enzyme biotech title page contents chapt1 and 2

8/13/2019 Enzyme Biotech Title Page Contents Chapt1 and 2

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N GRAY M CALVIN S C BHATIA

IAlkern Company S) Pte LtdSingapore

COLLEGE OF ENGINEERING LIBR RYUNIVERSITY O THE PHILIPPINES DILlMAN


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Enzymes iotechnology

ISBN: 9789810860882

First Edition: 2011

Alkem Company (S) Pte Ltd1 Penjuru Close, Singapore 608617email: [email protected]: (65)6261 7875Tel: (65) 6265 6666website: www.alkem.com.sg

opyright CBS Publishers Distributors Pvt. Ltd.

\Published by CBS Publishers Distributors Pvt. Ltd. in association withAlkem Company (S) Pte LtdAll rights reserved. No part of this book may be reproduced or transmitted inany form or by any means, electronic or mechanical, including photocopying,recording, or any information storage and retrieval system without permissionin writing from the authors and publishers.This book is published with the understanding that authors, publisher andprinter are neither responsible for the result of action taken on the basis of thiswork nor for any error or omissions.Printed by: Paras Offset Pvt. Ltd.


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refaceEnzymes are biological catalysts, or chemicals that speed up the rate of reaction between

substances without themselves being consumed in the reaction. As such, they are vital to suchbodily functions as digestion, and they make possible processes that normally could not occurexcept at high temperatures when they would threaten the well-being of the body. A type ofprotein, enzymes, sometimes work in tandem with non-proteins called co-enzymes. Among theprocesses in which enzymes play a vital role is in fermentation, which takes place in theproduction of alcohol or the baking of bread and also plays a part in numerous other naturalphenomena such as the purification of waste-water .

Enzymes are special kinds of proteins that are found in all living matter. Man, animals,plants-all living cells produce and need enzymes to live and grow. However enzymesthemselves are not living organisms. Enzymes are catalysts substances which speed upchemical reactions by being present in very small amounts and without being changed in thereaction.

Modern biotechnology techniques are utilized to improve microbial production strains toincrease to enzyme yields and to make minor amino acid changes that improve thefunctionality of the enzyme. These changes are not known to increase the ability of enzymes tocause allergies.

This reference textbook is divided into five sections. Section I discusses about principles ofindustria enzyme production and utilization. Chapter 1 in ~ i s section is devoted to basicconcepts of enzymes. Chapter 2 deals with general characteristics of enzymes, such as structure,mechanism and kinetics of enzymes. Chemical reactions, including those catalyzed byenzymes, depend on environmental conditions. The emphasis in Chapter 3 is on micro andmacro environmental effects on environmental activity and micro as well asmacroenvironmental effects. The dominant environmental factors affecting enzyme action arepH water activity and temperature. Chapter 4 concentrates on modern methods of enzymeexpression and design in a simplified and understandable manner- genetically engineeredproducts and amplification of genetic engineering for new food related applications. Chapter 5deals with fermentat ion process design. The task of the fermentation technologist is to provideand maintain the environment required for carrying out a desired biological process. Thus, awell designed growth environment is needed to obtain the maximum economic yield of theproduct and this task resolves into environmental design, determining the environmentalrequirements of the process, and fermentation engineering, providing the means for meetingthose requirements of the process, and fermentation engineering. Chapter 6 has a focus onextraction and purification of enzymes. Various mediums of extraction and purification arediscussed in detail.


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Section II concentrates on types of enzymes/ classification of enzymes. Chapter 7concentrates on carbohydrate hydrolyzing enzymes. Chapter 8 is devoted to proteases whichare enzymes that digest proteins. Various types of proteases, such as plant, animal, microbial,serine, etc. are discussed in detail. Chapter 9 deals with lipases which are enzymes thosecatalyses the hydrolysis of fat or breakdown of lipoproteins. The chapter discusses sources ofmicrobial and industrial sources of lipases along with processes of application of lipases.Chapter 10 concentrates on oxidoreductases which are enzyme catalyzing a reaction in whichtwo molecules of a compound interact so that one molecule is oxidized and the other reducedwith a molecule of water entering the reaction. Various types of oxidoreducatases along withtheir general characteristics are discussed.

