Enzymes Everywhere

ENZYMES

EVERYWHERE

J.G. SHEWALE

National Institute of Science Communication Dr K S Krishnan Marg New Delhi 110 012

CSIR GOLDEN JUBILEE SERIES

ENZYMES EVERYWHERE

Enzymes Everywhere J.G. Shewale

© 1996 National InstitUte of Science Communication First Edition: November 1996 ISBN: 81-7236-141-6 (Paperback)

CSIR Golden Jubilee Series Publication No. 19 Series Editor

Dr Bal Phondke

Volume Editor

Dr Sukanya Datta

Cover Design Illustrations

Pradlp Banerjee Neeru Sharma, Sushila Vohra, Neeru Vijan, Malkhan Sin?;h, Yogesh Kumar, Mohan Singh

Production

Shiv Kumar Marhkan, S P Singh, Ganesh Sahani, Khem Chand, RK. Kaushik, C P S Khari

Printing

Sudhir Chandra Mamgain, GC Porel, Bharat Sing] Rajbir Singh, Pramod Kumar Sharma

FOR SALE IN INDIA ONLY Price: Rs 30.00 (Paperback)

Designed, Printed and Published by National Institute of Science Communication Dr K S Krishnan Marg, New Delhi 110 012

Foreword The Council of Scientific & Industrial Research (CSIR), established in 1942, is committed to the advancement of scientific knowledge, and economic and industrial development of the country. Over the years CSIR has created a base for scientific capability and excellence spanning a wide spectrum of areas enabling it to carry out research and development as well as provide national standards, testing and certification facilities. It has also been training researchers, popularizing science and helping in the inculcation of scientific temper in the country. The CSIR today is a well knit and action oriented network of 41 laboratories spread throughout the country with activities ranging from molecular biology to mining, medicinal plants to mechanical engineering, mathematical modelling to l!letrology, chemitals to coal and so on. While discharging its mandate, CSIR has not lost sight of the necessity to remain at the cutting edge of science in order to be in a position to acquire and generate expertise in frontier areas of technology. CSIR's contributions to high-tech and emerging areas of science and technology are ~ecognised among others for precocious flowering of tissue cultured bamboo, DNA finger-printing, development of non-noble metal zeolite catalysts, mining of polymetallic nodules from the Indian Ocean bed, building an all-composite light research aircraft, high temperature superconductivity, to mention only a few. Being acutely aware that the pace of scientific and technological development cannot be maintained without a steady influx of bright young scientists, CSIR has undertaken a vigorous programme of human resource development which includes, inter alia, collaborative efforts with the University Grants Commission aimed at nurturing the budding careers of fresh science and technology graduates. However, all these would not yield the desired results in the absence of an atmosphere appreciative of advances in science

and technology. If the people at large remain in awe of science and consider it as something which is far removed from their realms, scientific culture cannot take root. CSIR has been alive to this problem and has been active in taking science to the people, particularly through the print medium. It has an active programme aimed at popularization of science, its concepts, achievements and utility, by bringing it to the doorsteps of the masses through both print and electronic media. This is expected to serve a dual purpose. First, it would create awareness and interest among the intelligent layman and, secondly, it would help youngsters at the point of choosing an academic career in getting a broad-based knowlepge about science in general and its frontier areas in particular. Such familiarity would not only kindle in them deep and abiding interest in matters scientific but would also be instrumental in helping them to choose the scientific or technological education- that is best suited to them according to their own interests and aptitudes. There would be no groping in the dark for them. However, this is one field where enough is never enough. This was the driving consideration when it was decide9 to bring out in this 50th anniversary year of CSIR a series of profusely illustrated and specially written popular monographs on a judicious mix of scientific and technological subjects varying from the outer space to the inner space. Some of the important subjects covered are astronomy, meteorology, oceanography, new materials, immunology and biotechnology. It is hoped that this series of monographs would be able to whet the varied appetites of a wide cross-section of the target readership and spur them on to gathering further knowledge on the subjects of their choice and liking. An exciting sojourn through the wonderland of science, we hope, awaits the reader. We can only wish him Bon voyage and say, happy hunting.

PREFACE Almost all people come to know about enzymes during their school days while learning about the process of digestion. However, few 'non-scientific' professionals remain aware of their impc:tance once formal education is over. Enzymes, are one of the few groups of macromolecules that control the existence of living matter on earth. The importance of enzymes becomes evident when one realizes that the synthesis of other crucial regulatory molecules such as DNA, hormones, and neuropeptides are catalysed by enzymes. Though the occurrence of enzymes has been known for centuries, the field of enzymes has seen a revolution only in the last 50 years or so with technological developments in diversified areas like fermentation, separation techniques, genetic engineering, immobilization and bioreactors. All these are covered by a single scientific discipline called Biotechnology. The methodologies are now so advanced that it is possible to selectively alter properties of an enzyme to suit a particular application. My purpose of writing this book is to share th~ new developments in the field of enzymes and their uses with those who do not consciously think about the manifold ,ways in which enzymes touch everyday lives. I hope that the readers come to the conclusion that enzymes play an indespensible role in keeping us healtheir, happier and more content.

ACKNOWLEDGEMENT My desire to write a popt:::larbook crystallised due to the inspiration given by Dr. C. SivaRaman, FNA who has always been a source of motivation for me. I am indeed, grateful to him. After reading the previous books in this series, I had madeup my mind not to miss the opportunity to be associated with a highly learned editor like Dr. G.P Phondke. I sincerely appreciate his suggestions, efforts and concern in bringing out this book in a presentable form. Mr. S.R. Tophkhane, a senior faculty at Institute of Management Education, Pune and a voracious reader has read this book thoroughly. His comments have given a different direction to my writing which is a must for a popular science book such as this. I thank him for his interest and time. Mr. A.K. Basu, Managing Director, and Dr. S.R. Naik, General Manager, R&D, Hindustim Antibiotics Ltd. have nurtured my interests throughout. I express my sincere thanks for their encouragement. I thank Miss. Aruna Deshpande and the most able artists at NISCOM who have created the pictures to make the subject more digestable and to keep-up the spirits of the readers. I also thank all my colleagues at HAL and at NISCOM wno have contributed to this work.

DEDICATED TO My wife MANISHA and

son SHANTANU

Contents

L

Prelude

I

Unknown toBread Known Made Back for to Supermarket the Each Future Other shelf Milking Glossary the Enzymes Our On the Daily Enzyme Curing Looking the Economics Good Ills Spirited Enzymes

86 79 1 30 40 45 51 61 13 24 70 5

what is 'life'? Have you Have everto think wondered ever you paused how

Prelude

the microscopic cells, the units of all life forms carryon their duties and so sustain 'life'? Just a moment's reflection would makeapparent the structural and biochemical complexity inherent in biological systems. This complexity is, however, masked by the synchronized, well orchestrated manner in which the multifarious cellular functions take place. But there is one underlying fundamental need that is common to all cellular functions. Each and every living cell needs energy to survive and conduct its metabolic functions. For providing this energy many chemical reactions take place simultaneously and the sum total of these cellular activities is called metabolism. This is a simple term for a host of complex reactions. For example, we eat food which is broken-down into easily assimilative compounds which are then converted into energy that allows us to carry out our day to day activities. The long drawn process beginning from the moment food is ingested to its final conversion to energy is summed up by the term metabolism. Metabolic

2

ENZYMES EVERYWHERE

Sul1strate

Mr. Enzyme

changes seem very simple but include several complex chemical reactions that are continuously and simultaneously carried out in each and every cell of the body. Trying to mimic similar chemical reactions in the test tubes has not been easy. The obvious question that arises is how are these chemical reactions performed at body temperature? Nature has answered this question in one simple word "Enzymes".

PRELUDE

3

Nearly all chemical reactions in biological system are catalysed by enzymes. A catalyst is a substance that alters the rate of reaction but remains chemically unaltered at the end of the reaction. Most catalysts accelerate the rate of reactions but some, known as negative catalysts, retard the rate of chemical reactions. Catalysts are classified into two groups: chemical catalysts and biological catalysts. Enzymes are biological catalysts or biocatalysts. The prefix 'bia' indicates the origin and the term 'catalyst' indicates the function of the substance. The catalytic efficiency of enzymes is very high. Enzymes can accelerate the rate of a chemical reaction at least 10,00,000 timesr One of the simplest reactions in biological systems involves the addition of a molecule of water to one molecule of carbon dioxide. It is catalysed by an enzyme called carbonic anhydrase. Just one molecule of carbonic anhydrase can add the necessary,water to 105 molecules of carbon dioxide per second and this rate is 107 times faster than one in which no catalyst is involved. The enormous catalytic power of enzymes is attributed to their capacity to bind to substrate molecules in precise configuration and their ability to make and break chemical bonds. Enzymes are unique catalysts and they offer many advantages over chemical catalysts. Enzymes are capable of catalysing reactions at prevailing cellular conditions of acidity or alkalinity, temperature and pressure. They are very efficient and their turnover number is very high which means more of the required end products is formed when an enzyme catalyses a reaction. Enzymes are specific towards substrate. Also, in enzyme catalysed reactions wastage is minimised as side product formation is either nil or minimal. Sometimes, the enzyme may be kept localized on a 'bed' over which the substrate is passed. In such immobilized enzyme preparations the enzyme can easily be separated and reused after the reaction is complete. Thus for large scale or industrial purposes immobilized enzymes spell economic benefits as well. Final products are purer in enzyme catalysed reactions. Such

4

ENZYMES EVERYWHERE

Inside a cell or out of it, enzymes keep on working for man

reactions provide simple, nonhazardous handling conditions and in industrial applications, the pollution load on effluents is reduced. So all in all, industrial houses have a lot to thank enzymes for. *'

agree, is nothing but a fa-

History as everyone will ble agreed upon. It relies

Unknown to

Known

a lot on arguments because the further back in time that one goes, the weaker does the evidence grow. However, the advent of the documentation era has made it easier to substantiate facts. Chronicling the history of enzymes is not an easy job because the constraints that mark the study of history are present here as welL Records of the early days when man first used enzymes or enzyme-like substances are lost in the mists of time. References to the use of substances similar to enzymes for making of cheese are found in Greek epic poems dating from about 800 B.C.The history of man's use of enzymes to suit his own purpose thus stretches quite a long way into the past. However, it was not till 1783, that investigative attempts were made to demonstrate that certain metabolic reactions could be carried out outside the body, in test tubes to be precise, by using catalysts present in cells. Lazzaro Spallanzani, the famous scientist of the time demonstrated that gastric juiCes could digest meat even outside the body. It was much later (1836) that Theodor

6

ENZYMES EVERYWHERE

Theodor Schwarm also formulated the cell theory, which explained that cells are the structural and functional building blocks of living beings

Schwarm named the active component in gastric juice as pepsin. These studies were followed by scientific arguments on whether alcohol fermentation was a monopoly of living cells. Th~ answer came in 1897, when glucose was successfully converted ,to alcohol and carbon dioxide by cell free extract of yeast. It is interesting to note that yeasts were one of the first organisms that were studied in the field of enzymology. The term 'Enzyme' itself was coined in 1876, by Prof.

