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Biocatalysis for Practitioners
Biocatalysis for Practitioners

Techniques, Reactions and Applications

Edited by

Gonzalo de Gonzalo
Iván Lavandera

­
Editors All books published by WILEY‐VCH are carefully
produced. Nevertheless, authors, editors, and
Prof. Gonzalo de Gonzalo publisher do not warrant the information
Universidad de Sevilla contained in these books, including this book, to be
Dpto. de Química Orgánica free of errors. Readers are advised to keep in mind
c/ Profesor García González 2 that statements, data, illustrations, procedural
41012 Sevilla details or other items may inadvertently be
Spain inaccurate.

Prof. Iván Lavandera Library of Congress Card No.: applied for

Universidad de Oviedo
Dpto. de Química Orgánica e Inorgánica British Library Cataloguing‐in‐Publication Data
Avenida Julián Clavería 8 A catalogue record for this book is available from
33006 Oviedo the British Library.
Spain
Bibliographic information published by the
Deutsche Nationalbibliothek
The Deutsche Nationalbibliothek lists this
publication in the Deutsche Nationalbibliografie;
detailed bibliographic data are available on the
Internet at <http://dnb.d‐nb.de>.

© 2021 WILEY‐VCH, GmbH, Boschstr. 12, 69469

Weinheim, Germany

All rights reserved (including those of translation

into other languages). No part of this book may
be reproduced in any form – by photoprinting,
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or translated into a machine language without
written permission from the publishers. Registered
names, trademarks, etc. used in this book, even
when not specifically marked as such, are not to be
considered unprotected by law.

Print ISBN: 978‐3‐527‐34683‐7

ePDF ISBN: 978‐3‐527‐82444‐1
ePub ISBN: 978‐3‐527‐82445‐8
oBook ISBN: 978‐3‐527‐82446‐5

Cover Design Adam-Design, Weinheim, Germany

Typesetting SPi Global, Chennai, India

Printing and Binding

Printed on acid‐free paper

10 9 8 7 6 5 4 3 2 1
 v

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
vi Contents

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
 Contents vii

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
viii Contents

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
 Contents ix

6.9.4.2  symmetric Reductive Amination of Acetophenone (14) Using Amine

A
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 Value-

Added 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
x Contents

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
 Contents xi

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 CO2 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
xii Contents