Section III is devoted to the application of enzymes in the industry. Enzymes are involved inthe formation of starch, gluten, hemicelluloses, and lipids during ripening and are the key todough fermentation and bread making, since they degrade these polymers. The activities ofenzymes in the flour used for bread-making depend on the climate, the type of grain, and theharvesting conditions. Chapter 11 focuses on milling and baking. Chapter 12 and 13 discussstarches, sugars and syrups and dairy products. Pectic enzymes occur in higher plants and areproduced by micro-organisms. Chapter 14 concentrates on pectic enzymes in fruit andvegetable juice manufacture. Chapter 15 deals with enzymes associated with savoury flavourenhancement. The focus of Chapter 16 and 17 is on wine and brewing. Chapter 18 is concernedwith fish processing and meat industry. Chapter 19 is concerned with textiles and laundrydetergents. Weaving textile fibers exposes the fabric s warp and weft strands to substantialmechanical stress. To prevent breakage of these strands they are generally reinforced withsizing. Sizing can consist of various materials. Chapter 2 and 21 deal with pulp and paper aswell as tanning industry. Chapter 22 and 23 concentrates on clinical analysis and organicsynthesis. Chapter 24 deals with enzymes and bioremediation. Biological methods orbioremediat ion promises to provide an eco-friendly solution to this menacing problem.

Section IV concentrates on instrumental techniques used in enzyme analysis. Chapter 25 isdevoted to instrumental techniques in enzymatic analysis. The course of enzyme-catalyzedreactions may be monitored by a variety of techniques, of which the most common at thepresent time are spectrophotometric, spectorfluorimetric and electrochemical procedures.Chapter 26 and 27 deal with enzyme thermistors and enzyme electrodes for food analysis.

Section V focuses on special topics. Chapter 28 focuses on legal considerations. Regardlessof whether enzyme preparations are defined as food additives or technical aids, it must beestablished that they are not health hazards. The purpose of this chapter is on legal aspects ofenzyme utilization and to inform the readers about the measures that the scientific communityand legislators have taken to provide maximum safety for both users and consumers of enzymepreparation. t should be noted that regulations for enzyme preparationsand their use are by nomeans uniform throughout the world. Chapter 29 deals with economic considerations for theuse of technical enzymes. The economic benefits of using technical enzyme preparations lies inlower process costs, often increasing product quality, reducing environmental impact, and


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making use of renewable resources in technology. Chapter 30 is devoted to patentingbiotechnology inventions. For biotechnology patents are the most important form ofintellectual property The chapter discusses number of reasons for issuing patents. Forexamples the patent holder obtains exclusive rights for the invention rights that are a rewardfor developing procedure compound or apparatus For a patent to be granted an inventionmust be novel not obvious and useful.

Glossary and index have been provided at the end for quick references. Diagrams figuresand tables supplement the text. All the topics have been covered in a cogent and lucid style tohelp the reader grasp the information quickly and easily.

The book should serve as an essential reference textbook of enzymes biotechnology anduseful reading for all students and teachers of engineering environment and life sciences. Theresearchers in analytical and instrumental techniques chemists and industria lists will also findit highly useful and informative.

N GrayM CalvinS C Bhatia


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ontents at a GlancePreface .................................................................................................................................................... v

SECTION IPrinciples of Industrial Enzyme Production nd Utilisation 1 99Chapter 1 Basic Concepts of Enzymes .......................................................................................... 3-7Chapter 2. General Characteristics of Enzymes ............................................................................ 8-31Chapter 3. Environmental Effects on Enzyme Activity ............................................................... 32-47Chapter 4. Modem Methods ofEnzyme Expression and Design ................................................. 48-68Chapter 5 Fermentation Process Design ..................................................................................... 69-85Chapter 6 Extraction and Purification ofEnzymes ..................................................................... 86-99

SECTIONTypes of Enzymes/Classification of Enzymes 101 272Chapter 7 Carbohydrate Hydrolysing Enzymes ........................................................................ 103-185Chapter 8 Proteases .................................................................................................................... 186-222Chapter 9 Lipases ...................................................................................................................... 223-231Chapter 10. Oxidoreductases ........................................................................................................ 232-272

SECTIONApplication of Enzymes in Industry 273 508Chapter 11 Milling and Baking .................................................................................................. 275-292Chapter 12 Starches Sugars and Syrups ................................................................................... 293-309Chapter 13. Dairy Products ................................. .................................. .................................. ..... 31 0-317Chapter 14. Pectic Enzymes in Fruit and Vegetable Juice Manufacture ..................................... 318-341Chapter 15. Enzymes Associated with Savoury Flavour Enhancement ..................................... 342-357Chapter 16. Wine ..................................................................... ..................................................... 358-367

