Enzymes in Industry

Production and Applications

Edited by Wolfgang Aehle

InnodataFile Attachment3527605010.jpg

Enzymes in IndustryEdited by W. Aehle

Also of Interest:

Liese, A., Seelbach, K., Wandrey, C.

Industrial Biotransformations

2000

3-527-30094-5

Biological Reaction EngineeeringDynamic Modelling and Simulation

Second, Completely Revised and Extended Edition

2003

ISBN 3-527-30759-1

Bisswanger, H.

Enzyme KineticsPrinciples and Methods

2002

ISBN 3-527-30343-X

Bisswanger, H.

Practical Enzymology

2003

ISBN 3-527-30444-4

Bornscheuer, U. T. (Ed.)

Enzymes in Lipid Modification

2000

ISBN 3-527-30176-3

Dunn, I. J., Heinzle, E., Ingham, J., P enosil, J. E.��

Enzymes in Industry

Production and Applications

Edited by Wolfgang Aehle

Dr. Wolfgang AehleGenencor International B. V.PO Box 2182300 AE LeidenThe Netherlands

r This book was carefullyproduced. Nevertheless,authors, editors, and publisher do not warrant theinformationcontained therein to be free of errors.Readers are advised to keep in mind that statements,data, illustrations,procedural details or other itemsmay inadvertently be inaccurate.

First Edition 1990Second, Completely Revised Edition 2004

Library of Congress Card No.: Applied for.British Library Cataloguing-in-Publication Data:A catalogue record for this book is available from theBritish Library

Die Deutsche Bibliothek –CIP Catalo guing-in-Publication-DataA catalogue record for this publication is availablefrom Die Deutsche Bibliothek

© 2004 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

All rights reserved (including those of translationinto other languages). No part of this book may bereproduced in any form – by photoprinting,microfilm, or any other means – nor transmitted ortranslated into a machine language without writtenpermission from the publishers. Registered names,trademarks, etc. used in this book, even when notspecifically marked as such, are not to be consideredunprotected by law.

Printed in the Federal Republic of Germany.

Printed on acid-free paper.

Typesetting Kühn & Weyh, Satz und Medien,FreiburgPrinting Strauss Offsetdruck GmbH,MörlenbachBookbinding Litges & Dopf Buchbinderei GmbH,Heppenheim

ISBN 3-527-29592-5

V

List of Contributors XV

Abbreviations XXI

1 Introduction 11.1 History 11.2 Enzyme Nomenclature 41.2.1 General Principles of Nomenclature 51.2.2 Classification and Numbering of Enzymes 61.3 Structure of Enzymes 71.3.1 Primary Structure 71.3.2 Three-Dimensional Structure 81.3.3 Quaternary Structure, Folding, and Domains 81.3.4 The Ribozyme 111.4 Biosynthesis of Enzymes 121.4.1 Enzymes and DNA 12

2 Catalytic Activity of Enzymes 132.1 Factors Governing Catalytic Activity 152.1.1 Temperature 152.1.2 Value of pH 162.1.3 Activation 172.1.4 Inhibition 172.1.5 Allostery 202.1.6 Biogenic Regulation of Activity 212.2 Enzyme Assays 222.2.1 Reaction Rate as a Measure of Catalytic Activity 222.2.2 Definition of Units 222.2.3 Absorption Photometry 232.2.4 Fluorometry 252.2.5 Luminometry 252.2.6 Radiometry 262.2.7 Potentiometry 26

Contents

VI

2.2.8 Conductometry 272.2.9 Calorimetry 272.2.10 Polarimetry 272.2.11 Manometry 272.2.12 Viscosimetry 272.2.13 Turbidimetry 282.2.14 Immobilized Enzymes 282.2.15 Electrophoresis 282.3 Quality Evaluation of Enzyme Preparations 302.3.1 Quality Criteria 302.3.2 Specific Activity 302.3.3 Protein Determination 312.3.4 Contaminating Activities 322.3.5 Electrophoretic Purity 322.3.6 High-Performance Liquid Chromatography 332.3.7 Performance Test 332.3.8 Amino Acid Analysis and Protein Sequence Analysis 332.3.9 Stability 342.3.10 Formulation of Enzyme Preparations 34

3 General Production Methods 373.1 Microbial Production 373.1.1 Organism and Enzyme Synthesis 383.1.2 Strain Improvement 403.1.3 Physiological Optimization 423.1.4 The Fermentor and its Limitations 443.1.5 Process Design 463.1.6 Modeling and Optimization 483.1.7 Instrumentation and Control 493.2 Isolation and Purification 503.2.1 Preparation of Biological Starting Materials 513.2.1.1 Cell Disruption by Mechanical Methods 523.2.1.2 Cell Disruption by Nonmechanical Methods 523.2.2 Separation of Solid Matter 523.2.2.1 Filtration 523.2.2.2 Centrifugation 543.2.2.3 Extraction 563.2.2.4 Flocculation and Flotation 563.2.3 Concentration 573.2.3.1 Thermal Methods 573.2.3.2 Precipitation 573.2.3.3 Ultrafiltration 583.2.4 Purification 603.2.4.1 Crystallization 603.2.4.2 Electrophoresis 61

