Table of Contents
Foreword xvii
Part I Enzyme Techniques 1
1 Techniques for Enzyme Purification 3
Adrie H. Westphal and Willem J. H. van Berkel
1.1 Introduction 3
1.2 Traditional Enzyme Purification 4
1.2.1 Ion Exchange Chromatography 7
1.2.2 Gel Filtration 9
1.2.3 Bio-affinity Chromatography 11
1.2.4 Hydrophobic Interaction Chromatography 14
1.2.5 Hydroxyapatite Chromatography 15
1.3 Example of a Traditional Enzyme Purification Protocol 17
1.4 Purification of Recombinant Enzymes 18
1.4.1 Immobilized Metal Affinity Chromatography 18
1.4.2 Affinity Chromatography with Protein Tags 20
1.5 Column Materials 22
1.6 Conclusions 24
References 25
2 Enzyme Modification 33
Antonino Biundo, Patricia Saénz-Méndez, and Tamas Görbe
2.1 Introduction 33
2.2 Practical Approach: Experimental Information, Analytical Methods, Tips and Tricks, and Examples 34
2.2.1 Directed Evolution 34
2.2.1.1 (Ultra)High-Throughput Screening and Selection 35
2.2.1.2 Applications of Directed Evolution Methodology 36
2.2.2 Semi-rational Design 37
2.2.2.1 Applications of Semi-rational Design Methodology 38
2.2.3 De Novo Enzyme Design 39
2.2.3.1 Applications of De Novo Enzyme Design Methodology 40
2.2.4 Rational Enzyme Design 40
2.2.4.1 Applications of Rational Design Methodology 41
2.3 Expectations and Perspectives 49
2.4 Concluding Remarks 50
References 51
3 Immobilization Techniques for the Preparation of Supported Biocatalysts: Making Better Biocatalysts Through Protein Immobilization 63
Javier Rocha-Martín, Lorena Betancor, and Fernando López-Gallego
3.1 Introduction 63
3.2 General Aspects to Optimize Enzyme Immobilization Protocols 64
3.2.1 Carrier Nature 64
3.2.2 Immobilization Chemistry 64
3.2.3 Protein Orientation 64
3.2.4 Multivalence of the Protein Attachment 65
3.2.5 Chemical and Geometrical Congruence 65
3.2.6 Enzyme Spatial Organization 65
3.3 Type of Carriers for Immobilized Proteins 66
3.3.1 Types of Materials 66
3.3.1.1 Organic Materials 66
3.3.1.2 Inorganic Materials 66
3.3.2 Geometry 67
3.3.2.1 Beads 67
3.3.2.2 Monoliths 67
3.3.2.3 Membranes 67
3.3.3 Dimensions 67
3.3.4 Commercially Available Porous Carriers for Enzyme Immobilization 68
3.4 Immobilization Methods and Manners 68
3.5 Evaluation of the Enzyme Immobilization Process 70
3.5.1 Considerations Before Immobilization 71
3.5.1.1 Preparation of the Enzymatic Solution to Be Immobilized 71
3.5.1.2 Stability of the Soluble Enzyme Under Immobilization Conditions 71
3.5.2 Parameters Required to Define an Immobilization Process 71
3.5.2.1 Immobilization Yield 72
3.5.2.2 Expressed Activity or Apparent Activity 72
3.5.2.3 Specific Activity of the Immobilized Biocatalyst 73
3.6 Applied Examples of Immobilized Enzymes 73
3.6.1 Characterization of the Immobilized Biocatalyst 74
3.6.1.1 Determination of the Catalytic Activity of the Final Immobilized Biocatalyst and Maximum Protein Loading Capacity 74
3.6.1.2 Apparent Kinetic Parameters of the Immobilized Enzyme 76
3.6.1.3 Biocatalyst Stability 77
3.6.1.3.1 The Half-life Time of Biocatalysts 78