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x ontents t a GlanceChapter 17. Brewing ..................................................................................................................... 368-381Chapter 18. Fish Processing and Meat Industry ............ ............ ............ ............ ........... ............ .. 382-389Chapter 19. Textiles and Laundry Detergents ................. .................. .................. ................... ..... 390-399Chapter 20. Pulp and Paper ........................... , ............................................................................. 400-413Chapter 21: Tanning Industry .......................................................................... ............................. 414-425Chapter 22. Clinical Analysis ........................... ............................. ............................. ................. 426-456Chapter 23. Enzymes in Organic Synthesis ............... ............... ................ ............... ............... .... 457-481Chapter 24. Enzymes and Bioremediation ................................................................................... 482-508

SE TION IVInstrumental Techniques Used in Enzyme nalysis ............................................................ 509 551Chapter 25. Instrumental Techniques in Enzymatic Analysis ............... ............... ............... ....... 511-523Chapter 26. Enzyme Thermistors for Food Analysis ................................................................. 524-534Chapter 27. Enzyme Electrodes for Food Analysis ...................... ............. ............. .............. ...... 535-551

SE TIONVSpecial opics 553 575Chapter 28. Legal Considerations ........................................................... ..................................... 555-565Chapter 29. Economic Considerations for the Use o Technical Enzymes ................................ 566-569Chapter 30. Patenting Biotechnology Inventions ......................................................................... 570-575

References ........................................................................................................................................... 577Index ............................................................................................................................................... 579-586


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ontentsPreface ................................................................................................................................................... v

ontents at a glance . . . . . . . . . . . . . . . . . . IX

SECTION IPrinciples of Industrial Enzyme Production nd Uti/isation l 991 Basic Concepts o Enzymes ...................................................................................................... 3-7

Introduction ................................................................................................................................... 3Enzymes Defined as Catalysts ....................................................................................................... 4History ofth Development ofTechnical Enzymes ........................................................................ 4Applications ofModem Enzyme Technology ................................................................................ 6

2. General Characteristics o Enzymes ..................................................................................... 8 31Introduction ................................................................................................................................... 8Important Role of Enzymes ........................................................................................................... 8Enzyme Structure and Action Mechanisms ................................................................................... 11Enzyme Analysis and Enzyme Units ........................................................................................... 12Enzyme Kinetics ........................................................................................................................... 17Inhibition and Activation .............................................................................................................. 22Non-Mi r;haelis-Menten Behaviour ............................................................................................... 26Kinetics of Multi enzyme Systems ........... ........... ............ ........... ........... ........... ........... ........... ...... 28Enzyme Utilisation Industry ........................................................................................................ 29

3. Environmental Effects on Enzyme Activity ........................................................................ 32 47Introduction ................................................................................................................................... 32Effect ofpH ................................................................................................................................... 32Effect of he Aqueous Environment .............................................................................................. 34Effect ofTemperature ................................................................................................................... 40Effect ofOther Environmental Conditions .................................................................................. 46

4. Modern Methods o Enzyme Expression and Design ......................................................... 48 68Introduction ................................................................................................................................... 48Background ................................................................................................................................... 48

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x ContentsGenetic Engineering ..................................................................................................................... 51Commercialisation ofGenetically Engineered Products .............................................................. 61Implications for the Future ........................................................................................................... 67

5 Fermentation Process Design ............................................................................................... 69 85Introduction ................................................................................................................................... 69Process Design ............................................................................................................................... 69Mode of Operation ....................................................................................................................... 72Adaptive Control ......................................................................................................................... 82Knowledge based Control ............................................................................................................ 82Statistical Optimisation ................................................................................................................ 83Variability ..................................................................................................................................... 85

6 Extraction and Purification of Enzymes ............................................................................ 86 99Introduction ................................................................................................................................... 86Extraction ofEnzymes .................................................................................................................. 86Purification of Enzymes .............................................................................................................. 90Determination ofMolecular Weights ofEnzymes ....................................................................... 97

SE TION IIype_s o Enzymes Classification of Enzymes 101 272

7 Carbohydrate Hydrolysing Enzymes ............................................................................... 103 185Introduction ................................................................................................................................... 103Action ofEnzymes ...................................................................................................................... 103Amylases ..................................................................................................................................... 105Carbohydrases Other Than Amylases ........................................................................................ 128Cellulose Degradation ................................................................................................................ 141Hemicellulases ..... ............. .............. .............. .............. .............. ............... .............. .............. .... 160Pectin Degradation ..................................................................................................................... 169.Pentosanases ............................................................................................................................... 179

