of middle lamellae, that leads most often to root tissue maceration (2), which suggests the production of pectic enzymes by FORL. These symptoms are considered to be intermediate between those of cortical rots and true wilts, which are associated with other formae speciales of this species. Evidence of pectic enzymes secreted by FORL species has been obtained (3). The total proteins of FORL subjected to isoelectric focusing were resolved into several bands widely distributed between pl 5.9 and 7.45. FORL (rz) presented one mayor band of activity with a pI of 7.0; however, FORL (r~3) presented one characteristic and principal band at 7.45, slightly more basic than these reported by others authors (4). The major activity of FORL (r~3) was purified by gel filtration and ion exchange chromatography. It had a Mw of 68 kDa, similar to that reported for other fungal exopolygalacturonases, Fusarium oxysporum f.sp. melonis (5), Fusarium oxysporum f.sp. ciceri (6) and Sclerotinia sclerotium (7), and higher than endopolygalaeturonases secreted by other plant pathogens, which are between 28 kDa and 43 kDa, a pH optimum of 5.6 and a optimum temperature of 60 ~ This activity was inhibited by calcium ions and it showed as exoenzyme (8). In the present work we study the influence of growing conditions in the control of polygalacturonase synthesis. It is important to know how polygalacturonase synthesis may be affected by growth conditions, since variation in enzyme number and activity has been observed in vitro depending on culture conditions (9,10). We used two diferents isolates of FORL (r2 and r~3) cultured on different carbon sources analyzing: enzyme activity, presence of extracellular polygalacturonase (using antibodies), general gene expresion pattern (by in vitro translation) and specific polygalacturonase gene expression.
MATERIALS AND METHODS
Organisms and culture conditions Two isolates of FORL (r2 and r~3) were provided by Dr. Tello (INIA, Madrid) from diseased tomato crops grown on the Spanish Mediterranean coast (11). Both isolates were maintained as stock cultures on potato-dextrose agar
883
(PDA) slants at 4~ The isolates were cultured in 100 ml Erlenmeyer flasks containing 20 ml liquid medium (12) with different carbon sources. The cultures were inoculated with mycelial disks cut from the margins of 7 day-old-colonies and incubated at 25~ under static conditions. The mycelia, of six days old culture, were harvested by filtration through Whatman paper n ~ 1 and kept at-80~ for RNA assays. The culture filtrates were used for enzyme assays. The substrates used as carbon sources were glucose (1.5 %, w/v) (Merck), sucrose (3 %, w/v) (Merck), apple and citric pectin (1%, w/v) (Fluka), polygalacturonic acid (1.5 %, w/v) (Sigma) and galacturonic acid (1.5 %, w/v) (Sigma). Enzyme activitv assays Polygalacturonase activity (PG) (EC 3.2.1.82) was assayed by following the release of reducing groups from 0 . 1 % sodium polypectate in 50 mM sodium acetate buffer pH 5.2, according to methods described by Somogyi (13) and Nelson (14). Enzyme and substrate controls were carried out. One unit of enzymic activity was defined as the amount releasing 1 #mol of galacturonic acid in 1 min. Estimation of glucose and protein concentration Glucose concentration was measured according to the Werner Method (15), with a Boehringer glucose oxidase kit. Protein concentration was determined by the Bradford method (16) using bovine serum albumin as a standard. Gel Electrophoresis SDS-PAGE was performed by the method of Laemmli (17), with 7.5 % polyacrylamide. Preoaration and characterization of Antibodies Polyclonal Antibodies against FORL r6 purified polygalacturonase were raised in white rabbits. For the first immunization 200 #g of purified protein in 300 #1 of distilled water was mixed with 200 #1 of PBS and 500 #1 of complete Freund's adjuvant and injected intramusculary into the leg. Two subsequent intramuscular injections, each containing 300 #g of protein in 1 ml of incomplete Freund's adjuvant were given at 1 month intervals. Finally, the rabbit was bled 1 week later. The antisera, separated from blood by incubation at 37 ~ were stored in 1 ml fractions at-20 ~ Specificity of the antisera was assessed by Western blotting. Electrophoretically separated proteins from culture filtrates were transferred to 0.45 #m nitrocellulose membranes. After transfer of proteins, membranes were
884
allowed to incubate in skimmed milk 5 % in PBS buffer, for 1 hour at room temperature. Nitrocellulose membranes were then incubated overnight with PG antiserum diluted 1/500 in PBS. Afterwards the membranes were rinsed twice with PBS and incubated for 2 hour in mouse peroxidase-eonjugated goat antirabbit inmunoglobulins diluted 1/500 in skimmed milk PBS. Antigen-Antibody reactions were visualized by using 10 mg of DAB and 15 mg of 4-chloro-1naphtol in 10 ml of methanol mixed with 40 ml of PBS containing 10 #1 of H202
RNA isolation Total RNA was isolated from mycclia cultured 6 days on PG-inducing and non- inducing conditions (apple pectin and glucose, respectively). The RNA extraction was performed according to the phenol-SDS method combined with selective precipitation using LiCI. RNA concentration was estimated by absortion at 260nm. R N A in vitro translation The wheat germ system (Promcga) and 35S-Mcthioninc were used for the in vitro translation, according to the manufacturer directions. 10 #g of total RNA were used for each reaction. The products were separated by monodimcnsional clcctrophorcsis (PAGE) with SDS (17). After fixation, gels were trcatexi with En3hance (Dupont) and dried. Gels were autoradiographod using X-ray film (Kodak X-OMAT AR). Exposure was made at -80~ for 6 days.
eDNA svnthcsis, amvlification and hvbridization. Total RNAs were used for reverse transcription-polymcrasc chain reaction. The cDNAs were synthesized using Pcrkin Elmer GcncAmp RNA PCR and RNA PCR Core kits the according to manufacturer protocols. Subsexlucntly the cDNAs were amplified using PCR with the 5' upper oligonuclcotidc A T C T ~ C A T G T C A T T G A and the 3' lower oligonucleotide GGTCGGCTTTCCAGTAGG based on the Polygalacturonasc sequence of Fusarium moniliforme (18). In all amplification experiments, oligonuclcotide primers were used at a f'mal concentration of 1 #M. PCR was performed for 35 cycles (1 min at 94~ - denaturation-, 1 min at 50~ -, 2min at 72~ -) followed by 5 min at 72~ of f'mal extension. The amplification products were clcctrophorcsed on agarosc gels. Blots were performed according to protocols supplied with Hybond m- N-nylon membrane (Amcrsham). A non-radioactively labeled DNA probe of 742bp was used for the hybridization. The probe was obtained from a gcnomic DNA from FORL r 2 using the same oligonuclex~idc above mentioned and the same conditions. The
885
probe was labelled with DIG dUTP by PCR using genomic DNA from FORL r 2 and the same amplification conditions and primers described previously. The membranes were hybridized according to the protocols supplied with the DIG Luminiscent Detection for Nucleic Acids Kit (Boehringer Mannheim) and they were exposed for 50 min at room temperature to Kodak X-OMAT AR film.
RESULTS
Polygalacturonases were strongly induced in FORL by apple pectin, citric pectin and polygalacturonic acid. When FORL was grown on glucose as carbon source no extracellular PG activity could be detected. Galacturonic acid did not look like a good inducer of PG since the increase of enzyme activity was delayed in comparison with the other carbon sources, after the 5th day of incubation, and also showed lower values (fig. 1). ulml 50-
40
/,.,re
30 20 I0 0
I 0
~" 1
2
3
4
5
6
7
t (days)
Figure 1. Time course of PG-production by FORL in culture media containing apple pectin (o), citric pectin (~), polygalacturonic acid (a), monogalacturonic acid (g-), glucose (~.) and sucrose (~). Since the best inducer for the polygalacturonase activity was apple pectin, the experiments analyzing expression were made in this conditions and as with glucose PG-activity could not be detected, it was used as control.
886
Fig-2 shows the patterns of total protein obtained by SDS-PAGE from culture filtrates ofmicelia growing on inducing (apple pectin) and non-inducing (glucose) conditions. All the lanes loaded contained the same amount of total proteins. A higher volume of culture filtrates was necessary to obtain this amount of protein in glucose than in apple pectin. All the protein patterns obtained were similar. A band of about 68 kDa was dearly seen in all the cases with no diferences in migration. It was also the main band in each lane. The intensity of the band was higher in inducing conditions than in non-inducing conditions. This band could correspond with PG, as it was later confirmed using antibodies raised against PG.
1
•l
23
~
4
:~
9i
56
m
~i 84184 ....
~
Figure 2. SDS-PAGE of a six days old culture. Lane 1 and 6 molecular mass standard, lane 2 and 4 apple pectin culture medium, lane 3 and 5 glucose culture medium. Lanes 2 and 3 to isolates r13, lanes 4 and 5 to isolates r2. The policlonal antibodies raised against the main band of PG also reacted with the 68 kDa band mentioned above. Total proteins from culture f'dtrates were obtained from both conditions and analyzed by SDS-PAGE. The gel was subsequently blotted and the filter hibridized with the PG antibodies (Fig-3). The results confirmed the presence of a extracellular PG with similar migration in inducing and non-inducing conditions.
887
1
2345
,".~
o
.~
9.
,
~.,o
..
9
.i". ~.~
Figure 3. Antigen-Antibody assay. Lane 1 molecular mass standard, lanes 2 and 4 glucose culture medium, lanes 3 and 5 apple pectin culture medium. Lanes 2 and 3 to isolate r2, lanes 4 and 5 to isolate r~3. In order to know if the presence of mature proteins correlated with the presence of mRNA, two differents experiments were carried out. In vitro translation of total mRNA isolated from mycelia growing in inducing and noninducing conditions showed similar patterns in both situations except for two bands present only in the former, between 29 and 33 kDa (Fig-4). These bands could correspond to the polypectate lyases. The patterns of both situations showed a common band, more intense in micelia grown in pectin, near 50 kDa wich could correspond to the deglicosilated PG of FORL with a Mr of 50 kDa.
1 ,~.~.
2 9
9 ..
3
4
. .
Figure 4. In vitro translation pattern of RNA. Lanes 2 and 4 from apple pectin
888 culture, lanes 1 and 3 glucose culture medium, lanes 1 and 2 correspond to isolate r~3 and lanes 3 and 4 to isolate rz. The second experiment was carried out with the same RNA isolated from mycelia growing on presence of glucose and apple pectin, cDNAs were obtained from those RNA by reverse trancription and used as template for PCR assays. Specific oligonucleotide primers for PG gene were used in the amplification reaction. The Fig-5 shows the results of this amplification experiment.
1
23
4
56
7
8
9
Figure 5. 1% agarose gel showing PCR amplification products for PG cDNA obtained from mRNA isolate. Lanes 1, 2, 4 and 6 apple pectin culture medium, lanes 3, 5 and 7 glucose culture medium, lanes 1, 2 and 3 to isolate r13 and lanes 4, 5, 6 and 7 to isolate r2. Lane 8 corresponds to the amplification of the 742 pb probe cloned in the vector PCR TM (In-vitrogen) digested with Eco RI and lane 9 ~. digested with PstI.
889
A subsequent blot of this gel was performed and hibridized with a probe of 742 bp for PG gene of FORL r2. Fig-6 shows the picture of the autorradiography obtained. The results indicate the presence of mRNA for PG gene in inducing as well as non inducing conditions.
12345
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m m m
.9 ~ ~
9 ~'
9 .~
.'.
9
m
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m
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, 9
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Figure 6. cDNAs amplification products hybridized with a DIG-labeled 742 bp probe. Lane 1, 2, 4 and 6 apple pectin culture medium, lanes 3, 5 and 7 glucose culture medium. Lanes 1, 2 and 3 to isolate r~3 and lanes 4, 5, 6 and 7 to isolate r2. Lane 8 corresponds to the hibridation with the 742 bp probe cloned in the vector PCR TM (In-vitrogen) digested with Eeo RI and lane 9 ~, digested with Pstl. Differences between both FORL isolates, r~3 and r2, were not observed in any of the experiments presented in this work.
890 DISCUSSION
It has been observed strong induction of PG activity by citric pectin, apple pectin and polygalacturonic acid, while galacturonic acid did not behave like a good inducer. This fact agree with Guevara (19) which found that unrestricted supply of galacturonic acid did not increased PG activity in FORL. However, galacturonic acid was good inducer when supplied in a restricted way. Blais (3) found, however, induction of the PG in presence of galacturonic acid this would be due to the organism or small variation on the culture conditions. Extracellular PG activity was not detected in cultures on glucose, a similar situation to that of Fusarium moniliforme (18). In contrast, all our data confirm the presence of the protein and the mRNA of PG in non-inducing conditions; our assays did reveal no differences between FORL PG growing on both conditions, regarding migration or other detectable characteristics that could justify the presence of the enzyme and its lack activity. It is posible that a very low concentration of the enzyme results in undetectable activity in enzyme assays. The detection of mRNA of PG gene(s) by cDNA-PCR amplification assays in non-inducing conditions would agree with the hypothesis of probable basal levels of mRNA and discard a de novo induction in presence of the inductor. However, a substantial increasing of mRNA and proteins and, subsequently, of activity were observed in presence of pectine. We can conclude that our results are compatible with a model for the control of PG synthesis at transcriptional level in response to the inducer but with certain levels of protein, apparently similar to that showing PG activity, and its corresponding mRNA in non-inducing conditions. Further studies in order to quantify the relative amount of these basal levels are on a course.
Acknowledgements This work was supported by DGICYT, Proyecto PB92-0205.
REFERENCES 1 W.R. Jarvis and R.A. Shoemaker, Phytopathology, 68 (1968) 1679. 2 P.M. Charest, G.B. Ouellette and F.J. Pauzi~, Can. J. Microbiol., 62 (1984)
891
1232. 3 P. Blais, P.A. Rogers and P.M Charest, Exp. Mycol., 16 (1992) 1. 4 N. Fern~_ndez, B. Patifio and C. V~quez, Mycol. Res., 97(4) (1993) 461. 5 M.J. Martfnez, M.T. Alconada, F. Guill6n, C. V~izquez and F. Reyes,FEMS Microbiol. Lett., 81 (1991) 145. 6 E. P6rez-Artes and M. Tena, J. Phytopathol. 124 (1989) 39. 7 Ch. Riou, G. Freyssinet and M. Fevre, Appl. Environ. Microbiol., 58 (1992) 578. 8 C. V~quez, B. Patifio and M.J Martinez, FEMS Microbiol. Lett., 110 (1993) 191. 9 W. Pagel and R. Heitefuss, Physiol. Mol. Plant Pathol., 37 (1990) 9. 10 G. De Lorenzo, G. Salvi, L. Degr~i, R. D'Ovidio and F. Cervone, J. Gen. Microbiol., 133 (1987) 3365. 11 J.C. Tello y A. Lacasa, Bol. San. Veg. Plagas 14 (1988) 307. 12 C. V~quez, M.J. Martfnez, R. Lahoz and F. Reyes, FEMS Microbiol. Lett., 37 (1986) 227. 13 M. Somogyi, J. Biol. Chem., 160 (1945) 61. 14 H. Nelson, J. Biol. Chem., 153 (1944) 375. 15 W. Werner, H.G Rey and H. Wielinger, Z. Anal. Chem., 252 (1970) 224. 16 M.M Bradford, Anal. Biochem., 72 (1976) 248. 17 M.K Laemmli (1970). Nature, 227 (1970) 680. 18 C. Caprari, A. Richter, C. Begmann, S. Lo Citer, G. Salvi, F. Cervone and G. De Lorenzo, Mycol. Res. 97 (4) (1993)497. 19 A. Guevara, M.T. Gonz,~lez-Ja6n, P. Est6vez (unpublished),
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J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
893
Endo-pectinase production by intraspecific hybrids of Aspergillus sp. CH-Y-1043 obtained by protoplast fusion S. Solis, E. Flores-S,~nchez and C. Huitr6n. Department of Biotechnology, Institute of Biomedical Research, National University of Mexico, UNAM. A.P. 70228, Mexico City, D.F. 04510
Abstract Protoplast fusion induced by polyethyleneglycol and Ca 2+ was carried out between two auxotrophic mutants of Aspergillus sp. CH-Y-1043. The hybrids obtained showed significant differences in endopectinase activity and morphology compared to the prototrophic strain. Strains grown on lemon peel showed production improvement with respect to the parental strain. Since H15 hybrid showed up to 90% higher endopectinase production than the wild type CH-Y-1043, kinetics of enzyme production in Fernbach flasks and Fermentor (14L) by H15 were determined.
1. INTRODUCTION Microbial pectinases have a great number of applications in food industry such as the preparation of fruits and vegetables pur~es and olive oil extraction. Their use is important in wine and fruit juice technology since pectinolytic enzyme action results.in greater yields of extracted juices and reduces the filtration time (1-2). The presence of endopectinases is essential for these applications. Commercial preparations of pectic enzymes mainly come from fungal sources, particularly from the genus Aspergillus. We isolated a strain of Aspergillus sp. CH-Y-1043 which grows better at 37~ and is capable to produce extracellularly variable amounts of inducible endo, exo-pectinases and pectin lyases when grown on pectin and a wide variety of materials containing pectin (3-6). Also, the presence of a constitutive conidial and cell bound exo-pectinase has been identified in this fungus (7). The specific activity of cell free filtrates of Aspergillus sp. CH-Y-1043 is two times higher than the best commercial pectinases preparations and the yield of clarifying apple juice by both is similar. Factors influencing enzyme production, such as carbon and nitrogen source, temperature and pH have been studied (6-9). Our work on regulatory aspects demonstrated induction by galacturonic acid and catabolic repression by glucose (9). Recently we have been working on the isolation of protoplasts from this and other pectinolytic fungi (10) and have used them for genetic fusion through intra and interspecific
894 protoplast fusion, because we are interested in improving endopectinase production. Obtention of hybrids by intra and interspecific protoplast fusion has shown higher yields of enzymes which are biotechnologically interesting in the genus Aspergillus (11-13). In this work we describe intraspecific protoplast fusion in Aspergillus sp. CH-Y-1043 mutant strains and the evaluation of endopectinolytic enzyme production by hybrids when were grown on lemon peel.
2. MATERIALS AND METHODS 2 . 1 . Strains.
Aspergillus sp. CH-Y-1043 was used as the prototrophic parental strain. The auxotrophic mutants A200 ade- (an adenine-requiring mutant) and A400 pyr- (a pyridoxine requiring mutant) were isolated from the parental strain by treatment with N-methyl-N'-nitronitrosoguanidina (NTG) as described previously (10). 2 . 2 . Media and culture conditions. Parental cells were routinely grown in potato dextrose agar (PDA) at 37~ and the mutant strains in complete medium (CM) containing (w/v): 2% glucose, 0.3% yeast extract and 0.3% bactopeptone. Minimal medium (MM) contained (w/v): 2% glucose, 0.1% K2HPO4, 0.05% MgSO4.7H20, 0.05% KCI, 0.001% FeSO4, 0.3% NaNO3, pH 4.5. CM and MM were solidified with 2% bactoagar. Enzyme production medium in Erlenmeyer flasks (EP1) containing: 0.2% (NH4)2SO4, 0.2% KH2PO4 and 0.2% K2HPO4 and 1% lemon peel as sole carbon source (sterilized separately). The initial pH was 3.0. The medium for enzyme production in fermentor and Fernbach flasks (EP2) contained: 0.4% (NH4)2SO4, 0.05% K2HPO4, 0.05% KH2PO4, 3% lemon peel and pH 2.8. 2 . 3 . Protoplast lation and fusion. The method and conditions employed for protoplast isolation and regeneration have been detailed elsewere (10). Fusion experiments were carried out with 106 protoplasts of two auxotrophs (ade-, pyr-) which were mixed and centrifuged 10 min at 2000 rpm. Pelleted protoplasts were resuspended in 1 ml of a solution containing (w/v) 30% polyethyleneglycol 3335 (PEG) and O.01M CaCl2 in O.05M glycine-NaOH buffer, pH 7.5. After 10 rain at room temperature, the suspension was centrifuged 10 rain at 2000 rpni. The pellet was resuspended in osmotic stabilizer solution (0.7M KCI) and serial dilutions were plated for regeneration on MM and CM containing 0.7M KC1. 2 . 4 . Enzyme production conditions. Experiments in 500 ml Erlenmeyer flasks and Fernbach flasks contained 200 ml and 1 L of EP1 and EP2 medium respectively. Inocula added to these cultures was 2 ml of spore suspension (5.0 optical density at 540 nm) for each 100 ml EP medium. All cultures were grown at 37~C in a shaking incubator (New Brunswik Sci. Co., USA), at 200 rpm. Then 10 ml of sample were withdrawn each 24 h during fermentation and immediately filtered through Millipore membranes of 0.45 ~m pore size; these cell-free filtrates were used for enzymatic assays and extracellular protein determinations by the Lowry method (14). Experiments in the 14 L fermentor (Microgen Fermentor New Brunswik Sci. Co., USA) were carried with 10L of fermentation medium EP2 and inoculum added was 1L of mycelium grown 24 h in
895
Fernbach flask with the same medium (EP2). Temperature was 37~ agitation was 1 vvm and 200 rpm respectively.
Aereation flow and
2 . 5 . Enzymatic assay Endo-pectinases were determined by reduction of viscosity of a 1% (w/v) pectin solution at 30~ and pH 4.2 using an Ostwald viscosimeter as previously described (9). One unit was defined as the amount of enzyme which reduced the initial viscosity of the solution by 50%.
2.6. Extracellular protein. Protein concentration was determined in cell free filtrates, according to the Lowry method (14) with bovine seric albumin (BSA) as standard.
3. RESULTS AND DISCUSSION Strains used in this study and characteristics of the mutants with respect to the parental strain are shown in Table 1. Although both A200 and A400 strains displayed a poor sporulation, their growth was different, A200 showed faster growth. Protoplasts were obtained from A200 and A400 mutants using Trictzoderma harzanium lytic enzymes. As shown in Table 1 the protoplast yield from A200 was 1.4 x 106 ml-~, this value was higher with respect to the protoplast number obtained from A400 mutant (0.5 x 106), in spite of incubation time of A200 mycelia being shorter. Protoplasts from mutants ade- and pyr- were fused with the addition of PEG and Ca2+ and many colonies developed after 5 days of incubation on MM plates with 0.7 M KCI, probably because they complemented auxotrophic requeriments. Of the total, twenty six hybrids were assayed for endo-pectinase production on lemon peel, because we are interested in the enzyme production from this raw material. The relative activity of the hybrids with respect to Aspergillus sp. CH-Y-1043 is presented in Table 2. The highest increases (up to 46%) were observed inHS, H6, H15, H l l , H13, H14, H10 and H25 hybrids. In some hybrids, lower values of endopectinases production with respect to parental strain were obtained.
Table 1 Characteristics of parental strain, mutants and conditions for protoplast isolation. protoplast isolation
Strains
Aspergillus CH-Y-1043 A200 A400
requirement sponllation
prototroph pyridoxine adenosine
good poor poor
growth
good good very slow
extracel, pigment
-brown greenish
mycelial age time yield (h) ( h ) 106ml"!
13 22
0.5 1-2
1.4 0.5
Protoplasts were isolated using 2 nag ml"~ Trichoderma harzanium lytic enzymes and 0.7 M KCI as osmotic stabilizer in 0.05 M phosphate buffer
896
T a b l e
2.
Comparison of relative endopectinolytic activity production between parental strain and hybrids strains ENDO-P(%) strains ENI~-P (%) strains ENDO-P(%)
Aspergillus CH-Y-1043 HI H2 H3 H4 H5
100
I-I6 II7 H8 H9 HI0 HI 1 HI2
84 75 96 78 136
134 118 91 118 146 132 54
H 13 I-I14 Hi5 H 16 I-t17 HIS HI9
136 139 143 92 112 136 90
strainsENDO-P(%) I-I20 H21 H22 I-I23 I-I24 H25 1-126
90 115 72 118 92 136 66
Determinations were carried out in cell free filtrates obtained at 96 h of fermentation on 1% lemon peel.
Four intraspecific hybrids were finally selected and they were grown to obtain the kinetics of endopectinases production and extracellular protein content. The course of endopectinase production by Aspergillus sp. CH-Y-1043 and hybrids during their growth on 1% lemon peel is s h o w n in Figure 1.
_
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_
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_ it15~
.