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
 Contents xiii

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 One-

pot 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
xiv Contents

14.2.4  equential Chemoenzymatic One-Pot Synthesis Combining a Reaction

S
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
 Contents xv

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
 xvii

Foreword

The application of biocatalysis to chemical processes has had an exponential growth in the
past 20 years. Biotransformations provide the central tools in industrial biotechnology
because they address the need for processes with less environmental impact in terms of
energy, raw materials, and waste production. Over the past few years, the use of enzymes
as biocatalysts for the introduction of enantiopure active compounds has become an estab-
lished manufacturing process in the specialty and pharmaceutical industry. It has demon-
strated that synthetic and computational tools can be exploited to generate new biocatalysts
with novel structure and chemical properties. The employment of biocatalysts is attractive
for synthetic organic chemists for producing optically active molecules.
In the present book “Biocatalysis for Practitioners,” the authors have attempted to cover
some of the most challenging areas in practical enzymatic catalysis through 16 interesting
chapters by different specialists of recognized prestige in their field. In my opinion, it is
important to recognize the work that the editors have carried out to cover in five sections
the most current trends in the field of biocatalysis, which makes this work very useful not
only for researchers in this field but also for students who are beginning to understand
biocatalysis, so it can be a good textbook for some careers.
The first section consists of four chapters where different techniques to improve and
discover new biocatalysts are described. Thus, purification, modification, immobilization,
and compartmentalization techniques are treated in each of the chapters with great suc-
cess, updating the reader on the progress of the different methodologies.
The second section also consists of four chapters of varied themes whose general title is
Enzymes Handling and Applications. The fifth chapter tries to explain formal aspects of the
so‐called catalytic promiscuity, where processes that in principle were difficult to imagine
can be nowadays catalyzed by different biocatalysts. Enzymes applied to the synthesis of
amines, some applications of oxidoreductases, and the use of glycosyltransferases for the
preparation of glycosides are the other chapters of this part.
Recently, many efforts are aimed to optimize the different biocatalytic processes by modi-
fying the reaction medium or improving the recycling of the cofactor. Thus, in Section 3,
entitled Ways to Improve Enzymatic Transformations, three very interesting reviews are
described on topics of great importance, such as Application of nonaqueous media in bio-
catalysis, non‐conventional cofactor regeneration systems, and Biocatalysis under continu-
ous flow conditions.
Exploring the Variety of Random
Documents with Different Content
insulted her and is expected to pay damages. If a man meets his
mother-in-law coming along the road and does not recognise her,
she will fall down on the ground as a sign, when he will run away. In
the same way a father-in-law will signal to his daughter-in-law; the
whole idea being that they are unworthy to be noticed till they have
proved that they can beget children.”79.2 However, if a wife should
prove barren for three years, the rules of avoidance between the
young couple and their parents-in-law cease to be observed.79.3
Hence the custom of avoidance among these people is associated in
some way with the wife’s fertility. So among the Awemba, a Bantu
tribe of Northern Rhodesia, “if a young man sees his mother-in-law
coming along the path, he must retreat into the bush and make way
for her, or if she suddenly comes upon him he must keep his eyes
fixed on the ground, and only after a child is born may they converse
together.”79.4 Among the Angoni, another Bantu tribe of British
Central Africa, it would be a gross breach of etiquette if a man were
to enter his son-in-law’s house; he may come within ten paces of the
door, but no nearer. A woman may not even approach her son-in-
law’s house, and she is never allowed to speak to him. Should they
meet accidentally on a path, the son-in-law gives way and makes a
circuit to avoid encountering his mother-in-law face to face.79.5 Here
then we see that a man avoids his son-in-law as well as his mother-
in-law, though not so strictly.
Among the Thonga, a Bantu tribe about
The custom of Delagoa Bay, when a man meets his mother-in-
avoiding mother-in-
law and wife of law or her sister on the road, he steps out of the
wife’s brother road into the forest on the right hand side and sits
among the Thonga down. She does the same. Then they salute each
of Delagoa Bay.
other in the usual way by clapping their hands.
After that they may talk to each other. When a man is in a hut, his
mother-in-law dare not enter it, but must sit down outside without
seeing him. So seated she may salute him, “Good morning, son of
So-and-so.” But she would not dare to pronounce his name.
However, when a man has been married many years, his mother-in-
law has less fear of him, and will even enter the hut where he is and
speak to him. But among the Thonga the woman whom a man is
bound by custom to avoid most rigidly is not his wife’s mother, but
the wife of his wife’s brother. If the two meet on a path, they carefully
avoid each other; he will step out of the way and she will hurry on,
while her companions, if she has any, will stop and chat with him.
She will not enter the same boat with him, if she can help it, to cross