Contents

VII

3.2.4.3 Chromatography 613.2.5 Product Formulation 693.2.6 Waste Disposal 703.3 Immobilization 703.3.1 Definitions 713.3.2 History 713.3.3 Methods 723.3.3.1 Carrier Binding 733.3.3.2 Cross-linking 763.3.3.3 Entrapment 763.3.4 Characterization 793.3.5 Application 80

4 Discovery and Development of Enzymes 834.1 Enzyme Screening 834.1.1 Overview 834.1.2 Natural Isolate Screening 844.1.3 Molecular Screening 864.1.4 Environmental Gene Screening 874.1.5 Genomic Screening 894.1.6 Proteomic Screening 914.2 Protein Engineering 924.2.1 Introduction 924.2.2 Application of Protein Engineering in Academia and Industry 974.2.3 Outlook 100

5 Industrial Enzymes 1015.1 Enzymes in Food Applications 1015.1.1 Enzymes in Baking 1015.1.1.1 Introduction 1015.1.1.2 Amylases 1025.1.1.3 Xylanases 1075.1.1.4 Oxidoreductases 1085.1.1.5 Lipases 1105.1.1.6 Proteases 1115.1.1.7 Transglutaminase 1135.1.2 Enzymes in Fruit Juice Production and Fruit Processing 1135.1.2.1 Introduction 1135.1.2.2 Biochemistry of Fruit Cell Walls 1135.1.2.3 Cell-Wall-Degrading Enzymes 1175.1.2.4 Apple Processing 1185.1.2.4.1 Apple Pulp Maceration 1185.1.2.4.2 Apple Juice Depectinization 1205.1.2.5 Red-Berry Processing 1215.1.2.6 Tropical Fruit and Citrus Processing 123

Contents

5.1.2.7 Conclusion 1245.1.3 Enzymes in Brewing 1255.1.3.1 Introduction 1255.1.3.2 Enzymes in Malting and Mashing 1275.1.3.3 Enzymes for Problem Prevention or Solving 1305.1.3.3.1 Bacterial -Amylase in Mashing 1305.1.3.3.2 Fungal -Amylase in Fermentation 1305.1.3.3.3 -Glucanase in Mashing 1315.1.3.3.4 Cysteine Endopeptidases (Postfermentation) 1315.1.3.3.5 Glucoamylase in Mashing 1315.1.3.4 Enzymes for Process Improvement 1325.1.3.4.1 Adjunct Brewing 1325.1.3.4.2 Improved Mashing Processes 1335.1.3.4.3 Shelf-Life Improvement 1345.1.3.4.4 Accelerated Maturation 1355.1.3.4.5 Starch-Haze Removal 1355.1.3.5 Special Brewing Processes 1365.1.4 Enzymes in Dairy Applications 1365.1.4.1 Introduction 1365.1.4.2 Cheesemaking 1375.1.4.2.1 Cheesemaking Process 1375.1.4.2.2 Mechanism of Renneting 1375.1.4.2.3 Types of Coagulants 1375.1.4.2.4 Properties of Coagulating Enzymes 1385.1.4.2.5 Cheese Ripening 1405.1.4.2.6 Cheese Flavors and Ripening Acceleration 1415.1.4.2.7 Lipase 1415.1.4.2.8 Lysozyme 1425.1.4.2.9 Milk Protein Hydrolysates 1435.1.4.2.10 Transglutaminase 1435.1.4.3 Milk Processing 1435.1.4.3.1 -Galactosidase 1435.1.4.3.2 Other Enzymes 1455.1.5 Other Food Applications 1455.1.5.1 Introduction 1455.1.5.2 Meat and Fish 1455.1.5.2.1 Meat Processing 1455.1.5.2.2 Fish Processing 1465.1.5.3 Protein Cross-linking 1475.1.5.4 Flavor Development 1485.1.5.4.1 Protein Hydrolysis 1485.1.5.4.2 Lipid Hydrolysis 1495.1.5.5 Egg Powder 1505.1.5.6 Oils and Fats 1515.1.5.6.1 Fat Splitting 151

ContentsVIII

��

�

�

5.1.5.6.2 Interesterification 1525.1.5.6.3 Esterification 1545.1.5.6.4 Oil Degumming 1555.2 Enzymes in Nonfood Applications 1555.2.1 Enzymes in Household Detergents 1555.2.1.1 Historical Development 1555.2.1.2 Laundry Soils 1575.2.1.3 Detergent Composition and Washing Process 1595.2.1.3.1 Washing Process 1595.2.1.3.2 Detergent Compositions 1595.2.1.4 Enzyme-Aided Detergency and Soil Removal 1615.2.1.5 Detergent Enzyme Performance Evaluation and Screening 1615.2.1.6 Enzyme Types 1655.2.1.6.1 Proteases 1655.2.1.6.2 Amylases 1695.2.1.6.3 Lipases 1725.2.1.6.4 Cellulases 1755.2.1.6.5 Mannanase 1775.2.1.7 Future Trends 1785.2.2 Enzymes in Automatic Dishwashing 1805.2.2.1 Introduction 1805.2.2.2 Characteristics of Enzymes for ADDs 1815.2.2.3 Proteases 1825.2.2.3.1 Proteins: The Substrate of Proteases 1825.2.2.3.2 Proteases for ADDs 1825.2.2.4 Amylases 1845.2.2.4.1 Starch: The Substrate of Amylases 1845.2.2.4.2 Amylases for ADDs 1855.2.2.5 Other Enzymes 1875.2.2.6 Automatic Dishwashing Detergents 1875.2.2.6.1 Composition of Automatic Dishwashing Detergents 1885.2.2.6.2 Application of Enzymes in ADD 1895.2.2.6.3 Stability and Compatibility 1945.2.3 Enzymes in Grain Wet-Milling 1945.2.3.1 Introduction 1945.2.3.2 Overview of the Conversion of Corn to HFCS 1945.2.3.2.1 Corn Steeping 1965.2.3.2.2 Coarse Grinding and Germ Removal by Cyclone Separation 1975.2.3.2.3 Fine Grinding and Fiber Removal by Screening 1975.2.3.2.4 Centrifugation and Washing to Separate Starch from Protein 1975.2.3.2.5 Hydrolysis with -Amylase (Liquefaction) 1975.2.3.2.6 Hydrolysis with Glucoamylase (Saccharification) 1985.2.3.2.7 Isomerization with Glucose Isomerase 1985.2.3.2.8 Fructose Enrichment and Blending 1995.2.3.3 -Amylase 199