8 Proteases .............................................................................................................................. 186 222Introduction ................................................................................................................................... 186Plant Proteases ........................................................................................................................... 187

Papaya Proteases ............................................................................................................... 187.Animal Proteases . . . ... . . . . .. . . . . . . . . 190Microbial Proteases: riefOverview ........................................................................................ 196Ester Cleavage: Fat Hydrolysis ................................................................................................. 211

9 Lipases ................................................................................................................................ 223 231Introduction ................................................................................................................................... 223Sources ofMicrobial Lipases ..................................................................................................... 223Lipases in Food Manufacturing Processing ................................................................................ 227Structured Lipid with the Aid ofLipases .................................................................................. 229


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ontents 10. 0 xi.doreductases .................................................................................................................... 232 2

Introduction ................................................................................................................................... 2Polyphenol Oxidase ....................................................................................................................... 2Peroxidases ................................................................................................................................... 2Lactoperoxidase ......................................................................................................................... 2Catalase ...................................................................................................................................... 2Sulphydryl Oxidase ........... ........... ............ ............ ........... ............ ............ ........... ............ ............ 25Glucose Oxidase ........................................................................................................................ 25Pyranose Oxidase ...................................................................................................................... 25Xanthine Oxidase ........................................................................................................................ 25Lipoxygenase .................................................................................. .......................................... 25Dehydrogenases ......................................................................................................................... 26Application ofOxidoreductases ................................................................................................. 26

SECTION IIIpplication of nzymes in Industry 273 50

11 Milling and Baking .............................................................................................................. 275-29Introduction ................................................................................................................................... 27Enzyme in Flour ........................................................................................................................ 27Use in the Baking Industry ........................................................................................................ 28Conclusion .................................................................................................................................. 29

12. Starches Sugars and Syrups ............................................................................................. 293-30Introduction ................................................................................................................................... 29Starch Production ....................................................................................................................... 29Starcb.froduction and Application .............................................................................................. 29Sweetener and Syrup Production ................................................................................................ 29Applications ofSweeteners and Syrups ...................................................................................... 30

13 Dairy Products ............... .................................................................................................. 310-31Introduction ................................................................................................................................... 31Natural Enzymes ofMilk .............................................................................................................. 31Milk-Clotting ............................................................................................................................... 31Cheese Ripening ......................................................................................................................... 31Lactase ....................................................................................................................................... 31Hydrogen Peroxide and Catalase Treatment ............................................................................... 31Miscellaneous Application ofEnzymes in Dairy Processing ..................................................... 31

14. Pectic Enzymes in ruit and Vegetable Juice Manufacture ........................................... 318-34Introduction ................................................................................................................................... 31Pectic Substances and Pectic Enzymes ...................................................................................... 31Occurrence and Microbial Production ofPectic Enzymes ......................................................... 32


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x v ContentsFood-Processing-Related Properties ofPectic Enzymes ............................................................ 321Methods Used in Technological Pectic Enzyme Research ........................................................ 325Use of Commercial Fungal Enzymes as Processing Aids .......................................................... 327Quality Aspects of Clear Fruit Juices Obtained by Enzyme Treatment of Juices or Pulp .......... 333Technological Functions ofEndogenous Pectic Enzymes ......................................................... 337

15 Enzymes Associated with Savoury Flavour Enhancement ............................................. 342 357Introduction ................................................................................................................................... 342Flavour Enhancers ..................................................................................................................... 342Enzymatic Hydrolysis of uclear Material ................................................................................ 344Commercial Production of Extracts Rich in Flavour Enhancers ............................................... 353Yeast Extracts ............................................................................................................................. 353Conclusion .................................................................................................................................. 356

16 Wine ................................................................................................................................... 358 367Introduction ................................................................................................................................... 358Endogenous Enzymes ..................................................................................................................... 358Use of Commercial Enzyme Preparations in Wine Making ........................................................ 361Future Developments ................................................................................................................. 366

17 Brewing ........................................................................................................................ ..... 368 381Introduction ................................................................................................................................... 368Malt and Its Reactions during Brewing ....................................................................................... 368Use of Brewer s Adjuncts .......................................................................................................... 372Brewing with Very High Adjunct Levels ..................................................................................... 374Keeping Beer Clear with Chillproofing Enzymes ....................................................................... 375Making New and Special Types of Beer with Enzymes ............................................................ 376Improving Filtration with Enzymes ........................................................................................... 379Extension of Shelf Life of c k g e d Beer ................................................................................. 380