a river. She will not eat out of the same dish. If he speaks to her, it is
with constraint and embarrassment. He will not enter her hut, but will
crouch at the door and address her in a voice trembling with
emotion. Should there be no one else to bring him food, she will do it
reluctantly, watching his hut and putting the food inside the door
when he is absent. It is not that they dislike each other, but that they
feel a mutual, a mysterious fear.80.1 However, among the Thonga,
the rules of avoidance between connexions by marriage decrease in
severity as time passes. The strained relations between a man and
his wife’s mother in particular become easier. He begins to call her
“Mother” and she calls him “Son.” This change even goes so far that
in some cases the man may go and dwell in the village of his wife’s
parents, especially if he has children and the children are grown
up.80.2 Again, among the Ovambo, a Bantu people of German South-
West Africa, a man may not look at his future mother-in-law while he
talks with her, but is bound to keep his eyes steadily fixed on the
ground. In some cases the avoidance is even more stringent; if the
two meet unexpectedly, they separate at once. But after the
marriage has been celebrated, the social intercourse between
mother-in-law and son-in-law becomes easier on both sides.81.1
Thus far our examples of ceremonial avoidance
The custom of between mother-in-law and son-in-law have been
avoiding the
mother-in-law drawn from Bantu tribes. But in Africa the custom,
among other than though apparently most prevalent and most
the Bantu tribes of strongly marked among peoples of the great Bantu
Africa.
stock, is not confined to them. Among the Masai of
British East Africa, “mothers-in-law and their sons-in-law must avoid
one another as much as possible; and if a son-in-law enters his
mother-in-law’s hut she must retire into the inner compartment and
sit on the bed, whilst he remains in the outer compartment; they may
then talk. Own brothers-in-law and sisters-in-law must also avoid one
another, though this rule does not apply to half-brothers-in-law and
sisters-in-law.”81.2 So, too, among the Bogos, a tribe on the outskirts
of Abyssinia, a man never sees the face of his mother-in-law and
never pronounces her name; the two take care not to meet.81.3
Among the Donaglas a husband after marriage “lives in his wife’s
house for a year, without being allowed to see his mother-in-law, with
whom he enters into relations only on the birth of his first son.”81.4 In
Darfur, when a youth has been betrothed to a girl, however intimate
he may have been with her parents before, he ceases to see them
until the ceremony has taken place, and even avoids them in the
street. They, on their part, hide their faces, if they happen to meet
him unexpectedly.81.5
To pass now from Africa to other parts of the
The custom of world, among the Looboos, a primitive tribe in the
avoiding relations
by marriage in tropical forests of Sumatra, custom forbids a
Sumatra and New woman to be in her father-in-law’s company and a
Guinea. man to be in his mother-in-law’s society. For
example, if a man meets his daughter-in-law, he
should cross over to the other side of the road to let her pass as far
as possible from him; but if the way is too narrow, he takes care in
time to get out of it. But no such reserve is prescribed between a
father-in-law and his son-in-law, or between a mother-in-law and her
daughter-in-law.82.1 Among the Bukaua, a Melanesian tribe of
German New Guinea, the rules of avoidance between persons
connected by marriage are very stringent; they may not touch each
other or mention each other’s names. But contrary to the usual
practice the avoidance seems to be quite as strict between persons
of the same sex as between males and females. At least the writer
who reports the custom illustrates it chiefly by the etiquette which is
observed between a man and his daughter’s husband. When a man
eats in presence of his son-in-law, he veils his face; but if
nevertheless his son-in-law should see his open mouth, the father-in-
law is so ashamed that he runs away into the wood. If he gives his
son-in-law anything, such as betel or tobacco, he will never put it in
his hand, but pours it on a leaf, and the son-in-law fetches it away. If
father-in-law and son-in-law both take part in a wild boar hunt, the
son-in-law will abstain from seizing or binding the boar, lest he
should chance to touch his father-in-law. If, however, through any
accident their hands or backs should come into contact, the father-
in-law is extremely horrified, and a dog must be at once killed, which
he gives to his son-in-law for the purpose of wiping out the stain on
his honour. If the two should ever fall out about anything, the son-in-
law will leave the village and his wife, and will stay away in some
other place till his father-in-law, for his daughter’s sake, calls him
back. A man in like manner will never touch his sister-in-law.82.2
Among the low savages of the Californian
The custom of peninsula a man was not allowed for some time to
avoiding relations
by marriage among look into the face of his mother-in-law or of his
the Indian tribes of wife’s other near relations; when these women
America. were present he had to step aside or hide
himself.83.1 Among the Indians of the Isla del
Malhado in Florida a father-in-law and mother-in-law might not enter
the house of their son-in-law, and he on his side might not appear
before his father-in-law and his relations. If they met by accident they
had to go apart to the distance of a bowshot, holding their heads
down and their eyes turned to the earth. But a woman was free to
converse with the father and mother of her husband.83.2 Among the
Indians of Yucatan, if a betrothed man saw his future father-in-law or
mother-in-law at a distance, he turned away as quickly as possible,
believing that a meeting with them would prevent him from begetting