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_
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-
-
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lo 1 2345
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,%
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,,
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~
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TIME (DAYS) Fig. 1 Comparison of endo-pectinase production between hybrids and parental strain grown on 1% lemon peel. In the parental strain enzymatic activity reached the m a x i m u m value at 96 h. The hybrids H5, H15 and H10 showed good activity compared to Aspergillus sp. CH-Y-1043. Also, a difference with respect to parental strain was detected, a slight drop of activity in hybrids at 120 h. This effect was not due to changes of the pH during fermentation because it was similar in all cultures. The causes of this response in hybrids are unknown. More experiments for to evaluate the presence of proteases in the culture m e d i u m should be performed. Table 3 presents the data of the higher levels of endopectinases produced by
897
hybrids, extracellular protein and specific activity. The optimal strain was HI5, which presented an increase of 90% with respect to the parental strain. Protein content in cell freefiltrates was also higher in hybrids and the specific activities were increased mainly in the H15 strain. Table 3.
Increases in endopectinolytic activity in hybrids with respect to Aspergillus sp. CH-Y-1043 Protein (rag ml l )
Strains
ENDO- P (U ml")
relative increase (%)
specific activity (U mg prot"l )
Aspergillus
0.310
27.17
-
87.36
CH-Y-1043 H5 H6 HI2 HI5
0.451 0.344 0.414 0.441
40.8 32.0 37.9 51.6
50 40 39 90
90.5 92.9 91.5 116.0
Determinations were carried out in cell free filtrates obtained at 96 h of fermentation on 1% lemon peel.
Due to the potential for endo-pectinase production shown by the hybrid H15 on lemon peel, studies on fermentor using 3% lemon peel were performed. As shown in Figure 2A, m a x i m u m endopectinolytic activity was 80.0 U ml -l which is higher (60%) than the activity produced by hybrid H15 in 500 lnl Erlenmeyer flasks we had previously observed in
120
A
100
r-~ :Z: r,..)
o., o z
8O 60 40
r~
20 I
12345
I
I
I
12345
TIME (days).
Fig. 2 Endo-pectinase production by the hybrid HI5 grown on 3% lemon peel in (A) 14 L fennentor mid (B) Fembach flask (2L)
898
Aspergillus sp. CH-Y-1043 an increase of endopectinase production when substrate concentration was increased up to 3% (5). On the other hand, simultaneous to the production in fermentor, kinetics of endopectinase in other Fernbach flask with the same medium and conditions of those used as inoculum for the fermentor was determined. In this case (2B) we found a maximum endo-pectinolytic activity of 119 U ml-1 which represent 132% higher than in Erlenmeyer flasks and 49% more than in the fermentor. It is important to mention the high viscosity problems observed at the beginning of fermentation in the fermentor, result in limitation of oxygen transference and then lower endo-pectinase production. However more experiments should be done about this point. Intraspecific hybrid H15 is regarded as a useful strain exhibiting high endopectinase production from agroindustrial byproduct compared to parental strain Aspergillus sp. CH-Y-1043. Also, intraspecific hybrids from Penicillium caseicolum have been reported which produce twice the lipolytic activity than the parental strain (15) and in Aspcrgillus sojae hybrids were obtained with high protease and glutaminase activity (16). Protoplast fusion, like intraspecific fusion, has been shown in this work, to be adequate for strain improvement of pectinolytic fungi. 4. ACKNOWLEDGEMENTS The authors express gratitude to Angeles L6pez for secretarial assistance and I. P6rezMontfort for traslation of the manuscript.
5 . REFERENCES
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
W.M. Fogarty and C.T. Kelly. In Microbial Enzymes and Biotechnology. W.M. Fogarty (ed.)Appl. Sci. Publishers London. 1983. W. Pilknik and E.M. Robmouts. In Enzymes in Food Processing. G.G. Birch, N. Brakenbrough and K.J. Parker (eds.) Appl. Sci. Publishers London. 1981. S. Saval and C. Huitr6n. Use of enzymes in food technology. P. Dupuy (ed.) Technique et Documentation Lavoisier. 1983. C. Huitr6n, S. Saval and M.E. Acu~a. Ann. NY. Acad. Sci., 434 (1984) 110. G. Larios, J.M. Garcia and C. Huitr6n. Biotechnol. Lett., 11 (1990) 729. L. Delgado, B. Trejo, C. Huitr6n and G. Aguilar. Appl. Microbiol. Biotechnol. 39 (1993) 515. G. Aguilar and C. Huitr6n. FEMS Microbiol. Left. 108 (1993) 127. G. Aguilar, B. Trejo, J. Garcfa and C. Huitr6n. Can. J. Microbiol. 37 (1991) 912. G. Aguilar and C. Huitr6n. Enzyme Microbiol. Technol., 9 (1986) 541. S. Solis, M. E. Flores and C. Huitr6n. Lett. Appl. Microbiol. 1996 in press. J.F. Peberdy. Mycol. Res. 93 (1989) 1. S. Ushijima, T. Nakadai and U. Kinji. Agric. Biol. Chem. 51 (1987) 2781. K. Ogawa, M. Tsuchimochi, K. Taniguchi and S. Nakatsu. Agric. Biol. Chem. 53 (1989) 2873. O.H. Lowry, N.J. Rosenbrough, A.L. Tart and R.J. Randall. J. Biol. Chem. 193 (1951) 265. P. Reymond, P. Veau and M. Fevre. Enzyme Microb. Technol. 8 (1986) 45. S. Ushijima. In Biotechnology Handbook. J.E. Smith (ed.) Plenum Pres New York. 1993.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
899
Candida boidinii- a new found producer of pectic enzymes complex. Eva Stratilov~i, Emilia Breierov~i and Ren~ita Vadkertiov~i
Institute of Chemistry SAS, Ddbravsk~i cesta 9, 842 38 Bratislava, Slovakia
Abstract
Candida boidinii is a further yeast producer of pectic enzymes complex. The production is induced by the presence of pectin as a C-source in the medium; the primary methabolic path is the utilization of methanol and the secondary the utilization of pectate chains. The pectic enzymes were bound on the cell walls or released on the cultivation medium. The main enzyme of pectic complex, polygalacturonase, was briefly characterized and the possibility to influence the production of its multiple forms discussed.
1. I N T R O D U C T I O N
The production of pectic enzymeshave been thoroughly studied and widely reported in plants, fungi and bacteria, because of their role in plant development, plant desease and their utilization in the food industry. The production, and hence the study, of this kind of enzymes is not so extensive in yeast. Only few yeast species producing these enzymes are known; these include Saccharomycesfragilis (synonym: Kluyveromyces marxianus) (Luh and Phaff, 1954; Wimborne and Rickard, 1978; Lim. et al., 1980, Barnby et al., 1990), Kluyveromyces lactis (Murad and Foda, 1992), Candida tropicalis (Luh and Phaff, 1951), Cryptoccocus albidus (Federici, 1985), Rhodotorula rubra (Vaughn et al., 1969), Geotrichum lactis (Pardo et al., 1991), Candida macedoniensis (Call et al., 1985), and Saccharomyces cerevisiae (Blanco et al., 1994; Gainvors et al., 1994). This study reports on the production of pectic enzymes and partial characterization of polygalacturonases produced by Candida boidinii. Candida boidinii belongs to the so called methylotrophic yeasts with famous utilization of methanol. Pectin, the natural substrate of peetie enzymes complex, can serve for mieroorganisms as a C - source by two different ways: after deesterification with peetinesterase as methanol and after hydrolytic eleavage with
900 polygalacturonase as D-galactopyranuronate units. Our results should also contribute to the studies of microorganisms adaptibility.
2. M A T E R I A L AND M E T H O D S
Candida boidinii (CCY 29-37-13) was obtained as an isolate from contaminated column of immobilized polygalacturonase, where 0.5% sodium pectate in 0.1 M acetate buffer, p H 4.6 was used as a substrate. Four other strains for comparison were obtained from Culture Collection of Yeasts, Institute of Chemistry (strains CCY 29-37-1, CCY 29-37-2, CCY 29-37-8, CCY 29-37-12). Candida boidinii was cultured at pH 3.51, 5.49 and 7.01, respectively. Czapek's Dox medium with citrus pectin (GENU Pectin, Denmark), sodium pectate or citrus pectin with 20% of D-galactopyranuronic acid (Fluka, Switzerland) as a carbon source were used. The growth curves were performed by measuring the optical density (OD) at 660 nm. The yeasts were removed by centrifugation from the cultivation medium and disrupped using quick freezing at -196 ~ in liquid nitrogen in 0.1 M acetate buffer pH 4.6. The cell walls were separated by centrifugation and the present proteins released by 1.5 M NaC1. The extracellular, cytosolic and wall-bound protein complexes were than obtained by the precipitation with ammonium sulphate (d.s.) and ethanol (1:4) followed by desalting on Sephadex G-25 Medium column (Pharmacia, Sweden) after 10 days of cultivation. The malt agar (10 ~ served as maintainanee medium. Polygalacturonase activity was assayed at 30 ~ in 0.1 M acetate buffer by measurement of the increase of reducing groups (Somogyi, 1952), using sodium pectate (0.5%) as the substrate and D-galactopyranuronic acid as a standard. Exopolygalacturonase activity was established by the same way using di(D-galactosiduronic) acid (1 ~tmol/ml) as a substrate. Activity of pectinesterase was performed by the continuous titration in a thermostatically controlled vessel with 0.01 M NaOH at pH 7.5 and 30 ~ under nitrogen atmosphere using a Radiometer pH-stat and autotitration set (Denmark); the substrate was 0.5% citrus pectin (d.c. 67%) in 0.15 M NaC1. Pectin lyase activity was determined by recording absorbance at 232 nm during incubation With 0.5% pectin (d.c. 93.8%) pH 5.2. The molecular masses of polygalacturonase and exopolygalacturonase were approximately determined by gel chromatography on Superose 12TM using FPLC device (Pharmaeia, Sweden) and the Calibration proteins II kit (Boehringer-M annheim, Germany). Ultrathin-layer isoeleetrie focusing in polyacrylamide gels on polyester films was performed as described (Radola, 1980). Polygalacturonase activity was detected by the print technique with a dyed substrate (Ostazin Brilliant Red-D-galaeturonan DP 10) (Markovi6 et al., 1992) or by the print technique with colouress D-galaeturonan DP 10 dyed additionally with ruthenium red (Sigma, Germany).
901 3. R E S U L T S AND D I S C U S S I O N
The contaminant of column with immobilized polygalacturonase, where 0.5% sodium pectate in 0.1 M acetate buffer at pH 4.6 was used as a substrate, was determined to be Candida boidinii Ramirez (Culture Collection of Yeasts, Institute of Chemistry, Slovakia). This result was a little bit surprising, because no report exists about the production of pectic enzymes by Candida boidinii and more - the isolated culture contained only one species and did not require any further purification. The simultaneous cultivation of this yeast together with Aspergillus niger on the malt agar showed a weak inhibition of the growth of fungus indicating the existence of killer activity of yeast. The cultivation of this yeast strain on pectin medium showed optimal grow conditions. The behaviour of this strain was compared with that of four strains of Candida boidinii from the Culture Collection of Yeasts. The grow curves of all strains on pectin medium showed marked plateau suggesting the presence of two existing C-sources in the pectin medium, requiting two different metabolic paths (Fig. 1).
0.6
go.4 o.z 0
0
2
.4 da6ys Time,
8
10
Fig. 1.: The grow curves of five strains of Candida boidinii on pectin medium. ( ~ - - - ~ ) - the new isolated strain; (IF----Q) - CCY 29-37-1; (C O ) - CCY 29-37-2, (~---Q) - CCY 29-37-8, ( ~ ) CCY 29-37-12.
The strain adapted to pectate showed lag phase by cultivation on this medium corresponding to the first phase of growth on pectin, but after short storage period (two weeks) on malt agar it lost completely the ability to grow on pectate (Fig. 2). There was a possibility to restore this ability to grow on pectate by primary cultivation on pectin. It seems, in conclusion, there is a primary utilization of methanol released from pectin by Candida boidinii and the secondary path, the utilization of D-galaeturonate chains, required an
902 adaptation time which was too long to have a possibility to alive it without other, more suitable, C-source.
0.6 o . 4
01/.
2
0
4 6 Time, days
8
10
Fig.2." The grow curves of new isolated strain of Candida boidinii on pectin and pectate medium. (~ ~ ) C. boidinii growing on pectin, ( ~ ) - the yeast precultured on pectin and cultured on pectate, ( O ' - ' - O ) - the strain on pectate after storage on malt agar.
The activities of pectic enzymes present in cultivation medium (98 mg of protein extracted from 2.5 1 of pectin medium) were poor, not leading to the clarification of cultivation medium indicating the cleavage of pectate chains, with values: 0.024 ~tmol/min.mg for polygalacturonase, 0.004 ~tmol/min.mg for exopolygalacturonase, 0.034 ~tmol/min.mg for pectinesterase and 0.005 l.tmol/min.mg for pectin lyase. The production of individual pectic enzymes was dependent on the C-source used in the cultivation medium (Tab. 1).
Table 1 The production of individual pectic enzymes by Candida boidinii - dependence on the C - source C-source
pectate pectin 20% MGA*
Activity (%) polygalacturonase
exopolygalaeturonase
pectinesterase
pectin lyase
100 67 40
34 28 100
24 100 14
traces traces traces
* 20% of D-galaetopyranuronie acid in pectin
903 The induction of polygalacturonase by the presence of deesterified groups of D-galacturonate and its repression by the presence of oligogalaetosiduronates, the induction of exopolygalaeturonase by the presence of oligogalactosiduronates and of pectinesterase by the presence of esterified groups of D-galacturonate was confirmed as it was by the fungus Aspergillus niger (Stratilov~i et al., 1995). The activity of the main enzyme of pectic enzymes complex, polygalacturonase, was dependent on the pH of the cultivation media; the highest activity was reached at pH 3.51 (natural pH of pectin medium), the activity decreased to 70% and 20% by the cultivation at pH 5.49 and pH 7.01, respectively. The isoelectrie focusing patterns showed the production of polygalacturonase multiple forms with isoelectric points varying from 3.5 to 7.5 (Fig. 3 A,B) with the possibility to influence their production with the change of the C-source and pH of the cultivation media.
A
.......
B
!L+: " ~:i~:::!i::!i!:
]iiiiiiiiiiii~i~i:: :.i~iii:
:iiiiiiiiii!iiii:ii:!i::i~:? 9 ~
e
d
c
b
a
e
d
c
b
a
Fig. 3.: Isoelectric focusing in ultrathin polyacrylamide layers (pH gradient 3 10) of multiple forms of polygalacturonase produced by Candida boidinii under different cultivation conditions; a - pectin, pH 3.51; b - pectin, pH 5.49; e pectin, pH 7.01; d - 20% of D-galaetopyranuronie acid in pectin; e - pectate. A - Activity detection with print technique on colouress D-galacturonan DP 10 dyed additionally with ruthenium red ( both exo- and polygalaeturonases) and B - activity detection with Ostazin Brilliant Red/D-galaeturonan DP 10 agar print (polygalacturonases).
904 The pH optima determination showed at least three pH regions with increased polygalacturonase activity (pH optima 4.0, 4.6 and pH 5.6); the pH 4.0 being the pH optimum of exopolygalacturonase (Fig. 4).
100
g r2
20 4.0
4.8 pH
5.6
Fig. 4.: pH optima of polygalacturonase [(O---O) - substrate 0.5% pectate] and exopolygalacturonase [(I~'--O) - specific substrate di(D-galactosiduronic) acid (1 ~tmol/ml)].
The approximate determination of molecular masses of polygalacturonases present in the medium (Fig. 5) showed two activity peaks corresponding the values of about 40 kDa for polygalacturonase and 50 kDa for exopolygalacturonase. The poor activities of pectic enzymes in the cultivation medium led us to prove the cell cytosole and the cell walls for these activities. The cytosole contains only traces of polygalacturonase activity, but the suspension of cell walls established the activity which seems to be widely sufficient for yeast growth and development. The characterization of this cell wall bound enzymes will be the object of our next studies.
Acknowledgements We thank Mrs. Helena (~igagov~ for excellent technical assistance.
905
1.0 4O O
1o.
J m
0
/o
30
Fraction, No.
5b
0
Fig. 5." The approximate molecular mass determination of polygalacturonase [(O---O) - substrate 0.5% pectate, pH 4.6] and exopolygalacturonase [(IF---O) - substrate 1.0 lxmol/ml of di(D-galactosiduronic) acid, pH 4.0] on Superose 12 column (FPLC device). Flow rate: 0.5 ml/min. System: 0.05 M phosphate buffer pH 7.0, 0.15 M NaC1. Standarts: Ferritin (450 kDa), Katalase (240 kDa), Aldolase (158 kDa), Albumin (68 kDa), Albumin (45 kDa), Chymotrypsinogen A (25 kDa), Cytochrome C (12.5 kDa).
4. R E F E R E N C E S
F.M. Bamby, F.F. Morphet, and D.L. Pyle, Enzyme Microb. Technol., 12 (1990) 891. P. Blanco, C. Sieiro, A. Diaz, and T.G. Villa, Can. J. Microbiol., 40 (1994) 974. H.P. Call, J.J. Walter, and C.C. Emeis, Food Biochem., 9 (1985) 325. F. Federici, Antonie van Leeuwenhoek, 51 (1985) 139. A. Gainvors, V. Fr6zier, H. Lemaresquier, C. Lequard, M. Aigle, and A. Belarbi, Yeast, 10 (1994) 1311. J. Lim, Y. Yamasaki, Y. Suzuki, and J. Ozawa, Agrie. Biol. Chem., 44 (1980) 473. B. S. Luh, and H.J. Phaff, Arch. Biochem. Biophys., 33 (1951) 213. B.S. Luh, and H.J. Phaff, Arch. Bioehem. Biophys., 48 (1954) 23. O. Markovi6, D. Mislovi6ov~i, P. Biely, and K. Heinrichov~i, J. Chromatogr., 603 (1992) 243. H.A. Murad, and M.S. Foda, Biores. Teehnol., 41 (1992) 247.
906 C. Pardo, M.A. Lapena, and M. Gacto, Can. J. Microbiol., 37 (1991) 974. B.J. Radola, Electrophoresis, 1 (1980) 43. M. Somogyi, J. Biol. Chem., 195 (1952) 19. E. Stratilov~, E. Breierov~, and R. Vadkertiov~, Biotechnol. Lett., (1995) (in press) R.H. Vaughn, T. Jakubezyl, J.D. McMillan, T.E. Higgins, B.A. Dave, and V.M. Crampton, Appl. Microbiol., 18 (1969) 771. M.P. Wimbome, and P.A.D. Rickard, Bioteehnol. Bioeng., 20 (1978) 231.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases
9 1996 Elsevier Science B.V. All fights reserved.
Cloning, sequence rhamnogalacturonase pectinolytic enzyme
907
and expression of the gene coding for (RHG) of Aspergillus aculeatus; a novel
M.E.G. Suykerbuyka, P.J. Schaap a, H. Stamb, W. Musters b and J. Visser a
a Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University,
Dreijenlaan 2, NL-6703 HA Wageningen, The Netherlands
b Unilever Research Laboratory, Olivier van Noortlaan 120, NL-3133 AT, Vlaardingen, The Netherlands
Abstract
Rhamnogalacturonase was purified from culture filtrate of Aspergillus aculeatus after growth on sugar-beet pulp. A gene coding for rhamnogalacturonase (rhgA) was isolated from a ~, cDNA expression library as well as from a recombinant phage ), genomic library. The cloned rhgA gene is interrupted by three introns and encodes a protein of 440 amino acids. Limited homology with A. niger polygalacturonase amino acid sequences is found. A. aculeatus and A. awamori strains overexpressing rhamnogalacturonase were obtained by transformation. The recombinant rhamnogalacturonase can degrade modified hairy regions and has a positive effect in the apple hot-mash liquefaction process.
1. INTRODUCTION
Pectin consists of 'smooth' regions of homogalacturonan and branched or 'hairy' regions with alternating galacturonic acid and rhamnose residues in the backbone. In the hairy regions most of the rhamnose residues are branched with complex, arabinose and/or galactose containing side-chains. The alternating galacturonic acid residues are methylated, acetylated or branched with xylose (1). Complete degradation of pectin is of great importance for various industrial processes such as clarification of fruit juices and requires the interplay of many enzymes (2). Rhamnogalacturonases (RHG) can degrade hairy regions by hydrolyzing ct-ngalacturonopyranosyl-(1,2)-c~-L-rhamn0pyranosyl linkages in the backbone. High RHG
908 activity was demonstrated in a commercial preparation (Ultra SP) from A. aculeatus, from which the enzyme was purified (3). Besides, other enzymes contributing to the degradation of hairy regions have been purified from UltraSP, among which rhamnogalacturonan ct-Lrhamnopyranohydrolase (4) and rhamnogalacturonan acetylesterase (5). Here we describe the purification of RHG, the cloning and characterization of the corresponding gene and (over)production of the enzyme in A. aculeatus and AspergiUus awamori.
2. PURIFICATION OF RHAMNOGALACTURONASE
For the purification of RHG, which has been described before (6), A. aculeatus was grown in sugar-beet pulp medium for 48 h. From a 2 1 culture containing 3.2 g protein, about 5 mg RHG was obtained after purification. Analysis by SDS-PAGE revealed only one band of about 55 kDa and thus the enzyme appeared to be pure. After N-glycanase treatment and SDS-PAGE analysis, a smaller protein band of about 46 kDa was visible (Fig.l). M
1
2
g
g o
W
i
Figure 1. SDS-PAGE of purified RHG before (lane 1) and after (lane 2) treatment with Nglycanase. M indicates standard MW markers.
909 3. ISOLATION AND CHARACTERIZATION OF CDNA AND GENOMIC CLONES ENCODING RHAMNOGALACTURONASE
A gene coding for rhamnogalacturonase (rhgA) was isolated by screening a lambda cDNA expression library with a mouse polyclonal antibody. However, the initial screening was not successful, because the antiserum did not react with native conformations of the protein. Therefore the inserts of ),ZAPII were excised and the colonies generated, containing recombinant pBluescriptSK(+) plasmids, were lysed in the presence of 8 M urea followed by screening with the polyclonal antiserum. Restriction analysis of four immunologically reactive clones revealed that two clones contained an insert of 1.5 kb and two clones contained an insert of 1 kb. All four clones contained the same 0.5 kb XhoI fragment, which was used as a probe to screen an A. aculeatus genomic library in ~,EMBL4. DNA of positive clones was subjected to Southern analysis, after which a limited restriction map could be made. Subcloning of a 3.9-kb BamHI-SalI fragment, containing the complete rhgA gene, in pBluescriptSK(+) resulted in plM803 (Fig.2B). Both strands of the 1.5 kb cDNA insert were completely sequenced and were found to comprise the entire coding region of the rhgA gene with a 24-bp leader sequence. The methionine at position 1 is followed by an open-reading frame comprised of 439 amino acids, encoding a putative protein with a deduced isoelectric point of 4.2 and a calculated molecular weight (MW) of 45962 Da. The calculated MW corresponds well to the value for RHG after N-glycanase treatment (see above), making it very likely that the protein is glycosylated. Three consensus sites for N-linked glycosylation are found. The DNA sequence of the internal 3.3-kb BamHI-HindIII fragment of the genomic clone pIM803 (Fig.2B) was determined in both strands and comprised the entire coding region of the rhgA gene and 1.2 kb of the promoter region. Comparison of the genomic DNA sequence with the cDNA sequence unambiguously identified the position and the size of three introns of respectively 64, 64 and 66 bp,
I rn
= ~ . . "I-
.
.
-~"I-
-x-r"
~~ v
o
,-X
o
,.. X
_ r~
"~ ,.. X
-
-
"o
.=_ "I-
_ -~ gO
Figure 2. Restriction map of the A. aculeatus rhgA genomic clone plM803, plM803 contains a 3.9-kb chromosomal BamHI-SalI fragment in pBluescriptSK(+). The open reading frame is indicated as closed bars. 5'- and 3'- untranslated sequences and introns are given as open boxes.
910 We compared the deduced amino acid sequence of A. aculeatus RHG with those of the A. niger polygalacturonases PGI, PGII and PGC (7). Whereas little overall homology is found (10.3 %) after alignment with the CLUSTAL program, residues which are conserved between RHG and the polygalacturonases are particularly located in those regions, that were designated as relevant for polygalacturonase activity (8). The possible candidates for carboxylate groups involved in catalysis in endo-PGII were postulated to correspond with aspartic acid residues at positions 180, 201 and 202 . These three negative charges are conserved in RHG (Asp-195, Asp-215 and Glu-216, Fig.3). Also, the nearby aspartic residue at position 183 in PGII is conserved in RHG (position 198). However, this residue was not included as a potential candidate because its equivalent is not found in tomato polygalacturonase (8). Furthermore, two of the eight cysteine residues that are conserved between RHG and the polygalacturonases, are located in this part of the sequence. The positively charged sequence Arg-Ile-Lys (residues 256-258) in PGII, postulated to play a role in substrate binding, is not fully conserved in RHG. The sequence reads in this case Met-Ile-Lys (residues 269-271). Another postulated active-site residue (His-223 in PGII), which is located in a conserved sequence Gly-His-Gly, present in all polygalacturonases, is absent in RHG at the corresponding position, but is present at position 289-291. The homology observed between RHG and endo-PG is relevant for further investigations of the catalytic mechanism and the different substrate specificities of these two pectinolytic hydrolases.