Contents IX

�

�

5.2.3.3.1 Origin and Enzymatic Properties 1995.2.3.3.2 Structure 2005.2.3.3.3 Industrial Use 2025.2.3.4 Glucoamylase 2025.2.3.4.1 Origin and Enzymatic Properties 2025.2.3.4.2 Structure 2035.2.3.4.3 Industrial Use 2045.2.3.5 Pullulanase 2055.2.3.5.1 Origin and Enzymatic Properties 2055.2.3.5.2 Structure 2055.2.3.5.3 Industrial Use 2065.2.3.6 Glucose Isomerase 2065.2.3.6.1 Origin and Enzymatic Properties 2065.2.3.6.2 Structure 2075.2.3.6.3 Industrial Use of Glucose Isomerase 2085.2.3.7 Use of Wheat Starch 2095.2.4 Enzymes in Animal Feeds 2105.2.4.1 Introduction 2105.2.4.2 Enzymes Used in Animal Feed 2115.2.4.2.1 Fiber-Degrading Enzymes 2115.2.4.2.2 Phytic Acid Degrading Enzymes (Phytases) 2155.2.4.2.3 Protein-Degrading Enzymes (Proteases) 2165.2.4.2.4 Starch-Degrading Enzymes (Amylases) 2175.2.4.3 Future Developments 2185.2.5 Enzymes in Textile Production 2195.2.5.1 Introduction 2195.2.5.2 Cellulose Fibers 2205.2.5.2.1 Desizing of Cotton Cellulose Fibers 2205.2.5.2.2 Scouring of Cotton 2215.2.5.2.3 Bleaching of Cotton 2215.2.5.2.4 Removal of Hydrogen Peroxide 2225.2.5.2.5 Cotton Finishing 2225.2.5.2.6 Ageing of Denim 2245.2.5.2.7 Processing of Man-Made Cellulose Fibers 2255.2.5.2.8 Processing of Bast Fibers 2265.2.5.3 Proteinous Fibers 2265.2.5.3.1 Wool Processing 2265.2.5.3.2 Degumming of Silk 2285.2.5.4 Textile Effluent Treatment and Recycling 2295.2.5.5 Outlook 2315.2.6 Enzymes in Pulp and Paper Processing 2325.2.6.1 Introduction 2325.2.6.2 Enzymes 2335.2.6.2.1 Cellulases 2345.2.6.2.2 Hemicellulases 234

ContentsX

5.2.6.2.3 Lignin-Modifying, Oxidative Enzymes 2365.2.6.3 Enzymes in Pulp and Paper Processing 2385.2.6.3.1 Mechanical Pulping 2385.2.6.3.2 Chemical Pulping 2395.2.6.3.3 Bleaching 2405.2.6.3.4 Papermaking 2425.2.6.3.5 Deinking 2445.3 Development of New Industrial Enzyme Applications 2445.3.1 Introduction 2445.3.2 Enzymes in Cosmetics 2465.3.2.1 Hair Dyeing 2475.3.2.1.1 Oxidases 2485.3.2.1.2 Peroxidases 2495.3.2.1.3 Polyphenol Oxidases 2495.3.2.2 Hair Waving 2505.3.2.3 Skin Care 2505.3.2.4 Toothpastes and Mouthwashes 2515.3.2.5 Enzymes in Cleaning of Artificial Dentures 2525.3.3 Enzymes for Preservation 2525.3.4 Enzymes in Hard-Surface Cleaning 2535.3.4.1 Enzymes in Membrane Cleaning 2535.3.4.1.1 Proteases 2535.3.4.1.2 Hemicellulases 2545.3.5 Enzymes Generating a pH Shift 2545.3.6 Enzymes in Cork Treatment 2555.3.7 Enzymes in Oil-Field Applications 2565.3.8 Enzymes in Wastewater Treatment 2565.3.9 Enzymes for Polymerisation: Wood Fiberboard Production 2575.4 Overview of Industrial Enzyme Applications 257

