Table of Contents
Preface xiii
Part I Overview of Industrial Enzyme Applications and Key Technologies 1
1.1 Industrial Enzyme Applications - Overview and Historic Perspective 3
Oliver May
1.1.1 Prehistoric Applications 3
1.1.2 Growing the Scientific Basis 5
1.1.3 The Beginning of Industrial Applications and the Emerging Enzyme Industry 12
References 21
1.2 Enzyme Development Technologies 25
Andreas Vogel
1.2.1 Introduction 25
1.2.2 Identification of Wild-Type Enzymes 26
1.2.2.1 Selection Parameters for Starting Enzymes 28
1.2.3 Enzyme Engineering 30
1.2.3.1 Types of Enzyme Modifications 30
1.2.3.2 General Engineering Strategies. Library Design and Generation 30
1.2.3.3 Screening for Better Enzymes 37
1.2.4 Impact of Enzyme Development Technologies Today and Tomorrow 38
Acknowledgments 41
References 41
1.3 Eukaryotic Expression Systems for Industrial Enzymes 47
Lukas Rieder, Nico Teuschler, Katharina Ebner, and Anton Glieder
1.3.1 Eukaryotic Enzyme Production Systems 47
1.3.2 Special Considerations for Working with Eukaryotic Expression Systems 47
1.3.2.1 Choice of Expression Host 47
1.3.2.2 Comparison of Cell Structure and Their Influence on Molecular Biology 49
1.3.3 Differences in Vector Design for Eukaryotic and Prokaryotic Hosts 51
1.3.4 Differences in Regulation of Gene Expression in Eukaryotes and Prokaryotes 56
1.3.4.1 Different Types of Promoters 58
1.3.5 Industrial Enzyme Production 58
1.3.6 Enzyme Production on Industrial Scale 61
1.3.6.1 Homologous Protein Production 61
1.3.6.2 Heterologous Protein Production 62
References 63
1.4 Process Considerations for the Application of Enzymes 71
Selin Kara and Andreas Liese
1.4.1 Biocatalyst Types Used in Industrial Processes 71
1.4.2 Enzyme Immobilization for Biocatalytic Processes 74
1.4.3 Reaction Medium Applied in Enzymatic Catalysis 76
1.4.3.1 Monophasic Systems - Organic Media 77
1.4.3.2 Multiphasic Systems - Liquid/Liquid Mixtures 80
1.4.3.3 Multiphasic Systems - Gas/Liquid Mixtures 83
1.4.3.4 Multiphasic Systems - Solid/Liquid Mixtures 84
1.4.4 Appropriate Reactor Types in Enzyme Catalysis 87
1.4.5 Assessment Criteria for Enzymatic Applications 90
References 92
Part II Enzyme Applications for the Food Industry 95
2.1 Enzymes Used in Baking 97
Joke A. Putseys and Margot E.F. Schooneveld-Bergmans
2.1.1 Introduction 97
2.1.2 The Baking Process -The Baker’s Needs 98
2.1.2.1 Flour Quality and Standardization 98
2.1.2.2 Mixing and Dough Handling 100
2.1.2.3 Fermentation and Dough Stability 105
2.1.2.4 Baking and Oven Spring 109
2.1.3 The Bread Quality - The Consumers’ Needs 111
2.1.3.1 Color and Flavor 111
2.1.3.2 Shelf Life 112
2.1.4 Trends and Opportunities for Baking Enzymes 116
2.1.4.1 Fine Baking and Confectionary 116
2.1.4.2 Consumer Preference: Health, Individual Values, and Convenience 117
2.1.5 Conclusion 118
References 119
2.2 Protein Modification to Meet the Demands of the Food Industry 125
Andrew Ellis
2.2.1 Food Proteins 125
2.2.2 Processing of Food Protein 127
2.2.3 Enzymes in the Processing of Food Proteins 127
2.2.4 Food Protein Value Chain 130
2.2.5 Recent Enzyme Developments 131
2.2.5.1 Simple Protein Modification (Value Level 3) 131
2.2.5.1.1 Developing Microbial Alternatives to Plant and Animal Enzymes 131
2.2.5.2 Specialized Enzyme Modification (Value Level 4) 134
2.2.5.2.1 Whey Protein Hydrolysates 134
2.2.5.2.2 Plant Protein Hydrolysates 134
2.2.5.3 Highly Specific Protein Modification (Value Level 5) 135
2.2.5.3.1 Gluten Modification 135
2.2.5.3.2 Acrylamide Reduction 135
2.2.5.3.3 Bioactive Peptides 136
2.2.6 Enzymes to Meet Future Needs 137
Acknowledgments 139
References 139
2.3 Dairy Enzymes 143
Peter Dekker
2.3.1 Introduction 143
2.3.2 Coagulants 145
2.3.2.1 Traditional Rennets 147