RHG PGI PGII PGC
- G G N E G G L .... D G I D V W G S N - I W V H D V E V T N K D E C V T V K S P A N N I L V E S SDGDDNG-GHNTDGFDISESTGVYISGATVKNQDDCIAINS-GESISFTG ADGDTQG-GHNTDAFDVGNSVGVNIIKPWVHNQDDCLAVNS-GENIWFTG TDGDTDDLAANTDGFDIGESTYITITGAEIYNQDDCVAINS-GENIYFSA .
RHG PGI PGII PGC
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
IYCNWSGGCAMGSLGADTD--VTDIVYRNVYTWSSNQMYMIKS-NGGSGT GTCSGGHGLSIGSVGGRDDNTVKNVTISDSTVSNSANGVRIKTIYKETGD GTCIGGHGLSIGSVGDRSNNVVKNVTIEHSTVSNSENAVRIKTISGATGS SVCSGGHGLSIGSVGGRDDNTVKNVTFYDVNVLKSQQAIRIKTIYGDTGS .
RHG PGI PGII PGC
.
231 222 216 236
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
. .
VSNVLLENF-IGHGNAYSLDIDGYW--SSMTAVAGDGVQLNNITVKNWKG VSEITYSNIQLSGITDYGIVIEQDYENGSPTGTPSTGIPITDVTVDGVTG VSEITYSNIVMSGISDYGVVIQQDYEDGKPTGKPTNGVTIQDVKLESVTG VSEVTYHEIAFSDATDYGIVIEQNYDDTSKT--PTTGVPITDFVLENIVG
278 272 266 286 .
325 322 316 334
Figure 3. Comparison of the amino acid sequence of RHG of A. aculeatus with polygalacturonases PGI, PGII and PGC of A. niger. The most homologous region of the proteins is shown. Numbering is from the first amino acid of the signal peptide. * Identical amino acids; 9 similar amino acids. The amino acid residues in bold type are referred to in the text.
911 4. OVEREXPRESSION OF THE RHGA GENE IN A. ACULEATUS AND A. A WAMORI
In order to verify the presence of a complete and functional rhgA gene in pIM803 and to generate a RHG-overproducing strain, an uridine auxotroph A. aculeatus strain was cotransformed with plM803 and the A. aculeatus pyrA gene. The resulting transformants were grown for 48 h on medium containing 1% sugar-beet pulp. According to a Western analysis of dilutions of the culture filtrate RHG was produced at a five to ten times higher level compared to the wild-type strain in most of the transformants (Fig.4, lanes 4-6). In order to achieve higher RHG expression, A. awamori was cotransformed with plM816, containing the rhgA gene under control of the A. awamori xylanase (exlA) promoter (9) and the homologous pyrA gene. Twelve transformants were grown in medium containing 1% xylose and the culture filtrates were analysed by Western blotting. Four of the analysed transformants (Fig.4, lanes 7-9) produced RHG at a level that was more than ten times higher than in the wild-type A. aculeatus strain grown on beet pulp or pectin. To investigate the regulation of expression of rhgA in A. aculeatus, the wild-type strain and a multicopy transformant were grown overnight in minimal medium (MM)/I% sucrose and transferred to MM/1% apple pectin. RNA was isolated from mycelium of both strains just before and 6 h and 24 h after a shift to apple pectin and compared by Northern analysis. No rhgA expression could be detected in either strain grown on sucrose (Fig.5A,B, lane 1). However, 6 h after the shift to apple pectin the rhgA mRNA level in the multicopy transformant was very high and declined to a low level after 24 h (Fig.5A, lanes 2,3). In the wild-type strain the rhgA mRNA was only visible 6 h after transfer, but at a lower level (Fig.5B, lanes 2,3).
1
2 3 4
5 6
7
8
910
.
..
Figure 4. Western analysis of culture filtrate of A. aculeatus wild type (lanes 1-3) and A. aculeatus (lanes 4-6) and A. awamori multicopy transformants (lanes 7-9). Medium samples were applied undiluted (lanes 1,4,7), 10 times diluted (lanes 2,5,8) or 100 times diluted (lanes 3,6,9). Lane 10 contains purified rhamnogalacturonase.
A
1
2
3
!i,! Figure 5A,B. Northern analysis of an A. aculeatus multicopy transformant (A) compared to the wild type (B). RNA was isolated from the mycelium before (lanes 1), 6 h after (lanes 2) and 24 h after (lanes 3) transfer of the corresponding strains to minimal medium (MM) with 1% apple pectin. The location of the rhgA transcript is indicated with an arrow.
912 5. RHAMNOGALACTURONASE ACTIVITY AND APPLICATION
RHG activity was demonstrated by modified hairy region degradation. One of the A. awamori transformants (NW208::plM816/3), in which high expression levels of A. aculeatus RHG were obtained under the control of the A. awamori exlA promoter (see
above), was grown on xylose medium. Culture filtrate of this transformant was incubated with modified hairy regions and analysed by HPAE chromatography analysis. Figure 7 shows the elution profile of isolated modified hairy regions without the addition of enzymes (Fig.6A), upon incubation with Biopectinase OS (Fig.6B) and upon incubation with RHG-containing culture filtrate of A. awamori transformant NW208::plM816/3 (Fig.6C). It is clear, that the isolated MHR can only be degraded by rhamnogalacturonase containing culture filtrate and not by Biopectinase. Using standard solutions it was shown that the peak at 23 min represents galacturonic acid. The other peaks of higher molecular mass could not be identified but presumably represent rhamnose and galacturonic acid containing oligomers as decribed before (10,11). As shown in Fig.4 the addition of RHG results in the appearance of various oligosaccharides from the substrate and hardly any galacturonic acid release, demonstrating the endo-RHG activity.
0.5
.
0
0.5B
.
.
I
.
.
.
.
t
.
i
"
t
I
. . . . . . . .
,
t
i
t
I
,
i
'
"1
t..._.J~
0.5 r
C 0 18
. ' i' 23
t...._.lL
J--_.L_J 28 33 Minutes
.J 38
Figure 6A-C. High-performance anion-exchange chromatography elution profile of isolated modified hairy regions without addition of enzymes (A), with Biopectinase OS (B) and with rhamnogalacturonase-containing culture filtrate from an A. awamori multicopy transformant (C). #C: #Coulomb. Data taken from Ref. 6.
913 Application Vials were performed by incubation of apple mash with Biopectinase LQ (Biopectinase OS supplemented with cellulases, 0.5 g/kg) in the presence or absence of culture filtrate of the RHG-producing wild-type or transformant strains. For this, A. aculeatus wild type and transformant (NW217::plM803/75) strains were grown on apple pectin and the A. awamor/ transformant (NW208::plM816/3) strain was grown on xylose for 24 h. Apple mash was incubated with samples of the culture filtrate after which the juice yield was measured. A 10% increase in yield at equal brix value was observed when RHG was added. The effect of addition of RHG in viscosity reduction of apple mash is shown in Fig.7. Obviously the presence of RHG has a positive effect on viscosity reduction especially in the initial phase of hot-mash liquefaction. The effect is most clearly demonstrated when RHG from the A. aculeatus and A. awamor/transformants is used as compared to that from the wild-type strain, reflecting the higher production levels of these recombinant strains.
35
n
~
o
30* o
~25 N ~ 20 15/-I 10
30
.....
I. . . . . . . . . . . 60
rain
120
Figure 7. The influence of rhamnogalacturonase (RHG) produced by wild-type and RHGoverproducing Aspergillus strains on viscosity reduction during apple hot-mash liquefaction. Apple mash was incubated with Biopectinase LQ alone (El) or in combination with RHG, produced by A. aculeatus wild type (O) and transformant strains (o) and an A. awamor/ transformant strain (m). Data taken from Ref. 6
914 6. REFERENCES 1
Schols HA, Posthumus MA, Voragen AGJ (1990) Structural features of hairy regions of pectins isolated from apple juice produced by the liquefaction process. Carbohydr Res 206:117-129 2 Whitaker JR (1984) Pectic substances, pectic enzymes and haze formation in fruit juices. Enzyme Microb Technol 6:341-349 3 Schols HA, Geraeds CJM, Searle-van Leeuwen MF, Kormelink FJM, Voragen AGJ (1990) Rhamnogalacturonase: a novel enzyme that degrades the hairy regions of pectins. Carbohydr Res 206:105-115 4 Mutter M, Beldman G, Schols HA, Voragen AGJ (1994) Rhamnogalacturonan c~-LRhamnopyranohydrolase. Plant Physiol 106:241-250 5 Searle-van Leeuwen MJF, Broek LAM van den, Schols HA, Beldman G, Voragen AGJ (1992) Rhamnogalacturonan acetylesterase: a novel enzyme from Aspergillus aculeatus, specific for the deacetylation of hairy (ramified) regions of pectins. Appl Microbiol Biotechnol 38:347-349 6 Suykerbuyk MEG, Schaap PJ, Stam H, Musters W, Visser J (1995) Cloning, sequence and expression of the gene coding for rhamnogalacturonase (RHG) of Aspergillus aculeatus; a novel pectinolytic enzyme. Appl Microbiol Biotechnol 43:861-870 7 Bussink HJD, Buxton FP, Fraaye BA, Graaff LH de, Visser J (1992) The polygalacturonases of Aspergillus niger are encoded by a family of diverged genes. Eur J Biochem 208:83-90 8 Bussink HJD, Buxton FP, Visser J (1991) Expression and sequence comparison of the Aspergillus niger and Aspergillus tubigensis genes encoding polygalacturonase II. Curr Genet 19:467-474 9 Hessing JGM, Rotterdam C van, Verbakel JMA, Roza M, Maat J, Gorcom RFM van, Hondel CAMJJ van den (1994) Isolation and characterization of a 1,4-fl-endoxylanase gene of A. awamori. Curr Genet 26:228-232 10 Schols HA, Voragen AGJ, Colquhoun IJ (1994) Isolation and characterization of rhamnogalacturonan oligomers, liberated during degradation of pectic hairy regions by rhamnogalacturonase. Carbohydr Res 256:97-111 11 Colquhoun IJ, Ruiter GA de, Schols HA, Voragen AGJ (1990) Identification by n.m.r, spectroscopy of oligosaccharides obtained by treatment of the hairy regions of apple pectin with rhamnogalacturonase. Carbohydr Res 206:131-144
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
915
Pectinase secretion by Aspergillus FP-180 and Aspergillus niger N.402 growing under stress induced by the pH of culture medium. Blanca A. Trejo-Aguilar a, Jaap Visser b and Guillermo Aguilar O a. a
Department of Food Science and Biotechnology, Faculty of Chemistry, National University of Mexico, UNAM. Conj. E, Fac. Quimica, CP 04510 Mexico D.F.
b
Section Molecular Genetics of Industrial Microorganisms, Agricultural University, Dreijenlaan 2, 6703-NL Wageningen, The Netherlands.
Abstract The effect of pH, ranging from 2.5 to 6.5, on growth and secretion of pectinases by two Aspergillus strains was evaluated at temperatures of 30 and 37~ The growth of Aspergillus niger N-402 at 30~ was abundant at all pH values tested and it was higher at this temperature that at 37~ where a maximum at 5.5 pH could be observed. On the other hand, the growth of Aspergillus FP-180 was seriously affected by initial pH particularly at 2.5 pHi and was also higher at 30~ than at 37~ In opposite way the activity secreted by Aspergillus FP-180 was higher than that secreted by Aspergillus niger N402 and particularly the endo activity at pH values of 2.5. It is apparent that stress induced by the pH is affecting the pattern of secretion of both strains. 1. INTRODUCTION Microorganism enzyme production, as well as that of the other metabolites, is affected by culture conditions, in particular by the pH of culture medium. Evidence is ample and involves different kinds of microorganisms (1-3). On the other hand, it is well known the ability of filamentous fungi to grow in a wide range of environmental conditions such as acidic or alkaline pH. In fact it has been reported that Aspergillus nidulans is able to grow in a media as acidic as pH 3 or as alkaline as 9. The pH of culture medium affects not only the cellular physiology but also gene expression, membrane potential and permeability, ionic exchange, protein synthesis and secretion and internal pH of the cell. The effect of pH on growth of
916
different microorganisms is well documented. However the effect of extreme pH conditions on growth and enzyme production are still poorly understood. In a previous work, we found an increased pectinase production under extreme acidic pH conditions. Since very acidic pH induced stress conditions in the cell it is interesting to evaluate the response of different Aspergillus strains toward stress induced by the pH of culture medium. In our laboratory we have studied the effect of extreme acidic pH on growth and secretion of pectinases by a wild white strain of Aspergillus and by Aspergillus niger N-402. 2. MATERIALS AND METHODS 2.1 Microorganisms: The strains used in this work were Aspergi//us niger N-402 and a white Aspergillus strain called Aspergillus FP-180 which was isolated from spoiled fruit in Mexico. 2.2 Culture medium and conditions: The strains were grown on basal medium, containing in g/l: K2HPO4, 2; KH2PO4, 2; (NH4)2SO4, 2 and 1% pectin as carbon source. Fermentation was carried out at 30 and 37*C at 100 strokes min-1. Samples were withdrawn at different intervals and used for the corresponding analysis. 2.3 Analytical Techniques: Cell growth was determinated as dry weight. Exopectinolytic activity was measured by the determination of the reducing sugars produced from 1% pectin solution after incubation at 45*C during 20 min. at pH 5.0. One Unit of Exo-activity was defined as the amount of enzyme which catalyzes the formation of l l~mol of galacturonic acid under the assay conditions. Endo-activity was measured by the relative change in viscosity of 1% pectin at 30 in an Ostwald viscometer. One Unit of endo activity was defined as the amount of enzyme which reduced the viscosity of 10 ml of pectin by 50% in 10 min. at pH 4.2.
2.4 SDS-PAGE and in situ Activity: Electrophoresis was carried out in denaturing conditions with a resolving gel containing 10% acrylamide, 2.7% bis-acrylamide and 20 l~g of bovine serum albumin per milliliter to enhance the renaturation of electrophoresed enzymes. Samples were boiled for 60 sec in sample buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 125 mM Tris-HCI, pH 6.8 and 0.005% bromophenol blue). Samples were electrophoresed at 15 mA for 1-1.5 h through 0.75 mm gel in a vertical slab gel SE-280 (Hoefer Sci. Ins.) Acrylamide gel were incubated for 1-1.5 h, with shaking, in two changes of 0.01 M Tris-HCI, pH 7.6. After this time the gels were placed onto agarose pectin or polygalacturonic acid overlays and incubated from 6 h to overnight. Subsequently, polyacrylamide gels were rinsed with distilled water and were protein stained with Coomassie blue R-250. The agarose-pectin overlays were prepared with 1% agarose and 0.1% pectin in 0.17 M acetate buffer, pH 5.0 and stained with 0.05% ruthenium red solution. The overlays were put onto a plastic sheet and dried at room temperature.
917
3. RESULTS AND DISCUSSION
Aspergillus niger N-402 was able to grow well despite the initial pH of culture
medium and temperature (Fig.l). The growth at 30~ at all pH values tested and it is clear that this strain was not affected by the lower initial pH at this temperature (Fig. 1A). However when the temperature was set at 37~ a decrease of around 35% in growth of A. niger N402 was observed at 2.5 and 3.5 pHi, respective to the maximum reached at a pH 5.5 at this temperature (Fig. 1B). 5 '.-- 4
E
v
E3
A -
B 4 ~'
1
E
m
3
v
E t--
r
2
-~9 2 E3
.-~ (9 E3
0
2.5 3.5 5.5 6.5
2.5 3.5 5.5 6.5
pH
pH
Figure 1. Maximum growth obtained by Aspergi//us FP-180 (open bars) and Aspergillus niger N402 (dashed bars) at different pH values on 1% pectin at 30~ (A) and 37~ (B).
On the other hand, Aspergillus FP-180, isolated from spoiled fruit in Mexico, showed a different pattern. At 30~ its growth was similar at pHi of 3.5 to 6.5 (Fig. 1A). However, at 2.5 pHi a decrease of 60% in relation to maximum was obtained. This reduction in growth was more accentuated that those occurred with A. niger. When the temperature was set at 37~ the same picture was observed (Fig 1B). However the effect of very acidic pHi (i.e. 2.5) was more evident with a reduction in cell growth of 80% at this temperature (Fig. 1B). An important decrease in growth was also found at 3.5 pHi. Cell growth of both strains was favored at 30~ Pectinolytic activity produced by both strains are shown in fig. 2A-2C. Exo pectinolytic activity (Fig.2A-B) secreted at 30~ was higher for the strain of A. FP180 at pH 2.5 to 5.5 and practically the same at 6.5 pH to that secreted by A. niger N402. The more important feature is that the higher activity was obtained at stressing conditions which seriously affects cell growth. This behavior was more noticeable at 37~ In fact Aspergillus FP-180 produced higher exo-pectinase levels at 2.5 and 3.5 that at 30~ The activity secreted at the higher temperature and 2.5
918
pHi was around twice that at the lower one. On the other hand, A. niger strain secreted exo-pectinase enzyme levels lower than that reached by the FP-180 strain irrespective temperature or pHi. For A. niger exo activity was found to be practically of same levels at both temperatures. There was not a clear effect of pH on the secretion of exo pectinase activity for this strain (Fig. 2 A-B). Endo pectinase activity was found to be very high for the strain FP-180 at a initial pH of 2.5 and 3.5 producing about 3 to 5 fold respectively in relation to A. niger, at 37~ (Fig.2C). At other pH values the level of endo-pectinase secreted diminished as pH increased, and was practically negligible at 6.5 pHi showing a clear relation between pHi and enzyme production or secretion. Endo pectinase produced by A. niger was very low as was the case of exo activity. However low production was accentuated at very acidic pH values ( i.e., 2.5 and 3.5). There was not a clear correlation in the production of this activity with the initial pH.
d
x-
40 _ A
B_40'
C
~ c~
~' 4 o E
g:D
E D
30
g
20
o
10
a0
-
3
-12
v
.>
. - -
o
~
0
t~. .m
o
rm
~ 0
~
[]_r-ca
2.5 3.5 5.5 6.5
2.5 3.5 5.5 6.5
2.5 3.5 5.5 6.5
pH
pH
pH
o
0 oo
Figure 2. Specific Exo (A and B)and Endo (C) pectinase activities produced by FP-180 (open bars) and Aspergillus niger N402 (dashed bars) at different pH values growing on 1% pectin at: 30~ (A) and 37~ (B, C).
Aspergillus
Electrophoreses of samples from the different pH values showed a very interesting differences in the band pattern of secreted proteins, both, between pH values for each strain and between strains itself (Fig.3). From SDS-PAGE it was observed, for both strains, a reduction in the number of band as the pHi diminished. At 2.5 pHi very few proteins were found to be secreted, particularly with Aspergillus FP-180. With Aspergillus niger N402 the effect on secretion was also observed at this pH, although, the growth was just slightly affected at this pH. The in situ activity overlays exhibit, for N402 strain, clearing and darker zones when polygalacturonic acid (PGA) and pectin, respectively, were used as substrates. These zones correspond to a proteins with a molecular mass of around 63-65 kDa
919
for clearing bands, probably due to a lyase activity, and to a proteins in the range of 48-52 kDa for darker zones in pectin overlay. This later being a pectinesterase. It could be observed that the clearing zones on PGA were very weak at 2.5 and 3.5 Pectin overlay
SDS-PAGE
PGA overlay
6.5
5.5
3.5
2.5
a
b
c
d
2.5
3.5
5.5
6.5
6.5
5.5
3.5
2.5
a
b
c
d
2.5
3.5
5.5
6.5
Figure 3. SDS-PAGE and in situ pectinase activity on pectin and polygalacturonic acid-agarose overlays of culture filtrates of Aspergillus niger N-402 (upper panel) and Aspergillus FP-180 (lower panel) at 2.5, 3.5, 5.5 and 6.5 pHi (Lanes a, b, c, and d, respectively). Electrophoresis on 10% acrylamide slab gel (14 X 8 cm) in the presence of SDS was according to Laemmli (6), run at 30 mA constant current for 2 hours. Crude cell-free samples were concentrated by lyophilization, dialyzed, boiled with sample buffer by 60 sec. and applied to each well. Polyacrylamide gel and overlays were incubated overnight with 0.17 acetate buffer at room temperature. pHi and of high intensity for samples from 5.5 and 6.5 pH which appeared also on pectin overlay (Fig. 3). On the other hand, FP-180 strain showed only clearing zones in overlays with both substrates in a range of molecular mass of 48-60 kDa (Fig. 3). In no case a pectinesterase activity was found for Aspergillus FP-180. However, the
920
clearing zones were of higher intensity for this strain than for Aspergillus niger N402. It is interesting to notice that Aspergillus FP-180 produced very high level of exo and endo activities as compared to A. niger under the conditions used in this work. It is possible that the complementation of both strains could improve the production of a complex and more efficient pectinolytic system. The above results showed that stress induced by the pH of culture medium affects the growth of Aspergillus FP-180 and to a lesser extent that of Aspergillus niger N 402. However pH not only affects growth, but also modified the pattern of pectinase production and/or secretion. It is believed that the reduced number of protein bands produced at 2.5 pH must be essential enzymes to degrade the outer substrate during fungal growth and as the pH turns less stressing factor the microorganisms could be able to produce a more complex mixture of extracellular enzymes. 4. ACKNOWLEDGMENTS Part of this work was supported by DGAPA/UNAM Project IN 209194 5. REFERENCES
1) E. Nahas, H.F. Terenzi and A. Rosi. J. Gen. Microbiol., 128 (1982) 1017-1021. 2) A. Rossi and H.N. Arst. FEMS Microbiology Lett. 66 (1990) 51-54. 3) G. Leone and J. van den Heuvel. Can. J. Bot. 65 (1986) 2133-2145. 4) G. Aguilar, B.A. Trejo, J.M. Garcia and C. Huitron. Can. J. Microbiol. 37 (1991) 912-917. 5) C. Dijkema, R.P. Rijken, H.C.M. Kester and J. Visser. FEMS Microbiology. Lett. 33 (1986) 125-131. 6) U.K. Laemmli. Nature 227 (1970) 680-685.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
S e l e c t i o n of a c o n s t i t u t i v e P e n i c i l l i u m strain
hyper-pectinolytic
921
mutant
from
a
Noomen Hadj-Taieb, Malika Ayadi and Ali Gargouri Centre de Biotechnologie de Sfax; BP "W"3038. Sfax-TUNISIA
ABSTRACT: The production of pectinases was studied in fungi. One strain, Penicillium occitanis, was chosen for nitrous acid mutagenesis. Based on Hexadecyl -trimethyl-ammonium bromide (CTAB) staining and after only one round of mutagenesis, an interesting mutant, CT1, was selected. It secretes about 20 times more pectinases than the CL100 wild-type strain but not cellulases or other hydrolases. In comparison with another already known mutant of the same strain, CT1 is, not only genetically stable and sporulating, but also able to secrete high amounts of pectinases on local substrates such as "orange peel". The most interesting feature of this mutant is its constitutivity: it produces the same specific activity of pectinases on citrus pectin as on glycerol, which is a potent repressor of pectinolytic activities in Penicillium and many other fungi.
1-INTRODUCTION: In biomass degradation, the biological decomposition of pectin is an important process which is accomplished by enzymes produced by a wide variety of saprophytic and phytopathogenic microorganisms (Rombouts and Pilnik, 1980). The enzymatic systems occur during the fruit ripening process causing softening. Pectinases hydrolyse pectins by different mechanisms, those of which act on principal chain of galacturonics and those acting on lateral chain or "hairy regions" (Schols et al. 1994). Pectinases play an important role within food industry since they improve the extraction, filtration and clarification of fruit juices (Pilnik and Rombouts, 1985). Their use in the olive oil and wine sectors has recently been reported (Servili et al., 1992; Gainvors et al.,1994). The synthesis of pectinases is a property shared by many different microorganisms, especially the Aspergillus genera. Many plant pectinases have been characterized and some of them cloned and now, in order to shed lights on the "plant-fungus" interaction, attention is turned on fungal enzymes. Many Erwinia and Aspergillus pectinases and the corresponding genes have been isolated and studied (Laing et a1.1993, Bussink et al, 1992). In this context, the selection of novel producer strains is an important step for phylogenetic comparisons and for biotechnological applications. One can even expect that strains producing "hairy regions" hydrolytic enzymes should be useful for some specific applications. It is also of a great benefit to search mutant strains producing enzymes at a much lower cost. Penicillium occitanis produces a wide range of extracellular enzymes such as cellulases and pectinases (Jain et al, 1990-a ). After multiple rounds of mutagenesis, a mutant named Po16 was selected from the CL100 wild strain and was shown to secrete very high amounts of pectinases and cellulases enzymes (Jain et al, 1990-b). Unfortunately, the fact that this mutant was poorly sporulating (Jain et al, 1990-a) raised serious problems for an industrial use and for an eventual continuation in the genetic improvement program. In the present study, we report the isolation of a new hyper-pectinolytic mutant from P.occitanis after a single round of nitrous acid mutagenesis. We present here comparisons with CL100 and Po16 strains and data indicating that our mutant is fully constitutive and that it can grow on cheap and local substrates.