6 Nonindustrial Enzyme Usage 2636.1 Enzymes in Organic Synthesis 2636.1.1 Introduction 2636.1.2 Examples of Enzymatic Conversions 2646.1.2.1 Syntheses by Means of Hydrolases 2646.1.2.2 Reduction of C—O and C—C Bonds 2686.1.2.3 Oxidation of Alcohols and Oxygenation of C—H and C—C Bonds 2696.1.2.4 C—C Coupling 2716.1.2.5 Formation of Glycosidic Bonds 2726.1.2.6 Enzymatic Protecting Proup Techniques 2746.1.3 Enzyme-Analogous Catalysts 2766.1.4 Commercial Applications 2786.1.4.1 General 2786.1.4.2 Nonstereoselective Biocatalytic Reactions 2806.1.4.3 Biocatalytic Resolution Processes 281

Contents XI

6.1.4.3.1 Enzymatic Acylation of Amino Groups 2866.1.4.3.2 Enzymatic Hydrolysis of Hydantoins 2876.1.4.3.3 Enzymatic Hydrolysis of Lactams 2896.1.4.3.4 Enzymatic Hydrolysis of C—O Bonds 2906.1.4.3.5 Enzymatic Hydrolysis of Nitriles 2916.1.4.3.6 Enzymatic Cleavage of threo Aldol Products 2926.1.4.4 Biocatalytic Asymmetric Synthesis 2926.1.4.4.1 Biocatalytic Reductive Amination of C—O Bonds 2936.1.4.4.2 Biocatalytic Hydrocyanation of C—O Bonds 2946.1.4.4.3 Biocatalytic Addition of Water to C—O Bonds 2946.1.4.4.4 Biocatalytic Amination of C—C Bonds 2946.1.5 Outlook 2956.2 Therapeutic Enzymes 2956.2.1 Requirements for the Use of Enzymes in Therapy 2956.2.2 Coping with Peculiar Protein Properties 2966.2.3 Sources of Enzymes and Production Systems 2996.2.4 Overview of Therapeutic Enzymes 3006.2.4.1 Oxidoreductases 3056.2.4.2 Transferases 3066.2.4.3 Esterases 3076.2.4.4 Nucleases 3076.2.4.5 Glycosidases 3086.2.4.6 Proteases 3106.2.4.6.1 Pancreatic and Gastric Proteases 3106.2.4.6.2 Plasma Proteases 3106.2.4.6.3 Coagulation Factors 3116.2.4.6.4 Plasminogen Activators 3136.2.4.6.5 Proteases from Snake Venoms 3156.2.4.6.6 Plant and Microbial Proteases 3166.2.4.7 Amidases 3166.2.4.8 Lyases 3176.3 Enzymes in Diagnosis 3176.3.1 Determination of Substrate Concentration 3186.3.2 Determination of Enzyme Activity 3196.3.3 Immunoassays 3206.4 Enzymes for Food Analysis 3226.4.1 Carbohydrates 3236.4.2 Organic Acids 3266.4.3 Alcohols 3306.4.4 Other Food Ingredients 3326.5 Enzymes in Genetic Engineering 3346.5.1 Restriction Endonucleases and Methylases 3376.5.1.1 Classification 3376.5.1.2 Activity of Class II Restriction Endonucleases 3476.5.1.2.1 Reaction Parameters 347

ContentsXII

6.5.1.2.2 Additional Structural Requirements Influencing Activity 3486.5.1.3 Specificity of Class II Restriction Endonucleases 3496.5.1.3.1 Palindromic Recognition Sequences 3496.5.1.3.2 Nonpalindromic Recognition Sequences 3526.5.1.3.3 Isoschizomers 3526.5.1.4 Changes in Sequence Specificity 3536.5.1.5 Novel Class II Restriction Endonucleases 3556.5.2 DNA Polymerases 3566.5.2.1 Escherichia coli DNA Polymerase I 3566.5.2.2 Klenow Enzyme 3586.5.2.3 T4 DNA Polymerase 3606.5.2.4 Reverse Transcriptase 3616.5.2.5 Terminal Transferase 3636.5.3 RNA Polymerases 3636.5.3.1 SP6 RNA Polymerase 3636.5.3.2 T7 RNA Polymerase 3656.5.4 DNA Nucleases 3666.5.4.1 DNase I 3676.5.4.2 Exonuclease III 3676.5.4.3 Nuclease S1 3686.5.4.4 Nuclease Bal 31 3696.5.5 RNA Nucleases 3706.5.5.1 RNase H 3706.5.5.2 Site-Specific RNases 3716.5.5.2.1 RNase A 3716.5.5.2.2 RNase CL3 3716.5.5.2.3 RNase T1Ribonuclease T1 3726.5.5.2.4 RNase U2Ribonuclease U2 3726.5.5.2.5 Nuclease S7 3726.5.5.2.6 Site-Specific RNases in RNA Sequence Analysis 3736.5.6 Modifying Enzymes 3776.5.6.1 Alkaline Phosphatase 3786.5.6.2 T4 DNA Ligase 3936.5.6.3 Escherichia coli DNA Ligase 3946.5.6.4 T4 Polynucleotide Kinase 3956.5.6.5 T4 Polynucleotide Kinase, 3́-Phosphatase-Free 3976.5.6.6 Methylase HpaII 397