2.3.2.2 Microbial Rennets 148
2.3.2.3 Fermentation Produced Chymosin 151
2.3.3 Ripening Enzymes 152
2.3.3.1 Proteases/Peptidases 153
2.3.3.2 Lipases/Esterases 154
2.3.4 Lactases 154
2.3.4.1 Neutral Lactase 156
2.3.4.2 Acid Lactase 158
2.3.4.3 GOS Production 158
2.3.5 Miscellaneous Enzymes 161
2.3.5.1 Oxidases/Peroxidases 161
2.3.5.2 Phopholipases 162
2.3.5.3 Cross-linking Enzymes 162
2.3.5.4 Preservation 163
2.3.6 New Developments 163
References 163
2.4 Enzymatic Process for the Synthesis of Cellobiose 167
Birgit Brucher and Thomas Häßler
2.4.1 Enzymatic Synthesis of Cellobiose 167
2.4.2 Cellobiose - Properties and Applications 168
2.4.3 Existing Routes for Cellobiose Synthesis 170
2.4.4 Enzyme Development 171
2.4.5 Process Development 173
2.4.5.1 Synthesis of Cellobiose 174
2.4.5.2 Purification of Cellobiose 174
2.4.6 Summary and Future Perspective 176
References 176
2.5 Emerging Field - Synthesis of Complex Carbohydrates. Case Study on HMOs 179
Dora Molnar-Gabor, Markus J. Hederos, Sebastian Bartsch, and Andreas Vogel
2.5.1 Introduction to Human Milk Oligosaccharides (HMOs) 179
2.5.1.1 Discovery and Function of HMOs 179
2.5.1.2 Structure of HMOs 180
2.5.1.3 HMO Production, Regulatory Authorizations, and Commercial Launch - Historical Overview 181
2.5.2 Glycom A/S Technologies Toward Commercial HMO Production 184
2.5.2.1 Whole Cell Microbial Fermentation to HMOs (In Vivo Process) 185
2.5.2.2 The Glycom In Vitro Concept to Diversify HMO Blends 187
2.5.2.3 Validation of the HMO Diversification Concept with Non-optimized Enzymes 187
2.5.3 Enzyme Development 189
2.5.3.1 Optimization of the α1-3/4 Transfucosidase 189
2.5.3.2 Optimization of the α2-6 Transsialidase 192
2.5.4 Applications of the Optimized Enzymes for the HMO Profiles 195
2.5.4.1 Scale-Up of the Lacto-N-fucopentaose III (LNFP-III), Sialyl Lacto-N-neotetraose (LST-c), and Sialyl Lacto-N-tetraose (LST-a) HMO Profiles 195
2.5.5 Conclusion and Perspective 197
References 198
Part III Enzyme Applications for Human and Animal Nutrition 203
3.1 Enzymes for Human Nutrition and Health 205
Yoshihiko Hirose
3.1.1 Introduction 205
3.1.2 Current Problems of Enzymes in Healthcare Business 205
3.1.3 Enzymes in Existing Healthcare Products 206
3.1.3.1 Digestive Enzymes 206
3.1.3.1.1 Digestive Enzymes in United States 206
3.1.3.1.2 Therapeutic Digestive Enzymes 207
3.1.3.2 Acid Lactase 207
3.1.3.3 α-Galactosidase (ADG) 208
3.1.3.4 Dextranase 208
3.1.3.5 Glucose Oxidase 208
3.1.3.6 Acetobacter Enzymes 210
3.1.3.7 Laccase (Polyphenol Oxidase) 210
3.1.4 New Enzyme Developments in Healthcare Products 211
3.1.4.1 Transglucosidase 211
3.1.4.2 Laccase 211
References 215
3.2 Enzyme Technology for Detoxification of Mycotoxins in Animal Feed 219
Dieter Moll
3.2.1 Introduction to Mycotoxins 219
3.2.2 Mycotoxin Mitigation Strategies 220
3.2.3 Enzyme Applications 224
3.2.4 FUMzyme® 225
3.2.4.1 The Substrate: Fumonisins 225
3.2.4.2 Enzyme Discovery 227
3.2.4.3 Enzyme Selection 230
3.2.4.4 Enzyme Activity Assays 232
3.2.4.5 Enzyme Characterization and Evaluation 233
3.2.4.6 Enzyme Feeding Trials and Biomarker Analysis 234
3.2.4.7 Enzyme Engineering 237
3.2.4.8 Enzyme Production 238
3.2.4.9 Enzyme Registration 239
3.2.5 Future Mycotoxinases 240
3.2.6 Conclusions 242
References 243
3.3 Phytases for Feed Applications 255
Nikolay Outchkourov and Spas Petkov
3.3.1 Phytase As a Feed Enzyme: Introduction and Significance 255
3.3.2 Historical Overview of the Phytase Market Development 256
3.3.3 From Phytate to Phosphorus: Step by Step Action of the Phytase 259
3.3.3.1 Properties of Phytate 259
3.3.3.2 Phytases Structural and Functional Classification 260
3.3.3.2.1 Phytases from the Histidine Acid Phosphatases (HAP) Superfamily 261