UNKNOWN TO KNOWN

7

Wilhelm Friedrich Kuhne from Greek words meaning 'In yeast'. Today, the field of enzymology has expanded beyond the expectations of the early investigators. More than 2000 different enzymes are now known and the list is still growing. This has given rise to a piquant situation. Giving a unique name to each and every enzyme so as to ensure for it a distinct identity is proved to be a challenging task. To bring about a degree of uniformity and to lay down internationally respected rules of nomenclature it became necessary to appoint a Commission called "Enzyme Commission". The Commission was appointed in 1956 and its mandate was to provide a systematic platform for the nomenclature of enzymes. It seemed reasonable to classify the enzymes in accordance with the type of reaction they catalyse. Today, all known enzymes are classified into six classes and each class is further divided into subclasses. Each subclass has sub-sub classes listed under it. Any given sub-sub class may have many enzymes each with its own unique number known as tl~e Enzyme Commission (E.C.) number. Trypsin, for example is designated as E.C. 3.4.21.4. The numbering is analogous to the way a student may be identified in school - Class V, Section A, Roll No 46. The difference is that a comma separates the elements here and a point separates the elements in the E.c. number. Usually, however, enzymes have a common name as well as a systematic name. Names are based on the nature of the enzyme. Its substrate, end product or the type of reaction it catalyses guides the selection of name. Cellulase is so named because it acts on cellulose. Similarly pectinase gets its name from its substrate pectin. Most enzymes have names ending with - 'ase'. Urease, lipase, pectinase and cellulase are examples of enzymes whose names have been coined by suffixing 'ase' to their respective substrates. Trypsin, renin, lysozyme and pepsin, however, are "exceptions that prove the rule."

8

ENZYMES EVERYWHERE

UNKNOWN TO KNOWN

9

Once order was restored to the hitherto chaotic field of enzyme nomenclature, scientists shifted their attention to another quest. This was the sea.rch to discover the nature of enzymes. The search ended in 1926, when James B. Sumner crystallized urease heralding in subsequent reports on isolation and purification of other enzymes. Soon, the statement "Enzymes are proteins" became standard textbook material. Although the sweeping statement has come in for scrutiny and criticism in recent times, it seemed factual enough in the early days of enzymology because it so happened that all the enzymes characterised subsequent to urease were proteins. Or else, they were proteins associated with <;arbohydrates (glycoproteins) or lipids (lipoproteins). The axiom, "All enzymes are proteins but not all proteins are enzymes", ruled scientific literature for the next 40 years or so, till in the 1980's when Arthur J. Zaug and Thomas R. Cech established the catalytic ability of ribonucleic acid (RNA) in joining several short RNA molecules to form a long chain. Self-splicing of 'RNA molecules was observed in ribosomal RNA of protozoans at about the same time. The discovery of non-protein molecules that show catalytic ability well within the framework of the traditional definition of enzymes has forced enzymologists to shed their complacency about the nature of enzymes and has encouraged them to investigate non-protein molecules for catalytic abilities. However, examples of non-protein molecules functioning as enzymes are still quite rare and almost all known enzymes are proteinaceous in. nature. Enzymes are made up of a string of amino acids. As the name indicates, amino acid molecules have at least one amino group and one carboxyl group. The total number of amino acids on the enzyme molecule varies from enzyme to enzyme, as do the kinds of amino acids that make it up. It is this difference that characterizes the protein (enzyme). The discrepancy in enzyme sizes can be appreciated better when the two enzymes ribonuclease and beta-galactosidase are

10

ENZYMES EVERYWHERE

compared. Each molecule of the pancreatic enzyme ribonuclease contains 120 amino acids whereas every molecule of beta-galac;tosidase isolated from the bacterium Escherichia coli contains 1021 amino acids. An amino acid joins with another amino acid by means of a peptide bond. The chain of amino acids so generated is called a polypeptide chain. The term is self explanatory because as everyone knows, 'poly' means many. In an enzyme, the amino acids are linked with one another in a very strict and disciplined manner. The position of each amino acid in the polypeptide chain of an enzyme is fixed. The polypeptide chain is unbranched. The sequence of amino acids in a polypeptide chain is called amino acid sequence. Enzymes are composed of one or more polypeptide chains which mayor may not be identical. The amino acid sequence or the order of amino acids in which they are present is again a characteristic of an enzyme. The sequence of amino acids

A polypeptide is made up of three or more amino acids

UNKNOWN TO KNOWN

11

in a protein is controlled at the level of the gene cfndit changes only when the structure of the gene is altered. The polypeptide chain is very flexible and can be folded in many ways. However, the folding of the chain must be such that the resultant configuration is a stable one. The folding of the polypeptide in an enzyme takes place in a very orderly fashion and is determined by the chemical nature of side chains of the amino acids and their functional or structural role. The folding of polypeptide chain gives different shapes to enzyme molecules which may vary in shape from round or oval to rod shaped. In an folded enzyme molecule relatively few amino acids are located on the surface. Most amino .acids are located in the interior of the molecule.

12

ENZYMES EVERYWHERE

Folding of the polypeptide chain takes place during (or immediately after) its synthesis. Considering the size of the peptide and taking into account its flexible nature, we may speculate on the unlimited number of possibilities of how a polypeptide can be folded. But in folding it into its correct form at the first attempt lies Nature's beauty. It is estimated that the time to search manually for the co:r:rectfolding of the most simple enzyme ribozyme (124 amino acid residues) may take 1095 years. Even a computer would take some time to arrive at the correct configuration. Biological systems do it routinely in a few milliseconds. The domains formed as a result of folding are not merely a physical and chemical part of the structure. These are essential requisites for the smooth functioning of the enzyme as they act as "coded signals". Each code is different and needs to be decoded by a molecule concerned specifically with the task. Some areas governed by these codes are: transport of the enzyme to desired locus of action, substrate binding, folding of nascent chain, site antigenicity and metal binding.

*

for each other" explains the Therelationship popular between saying, "Made an enzyme and its substrate. In a biological system, each reaction is catalysed by a different enzyme. In an enzymatic reaction, the substrate first binds to the enzyme to form an enzyme-substrate complex. Only later is the product rele",;sed from the enzyme which now becomes free to bind to another molecule of the substrate.

Made for Each Other

The area of the enzyme where the substrate binds and catalysis takes place is called .the 'active site'. Only a part of enzyme constitutes the active site. Other areas of the enzyme molecule contribute towards maintaining the conformation of the active site. Only compounds that can fit into the active site are accepted as a substrate by a given enzyme. The mechanism resembles a nursery school jigsaw game wherein a triangular piece can fit only into a triangular slot, a square piece into a square slot and a circular piece into a circular slot. The model that explains the binding of substrate to enzyme ts also known as lock and key hypothesis because just as a specific lock needs its own key to be opened, each substrate requires a specific

14

ENZYMES EVERYWHERE

Enzymes and their substrates are 'made for each other'

enzyme to break it down. Because of the tailor-made requirementin forming enzyme-substrate complex, all enzymes are highly specific. Catalysis by an enzyme involves targeted interaction between the substrate and the enzyme molecule. For this, the conformation of enzyme molecule should also be maintained in the desired form. Thus, any change in the environment or structure of the enzyme molecule results in an alteration in the rate of enzyme activity. The end-products of certain enzymes can also cause undesirable changes in metabolic activity. Therefore the formation of such products or in .other words, the activity of enzymes generating such products needs to be regulated.

MADE FOR EACH OTHER

15

60' 50'

Temperature and pH are two of the most common physical parameters that affect enzyme activity. Each enzyme has its own optimum pH and temperature at which its rate of enzymatic reaction is maximum. Optimum pH and temperature for most of the enzymes range between 5 to 7 and 40° to 60°C respectively. However, an ample number of enzymes have optimum pH and temperature outside this range. A chemical entity that affects almost all enzymes is their respective substrate. This is because the substr;:l.te binds

16

ENZYMES EVERYWHERE

physically to the enzyme. Besides substrates, many chemical effectors alter the Qatalyt~cactivity of the enzyme. The compounds that increase the enzyme activity are called activators and those that decrease the activity are called inhibitors. Such effectors include metal ions, substrate analogues, end-products and certain organic compounds. In many enzymatic reactions, the enzyme alone is not capable of performing the reaction and some cofadors are essential to its optional functioning. The cofactors may be metal ions, co-enzymes or proteins. In the normal process of metabolism, enzyme activity is highly regulated. Cellular regulation of enzyme action is a complex phenomenon although we may tend to take it for granted. The most common demonstration of enzyme action" that we can see happens when we hurt ourselves. It is an experience every one of us has had. A wound or a cut begins to bleed but after a while, the bleeding stops and a blood clot is formed. The formation of blood clot is a result of a series of reactions wherein specific proteases are activated in a sequential manner. In a cascade reaction the product of previous reaction catalyses the next reaction. The regulation of the enzymatic sequence resembles an automobile assembly line wherein the product of one shop is the raw material for the next. Another interesting example of enzyme regulation is seen during the synthesis of lactose or milk sugar. Lactose is "synthesized in the mammary gland by an enzyme termed lactose synthase. Lactose synthase is a complex molecule made up of the enzyme galactosyltransferase and a protein alpha-lactalbumin. Galactosyltransferase alone cannot synthesize lactose though it catalyses transfer of galactose to various proteins in many tissues. Its ability to transfer galactose to glucose forming lactose is very low. Alpha-lactalbumin is a milk protein, synthesized and present only in mammary glands. Alpha-lactalbumin joiJ:lshands, so to say with galactosyltransferase to form lactose synthase, which

MADE FOR EACH OTHER

17

Red blood cells

White blood cells

Clotting of blood is a chain reaction brought about by enzymes

can now very efficiently catalyse transfer of galactose to glucose to form lactose. Enzymes are made in the living cells by the process of protein synthesis. The amino acid sequence of an enzyme is encoded by a stretch of DNA that is designated as a gene. It is extremely interesting that although a cell contains all the genetic information needed to produce all the proteins, it does not produce the proteins continuously at all times. A normal cell produces the necessary protein(s) and that too, at a regulated rate. The sequence of nucleotide bases in the DNA molecule determines the structures of proteins produced. The informa-

18

ENZYMES EVERyWHERE

tion encoded in the sequence of the DNA molecule is copied on to the strands of a messenger molecule called messenger RNA. This process is called transcription. It involves copying on the mRNA, the base sequence present on the DNA segment. However, there is one important difference between the DNA message and its RNA copy. RNA has the base uracil wherever the base thymine is present on the corresponding' DNA segment. The synthesis of a messenger RNA transcript is catalysed by the enzyme RNA polymerase which carries out multiple A

ChainB~.