922 2. M A T E R I A L S AND M E T H O D S
2.1 Microorganisms" Some of them were originally isolated in our laboratory and others kindly provided by others research laboratories. These strains were maintained on potato dextrose agar (PDA) at 4~ or as spores in 20% glycerol at -20~ Pol6 is a mutant of the wild type strain Penicillium occitanis CL100, both kindly provided by Professor G. Tiraby, Toulouse University. Three strains, two Aspergillus niger : A1 and F38 (Hamdi et al, 1991) and one Trichoderma inhamatum:: OL1J, have been isolated in our laboratory.
2.2 Mutagenesis and Screening conditions" The strain was grown on PDA plates for 7 days and spores were resuspended in water containing 0.1% triton. 0.4 ml (108 sp./ml ) are suspended in 5 ml of citrate buffer pH 3, in ice. 501.tl of Na NO 2 0.2 M were added at time "zero". 0.5 ml were taken at various time and were neutralized with an equal volume of K2HPO4 (0.2M). Spores are then diluted and plated on PDA in order to plot the viability curve. A survival rates of 10 % andl % were obtained for 40 and 80 mn of treatment respectively and were considered to be suitables for the screening of mutants.The basic screening medium was Hankin (Hankin et al. 1971) and BSM (2.5 g/1 (NH4)2SO4, 1 g/1 KH2PO 4, 0.3 g/1 MgSO4, 0.2 g/l CaCL2, 5 mg FeSO 4, 2 mid Tween 80 and 1 ml/1 oligoelement solution, pH 5.5) supplemented with 0.5% of the appropriate carbon source and 0.1% v/v Triton X 100 to restrict growth. CTAB 1% or iodine solution were added on colonies. The selection of pectinase-producing strains was based on the detection of clearing halo surrounding the colonies. Total pectinase, cellulase and lipase activities secreted by colonies were detected on BSM plates containing respectively 1% of citrus pectin, 2% Walseth cellulose and 1% olive oil + rhodamine. After few days at 30~ pectin plates were covered by 1% CTAB for lhour, positive colonies became surrounded by a clear halo; walseth plates are not stained: the halo is visible directly on positive clones; lipase activity is revealed under UV on oil-rhodamine plates.
2.3 Production of enzymes in flasks: The basal medium of Mandels (Mandels et al., 1976) was used with the following modifications: it was buffered with 3 g/l of sodium nitrate to pH 5.5 and supplemented with 1% w/v citrus pectin " Sigma" or other carbon sources. For enzyme production, 50 ml medium in 250 ml erlemneyer flasks were inoculatedwith spores (106 spores/ml ) exept for the non sporulating Pol 6 strain, where mycelium was used. The culture were incubated at 30 ~ C on a rotary shaker (150 rev mn -1) for 5 days. The culture broth was filtered (Millipore 0.45 ktm ) and the supernatant was analysed for pectinolytic activities, reducing sugars and proteins.
923
2.4 Enzyme Assays : Reducing sugars in the culture filtrate were determined using the DNS method ( Miller, 1959) using the galacturonic acid as standard. Exo and endo-pectinolytic activities were determined by measuring the formation of reducing sugars and by the relative change in viscosity, respectively. Exo-pectinase and exo-polygalacturonase activities were determined as follows: 0.5ml of culture filtrate were added to 0.5 ml of 0.9% citrus pectin 75% DM (Sigma) or 1% Polygalacturonic acid respectively in 50 mM citrate buffer pH4.8. After lh incubation at 45~ the reducing sugars liberated by the enzyme were determined using the DNS method. One unit is defined as the amount of enzyme liberating one ktmole galacturonic acid per minute. Endo-polygalacturonase and Endo-pectinase activities were determined via the reduction in viscosity of 0.5 % polygalacturonic acid solution and 0.25% citrus pectin solution respectively. The relative change in viscosity was measured using an Ostwald viscosimeter as described by Sakai T. (1988) : 6 ml of 0.25% citrus pectin or 0.5% polygalacturonic acid in 50 mM citrate buffer pH 4.8 was incubated at 37 o C for 3 mn and then 1 ml of culture filtrate was added, the mixture was incubated at 37 ~ C for 5 mn. The rate of viscosity reduction (A) is calculated using the equation: A = (Ta - T / Ta - To ) x 100, where T is the flow time (sec) of the reaction mixture, Ta is the flow time (sec) of polygalacturonic acid solution or pectin citrus solution added to the heat-inactivated enzyme, and To is the flow time (sec) of water added to the heat-inactived enzyme. One unit of enzyme activity is defined as the activity reducing the viscosity by 50%. Proteins concentration was estimated with the Biorad reagent by the method of Bradford (Bradford, 1976), with crystallin bovine serum albumin as standard.
3. R E S U L T S
3.1 Penicillium occitanis as a pectinases producer strain: Among more than 20 fungal strains tested on agar plates, we selected some of them to be tested on liquid cultures: two Aspergillus niger (A1 and F38), one Trichoderma inhamatum (OL1J), one Penicillium occitanis (CL100) and its hyperproducer mutant Po16 (Jain et al, 1990-b). As it is shown in table 1, the Po16 mutant is the best producer of pectinases. Moreover, this mutant secretes a wide variety of hydrolytic enzymes such as cellulases, hemicellulases and even lipases. The major disavantage of Po16 is that it can not sporulate. We shall note that this mutant was selected after 8 rounds of mutagenesis and selection ( Jain et al 1990-b): it should have accumulated many mutations responsibles of this particular state. Table 1 Qualitative comparison between fungal strains Strains(a)
Pectinases (b)
Cellulases (b)
Lipases (b)
Sporulation
P.occitanis (CL100) w.t P.occitanis (Po16) mutant A.niger (A1) w.t A.niger (F38) w.t T.inhamatum (OL1J) w.t
+ +++ + + +
+ +++ +++-
+++ +++-
++ -++ ++ ++
(a): the name of the strain is indicated between (b): enzymatic activities were determined qualitatively on plates as in Materials and Methods with +++>++>+>+->--. Sporulation was estimated on PDA plates.
924
3.2 Mutagenesis strategy CTI mutant:
of P. occitanis
parental strain and selection of the
In order to select a sporulafing hyperproducer mutants from P.occitanis, we planned to mutagenize the wild type strain by Nitrous Acid. After two times of treatment, 40 and 80 mn, leading to 10% and 1% of viability, mutagenized spores were plated on either PDA or Hankin medium containing 1% of citrus pectin. Two ways of revealing were adopted: 1) Indirect staining with iodine: colonies were arranged in duplicate grids one on PDA and the second on BSM+pectin, the latter ones were stained by iodine. 2) Direct staining by CTAB (Hexadecyl -trimethyl- ammonium bromide ): the colonies, which arose from mutagenized spores on Hankin medium, were directly stained by CTAB and the interesting mutants having a big halo were picked up and sub-cloned on a fresch plate. All the primary clones were then subcloned giving a set of secondary clones which were tested again by the same staining procedure. From the interesting ones, three independant colonies were cultured in liquid and their enzymatic capacities were assessed. From 800 colonies tested by the "indirect iodine procedure", only one interesting mutant was selected but after sub-cloning, it was discarded due to its instability. From 2000 colonies tested by the "CTAB procedure", one mutant named CT1 was selected. Surprisingly, it exhibited, as a primary clone, a big precipitate or white halo but not a clear halo. After sub-cloning on plate to purify it from the potential contaminating spores and after multiple round of growth and sporulation, it became clear that the CT1 mutant was pure, still sporulating and genetically stable.
3.3 In comparison to the Po16 mutant, CT1
hypersecretes only pectinases.
The wild type CL100 and the mutant CT1 strains were cultured in liquid Mandels media containing 1% citrus pectin. It was already known that the wild strain CL 100 produces very low amounts of both pectinases and cellulases and that the mutant Po16 secretes very high amounts of both cellulases and pectinases (Jain et al, 1990 b) but the CT1 mutant exhibits a particular feature in sense that it hypersecretes only pectinases. In fact, CT1 hyper-produces all classical pectinolytic activities such as endo-polygalcturonase (endoPG), endo-pectinase (endoPC), exopolygalacturonase (exoPG), exo-pectinase (exoPC), pectin methyl-esterase (PE), polygalacturonic acid lyases (PGlyases) and pectin lyases (PClyases); these two latter activities are not shown here. We don't yet know if CT1 secretes also debranching pectinases. Cultured grown on pectin or on cellulose substrate, the CT1 mutant didn't produce more cellulases than the wild strain, compare on table2 the fp (filter paper) activity of both strains. Table 2 Enzymes produced by parental (CL100) and mutant (CT1) strains cultured on citrus pectin Strain
Prot(a)
ExoPC(b)
EndoPC(b)
CT1 CL100
55 23
47 2.3
100 2.1
ExoPG(b) 250 6.5
EndoPG(b) 80 1.5
PE(b)
fp(C)
4 0.6
0.22 0.05
The strains were cultured on Mandels medium + 1% citrus pectin for 5 days and the enzymatic activities of culture filtrates were determined on three substrates: citrus pectin, polygalacturonic acid and filter paper. (a): extracellular proteins are in ktg/ml. (b): pectinolytic activities on pectin (PC) and on polygalacturonic acid (PG) and Pectin esterase (PE) are in units/ml. (c): total cellulolytic activity (filter paper, fp) are in mg of liberated reducing sugars/ml.
925
3.4 C T I h y p e r p r o d u c e s peel" local substrate.
pectinases on "citrus pectin "
as well as on "orange
One of the important criteria taken into account for the choice of an industrial producer strain is its ability to secrete enzymes on cheap and local substrate. Thus, we cultured our mutant as well as the Po16 mutant on a local substrate: milled "orange peel", at the same concentration as citrus pectin in the liquid medium. The results summarized on table 3 show a net difference between both strains: the CT1 mutant is able to produce high amounts of endo and exopectinases on both substrates whereas Po16 is unable to hyper-produce both pectinases on "orange peel". Table 3 Comparison of Enzymes production by CT1 and Po16 strains cultured on citrus pectin and orange peel Citrus Pectin Strain CT1 Po16
Prot 55 76
Orange peel
ExoPC 47 25
EndoPC 100 23
Prot 145 118
ExoPC
EndoPC
54 3.5
100 2.8
Proteins are in gg/ml and pectinases are in U/ml. 3.5 CT1 is a constitutive mutant"
In order to better characterize our mutant, we compared its pectinases production (endo and exoPG, endo and exoPC) on different carbon sources with that of the CL100 mother strain as well as the Po16 mutant and the Aspergillus niger F38 strain. We cultured these strains on Mandels medium contaning 1% of one of the following substrates: citrus pectin, orange peel, polygalacturonic acid, glucose and glycerol. Figure 1 summarizes all the results and shows clearly that the CT1 mutant is a constitutive one. Indeed, it produced the same amounts of specific activites of all pectinolytic activities on inducer substrates as well as on the repressor ones (glucose and glycerol) although it was more derepressed on glycerol than on glucose. Note that the scale of ordinates on figure 1 is very different between CT1 and the other strains and strengthens the fact that even on glucose the CT1 mutant produced relatively more pectinases than CL100 and Po16 strains. On the other hand, the pectinase production of all three remaining strains are repressed by glucose and glycerol, what is currently known about Aspergillus (Solis et a1.1990; Aguilar et al., 1990). Other hydrolytic enzymes, such cellulases, are still repressed by glucose and glycerol in the CT1 mutant (data not shown). Figure 1 Extracellular pectinases (expressed as specific activities) produced by strains grown on different substrates: The substrates used were: PC: citrus pectin; E: orange peel; G: glucose, Gly: gycerol, APG: polygalacturonic acid. Results of two cultures (1 and 2) are presented. On ordinates, specific activities (Activity Units par gg of extracellular proteins) are presented. Note: the ordinates scale differs from one histogramme to another.
926
CT1 10 o
8 =L
1 ExoPC m EndoPC I! ExoPG !~ EndoPG rIPE
6 ,,m
2
om
O, 9
PC1
PC2
E1
E2
G1
,
9
|
62 GLY1GLY2 APG1 APG2
Carbon source
CLIO0 0,3
0,2
,H
~
0,1
,m
o
0,0 PC1
PC2
E1
E2
G1
n
n
,-.,~n...r
G2 GLY1 GLY2 APG1 APG2
Carbon source
1 ExoPC [I EndoPC Im ExoPG IrA EndoPG rIPE
927
Po16 ~
0,8
0
0,6
"~.
0,4
m m m Q
0,2"
ExoPC EndoPC ExoPG PE
O,
0,0 PC1
PC2
E1
E2
G1
G2 GLY1 GLY2APG1 APG2
Carbon source
F38 0,3 o
0,2" o~
1 m m D
o~
o_,
0,1"
5
ExoPC EndoPC ExoPG EndoPG
OPE 0,0
. . PC1 PC2
. E1
. E2
. . . . . G1 G2 GLY1 GLY2 APG1 APG2
Carbon source
928 4. DISCUSSION We have tested various fungal strains for their secretion of pectinases in order to apply these extracellular juices in the improvement of olive oil extraction. The hypercellulolytic mutant strain Po16 secreted high amounts of pectinases (Jain et al, 1990-a,b) but didn't sporulate. Therefore, we decided to select a novel mutant from the wild type mother strain CL100. Indeed, after Nitrous acid treatment and selection on pectin plates stained with CTAB, we selected a very interesting mutant named CT1. It has the same enhanced capacity to secrete pectinolytic enzymes as the Po16 mutant but it sporulates very well and does not hyperproduce other hydrolases such as cellulases or lipases. The most interesting result concerns its ability to produce high specific activities of pectinases on glycerol and glucose whereas the mother (CL100) and the mutant (Po16) strains have to be cultured on pectin based substrates to produce pectinolytic enzymes. This result suggests that CT1 should be a regulation mutant and henceforth a constitutive one. The fact that the CT1 mutant was selected after only one round of mutagenesis suggests that it has been touched by a single mutation, which is an ideal situation for fondamental studies to discover the nature of this mutation. This mutant is deregulated for more than one pectinolytic enzyme ,but not for the other hydrolytic enzymes such as cellulases, subsequently we believe that the responsible mutation should probably lie in a regulatory gene encoding a trans-acting factor involved in the specific expression of pectinolytic enzymes. The CT1 mutant can produce high amounts of pectinases on local "orange peel" substrate whereas Po16 hyper-secretes pectinases only on citrus pectin or polygalacturonic acid. Concerning the mother strain, even if it produces much lower amounts of pectinases, its behaviour resembles that of Po16. This result suggests that the mutation in CT1 would have turned on the expression of a gene whose product is involved in the degradation of orange peel and/or in the uptake of some nutrients. Whatever the explanation is, since the CT1 mutant secretes high amounts of pectinases on cheap local substrates, it should be very useful for industrial applications. We have already successfully tested it in the improvement of oil extraction from olives. In this work, we focussed our attention on some pectinolytic enzymes in order to compare the various strains studied. Other activities such as lyases and "hairy regions" specific enzymes have to be assessed in order to get more detailed on the capacities of our mutant and the other strains. 5. R E F E R E N C E S
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Rombouts F.M. and Pilnik W. Microbial Enzymes and Bioconversions, (1980) 227-281 Schols H.A., Voragen A.G.J. and Colquhoun I.J. Carbohydr. Res. 256 (1994) 97-111 Pilnik W. and Rombouts F.M. Carbohydr. Res., 142 (1985) 93-105 Servili M., Begliomini A.L.and Montedoro G. J. Sci. Food Agric. 58, (1992) 253-260 Gainvors A., Karam N., Lequart C. and Belarbi A. Biotechnol. Letters 16, 12 (1994) 1329-1334 Laing E., Pretorius I.S. Appl. Microbiol. Biotechnol. 39, (1993) 181-188 Bussink H.J.D., van den Hombergh J.P.T.W., van den Ijssel P.R.L.A. and Visser J.. Appil Microbiol Biotechnol 37 (1992) 324-329 Jain S., Parriche M., Durand H. and Tiraby G. Enzyme Microbiol. Technol., 12 (1990-a) 691-696 Jain S. Durand H. and Tiraby G. Appl. Microbiol. Biotechnol., 34 (1990-b) 308-312 Hamdi M., Bouhamed H. and Ellouz R. Appl. Microbiol. Biotechnol.36,(1991) 285-288 Hankin L., Zucker M. and Sands D.C. Applied Microbiology, Aug.(1971) 205-209 Mandels M., Andreoti R. and Roche C. Biotechnol. Bioeng., 6 (1976) 21-23 Miller G.L. Anal. Chem. 31 (1959) 426-428 Sakai T. Methods in Enzymology 161 (1988) 335-350 Bradford M. Anal. Biochem. 72 (1976) 248 Solis S., Flores M.E. and Huitron C. Biotechnol.Letters, 10 (1990) 751-756 Aguilar G. and Huitron C. Biotechnol. LetterS 12, 9, (1990) 655-660
APPLICATIONS.
A) DEVELOPMENTS IN PECTIN MANUFACTURING AND APPLICATIONS
This Page Intentionally Left Blank
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
Production pectin
931
of hypocaloric jellies of grape juice with
sunflower
M.L. Alarc&o-Silva a, H. Gil Azinheira b, M. I. N. Janu&rio c, M.C.A.Leit~o b and T. C. Curado d
a Centro de Microbiologia e IndQstrias Agricolas, Instituto Superior de Agronomia, Tapada da Ajuda, 1399 Lisboa, Portugal. b Centro de Estudos de Produg&o e Tecnologia Agricolas, Instituto de Investigas Tapada da Ajuda, 1301 Lisboa, Portugal.
Cientifica Tropical,
c Secs Aut6noma de Agronomia Tropical e Sub-Tropical, Instituto Superior de Agronomia, Tapada da Ajuda, 1399 Lisboa, Portugal. d Estas Nacional de Tecnologia de Produtos Agrarios, Instituto Nacional de Investiga;&o Agraria, Quinta do Marquis, 2780 Oeiras, Portugal
Abstract The increasing interest of low-caloric foods in diet is well known. This fact has led to the study of Iow-methoxyl pectins that allow to obtain jellified products (such as jams, jellies, marmalade) by using very small quantities of sugar. Sunflower head residues are one of the richest sources of Iow-methoxyl pectin, their most important property being the ability to form gels, if correct proportions of divalent ions (usually calcium) are available. Following earlier studies about physico-chemical characterization of sunflower pectin (Alarc~o-Silva, 1990; Leit~o et a/, 1995) and technological utilization in the manufacture of low-caloric gels (Alarc~o-Silva eta/, 1992), we intend with this contribution to study the behaviour of this pectin in the confection of grape juice jellies and the evaluation of their organoleptic characteristics.
Keywords: hypocaloric jelly; Iow-methoxyl pectin; sunflower; texture profile analysis; sensory evaluation. 1. INTRODUCTION
The importance of some pectic substances (pectin or pectins) either due to the great amounts that certain raw materials contain, or the existence of any specific food use, as well as its hypocholesterolemic effect (based in in vitro and in vivo studies) would explain the increasing interest on deepening physico-chemical studies of pectic substances.
932
Sunflower plant (Helianthus annuus L.)is an important crop in Portugal and its head residues, remaining on soil after the seeds have been removed for oil industry, are one of the richest sources of Iow-methoxyl pectin (ca 19% original dry matter), the most important property being the ability to form gels even without sugar addition, if correct amounts of divalent ions (usually calcium) are present. In our country, as well as in other regions of the European Union where vineyard and wine consumption are traditional there is a great interest in diversifying grapederivated products, especially those varieties of less enological potential. The manufacture of reduced calorie jellies and/or jams is one of the possible items to be considered. Today such products are gaining importance as an interesting segment of food market, because people are more and more aware of their beneficial effects on health. Following previous works on physico-chemical characterisation of sunflower Iowmethoxyl pectins (Alarc~o-Silva, 1990, Leit~o et al., 1995) and technological utilisation in the manufacture of low calorie gels (Alarc~o-Silva et aL, 1992), this investigation was carried out to test the suitability of that pectin to the confection of grape juice reduced calorie jellies in comparison with two types of commercial pectin. Aiming at the optimisation of low-calorie jelly formula, based on consumers' preferences, the jellies were submitted to a sensory panel test judgement and instrumental texture-analysis.
2. MATERIALS AND METHODS 2.1. Sample composition Grape juice was obtained from a 1:1 mixture of two red cultivars, "Benfica" and "Piriquita". After harvest the grapes were washed, sorted out, crushed, treated with SO2, pressed and kept at 7~ for three days. The juice was filtered and stored at -3 to 0 ~ using 0.06% potassium sorbate as preservative. The soluble solids content was 22 ~ the pH 3.3, calcium, potassium and magnesium contents were 125, 1170 and 240 mg dm 3, respectively. Experimental Iow-methoxyl pectin was obtained from dry heads (without seeds) of sunflower (Helianthus annus L.). The extraction of pectin was carried out according to the method of Lin eta/. (1975) with slight modifications. Only oxalate-soluble fraction which was submitted to consecutive treatments of purification as described previously was considered (Leit~o et a/., 1995). The standard Iow-methoxyl commercial pectins used were Violettband D-075 (amidated pectin) and Violettband Rein (non-amidated pectin) provided by OBIPEKTIN AG (Switzerland). 2.2. Jellies preparation The grape jellies were made using the pectin concentrations of 0.5, 0.75, and 1% for the three types of essayed pectin.
933
The formula (Table 1) and the general procedure used in the preparation of the low-calorie jellies was as follows: the pectin was blended with 25% its weight in sugar, dispersed in hot water with magnetic stirring and added to the grape juice previously heated at 60-70 ~ The mixture juice-pectin was heated with continuous stirring to ensure full suspension of pectin. The remaining sugar was incorporated when temperature approaches 70 ~ and heating stopped after the mixture reached 39 ~ (refractometer reading). Then it was poured into glass containers, kept at18~ for 15 minutes and stored at 4~ until evaluation of jellies quality characteristics. Table 1 Jellies experimental Ingredients Pectin Sugar Water Grape juice
formula Amounts (g) 1; 1.5; 2 52 50 100
2.3. Sensory evaluation
The jellies (20 sets) were submitted to a sensory panel (ten panellists from the laboratory staff with some experience in sensory evaluation) requested to give a score (from /ow to high in a non-structured 10 cm scale) to each of the following characteristics: aroma (intensity), taste (sweet, acid and intensity), texture (hardness, spreadability) and overall acceptance. Tests took place in a standardised test room provided with individual booths and the trials assessed in four sessions with five randomly grouped samples at each time.
2.4. Texture-profile analysis
The textural characterisation of the jellies was made by using the empirical technique of Texture-Profile Analysis (TPA) that allows the evaluation of the following parameters: fracturability, hardness, cohesiveness, adhesiveness, springiness, gumminess and chewiness. The samples were analysed on a TAX T2 Texture Analyser programmed for the following conditions: a cylindrical plunger with 1 in. diameter; contact force of 5 g, contact area of 284.88 mm2, speed of 2 mms 1 and a 5 s interval between the first and second bites. The experiments made in triplicate were performed at room temperature.