children.83.3 Among the Arawaks of British Guiana a man may never
see the face of his wife’s mother. If she is in the house with him, they
must be separated by a screen or partition-wall; if she travels with
him in a canoe, she steps in first, in order that she may turn her back
to him.83.4 Among the Caribs “the women never quit their father’s
house, and in that they have an advantage over their husbands in as
much as they may talk to all sorts of people, whereas the husband
dare not converse with his wife’s relations, unless he is dispensed
from this observance either by their tender age or by their
intoxication. They shun meeting them and make great circuits for
that purpose. If they are surprised in a place where they cannot help
meeting, the person addressed turns his face another way so as not
to be obliged to see the person, whose voice he is compelled to
hear.”83.5 Among the Araucanian Indians of Chili a man’s mother-in-
law refuses to speak to or even to look at him during the marriage
festivity, and “the point of honour is, in some instances, carried so
far, that for years after the marriage the mother never addresses her
son-in-law face to face; though with her back turned, or with the
interposition of a fence or a partition, she will converse with him
freely.”84.1
It would be easy to multiply examples of similar
The custom of customs of avoidance between persons closely
avoiding relations
by marriage cannot connected by marriage, but the foregoing may
be separated from serve as specimens. Now in order to determine
the similar custom the meaning of such customs it is very important
of avoiding relations
by blood; both are to observe that similar customs of avoidance are
probably practised in some tribes not merely between
precautions to
prevent improper
persons connected with each other by marriage,
relations between but also between the nearest blood relations of
the sexes. different sexes, namely, between parents and
children and between brothers and sisters;84.2 and
the customs are so alike that it seems difficult or impossible to
separate them and to offer one explanation of the avoidance of
connexions by marriage and another different explanation of the
avoidance of blood relations. Yet this is what is done by some who
attempt to explain the customs of avoidance; or rather they confine
their attention wholly to connexions by marriage, or even to mothers-
in-law alone, while they completely ignore blood relations, although
in point of fact it is the avoidance of blood relations which seems to
furnish the key to the problem of such avoidances in general. The
true explanation of all such customs of avoidance appears to be, as I
have already indicated, that they are precautions designed to
remove the temptation to sexual intercourse between persons whose
marriage union is for any reason repugnant to the moral sense of the
community. This explanation, while it has been rejected by theorists
at home, has been adopted by some of the best observers of savage
life, whose opinion is entitled to carry the greatest weight.85.1
That a fear of improper intimacy even between
Mutual avoidance of the nearest blood relations is not baseless among
mother and son, of
father and daughter, races of a lower culture seems proved by the
and of brother and testimony of a Dutch missionary in regard to the
Battas or Bataks of Sumatra, a people who have
sister among the attained to a fairly high degree of barbaric
Battas.
civilization. The Battas “observe certain rules of
avoidance in regard to near relations by blood or marriage; and we
are informed that such avoidance springs not from the strictness but
from the looseness of their moral practice. A Batta, it is said,
assumes that a solitary meeting of a man with a woman leads to an
improper intimacy between them. But at the same time he believes
that incest or the sexual intercourse of near relations excites the
anger of the gods and entails calamities of all sorts. Hence near
relations are obliged to avoid each other lest they should succumb to
temptation. A Batta, for example, would think it shocking were a
brother to escort his sister to an evening party. Even in the presence
of others a Batta brother and sister feel embarrassed. If one of them
comes into the house, the other will go away. Further, a man may
never be alone in the house with his daughter, nor a mother with her
son. A man may never speak to his mother-in-law nor a woman to
her father-in-law. The Dutch missionary who reports these customs
adds that he is sorry to say that from what he knows of the Battas he
believes the maintenance of most of these rules to be very
necessary. For the same reason, he tells us, as soon as Batta lads
have reached the age of puberty they are no longer allowed to sleep
in the family house but are sent away to pass the night in a separate
building (djambon); and similarly as soon as a man loses his wife by
death he is excluded from the house.”85.2
In like manner among the Melanesians of the
Mutual avoidance of Banks’ Islands and the New Hebrides a man must
mother and son and
of brother and sister not only avoid his mother-in-law; from the time
among the when he reaches or approaches puberty and has
Melanesians. begun to wear clothes instead of running about
naked, he must avoid his mother and sisters, and
he may no longer live in the same house with them; he takes up his
quarters in the clubhouse of the unmarried males, where he now
regularly eats and sleeps. He may go to his father’s house to ask for
food, but if his sister is within he must go away before he eats; if she
is not there, he may sit down near the door and eat. If by chance
brother and sister meet in the path, she runs away or hides. If a boy,
walking on the sands, perceives footprints which he knows to be
those of his sister, he will not follow them, nor will she follow his. This