~~>7~ ChainA

.:f\~!

./2,.~...~ nucleoside Building triphospnate' 'tmits of ~ Released phosphate

RNA-polymerase helps in transcription

MADE FOR EACH OTHER

19

functions in the cell. It recognises a molecular 'start' signal which indicates to it the beginning of a gene. It then 'reads' the gene. Finally it recognises the 'stop' signal which indicates the end of the gene and thus end of transcription for that particular sequence. The DNA sequence that RNA polymerase recognises as a start signal is called 'promoter'. The same or similar promoter sequences exist at the start of most genes so that the same RNA polymerase can be used to transcribe a large number of genes. Only one of the two DNA strands is transcribed and the final mRNA molecule contains a copy of all the information originally present in the DNA molecule. However, it still needs enzymatic processing or modification before the functional finished products are released. In the early days of molecular biology it was thought that the sequence of nuc1eotides transcribed from the gene was completely represented in the mRNA which in turn, was completely translated into the amino acid language of proteins. With better techniques for gene analysis, this idea has had to be revised .. In higher organisms the genes are found to contain stretches of coding sequences interspersed with pieces of DNA sequences which do not apparently make sense. Such genes are called 'interrupted' or 'split' genes. It is surprising that entire sequences, irrespective of whether 'junk' or not, are copied during transcription. The meaningless sequences that occur within genes are called 'introns'. The sequences of mRNA coding for them are edited out by cellular enzymes. Such post-transcriptional modifications create a continuous, meaningful mRNA message. The portions retained and which finally translate into the amino acid language of proteins are termed 'exons'. The mRNA that finally contains the entire coded informa-· tion has to undergo further modifications before the second step for protein synthesis can begin. In this post-transcriptional processing, the mRNA is given a 'tail' made of many adenine molecules. This 'poly A: tail is believed to help

20

ENZYMES EVERYWHERE

Exon

Intron

Exon

Intron

Exon

DNA

Full length mRNA

1

1I£_og 10'''''''

Processed mRNA

1

TMo,'ation

Protein

Split genes need tailoring before functional enzymes can be made

transfer the mRNA from the nucleus where it is formed tp the cytoplasm where it carries out its function. The 'tail' is sub'sequently removed before the second step in protein synthe.sis can begin. It is called translation, because it involves a change from the nucleotide language of mRNA to the amino acid language of proteins. The mRNA transcript is the intermediary through which the genetic code is conveyed to the protein synthesising machinery of the cell for decoding. In this process, the mRNA gets associated with ribosomes which are compact cellular organelles with their own RNA (ribosomal or rRNA) and a defined set of ribosomal proteins. Another kind of RNA

MADE FOR EACH OTHER

21

Anticodon tRNA has a clover-leaf structure. Code words for amino acids (inset)

called transfer RNA (tRNA) also plays an important role in translation and is considered to be the I adapter molecule' that translates the genetic code into amino acid language. Each mRNA strand becomes associated with ribosomes to form a polyribosome or polysome. The ribosomes associate with the mRNA at a specific end (5' end) and read the

22

ENZYMES EVERYWHERE

message, triplet by triplet. Since the mRNA coded language is written as a series of triplet bases or 'words' it is evident that the nature of the message would change if the reading frame of the code shifted by even a single ba(5e.The first triplet base or codon is called the initiation codon and its spelling is always AVe. It codes for the amino acid methionine. All newly formed protein chains begin with methionine which is later enzymatically cleaved. Each subsequent codon pairs with the complementary 'anticodon' of fhe tRNA and a ,checking factor' ensures that there is a correct fit between the codon and the anticodon. The sequence of codons on an mRNA molecule determines the sequence of anticodons of the "different tRNA molecules, and thus, the different amino acids. There are as many kinds of transfer RNAs in the cell as there are codons. In all there are 61 of them, since these codons are nonsense ones that do not code for any amino acids. The nonsense codons are VAG, VGA and VAA and each of the codons stands for a molecular 'stop' sign indicating the end of translation. Sometimes there may be more than one tRNA for a partkular amino acid. Since the triplet codons of mRNA which are ultimately translated into proteins are dependent on the DNA molecule, it is evident that the DNA molecule holds the key to protein structure. In fact, the central dogma of molecular biology sums it up in one succint sentence. DNA transcription RNA translation protein. Grammatically stated it would read: DNA makes RNA and RNA makes protein. Once the newly formed polypeptide chain is released it ~s folded into the structurally stable molecular conformation. The molecule thus formed is subjected to other modifications as well and only then it is transported to a location where its activity is required by the body. The entire system by which coded genetic information is processed, is replete with scope for error. However, the 'proof-reading' enzymes and 'repair' enzymes ensure that errors are detected and elimin~ted by

23

MADE FOR EACH OTHER

Replication

r@ ~

~)

Translation ~~~ Protein RNA

DNA

Original dogma,

.~

Intron

Transcription

~

processlngZT' L::::::t> m~A~ranSCriPtio~

Reverse Transcription Pre-mRNA

<7

Exon

~

..... '.

I..•

Protem mRNA

Revised dogma

The central dogma of molecular biology has been modified over the years

the built-in systems of the cells. The normal functioning of a cell is a tribute to multifarious roles enzymes play. q.

of enzymes is not new. The The . usecommercial of yeasts as aexploitation biocatalyst

Enzyme Econo•

mlCS

dates back to about 6000 B.c. However, the production of enzymes on a large scale cannot boast of so ancient a history. The first large-scale production of enzyme came about only in 1874, with the first industrial batch of chymosin. After that there was no looking back. From the 1913's onwards the detergent industry be. came a prime consumer of enzymes. A similar trend was set by t~e leather industry in 1917 and the starch industry in 1950. The last three decades have been marked by an explosive advancement in the field of commercial production of enzymes. The advancements in the techniques of genetic engineering .which permit the manipulation of cellul~r DNA, have led to the opening up of a new field called protein engineering. The structure of a protein can now be altered by effecting specific and precise changes in the DNA molecule; changes which ultimately will be reflected in the protein formed. The structurally altered enzyme thus obtained has different physicochemical properties which distinguishes it from its

ENZYME ECONOMICS

25

The future of the enzyme industry is bright

normal cellular counterpart. The physicochemical differences engineered into the enzyme would, of course, depend on the requirement(s) of the industry which would be putting it to use. For example, an alkaline protease with enhanced resistance 'to heat would be ideally suited for the detergent industry. It has also been possible to increase manifold, production of microbial enzymes by inserting extra copies of the gene responsible for producing the enzymes into the microbe's genome. The extra copies of the gene result in hyperproduction of the enzyme and this increased yield means extra profit for the industry. That we now have the capability to use microbes to express important enzymes of animal and plant origin is a feather in the cap of biotechnology. It is, however, difficult to obtain the exact sales-figure of enzymes annually on a global scale: A conservative estimate has placed

26

ENZYMES EVERYWHERE

the world-wide sale of enzymes for the year 1990 at about 720 million US dollars. Since then, the market has grown and diversified. The question that naturally arises is, 'from where do all these enzymes come?' Or in other words, 'What is the source of commercially exploited enzymes '? Any living organism can be source of an enzyme. Major sources of enzymes include plants, animals and even. microorganisms.Traditionally, microorganisms have been the most favoured source of enzymes, mainly because of the ease with which they can be cultivated in large fermentation vats. But first cultivating a microbial or animal or plant species is not enough. A number of operations have to be undertaken prior to being able to use an enzyme industrially. The operations that need to be carried out dep.end on whether animal or plant or microbial species is the source for the enzyme. If the source is an animal or plant, the organ or plant-part that is the source of the enzyme has to be isolated first and then, the enzyme has to be purified from the extract. If the enzyme is from a microbial source the nature of the species influences the way in which the enzyme is to be isolated. Certain microbes secrete the enzyme into the cultivation medium while others do not. In the former case, the culture medium is the source of the enzyme, while in the latter microbial extract has to be prepared first. The-enzyme(s) present in either the extract or the culture medium has to be purified by using various separation methods. The level of purification depends on the ultimate end use of the enzyme. The cost of the purified enzyme also has to be within reasonable limits and this criterion also affects the level of purification. For example, amylases and pectinases used in the food industry are crude enzyme preparations whereas urokinase and streptokinase used as drugs are highly purified preparations. The form in which an enzymes is used depends on the nature of its application, properties, manufacturing cost and also on the properties of its substrate. The different prepara-

ENZYME ECONOMICS

27

The first ever enzyme bank!

tions of enzymes that are currently in use can be grouped into four types; soluble enzyme, whole cells, immobilized enzyme and immobilized cells. Soluble enzyme is the crude extract, clarified broth or purified enzyme preparation. Enzymes used in this form are low cosJ preparations used mainly for hydrolysis of carbohydrates, proteins and fats. Intact cells containing desired enzymes are often used as enzyme capsules. These are the 'whole cells'. This approach is used

28

ENZYMES EVERYWHERE

mainly when the enzyme becomes unstable after extraction from cells or the process for purification is not economical enough. Enzymes can be reused if they are separated from the reaction mixture after completion of the reaction, Enzymes, being large molecules and soluble in water are very difficult to recover from reaction mixtures but enzymes particuarly the expensive ones need to be recovered and reused to make a process economically attractive .. A brilliant approach called immobilization was developed in the 1960s which is now routinely used in various processes. Immobilization of enzymes, means what it implies. It means the attachment or confinement of an enzyme to an insoluble support so that it is capable of catalysing the reaction and once that is done, it can be separated from the reaction mixture by simple means. The immobilization of the enzyme may be brought about by physical or chemical means. Enzymes can be reused many times in this form. The benefits of this method become evident when immobilized glucose isomerase is used for more than 200 days continuously and immobilized penicillin acylase more than 1000 times. Little wonder the,n, that this is a popular method. When microbial cells containing enzyme are confined to a solid support, the catalyst preparation is called immobilized cells. For performing an enzymatic reaction the substrate and enzyme have to be mixed under appropriate conditions so that the enzyme is active and stable, and the substrate and product are not degraded. The vessel in which the enzymatic reaction is performed is called the enzyme reactor. Different applications demand different enzyme reactors. The design and the choice of a reactor is determined by various issues, such as properties of enzyme, form of enzyme, properties of the support (if immobilized), properties of the substrate and product, reaction parameters, reaction kinetics, batch size and schedule of replacement of enzyme. Operational parameters such as pH, temperature and concentration of sub-

ENZYME ECONOMICS

29

strate are so maintained that the catalytic efficiency of an enzyme remains at its maximum . ••

tegral part of human life. an inproducts that have been obtained by using enzymes either directly or indirectly. Enzymes are used in different industries for making a number of products. The emphasis on the use of enzymes in industry has come about because enzymes have unique properties. Catalysis of chemical reactions at • very high rates, operations at ambient conditions, selectivity of substrate, minimal side reactions, simple operations, availability in large amounts from microbial sources, reusability if immobilized, non-toxic nature and nonpolluting effluent generation are only some of them. These properties make enzymatic processes cost effective and energy saving when oompared to chemical processes. Further, special equipment such as containers resistant to corrosion, heat or pressure are not required for enzymatic processes.