3.7 Challenges and Opportunities in Enzyme Immobilization 79
3.8 Conclusions 81
List of Abbreviations 82
References 82
4 Compartmentalization in Biocatalysis 89
Robert Kourist and Javier González-Sabín
4.1 Introduction 89
4.2 Cell as a Compartment 93
4.3 Compartmentalization Using Protein Assemblies 95
4.4 Compartmentalization Using Emulsion and Micellar Systems 96
4.5 Compartmentalization Using Encapsulation 100
4.6 Compartmentalization Using Tea Bags and Thimbles 103
4.7 Separation of Reaction Steps Using Continuous Flow 105
4.8 Conclusions and Prospects 107
References 108
Part II Enzymes Handling and Applications 113
5 Promiscuous Activity of Hydrolases 115
Erika V. M. Orozco and André L. M. Porto
5.1 Introduction 115
5.2 Catalytic Promiscuity 116
5.3 Hydrolases 117
5.3.1 Applications of Hydrolases to Organic Synthesis 118
5.3.2 Lipases and Their Hydrolysis Mechanism 122
5.3.3 Catalytic Promiscuity of Hydrolases 122
5.3.4 Promiscuous Aldol Reaction Catalyzed by Hydrolases 130
5.3.5 Aldol Reaction Between 4-Cyanobenzaldehyde and Cyclohexanone Catalyzed by Porcine Pancreatic Lipase (PPL-II) and Rhizopus niveus Lipase (RNL) 135
5.4 Conclusions 136
References 137
6 Enzymes Applied to the Synthesis of Amines 143
Francesco G. Mutti and Tanja Knaus
6.1 Introduction 143
6.2 Hydrolases 145
6.2.1 Practical Approaches with Hydrolases 145
6.2.1.1 Kinetic Resolution 145
6.2.1.2 Dynamic Kinetic Resolution 146
6.2.2 Practical Examples with Hydrolases 148
6.2.2.1 Kinetic Resolution of Racemic α-Methylbenzylamine Through the Methoxyacetylation Catalyzed by a Lipase 148
6.2.2.2 Dynamic Kinetic Resolution for the Synthesis of Norsertraline 149
6.3 Amine Oxidases 149
6.3.1 Practical Approaches with Amine Oxidases 150
6.3.1.1 Kinetic Resolution and Deracemization 150
6.3.2 Practical Examples with Amine Oxidases 151
6.3.2.1 One-pot, One-enzyme Oxidative Pictet-Spengler Approach Combined with Deracemization 151
6.3.2.2 Desymmetrization of meso-compounds 152
6.4 Transaminases (or Aminotransferases) 152
6.4.1 Practical Approaches with Transaminases 153
6.4.2 Practical Examples with Transaminases 153
6.4.2.1 Kinetic Resolution and Deracemization 153
6.4.2.2 Asymmetric Synthesis from Prochiral Ketone 155
6.5 Amine Dehydrogenases, Imine Reductases, and Reductive Aminases 155
6.5.1 Practical Approaches with Amine Dehydrogenases, Imine Reductases, and Reductive Aminases 156
6.5.2 Practical Examples with Amine Dehydrogenases, Imine Reductases, and Reductive Aminases 160
6.5.2.1 IRed-Catalyzed Reductive Amination of an Aldehyde Combined with KR of a Racemic Amine 160
6.5.2.2 Asymmetric Reductive Amination Catalyzed by AmDH 162
6.6 Ammonia Lyases 162
6.6.1 Practical Approaches with Ammonia Lyases 163
6.6.1.1 Aspartase, 3-Methylaspartate Ammonia Lyase, and Related Enzymes 163
6.6.1.2 Aromatic Amino Acid Ammonia Lyases and Mutases 165
6.6.2 Practical Examples with Ammonia Lyases 166
6.6.2.1 Chemoenzymatic Synthesis of (S)-2-Indolinecarboxylic Acid 166
6.6.2.2 Synthesis of L-Aspartate from Fumarate 166