3.3.3.2.2 β-Propeller Phytase (BPP) 261
3.3.3.2.3 Cysteine Phytase (CPhy) 263
3.3.3.2.4 Purple Acid Phytases (PAPhy) 263
3.3.3.2.5 Classification of the Phytases Based on Phytate Dephosphorylation Steps 263
3.3.4 Nutritional Values of Phytase in Animal Feed 265
3.3.5 Phytase Application As Feed Additive 265
3.3.6 Effective Phytate Hydrolysis in the Upper Digestive Tract of the Animal 266
3.3.7 Kinetic Description of Ideal Phytases 269
3.3.8 Resistance to Low pH and Proteases 271
3.3.9 Temperature Stability 271
3.3.10 In lieu of Conclusion: Lessons from Phytase Super Dosing Trials 274
References 275
Part IV Enzymes for Biorefinery Applications 287
4.1 Enzymes for Pulp and Paper Applications 289
Debayan Ghosh, Bikas Saha, and Baljeet Singh
4.1.1 Refining and Fiber Development Enzyme 290
4.1.1.1 Microscopic Evaluation 291
4.1.1.2 Evaluation of Enzyme-Treated Handsheets 293
4.1.1.2.1 Case Study 1 293
4.1.1.2.2 Case Study 2 295
4.1.2 Drainage Improvement Enzyme 296
4.1.2.1 Case Study 3 299
4.1.2.2 Case Study 4 300
4.1.3 Stickies Control Enzyme 301
4.1.3.1 Case Study 5 303
4.1.4 Deinking Enzymes 306
4.1.4.1 Case Study 6 307
4.1.5 Hardwood Vessel Breaking Enzyme 308
4.1.5.1 Fiber Tester Image Analysis 308
4.1.6 Native Starch Conversion Enzyme 310
4.1.7 Bleach Boosting Enzyme 312
4.1.7.1 Common Bleaching Agents 312
4.1.7.1.1 Case Study 7 313
4.1.7.2 Overcoming Challenges Faced by Bleaching Enzymes in Pulp and Paper industry 315
4.1.8 Paper Mill Effluent Treatment Enzymes 315
4.1.8.1 Case Study 8 316
4.1.9 Slushing Enzyme 317
4.1.9.1 Case Study 9 317
4.1.9.2 Role of Enzymes in Pulp and Paper Industry - End Note! 318
References 319
4.2 Enzymes in Vegetable Oil Degumming Processes 323
Arjen Sein, Tim Hitchman, and Chris L.G. Dayton
4.2.1 Introduction 323
4.2.2 General Seed Oil Processes 324
4.2.2.1 Phospholipids 325
4.2.2.2 A Molecular View of the Degumming Process 327
4.2.3 Enzymatic Degumming 330
4.2.3.1 Phospholipase C 331
4.2.3.2 Ways to Cope with Poor Conversion/Poor Quality Oils in PLC-Based Processes 333
4.2.3.3 Phospholipase A 336
4.2.4 Enzymatic Degumming in Industrial Practice 337
4.2.4.1 Introduction Hurdles 341
4.2.5 Other Applications of Enzymes in Oil - Outlook 343
4.2.5.1 Enzymatic Interesterification of Triglyceride Oils 343
4.2.5.2 Biodiesel 344
4.2.5.3 Enzyme-Assisted Decoloring 344
4.2.5.4 Enzyme-Assisted Oil Extraction 344
4.2.6 Conclusion 345
Acknowledgments 345
References 345
Part V Enzymes used in Fine Chemical Production 351
5.1 KREDs: Toward Green, Cost-Effective, and Efficient Chiral Alcohol Generation 353
Chris Micklitsch, Da Duan, and Margie Borra-Garske
5.1.1 Introduction 353
5.1.2 Ketoreductases 355
5.1.3 Cofactor Recycling 356
5.1.4 CodeEvolver® Protein Engineering Technology 358
5.1.5 Reduction of a Wide Range of Ketones/Aldehydes 358
5.1.6 Critical Selectivity Tools for Enantiopure Asymmetric Carbonyl Reduction 364
5.1.7 Examples of Improved KREDs for Improved Manufacturing 369
5.1.8 KREDs: Going Green and Saving Green 373
References 377
5.2 An Aldolase for the Synthesis of the Statin Side Chain 385
Martin Schürmann
5.2.1 Introduction - Biocatalysis 385
5.2.1.1 Enzymes as Biocatalysts in Chemical Process 385
5.2.1.2 Biocatalytic Routes to the Statin Side Chain 387
5.2.2 The Aldolase DERA in Application 387
5.2.2.1 DERA-Catalyzed Aldol Reactions 387
5.2.2.2 Feasibility Phase of DERA-Enabled Statin Side Chain Process 390
5.2.3 Directed Evolution and Protein Engineering to Improve DERA 392
5.2.3.1 Rational Design 392
5.2.3.2 Directed Evolution of DERA 394
5.2.3.3 Other Approaches to Suitable or Improved DERAs 396
5.2.3.4 Other Applications of Process Intermediates and the DERA Technology 397
5.2.4 Conclusions 398
Acknowledgments 400
References 401
Index 405