Enzymes Everyonehave of become us uses

Spirited Enzymes

During the early days of enzyme technology development was slow. This was due to impediments in the fields of enzyme stabilization, production on large scale, co-factor regeneration and lack of enzyme immobilization faciiities. Advancements in vari-

SPIRITED ENZYMES

31

ous areas of biotechnology have overcome most of the hurdles and currently, the pace of development of enzyme technology is quite rapid. It is very difficult to list applications of all enzymes, simply because of the exhaustive nature of such a compilation. Further, some enzymes like amylases, proteases and cellulases are routinely used in many different industries to derive more than one product although not all industries rely equally on the some enzyme to make their products~ Of all industries that use enzymes, food and dairy industry, pharmaceutical industry and detergent industry seem to rely the most on enzymes to produce products for mass consumption. The textile industry and paper industry also use enzymes for various processes. Ancient man no doubt relished the spontaneously fermented slurries of fruits, honey and cereals but it took some

Ancient painting showing wine-making

32

ENZYMES EVERYWHERE

time for him to eventually work out methods that would yield similar results. It is likely that in the early days at least, the mechanism behind fermentation was not quite understood. By the time Egyptian, Greek and Roman civilizations reached their respective pinnacles, however, brewing alcohol had become common practice. The earliest references to wine date back about 5000 years. Ancient Egyptian panels showing people harvesting, crushing grapes and storing wine have been discovered. During the Middle Ages, certain monasteries earned fame because of their devotion to the art. Beer, wine and other alcoholic spirits were produced by the gallon much before the scientific facts behind fermentation by yeast was worked out in 1876, by Louis Pasteur. In fact, the production of alcoholic wine by yeast fermentation is considered to be the oldest fermentation process known to man. Today, of course, the science of brewing has made incredible progress and innovations have .produced a plethora of rare spirits. However, the basic process of brewing alcohol remains unaltered. Malt and hot water are taken in a tank to which select cereals are added and the ingredients are mashed up. During the process of mashing, enzymes present in the malt degrade the proteins, starch and other polysaccharides present in the malt and other cereals. The syrup obtained from the mash is called wort. It is filtered and transferred to a fermenter. The conditions in the fermenter encourage the growth of yeasts which bring about fermentative changes resulting in the production of alcohol. Yeasts are a kind of fungus and are found almost everywhere in nature. Since they lack the green colouring matter or chlorophyll that enables plants to synthesize their own food, yeasts depend on external sources for food. They feed on sugar obtained from a variety of natural sources such as fruit, cereals, nectar and molasses. Yeast enzymes or ferments break down food substrate so as to assimilate it easily. Different species of yeast produce different kinds of enzymes. Those yeasts that have enzymes that can break down sugar

SPIRITED ENZYMES

33

Malt kiln Maltbin$

Spirit receiver

Making whisky

into alcohol and carbon dioxide are much in demand by brewers to make wine. Yeast used by the beer industry is called brewer's yeast. It is different from baker's yeast. Brewer's yeast cannot act directly on the cereal used to brew beer. Brewers need to convert the starch in the grain to sugar by the process of malting. The yeast then converts the sugar to alcohol by the process of fermentation. Purified enzymes can replace malt as a source of enzyme. Purified preparations of enzymes such as alpha-amylase, glucanases, proteases and amylo glucosidase are added at the mashing stage for the degradation of polysaccharides and proteins. Malt is also

ENZYMES EVERYWHERE

34

Yeasts

replaced by barley and seasonal cereals used as supplement in breweries. Proteins are also added to the syrup obtained after mashing. This protein maintains the ratio between carbon and nitrogen in the syrup, an essential requirement for the growth of yeast. However, these adjuvents are not metabolised by the yeast. The next step in brewing of alcohol necessitates filtration. However, the presence of un-degraded polysaccharides make the syrup thick and viscous which makes filtration, a time consuming process. Prolonged filtration of the syrup adversely alters the quality of beer, particularly its flavour and stability. A cocktail of enzymes consisting of beta-glucanases, pentosanase xylanase, arabinase and beta-amylase is used to completely break down the complex carbohydrates. This leads to easier filtration of the syrup. Alpha-

35

SPIRITED ENZYMES

fE~y~

Ai?plkation/prodtict

Remo!al ofstarcl1 frO!llJ2La:.~.,,1

I~~p~n

~emoval of turbidity

11P~~tinase+Ce~ula~ .",

Maceration an41iquification :.•............. 'd..... ' ··.,..'-."L ..••...__.~ . ... ~---~-~------~

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'Protease ~--

Enzymes in fruit juice and wine industry

amylases are sometimes used to hydrolyse residual starch molecules to improve the fermentability of the syrup. Once the beer is brewed, special attention is p3.id to 'details'. Proteins and tannic substances present in beer form undesirable colloidal precipitate when beer is chilled before consumption. The formation of this "chillhaze" may be prevented by degradation of the protein in the beer. Papain, an enzyme isolated from papaya is used to break down the remnants of protein in beer. Tannins may be removed by treating the beer with polyvinylpyrolidone. However, the process is tedious and also expensive.

36

ENZTh1ES EVERYWHERE

Normally beer contains a small quantity of dextrins which are soluble polysaccharides produced as a result of hydrolysis of starch. Dextrins increase the caloric value of beer, a fact that deters diabetics. The use of enzymes such as amyloglucosidase, pullulanase and amylase removes the dextrins'and so these enzymes are used to prepare low caloric beer for th~ diet conscious. Sometimes beet sugar is used as a source of carbohydrate in brewing. Beet sugar contains raffinose, a trisaccharide which yields glucose, fructose and galactose on hydrolysis. The enzyme alpha-galactosidase is used to hydrolyse raffinose whenever beet sugar is used in brewing. Once alcohol has been brewed it has to mature for optimal development of flavours. The use of another enzyme, alphaaceto acetate decarboxylase can shorten or even completely eliminate the maturation period. Wine is another alcoholic beverage. It is mostly made from grapes but it can also be made from fruits such as apples and pears. Wines are usu~lly classified according to their colours into red, white or rose wines. The colour comes from the skin of crushed grapes of which wine is made. When grape skins have little contact with the juice a pale wine called blanc de noir is produced. Wine-making, like the brewing of beer is a multistep process. Grapes are harvested as soon as they are ripe and have optimal acid content, flavour and aroma. Then grapes are cleaned and crushed by a machine. The crushed grapes and the juice is called must. Depending on the actual colour of the wine, the wine-maker allows the skin and juice to remain in contact for varying periods of time. To make red wine, the seeds and skins are allowed to enter the fermentation tank along with the juice.For white wine, the skin and pulp are filtered out and only the juice is allowed to eJlter the fermenter. During the process of fermentation, the yeasts change the glucose and fructose present in the juice to ethanol and

SPIRITED ENZYMES

37

Filter

Wine making

38

ENZYMES EVERYWHERE

carbon dioxide. Yeasts also produce some by-products that contribute to the wine's flavour and aroma. The fermentation of red wine takes 4-6 days and white wine fermentation takes thrice as long. Most red table wines and some white table wines undergo a second round of fermentation, this time by bacteria. In this second fermentation maleic acid present in the wine is converted into lactic acid. The process is called malolactic fermentation. Although fruits find maximal use in the preparation of fruit- juices, juice-concentrates and fruit-pulp, some fruits are used to make wine as well. The first step in any process that uses fruit is, of course, crushing it to obtain an extract. However, the presence of pectin - a substance that forms the matrix of plant cell walls and which cements cellulose fibres together, reduces extraction efficiency. The soluble pectins present in the juice imparts a cloudiness to it. It also retards filtration due to high viscosity of the extract. The hurdle is overcome by using pectinases. Pectinases hydrolyse the insoluble pectin thereby increasing the yield of juice extraction and thus the rate of filtration of juices and wine is increased. Clearer and thinner juices are less likely to clog filters or tubings. Blockages caused by bulky pulps, in contrast, can sometimes wreak havoc in bottling plants. Food technologists, Harvey T Chan and his colleagues at the University of Hawaii, have isolated pectinases from the fungus Aspergillus niger which when raised in indoor vats proved to be a prolific producer of the enzyme. Pectinases are also used in the fermentation of red wine, and production of homogeneous purees and nectar bases. The enzyme finds use in the extraction of clear juice from many fruits. Apart from pectinase, many other enzymes such as papain, naringinase, proteases, arabinase, cellulase and amyloglucosidase are roped in by the fruit juice and wine industry. During the extraction of coffee from coffee seeds, the flesh of the coffee fruit is removed from the seed (coffee bean). The process is very similar to the extraction of juice or pulp from

Enzymes in coffee and tea industry

other fruits. Naturally, the pectin present in coffee fruit interferes with the process of extraction and lowers the yield of coffee. Pectinases are used to degrade the pectin in coffee fruit pulp for increasing the extraction efficiency. For preparation of instant coffee, the coffee extract is highly concentrated and then spray-dried or freeze dried. A special type of polysaccharide called galactomannan which is present in the coffee extract contributes to the viscosity of the extract and limits the extent to which it can be concentrated. The galactomannans are degraded by using the enzyme galact(;!l1annase. In tea it is desirable to develop a colour without extracting too much of tannins in the brew. Phenol oxidases are used in tea processing to oxidize certain aromatic compounds giving the colour when the drink is prepared .. It would seem that nature has provided all the enzymes man may need in his commercial endeavours .