934
3. RESULTS AND DISCUSSION 3.1. Physical characteristics of low-calorie jellies In this study we intend to investigate if a correlation between sensory evaluation and instrumental measurements of the Iow-methoxyl pectin jellies could be established. Texture profile analysis is an empirical technique of double-penetration that simulates two bites of the jaw action. Data obtained from the Force-time plots enable the evaluation of seven texture parameters (Figure 1). Hardness is an estimation of the required force to penetrate jelly (peak force during the first bite). The results showed that jellies prepared with non-amidated pectin had such a low hardness that values could not be measured in the used instrumental conditions. Therefore the non-amidated pectin will not be considered in the other parameters interpretation. This fact agrees with the general information that non-amidated pectins usually require more calcium ions than those already present in the juice for a good gelation (Pedersen, 1980; Pilgrim et al, 1991). As far as amidated pectin and the sunflower pectin for range concentration under study (0.5, 0.75 and 1%) are concerned, the behaviour of jellies was similar, hardness values increasing for higher concentration levels (Figure 2). The lowest value was registered for 0.5% amidated pectin and a maximum was reached at 1% of the same pectin. The cohesiveness that represents the work required to overcome the internal bonds of the sample (jelly) shows an increase with the pectin content of jelly and there are no apparent differences between the two types of pectin considered (Figure 2). Similar behaviour was observed for springiness (elasticity) which is given by the time that the material spends to recover its non-deformed condition after the first bite (Figure 2). Adhesiveness, defined as the work necessary to overcome the attractive forces between the surface of the sample and the surface of other materials with which the food comes into contact, e.g. tongue, teeth, palate, etc. (Szczesniak, 1963), is given on the texturometer curve by the negative force area, representing the work needed to pull out the plunger from the sample. This parameter's value may be considered an evaluation of stickiness of jelly. Fracturability, also called brittleness, is given by the measure (%) of the plunger path into the jelly when it breaks. The jellies analysed had no stickiness at all and were non-brittle for the levels tested. This finding, especially as far as sunflower pectin is concerned is quite different from what could be expected, based on Chang's results (1992). The evaluation of two other parameters results from simple arithmetics: gumminess is the product of hardness x cohesiveness and chewiness is given by the product of hardness x cohesiveness x springiness. The results obtained for the analysed jellies are shown in Figure 3. As one would expect, both parameters increase with pectin concentrations as it was likewise observed with hardness, cohesiveness and springiness.
935 M1 /~eas (g s) 1. 16.509 2. 14.SS2 3. N/A
!
2
Peaks (4~) 1, 15.6 2. 14.7 3. N/A
I
-0.0
S~'oJn : N/A
ll~ ~ |llVintJPllill II
,+0
0.
l
,Ill 32 +0
,,.
I n ~ l l Stress : 3.997E§ lll/n.+ Ploduot Height : N/A Compression : 2.000 mm Resilienoe : N/A
Springiness : -0.900
Gumminess :
Cohesiveness :0.877
Adhesiveness : N/A
Chewiness :
In~ad Modulus : N/A
12.314
13.683
Fraoturabilil~ : N/A
N/A
Hardness:
N/A
IS.6 g
Figure 1. TPA profile (Sunflower pectin 1%)
18,000
Q Springiness
] i El Cohesiveness 1 G Hardness
16,000 14,000 12,000 10,000 8,000 6,000 4,000 2,000 0,000
-o
~
~
of)
(f)
c/)
Figure 2 Hardness, Springiness and Cohesiveness vs. pectin concentration
936
16,000
EIChewiness I
14,000
i OGumminess
12,00010,0008,000
6,000 4,0002,000-
0,000-
e
9 ~n
. . . . . . . .
o ~ ,~.-
Figure 3 Gumminess and Chewiness
0,8
IA50
I,~
Ln
pectinconcentration
vs.
l
i
o,6 +
! I
I 0,4 4
I
0,2 !
IIA75 9
XS50 X AC
,
/t
/
"
..
XTI
$100
'
/ / ,,
/ '
/ ,, -0,2 '-:-, "~ { .
..04•
-/ " sXw X F l a
~,
$75
'
~ ~
".
'
~"OHad
".
~ x
".
~ '
06"I"
A_~I100
"
XHS ~ "xOA
I
i
Figure 4. Plotting of jellies (6) and variables (12) on a plane formed by the first and second principal components, F1F2. Had= Hardness; Spr.= Springiness; Che= Chewiness; Gum= Gumminess; Coe= Cohesiveness; HS= Sensory hardness; SP= Spreadability; Ac= Acid; Sw= Sweet; Fla= Flavour; TI= Taste intensity; OA= Overall acceptance.
t
937
3.2. Sensory analysis The evaluation of some parameters was quite discrepant probably due to the resort to a non-trained panel. Nevertheless all the answers were considered (maximum value for standard deviation being ___3.0,results not shown). The data from sensory evaluation and texture profile analysis of the jellies made with amidated pectin and sunflower pectin were subjected to Principal component analysis (PC) using the statistical software based on Jacobi method (Univac, 1973). The results of PC analysis are shown in figure 7. The plane of two principal components (F1,F2) explain 89,75 % of the variance contained in the original data. The attributes related with textural evaluation are highly correlated with the first principal component (Had.=0.95, Spr.=0.97, Che.=0.98, Gum.=0.95, Coe=0.98, HS=0.82 and SP=-0.93). As it could be expected, spreadability increases along the negative side of the axis unlike other textural parameters. The overlapping of textural attributes suggests that characterisation of this kind of jellies could be based on the evaluation of a single parameter. The concept of hardness being the easiest to apprehend and due to its close relation with the same sensory attribute, we believe that when jellies are to be appreciated from a textural point of view, hardness may be measured on its own. Overall acceptance is an attribute that seems to be influenced either by textural attributes or the flavour ones. However it is possible to establish correlations between overall acceptance and hardness (Figures 5, 6, 7, 8). The jellies made with sunflower pectin and amidated pectin (level 1%) are very similar (in so far as texture is concerned) but as the polynomial correlation (degree 2) suggests, above 0.7% of sunflower pectin the overall acceptance decreases probably due to the appearance of perceived in-mouth sensations described as "greasy" and "clammy" and leading to an unfavourable appreciation. A decrease in the perception of taste and aroma of the jellies has been noted when hardness increases, which agrees with Chai eta/. (1991) who refer a reduction in the perception of flavour intensity with an increase of gel rigidity which may be related with the available surface area of gel exposed on chewing.
4. CONCLUSION From the results of this study it appears that commercial amidated and experimental sunflower pectins have similar behaviours and from a consumer's point of view there are only small differences mainly related with a visual evaluation of the jellies, and "greasy" and "clammy" tastes. Jellies made with sunflower pectin show small air bubbles that could be responsible for the slight opacity observed; with amidated pectin jellies are very transparent as it is also referred by several authors.
I
y = - 0 , 0 8 9 9 ~+ ~2 , 3 3 8 9 ~ - 9,7961
I
R2 = 1
4 9 -4 B -4.7 ~46 0
5.4
,
I I
4
r
b
938
5.5
/h e o u e l d e o o e IleJeAO
eoum, d e o o e IleJeAO
5.5 5.4 5,3 5.2 --
y = -0,742+ ~ ~8 , 2 0 0 8 ~ - 17,203 R2 = 1
m 5
10
15
20
4,7
Hardness
46
2
0
Figure 5 Correlation between overall acceptance and hardness (jellies made with sunflower pectin)
a
4 6 Sensory hardness
Figure 6. Correlation between overall acceptance and sensory hardness (jellies made with sunflower pectin)
6
3.-
N
y = 0.781+ ~ 0,759 R2= 0,9998
O
2.-
1
-
y = 0 . 4 5 2 7 ~ 2.0352 R2 = 0,9912
IIRJeA
e0ue%de00e IleJeA O
eoue%deooe
5
~~
0
0-
Figure 7. Correlation between overall acceptance and hardness. (Jellies made with amidated pectin)
2
4
6
8
Sensory hardness
Figure 8. Correlation between overall acceptance and sensory hardness. (Jellies made with amidated pectin)
939
The good results obtained in the production of jellies with experimental sunflower pectin are all the more interesting as this pectin has not suffered any standardisation process like the commercial ones. Such a process could eventually overcome the undesirable characteristics mentioned above. Although this study needs further work it is thought to give some contribution to a better understanding of the behaviour of Iow-methoxyl sunflower pectin in food technology namely in reduced calorie jellies or jams. The results obtained corroborate the interest, already suggested by the authors, in using sunflower agricultural wastes as an important source of natural Iow-methoxyl pectin for these purposes.
5. REFERENCES
Alarc~lo-Silva, M. L. Characterization of a pectin from sunflower head residues. Acta Alimentaria, 19, 1 (1990) 19-26. Alarc~.o-Silva, M.L.; Curado, T.C.; Sousa, I.M.N. Gelificados hipocal6ricos de um sumo de uva por incorpora~o de pectina com baixo teor de metoxilo extraida de residuos de girassol. In: Actas das I Jornadas das Ind0strias Agro-Alimentares, ISA, Lisboa, 1992. Chai, E.; Oakenfull, D.G.; McBride, R.L.; Lane, A.G. Sensory perception and rheology of flavoured gels. Food Australia, 43, 6 (1991) 256-257. Chang, K.C.; Miyamoto, A. Gelling characteristics of pectin from sunflower head residues. J. Food Sci., 57(1992)1435-1443. Leit,~o, M. C. A.; Alarc&o-Silva, M. L.; Janu&rio, M. I. N.; Azinheira, H. G. Galacturonic acid in pectic substances of sunflower head residues: quantitative determination by HPLC. Carbohydr. Polym.,26(1995)165-169. Pedersen, J.K.. Carrageenan, pectin and xanthan/Iocust bean gum gels. Trends in their food uses.Food Chem. 6 (1980) 77-88. Pilgrim, G.W.; Walter, R. H.; Oakenfull, D.G. Jams, jellies and preserves. In: The chemistry and technology of pectin . Reginald H. Walter (ed.) Academic Press, London, 1991, 23-50. Szczesniak, A. S. Classification of textural characteristics. J. Food Sci., 28, 4 (1963) 385-389. Univac. Large scale systems, STAT-PACK, FACTAN - Factor and principal component analysis (1973) 33-39.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
941
Influence of microwave pretreatment of fresh orange peels on pectin extraction
M.Kratchanova a, E.Pavlova a, I.Panchev b, Chr.Kratchanov b aBulgarian Academy of Sciences, Institute of Organic Chemistry, with Centre of Phytochemistry, 95 V.Aprilov Str, P.O.Box 27, LBAS - Plovdiv, 4002, Bulgaria bHigher Institute of Food and Flavour Industries, 26 Maritza Blvd., Plovdiv, 4002 Bulgaria
Abstract Laboratory studies on the extraction of pectin from orange peels, pretreated in an electromagnetic field of hyper frequency, were carried out. The influence of intensity of microwave treatment (Pw) and time on pectin yield and pectin quality was investigated. It was established that the increase of Pw and time lead to increase in the pectin yield with 180 240 % in comparison with the control. Apparently, the microwave treatment leads to a considerable increase in the soluble form of pectin, characterized by increase in the jelly strenght and in the polyuronic content.
Introduction In a previous publication (1) we reported that the pretreatment of fresh fruit waste using microwave heating ensured a better extraction of pectin, resulting in an increase in the yield of pectin from 10 to 50 %. It was established that this microwave pretreatment ensured retention of the degree of esterification of the extracted pectin, better expressed in citrus peels. The aim of the present investigation is to study the influence of microwave pretreatment of fresh orange peels on the extraction time of orange pectin and the influence of the intensity and duration of microwave heating on pectin yield. Materials and Methods Two lots of oranges of the Navel I and II type imported from Greece, were used in this study. The peels were removed, then finely cut and processed following two procedures: part of them were dried in the laboratory drier at 60~ while the rest were pretreated in a microwave oven and then dried in a laboratory drier at 60 ~ Microwave heating of the fresh orange peels The orange peels (200g) were placed in a glass vessel and heated in a microwave oven Samsung with different duration of exposure: 2,5,10,15 and 20 min and with different intensity: 0.45, 0.63 and 0.9 kW.
942 Extraction of pectin The dry mass, obtained atter drying 200 g of fresh orange peels, was subjected to extraction by adding 2.5 L water. By applying 0.5 N hydrochloric acid, pH was adjusted to 1.5. The mixture was then heated to 80 - 82 ~ and extraction was carried out with continuously stirring in a laboratory stirrer for 1 hr (Table 3). The experiments quoted in Table 2 were conducted with 30, 60, 90 and 120 min duration of extraction. The hot mass was filtered through cloth. After cooling, the filtrate was coagulated using an equal volume of 96 % ethanol and let~ for an hour. The coagulated pectin was separated by filtration, washed once with 70 % hydrochloric acid ethanol, then with70 % ethanol to neutral reaction and finally with 96 % ethanol. It was dried at 60 ~ in a laboratory drier. Methods of Analysis The anhydrouronic acid content (AUAC) of the initial material was determined by .the method of Gee (2). Analysis for pectin was implemented by the method of Owens et al. (3). The gel strength was determined by the Tarr-Baker method according to the procedure described by Bender (4). Intrinsic viscosity [~1] and Huggins' constant KH were calculated according to Huggins' equation qsp/C = [vi] + K'H [q]2c according to (5). The average molecular mass Mv was determined by solving the equation following the methods in (6). Results and discussion Data on the pectin content of the initial material (AUAC and DE) is presented in Table 1. It is evident that the main difference between the two lots of oranges was in their anhydrouronic acid content. Observations from previous research (1) were confirmed that microwave heating of the fresh material ensured better drying conditions by inactivating pectolitic enzymes, particularly pectinesterase. Thus a better retention of the degree of esterification of pectin was ensured. Table 1 Analysis of initial materials Kind of materials AUAC, % DE, % Orange peels Navel I 4.8 76,9 Fresh 12.1 68.8 Dry 12.7 72.7 Dried a~er 10-min microwave heating Orange peels Navel II 3.4 73.8 Fresh 13.5 71.7 Dry 14.0 72.8 Dried after 10-min microwave heating
In the first series of experiments (Table 2) the influence of microwave pretreatment on extraction of pectin was followed. Microwave pretreatment had a slight positive effect on the
rable 2 Influence of extraction time on the yield and characteristics of pectin from fresh Navel I orange peels pretreated by microwave heating Sample Nr
Kind of initial material for pectin extraction
1
Dried material - control sample Dried after 10 min microwave heating Dried material - control sample Dried after 10 min microwave heating Dried material - control sample Dried after 10 min microwave heating Dried material - control sample Dried afker 10 min microwave
2
3 4 5 6 7 8
Yield of pectin, g per 200gfresh material
AUAC, %
30 30
3.5 8.7
60.6 68.9
60
3.9 9.4
90 120 120
Extraction time min
60 90
DE, %
Molecul mass
Huggins constant
Gel. strength
M V
KH
Intrinsic viscosity [q]d1.g''
63.3 70.3
72 000 65 000
0.5 0.5
4.5 3.9
175 200
68.6 66.3
64.6 69.4
64000 59 000
1.o 1.1
3.8 3.4
195 215
4.8 9.2
65.8 66.4
63.1 67.8
66 000 60000
0.7 0.6
4.0 3.5
200 225
4.3 8.6
64.5 73.2
60.1 68.2
53 000 57000
1.0 1.o
3.0 3.3
190 205
~~
"TB
943
944
Table 3 Influence of intensity and duration of microwave heating of fresh Navel I1 orange peels on pectin yield and characteristics Time of Yield of pectin, g AUAC, DE, % Molecul Huggins Intrinsic Intensity of viscosity constant KH mass microwave % per 200 g fresh microwave treatment K, heating min material M" [q]d1.g-l 61 600 3.1 3.7 64.3 64.0 2.8 control sample 0.45 0.45 0.45 0.63 0.63 0.63 0.63 0.90 0.90 0.90 0.90
10 15 20 2 5 10 20 2 5 10 15
7.1 7.5 8.5 6.1 7.4 8.4
Gel. strength
"TB 155
63.4 70.7 70.2 67.3 68.9 69.1
70.6 71.0 68.7 67.0 71.3 72.4
60 000 65 000 61 000 60 000 66 000 61 000
3.6 1.4 1.3 2.8 1.2 1.7
3.5 3.9 3.6 3.5 4.0 3.6
165 185 185 165 185 185
70.4 71.8 72.0
70.4 72.6 72.7
54 600 54 500 55 000
3.5 2.5 3.3
3.1 3.1 3.2
190 180 175
material bruns 7.3 8.0 8.7
material bruns
945 duration of extraction - maximal yield of pectin was achieved for ca 60 min; 90 min for the control samples.It may be concluded that duration of extraction has a stronger degradation effect on pectin molecules than microwave treatment of the fresh material. It is worth noting the considerable difference between the yield of pectin in the experimental samples and in the control samples. Apparently, microwave heating of the fruit tissue affects mainly the state of the protopectin - it destroys the bonds of the pectin macromolecules with the other polymers and thus protopectin turns into a water-soluble form. At the same time there is a slight positive effect on the capillary permeability of the fruit tissue and hence on the following process of penetration of the molecules of the solvent. It is worth noting the data on the intrinsic viscosity and the molecular mass. Data for experiments 1-6 show that microwave treatment rather leads to disintegration of the association bonds than to distinct depolymerization effect on the pectin macromolecules. This is observed in terms of a slight decrease in the molecular mass and a distinct increase in the gel strength force of pectin. Data from experiments 7 and 8 (long-lasting extraction) are a deviation from this dependence which is in support of the conclusion that long-lasting extraction in an acid medium has a strong negative effect on the quality of pectin. A sharp decreased yield of pectin was also observed in the control samples. The next series of experiments was dedicated to studying the effect of the intensity of the microwave field and duration of microwave exposure on the yield and quality of pectin. Data from oranges of the Navel II type (Table 3) show there is an inverse correlation between the field intensity and duration of exposure mainly expressed for 0.45 and 0.63 kW intensity. In case of a weaker field, longer microwave treatment is needed. According to expectation, a stronger destructive effect of microwave heating was observed at the top intensity of 0.90 kW: the molecular mass of pectin decreased by 10 %, accompanied by a slight increase in the yield. Duration of acceptable microwave heating was reduced for the higher values of the field intensity because of burn of material. The results presented confirmed previous observations by our team (1) and other authors (7) about the favourable influence of microwave heating of pectin row material on the yield and quality of extracted pectin. Data from the present experiments show that microwave destruction processes on biopolymers in the flesh fruit tissue can be overcome by choosing the intensity of the field and duration of exposure. Microwave effect is rather in terms of a denaturation process on the protein molecules, polysaccharide associates and protopectin, than a depolymerization process. An evidence that speaks for itself is the increase in the gel strength of the pectin, obtained from microwave pretreated materials, compared to that for the control sample: a sharp increase in the gel strength at the same or slightly lower molecular mass. On the other hand, comparison with data from Manabe's publication (7) shows that a more favourable microwave effect is accomplished when the raw material is treated before extraction instead of during pectin extraction. This conclusion is more favourable for industrial production of pectin- the orange raw materials should be. first subjected to microwave heating and then dried. Acknowledgement: The authors thank the Research Foundation of Bulgaria for the financial support of this work (project CC-457).
946 REFERENCES 1. M.Kratchanova, I.Panchev, E.Pavlova, L.Shtereva. Carbohydrate Polymers, 25 (1994) 141. 2. M.Gee, E.A.Mc Comb, R.M.Mc Cready. J.Food Sci., 23 (1958) 72. 3. H.S.Owens, R.Credy, A.Chepheral, T.Shultz, E.Pippen, N.Swenson, J.Miers, F.Erlander, W.Maclay. AIC Report 340, Western Regional Research Laboratory, Albany, CA, 1958. 4. W.A.Bender, Analyt.Chem., 21 (1949) 408. 5. H.Moravettz, Macromolecules in solution Interscierice Publishers, New Yormk, (1967) 254. 6. H.Anger, G.Berth. Carbohydrate Polymers, 6 (1986) 193. 7. M.Manabe, I.Naohara, T.Sato, J.Okada. Nippon Shokuhun Kogyo Gakkaishi, 35 (1988) 497.
Visser and A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
J.
P r o p e r t i e s of p e c t i n e s t e r a s e from Penicillium new developments in p e c t i n a p p l i c a t i o n s
947 fellutanum
V.L. Aizenber 9, S.A. Syrchin, S.A. Sedina, V.N. Vasil'ev, Demchenko, P.N.Vitte
Biourge
L.N. Shinkarenko,
and
P.I.
Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine, Kiev, Ukraine Abstract Some properties of Penicillium fellutanum pectinesterase were studied. The optimum of pectinesterase action was detected at pH 5 and 45 ~ The enzyme was stable at pH 4 - 5 and 40 ~ (pH 5)"for 240 min. and was specific towards lemon pectin. An enzyme preparation composed mainly of pectinesterase was partially purified by gel filtration. Pectinesterase activity was accumulated in one of the obtained fractions. Molecular weights of fraction determined were found to be 46,000 and 1,200. Disk electrophoresis in polyacrilamide gel of the purified preparation revealed two protein bonds with one active component. The partially purified enzyme had the kinetic characteristics: Vm = 14.7"10 -5 M*min -1', Km = 5.56"10 -3 M; Ks = 0,22 M. New preparation of pectinesterase is recommended for production of lowmetoxilated pectin example for medicine. 1. I N T R O D U C T I O N
Pectinesterases are formed by numerous fungi but knowledge about fungal pectinesterase is limited [1], Capacity to synthesize extracellular enzymes depolymerising pectin was studied in 340 fungi isolates of the genera Penicillia belonging to 38 species of five sections (according to classification of Pidoplichko} [2]. There are a few data in the literature concernin 9 ability of Penicillia to split pectin substances in c o m p a r a t i v e - t a x o n o m i c aspect. The species, which were not described previously (such as P. fellutanum, P. thomii and P. multicolor) have been found among these possessing pectolytic activity. Such species of fungi as P. wortmanii and P. velutinum were found to be potentially low active. New n o n - t o x i c strain P. fellutanum Biourge from the soil of Kiev region was selected, It was characterized by the predominant pectinesterase synthesis [3]. Subject of the present message is study of the conditions for Penicillium fellutanum cultivation (that is a producer of pectinesterase) and analysis of some properties of pectinesterase enzymatic preparation. Miller and Macmillan [4] carried out purification of pectinesterase from Fusarium oxysporum f. sp. vasinfectum culture fluid (fivefold degree of purification). According to the obtained data the purified enzyme possessed very low polygalacturonatlyase one. Disk electrophoresis at pH 4.3 revealed two protein components. The authors did not study distribution of pectinesterase activity in these components. Molecular weight of fungal pectinesterase determined using gel filtration on Sefadex G - 75 was found to be 35,000. It was reported [5] that 4 active components with molecular weight of 56, 000, 30,000, 10,000 and 1,600 were obtained by separation of complex enzyme preparation from Penicillium citronum usin 9 a Sephadex G - 7 5 column. Three components revealed both pectinmethylesterase and pectinlyase activities, and the last one mentioned revealed only pectinlyase activity.
948 2. MATERIALS AND METHODS In the study culture of p. fellutanum, strain 57599 was used, which was grown by d e e p - g r o w t h method in flasks, dia 750 ml on Chapec medium with addition of beet pectin (1%) as a source of carbon or beet pulp (4%). It was grown on the rockers (220- 240 rev/min.} at 2 6 - 28 ~ For study of substrate specificity pectin with various degrees of metoxilation (expressed as a percentage) were used: beet substrate-37.8, apple s u b s t r a t e - 7 0 , l e m o n - 8 2 . Specificity of pectinesterase action was analyzed under optimum temperature and acidity of the medium using beet, apple and lemon pectin according to the speed of methanol formation (M 910 .5 ' rain.Enzymatic preparation with predominant content of pectinesterase (obtained from Penicillium fellutanum culture liquid by isolation by acetone was purified. Primary enzymatic preparation was r e - p r e c i p i t a t e d by three volumes of ethyl alcohol and centrifuged (6000 rev/min.} during 20 rain. The obtained precipitate of partially purified pectinesterase preparation was dried in v a c u u m - d e s i c c a t o r . Sephadexes G 50, G - 7 5 , G - 1 0 0 , G - 2 0 0 "LKB" (Sweden) and Toyopearl H W - 5 5 (Japan) were used for separation of enzymatic complex by gel-filtration. The specimens were analyzed at spectrophotometer at 280 nm. Preparations were purified from salts by dialysis. Protein concentrations in the initial and purified enzyme preparations were determined by Lowry method [6]. Molecular weight of the components of the enzymatic complex was determined using a Sephadex G - 7 5 column after its calibration by dextrans with molecular weight equal to 10,000, 40,000 and 70,000 and rafinose with molecular weight of 504. Fractions were also analyzed by the disk-electrophoresis method in PAAG [7] using 7.5% polyacrilamide gel (pH 4.3). Activity of pectinesterase was determined by titrometric method [8]. The enzymatically released methanol analyzed by g a s - l i q u i d chromatography [9]. 1% solution of highlymetoxilated beet pectin (made by "Biochimreactiv" co.) was used as a substrate for enzymatic activity determination (degree of metoxilation is equal to 37.8%}. Pectinesterase activity expressed as a unit corresponding to the microequivalent of ester bonds of pectin molecule, which were hydrolyzed during 1 rain. at 45 ~ and pH 5.0 under the conditions, which were optimum for these enzymes. Endopolygalacturonase and exopolygalacturonase activities were determined using a technique determined by Lifshitz [8]. Activity of pectintranseliminase was determined by procedure [ 10]. Speed of pectin hydrolysis catalyzed by pectinesterase was measured according to the quantity of alkali, used for titration of the free carboxyl groups during one minute (M'min-1} at 45 ~ Acidity of the reaction mixes after incubation increased as the activity of the probe r o - s e during determination of pectinesterase activity of the samples.It was caused by the f o r - m a t i o n of carboxyl groups as a result of pectin ester bonds hydrolysis under pectinesterase a c - t i o n . T h a t is why kinetic characteristics of substrate hydrolysis were measured according to the speed of pectin hydrolysis by continuously recorded titration of the free carboxyl groups [11]. After Michaelic constant (Kin} determination pectin concentration with known esterification degree was expressed in m o l / M according to the content of anhydrogalacturoni c acid. Kinetic parameters of the reaction of pectin hydrolysis (catalyzed by analyzed pectinesterase) were obtained by Z i n e w e a v e r - B e r k method [12].