mutual avoidance lasts through life. Not only must he avoid the
persons of his sisters, but he may not pronounce their names or
even use a common word which happens to form part of any one of
their names. In like manner his sisters eschew the use of his name
and of all words which form part of it. Strict, too, is a boy’s reserve
towards his mother from the time when he begins to wear clothes,
and the reserve increases as he grows to manhood. It is greater on
her side than on his. He may go to the house and ask for food and
his mother may bring it out for him, but she will not give it to him; she
puts it down for him to take. If she calls to him to come, she speaks
to him in the plural, in a more distant manner; “Come ye,” she says,
not “Come thou.” If they talk together she sits at a little distance and
turns away, for she is shy of her grown-up son. “The meaning of all
this,” as Dr. Codrington observes, “is obvious.”86.1
Mutual avoidance of
a man and his
When a Melanesian man of the Banks’ Islands
mother-in-law marries, he is bound in like manner to avoid his
among the mother-in-law. “The rules of avoidance are very
Melanesians.
strict and minute. As regards the avoidance of the
person, a man will not come near his wife’s mother; the avoidance is
mutual; if the two chance to meet in a path, the woman will step out
of it and stand with her back turned till he has gone by, or perhaps if
it be more convenient he will move out of the way. At Vanua Lava, in
Port Patteson, a man would not follow his mother-in-law along the
beach, nor she him, until the tide had washed out the footsteps of
the first traveller from the sand. At the same time a man and his
mother-in-law will talk at a distance.”87.1
It seems obvious that these Melanesian
It is significant that customs of avoidance are the same, and must be
mutual avoidance
between blood explained in the same way whether the woman
relations of opposite whom a man shuns is his wife’s mother or his own
sexes begins at or mother or his sister. Now it is highly significant that
near puberty.
just as among the Akamba of East Africa the
mutual avoidance of father and daughter only begins when the girl
has reached puberty, so among the Melanesians the mutual
avoidance of a boy on the one side and of his mother and sisters on
the other only begins when the boy has reached or approached
puberty. Thus in both peoples the avoidance between the nearest
blood relations only commences at the dangerous age when sexual
connexion on both sides begins to be possible. It seems difficult,
therefore, to evade the conclusion that the mutual avoidance is
adopted for no other reason than to diminish as far as possible the
chances of sexual unions which public opinion condemns as
incestuous. But if that is the reason why a young Melanesian boy, on
the verge of puberty, avoids his own mother and sisters, it is natural
and almost necessary to infer that it is the same reason which leads
him, as a full-grown and married man, to eschew the company of his
wife’s mother.
Similar customs of avoidance between mothers
Mutual avoidance of and sons, between fathers and daughters, and
mother and son, of
father and daughter, between brothers and sisters are observed by the
and of brother and natives of the Caroline Islands, and the writer who
sister in the records them assigns the fear of incest as the
Caroline Islands.
motive for their observance. “The prohibition of
marriage,” he says, “and of sexual intercourse between kinsfolk of
the same tribe is regarded by the Central Caroline natives as a
divine ordinance; its breach is therefore, in their opinion, punished by
the higher powers with sickness or death. The law influences in a
characteristic way the whole social life of the islanders, for efforts are
made to keep members of families of different sexes apart from each
other even in their youth. Unmarried men and boys, from the time
when they begin to speak, may therefore not remain by night in the
huts, but must sleep in the fel, the assembly-house. In the evening
their meal (âkot) is brought thither to them by their mothers or
sisters. Only when a son is sick may his mother receive him in the
hut and tend him there. On the other hand entrance to the assembly-
house (fel) is forbidden to women and girls except on the occasion of
the pwarik festival; whereas female members of other tribes are free
to visit it, although, so far as I could observe, they seldom make use
of the permission. Unmarried girls sleep in the huts with their
parents.
“These restrictions, which custom and tradition have instituted
within the family, find expression also in the behaviour of the
members of families toward each other. The following persons,
namely, have to be treated with respect—the daughters by their
father, the sons by their mother, the brothers by their sisters. In
presence of such relations, as in the presence of a chief, you may
not stand, but must sit down; if you are obliged on narrow paths to
pass by one of them you must first obtain permission and then do it
in a stooping or creeping posture. You allow them everywhere to go
in front; you also avoid to drink out of the vessel which they have just
used; you do not touch them, but keep always at a certain distance
from them; the head especially is deemed sacred.”88.1
In all these cases the custom of mutual
Mutual avoidance of avoidance is observed by persons of opposite sex
male and female
cousins in some who, though physically capable of sexual union,
tribes. are forbidden by tradition and public opinion to
have any such commerce with each other. Thus
far the blood relations whom a man is forbidden to marry and
compelled to avoid, are his own mother, his own daughter, and his
own sisters. But to this list some people add a man’s female cousins
or at least certain of them; for many races draw a sharp line of
distinction between cousins according as they are children of two