•

'staff of life' because in

Bread called some has formbeen or the otherthe it forms the staple diet of the world. It is thought that ancient Egyptians first learnt to crush grain, add water to it to form a dough and then bake it to make bread some 3000years before Christ. It was also the Egyptians who discovered, probably by chance, that by using yeast they could have lighter, fluffier 'loaves of bread'.

Our

Daily Bread

Today too, the two main types of bread continue to be leavened and unleavened. Leavened bread usually has yeast added to the dough so that it is light and fluffy. Unleavened bread does not have yeast added to the dough and is therefore, always dry and hard. Most of the bread available today is made from wheat because wheat contains substance called gluten which helps the bread to 'rise' so that the loaf made from wheat is lighter and fluffier than bread made with other cereals. Gluten is a reserve protein found in cereals and the 'sticky substance that acts as the binding agent in drugs. Rye flour, for example, does not have gluten so wheat flour is blended with it to give fluffy bread. Unblended rye

OUR DAILY BREAD

41

Give us this day our daily bread

42

ENZYMES EVERYWHERE

flour gives crisp, hard biscuit-like bread when baked. Bakeries commercially producing bread rely on enzymes to facilitate the process of baking and to enhance the quality of the bread. The process of bread-making starts with milling of grains. Grains contain small amounts of enzymes such as amylases and proteases. Amylases degrade the starch in the flour to sugars. Once the flour is ready, a dough is made by • mixing the ingredients which comprise flour, yeast, salt, sugar, fat, water, flavours and additives. Once added to the dough, the yeast starts utilising the sugars present in the dough. This process releases carbon dioxide. The carbon dioxide produced by the yeast makes the dough rise. The protocol runs smoothly if the grain contains sufficient amylases to degrade starch. However, the quantity of amlyases present in the flour varies with geographical variation, seed quality of the grains, cultivation conditions, milling temperature and storage of flour. Usually the amylase content of flour is very low. This leads to lesser starch degradation and hence inferior quality of bread. But all hope need not be abandoned. Addition of extra enzymes to compensate for the low amylase content is one option that bakers exercise so as to get bread of superior quality. The other option is to supplement the dough with sugars. The addition of amylase has the inl:terent advantage that its action generates sugars at a rate that is optimal for consumption by the yeast. The addition of amylase at a controlled rate thus not only caters to the needs of the yeast but also permits standardization of the enzyme content of different batches of flour. A standardized starting material obviously ensures final products of uniform quality especially if the subsequent steps are standardized as well. Bread made from wheat and rye flour need the helping action of another enzyme as well. This enzyme is pentosanase and as its name indicates, it acts on pentosans present in the ~our. Pentosans are polysaccharides made of condensed pentose units. Pentosans adversely interact with gluten and the result is poor loaf structure. Bakers, thus, prefer to degrade

43

OUR DAILY BREAD

Enzymes used in baking industry

pentosans present in the dough so that larger loaf volume and improved crumb structure can be achieved. The aroma wafting from freshly baked bread is no doubt a delightful experience. No less delightful is the visual experience of sighting the nicely browned crust of the load. This brown colour, so tempting to look at, is the result of Maillard reaction which takes place when an amino acid and a sugar react. It also contributes to a characteristic flavour. Bakers enhance the browning of the crust by increasing the glucose content of the bread by using the enzyme amyloglucosidase. Baking biscuits, however, entails attention to qualities other than lightness. People appreciate a certain' chewiness' in biscuits, crackers and cookies. To achieve this, it becomes necessary to have a dough that is soft and which has pronounced plasticity. Plastic properties of dough are determined by the gluten content and selected enzymes are used

44

ENZYMES EVERYWHERE

to control the degradation of gluten so as to obtain desired plasticity. The result is evident when one bites into a premium-quality biscuit that goes 'crunch' in the mouth.

*

been part of man's Milkhave products diet and since milk the early days

Milking the \

Enzymes

of civilization. Cheese, fermented drinks and curds are only a few examples of the many ways in which milk may be enjoyed. Man discovered quite early in the history of dairy science that raw milk of good quality develops a clean, sour flavour if kept under conditions that permit bacterial growth. The Bible has references to fermented milk and no doubt, this beverage gave consumers the opportunity to taste a drink with a distinctive and desirable flavour. Today purified enzymes help dairy scientists to create milkproducts of high quality. The many different kinds of cheese that line supermarket shelves bear testimony to that. The manufacture of all cheese depends on the activity of microorganisms. Several hundred varieties of cheese are manufactured, and interestingly, most can be made from the same batch of milk by varying the conditions to suit the selected microorganism. The first step in cheese production calls for the clotting of milk to give a firm curd consisting mainly of coagulated casein. Clotting milk is

46

ENZYMES EVERYWHERE

Say Cheese!

probably an ancient practice and is still prevalant. In the ancient days (800 Be) the stomach of a freshly slaughtered goat was used to carry out this process. Later on (19th century), the use of stomachs of young animals such as calves and lambs replaced the use of goat stomachs. The reason behind this usage was the natural presence of the enzyme rennin which coagulates milk. Freshly slaughtered animals were used so that the rennin present in the stomach could be used to coagulate the milk before degradative chances that set in after death deactivated the enzyme. Of course, the ancients did not know how rennin worked or even what it was. For them it was a tradition that had to be followed.

MILKING THE ENZYMES

47

Today rennin extracted from microorganism are available for commercial purposes and young animals are not slaughtered for rennin any more. Rennin is a protease and it breaks the peptide bonds on a protein molecule. However, rennin does not break all the peptide bonds in the protein molecule rather, its action is restricted to specific bonds in the casein molecule. Since it selectively breaks certain bonds and not others, it brings about limited cleavage in the protein molecule which then precipitates in the milk as a mass with a smooth, gel-like appearance. Addition of certain other enzymes can cause new flavour to develop in cheese. Lipases that convert milk fat into fatty acids are used to develop some flavours. Cheese . is also hydrolysed further by other proteases to obtain' enzyme-modified' cheese which is used in flavouring of soups, dressings, dips and snack-foods. Most cheese require 'ripening' by bacteria or molds after the coagulated casein is pressed into characteristic size and shape. Depending on whether the cheese is to be the 'hard' or the 'soft' type, ripening can be of two types. For hard cheese like Swiss or Cheddar, the bacteria are distributed throughout the interior of the casein to be 'ripened'. For soft cheese such as Limburger and Camembert, the bacteria are encouraged to grow on the surface. Since the microbial enzymes must diffuse from the surface and then 'seep' in, the soft cheeses are usually made in small sizes. The process of 'ripening' is a complex one and it is difficult to distinguish all the biochemical· changes that takes place. It is even more difficult to link the changes to specific microbial species. However, it is evident that during the process of 'ripening' much of the lactose in the curdled milk is degraded to lactic acid and small amouI1ts of volatile acids and alcohols are also produced. Proteolysis degrades proteins to amino acids which is turn may be further broken down to water soluble compounds as in the case of Camembert and Limburger

ENZYMES EVERYWHERE

48

cheese. Hard cheese, such as Swiss cheese, ~hows a lesser degree of proteolysis.

Lactase

Protease Milk.wotein ~meDJo('H6ed~cation Ckeese Membrane deiIDing ~~:~~+:"""""~ Lipase

Develops' [email protected]~.ese Hydrolyses-bo.tter fat R~moveshydrog~np~, .!1i

Milk flavour improVe: Preservation

Enzymes used in the dairy industry

MILKING THE ENZYMES

49

During the ripening of Roquefort and Blue cheese, the mold Penicillium roqueforti releases lipases that hydrolyse the fat, releasing butyric, caproic, caprylic, capric and higher fatty acids. Some of these are oxidized forming compounds that contribute to the characteristic flavour of these cheeses. Milk products enjoy wide popularity not only because of the_irtaste and flavour. It is estimated that about 66 per cent

All this and more

50

ENZYMES EVERYWHERE

of the world's population has lactose intolerance, that is, they cannot digest lactose, the sugar present in milk and so cannot enjoy milk unless it is processed into a product. In order to overcome this problem, lactase is used to break down milk sugar lactose to glucose and galactose. Milk and whey treated with lactase become free of lactose. The enzyme action also improves other properties of milk. The texture of milk is improved and its tendency to crystallize during the making of ice-cream is reduced so that smoother ice cream can be made. Milk needs to be 'improved' by controlled enzymatic reactions because its final properties have a crucial role to play in the production of bakery and meat products. Emulsifying capacity, solubility and whipping properties of milk proteins are some parameters that must be rigidly standardized if quality products are to be made. Controlled hydrolysis of milk proteins by pro teases is used to develop these charac. teristics. Trypsin and sulfhydryl oxidase are used for flavour development. Esterases are used in the hydrolysis of butter fat to obtain low fat drinks. These drinks enjoy a large market in today' s calorie conscious' world. Apart from enhancing the quality of milk, enzymes play an important role in cleaning the membranes used in the dairy for filtering milk and its components. A coating of protein often fouls these membranes and proteases are used to break them down, cleaning the membranes in the process. Cleanliness is thus achieved enzymatically. Preservation of milk products is also done by using selected enzymes. Glucose oxidase prevents oxidation and/or microbial growth and ensures that the milk remains fresh and tasty till the last drop.

*

It

On the Supermarket Shelf

quantify how dependent the would be difficult to truly food industry is on enzymes. A walk down the supermarket aisle or even a peep into the local store might give a hint as to why the job would be almost impossible. There is virtually no processed product that does not use enzymes and some use more than one enzyme. Indeed certain rawmaterials would have had only limited use had not enzymes converted them to. compound with versatile uses. Starch a polymer of glucose, is a storage polysaccharide in nature. Starch as such has limited applications unless hydrolysed to glucose. The glucose syrup so obtained can be used in various products such as soft drinks, confectionary, ice-creams, baked products, meats, sauces, canned fruits, baby food, and alcohol. Different grades of syrups suitable for use by different industries are generated by using different enzymes. It is easy to degrade starch. In the good old days starch was hydrolysed using acids at elevated temperatures of 140° to 150°C. However, this resulted in low quality of syrup. Today, enzymes make a better job of it.