949 3. RESULTS AND DISCUSSION Peak of relative activity was observed at pH 5.0 when it was determined pH optimum of P. bellutanum pectinesterase action. Under pH 4 and 6 the above mentioned activity accounted for 60% of its maximal value. Study of the temperature optimum of pectinesterase activity showed, that peak of pectinesterase activity was observed at the temperature equal to 45 ~ It is shown on Figure 1 that pectinesterase was stable at pH 4 - 5 . At pH 2 activity of the enzyme reduced by 25% in 60 min., at pH 3 and pH 6 it decreased by 6 - 8 % . At pH 8 the activity decreased by 90.7% during the same time. At pH 9 the enzyme activity was inactivated during 15 min. Fig. 1
Determination of pH-stability pectinesterase from P.fellutanum
100 90
pH 2
80 >
pH 3
7o
---)le--
pH6 ,...,
o >
pH 7
E:
pH8
10
,
0
.
10
.
.
.
20 30 40 Incubation time, min
60
.
.
.
.
.
,It was found out that P. fellutanum pectinesterase was sensitive to the temperature of 5 0 - 6 0 ~ At this temperature gradual decrease of the activity was observed. For example at 50 ~ the initial activity of the enzyme decreased by app. 43% in 60 min., at 60 ~ it decreased by 94% during the same time. At 65 ~ the enzyme was nearly totally deactivated yet in 20 min. The obtained data show kinetics of enzymatic activity at various temperatures within various time intervals. Pectinesterase was stable at the temperature of 40 ~ (pH 5) during 240 min. (Figure 2}.
950 Fig 2.
Determination of termostability of pectinesterase from P.fellutanum
100~=
'
9oi
=
-
= -
---
= .... =
=
=--
=---
............................................................................................... -]
80
..................................................................... -------...._...._..
70
~ >~
40
40 ~ §
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0 50
m
50 ~
.................................................. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
..............
30
60 ~
. . . . . . . . .
0
...........................................................................................
10
.......
10
2()
30
40
50
60
90
120
240
Time, minutes
Data given on Figure 3 show that optimal concentration of studied pectin varied from 0.75 to 1%. Under this concentration maximal speed of released methanol (caused by pectinesterase) was observed. It was equal to 1 , 8 2 - 2 . 0 2 M*I0 - s * rain.-1. Both low (0.25?/o) and high (3%) concentrations hindered methanol release. Fig 3,
Speed of methanol formation during enzyme action on various substrate ._c E 2.2! / u~"/ '0 2"
.........................
~ .............
9
/
o
1.8 -!
0
/
E
............................................................
1.6.i 1.4-
~0 1.2-
pectin=
/
/
_ / .... / .
/
/
/ , . /
~e 0.8 /00.25u.5'0.75' 1 "1,25' 1.5 '1.75' 2 ' 3 Pectin concentration, %
;eet '~le
951 Degree of pectin metoxylation is the main factor that determines the pectinesterase action. Lemon pectin was hydrolyzed most easily among all pectin studies: the speed of methanol release was 2.02 M* 10 -5 * min.-1. This is accounted by the fact, that the degree of the lemon pectin metoxylation is higher than in case of beet and apple pectin, All above mentioned shows, that pectinesterase from P. fellutanum possesses specificity towards lemon pectin (Figure 3}. P. fellutanum 57699 is characterized by the predominant synthesis of pectinesterase. Enzymatic complex produced by the fungus is distinguished from known industrial producers for increased content of extracellular pectinesterase of pectolytical complex. After purification of the enzyme preparation both at Sephadexes of various types and at Toyopearl H W - 5 6 two fractions were obtained (fractions 1 - 2}. One of them (fraction 1} possessed pectinesterase activity under the condition of absence of the activity of other pectolytic enzymes. Molecular weights of fractions I - 2 determined using a Sephadex G - 7 5 column (after its' calibration by various dextranes} were found to be 46,000 and 1,200. The results obtained after purification of pectinesterase preparation using columns with various gels certified that the active fraction (fraction 1 has greater molecular weight} was not subjected to further separation on the tested gels. Components of fraction 1 increased an activity of elute. That can be explained by their acid properties. Pectinesterase activity was accumulated in fraction 1. Activity of the other components of pectolytic complex was not found in the other studied probes of fraction 1. Disk electrophoresis o f pectinesterase enzyme preparation in polyacrilamide gel revealed 4 protein bands in the fraction 1 after its g e l - f i l t r a t i o n through Toyopearl H W - 5 5 . One of the two obtained bands (a wider one} possessed pectinesterase activity, while the second one did not reveal it. Partial purification of pectinesterase preparation enlarged to some extent its specific activity. A number of kinetic characteristics were obtained with preparation of the fraction 1. It was found out that reaction of the hydrolysis of highlymetoxilated beet pectin (catalyzed by P. fellutanum pectinesterase} obeyed M i c h a e l i s - M e n t e n equation only under low substrate concentrations (up to 1.2%}, when graph of the dependence of reaction speed was hyperbolic in form. In case of t w o - s t a g e enzymatic reactions, which did not obey M i c h a e l i s Menten e q u - a t i o n reaction speed was a t its maximum and then decreased.Graph of speed of substrate h y d - r o l y s i s against In concentration acquired a shape of symmetric or asymmetric bell (Figure 4}.
952 Fig 4
10g 8 7 5
ZnZopt,
q -6
-5
-q
-3
Is] Figure 4. Initial speed of the pectin hydrolysis reaction catalyzed by pectinesterase: 1 - theoretically calculated symmetric bell, 2 - e x p e r i m e n t a l l y obtained curve.
In our study the traditional pattern was changed when the concentration of substrate was enlarged. Figure 4 shows, that speed of the reaction of hydrolysis plotted against concentration of substrate yields a line in the shape of asymmetric bell. R i g h t p a r t of the plot has the smaller slope at great concentrations of substrate as against calculated one for t w o - s t a g e reaction. All above stated is the evidence of the formation of the triple e n z y m e substrate complex. It possesses some activity but its activity is lower than that of e n z y m e - s u b s t r a t e complex, i.e. B is other than zero (0
953
Fig 5.
1 8000 ..o~..V
..... :.k ..... -~.'..:........... 3 ....... :.'.'.,.w.......
6000 .~
~
<
4000
:~
.:~j,,.--/;~" 1
2000
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~.o
,,
h .-''~
9 i.~,:
" ' " : : " " : ............... ,..-r
..... w - , W . = ' = " : ' " ' " - - - ~
..................
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5 glr'-~...4[l" ...........
. .. ,...,.V
" "
-
,"
I
I
5
10
I .....
15
l
I
20
25
'1
L__
30
Time, days
Fig. 5. Accumulation of 1 2 3 4 5 6
-
-
-
-
---
85 Sr in the rats ( Daily dose 1300 per/animal )
conf~oI; pecan; acb've c a r b o n e ; c a r b o n e - - p e c t i a e - - vitam#2 c o m p o s i t i o n sodium algiaate; pec~'ne--vitamkle composidon. .
tablets;
Proposed a reasonable approach of i n t e r v e n t i o n by pectincontaining e n t e r o s o r b e n t s a n d carried it o u t a m o n g the c h i l d r e n of Kiev C h i l d r e n g a r t e n ' s ("Malyatko") in m o r e t h a n I00 children. A p p l y of this c o m p l e x allow essentially b r i n g to lower c o n t e n t s of t h e 137 Cs in t h e b o d y (fig. 6 ), It is r e c o m m e n d e d for t h e p o p u l a t i o n (first of all of the children) w h o r e s i d e n t in ecologically a d v e r s e c o n d i t i o n s to u s e of p e c t i n c o n t a i n i n g p r a p e r a t i o n forms [ 13]. T h e r e are the following i n t e r v e n t i o n levels for u s e of the different p r e v e n t i v e measures: l} p e r s o n u n d e r study; 2} e n t e r p r i s e or children cohort in childrengarten; 3) r e g i o n or district; 4) n a t i o n a l level. For all risk factors a n d their c o m b i n a t i o n s w e will use both routine and specially d e s i g n e d a p p r o a c h e s to risk m a n a g e m e n t with d u e a c c o u n t of n e w c o m b i n a t i o n s of t h e s e factors. T h e p r o p o s e d a p p r o a c h will allow to d e v e l o p s t r a t e g y of p r e v e n t i v e i n t e r v e n t i o n into risk m a n a g e m e n t of t h e c o h o r t s w h o w o r k i n g or r e s i d e n t in ecologically adverse conditions to use of p e c t i n c o n t a i n i n g p r a p e r a t i o n forms.
954 Fig. 6
PREVENTIVE ACTIVITY WITH PECTINE EFFECTIVITY
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DECREASE OF/37Cs C O N T E N T IN THE BODE % We are very thankful to Mr.G.A.Nikitin, Professor of Ukrainian University of Food Technologies and to Mr.S.L.Upolnikov, the Director of CDK "CIET" company for their assistance in preparation of this paper
4. REFERENCES i. Rombants FM, Pilnik W. Microbial Enzymes and Bioconversions, 5, 1980. 2. Pidoplichko NM. Penicillia. Ukraine, Kiev, 1972. 3. Aizenberg VL, Bilay TI, Vasil'ev et al. USSR Patent No. 985022 (1982}. 4. Miller l, Macmillan LD. Biochemestry, 10, 4, 1971. 5. Olntida PO, Akintude OA. Trans. Brit. Mycol. Soc., 72, 1, 1979. 6. Lowry OH, Rosenbrough NI et al. J. Biol. Chem, 193, 1, 1951. 7. Davis BI Ann.N.I. Acad. Sci., 121, 2, 1964. 8. Lifshits DB. Russia, Moscow, Viniti publ. house, 1971. 9. Baron A, Rombouts FM, Drilleau I, Pilnik W. Z e b e n s - W i s s . Technol., 13, 6, 1980. 10.Nagel CW, Vaughn RN. Arch. Biochem. and Biophys., 94, 1961. 1 1 . R e x o v a - B e n k o v a L, Markovic O. Advan. Carbohydr. Chem. and Biochem., 33, 1976. 12.Beresin IV, Klesov AA. Russia, Moscow, MGU, 1976. 13. Traktenberg I.M.I Litenko V.A., Dereviago I.B., Demchenko P.I., Mikhailovskij C.V. Vrachebnoje Delo, 5, 1992
APPLICATIONS.
B) APPLICATION OF PECTINASES IN BEVERAGE, FOOD, FEED AND NOVEL TECHNOLOGIES
This Page Intentionally Left Blank
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
Enzymatic maceration of apple parenchyma degradation
957
: modelling
of
the
A. Baron, P. Massiot, C. Ella Missang & J.F. Drilleau Institut National de la Recherche Agronomique. Station de Recherches Cidricoles, Biotransformation des Fruits et IMgumes, BP 29, 35650 Le Rheu, France.
Abstract The work aimed to study the phenomena which took place in an enzymatic reactor where dices of apple parenchyma tissue were macerated by polygalacturonase. The degradation rate was very fast at the beginning of reaction (74 mg of AIS / h) but slowed down with time (2.6 m g / h after 7 h). Loss of enzyme efficiency was mainly due to the enzyme distribution in the different compartments resulting from the enzymatic maceration. This study lead to a kinetic model describing the tissue degradation during the reaction time. It was supposed that two mechanisms were involved in the degradation process. The first one is very fast, affecting the peripheric part of the parenchyma dices where most of the enzyme is adsorbed while the second mechanism is slow for it affects the deeper zone of the tissue where the enzyme amount is low.
1. INTRODUCTION The growing importance of enzymatic treatment in the industrial processing of fruit requires basic studies and control of the enzymatic mechanism reaction. Acting alone, polygalacturonase (PG) degrades pectic substances from middle lamella causing the release of single cells and soluble pectin. This process, called "maceration", is used to desintegrate tissues for nectar procuction. The object of this research was to study the phenomena which occured in an enzymatic reactor where dices of apple parenchyma tissue were macerated by PG.
2. MACERATION KINETIC During the maceration, three products were distinguished : (1) residual parenchyma, (2) pulp which consisted of free ceils, and (3) juice which containted soluble pectins and oligouronides. Each of these products was estimated by its content of Alcohol Insoluble Solid (AIS).
958
100, 80 C'D Or~ ~ ~
Parenchyma
601
Pulp
40
20]/
Juice
0
10 ,,
,
20
Time (h) ,
,,,
30 ,
,
Figure 1 Maceration kinetic of apple parenchyma by polygalacturonases. The open symbols are the observed values. The continuous line are the values predicted by the model (see Fig 3 and table 1).
The main product was pulp (up to 50 % of total AIS), and the precipitable pectin of the juice reached 17 % of the total AIS (Fig. 1). The degradation rate of the tissue was very fast at the beginning of the reaction (74 mg of AIS / h) but slowed down later (2.6 m g / h after 7 h).
3. P O L Y G A L A C T U R O N A S E
DISTRIBUTION
Loss of enzyme efficiency was due to short and long-term PG distribution between the different products (Fig. 2).
959
A
IO0
"~
IO0
80
80
60
60
40
40
JUiCe
20
O~r 0
9
, 1
9
, 2
9
, 3
B Pulp Juiee
20 9
,
,
4
Parenchyma
, 5
1
Time (mini
2
3
4
5
6
Time (h)
Figure 2. Recovered PG activity in the different compartments. A" Short-term distribution B" Long-term distribution
3.1. Short-term distribution During the early stage of the maceration (up to 5 min approx.), before any cells had been released, 70 % of the enzyme poured into the reactor was adsorbed into the tissue resulting in a very high enzyme activity. 3.2. Long-term distribution As the reaction went on, the activity in the tissue fell (12 % after 30 min and 3 % after 6 h) while it increased in the pulp (55 %) and in the juice (from 32 % to 40 % between 30 min and 6 h of maceration) thus limiting the enzyme fraction available for the continuation of the tissue maceration.
4. MODELLING OF THE KINETIC 4.1. Hypotheses The first assumption had been that there were two mechanisms involved in the degradation process. (Fig. 3) The first one was very fast (rate constant kl) affecting the peripheric part of the parenchyma (M1) where most of the enzyme was initialy adsorbed. The second mechanism was slow (rate constant k2) for it affected the deeper zone of the tissue (M2) where the enzyme amount was low. The second assumption was that the maceration released cells C 1 and hydrosoluble pectin P1. The cell wall of these cells may have been partially degraded to produce non-degradable cells C2 and pectin P1. P1 was also partially degraded to ultimate alcohol precipitable pectins P2 and oligouronides O.
960 Parenchyma degradation kl
M2
k2
,,~
P Cl
~.-
qC1 + (1-q) P1
+
(l-p) P1
C~ free cells degradation C
k3
~
r C2 +
( l - r ) P1
P~ solubilized pectin degradation P1
k4
-~"-
sP2
+
(1-s) O
Figure 3. Model of apple parenchyma maceration by polygalacturonases M, C, P and O are the weights of parenchyma, free cells, soluble pectin and oligouronides, respectively
4.2. Mathematics T h e s i m u l t a n e o u s d i f f e r e n t i a l e q u a t i o n s are as f o l l o w s 9 - Parenchyma compartment
dM, - ~ - - -k,M, d-~Mt--J'- - k, M~ M - M, + M,
- Pulp compartment
dC, -~- = pk, M, + qk, IVl,-k,C, ~ t " rk. C, C= C, +C.
961 - Juice compartment
dE',
-~- - (1- p)k, M , . (1- q)k, M= + (1- r)k,C, - k,P, --~Pt'- sk, P, P-P, +P=
4.3. Model verification
Table 1 summerizes the values of the parameters used to fit the data of the Fig. 1.
Table 1 Parameters values used to fit the maceration kinetic data Initial AIS (m~;) Rate constants Mol Mo-z kl k2 k3 k4 37.2 62.8 1.55 .27 9.98 20.21 i
,,,,
,,,
Products ratio q r .57 .85
,,
,,
p .68
S
.32
C1 and P1 were transitory compartments (Fig. 4). C2 and P2 were ultimate ones.
10
."~ ~~:: '~20,10. r./3 0
0
2~~cC12 C 9
1
P2
A ,
2
=
3
=
4
,
5
,
6
0
1
B 2
T i m e (h)
3
4
5
6
Time (h)
Figure 4. Predicted composition of the pulp and juice compartments A" Pulp composition ; B: Juice composition Modifications of the cell wall composition of released cells occured during the maceration (Fig. 5). In pectic polysaccharide, galactosc increased whereas galacturonic acid and arabinose decreased. Cellulose and hemicellulosc compositions were not modified.
962
Pectic sugars 100 %
100 %
Non-pectic sugars
Galactose
Glucose
Arabinose
50 %
5O %
Rhamnose Galacturonic acid
0% 0.51
2
4
0.51 2 4 8 Time (h)
8
Time (h)
Manose Xylose
0%
Figure 5. Sugar composition of cell wall polysaccharides from pulp
,_ 28h:_ ....... ~ E a)
I
I
1
2h
E/3
o 03 eL)
lh -
r ~ f.,.-4
~
E o o
_
_--
0.5 h
. . . . . . . .
Control
./ 0
Kav
1
Figure 6. High-Performance Size-Exclusion Chromatography of solubilized pectin from the juice. Control is CDTA extracted pectin from parenchyma. The molecular weight of solubilized pectins in the juice (Fig. 6) progressively decreased and became more dispersed. These two observations were conform with the hypothesis.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
963
Enzymatic treatment in the extraction of cold-pressed lemon peel oils L. Coil, D. Saura, J.M. Ros, M. Moliner and J. Laencina. Department of Food Science and Technology, CEBAS-CSIC, PO Box 4195, 30080 Murcia, Spain. & Department of Food Technology, Campus de Espinardo, University of Murcia, 30071 Murcia, Spain.
Abstract Cold-pressed essential oils from the peel are the first by-products to be recovered during the processing of citrus fruits and any improvement in their recovery is of great interest for the lemon processing industry. An oil recovery system which does not involve recycling results in high water consumption and high volumes of waste water. However, enzymatic treatment allows the aqueous discharges from desludger and polisher centrifuges to be recycled to the extractors, increasing the yield of essential oil and reducing water consumption and waste without harming oil quality. In addition, enzymatic treatment leads to the production of a less environmentally damaging waste.
INTRODUCTION Cold-pressed essential oils from the peel are some of the most important by-products recovered during the processing of Citrus fruits. The presence of limonene in the aqueous discharges, with its antimicrobial activity [1], decreases the effectiveness of the waste treatment system and increases the time necessary for the biological breakdown of the organic matter produced in the peel oil recovery system [2,3]. Additional recovery of essential oils from waste water would increase industry's returns and reduce the pollution problems associated with the disposal of waste water [4,5]. Several methods for reducing the levels of residual essential oils in the aqueous effluent have been developed over the years [6-11]. To remove the essential oil from the peel of citrus fruits, the oil glands, which are located in the flavedo (the outer coloured portion of the peel), are ruptured by mechanical systems. The oil is washed away with a spray of water to produce an oil-in-water emulsion with small peel particles. To prevent absorption of the essential oil by the spongy albedo (the inner white portion of the peel), this emulsion is passed through a screening device (finisher) of 0.5 to 0.7 mm in diameter, which removes the coarsest particles of the fruit peels [ 12]. The essential oil is recovered from the oil-in-water emulsion by a two-stage centrifugation process. The first-stage oil separator, the desludger, breaks down the emulsion by a high-force centrifuge, which separates the oil-in-water emulsion into three phases: an aqueous phase with a very low oil content (aqueous discharge), a solid phase containing the fine peel particles in water (sludge), and an oil-rich emulsion. The oil-rich emulsion is fed into a high-speed
964 centrifuge (polisher), which removes the remaining water and separates the very fine peel particles. With this system it is possible to recover the essential oil from the oil-in-water emulsion with only minimal losses [ 13-15]. In a recycling system, the aqueous discharge effluent from both centrifuges is returned to the extractors for additional oil recovery, the water being reused. During this extraction process the viscosity of the emulsions increases because peel polysaccharides, mainly pectins, are transported with the emulsion. Enzymatic breakdown of the internal links of the pectin, catalysed by endopolygalacturonase activity, produces an important decrease in the viscosity of the emulsion [16]. In addition, enzymatic treatment removes pectins from the emulsion and contributes to it destabilization [ 17]. The objectives of this study were to compare the yields of cold-pressed essential oil, water consumption, material balance and efficiency of the process in a typical citrus peel oil recovery plant with and without recycling system. The different emulsions and aqueous discharges from these processes were also characterized.
MATERIALS AND METHODS To compare a recycling and a non-recycling cold-pressed essential lemon oil recovery system, two experiments in the same lemon processing plant (CITRIMUSA, a Kas-Pepsico Company in Murcia, Spain) were carried out during the processing of mid-season lemons cv Rodrejo. The assays were performed on two consecutive days in order to avoid difficult to control parameters such as place of origin, lemon ripeness, maintenance conditions of the machines, room temperature, etc. The plant consisted of four FMC In-line extractors (FMC, FL, USA). After extraction, the oil-in-water emulsion passed through a finisher. The finisher emulsion was discharged into a 500 L water-tank that fed two desludger separators. The oil-rich emulsions from the desludgers were fed into a second stage centrifuge (polisher). Both desludger and polisher centrifuges were from Alfa-Laval (Lund, Sweden). In the recycling system, the aqueous effluents from the desludgers (aqueous discharge I) and the polisher (aqueous discharge II) were discharged into a holding water-tank, from which they were recycled to the extractor. The holding time of the aqueous effluent in this tank was approximately 30 min. To start the recycling system assay, 1200 L of water and 600 mL of the technical enzyme (0.5 mL enzyme/L of water) were added to the 2500 L holding tank. A periodical addition of water and enzyme at the same concentration was necessary to compensate for losses caused by the peel residues and centrifugation. The open system assay was performed in the normal way as reference, without recycling or enzyme addition. The technical enzyme used was Novoferm 14 (NV14) from Novo Nordisk Ferment Ltd. (Dittingen, Switzerland). This preparation is produced by a selected strain of Aspergillus niger and contains a mixture of different enzymatic activities, mainly pectinases, arabanases, hemicellulases and cellulases. Within the pectinases, the preparation contains only very little pectinesterase activity and the polygalacturonase/pectinesterase ratio is very high. NV14 has a optimum pH for polygalacturonase activity of approximately 5.5 [ 18]. Emulsions and aqueous discharges were analyzed for pH, soluble solids (~ recoverable oil with the Clevenger method [19], total pectin substances as anhydrogalacturonic acid [20,
965 21] and viscosity. The viscosity of the emulsion samples was measured at 30~ with a DV II viscometer from Brookfield (Stoughton, MA, USA). Both the low concentration emulsions and those containing high concentrations of pectin hydrolysed by the endopolygalacturonase activity were of low viscosity. Since both types of emulsion exhibited a Newtonian behaviour a Brookfield viscometer was adequate for measuring their apparent viscosity. Emulsion flow and that of the aqueous discharges were measured volumetrically in holding tanks. To make the material balance, the essential oil levels of the peel were measured before extraction by coldpressing. To control the quality of the lemon oil, the cold-pressed oil was analyzed in a HewlettPackard 5890A (USA) gas chromatograph equipped with a Hewlett-Packard 7673 autoinjector and a flame ionization detector. Samples (0.6~tl) were separated on a fused silica capillary column OV-101 (50m x 0.25ram). Retention time and percentage composition were determined in a Shimadzu CR-1A integrator (Japan). Specific gravity, angular rotation and refractive index were examined according to the Food Chemicals Codex [22] and United States Pharmacopeia [23].