brothers or of two sisters or of a brother and a sister, and while they
permit or even prefer marriage with certain cousins, they absolutely
forbid marriage with certain others. Now, it is highly significant that
some tribes which forbid a man to marry certain of his cousins also
compel him to adopt towards them the same attitude of social
reserve which in the same or other tribes a man is obliged to
observe towards his wife’s mother, his own mother, and his own
sisters, all of whom in like manner he is forbidden to marry. Thus
among the tribes in the central part of New Ireland
Mutual avoidance of (New Mecklenburg) a male and a female cousin,
male and female
cousins in New the children of a brother and a sister respectively,
Ireland. are most strictly forbidden by custom to marry
each other; indeed this prohibition is described as
the most stringent of all; the usual saying in regard to such relations
is, “The cousin is holy” (i tábu ra kókup). Now, in these tribes a man
is not merely forbidden to marry his female cousin, the daughter of
his father’s sister or of his mother’s brother; he must also avoid her
socially, just as in other tribes a man must avoid his wife’s mother,
his own mother, his own daughter, and his own sisters. The cousins
may not approach each other, they may not shake hands or even
touch each other, they may not give each other presents, they may
not mention each other’s names; but they are allowed to speak to
each other at a distance of some paces. These rules of avoidance,
these social barriers erected between cousins, the children of a
brother and a sister respectively, are interpreted most naturally and
simply as precautions intended to obviate the danger of a criminal
intercourse between persons whose sexual union would be regarded
by public opinion with deep displeasure. Indeed the Catholic
missionary, to whom we are indebted for the information, assumes
this interpretation of the rules as if it were too obvious to call for
serious discussion. He says that all the customs of avoidance “are
observed as outward symbols of this prohibition of marriage”; and he
adds that “were the outward sign of the prohibition of marriage, to
which the natives cleave with genuine obstinacy, abolished or even
weakened, there would be an immediate danger of the natives
contracting such marriages.”90.1 It seems difficult for a rational man
to draw any other inference. If any confirmation were needed, it
would be furnished by the fact that among these tribes of New
Ireland brothers and sisters are obliged to observe precisely the
same rules of mutual avoidance, and that incest between brother
and sister is a crime which is punished with hanging; they may not
come near each other, they may not shake hands, they may not
touch each other, they may not give each other presents; but they
are allowed to speak to each other at a distance of some paces. And
the penalty for incest with a daughter is also death by hanging.90.2
Amongst the Baganda of Central Africa in like
Mutual avoidance of manner a man was forbidden under pain of death
certain male and
female cousins to marry or have sexual intercourse with his
among the cousin, the daughter either of his father’s sister or
Baganda; marriage of his mother’s brother; and such cousins might
or sexual
intercourse not approach each other, nor hand each other
forbidden between anything, nor enter the same house, nor eat out of
these cousins under
pain of death.
the same dish. Were cousins to break these rules
of social avoidance, in other words, if they were to
approach each other or hand each other anything, it was believed
that they would fall ill, that their hands would tremble, and that they
would be unfit for any work.90.3 Here, again, the prohibition of social
intercourse was in all probability merely a precaution against sexual
intercourse, for which the penalty was death. And the same may be
said of the similar custom of avoidance which among these same
Baganda a man had to observe towards his wife’s mother. “No man
might see his mother-in-law, or speak face to face with her; she
covered her face, if she passed her son-in-law, and he gave her the
path and made a detour, if he saw her coming. If she was in the
house, he might not enter, but he was allowed to speak to her from a
distance. This was said to be because he had seen her daughter’s
nakedness. If a son-in-law accidentally saw his mother-in-law’s
breasts, he sent her a barkcloth in compensation, to cover herself,
lest some illness, such as tremor, should come upon him. The
punishment for incest was death; no member of a clan would shield
a person guilty thereof; the offender was disowned by the clan, tried
by the chief of the district, and put to death.”91.1
The prohibition of marriage with certain cousins
Marriage between appears to be widespread among African peoples
certain cousins
forbidden among of the Bantu stock. Thus in regard to the Bantus of
some South African South Africa we read that “every man of a coast
tribes but allowed tribe regarded himself as the protector of those
among others.
females whom we would call his cousins, second
cousins, third cousins, and so forth, on the father’s side, while some
had a similar feeling towards the same relatives on the mother’s side
as well, and classified them all as sisters. Immorality with one of
them would have been considered incestuous, something horrible,
something unutterably disgraceful. Of old it was punished by the
death of the male, and even now a heavy fine is inflicted upon him,
while the guilt of the female must be atoned by a sacrifice performed
with due ceremony by the tribal priest, or it is believed a curse will
rest upon her and her issue.… In contrast to this prohibition the
native of the interior almost as a rule married the daughter of his
father’s brother, in order, as he said, to keep property from being lost
to his family. This custom more than anything else created a disgust