18 Fish Processing and Meat Industry ................................................................................ 382 389Introduction ................................................................................................................................... 382Traditional Processes ................................................................................................................. 382New Applications ofEnzymes in Fish Processing ..................................................................... 385Enzymes in the Meat Industry ................................................................................................... 388

19 Textiles and Laundry Detergents ..................................................................................... 390 399Introduction ................................................................................................................................... 390Desizing Enzymes ...................................................................................................................... 390Enzymes in Laundry Detergents ................................................................................................ 394

20. Pulp and Paper .................................................................................................................. 400 413Introduction ................................................................................................................................... 400Process and Production of Pulp for Paper .................................................................................. 4 1


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ContentsRecent Advances in the Application of Enzymes for Paper and Pulp ......................................... 40Prospects of Genetic Engineering in Bioconversion .................................................................. 41

21. Tanning Industry ................................................................................................................ 414-42Introduction ................................................................................................................................... 41Skin Structure ............................................................................................................................. 41Enzymes for Leather Processing ................................................................................................. 41

22 Clinical Analysis ................................................................................................................ 426-45Introduction ................................................................................................................................... 42Measurement of Substrate Concentration with Enzymes ............................................................ 42Measurement of Enzymes .......................................................................................................... 43Immobilised Enzymes for Measuring Substrate Concentrations ............................................... 43Enzyme Immunoassay EIA) ..................................................................................................... 44Future ......................................................................................................................................... 45

23. Enzymes in Organic Synthesis ........................................................................................... 457-48Introduction ................................................................................................................................... 45Hydrolytic Enzymes .................................................................................................................. 45Oxidative-Reductive Enzymes ................................................................................................... 46Carbon-Carbon Bond-forming Enzymes .................................................................................... 47

24 Enzymes and Bioremediation ........................................................................................... 482-50Introduction ................................................................................................................................... 48Xenobiotic Compounds .................................................................................................................. 48Toxicity ...................................................................................................................................... 48Adaptive Responses ................................................................................................................... 48Physico-chemical Factors Influencing Biodegradation .............................................................. 48Hydrocarboo Degradation .......................................................................................................... 48Enzymology of n-Alkane Oxidation ........................................................................................... 49Genetic Regulation ofAlkane Oxidation ................................................................................... 49Biodegradation ofAromatic Compounds .................................................................................. 49Mechanism ofMonocyclic Hydrocarbon Degradation .............................................................. 49Enzymes of the Lower Pathway ................................................................................................. 50

SE TION IVInstrumental Techniques Used in Enzyme nalysis 509 5525 Instrumental Techniques in Enzymatic Analysis ............................................................ 511-52

Introduction ................................................................................................................................... 51Principles of he Available Detection Techniques ........................................................................ 51Automation in Enzymatic Analysis ........................................................................................... 51


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xvi Contents26. Enzyme Thermistors for Food Analysis ........................................................................... 524-534

Introduction ........................ ................................................................................................... .... 524Measuring Principle .................................................................................................................... 525Instrumentat ion .......................................................................................................................... 5 6Applications ............................................................................................................................... 527

27. Enzyme Electrodes for Food Analysis .............................................................................. 535-551Introduction ................................................................................................................................... 535Fundamentals ofAmperometr ic Devices ...................................................................................... 536Electrode Materials and Fabrication .......................................................................................... 537Development ofAnalytical Devices ........................................................................................... 538Examples of Enzyme Electrodes Applied to Food Analysis ...................................................... 542Enzyme Electrodes for Alcoholic Compounds ........................................................................... 548

SECTIONVpecial Topics ................................................................................................................................ 553 575

28. Legal Considerations ......................................................................................................... 555-565Introduction ................................................................................................................................... 555Regulatory Requirements for Enzyme Preparations .................................................................... 556Who is Involved with Enzymes? ............................................................................................... 556Toxicity and Allergies ................................................................................................................. 557Toxins ......................................................................................................................................... 557Documentation ....... ................................................................................................................... 559Some National Regulations ........................................................................................................ 560

29. Economic Considerations for the Use of Technical Enzymes ........................................ 566-569Introduction ................................................................................................................................... 566Increasing Yields and Improving Raw-materials Utilisation ..................................................... 566Lowering Costs .......................................................................................................................... 567Altering Technical Properties ....................................................................................................... 567Improving Preservat ion Flavour and Cleansing Action ............................................................ 567Market ofTechnical Enzyme Preparations ................................................................................. 569