7 Enzyme Safety and Regulatory Considerations 3997.1 Safe Handling of Enzymes 3997.1.1 Possible Health Effects 4007.1.2 Control Technology 4007.2 Product Regulatory Considerations 4037.2.1 Food-Use Enzymes 4047.2.2 Feed-Use Enzymes 408

Contents XIII

7.2.3 Industrial-Use Enzymes 410

References 413

Index 465

ContentsXIV

Editor

Dr. Wolfgang AehleGenencor International B. V.Research & DevelopmentP.O. Box 2182300 AE LeidenThe Netherlands

Authors

Dr. Wolfgang AehleGenencor International B. V.Research & DevelopmentP.O. Box 2182300 AE LeidenThe NetherlandsChapter 1, Sections 4.2, 5.4

Dr. Richard L. AntrimGrain Processing Corporation1600 Oregon StreetMuscatine, IA 52761-1494USASection 5.2.3

Todd BeckerGenencor International, Inc.925 Page Mill RoadPalo Alto, CA 94304-1013USASection 3.2.5

Dr. Rick BottGenencor International, Inc.925 Page Mill RoadPalo Alto, CA 94304-1013USASection 4.2

Dr. Johanna BuchertVTT BiotechnologyP.O. Box 15002044 VTTFinlandSection 5.2.6

Dr. Heidi BurrowsFinfeeds InternationalP.O. Box 777Wiltshire, SN8 1XN MarlboroughUKSection 5.2.4

Alice J. CaddowGenencor International Inc.925 Page Mill RoadPalo Alto, CA 94304-1013USAChapter 7

Dr. Gopal K. ChotaniGenencor International, Inc.925 Page Mill RoadPalo Alto, CA 94304-1013USASection 3.1

XV

List of Contributors

Beth ConcobyGenencor International Inc.925 Page Mill RoadPalo Alto, CA 94304-1013USAChapter 7

Dr. Hans de NobelGenencor International B. V.Research & DevelopmentP.O. Box 2182300 AE LeidenThe NetherlandsSection 4.1

Dr. André de RoosDSM Food SpecialitiesP.O. Box 12600 MA DelftThe NetherlandsSection 5.1.4

Dr. Carlo DinkelSiChem GmbHBITZFahrenheitstr. 128359 BremenGermanySection 6.1

Timothy C. DodgeGenencor International, Inc.925 Page Mill RoadPalo Alto, CA 94304-1013USASection 3.1

Prof. Dr. Karlheinz DrauzDegussa AGFine ChemicalsRodenbacher Chaussée 463457 Hanau-WolfgangGermanySection 6.1

Prof. Dr. Saburo Fukui †formerly Department ofIndustrial ChemistryFaculty of EngineeringKyoto University606-8501 KyotoJapanSection 3.3

Dr. Christian GölkerBayer AGAprather Weg42096 WuppertalGermanySection 3.2

Dr. Catherine GrassinDSM Food Specialties15, rue des ComtessesP.O. Box 23959472 Seclin CedexFranceSection 5.1.2

Dr. Harald GrögerDegussa AGProject House BiotechnologyRodenbacher Chaussée 463457 Hanau-WolfgangGermanySection 6.1

Dr. Meng H. HengGenencor International, Inc.925 Page Mill RoadPalo Alto, CA 94304-1013USASections 3.2.2.4, 3.2.4.1

Dr. Günther Hennigerformerly Roche Diagnostics GmbHNonnenwald 282377 PenzbergGermanySection 6.4

List of ContributorsXVI

Dr. Ivan HerbotsProcter & Gamble Eurocor S.A.Temselaan 1001853 Strombeek-BeverBelgiumSection 5.2.1

Dr. Marga HerweijerDSM Food SpecialtiesResearch and DevelopmentP.O. Box 12600 MA DelftThe NetherlandsSection 5.1.2

Dr. Brian JonesGenencor International B. V.Research & DevelopmentP.O. Box 2182300 AE LeidenThe NetherlandsSection 4.1

Dr. Albert JonkeRoche Diagnostics GmbHNonnenwald 282377 PenzbergGermanyChapter 2

John KanGenencor International, Inc.925 Page Mill RoadPalo Alto, CA 94304-1013USASections 3.2.2.4, 3.2.4.1

Dr. Christoph KesslerRoche Diagnostics GmbHNonnenwald 282377 PenzbergGermanySection 6.5

Dr. Beatrix KottwitzR&D / Technology Laundry andHome CareHenkel KGaAHenkelstr. 6740191 DüsseldorfGermanySection 5.2.2

Dr. Karsten M. KraghDanisco A/SEdwin Rahrs Vej 388220 BrabrandDenmarkSection 5.1.1

Dr. Georg-Burkhard KresseRoche Diagnostics GmbHPharma Research / BiologyNonnenwald 282377 PenzbergGermanySection 6.2

Dr. Herman B. M. LentingTNO IndustryCentre for Textile ResearchP.O. Box 3377500 AH EnschedeThe NetherlandsSection 5.2.5

Dr. Karl-Heinz MaurerHenkel KGaADepartment of Enzyme TechnologyHenkelstr. 6740191 DüsseldorfGermanySection 5.3

List of Contributors XVII

Dr. Gerhard Michalformerly Boehringer MannheimGmbHResearch68298 MannheimGermanyChapter 2