52

ENZYMES EVERYWHERE

Take your pick

The enzymes used in hydrolysis of starch are alpha-amylase, amyloglucosidase, pullulanase and isoamylase. Alphaamylase liquifies starch, generating smaller fragments of glucose chains or dextrins. Amyloglucosidase hydrolyses dextrins by a process called saccharification, to form glucose syrup. Pullulanase and isoamylase enhance the saccharification by generating amylose fragments. Alpha-amylases which retain their activity at temperatures as high as 105° to 110°C have been developed and are available in the market.Glucose syrup. thus obtained from starch can be fermented to form alcohol. However, its major use is in the food industry which

ON THE SUPERMARKET SHELF

53

Enzymes in the starch industry

demands a certain degree of sweetness in its basic raw material. Glucose as such is not a very sweet sugar. An enzyme called glucose isomerase is used to increase the sweetness of glucose syrup. This enzyme converts glucose into fructose. Fructose, commonly called fruit sugar because it occurs in ripe fruits, is about twice as sweet as glucose. Glucose syrup after treatment with glucose isomerase contains both glucose and fructose. This is because the reaction catalysed by glucose isomerase is a reversible one. This means that just as glucose is converted to fructose in the forward reaction, the reverse also takes place simultaneously, with some fructose being reconverted to glucose. Once an equilibrium stage is reached it is possible to obtain a syrup containing 55% fructose. Such syrups are called high fructose com syrup (HFCS) and are as sweet as ordinary cane sugar and have equal energy content. HFCS has replaced sugar (sucrose) in many food products. Another industry that uses enzymes in a big way is the protein industry. It constitutes the sectors whose final products are proteins and which also deal with the processing of

54

ENZYMES EVERYWHERE

Enzymes in the protein industry

proteinaceous raw materials. Since protein is a basic constituent of plants and animals, its processing forms the backbone of many industries. Leather industry, dairy industry and breweries are only some examples of the protein industry.Enzymes help the protein industry to convert bones collected from slaughter houses to gelatin, a product with many uses. Such bones usually have some meat attached to them. This residual meat that remains adhering to the bone is removed by protein digesting enzymes. The cleaned bones thus obtained are used to prepare pure gelatin and the meat extract is used in canned meat products and soups. So nothing is wasted after all. Even the blood collected from slaughter houses is used to make products that have economic value.

ON THE SUPERMARKET SHELF

55

Red blood cells (RBC)account for 75 per-cent of total blood proteins. However, the use of RBCs is discouraged because of their red colour. So the RBCs are treated with proteases to obtain blood cell hydrolysate (BCH) which is a pale liquid and which becomes a white powder when dried. BCH has several applications: water binding agent for chopped meat and meat extender in minced meat being the two most common uses.Certain fish oils have medicinal value. Enzymes are used to extract these oils from fish proteins. The extraction process is accelerated by using the protein degrading enzyme papain. The indirect use of papain is known to every cook who has used slices of raw papaya to tenderize meat. The self-same enzyme in its pure form is used by the industry to hasten fish protein digestion. The fish- industry has other novel uses for enzymes too. 'Stick water' is a term used in the fish industry for the supernatant that remains after cooking and processing of fish. Stick water has some soluble proteins in it which are not allowed to go waste but recovered by the process of evaporation which gets rid of the extra water. This process is, however, slow after the extract attains a particular viscosity. Hydrolysis of proteins by proteases reduces the viscosity of the liquid undergoing evaporation and this makes the process of recovery more efficient. Vegetable proteins too have found application in various food products. In order to obtain the concentrated proteins from vegetables, polysaccharides are removed from the vegetables by using appropriate enzymes. Those most commonly used are the cellulases and amylases. Protein hydrolysates, prepared by using proteases are used to enrich food products such as juices. The addition of protein makes these juices more nutritive and this value-addition is made possible only by the judicious use of enzymes that helped produce the protein hydrolysate. Certain proteins and peptides generated by specific proteolysis possess unique properties which are utilized in prepa-

56

ENZYMES EVERYWHERE

ration of various products. Human beings are not the only ones to profit from the clever use of enzymes. Animals too are beneficiaries of products made by using enzymes. Many animals, specially the young ones, are unable to digest the food components of different artificial feeds. This is of great concern to farmers and animal breeders since only a healthy well- fed animal can lead to profits. Attention is given to the feed of dairy, poultry and meat animals sinceJ:he growth of such animals is directly related to business. Digestive enzymes, specially proteases are used to overcome problems of digestion. These enzymes are sometimes added to the . feed, at other times they are given as protein supplementing materials.

ON THE SUPERMARKET SHELF

57

Proteases are also used to improve palatability of pet foods. Hydrolysis of protein increases its absorption in the intestine. The peptides and amino acids generated in the food as a result of protease action give a brothlike savoury flavour which the pets like. Hydrolytic enzymes like cellulases, xylanases, pectinases, amylases and proteases are used in proper combination in poultry feed preparations as well . Enzymatically treated cereal feed is absorbed better and the birds grow faster which is the ultimate goal. Further, hydrolysis of polysccharides like beta-glucans in the feed reduces wet and sticky droppings. Since sticky droppings increase the chances of infection and reduce the growth rate, this means better health for the birds and more profits for the owners. But profit is not the only consideration of the food industry as a part of statutory requirement and standard operating procedure, testing of raw materials, analysis of intermediate products and testing of final products are routinely carried out in food industry. Testing of raw materials and inprocess intermediates forms in part of quality assurance, control of production processes, maintaining the process yield and quality of product. Testing a product before releasing it in the market is a statutory requirement and this is enforced from time to time by concerned authorities. Legal agencies like Food and Drug Administration, Custom Inspectors and Military Inspectors have laid down maximal limits for usage of certain ingredients in food products. Products are permissible for sale, export or import only if they conform to the desired levels of ingredients. Enzymes because of their specificity and easy availability have become very handy tools in analytical methods used for food analysis. Enzymes are used for the determination of the quality and quantity of a varied spectrum of compounds such as polysaccharides, sugars, organic acids, alcohols, fats and cholesterol in food. Many processes used in the food industry need to be monitored at scheduled time intervals for achieving high

58

ENZYMES EVERYWHERE

A few enzymes used in food analyses

ON THE SUPERMARKET SHELF

59

throughputs and reliability. As a result routine analysis of number of samples becomes part of the process. Automatic analyses systems in which all operations of a test are performed sequentially and continuously by robot type operations controlled by a computer have been developed to meet such needs. In these systems the enzymes are 'immobilized' before use. This novel way of using enzymes ensures that the enzymes used is not wasted but that it can be recovered for subsequent use. Immobilized enzymes are simply those enzymes which have been physically confined to one place. It is a way of chemically tying the enzyme to an inert support material such that the enzyme retains all its catalytic power. High fructose syrups which find use as sweetner in soft drinks are commercially prepared by immobilizing the enzyme glucose isomerase on a cellulose polymer. Polystyrene resins, kaolinite, collagen, alumina, glass and silica gel are all used, with varying degrees of effectiveness, as support materials. Another method used for immobilization is one in which the enzyme is enclosed in a net like polymer envelope. The pores in the net are too small to allow the enzyme to escape but large enough to let the substrate molecule enter. There are two types of analytical applications o{immobilized enzymes: the automated analyser and the enzy'me electrode. In automated analysis the immobilized analyser systems where in enzymes bound to the inside walls of the tubes are used in autoanalysers. Enzyme electrodes are probes capable of generating an electrical impulse as a result of reaction catalysed by an immobilised enzyme that is fixed onto or around the probe. The use of immobilized enzymes has markedly decreased costs and the results are also at par with older, more traditional methods of using soluble enzymes. Enzymes also help the food industry to lessen the load on the environment. Effluent treatment in today's environmentally aware world is an important exercise. Enzyme technolo-

60

ENZYMES EVERYWHERE

gies have been developed to treat effluents so that the discharge from the industries is non-polluting in nature. For example, biological oxygen demand (BOD) reduction of milk industry waste is accomplished by hydrolysis of lactose by lactase. Similarly, enzymes are used for removal of pesticides, detergents and other toxic substances. if

metabolism of a cell in-

Whenvolvesit istheknown that the orchestrated

Curing the Ills

participation of many enzymes, it is not surprising to learn that some enzymes are used as theraputic agents. However, this is a relatively unexplored field and much of its potential is still to be exploited. The pharmacetucal industry uses enzymes directly as therapeutic agents and also as catalysts in the production of bulk drugs/ drug intermediates. However, the total number and turnover of therapeutic enzymes is less than one would expect. This is because there are some bottlenecks in the use of enzymes as therapeutic agents. Processing an enzyme to the acceptable level of purity is in itself a hard task and the purified enzyme must not induce allergic reactions in the recipient. Translocation of the enzyme in an active form to the site of action and monitoring its levels in the body continue to be thorns in the skins of the biochemists. Certain methods have been designed to overcome some of these problems. Immobilization and encapsulation of enzymes are two ways in which the bottleneck has been eased.

62

ENZYMES EVERYWHERE

Many enzymes are used as medicines

CURING THE ILLS

63

The most common use of enzymes as an orally administered medicine is as aids to digestion. A large number of people, particularly the kids and the elderly suffer from pancreatic insufficiency which results in digestive disorders. The partial hydrolysis of food constituents, proteins, polysaccharides and fats in impaired digestion leads to diahorrea and weakness. Hydrolases such as chymotrypsin, trypsin, pepsin, papain, lipases, amylases, cellulases, hemicellulases and diastase used in various combinations ensure complete hydrolysis of food and bring relief. Problems of the circulatory system can also be solved using enzymes. Usually blood doesnot form clots inside the vessels but occasionally a small clot may be formed. Such a clot . interferes with the normal blood flow and should it be large enough to obstruct the flow totally, the patient may even collapse. Such blocks in blood flow amplifies the clinical symptoms of heart attacks~ arterial occlusion and acute pulmonary embolism, often with fatal results. The clot may be dissolved by thrombolytic enzymes before a serious condition is precipitated. In plasma and serum a substance' called plasminogen or profibrinolysin may be activated to an enzyme called plasmin or fibrinolysis which lyses the clot. This activation may be brought about in various ways and certain proteins isolated from bacteria have proved to be good activating agents. Examples of bacterial activators for plasminogen are streptokinase and staphylokinase. Streptokinase forms a complex with plasminogen. This complex is catalytically active and activates plasminogen to form plasmin which breaks down the blood clot and restores smooth blood flow in the vessels. Inflammation, a condition characterized by pain, swelling, redness and heat, is the result of tissue destruction due to degradation of tissue matrix substances such as hyaluronic acids, collagen, proteoglycans and cell lysis. Anti-inflammatory enzymes cure the inflammation and associated edema by hydrolysis and by destroying proteinaceous tissue debris.