30. Patenting Biotechnology Inventions ................................................................................. 570-575Introduction ................................................................................................................................... 570What Constitutes a Patent? ......................................................................................................... 571Patenting in Different Countries ................................................................................................. 573Patenting Multicellular Organisms ............................................................................................. 573Patenting and Fundamental Research ......................................................................................... 574

References 577Index ............................................................................................................................................... 579-586


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SE TION IPrinciples of Industrial nzymeProduction and Utilisation

1 Basic Concepts of Enzymes 32 ~ ~ e r a l Characteristics of Enzymes 83 Environmental Effects on Enzyme ctivity 34 Modern Methods of Enzyme Expression and Design 485 Fermentation Process Design 696 Extraction and Purification of Enzymes 86


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asic Conceptso EnzymesINTRODUCTIONLife on earth is based on constant change. Inorganic matter forms the complicated structures that makeup the living world, which will again decay into lifeless matter. The lives o plants, animals and humanbeings are included in this great cycle. The inert substances that form the foundation o life have a verysimple structure. Water. carbon dioxide and nitrogen are the basic ingredients that, with the ~ n r g ysupplied by sunlight, permit plants to synthesise the molecular moieties that support life. These compoundsare 1:1en used to provide more elaborate compounds required by animal life.

The use of enzymes and micro-organisms in processing raw materials from plants and animals hasoeen practiced for a long time. At one time, living micro-organisms were used predominantly. Traditionalprocesses, such as the production o alcoholic beverages and yeast-fermented doughs in baking bread,are displayed in Egyptian wall paintings. Further examples are the processes for preserving food, suchas vegetable conservation by fermentation with lactobacilli, or preserving milk by making cheese.

Chemical xperiments have shown how specific compounds. namely the essential amino acids, can be generated from simple mixtures o carbon, nitrogen, oxygen and hydrogen when subjected to extremepressures and temperatures. Once formed, less extreme conditions can cause the degradation of thesecomplex structures as well. Under the current global conditions, such processes proceed only veryslowly. What then, causes the rapid synthesis o these compounds that are essential for life and thedegradation ofthe high-molecular-weight and highly organised structures that make up life? The catalystsfor this assembly and disassembly were for a long time thought to be life itself, or rather a fundamentalprinciple o living cells.

The first observation o an enzymatic degradation reaction was by Spallanzani, a priest and naturalistfor Padua, Italy. After placing meat in small porous capsules, he examined the regurgitated pellets o thehawks who had eaten this material and found the capsules to be empty proving that the meat had beenrapidly liquefied by the stomach juices o these birds o prey.

Later, Kirchhoff found that barley contained a substance that was capable o liquefying starch pasteinto sugar. He assumed that the reaction was caused by the gluten protein of the barley. Subsequently,Payen and Persoz termed the working principle o this saccharification di st se (Greek for separation),a term still used for the amylases in the brewing industry. The degradation o starch, which is composedo sugar moieties and the subsequent process o alcoholic fermentation o the sugar into carbon dioxideand alcohol occupied much o this nineteenth-century chemist's time.

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4 Enzymes BiotechnologyPasteur showed that fennentation is closelv associated with live veast. He distinguished between the

Lactions of 'organised ferments' (cellular) and the 'unorganised ferments (soluble). These solubleferments'. which are not bound to the living ceiL were labelled enzymes by KUhne. This tennis derivedfrom the Greek enzyme. meaning 'in sour dough. Concrete evidence for this assumption was providedby E Buchner, as he showed that the cell-free extract from yeast cells could also produce alcohol fromsugars. The enzyme active in fennentation was tenned zymase. The first book summarising the wonderfulmechanism of the enzymes known at that time and outlining the history of their discovery was writtenby Green and is still read with interest today.ENZYMES DEFINED S C T LYSTSFriederich Wilhelm Ostwald first coined the now-familiar definition of a catalyst. specifically. that acatalyst is a substance that alters the rate of a chemical reaction without being present in the reactionproducts. Thus. Ostwald recognised enzymes as catalysts.