Dr. Marja-Leena Niku-PaavolaVTT BiotechnologyP.O. Box 15002044 VTTFinlandSection 5.2.6

Prof. Dr. Richard N. PerhamUniversity of CambridgeDepartment of Biochemistry80 Tennis Court RoadCB2 1GA CambridgeEnglandChapter 1

Dr. Charlotte Horsmans PoulsenDanisco A/SEdwin Rahrs Vej 388220 BrabrandDenmarkSection 5.1.1

Prof. Dr. Peter J. ReillyIowa State UniversityDepartment of Chemical Engineering2114 Sweeney HallAmes, IA 50011-2230USASection 5.2.3

Dr. Andrea SaettlerHenkel KGaADepartment of Enzyme TechnologyHenkelstr. 6740191 DüsseldorfGermanySection 5.3

Dr. Rainer SchmuckRoche Diagnostics GmbHDiagnostic ResearchNonnenwald 282377 PenzbergGermanySection 6.3

Dr. Carsten SchultzEMBLMeyerhofstr. 169117 HeidelbergGermanySection 6.1

Dr. Jorn Borch SoeDanisco A/SEdwin Rahrs Vej 388220 BrabrandDenmarkSection 5.1.5

Jens Frisback SorensenDanisco A/SEdwin Rahrs Vej 388220 BrabrandDenmarkSection 5.1.1

Dr. Anna SuurnäkkiVTT BiotechnologyP.O. Box 15002044 VTTFinlandSection 5.2.6

Prof. Dr. Atsuo TanakaDepartment of Industrial ChemistryFaculty of EngineeringKyoto University606-8501 KyotoJapanSection 3.3

List of ContributorsXVIII

Dr. Andreas HermanTerwisscha van ScheltingaDSM-Gist Research and DevelopmentAlexander Fleminglaan 12613 AX DelftThe NetherlandsSection 3.1

Dr. Liisa ViikariVTT BiotechnologyP.O. Box 15002044 VTTFinlandSection 5.2.6

Prof. Dr. Herbert WaldmannMax-Planck-Institut für molekularePhysiologieOtto-Hahn-Str. 1144227 DortmundGermanySection 6.1

Dr. Jan WilmsAntonine der KinderenRietschoot 1791511 WG OostzaanThe NetherlandsSection 5.1.3

Dr. Karl WulffBoeringer Mannheim GmbHBahnhofsstr. 9-1582327 TutzingGermanySection 6.3

List of Contributors XIX

XXI

A: adenosineACA: acetamidocinnamic acidACL: -amino- -aprolactamADH: alcohol dehydrogenaseADI: acceptable daily intakeADP: adenosine 5́ -diphosphateAla: alanineArg: arginineAMP: adenosine 5́ -monophosphateATC: d,l-2-amino-D2-thiazoline-4-carboxylic acidATP: adenosine 5́ -triphosphate

C: cytidinecDNA: copy DNACL: citrate lyaseCMP: cytidine 5́ -monophosphateCoA: coenzyme ACS: citrate synthetaseCTP: cytidine 5́ -triphosphate

d: deoxydam: gene locus for E. coli DNA adenine methylase (N6-methyladenine)dcmI: gene locus for E. coli DNA cytosine methylase(5-methylcytosine)dd: dideoxyddNTP: dideoxynucleoside 5́-triphosphateDE: dextrose equivalentDEAE: diethylaminoethylDNA: deoxyribonucleic acidDNase: deoxyribonucleasedNTP: deoxynucleoside 5́-triphosphateDOPA: 3-(3,4-dihydroxyphenylalanine) [3-hydroxy-l-tyrosine]dpm: decays per minuteds: double-stranded

Abbreviations

� �

XXII

E.C.: Enzyme Commission

F6P: fructose 6-phosphateFAN: free alpha amino nitrogen, i.e., a measure of peptides/amino acids

available for yeast to be used as nutrientfMet:FMN: flavin mononucleotideFMNH2: flavin mononucleotide, reduced

G: guanosineGDP: guanosine 5 -́diphosphateGlu: glutamic acidGly: glycineGMP: guanosine 5 -́monophosphateGOD: glucose oxidaseGOT: glutamate oxaloacetate transaminaseG6P: glucose 6-phosphateGPT: glutamate pyruvate transaminaseGTP: guanosine 5 -́triphosphate

3-HBDH: 3-hydroxybutyrate dehydrogenaseHFCS: high-fructose corn syruphsdM: E. coli gene locus for methylationhsdR: E. coli gene locus for restrictionhsdS: E. coli gene locus for sequence specificity

IDP: inosine 5́ -diphosphateIle: isoleucineINT: iodonitrotetrazolium chlorideITP: inosine 5́ -triphosphate

LDH: lactate dehydrogenaseLys: lysine

m(superscript): methylatedMDH: malate dehydrogenaseMet: methionineM6P: mannose 6-phosphatemRNA: messenger RNAMTT: 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide

N: any nucleotideNAD: nicotinamide – adenine dinucleotideNADH: nicotinamide – adenine dinucleotide, reducedNADP: nicotinamide – adenine dinucleotide phosphate