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Ointments containing such enzymes need 'only to be applied . to the skin for their effect to show. Enzymes find use in cancer therapy as well. Their use hinges on the fact that the metabolism of normal cells is different from that of tumour cells. In simple terms it can be said that tumour cells have specific requirements for some aminoacids which they cannot synthesize. For example, tumour cells require an abundant supply of L - asparagine. They would thus be vulnerable to attacks on the availability of this

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amino acid and it is this reason why the enzyme L - asparginase is popular as an anticancer agent. L - asparginase can break L- aspargine into L-aspartic acid and ammonia. Treatment with L-asparginase deprives the tumour cells of Laspargine and hence reduces its rate of growth. Other novel approches in treating tumour cells involves degradation of the membrane polysaccharides, structural or functional proteins and nucleic acids by using enzymes. While cancer is in many ways an acquired disorder of the body, there are certain "inborn errors of metabolism" that manifest as congenital abnormalities that persist throughout life. Most metabolic activities are multi-step processes with each step being governed by a specific enzyme. Should even one enzyme be lacking or defective, the entire pathway is compromised. The administration of such an enzyme would,

CF patients need special care

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of course, be a logical step to take under the circumstances, and this approach has been explored for treating patients with "inborn errors of metabolism". One such congenital or 'inborn' error is cystic fibrosis. In cystic fibrosis, sticky and heavy mucus clogs the respiratory tract and the narrow airways of the lungs become infected by bacteria. Repeated infections cause the lungs to become weak and patients cough up blood when major blood vessels rupture. In another form of cystic fibrosis the pancreas is affected as well. As a result, these patients suffer from malabsorption of nutrients and hence malnutrition and other associated disorders occur. To overcome the problem of malabsorption, extracts of pancreas (pancreatin) containing various digestive enzymes such as, trypsin and lipase are used. Another congenital disorder is hemophilia. It is a disorder in which the factor responsible for the clotting of blood is not synthesized by the body. Hemophilics therefore, are at risk of bleeding to death even from a minor wound. An antihemo-, philic factor is administered either by injection or by infusion to prevent bleeding. Deficiency of beta-glucocerebrosidase leads to Gaucher's disease. Due to the absence of this enzyme, certain cells, aptly named Gaucher's cells, are formed in the liver, spleen and bone marrow. Gaucher's disease is characterized by enlargement of liver, weakening of bone (osteoporosis) and loss of bone tissue (Osteonecrosis). Supplementary doses of betaglucocerebrosidase is used to treat this disease. Enzymes that are used as drugs specially for parenteral .applications undergo a series of quality control tests to ensure that the enzyme protein is not degraded, that it retains its activity and that it does not have any contaminating molecules that may lead to complications. Enzymes are used in manufacture of pharmaceutical drugs too. Enzyme technologies have become very handy in pharmaceutical industries for manufacture of bulk drug in-

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Enzymes used in pharmaceutical bulk products

termediates. Penicillin, the wonder drug, is used for controlling certain bacterial diseases. However, only 15% of the penicillin produced is used directly as a drug and the rest is

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Enzymes

used in therapy

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69

used to produce other broad spectrum antibiotics such as ampicillin, amoxycillin and cephalexin. The enzyme penicillin acylase is used industrially to produce 6-aminopenicillanic acid(6-APA) and 7- aminodesacetoxycephalosporanic acid(7-ADCA). from penicillin.Similarly, sequential actions of the enzymes, D-amino acid oxidase and glutaryl acylase are used for production of 7- amino cephalosphocanic acid (7ACA) from cephalosporin C. Cephalosporin C as such does not have any antibacterial activity but semisynthetic cephalosporins prepared from 7-ACA are potent antibiotics. Production of 6-APA, 7-ADCA and 7-ACA is probably the most significant contribution of enzymes in controlling various diseases. Besides these, many other enzymes are also used for production of drug intermediates. The medical industry also benefits from enzymes in other ways. Diagnostic tests have become an integral part of medical diagnosis. Very often a doctor advises the patient to get routine tests done in a clinical laboratory in order to better understand the exact nature of the disease. These tests are nothing but standardized methods to detect products of certain metabolic pathways. In any metabolic disorder, the altered metabolism is reflected in a changed level of metabolic product. For example, the blood glucose level goes up in diabetes and the level of serum bilirubin also shows a marked increase in jaundice. These changed levels give doctors the clues necessary to arrive at a diagnosis. Enzymes are used in three different ways for carrying out diagnostic tests in clinical laboratory: to determine the concentration of a substance directly, to determine the level of enzymes present in biological system and as markers for determination of substances that are enzymatically inert. ••

It

open a book, buy a sari, wear is not and very wash apparent whenthat we shoes clothes enzymes have played a key role in the production of these disparate products.

Looking Good

Making paper from wood requires separation of the wood fibres from each other before reforming them into a sheet. The resinous constituents of wood is termed pitch and triglycerides make up much of it. Being sticky in nature it adheres to machine parts and creates spots and holes in the paper. Such blemishes lower the quality of paper. Lipases that digest the triglycerides are used to treat the pulp in order to overcome these problems. During the process of making pulp much of the lignin is removed but the residual lignin has to be removed by bleaching.Bleaching involves chlorination and so the effluent from the bleaching plant is rich in chlorinated substances and highly polluting. Now wood is cellulose, lignin and hemicellulose complexed together to form a highly crystalline structure. This very structure of wood makes it possible for industrialists to reduce pollutant load in effluent by enzymatic means. When pulp is treated with xyla-

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71

nases, these enzymes degrade the hemicelluloses, an action that loosens the lignin-hemicellulose-complex. This reduces the use of chlorine by as much as 66 per cent during bleaching and obviously therefore, lessens the pollutiOh load. Effluent treatment is of major concern today and even an apparently innocuous pollutant like starch is monitored in the effluents generated by the paper and textile industries which use the enzyme alpha- amylase to remove it. Removal of starch improves the settling characteristics of the waste. While on one hand, industrialists find ways and means to remove starch from effluents, on the other, they use the same substance to enhance the quality of fabrics. The fibres of cotton and synthetic material are coated with starchy substances called 'si?:ing material' to give strength to the fabric during weaving. Once weaving is over, however, the sizing material has to be removed so t~at finishing processes such as bleaching, dyeing and printing may be better performed. The process of removal of sizing material is called' desizing' and must be carried out without any damage to the fabric. Earlier desizing was carried out by using acids, alkalies or oxidizing agents which, unfortunately, had an adverse effect· on the fabrics. Today, efficient enzymes such as amylases are used for desizing without damaging the fabrics. Fashion dictates that certain fabrics especially denim, should look 'worn' even when new. Traditionally, this finishing was achieved by the abrasive action of pumice stones, hence the term' stone-wash jeans'. Treatment by cellulase has replaced this tedious operation. However, the enzyme is allowed to attack only the surface of the fabric so that the strength of the fabric is maintained even as it begins to look 'worn'. If the garments have been dyed using indigo blue before enzyme treatment, cellulase action also makes them look 'faded'. E~nce is enhanced by the softness and smoothness of the fabric and the textile industry values these qualities very highly. Cellulases help give a smoother surface to fabrics by

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Enzymes are used to facilitate production of many consumer goods

LOOKING GOOD

73

Enzymes used in the textile industry

hydrolysing protruding microfibrils. This process is termed 'biopolishing'. This treatment also reduces the tendency of microfibrils to form tiny ball-like knots. Piling, as this tendency is called, detracts from the quality of the finished product. So, cellulase treatment has a two-in-one action that the textile industry exploits. Some other enzymes are used by the textile industry too. Proteases for example, are used to remove sericin, the proteinaceous substance that covers the silk fibres in a process called deguming of raw silk. Once the fabric has reached the consumer, the question of cleanliness arises. Protein stains on clothes commonly come from sweat, blood or crushed grass. These stains bind strongly to the fibres of the clother. In addition, these serve as binders for dirt particles and pigments. Repeated washing of clothes without detergents result in permanent stains due to oxidation and denaturation of the organic matters. Proteases, are incorporated in detergents for removal of such stains. Synergistic effect of proteases and detergents remove these tough stains. Besides proteinous matter, residues of starchy food and oily and fatty matter contribute to stains too. So amylases and lipases are also incorporated in the detergent for removal of starchy and greasy stains, respectively.

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Enzymes used in detergent industry

After repeated washing, microfibrils separate from core fibres. These separated fibrils give roughness to cloth, trap the dirt particles and give a dull appearance to colours. To overcome these effects, micro fibrils are degraded by cellulases which are also incorporated in the detergent. Now, it . would seem that since cellulase digests cellulose, cotton being pure cellulose would be totally degraded and rendered soluble by the enzyme. Imagine the shock when one dips a cotton shirt or a sari in a pail of cellulase-detergent and the clothes dissolve! Thankfully such is not the case. In fact, as far back as 1970, H.R. Browning working for the commercial giant Unilever, had applied for a patent for using cellulase to prevent fabrics from becoming harsh as a result of repeated washing. This apparent dichotomy between the expected and observed behaviour of cellulases is because there are cellulases which have the ability to attack only those parts of the fibres. which have been damaged in .previous washes. Since only these damaged bits are responsible for the harshness of the fabrics, their destruction by cellulases restores the texture of the fabric without destroying its strength.

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Use of enzymes in detergent is gaining popularity. In Europe today, about 70% of the heavy duty laundry detergents contain enzymes. However, the idea of using enzymes to wash clothes is not a new one. This innovative concept is at least 83 years old. Conceptually the idea originated in 1913 with Otto Rohm, founder - partner of Rohm and Hass in Germany. In 1913, he launched the first enzyme-containing detergent, Bumus, which was marked and it continued to be sold all over Europe for the next 50 years. Burnus contained pancreatin and it cleaned fabrics in a shorter time with lesser exertion than conventional detergents. In 1958, Novo Industri was faced with the' problem of cleaning the blood and fat soiled aprons of workers in the meat and fish industries. Novo solved the problem by using the enzyme alcalase, a fermentation product of Bacillus licheniformes. Alcalase was marketed in 1960 and since then, a battery of enzymes have become available for use either separately or blended with one another. Fatty stains are stubborn and especially problematic but in 1987, Novo again made another breakthrough when it genetically engineered Aspergillus to give fat-splitting enzymes or lipases. The enzyme lipolase was selected because it was compatible with other enzymes and blends of proteases and lipolase could be made. However, this enzyme does not act when the material is being washed for the first tirne. During the drying phase, when the fabric does not retain quite so much moisture, this enzyme becomes active. Thus, the material becomes clean of all greasy stains in the second wash cycle. Starch containing stains such as food stains that arise from gravy, chocolate and baby food may be removed by antistarch enzymes or amylases commercially available from the bacteria B. amyloliquefaciens and B. licheniformes. Amlyases have been successfully utilized not only in household laundry detergents but also in industrial laundering and machine dishwashing. However, not all enzymes can be used in detergents. Only those enzymes that are active in alkaline condi-

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tions and at elevated temperatures can be used. They should also be compatible with the components (binders and surfactants) of detergent. The enzymes used in detergents are in granulated form to avoid possible allergic reactions in people who handle them.In liquid detergents, enzymes are coated in.silicone oil.