6.6.2.3 Enzymatic and Chemoenzymatic Synthesis of Toxin A and Aspergillomarasmine A and B 166
6.7 Pictet-Spenglerases 167
6.7.1 Practical Approaches with Pictet-Spenglerases 167
6.7.2 Practical Examples with Pictet-Spenglerases 169
6.7.2.1 Biocatalytic Synthesis of (R)-Harmicine 169
6.7.2.2 Biocatalytic Synthesis of (S)-Trolline and Analogs 169
6.8 Engineered Cytochrome P450s (Cytochrome “P411”) 169
6.8.1 Practical Approaches with Engineered Cytochrome P450s 170
6.9 Protocols for Selected Reactions 171
6.9.1 Hydrolases 171
6.9.1.1 Kinetic Resolution rac-Methylbenzylamine (rac-1) 171
6.9.1.2 Dynamic Kinetic Resolution of Norsertraline Intermediate (rac-3) 171
6.9.2 Monoamine Oxidases 172
6.9.2.1 Chemoenzymatic Deracemization of Harmicine (rac-8) 172
6.9.3 ω-Transaminases 172
6.9.3.1 Deracemization of Mexiletine (rac-9, Kinetic Resolution, Followed by Formal Reductive Amination) 172
6.9.4 Imine Reductases and Amine Dehydrogenases 172
6.9.4.1 Reductive Amination of Aldehyde (11) with Kinetic Resolution of Amine Nucleophile (rac-trans-12) 172
6.9.4.2 Asymmetric Reductive Amination of Acetophenone (14) Using Amine Dehydrogenase 173
6.9.5 Ammonia Lyases 173
6.9.5.1 Asymmetric Ammonia Addition to 2′-Chlorocinnamic Acid (17) 173
6.9.6 Pictet-Spenglerases 173
6.9.6.1 Asymmetric Pictet-Spengler Reaction with Strictosidine Synthase 173
6.9.7 Engineered Cytochrome P450s 174
6.9.7.1 Intermolecular Alkane C-H Amination Using Cytochrome P411 174
6.10 Conclusions 174
Acknowledgments 175
References 175
7 Applications of Oxidoreductases in Synthesis: A Roadmap to Access ValueAdded Products 181
Mélanie Hall
7.1 Introduction 181
7.2 Reductive Processes 184
7.2.1 Reduction of C═O Bonds 184
7.2.1.1 Selection of Alcohol Dehydrogenase (ADH) for Stereoselective Reduction Reactions 185
7.2.1.1.1 Absolute Configuration of the Product 185
7.2.1.1.2 Substrate Type 186
7.2.1.1.3 Thermostability 187
7.2.1.1.4 Cofactor Preference 187
7.2.1.1.5 Kits 187
7.2.1.2 Practical Approach 187
7.2.1.2.1 Montelukast 188
7.2.1.2.2 Atorvastatin 189
7.2.1.2.3 Dynamic Kinetic Resolutions 189
7.2.1.2.4 Disproportionation 190
7.2.1.2.5 Redox Isomerization 190
7.2.2 Reduction of C═C Bonds 191
7.2.2.1 Mechanism 191
7.2.2.2 Enzymes and Substrates 193
7.2.2.2.1 Enzymes 193
7.2.2.2.2 Substrates 193
7.2.2.3 Practical Approach 196
7.2.2.3.1 Stereocontrol 196
7.2.2.3.2 (Dynamic) Kinetic Resolution 197
7.3 Oxidative Processes 198
7.3.1 Oxygenations 198
7.3.1.1 Baeyer-Villiger Oxidations 198
7.3.1.1.1 Regiopreference 200
7.3.1.1.2 Stereoselectivity 201
7.3.1.1.3 Practical Approach 203
7.3.1.2 Epoxidation of Alkenes 204
7.3.2 Heteroatom Oxidation 206
7.3.2.1 Reaction 206
7.3.2.2 Substrates 207
7.3.3 Peroxygenases: One Catalyst - Many Reactions 207
7.4 Protocols for Selected Reactions Employing Oxidoreductases 209
7.4.1 Alcohol Dehydrogenase (ADH): Disproportionation of rac-2-Phenylpropanal 209
7.4.1.1 Biotransformation 209
7.4.1.2 Product Recovery and Purification 210
7.4.2 Ene-reductase/Old Yellow Enzyme (OYE): Dynamic Kinetic Resolution of a γ-substituted Lactone 210