Today, catalysts and catalysis are familiar terms. The catalysts in internal-combustion enginesaccelerate the conversion of the pollutants carbon monoxide and nitrogen oxides into the less pollutingcarbon dioxide and nitrogen, without any change in the catalysts during the process. According to thebasic research of Tammann and Van 't Hoff, a catalyst must be able to regenerate the initial substratefrom its products. Enzymatic reactions also have an equilibrium. An example is the fonnation an(scisson of an ester that forms on reaction between an alcohol with an acid:

SvntheSISAcetic acid + alcohol ~ = = = = = = acetate ester + waterHvdrolys sIf acetic acid and alcohol are mixed in equimolar amounts. some ofthe molecules will react to fonn

an ester and water (synthesis). Yet. there still will be significant quantities of acetic acid and alcohol inthe reaction mixture as ; ell as the products generated, este r and water. A mixture of the same compositioncan also be obtained when starting with water and an ester (hydrolysis). Even in the absence of acatalyst. the reactions reach equilibrium between ester and synthesis and ester hydrolysis.

When forming and splitting butyl ester. it was found that the equilibrium was rapidly establishedfrom either direction on the addition of porcine pancreatic powder (pancreatic lipase). Thus. for life toexist, the biochemical equilibria of essential and exceedingly complex reaction systems must beestablished rapidly. Therefore, enzymes are the vital catalysts in all life processes.HISTORY OF THE DEVELOPMENT OF TECHNIC L ENZYMESEnzyme preparations. from extracts of plants or animal tissue. were used well before much was knownabout the nature of properties of enzymes. The development oftoday s industrially important microbialenzymes began with the Japanese Jokichi Takamine. Born in Japan and later a US immigrant. Takaminestudied the production ofenzymes from moulds. He obtained a process patent for making a diastaticenzyme preparation from-fungi. He named the product Takadiastase. a mixture of carbohydrases andproteolytic enzymes.

The production occurred either via a surface culture, a semisolid culture, or the koji process on moistwheat bran with nutrient salts and buffers added. The sterile culture media. mixed with spores fromAspergillus oryzae was poured onto metal trays to a thickness of a few centimetres and incubated inclimate-controlled culture chambers. The culture media, permeated with fungal mycelia, are groundand extracted with water. In a modified fonn, this process is still used by various manufacturers. although


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Basic Concepts o Enzymesit has largely been replaced by the development of the submerged fermentation process carried out inlarge fermenters.

Auguste Boidin discovered a new process for the manufacture of alcohol from cereals. Known asthe amyloprocess this method involves the extraction of cereal polysaccharides by boiling, theninoculation with a fungus that, in turn. produces saccharifying enzymes and subsequently yeasfermentation of the sugars. Boidin and Professor Jean Etfront who had studied enzymes involved inalcohol production since 1900, founded the Societe Rapidase in 1920.

Enzyme preparations from animal organs also continue to play an important industrial role. OttoRohm discovered the effectiveness of pancreatic proteases in liming and bating of hides in leathemanufacture. Until then, slurries of dog feces had been used in bating. Rohm concluded that the batingeffect of dog feces was due to excreted pancreatic proteases and subsequently developed the firststandardised enzymatic bating agent that marketed under the name oropon , offered the tanner greatercontrol and reliability in the sensitive leather manufacturing process.

Otto Rohm developed additional uses for the pancre1tic enzymes he manufactured in Darmstadtincluding the degumming of raw silk. The importance of these enzymes for the silk industry at that timecan be shown by the following cost computation: One kilogram Degomr a S pancreatic protease)with a value of 4 [Reichsmark] can degum the same amount of silk as I 0 kilograms of expensiveMarseilles soap at that time 2-3 reichsmarks per kilogram). Rohm introduced the first commerciallyenzymatic detergent. The product, named Burnus . rapidly became known as a good prewash andsoaking agent. In addition to pancreatic enzymes, it contained sodium carbonate and sodium bicarbonate,which held the pH in the wash water below 9 The pancreatic proteases are sufficiently active and stableat low temperatures in this pH range. However, pancreatic enzymes are inactive in new detergentformulations that contain sequestrants, anionic detergents and oxidising agents.

Jaag in Switzerland developed a detergent that contained, as active components, proteases fromBacillus subtilis. The Novo Company [NO] in Copenhagen deserves credit for the subsequentdevelopment of this enzyme. This will be discussed in more detail later. Plant enzymes, especially theproteases fr8m papaya fruits i.e. papain), are industrially important. Wallerstein was the first to usepapain for stabilising beer. Beer stabilisation. which prevents protein flocculation or hazing onrefrigeration, is particularly important in the United States, where beer is cold-stored.

Boidin and Effront obtained a process patent for manufacturing an enzyme preparation from culturesof B subtilis grown on the surface of a liquid nutrient medium. The preparation primarily containedamylase, which was able to replace the malt that previously had been used in textile sizing.