RESULTS AND DISCUSSION Evolution of pH In the non-recycling system, the pH values of all the samples become stabilized at around 3.4-3.5 atter 180 minutes. However, the pH values were lower (3.2-3.4) in the recycling system because recycling of the aqueous discharges had the effect of concentrating the aqueous phase of the organic acids. In both systems the aqueous discharges from the desludger show the lowest pH values of the respective assays. The optimum pH for the polygalacturonase activity of NV 14 is 5.5. At pH 3.5, NV14 has only about 30% of the polygalacturonase activity shown at pH 5.5 [ 18]. Evolution of soluble solids The soluble solids in the non-recycling system were very stable, with a value lower than 2.0~ In addition, the soluble solids content of the oil-rich emulsion was lower than in the other emulsions (Figure l a). Soluble solids in the aqueous discharge from the desludger (1.5~ were lower than those reported by Steger [4] and Parish et al. [5] in the aqueous discharges from a non-recycling orange peel oil recovery system (2.2 and 3.8~ respectively). In the recycling system, soluble solid content of all the samples was bigger than in the nonrecycling system (Figure lb). The recycling system presented the higher value for the oil-rich emulsion from the desludger, while the levels of the other emulsions tended to fluctuate.
966 8
8
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6
o
o
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Time (min) -e- Oil Finisher Emulsion
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Time (rain)
-I-Aqueous Discharge I
-e-Oil Finisher Emulsion
--]-Aqueous Discharge I
I
~ Oil Rich Emulsion
~
Oil Rich Emulsion
Aqueous
Discharge
II
Figure 1a Evolution of soluble solids. Non-recycling system.
Aqueous
Discharge
II
Figure lb Evolution of soluble solids. Recycling system.
Evolution of pectic substances In the non-recycling system, the pectic substance content of the different samples were very stable (Figure 2a). At the end of the assay the oil-in-water emulsion from the finisher and both aqueous discharges had very similar values of around 0.2 g AGA/L, similar to that reported by Parish et al. [5] for the aqueous discharge from an orange peel oil recovery system. However, the oil-rich emulsion had higher values (around 4.0 g AGA/L). 12
12
9 0
0
60
120
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240
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360
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480
0
,
60
.
,
.
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Time (rain) -O- Oil Finisher Emulsion Oil Rich Emulsion
,
180
.
.
.
.
240
.
300
,
i
360
,
420
.
480
Time (min)
-~--Aqueous Discharge I
Oil Finisher Emulsion
--+-Aqueous Disclmrge I
D
Oil Rich Emulsion
~
Aqueous
Discharge
II
Figure 2a. Evolution of pectic substances. Non-recycling system.
Aqueous
Discharge
Figure 2b. Evolution of pectic substances. Recycling system.
II
967 The continuous accumulation of peel polysaccharides in the recycling system meant that the content in pectic substances was higher than in the same samples for the non-recycling system (Figure 2b). The oil finisher emulsion and aqueous discharges from the desludger and polisher showed a similar evolution, with final values of around 1.3-1.8 g AGA/L. The oil-rich emulsions had the highest levels of AGA, which stabilized around 300 minutes after the beginning of the experiment at about of 10 g AGA/L. In both experiments the pectic substance content of the oil finisher emulsion was higher than in the aqueous discharges. Evolution of viscosity In the non-recycling system all samples had a stable viscosity, the oil-rich emulsion having the highest viscosity with values of around 32 mPa.s (Figure 3a). In the recycling system, the oil rich emulsion also had the higher viscosities although with a wavering profile (Figure 3b). This appears to be a general behaviour in a recycling system using enzymatic treatment [24]. From an initial viscosity, during the first 90 minutes, of around 20 mPa.s, its viscosity increased to reach a maximum of about 141 mPa.s at 300. After 300 minutes the viscosity of the oil-rich emulsion decreased again before stabilizing at around 50 mPa.s. This behaviour appears to be related with the time necessary for the pectinase activity to reach the oil-rich emulsion from the desludger and hydrolyse the pectic materials [25]. ' In both experiments constant and low viscosities (0.9-2.5 mPa.s) were observed for the oilpoor emulsions (oil finisher emulsions and aqueous discharges from centrifuges). 160
160 140 120
,~,
100
100
.~
80
80
~
6o
60
40
~
2O 0
0
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."i'm.
mira _.
,Imam:
6T ,~0"q80-~40 300-360 420
,dk .,k mmm
480
0
60
120
Tune (rain) Oil Finisher Emulsion
-~- Oil Rich Emulsion
m
240
300
mlm
360
m
420
480
Time (nan)
--'~Aqueous Discharge 1 m
m
180
Aqueous Discharge II
Figure 3a. Evolution of viscosity. Non-recycling system.
- 0 Oil Finisher Emulsion 4~- Oil Rich Emulsion
-4-Aqueous Discharge ! m Aqueous Discharge II
Figure 3b. Evolution of viscosity. Recycling system.
Material balance During the non-recycling experiment 40200 Kg of lemon fruit were processed, with an average of 0.823% in weight of recoverable oil. Therefore, the total available oil in the lemons was 330.8 Kg. The oil finisher emulsion had an average of 0.995% (w/w) of recoverable oil,
968 and the efficiency of the extractors with the finisher was 87%. The aqueous discharges from desludger and polisher had an average of 0.070% and 0.630% (w/w) of recoverable oil, respectively. A total of 21.9 kg of essential oil were lost though aqueous discharges. The efficiency of the desludger was 72% and that of the polisher 99%. At the end of the assay 203 Kg of essential oil had been recovered and the yield of the system was 61%. The water consumption was 721 L/ton of processed fruit. Material balances for the non-recycling and recycling system are shown in Table 1. Table 1. Material balance in the non-recycling and recyclin~ lemon peel oil recovery systems. Non-recycling system Recycling system Lemon fruit Total weight (Kg) 40200 43200 Recoverable oil % (w/w) 0.823 0.816 Total available oil (Kg) 330.8 352.5 Oil finisher emulsion Flow (L/h) 2880 2700 Recoverable oil % (w/w) 0.995 0.980 Total available oil to desludger (Kg) 286.6 264.6 Efficiency finisher + extractors (%) 87 75 Aqueous discharge from desludger Flow (L/h) 2800 2600 Recoverable oil % (w/w) 0.070 0.065 Total available oil (Kg) 19.6 16.9 Efficiency of the desludger (%) 72 87 Aqueous discharge from polisher Flow (L/h) 36 144 Recoverable oil % (w/w) 0.630 0.945 Total available oil (Kg) 2.3 13.6 Efficiency of the polisher (%) 99 94 Yield of the system Recovered total oil (Kg) 203.0 215.5 Yield % (w/w) 0.505 0.498 Efficiency (%) 61 61 Hours working 10 10 Water consumption (L) 29000 6000 Novoferm 14 consumption (L) 3 In the experiment involving the recycling system 43200 Kg of lemons with an average of 0.816% (w/w) of recoverable oil (352.5 Kg of available essential oil in lemons) were processed. During the extraction and finishing process 88 Kg of essential oil were lost, meaning that the efficiency of both stages together was 75%. The aqueous discharges from desludger and polisher had an average of 0.065% and 0.945% (w/w) of recoverable oil and their efficiencies were 87% and 94%, respectively. With the recycled recovery system, there were minimal losses of essential oil though the aqueous discharges, because the effluents were
969 returned to the extractor for oil recovery. During this experiment 215.5 Kg of essential oil were recovered and the yield of the system was the same as that of the non-recycling system (61%). Water consumption was 139 L/ton of processed fruit and NV14 consumption, to maintain an enzyme concentration of 0.5 mL/L of water throughout the experiment, was 70 mL/ton of processed fruit. Both recovery systems showed the same overall efficiencies although the partial efficiencies for the extractors and finisher considered together, the desludger and the polisher are different. The aqueous discharges from the centrifuges which return to the extractors produce emulsions with different characteristics and this results in different machinery efficiencies. Insoluble solids in the aqueous discharge recycled to the extractors reduce the effectiveness of the spray of water to wash away the essential oil in the extractors. The efficiency of the desludger in the recycling system is higher than in the non-recycling system because enzymatic treatment hydrolyses pectins from the emulsion and contributes to destabilizing the emulsion. Some modifications to improve the yield in the recycling system, such as the use of extractors with spray tings of 0.063" (larger than the 0.049" currently used) and the filtering of the aqueous discharges from the centrifuges are proposed. In this way, the efficiency of the extractors and finisher will be similar to those in the non-recycling system. Emulsions with stabilized and reduced viscosity are essential for centrifuges to work efficiently [14, 25]. A holding tank for the oil-rich emulsion from the desludger would stabilize this emulsion before it is fed to the polisher, improving the efficiency of the polisher in the recycling system. With these modifications the essential oil yield in the recycling system will be between 70-75% of the total available oil in the lemon peels. Both recycled and non-recycled essential oils met the standards for specific gravity, angular rotation and refractive index of the Food Chemicals Codex [22] and United States Pharmacopeia [23] for cold-pressed lemon peel oil. Results of gas chromatography analysis show the same compounds and levels in both systems, specially for the oxygenated compounds as citral (neral and geranial). Therefore, recycling the aqueous discharges to the extractor does not cause undesirable modifications.
ACKNOWLEDGEMENTS The authors wish to thank CITRJMUSA (Murcia, Spain) for their collaboration in the industrial experiments, and Novo Nordisk Ferment Ltd. (Dittingen, Switzerland) for their support in enzymatic preparation.
REFERENCES
Murdock, D.E. y Allen, W.E. 1960. Food T echnol. 14:441. McNary, R.R.; Wolford, R.W. and Patton, V.D. 1951. Food Technol. 8:319. Ratcliff, M.W. 1977. In: Citrus Science and Technology. Nagy, S. (Ed.). AVI Pu. Co. Westport, CT. Steger, E.S. 1979. The Citrus Industry Magazine, 60 (8): 26. Parish, M.E.; Braddock, R.J. and Graumlich, T.R. 1986. J. Food Sci. 51" 431.
970 6 7 8 9 10 11 12 13 14 15 16 17
18 19 20 21 22 23 24 25
Platt, W.C. and Poston, A.L. 1962. U.S. Patent Number 3.058.887; Oct. 16. Veldhuis, M.K.; Berry, C.J.; Wagner, C.J.Jr., Lund, E.D. and Bryan, N.L. 1972. J. Food Sci. 37: 108. Lund, E.D and Bryan, W.L. 1976. J. Food Sci. 41:1194. Earhart, J.P. and King, C.J. 1976. J. Food Sci. 41: 1247. Braddock, R.J. and Adams, J.P. 1984. Food Technol. 38 (12): 109. Ericson, A.P. 1992. J. Food Sci. 57:186. Kesterson, J.W.; Hendrickson, R. and Braddock, R.J. 1971. Fla. Agr. Exp. Sta. Tech. Bull. 749. Matthews, R.F. and Braddock, R.J. 1987. Food Technol. 41 (1): 57. Dickenson, C. 1987. In: Filters and Filtration Handbook. 2nd Ed. The Trade & Technical Press Limited. Surrey. England. Bott. E.W. and Sch6ttler, P. 1989. Technical-Scientific Documentation No.14. Westfalia Separator AG, Westfalia. Fogarty, W.M. and Kelly, C.T. 1983. In: Microbial Enzymes and Biotechnology. Fogarty, W.M. (Ed.). Applied Science Publishers, London. Robins, M.M. 1991. In: Microemulsions and emusions in foods. EI-Nokaly, M. and Cornell, D. (Ed.). ACS Symposium series n~ 448. American Chemical Soc. Washington, DC. Novo. 1991. Technical Scientific Documentation. Novo Nordisk Ferment Ltd., Dittingen. Reed, J.B., Hendrix, C.M. Jr., Hendrix, D.L. 1986. In: Quality Control Manual for Citrus Processing Plants. Volume I. INTERCIT, Inc. Safety Harbor, FL. Scott, R.W. 1979. Anal. Chem. 51: 136. McFeeters, R.F and Armstrong, S. 1984. Anal. Biochem. 139: 212. Food Chemicals Codex. 1981.3rd Ed. National Academic Press. Washintong, D.C. The United States Pharmacopeia 19th Revision. United States Pharmacopeial Convention, Inc, Rockvill, Md. Coil, L. 1992. Tesis de Grado. Faculty of Biology. University of Murcia. Coil, L.; Saura, D.; Ruiz, M.P.; Ros, J.M.~ Chnovas, J.A.and Laencina, J. 1995. Food Control. 6 (3): 143.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All fights reserved.
971
Immobilised pectinase efficiency in the depolymerisation of pectin in a model solution and apple juice C. Dinnella a, A. Stagni ", G. Lanzarini b and M. Laus" aUniversita di Bologna, Dipartimento di Chimica Industriale e dei Materiali, Viale Risorgimento 4, 40136 Bologna, Italy. bUniversit& della Basilicata, Dipartimento di Biologia, Difesa e Biotecnologie Forestali ,Via N. Sauro 85, 85100 Potenza, Italy. Abstract
A commercial pectinase, immobilised on appropriately functionalised 7-alumina spheres, was loaded in a packed bed reactor and employed to depolymerise the pectin contained in a model solution and in the apple juice. The activity of the immobilized enzyme was tested in several batch reactions and compared with the one of the free enzyme. A successful apple juice depectinisation was obtained using the pectinase immobilised system. In addition, an endopolygalacturonase from Kluyveromyces marxianus, previously purified in a single-step process with coreshell microspheres specifically prepared, was immobilised on the same active support and the efficiency of the resulting catalyst was tested. 1. INTRODUCTION
Since many years, pectolytic enzymes have been widely used in industrial beverage processing to improve either the quality and the yields in fruit juice extraction or the characteristics of the final product [1,2]. To this purpose, complex enzymatic mixtures, containing several pectolytic enzymes and often also cellulose, hemicellulose and ligninolytic activities, are usually employed in the free form. The interactions among enzymes, substrates and other components of fruit juice make the system very difficult to be investigated and only few publications are devoted to the study of enzymatic pools [3-5]. An effective alternative way to carry out the depectinisation process is represented by the use of immobilized enzymes. This approach allows for a facile and efficient enzymatic reaction control to be achieved. In fact, it is possible to avoid or at least to reduce the level of extraneous substances originating from the raw pectinases in the final product. In addition, continuous processes can be set up. This work deals with the preparation and study of a biocatalyst prepared by immobilising a commercial pectinase on a tailor made support, and its use in a
972
Table 1 Main characteristics of the enzymes present in Pectolyase Y23
a)
Enzyme
UE/mg
Optimum pH
Iso-electric point
Molar mass
PL
1.0
6.0
7.7
32000
PG
27
4.5
-a
35500
PE
7.7
4.7
-a
-a
Not known
packed bed reactor for the depectinisation of a cloudy apple juice. In addition, an endopolygalacturonase from yeast culture broth, purified using protein friendly coreshell polystyrene microspheres [6], was also immobilised and studied for its pectin catalytic activity. The yeast chosen, the Kluyveromyces marxianus, is a GRAS (generally recognised as safe) microorganism and was demonstrated to be a potential source of endopolygalacturonase to be used in food industry. Furthermore, it is reported as a constitutive producer of the endopolygalacturonase [7].
2, EXPERIMENTAL 2.1. Materials Pectolyase Y-23 (Seishin Corporation) was used without further purification as endopectinlyase (EC 4.2.2.10) (PL), endopolygalacturonase (EC 3.2.1.15) (PG) and pectinesterase (EC 3.1.1.11 ) (PE) source (Table 1). The Kluyveromyces marxianus strain (ATCC 36907) was grown in a aqueous medium containing 1% (w/v) yeast extract, 1% (w/v) bacteriological peptone and 2% (w/v) glucose and was used as endopolygalacturonase producer, y-alumina Spheralite 531 (o = 2.5 - 4.0 mm) was supplied by Rh6ne-Poulenc. 2.2. Preparation of the core-shell microspheres The preparation by dispersion polymerization of the microsphere sample employed in this study was previously described [8]. The microsphere sample utilized in this study has a monomodal diameter distribution with mean diameter value d= 3.09 IJm and standard deviation dsdev = 0.74 IJm. The microsphere surface is covered by a poly(methacrylic acid-co-ethylacrylate) whose percent by weight is 1.1 %. 2.3. Enzymatic activity assay PL activity was tested [9] by detecting the formation of unsatured oligomer,~, derived from the enzymatic depolymerisation of 0.5% (w/v) buffered pectin solution~ through the absorbance increase at 235 nm.
973
The PG activity was measured [10] by detecting the increase of reducing groups during the depolymerisation of 1% (w/v) polygalacturonic acid buffered solutions, by a colorimetric method based on the 3,5-dinitrosalicylic acid. The PE activity was measured [11] by detecting titrimetrically the increase of carboxilic groups during the hydrolysis of 0.5% (w/v) pectin solutions. The total activity of the enzymatic mixture was determined from the percentage reduction of viscosity [12] during the depolymerization of solutions, buffered at different pH values, containing 1% (w/v) of the polygalacturonic acid or 0.5% (w/v) of pectin. A Cannon-Fenske viscosimeter, (ASTM series n ~ 150) kept at the constant temperature of 25 ~ was used to carry out the tests.
2.4. Protein assay The protein content was determined using a commercial assay kit (Bio-Rad Protein Assay kit) with Bovine Serum Albumin as standard, following the procedure described by Bradford [13]. 2.5. Endopolygalacturonase purification procedure The culture broth was recovered after 72 h of fermentation, the biomass removed and the total protein content measured. Broth aliquots with a protein content of 1 mg were collected and their pH regulated at different values ranging from 3.5 to 8.0. To each broth fraction, 50 mg of the microspheres sample, previously equilibrated at the corresponding pH, was added and the suspension left under stirring overnight. Then, the microspheres were removed by centrifugation and the protein content and the PG activity were assayed on the resulting supematant. 2.6. Enzyme immobilisation The Pectolyase Y-23 and the purified yeast PG were immobilised as described by Coletti-Previero et al. [14]. 5 g of ~/-alumina spheres, previously equilibrated in 200 mL of a buffered solution at pH 6.0, was treated first with 30 mL of 0.04 M ophosphorylethanolamine and then with 30 mL of 0.56 M glutaraldehyde. These two reactions were performed at 25 ~ and pH 6.0, for 1.5 h and were followed by several washings with-abundant distilled water. Finally, 15 mL of 10 mg/mL Pectolyase Y-23 solution or 25 mL of 0.5 mg/mL of purified yeast PG solution, both buffered at pH 6.0, were added and left to react for 2 h at 25 ~ 1r-alumina spheres were then washed with 450 mL of distilled water. The reaction solution was tested for protein content and enzymatic activities. 2.7. Model solution depolymerization in batch reactions The enzymes immobilized on y-alumina spheres were loaded in a reactor consisting of a thermostatated column where the reagents were recycled by means of a peristaltic pump with a flow of 3.0 mL/min. 15 mL of 0.5% (w/v) pectin or 1% (w/v) polygalacturonic acid buffered solutions were loaded and recycled for 30 rain at 25 *C. The reaction mixture was collected and analysed as above described to determine the percentage reduction of viscosity and the concentration of reducing groups and unsatured oligomers. The biocatalyst was abundantly washed with distilled water before performing the following batch reaction.
974
100
5
80 "~ o r
g]
4
60 "~.
o
o
3
40 .
,I--I
~ O
2
2o 0 0
40
80 t/min
120
~
160
Figure 1. Trend of the concentration of the reducing (O) and unsatured (11) groups and percentage reduction of viscosity (A) of the pectin solution as a function of the reaction time, at pH 6.0.
2.8. Cloudy apple juice preparation and depectinisation
Pulp from ripe Golden apple was pressed in a mortar and filtered, thus obtaining a cloudy and dense juice. Potassium metabisulphite was added as antioxidant at a final concentration of 0.15 mg/mL. The pH of the prepared apple juices was 4.1 + 4.3. Depectinisation experiments were carried out loading the juice in the packed bed reactor and recycling for 30 rain at 25 ~ The reaction mixture was then collected and the percentage reduction of viscosity measured as above described. The y-alumina spheres were abundantly washed with distilled water before performing the successive batch reaction. Keeping the treated juices at 4 ~ for 24 h, they presented two separate phases: a dense precipitate and a clear supematant. The latter was used for the alcohol tests. 1 mL of a 90% ethanol solution containing 0.05 M hydrochloric acid was added to 0.5 mL of the supernatant. The depectinisation efficiency is qualitatively estimated from the presence and the characteristics of the precipitate.
975 100 o~-I
o
O
80 60
O 9,-~
40
~
20
~
0
I I
0
1 2 3 4 [Reducing G r o u p s ] / m M
5
Figure 2. Trend of the percentage reduction of viscosity of the pectin solution at pH 6.0 (Q) and pH 3.0 ( I ) and of the polygalacturonic acid at pH 4.1 (A) as a function of the concentration of the reducing groups. 3. RESULTS AND DISCUSSION
The Pectolyase Y-23 catalytic activity was studied on several solutions of pectin at pH 3.0 and 6.0 and of polygalacturonic acid at pH 4.1. The enzymatic action was detected following the formation of unsatured oligomers and reducing groups. The percentage reduction of viscosity was also measured. During the pectin depolymerization at pH 6.0 (Figure 1), the PG activity is about 20 times higher than the one of the PL, in agreement with the known specific activity of the enzymes present in the pectinase sample (Table 1). In addition, it should be observed that the PL activity reaches its saturation value after about 30 min whereas the PG activity increases regularly with time. This dual behaviour is probably connected to the competitive hydrolytic action of PE that quickly transforms the pectin into the polygalacturonic acid. Accordingly, the reducing group formation is mainly clue to the PG action and the pectin depolymerisation derives from the sequential action of PE and PG. At pH 3.0, all enzymatic activities are lower than the ones at pH 6.0. This effect is especially marked for the PL because its optimum pH value is definitely higher than those of PG and PE (Table 1). PG is very efficient in depolymerising the polygalacturonic acid. The solution viscosity reaches its minimum value, which corresponds to a rather low molar mass, after 40 min at both pH 4.1 and 6.0. Figure 2 reports the trend of the percentage reduction of viscosity as a function of the concentration of the reducing groups for the depolymerisation of pectin at pH 3.0
976
Asf/Asi
1 0
3
I
I
4
5
I
6 pH
I
i
7
8
9
Figure 3. Ratio between the PG specific activity measured after the purification procedure (ASf) and the initial PG specific activity (ASi). and 6.0 and of polygalacturonic acid at pH 4.1. Within this data representation, the higher is the slope and the more effective is the enzyme in reducing the molar mass of the biopolymers. At pH 6, the pectin molar mass reduction is higher than at pH 3.0, keeping constant the number of reducing groups. Although the combined effects of several factors should be taken into account, this result seem to suggest that the enzyme operates a preferential cleavage at the end of the pectin chain at pH 3.0, whereas a more random cleavage occurs at pH 6.0. The catalytic action of the pectinase immobilised on y-alumina was studied at 25 "C measuring the viscosity percentage reduction after a reaction time of 30 min. The immobilised biocatalyst was used for several consecutive reactions. The three enzymes present in the pectinase sample were immobilised in the active form. In fact, both reducing groups and unsatured oligomers were detected in the final reaction mixture. Furthermore, in all the experiments performed at different pH values, there was a good activity retention. After five reaction cycles, at least 66% of the initial activity was detected on the biocatalyst and no activity was released in the medium. The immobilised Pectolyase Y-23 was used to perform consecutive depectinisations of a cloudy apple juice in batch reactions. After five reaction cycles, the percentage reduction of viscosity was greater than 90%. The viscosity of the apple juice feed through a bed of inactive y-alumina spheres remains unchanged thus confirming that the viscosity loss attributable to pectin adsorption on the carrier is negligible. The alcohol tests, performed on the juices previously treated with the
977
immobilized biocatalyst, were negative. Accordingly, from a technological point of view, the depectinisation of the apple juice was complete. The endopolygalacturonase obtained from a Kluyveromyces marxianus culture broth was purified through the addition of specifically designed core-shell microspheres consisting of an inner polystyrene core and an outer shell constituted by a poly(methacrylic acid-co-ethylacrylate) statistical copolymer. These microspheres were previously found very effective in purifying the pectinlyase within a commercial pectinase sample [15]. Several scouting experiments were performed to find the best pH conditions. Figure 3 reports the ratio between the PG specific activity measured after the purification procedure (ASf) and the initial PG specific activity (ASi). At pH 3.5, the microspheres are able to remove from the broth the major part of the protein without PG activity, thus providing a four time increase of the enzyme specific activity. The purified PG from Kluyveromyces marxianus was immobilised following the above procedure. Batch reactions in the packed bed reactor were done to evaluate the biocatalyst stability. After an initial loss, due to enzyme release, the residual PG activity reaches a plateau value corresponding to about 40% of the initial activity. Probably, some broth component interfered during the immobilisation reaction weakening the protein-carrier interactions. 4. CONCLUSIONS The Pectolyase Y-23 catalytic activity was studied on several solutions of pectin at pH 3.0 and 6.0 and of polygalacturonic acid at pH 4.1. During pectin depolymerisation, the PL activity is about 20 times lower than the one of the PG and reaches its saturation value after about 30 min whereas the PG activity increases regularly with time. This dual behaviour is probably connected to the competitive hydrolytic action of PE that quickly transforms the pectin into the polygalacturonic acid. Accordingly, the reducing group formation is mainly due to the PG action and the pectin depolymerisation derives from the sequential action of PE and PG. The pectinase was supported on 7-alumina and the three enzymes present in the pectinase sample were found still active after the immobilisation. The supported biocatalyst was used in several reaction cycles to perform consecutive depectinisations of a cloudy apple juice with a negligible loss of biocatalytic activity. Finally, core-shell microspheres were found very effective in purifying the endopolygalacturonase, obtained from a Kluyveromyces marxianus culture broth, which was immobilised on 1r-alumina in the active form. ACKNOWLEDGEMENT Research supported by National Research Council of Italy, Special Project RAISA, subproject 4.