and contempt for them by the people of the coast, who term such
intermarriages the union of dogs, and attribute to them the insanity
and idiocy which in recent times has become prevalent among the
inland tribes.”91.2
Among the Thonga, a Bantu tribe about
Marriage between Delagoa Bay, marriages between cousins are as a
cousins allowed in
some African tribes rule prohibited, and it is believed that such unions
on condition that an are unfruitful. However, custom permits cousins to
expiatory sacrifice marry each other on condition that they perform an
is offered.
expiatory ceremony which is supposed to avert the
curse of barrenness from the wife. A goat is sacrificed, and the
couple are anointed with the green liquid extracted from the half-
digested grass in the animal’s stomach. Then a hole is cut in the
goat’s skin and through this hole the heads of the cousins are
inserted. The goat’s liver is then handed to them, quite raw, through
the hole in the skin, and they must tear it out with their teeth without
using a knife. Having torn it out, they eat it. The word for liver
(shibindji) also means “patience,” “determination.” So they say to the
couple, “You have acted with strong determination. Eat the liver now!
Eat it in the full light of the day, not in the dark! It will be an offering to
the gods.” Then the family priest prays, saying: “You, our gods, So-
and-so, look! We have done it in the daylight. It has not been done
by stealth. Bless them, give them children!” When he has done
praying, the assistants take all the half-digested grass from the
goat’s stomach and place it on the wife’s head, saying, “Go and bear
children!”92.1 Among the Wagogo of German East Africa marriage is
forbidden between cousins who are the children of two brothers or of
two sisters, but is permitted between cousins who are the children of
a brother and sister respectively. However, in this case it is usual for
the wife’s father to kill a sheep and put on a leather armlet, made
presumably from the sheep’s skin; otherwise it is supposed that the
marriage would be unfruitful.92.2 Thus the Wagogo, like the Thonga,
imagine that the marriage of cousins is doomed to infertility unless
an expiatory sacrifice is offered and a peculiar use made of the
victim’s skin. Again, the Akikuyu of British East Africa forbid the
marriage of cousins and second cousins, the children and
grandchildren of brothers and sisters. If such persons married, they
would commit a grave sin, and all their children would surely die; for
the curse or ceremonial pollution (thahu) incurred by such a crime
cannot be purged away. Nevertheless it sometimes happens that a
man unwittingly marries a first or second cousin; for instance, if a
part of the family moves away to another district, it may come about
that a man makes the acquaintance of a girl and marries her before
he discovers the relationship. In such a case, where the sin has
been committed unknowingly, the curse can be averted by the
performance of an expiatory rite. The elders take a sheep and place
it on the woman’s shoulders; there it is killed and the intestines taken
out. Then the elders solemnly sever the intestines with a sharp
splinter of wood taken from a bush of a certain sort (mukeo), “and
they announce that they are cutting the clan kutinyarurira, by which
they mean that they are severing the bond of relationship which
exists between the pair. A medicine man then comes and purifies the
couple.”93.1 In all these cases we may assume with a fair degree of
probability that the old prohibition of marriage between cousins is
breaking down, and that the expiatory sacrifice offered when such a
marriage does take place is merely a salve to the uneasy conscience
of those who commit or connive at a breach of the ancient taboo.
Thus the prohibition of marriage between
The mutual cousins, and the rules of ceremonial avoidance
avoidance of male
and female cousins observed in some tribes between persons who
is probably a stand in that relationship to each other, appear
precaution against both to spring from a belief, right or wrong, in the
a criminal intimacy
between them. injurious effects of such unions and from a desire
to avoid them. The mutual avoidance of the
cousins is merely a precaution to prevent a closer and more criminal
intimacy between them. If that is so, it furnishes a confirmation of the
view that all the customs of ceremonial avoidance between blood
relations or connexions by marriage of opposite sexes are based
simply on a fear of incest.
 The theory is perhaps confirmed by the
The mutual observation that in some tribes the avoidance
avoidance between
a man and his between a man and his wife’s mother lasts only
wife’s relations until he has had a child by his wife;94.1 while in
seems to be partly
grounded on a fear others, though avoidance continues longer, it
of rendering the gradually wears away with time as the man and
wife infertile.
woman advance in years,94.2 and in others, again,
it is observed only between a man and his future mother-in-law, and
comes to an end with his marriage.94.3 These customs suggest that
in the minds of the people who practise them there is a close
connexion between the avoidance of the wife’s relations and the
dread of an infertile marriage. The Indians of Yucatan, as we saw,
believe that if a betrothed man were to meet his future mother-in-law
or father-in-law, he would thereby lose the power of begetting
children. Such a fear seems to be only an extension by false analogy
of that belief in the disastrous consequences of illicit sexual relations
which we dealt with in an earlier part of this chapter,94.4 and of which
we shall have more to say presently.94.5 From thinking, rightly or
wrongly, that sexual intercourse between certain persons is fraught
with serious dangers, the savage jumped to the conclusion that