Rohm and Haas began producing fungal proteases with semisolid cultures. These enzymes producedgentle bating preparations suited tor special applications in leather manufacture. About 1934, the firstpectinase preparations for clarifying fruit juices and improving juice extraction from grapes were producedby the same method.

In 1958, Underkot1er published a list of commercial enzyme preparations. The most important productswere the amylases that were used in bakeries and breweries and in the manufacture of starch hydrolysates.Second were the microbial proteases, which were used in leather manufacture and in removing ~ z e sfrom cold beer. Other enzymes such as pectinases, lactases, invertases. lipases, and cellulases were.however. produced only in small quantities. The years following World War II saw a rapid developmentof submerged fermentation technology, triggered by the then-extant antibiotic manufacturing methods.Bacterial and fungal amylases could now be manufactured inexpensively. This had a great influence onthe commercial development of enzymatic starch conversion to dextrins and glucose.


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Enzymes BiotechnologyIn 1960, Novo Nordisk [NO] began production of alkaline bacterial proteases from acillus

licheniformis for use in detergents. A flurry of activity followed that led to a wave of innovations in theentire field of industrial enzymes.

The commercial development of alkaline bacterial proteases was briefly interrupted when cases oflung disease, due to allergic reactions, were reported in the United States. These allergies occurred inworkers who h d direct cont ct with dusty enzyme concentrates during detergent manufacture.Granulating the concentrates helped avoid these risks. In 1971, The National Academy of SciencesUSA) confirmed that the use ofdetergents containing enzymes posed no health hazard to the consumer.

Proteases from B lichemformis are currently the commercially most important microbial enzymes.They represent approximately 60 per cent of all the industrial enzymes, followed by the carbohydratesmaking up about 30 per cent. The latter include the amylases, glucose isomerases, pectinases andcellulases.The development of other industrial enzymes will be discussed in subsequent chapters.

PPLIC TIONS OF MODERN ENZYME TECHNOLOGYMajor targets of modern enzyme technology continue to be preservation of foods and food componentse.g. vitamins), more efficient use of raw materials and improvement offood quality such stexture and

taste. Enzymes are also used in the food industry for process optimisation to reduce process costs,including energy requirements. Other current objectives include the utilisation of new raw materials forfeeding humans and animals, the manufacture ofdietetic foods and eliminating antinutritive substancesfrom certain nutritional raw materials.

Biotechnological methods have also replaced some of the traditional chemical processes. Enzymaticmethods, which include the most recent procedures with immobilised enzymes and immobilised microorganisms, constitute only one aspect of modern biotechnology. Biotechnology encompasses manyprocesses for producing biologically active compounds, such as amino acids and antibiotics as well asprocessing waste-water..,Examples o new biotechnological processes include:

1 The use of enzymes in nonaqueous media for the production of chiral compounds and thesynthesis of special polymers.

2. The use of enzymes for the synthesis of amino acids, peptides and antibiotics.3 The use of enzymes for recycling food wastes and in waste-water treatment.4 The production ofsweeteners, such as aspartame, using combined microbiological and enzymatic

methods.5. The production of cyclodextrins from starch with specific enzymes.6 The use of cellulases and lipases as active components of detergents.7 The production of rare and expensive mammalian enzymes by micro-organisms. For example,

pure c lf stomach rennet is currently not available in sufficient quantities.8 The production of tailored enzymes to serve as specific, process-adapted catalysts. Theseprocedures are part of the so-called protein engineering, which means the design of proteinswith specific functions.

9 Large-scale production of enzymes by gene technology is to be expected in future years;recombinant chymosin was a pioneering first. It has since overcome the world shortage ofchymosin in the cheese industry. Thus, many limited or costly traditional enzyme sources willbe replaceable.


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Basic Concepts o EnzymesIn the past obtaining the desired metabolites and improved performance depended on the conventional

genetic methods o inducing mutations and hybridising sexually propagating micro-organisms. Inasexually reproducing micro-organisms physical and chemical mutagenesis produced the necessaryvariability. The sought-after characteristic or product was subsequently found by screening large numberso progeny. Mutations randomly alter the genetic or hereditary information. Success dependson whetherone o the random mutations occurs at the correct site within the genome; only then will it be possibleto develop better microbial strains. The extensive screening required makes t very expensive to determinewhether such randcru mutations have produced the changes sought in the productiono the desiredsubstance. Now gene technology permits site-specific changes and yields productso great purity undereconomically feasible conditions

enzyme biotech title page contents chapt1 and 2

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