978
5. REFERENCES
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
A. H. Rose (ed), Economic Microbiology, vol. 5: Microbial Enzymes and Bioconversion, Academic Press, London, (1980). C. Cantarelli and G. Lanzarini, Biotechnological Application in Beverage Production, Elsevier Applied Science, (1989). P. Lozano, A. Manjion, F. Romajaro and J. L. Iborra, Process Biochem., June 1988 75. P. Lozano, A. Manjon, J. L. Iborra, M. Canovas and F. Romajaro, Enzyme Microb. Technol., 12 (1990) 499. M. Kminkova and J. Kucera, Enzyme Microb. Technol., 5 (1983) 204. M. Laus, C. Dinnella, G. Lanzarini and A. Casagrande, Polymer (1995), in press. F.M. Barnby, F. F. Morphet and D. L. Pyle, Enzyme Microb. Technol., 12 (1990) 891. C. Dinnella, G. Lanzarini, M. Zannoni and M. Laus, Makromol. Chem. Rapid Comm., 15 (1994) 909. P. Albersheim and U. Killias, Arch. Biochem. Biophys., 97, (1962) 191. M. F. Chaplin and J. F. Kennedy (eds.), Carbohydrate Analysis, a Practical Approach, I.R.L. Press, Oxford, (1986). Z. I. Kertesz, J. Biological Chem., 121 (1937) 589. J. G. Hancock, R. L. Millar and J. W. Coerber, Phytopathol., 54 (1964) 928. M. M. Bradford, Anal. Biochem., 72 (1976) 248. M.A. Coletti-Previero and A. Previero, Anal. Biochem., 180 (1989) 1. C. Dinnella, M. Laus, G. Lanzarini and M. Doria, Biotechnol. and Applied Biochem., in press.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
979
Pectinases in w o o d d e b a r k i n g
Marjaana R~itt6 and Liisa Viikari VTT Biotechnology and Food Research P.O. Box 1501, FIN-02044 VTT, FINLAND
Abstract In debarking the bark is removed from wood along the cambial layer. Pretreatment with pectinases improves the efficiency of mechanical debarking measured as reduction of energy consumption in a laboratory scale debarker. The effects of different pectinases vary significantly depending on the side activities of the enzyme preparations The main carbohydrate components identified in spruce cambium were galacturonic acid, glucose, galactose, arabinose and xylose Purified polygalacturonases solubilized galacturonic acid from isolated cambium, but neutral sugars, especially glucose were not solubilized as efficiently as by the commercial pectinase preparation shown to be effective in the preteatments.
INTRODUCTION
Extensive debarking is needed for the production of high quality mechanical and chemical pulps because even low amounts of bark residues cause darkening of the product. Debarking of pulpwood is mainly performed in debarking drums. Complete debarking leads to losses of raw material and to increased energy consumption due to prolonged treatment in the mechanical drums. In mechanical debarking the bark is removed from wood along the cambial layer. Cambial cells divide continuously and have a lower mechanical strenght than that of the other wood cells. Charasteristic to the cambium are high pectin and protein contents and the absence or low concentration of lignin (Simson and Timell 1978). Treatment of wood with pectinolytic enzymes has been found to facilitate debarking (R~itt6 et al 1993). Enzymatic treatment is mainly applicable to poorly debarked wood selected after preliminary mechanical debarking as intact bark in untreated logs forms an effective barrier to the enzyme. The relatively high prize of the enzymes is one factor hindering application of the method in practical scale. In this the work composition of the cambium and the effects of enzymes on isolated cambium were studied in order to determine the optimal enzyme composition for debarking.
980 MATERIALS AND METHODS
Debarking Improvement of debarking efficiency was measured as the decrease in energy comsumption in a laboratory scale debarker. The enzymatic treatments and the debarking were carried out as described earlier (Riitt6 et al 1993). Enzymes Pectinex Ultra SPL (Novo Nordisk), endo-polygalacturonase (MegaZyme) and a partially purified polygalacturonase from Aspergillus niger(Bailey and Ojamo 1990) were used. Isolation of the cambium The cambium was isolated from spruce (Picea abies) logs felled in October-November. The bark was stripped off, and the cambium was removed by gentle scraping. The isolated cambium was immediately frozen in liquid nitrogen and freeze-dried. The carbohydrate composition of the isolated cambium was analysed by I-IPLC after enzymatic hydrolysis using Pectinex Ultra and a mixture of cellulases and hemicellulases (Buchert et al 1993). Hydrolysis of the isolated cambium The enzymatic hydrolysis of the isolated cambium were carried for 24 hours out at 40~ in 50 mM acetate buffer, pH 5. The enzymes were dosed according to the polygalacturonase activity. The reference samples were incubated in buffer without enzymes. The oligomers liberated in the enzymatic treatments of the cambium were further hydrolysed to monomers using Pectinex Ultra and a mixture of cellulases and hemicellulases (Buchert et al 1993) and analysed for monomeric sugars and galacturonic acid by HPLC.
RESULTS AND DISCUSSION
Pretreatment of wood with pectinolytic enzymes facilitates the debarking. The energy consumption during debarking of spruce in a laboratory scale debarker is clearly decreased after treatmeiat with Pectinex Ultra (Table 1). Several hours is needed for effective preteatment (Table 2). Table 1. Effect ofpretreatment with Pectinex Ultra (24 h at 20~ on debarkin~ of spruce. Enzyme dosage (nkat/ml) Improvement of debarking (%) 0 0 40 25 185 50 900 80
981 Table 2 The effect of pretreatment time on the debarking of spruce. The enzyme was Pectinex Ultra., 185 nkat/ml. Improvement of debarking (%) Pretreatment time (h) 0 0 4 4 12 40 24 50
The effect of the partially purified polygalacturonase was low as compared to that of the commercial pectinase preparation, Pectinex Ultra, containing various side activities in addition to polygalacturonase (Table 3).
Table 3. Improvement of debarking efficiency by pretreatment with pectinase preparations containing different levels of side activities. Enzyme Enzymatic activities (nkat/ml) Improvement of debarking endoPL endo0t-ara endoendo(% of control) PG ara glu xyl Pectinex Ultra 185 0.86 7.3 1.5 7.3 2.8 50 Partially 185 <0.01 0.06 0.02 0.03 0.39 13 purified PG
On the basis of the available information on chemical composition of the cambium, pectinolytic enzymes were originally chosen for loosening the bark-to-wood bond. To gain more information on the side activities essential for cambium solubilization in addition to polygalacturonase, cambium composition was analysed. The problems in mechanical debarking occur during cambium dormancy in the wintertime. Therefore, cambial tissue for analysis was isolated from spruce logs felled during the winter. The main carbohydrate components identified in the isolated cambium were glucose, galactose, arabinose, xylose and galacturonic acid (Table 4). In visual inspection the non-hydrolysed residue was found to contain wood fibres. Table 4. Carbohydrate composition of the isolated cambium. Carbohydrate Glucose Galactose Arabinose Xylose Galacturonic acid Non-hydrolysed
% of cambium d.w. 16.2 8.0 10.0 3.7 9.8 29.0
982 In hydrolysis of isolated cambium the purified polygalacturonases solunbilized as much galacturonic acid as Pectinex Ultra, whereas solubilization of the neutral sugars arabinose, galactose and glucose was lower. The most striking difference between Pectinex Ultra and the purified polygalacturonases was solubilization of glucose. (Table 5). Table 5. Hydrolysis of isolated cambium with pectinases. Enzyme dosage 10000 nkat/g. Enzyme Solubilized carbohydrates (%) glu 8al ara xyl Pectinex Ultra 12.0 4.3 8.8 2.6 Endo-PG 5.4 2.7 6.4 2.3 Partially purified PG 5.4 2.7 6.1 2.7 ref 4.9 0.5 1.2 2.2
gal-u 7.4 7.8 7.2 <0.1
CONCLUSIONS Preteatment with pectinases improves mechanical debarking of wood significantly. In addition to polygalacturonase, enzymes hydrolysing the neutral polymers in cambial tissue (arabinanase, galactanase, and glucanase) are needed for effective solubilization of spruce cambium.
REFERENCES Bailey M and Ojamo H (1990) Bioseparation 1:133. Buchert J, Siika-aho M, Bailey M, Puls J, Valkeaj~irvi A, Pere J and Viikari L. Biotechnology Techniques 7:785. R~itt6 M, Kantelinen A, Bailey M and Viikari L (1993) Tappi Journal 76:125. Simson BW and Timell TE (1978) Cellulose Chem. technol 12:39.
J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.
983
Oligouronides production in a membrane reactor by enzymatic degradation of pectins from Citrus peel. A preliminary study J.M. Ros, D. Saura, L. Coil, M. Moliner and J. Laencina Department of Food Technology, University of Murcia, Campus de Espinardo, Murcia, Spain & Department of Food Science and Technology, CEBAS (CSIC), Avda de la Fama 1, 30003 Murcia, Spain
Abstract
The production of oligouronides in a membrane reactor combining the enzymatic degradation of the substrate with the separation of the reaction products by cross-flow filtration has been studied. Polygalacturonic acid from Citrus peel was degraded by an endopolygalacturonase from Rhizopus nigricans. The monitoring of the viscosity indicated that only aider 4 hours of incubation was the viscosity of the system adequate to be cross-flow filtered. At that moment, the cross-flow device was connected with the enzymatic reactor and cross-flow filtration was carried-out for 8 h. Enzyme free-oligouronides were obtained in the permeate current. Oligouronides were obtained in gram amounts after freeze-drying of the collected permeate. An analysis of the composition indicated that galacturonic acid oligomers from mono to hexa were present.
INTRODUCTION Oligouronides are oligomers of galacturonic acid with a degree of polymerisation up to 25. Several applications and bioactivities of the oligouronides have been reported according to their degree of polymerisation, for instance studies of the mechanism of action of pectic enzymes and fundamental studies in both plant biochemistry and pathology [ 1-4]. If available in great amounts it can be used for several purposes and the development of new applications. Oligouronides are obtained by both chemical [5] and enzymatic degradation of higher polymers of galacturonic acid, for instance pectins with different degrees of methylation [2,3,6-10]. After the degradation step, purification is performed by size exclusion or ion exchange chromatography. Membrane technology opens new possibilities in the production of oligouronides, since it allows a continuous separation of the oligouronides from the not yet degraded substrate and the enzyme. This feature is of special interest since oligouronides are also substrates of the enzyme. Recycling of the enzyme is possible too. On the present subject no references were found in the literature. Our work deals with the production of
984 oligouronides in a membrane reactor combining the enzymatic degradation of the substrate with the separation of the reaction products by cross-flow filtration.
EXPERIMENTAL
Enzyme.mPolygalacturonase (PG) was obtained from a culture of Rhizopus nigricans using Citrus pectin as carbon source [11,12]. The enzyme used for oligouronides obtention was the residual activity after a thermal treatment (100~ 60 sec) of the native Rh. nigricans endo and exoPG, since the endo-enzyme was thermoresistant while the exo-enzyme was thermolabile [ 13]. Substrate.mPoly-D-galacturorfic acid (PGA) from Citrus peel (Serva, Heidelberg, Germany) was used for obtaining oligouronides. The PGA was previously characterised. The total sugar content was 78% w/w. The composition (in mol%) was as follows: rhamnose 1, arabinose <1, xylose <1, mannose 1, galactose 3, glucose <1 and galacturonic acid 94. Fucose was not detected. Methylation and acetylation were both less than 1%. The average molecular weight was 25000 daltons with a polydispersity index of 1.5. Reaction conditions.~The reaction volume used was 2000 mL. A substrate concentration of 20 mg/mL and an enzym~itic activity of 1 U/mL were used. The enzymatic incubation was performed at pH 4.0 (without buffer) and 40~ Cross-flow ultrafiltration equipment.raThe device used is shown in Figure 1. It included a glass reactor (R) with temperature, pH and stirring control, a Minitan TM pump (P) (Millipore, Bedford, USA), a Harp TM hollow fiber membrane cartridge (M) (Romicon-Supelco, Bellefonte, USA) with a cut-off of 2000 daltons, and a permeate exit (f) for fraction collection. The retentate (r) was returned to the reactor. Analytical methods.~Viscosity measures were performed in an Ostwald type capillary viscosimeter. The relative viscosity was calculated according to Pharr and Dickinson [14]. Samples from the retentate and the permeate were taken at time intervals and reducing ends [ 15] and uronic acids [ 16] were determined using galacturonic acid (Sigma, St. Louis, USA) as standard. An average degree of polymerisation (DP) was calculated as the ratio between monogalacturonic acid and reducing ends. The materials were characterised and the uronic acid content [17], neutral sugar content [18] and degrees of methylation and acetylation [19] were determined. Molecular weight distribution was determined by high performance size exclusion chromatography (HPSEC) as described by Schols et al. [20]. Oligouronides were analysed by high performance liquid chromatography (HPLC) and by high performance anion exchange chromatography (HPAEC). HPLC was performed in an ORH-801 column (Interaction, Mountain View, USA) using 5 mM sulphuric acid as eluent, an isocratic flow of 0.6 mL/min, room temperature, and UV detection at 210 nm. Tri-, di- and monogalacturonic acid (Sigma, St. Louis, USA) were used as standards. HPAEC was performed as described by Schols et al. [21].
985 RESULTS
Viscosity evolution.---Figure 2 shows the evolution of the relative viscosity of the reaction medium during the reaction time for a reactor without membrane. Relative viscosity decreased because of the depolymerising activity of the enzyme. After 6 hours of reaction the viscosity became almost constant, which suggested that almost no molecules (polymers) were present in the reactor to contribute to the viscosity. Relative vi~osity
R
P
M
o
~
~ Reaction time (h)
Figure 1. Cross-flow ultrafiltration equipment.
Figure 2. Viscosity evolution.
The viscosity of the 20 mg/mL PGA solution was too high to be filtered by the 2000 daltons cut-off membrane. In direct filtration or filtration experiments at the earliest stages of the enzymatic degradation, a gel appeared between the hollow fibers of the cartridge, which meant that we had to keep the reaction without filtration for 6 hours (until the viscosity of the system was adequate). Oligouronides evolution in a reactor without cross-flow ultrafiltration membrane.---Since the presence of oligouronides in the reaction medium depended of the reaction time, a study of the their evolution during the reaction without cross-flow ultrafiltration (UF) membrane was performed. The concentration of PGA was 2.5 mg/mL. Samples taken from the reactor at different time intervals were analysed by HPLC for their oligouronide content. This analytical system only permited the chromatographic separation up to tetragalacturonic acid. Figure 3a shows the evolution of the oligouronide content during the reaction when native Rh. nigricans endo and exoPG was used. Mono-, di-, tri- and tetragalacturonic acid were found from the enzymatic action within the PGA. Mono- and digalacturonic acid continued to increase, while trigalacturonic acid became stable and tetragalacturonic acid, after reaching a maximum of concentration, disappeared totally. Tetragalacturonic acid is substrate of the endo and exoPG [22] and so its level in the reaction medium is the balance between its appearance from the degradation of higher oligouronides and polyuronides, and its disappearance caused by the endo and exoPG in mono- and trigalacturonic acid [23]. A similar explanation can be given for other oligouronides. It should be remembered that higher oligouronides are easier degraded than smaller ones [22]. Mono-
986 and digalacturonic acid are final degradation products, for which reason their concentrations increased continuously. In an effort to improve the enzymatic production of oligouronides, especially those of high DP, PGA was also incubated with the residual activity after a thermal treatment of the native Rh. nigricans endo and exoPG. This enzyme showed only endoPG activity [13]. Figure 3b shows the evolution of the oligouronide content during the reaction when pasteurised Rh. nigricans endoPG was used. 2000
Oli8ouronides (ttM)
1500
1500
1000
1000
500
500
0
,in 0
0
50
l O0
Oli8ouronides (~tM)
2000
150
200
250
Reaction time (rain) -O- MonoGA ~'~ TriGA
--~-DiGA I TetraGA
300
350
-
i i l
o
|
50
|
I
100
150
|
I
200
250
- - i
I
300
350
Reaction time (min) O- MonoGA
-~-DiOA
~'~ TriGA
I
TetraGA
Figure 3. Oligouronides evolution in a reactor without cross-flow ultrafiltration membrane. (a) Native Rh. nigricans endoPG. (b) Pasteurised Rh. nigricans endoPG. The general trend is similar to Figure 3a, but here the tendency of trigalacturonic acid to continue increasing is clearer than in Figure 3a. Also, since the endoPG has more difficulty in degrading tetragalacturonic acid, the tetragalacturonic acid level in Figure 3b was higher and it was present for longer than in Figure 3a. It, too, finally disappeared. These results indicated the convenience of using only the endo-enzyme to obtain high DP oligouronides.
Cross-flow UF equipment and oligouronide mixture obtention.--We used a cross-flow UF membrane (Figure 1). Continuous filtration of the reaction medium made it possible to separate the oligouronides from the enzyme, which can degrade these oligouronides several times. Oligouronides smaller than the cut-off of the membrane escape from the enzyme and are recovered in the permeate reservoir, while the retentate is returned to the reactor. Since the cut-off of the membrane was 2000 daltons, only oligouronides of DP up to 10 can be expected. No buffer was present in the reaction medium, which made it possible to obtain the oligouronides as partial sodium salts after freeze-drying of the collected permeate. The reaction conditions used were a compromise between maximum enzymatic activity for PGA degradation, minimum enzymatic activity within the new oligouronides produced and minimum enzyme inactivation so that it could be re-used [24]. The enzyme/substrate ratio was chosen
987 after monitoring degradation studies of PGA in which the decrease of the viscosity and the appearance of oligouronides occurred in a few hours [24]. Figure 4 shows the evolution of the permeate flux during the reaction. Permeate flux (mL/h) 40O
300
200
100 -
Reaction time (h)
Figure 4. Evolution of the permeate flux. Although the trend in the viscosity of the system tended to decrease, which facilitated filtration, a gel occasionally appeared during the filtration and a polarisation layer was always formed. The first line (1-5 h) of Figure 4 is based on an assay in which the filtration started after a reaction time of 1.5 hours, when there were still polymers and the viscosity of the system was high. A sharp decrease in the permeate flux was obtained because a gel appeared among the hollow fibers, as did a polarisation layer. The second line (4-11 h) of Figure 4 is from an assay in which the filtration started after a reaction time of 4.5 hours, when there were no polymers and the viscosity of the system was low. In this experiment no sharp decrease in the permeate flux was obtained and no gel appeared between the hollow fibers. Only the appearance of the polarisation layer was responsible for the decrease of the flux from 250 mL/h (initial flux) to almost 200 mL/h. Results reported so far indicate that to obtain oligouronides using the above described conditions and equipment, filtration should be started after 4 hours of reaction. Figure 5a shows the evolution of the average DP of the substrate and degradation products at the retentate side of the membrane. The initial DP was estimated at around 150 and decreased to 40 after 4 hours of reaction. At this moment the cross-flow filter was connected and filtration carried-out till the volume of the retentate was about 100 mL. The DP continued to be brought down by the action of the endoPG. The different slopes (ADP/At) of the line for the reaction times 4-5 hours and 5-8 hours indicate that the ability of the endoPG to degrade the substrate varied, being higher within longer polyuronides. Galacturonic acid and reduced ends increased as an effect of the concentration. At the beginning of filtration the average DP was around 10
988 while at the end it was around 5. Figure 5b shows the evolution of the average DP of the oligouronides at the permeate side of the membrane. At the beginning of filtration the average DP was 6, decreasing to 5 and finally to 4. There was a good correlation between the analysis carried-out at both sides of the membrane. DP
50
GA (mM)
250
7
DP
G A (huM)
140
(b)
40
6
120
5
lO0
4
80
3
60
- 200
-
30
150
20
-100
10
0
0 4
5
6
7
4
6
Reaction time (h) ~It Reducing end
A - Galacturonic acid
~
lo
12
Reaction time (h) O-DP
Reducing end
~
Galacturonic acid
-e- DP
Figure 5. Evolution of the average DP of (a) the sustrate and degradation products at the retentate side of the membrane and (b) the oligouronides at the permeate side of the membrane. Figure 6 shows the change in molecular weight distribution of PGA before the action of the endoPG and after degradation followed by cross-flow filtration. There was a good degree of degradation. Using SEC columns calibrated with pectin standards [25], an average molecular weight of 25000 daltons was calculated for PGA. The late retention time of the oligouronides indicated its low molecular weight. Finally the collected filtrate containing the oligouronides was freeze-dried. These were salt and enzyme free. The yield of the process was 1A g of oligouronides/g of PGA. Oligouronides mixture characterisation.--The mixture of oligouronides was characterised. Figure 7 shows that the mixture was formed of mono- up to hexagalacturonic acid. This chromatogram was obtained with a Dionex TM HPAEC system, which had better chromatographic resolution than HPLC. The total content of sugars was 70% w/w. The composition (in mol%) was as follows: rhamnose <1, arabinose <1, mannose 2, galactose 1, and galacturonic acid 97. Fucose, xylose and glucose were not detected.
989
MonoGA o.._.
TriGA
I I ,
I
TetraGA
i,. F 23
28
33
38
Retentiontime(rain)
Figure 6. HPSEC chromatogram.
I PentaGA
,; ,; 20 2; 3; 3; 4; 4; 50 Retentiontime(rain)
Figure 7. HPAEC chromatogram.
DISCUSSION Initial viscosities do not allow direct ultrafiltration of the medium. Less concentrated solutions are permited, but in this case, the yield of the process is too low. This is a very interesting point for the development of a continuous process, although so far our work has only been with batches. The use of a membrane allows the enzyme to be re-used. When oligouronides were obtained enzymatically with no membrane, reactions were stopped by heating, and it was impossible to re-use the enzyme. Alternatively, an immobilised endoPG could be used, although problems arise. PGA diffusion from the medium to the immobilised enzyme is difficult because of the polymeric nature of PGA [26]. Moreover, the reaction products act as a competitive inhibitor of the enzyme [22], so that it is advisable to use a membrane device to establish a quick separation between the oligouronides and the enzyme. The fact that oligouronides are also substrates of the enzyme employed for their obtention also supports the introduction of a membrane. Further degradation of the oligouronides obtained is thus avoided. With the design proposed in this work oligouronides escape from the enzyme, which means that it is possible to obtain "tailor-made" oligouronides by using several membranes of different cut-off. This point is of special interest for obtaining higher oligouronides. The use of a membrane also allows better use of the substrate. The use of a pure endoPG is very important since it facilitates the obtention of higher oligouronides. The use of a membrane permits a continuous addition of fresh PGA, the obtention of oligouronides and scaling-up of the process for industrial purposes. In our conditions it was necessary keep for 4 hours the system operating before starting the filtration. If filtration is started earlier, there is a strong decrease in the flux. A polarisation layer always appeared and this phenomena appears to be intrinsic to all UF operations. By waiting 4 hours before starting filtration a smaller polarisation layer appears and there is no gel. Because of the endoPG activity the DP at the retentate side of the membrane tends to decrease and only if oligouronides are continuously removed from the medium is it possible to obtain them without further degradation. In this preliminary work oligouronides up to hexagalacturonic acid were obtained. For the further isolation of each pure oligouronide
990 several procedures have been described [ 1-5,10,27-29]. Since it is of special interest to obtain higher oligouronides, the use of several membranes with different cut-off, the optimisation of the reaction conditions and the use of an enzymatic system involving a pectinesterase (PE) coupled to the endoPG are now the subjects of research. The use of a PE and endoPG system means that pectin can be used as a cheaper substrate than PGA.
Acknowledgements. The authors thank the Division of Food Chemistry, Department of Food Science and Technology, Wageningen Agricultural University, The Netherlands, for the possibility of carrying-out the characterisation of materials. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
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