7.4.2.1 Biotransformation 210
7.4.2.2 Product Recovery and Purification 210
7.4.3 Baeyer-Villiger Monooxygenase (BVMO): Kinetic Resolution of a Racemic Ketone 210
7.4.3.1 Biotransformation 211
7.4.3.2 Product Recovery and Purification 211
7.4.4 Baeyer-Villiger Monooxygenase (BVMO): Asymmetric Sulfoxidation 211
7.4.4.1 Biotransformation 211
7.4.4.2 Product Recovery and Purification 211
7.5 Conclusions 211
Acknowledgments 212
References 212
8 Glycosyltransferase Cascades Made Fit For the Biocatalytic Production of Natural Product Glycosides 225
Bernd Nidetzky
8.1 Introduction: Glycosylated Natural Products and Leloir Glycosyltransferases 225
8.2 Glycosylated Flavonoids and Nothofagin 227
8.3 Glycosyltransferase Cascades for Biocatalytic Synthesis of Nothofagin 229
8.4 Enzyme Expression 230
8.5 Solvent Engineering for Substrate Solubilization 232
8.6 Nothofagin Production at 100 g Scale 233
8.7 Concluding Remarks 237
References 237
Part III Ways to Improve Enzymatic Transformations 245
9 Application of Nonaqueous Media in Biocatalysis 247
Afifa A. Koesoema and Tomoko Matsuda
9.1 Introduction 247
9.2 Advantages and Disadvantages of Reactions in Nonaqueous Media 248
9.3 Nonaqueous Media Used for Biocatalysis 248
9.4 Enzymatic Activity and Inactivation in Nonaqueous Media 251
9.4.1 Enzymatic Activity in Nonaqueous Media 251
9.4.2 Factors Causing Inactivation of Enzymes in Nonaqueous Media 252
9.5 Practical Approaches to Stabilize Enzymes in Nonaqueous Media 252
9.5.1 Utilization of Nonaqueous Media-Tolerant Enzymes or Host Cells 252
9.5.2 Enzyme Immobilization 253
9.5.3 Modification of the Enzyme Preparation 254
9.5.4 Protein Engineering 255
9.6 Examples of Biocatalyzed Reactions in Solvent-Free Systems 256
9.7 Examples of Reactions in Micro-aqueous Systems 258
9.8 Examples of Reactions in Bio-Based Liquids 260
9.8.1 2-Methyltetrahydrofuran (MeTHF) 260
9.8.2 Cyclopentyl Methyl Ether (CPME) 261
9.8.3 Potential Application of other Bio-based Liquids 262
9.9 Examples of Reactions in Liquid CO+ 262
9.10 Examples of Reactions in CO2-Expanded Bio-based Liquids 264
9.11 Examples of Reactions in Natural Deep Eutectic Solvents 265
9.12 Conclusions and Future Perspectives 267
References 267
10 Nonconventional Cofactor Regeneration Systems 275
Jiafu Shi, Yizhou Wu, Zhongyi Jiang, Yiying Sun, Qian Huo, Weiran Li, Yang Zhao, and Yuqing Cheng
10.1 Introduction 275
10.2 Basics of Photocatalytic NADH Regeneration 279
10.2.1 Processes and Mechanism Associated with Photocatalytic NADH Regeneration 279
10.2.2 Aspects of Measuring Photocatalytic NADH Regeneration 281
10.3 Advancements in Photocatalytic NADH Regeneration 282
10.3.1 Nature Photosensitizers 282
10.3.2 Organic Molecular Photosensitizers 282
10.3.3 Inorganic Semiconductors 285
10.3.4 Organic Semiconductors 288
10.4 Expectations 290
10.5 Conclusions and Prospects 292
10.5.1 Conclusions 292
10.5.2 Prospects 292
List of Abbreviations 292
References 293
11 Biocatalysis Under Continuous Flow Conditions 297