Abbreviations

N-formylmethionine

XXIII

NADPH: nicotinamide – adenine dinucleotide phosphate, reducedNMN: nicotinamide mononucleotideNTP: nucleoside 5́-triphosphate

p: phosphate groups32P: phosphate groups containing 32P phosphorus atomspi: inorganic phosphate°P: degree Plato; i.e., sugar content equivalent to 1 % sucrose by weightPEP: phosphoenolpyruvate6-PGDH: 6-phosphogluconate dehydrogenasePhe: phenylalaninePMS: 5-methylphenazinium methyl sulfatepoly(dA): poly(deoxyadenosine 5́-monophosphate)ppi: inorganic pyrophosphatePro: prolinePRPP: phosphoribosyl pyrophosphatePu: purinePy: pyrimidine

r: riboRNA: ribonucleic acidRNase: ribonuclease

SAM:SMHT: serine hydroxymethyltransferasess: single-stranded

T: thymidineTMP: thymidine 5́ -monophosphatetRNA: transfer RNATTP: thymidine 5́ -triphosphate

U: uridineUMP: uridine 5́-monophosphateUTP: uridine 5́-triphosphate

Val: valine

Plasmids:pBR322pBR328pSM1pSP64pSP65pSPT18, pSPT19

Abbreviations

S-adenosylmethionine

pT7–1, pT7–2pUC 18, pUC 19pUR222

Bacteriophages:fdghlM13N4PBS1PBS2SPO1SP6SP15T3T4T5T7XP12gt11SM11X174

Eukaryotic viruses:Ad2SV40

AbbreviationsXXIV

1

Enzymes are the catalysts of biological processes. Like any other catalyst, an enzymebrings the reaction catalyzed to its equilibrium position more quickly than wouldoccur otherwise; an enzyme cannot bring about a reaction with an unfavorablechange in free energy unless that reaction can be coupled to one whose free energychange is more favorable. This situation is not uncommon in biological systems,but the true role of the enzymes involved should not be mistaken.

The activities of enzymes have been recognized for thousands of years; the fer-mentation of sugar to alcohol by yeast is among the earliest examples of a biotechno-logical process. However, only recently have the properties of enzymes been under-stood properly. Indeed, research on enzymes has now entered a new phase with thefusion of ideas from protein chemistry, molecular biophysics, and molecular biol-ogy. Full accounts of the chemistry of enzymes, their structure, kinetics, and techno-logical potential can be found in many books and series devoted to these topics[1–5]. This chapter reviews some aspects of the history of enzymes, their nomencla-ture, their structure, and their relationship to recent developments in molecularbiology.

1.1History

Detailed histories of the study of enzymes can be found in the literature [6], [7].

Early Concepts of Enzymes. The term “enzyme” (literally “in yeast”) was coined byKühne in 1876. Yeast, because of the acknowledged importance of fermentation,was a popular subject of research. A major controversy at that time, associated mostmemorably with Liebig and Pasteur, was whether or not the process of fermenta-tion was separable from the living cell. No belief in the necessity of vital forces, how-ever, survived the demonstration by Buchner (1897) that alcoholic fermentationcould by carried out by a cell-free yeast extract. The existence of extracellularenzymes had, for reasons of experimental accessibility, already been recognized. Forexample, as early as 1783, Spallanzani had demonstrated that gastric juice coulddigest meat in vitro, and Schwann (1836) called the active substance pepsin.

1

Introduction

1 Introduction

Kühne himself appears to have given trypsin its present name, although its exis-tence in the intestine had been suspected since the early 1800s.

Enzymes as Proteins. By the early 1800s, the proteinaceous nature of enzymes hadbeen recognized. Knowledge of the chemistry of proteins drew heavily on theimproving techniques and concepts of organic chemistry in the second half of the1800s; it culminated in the peptide theory of protein structure, usually credited toFischer und Hofmeister. However, methods that had permitted the separationand synthesis of small peptides were unequal to the task of purifying enzymes.Indeed, there was no consensus that enzymes were proteins. Then, in 1926, Sum-ner crystallized urease from jack bean meal and announced it to be a simple pro-tein. However, Willstätter argued that enzymes were not proteins but “colloidalcarriers” with “active prosthetic groups”. However, with the conclusive work byNorthrop et al., who isolated a series of crystalline proteolytic enzymes, beginningwith pepsin in 1930, the proteinaceous nature of enzymes was established.

The isolation and characterization of intracellular enzymes was naturally morecomplicated and, once again, significant improvements were necessary in the sepa-ration techniques applicable to proteins before, in the late 1940s, any such enzymebecame available in reasonable quantities. Because of the large amounts of accessi-ble starting material and the historical importance of fermentation experiments,most of the first pure intracellular enzymes came from yeast and skeletal muscle.However, as purification methods were improved, the number of enzymes obtainedin pure form increased tremendously and still continues to grow. Methods of pro-tein purification are so sophisticated today that, with sufficient effort, any desiredenzyme can probably be purified completely, even though very small amounts willbe obtained if the source is poor.

Primary Structure. After the protein nature of enzymes had been accepted, the waywas clear for more precise analysis of their composition and structure. Most aminoacids had been identified by the early 20th century. The methods of amino acid anal-ysis then available, such as gravimetric analysis or microbiological assay, were quiteaccurate but very slow and required large amounts of material. The breakthroughcame with the work of Moore and Stein on ion-exchange chromatography ofamino acids, which culminated in 1958 in the introduction of the first automatedamino acid analyzer [8].