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Enzymes used in the leather industry

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ENZYMESEVERY~ERE

Use of enzymes in detergents has not only reduced the consumption of chemical and thermal energy but made the entire process of washing clothes so much more easy. Good shoes complement clean clothes of good quality. Enzymes help the leather industry to produce items Qfquality. Animal skin goes through a series of operations prior to making of various leather goods: curing, soaking, liming or unhairing, deliming, bating, pickling and tanning. Enzymes are mainly used in soaking, unhairing and bating processes. Hides are first soaked for rehydration before further processing. The better the rehydration, the superior the leather. Proteins between the fibres of the hide are removed by proteases. The removal of proteins disturbs the molecula.r structure and enables faster hydration or soaking'. Then again, proteases are used in unhairing of the leather. These are used as supplement in chemical dehairing process. The enzyme removes hair by disturbing the proteinaceous matter present at the base of the hair. Bating is a process in which certain protein components in leather are removed so that the l~ather becomes pliable. The degree of bating required depends on the type of final product. Soft leather goods such as purses and gloves need strong bating. Trypsin is used for hydrolysis and removal of proteins in bating. Use of enzymes in bating is one of the early industrial 'applications of enzymes and first patent was taken by Dr. Otto Rohm in 1908. Since those early days, enzymes have seen innovative uses in the industry and are silent, behindthe-scene workers helping to turn out a well-groomed person .

••

that enzymes are Porimpression a long time there an labile molecules and was active

Back to the Future

only in aqueous media. In the last decade or so, this concept has been revised as it has been demonstrated that certain enzymes can catalyse a reaction in partially Or totally nonaqueous media. This finding has considerably widened the scope of enzyme usage and the most popular members of this group of enzymes are the lipases. The newly discovered properties of enzymes coupled with their characteristic features have made them even more popular with those who use enzymes as tools. The unique properties of enzymes, the most important of which is their substrate specificity, are advantageous to researchers in various fields. In fact, the fields of molecular biology and genetic engineering owe a lot to the availability of the enzyme restriction endonucleases. Restriction endonucleases break nucleic acid molecules at specific sites. Other enzymes, such as ligases, can 'glue' cut ends of nucleic acid molecules. Thus, tailored modification of genes is possible, because researchers can now 'cut and join' DNA frag-

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80

Foreign DNA

E.coli Cells divide

Making a molecular ferry

ments almost at will. This approach .can be used to develop an enzyme with the desired altered properties. If one were to cut up the microbial genome and insert a gene responsible for the elaboration of a desired protein and then 'sew' up the foreign gene such that it now became part of the genome, one would have a genetically tailored organism that would now produce the protein of choice. Today, researchers are doing just this to produce genetically engineered microbes which produce proteins in sub-

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stantial quantities. Hundreds of restriction endonucleases and ligases available commercially for 'cutting and stiching' genes. It is clear that genetic engineers will rely more heavily on enzymes in the days to come. Also microbial production of protein is comparatively pollution free and the need of the hour is to achieve significant industrial growth without adversely affecting the environment. Concern for the environment has increased after the world has witnessed the disastrous effects of pollution. E~yme technology is probably the answer for tomorrow. Compared to chemical technology, enzyme technology .consumes very low energy, avoids use of hazardous and polluting chemicals, does not yield side products, gives better stability to reactants and products, and generates purer final products. The waste is biodegradable and the effluent nonpolluting. Because of these advantages, enzyme technology has come to be known as Green Technology. The only apparent drawback of enzyme technology is the use of microbes that are cultivated on a large scale for the production of enzymes. However, the organisms used are not disease causing ones. The risk is further reduced since they are grown under controlled conditions and on specialised nutritive media. Most of the industrial strains are developed for production of a particular product and are highly sensitrve. Hence they do not grow under normal environmental conditions. The genetically engineered organisms that are used industrially undergo scrutiny, so that provisions laid down for production conditions, worker's protection-and product approval are not ignored. Scientists are aware of the awesome powers of enzymes and are always on the look out for new enzymes with unique qualities. But, where do they find enzymes? Since enzymes are biocatalysts, the best place to look for un\lsual enzymes is to identify unusual life forms. For example, in the past few years, the upper temperature limit for life has had to be pushed back by the discovery of extraordinary microbes

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ENZYMES EVERYWHERE

On to Mars!

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83

which thrive in boiling water! Heat-loving or thermophilic bacteria have been identified in geysers, volcanoes, under-sea hydrothermal vents and even in .the boiling out flows of geothermal power plants. The finding has upset the traditiona I line of thought which said boiling kills all microbes. Clearly not all microbes can be killed by boiling, although most microbes are destroyed at 100°C and above. Since heat-loving microbes were first discovered by Thomas Brock of the University of Wisconsin (USA) and Jin Brierly of Montana State University (USA) in the late sixties, these thermophilic bacteria have mystified 'and delighted scientists. The question as to how their biomolecules remain stable at such high temperatures still remains a mystery. However, because the enzymes of these species can "beat the heat", the scope for industrial applications appears bright. Thermophilic enzymes have been used in polymerase chain reaction (PCR), a technique that amplifies DNA and which has diagnostic and forensic value. Hydrogenases obtained from thermophilic bacteria are being considered for future commercial use. The detergent industry is tentatively testing the idea of using heat- stable proteases. Another possible use would be sugar production by hydrolyzing starch. Traditionally this requires high temperature but' most enzymes do not function optimally at elevated temperatures. Since thermophilic enzymes are heat- stable, they are expected to prove advantageous for sugar producers. Thermophilic bacteria are also being considered as environment-friendly candidates for making clean-coal. Some of these bacteria can transform sulphur into hydrogen sulphide. So, they could use up the sulphur in coal to give a coal with little sulphur content. Such a coal would burn better with little emission fo pollute environment. Another variety of enzymes with enormous potential are the exoenzymes secreted by.alkali-Ioving microbes. These enzymes secreted into an alkaline medium initiate the breakdown of complex polymeric substances. Proteases extracted

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ENZYMES EVERYWHERE

l

p

__

r© ~'

And$o~tt peR is a cyclic process

from alkalopohilic bacillus have been used in washing powders and liquids as they are stable in the presence of detergents. Fungal enzymes are another variety of enzymes with immense potentiaL Gluea amylase, one such fungal enzyme, is capable of hydrolysing starchy substances to glucose. It is extensively used in the industry for the preparation of crystalline glucose or glucose syrup and high fructose syrup. Efforts by Indian scientists have led to the isolation of a strain of Aspergillus terreus that could break down starchy sub-

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stances to glucose. It was first tested on a variety of starchy wastes easily available in India. It was grown on cheap agricultural residues such as wheat bran, rice bran, spoiled potatoes and bananas. The strain was found to be an economically feasible prospect for large scale production of the enzyme. Scientists at the Indian'Institute of Technology, Kharagpur, have reported the successful use of protease extracted from a fungal species isolated in the early nineties. Since proteases account for nearly 60 per cent of total enzyme sales, the importance of this finding is self-evident. In the world today, the reach of speciality enzymes is growing by the day, and the market for speciality enzymes is likely to cross $600 million. Current levels of expertise offer a means to engineer microbes to mass produce enzymes at lower costs. As techniques are developed to restructure and redesign enzymes for greater sensitivity and higher activity, their reach and scope is likely to leave few processes untouched. It would not be a tall claim to say that the catalytic power of enzymes has and will continue to protect the environment and maintain the natural balance of the earth . ••

•

GLOSSARY Amino acids: Organic compounds that bear atleast one amino group and one carboxyl group. They have both basic and acidic properties. Amino group: The basic -NH2 group. Biological oxygen demand (BOD): The amount of oxygen required by the living organisms engaged in neutralisation and ultimate destruction or stabilisation of organic matter. Carboxyl group: The basic -COOH group. Cofactor: A small molecule required for enzyme activity. It could be organic or inorganic in nature. Collodial: A system of particles dispersed in a liquid, which do not separate because of their small size . .Derivatization: A process by which an organic compound is obtained fron another organic compound. Domains: Compact local units formed by folding of the polypeptide chain. Edema: Excessive 'accumulation of fluid in the tissue spaces. Genome: The total genetic content of a cell or species. Hydrothermal vents: Hot water springs. Many are found at great depths in the oceans. Lignin: A complex compound present in wood. pH: A numerical scale from 0 to 14, used to express the acidity or alkalinity of a solution. pH below 7 indicates an acidic solution while a value above 7 indicates an alkaline solution. Protozoans: Single celled animals.

GLOSSARY

Substrate analogues: Compounds whose chemical structure or some portions of the structure aJ;'esimilar to the chemical structure of the substrate. Trisaccharide: Composed of three sugar units. The word tri ~eans three and saccharide refers to sugar.

D ISTI N G U ISH ED

by their versatility and extreme efficiency, enzymes are catalysts par excellence. Their superior qualities and non-polluting nature makes them profitable choices for industrial use in fields as diverse as breweries, leather, medicine, detergent, dairy, and scientific research. The domestic kitchen is another arena where naturally extracted fresh enzymes help create culinary miracles. The tip-top, well groomed look of the individual too owes a debt to enzyme action. The enzymes are isolated from exotic Iifeforms inhabiting a bewildering variety of habitats--from volcanic vents to depths of the deep sea, no part of the earth is too hot, too cold, too acidic or too alkaline for lifeforms to find conditions too harsh to survive. This extensively illustrated, lucidly detailed book identifies the amazingly diverse spheres where enzymes are employed and drives home the point that there truly are, Enzymes Everywhere. About the Author Dr Jaiprakash G. Shewale obtained his Ph.D. from the National Chemical Laboratory, University of Pune, and Diploma in Management from All India Management Association, New Delhi, Presently he is Group Manager at R&D of Hindustan Antibiotics LId (HAL), Pimpri. During his stay in the US, he has contributed to the deciphering of amino acid sequences of several proteins of physiological importance. At HAL, he has played a key role in the development of immobilized penicillin G acylase and 6-APA technology and has developed other important enzymes useful in the interconversion of penicillins and cephalosp?rins. Dr. Shewale has over 60 publications and 11 patents to his credit. He is a Fellow of Maharashtra Academy of Sciences. He is also a recipient of Meritoriallnvention Award from National Research Development Corporation, New Delhi. -

J

78817211 361419

ISBN: 81 - 7236-141-6