Bruna Goes Palma, Marcelo A. do Nascimento, Raquel A. C. Leão, Omar G. Pandoli, and Rodrigo O. M. A. de Souza
11.1 Introduction 297
11.2 Practical Approach for Biocatalysis Under Continuous Flow Conditions 299
11.2.1 Esterification 299
11.2.1.1 Experimental Procedure 301
11.2.2 Transesterification 302
11.2.2.1 Experimental Procedure 303
11.2.3 Kinetic Resolutions 303
11.2.3.1 Kinetic Resolution of Amines Employing Lipases 304
11.2.3.1.1 Experimental Procedure 304
11.2.3.2 Kinetic Resolutions Employing ω-Transaminases 305
11.2.3.2.1 Experimental Procedure 305
11.2.3.3 Kinetic Resolution of Alcohols Using Lipases 307
11.2.3.3.1 Experimental Procedure 307
11.2.4 Dynamic Kinetic Resolutions 308
11.2.4.1 Experimental Procedure 309
11.2.5 Asymmetric Synthesis 309
11.2.5.1 Experimental Procedure 311
11.2.5.1.1 Protein Immobilization 311
11.2.5.1.2 Ion Exchange of NADPH on Ag-DEAE 311
11.2.5.1.3 General Procedure for the Continuous Asymmetric Reduction 311
11.3 Conclusions and Perspective 311
References 312
Part IV Recent Trends in Enzyme-Catalyzed Reactions 317
12 Photobiocatalysis 319
Martín G. López-Vidal, Guillermo Gamboa, Gabriela Oksdath-Mansilla, and Fabricio R. Bisogno
12.1 Introduction 319
12.2 Oxidative Processes 321
12.2.1 Baeyer-Villiger Oxidation 321
12.2.2 Alkane Hydroxylation 322
12.2.3 O-Dealkylation 326
12.2.4 Decarboxylation 327
12.2.4.1 Alkene Production 327
12.2.4.2 Alkane Production 328
12.2.5 Epoxidation 330
12.3 Reductive Processes 332
12.3.1 Carbonyl Reduction 332
12.3.2 Olefin Reduction 336
12.3.3 Imine Reduction 342
12.3.4 Reductive Amination 344
12.3.5 Dehalogenation 345
12.3.6 Deacetoxylation 347
12.4 Combination of Photooxidation and Enzymatic Transformation 348
12.5 Summary and Outlook 352
Abbreviations 352
References 354
13 Practical Multienzymatic Transformations: Combining Enzymes for the Onepot Synthesis of Organic Molecules in a Straightforward Manner 361
Jesús Albarrán-Velo, Sergio González-Granda, Marina López-Agudo, and Vicente Gotor-Fernández
13.1 Introduction 361
13.2 Non-stereoselective Bienzymatic Transformations 363
13.2.1 Amine Synthesis 363
13.2.2 Bienzymatic Linear Cascades Toward the Production of Other Organic Compounds 365
13.3 Stereoselective Bienzymatic Transformations 367
13.3.1 Stereoselective Amine Synthesis Through Concurrent Processes 368
13.3.1.1 Amination of Alcohols 368
13.3.1.2 Deracemization of Amines 371
13.3.1.3 Amino Alcohol Synthesis 372
13.3.1.4 Other Bienzymatic Stereoselective Synthesis of Amines 374
13.3.2 Stereoselective Bienzymatic Cascades Toward the Production of Other Organic Compounds 377
13.3.2.1 Synthesis of Organic Compounds Other Than Amino Acids 377
13.3.2.2 Amino Acid Synthesis 383
13.4 Multienzymatic Transformations: Increasing Synthetic Complexity 386
13.5 Summary and Outlook 395
References 395
14 Chemoenzymatic Sequential One-Pot Protocols 403
Harald Gröger
14.1 Introduction: Theoretical Information and Conceptual Overview 403
14.2 State of the Art in Sequential Chemoenzymatic One-Pot Synthesis: Selected Examples and Historical Overview About Selected Contributions 406