The more complex question – the arrangement of the constituent amino acids ina given protein, generally referred to as its primary structure – was solved in the late1940s. The determination in 1951 of the amino acid sequence of the -chain of insu-lin by Sanger and Tuppy [10] demonstrated for the first time that a given proteindoes indeed have a unique primary structure. The genetic implications of this wereenormous. The introduction by Edman of the phenyl isothiocyanate degradation ofproteins stepwise from the N-terminus, in manual form in 1950 and subsequentlyautomated in 1967 [11], provided the principal chemical method for determiningthe amino acid sequences of proteins. The primary structures of pancreatic ribonu-clease [12] and egg-white lysozyme [13] were published in 1963. Both of these

2

�

1.1 History

enzymes, simple extracellular proteins, contain about 120 amino acids. The firstintracellular enzyme to have its primary structure determined was glyceraldehyde3-phosphate dehydrogenase [14], which has an amino acid sequence of 330 residuesand represents a size (250 – 400 residues) typical of many enzymes. Protein sequen-cing is increasingly performed by liquid chromatography/mass spectrometry(LC/MS) techniques, and several tools and software packages are now available forprotein identification and characterization. The methods of protein sequence analysisare now so well developed that no real practical deterrent exists, other than time orexpense, to determination of the amino acid sequence of any polypeptide chain [9].

A more recent fundamental concept called proteome (protein complement to agenome) will enable researchers to unravel biochemical and physiological mecha-nisms of complex multivariate diseases at the functional molecular level. A new dis-cipline, proteomics, complements physical genome research. Proteomics can bedefined as “the qualitative and quantitative comparison of proteomes under differ-ent conditions to further unravel biological processes” [15].

Active Site. The fact that enzymes are highly substrate specific and are generallymuch larger than the substrates on which they act quickly became apparent. Theearliest kinetic analyses of enzymatic reactions indicated the formation of transientenzyme – substrate complexes. These observations could be explained easily if theconversion of substrate to product was assumed to occur at a restricted site on anenzyme molecule. This site soon became known as the active center or, as is morecommon today, the active site.

Particular compounds were found to react with specific amino acid side chainsand thus inhibit particular enzymes. This suggested that such side chains mighttake part in the catalytic mechanisms of these enzymes. An early example was theinhibition of glycolysis or fermentation by iodoacetic acid, which was later recog-nized as resulting from reaction with a unique cysteine residue of glyceraldehyde 3-phosphate dehydrogenase, which normally carries the substrate in a thioester link-age [16].

Many such group-specific reagents have now been identified as inhibitors of indi-vidual enzymes; often they are effective because of the hyper-reactivity of a function-ally important side chain in the enzyme's active site. However, a more sophisticatedapproach to the design of enzyme inhibitors became possible when the reactivegroup was attached to a substrate; in this way, the specificity of the target enzymewas utilized to achieve selective inhibition of the enzyme [17]. Such active-site-direct-ed inhibitors have acquired major importance not only academically in the study ofenzyme mechanisms but also commercially in the search for a rational approach toselective toxicity or chemotherapy.

Three-Dimensional Structure. Chemical studies showed that the active site of anenzyme consists of a constellation of amino acid side chains brought together spa-tially from different parts of the polypeptide chain. If this three-dimensional struc-ture was disrupted by denaturation, that is, without breaking any covalent bonds,the biological activity of the enzyme was destroyed. In addition, it was found that all

3

1 Introduction

the information required for a protein to fold up spontaneously in solution andreproduce its native shape was contained in its primary structure. This was part ofthe original “central dogma” of molecular biology.

The X-ray crystallography of proteins [18] demonstrated unequivocally that a givenprotein has a unique three-dimensional structure. Among the basic design princi-ples was the tendency of hydrophobic amino acid side chains to be associated withthe hydrophobic interior of the folded molecule, whereas charged side chains werealmost exclusively situated on the hydrophilic exterior or surface. The first high-res-olution crystallographic analysis of an enzyme, egg-white lysozyme, confirmed theseprinciples and led to the proposal of a detailed mechanism [19]. The active site waslocated in a cleft in the structure (Fig. 1), which has subsequently proved to be acommon feature of active sites. According to this, the enzymatic reaction takes placein a hydrophobic environment, and the successive chemical events involving sub-strate and protein side chains are not constrained by the ambient conditions of aque-ous solution and neutral pH.

Figure 1. A molecular model of the enzyme lysozyme:the arrow points to the cleft that accepts thepolysaccharide substrate (Reproduced by courtesy ofJ. A. Rupley)

1.2Enzyme Nomenclature

Strict specificity is a distinguishing feature of enzymes, as opposed to other knowncatalysts. Enzymes occur in myriad forms and catalyze an enormous range of reac-tions. By the late 1950 s the number of known enzymes had increased so rapidlythat their nomenclature was becoming confused or, worse still, misleading becausethe same enzyme was often known to different workers by different names; in addi-tion, the name frequently conveyed little or nothing about the nature of the reactioncatalyzed.

To bring order to this chaotic situation, an International Commission on Enzymeswas established in 1956 under the auspices of the International Union of Biochem-

4