14.2.1 Sequential Chemoenzymatic One-Pot Synthesis Combining a Metal-Catalyzed Reaction with a Biotransformation 406
14.2.2 Sequential Chemoenzymatic One-Pot Synthesis Combining an Organocatalytic Reaction with a Biotransformation 411
14.2.3 Sequential Chemoenzymatic One-Pot Synthesis Combining a Reaction Catalyzed by a Heterogeneous Chemocatalyst with a Biotransformation 416
14.2.4 Sequential Chemoenzymatic One-Pot Synthesis Combining a Reaction Catalyzed by a Heterogeneous Biocatalyst with a Chemocatalytic Transformation 417
14.2.5 Sequential Chemoenzymatic One-Pot Synthesis Combining More than Two Reactions 418
14.3 Practical Aspects of the Development of Sequential Chemoenzymatic One-Pot Syntheses 420
14.4 Conclusions and Outlook 423
References 424
Part V Industrial Biocatalysis 427
15 Industrial Processes Using Biocatalysts 429
Florian Kleinbeck, Marek Mahut, and Thierry Schlama
15.1 Introduction 429
15.2 Biocatalysis in the Pharmaceutical Industry 430
15.2.1 Pregabalin 431
15.2.2 Vernakalant 432
15.2.3 Sitagliptin 433
15.2.4 Esomeprazole 435
15.2.5 Montelukast 436
15.2.6 Boceprevir 439
15.3 Aspects to Consider for Development of a Biocatalytic Process on Commercial Scale - A Case Study 442
15.3.1 Identification of a Suitable Enzyme 443
15.3.2 Process Development 443
15.3.3 Control Strategy and Regulatory Considerations 445
15.3.3.1 Impurities 446
15.3.3.2 Types of Biocatalysts 450
15.3.3.3 Type of Expression System 451
15.3.3.4 Route of Administration 451
15.3.3.5 Position of the Biocatalytic Step in the Synthesis and Downstream Transformations 451
15.3.3.6 Summary of the Case Study 452
15.3.4 Health, Process Safety and Environmental Aspects 453
15.3.4.1 Health 453
15.3.4.2 Process Safety 453
15.3.4.3 Environmental Aspects 454
15.3.5 Equipment Utilization and Throughput Time 455
15.3.6 Equipment Cleaning 455
15.3.7 Enzyme Release Testing 456
15.3.8 Transport and Storage 457
15.4 Conclusions, Expectations, and Prospects 458
Acknowledgments 460
List of Abbreviations 460
References 461
16 Enzymatic Commercial Sources 467
Gonzalo de Gonzalo and Iván Lavandera
16.1 Introduction 467
16.2 European Companies 468
16.2.1 AB Enzymes 468
16.2.2 Almac 468
16.2.3 Biocatalysts 469
16.2.4 c-Lecta GmbH 469
16.2.5 Enzymicals 470
16.2.6 Evoxx Technologies GmbH 470
16.2.7 GECCO 471
16.2.8 Inofea AG 472
16.2.9 Johnson-Matthey 472
16.2.10 Metgen Oy 473
16.2.11 Novozymes 474
16.2.12 Prozomix 474
16.2.13 Royal DSM 475
16.3 American Companies 475
16.3.1 Codexis Inc. 475
16.3.2 Dupont Nutrition and Biosciences 476
16.3.3 IBEX Technologies 476
16.3.4 MP Biomedical 477
16.3.5 Sigma-Aldrich 477
16.3.6 Strem Chemicals, Inc. 478
16.3.7 Worthington Biochemical Corp 479
16.4 Asian Enzyme Suppliers 480
16.4.1 Advanced Enzymes Technologies, Ltd. 480
16.4.2 Amano Enzyme Co., Ltd. 480
16.4.3 Aumgene Biosciences 481
16.4.4 EnzymeWorks 481
16.4.5 Meito Sangyo Co., Ltd. 481
16.4.6 Oriental Yeast Co., Ltd. 482
16.4.7 Takabio 482
16.4.8 Toyobo Co., Ltd. 482
16.5 Outlook 483
References 484
Index 487