social intercourse between them may be also perilous by virtue of a
sort of physical infection acting through simple contact or even at a
distance; or if, in many cases, he did not go so far as to suppose that
for a man merely to see or touch his mother-in-law sufficed to blast
the fertility of his wife’s womb, yet he may have thought, with much
better reason, that intimate social converse between him and her
might easily lead to something worse, and that to guard against such
a possibility it was best to raise a strong barrier of etiquette between
them. It is not, of course, to be supposed that these rules of
avoidance were the result of deliberate legislation; rather they were
the spontaneous and gradual growth of feelings and thoughts of
which the savages themselves perhaps had no clear consciousness.
In what precedes I have merely attempted to sum up in language
intelligible to civilized man the outcome of a long course of moral and
social evolution.
 These considerations perhaps obviate to some extent the only
serious difficulty which lies in the way of the theory here advocated.
If the custom of avoidance was adopted in order to
The mutual guard against the danger of incest, how comes it
avoidance between
persons of the that the custom is often observed towards persons
same sex was of the same sex, for example, by a man towards
probably an his father-in-law as well as towards his mother-in-
extension by false
analogy of the law? The difficulty is undoubtedly serious: the only
mutual avoidance way of meeting it that I can suggest is the one I
between persons of
different sexes.
have already indicated. We may suppose that the
deeply rooted beliefs of the savage in the fatal
effects of marriage between certain classes of persons, whether
relations by blood or connexions by marriage, gradually spread in his
mind so as to embrace the relations between men and men as well
as between men and women; till he had worked himself into the
conviction that to see or touch his father-in-law, for example, was
nearly or quite as dangerous as to touch or have improper relations
with his mother-in-law. It is no doubt easy for us to detect the flaw in
this process of reasoning; but we should beware of casting stones at
the illogical savage, for it is possible or even probable that many of
our own cherished convictions are no better founded.
Viewed from this standpoint the customs of
The custom of ceremonial avoidance among savages assume a
mutual avoidance
between near serious aspect very different from the appearance
relations has of arbitrariness and absurdity which they are apt to
probably had the present to the civilized observer who does not look
effect of checking
the practice of below the surface of savage society. So far as
inbreeding. these customs have helped, as they probably
have done, to suppress the tendency to
inbreeding, that is, to the marriage of near relations, we must
conclude that their effect has been salutary, if, as many eminent
biologists hold, long-continued inbreeding is injurious to the stock,
whether animal or vegetable, by rendering it in the end infertile.95.1
However, men of science are as yet by no means agreed as to the
results of consanguineous marriages, and a living authority on the
subject has recently closed a review of the evidence as follows:
“When we take into account such evidence as there is from animals
and plants, and such studies as those of Huth,95.2 and the instances
and counter-instances of communities with a high degree of
consanguinity, we are led to the conclusion that the prejudices and
laws of many peoples against the marriage of near kin rest on a
basis not so much biological as social.”96.1 Whatever may be the
ultimate verdict of science on this disputed question, it will not affect
the result of the present enquiry, which merely affirms the deep and
far-reaching influence which in the long course of human history
superstition has exercised on morality. Whether the influence has on
the whole been for good or evil does not concern us. It suffices for
our purpose to shew that superstition has been a crutch to morality,
whether to support it in the fair way of virtue or to precipitate it into
the miry pit of vice. To return to the point from which we wandered
into this digression, we must leave in suspense the question whether
the Australian savages were wise or foolish who forbade a man
under pain of death to speak to his mother-in-law.
I will conclude this part of my subject with a few
Other examples of more instances of the extreme severity with which
the severe
punishment of certain races have visited what they deemed
sexual crime. improper connexions between the sexes.
Among the Indians who inhabited the coast of
The Indians of Brazil near Rio de Janeiro about the middle of the
Brazil. sixteenth century, a married woman who gave
birth to an illegitimate child was either killed or
abandoned to the caprice of the young men who could not afford to
keep a wife. Her child was buried alive; for they said that were he to
grow up he would only serve to perpetuate his mother’s disgrace; he
would not be allowed to go to war with the rest for fear of the
misfortunes and disasters he might draw down upon them, and no
one would eat any food, whether venison, fish, or what not, which
the miserable outcast had touched.96.2 In Ruanda,
The natives of
Ruanda.
a district of Central Africa, down to recent years
any unmarried woman who was got with child
used to be put to death with her baby, whether born or unborn. A
spot at the mouth of the Akanyaru river was the place of execution,
where the guilty women and their innocent offspring were hurled into
the water. As usual, this Puritanical strictness of morality has been
relaxed under European influence; illegitimate children are still killed,
but their mothers escape with the fine of a cow.97.1
The Saxons.
Among the Saxons down to the days of St.
Boniface the adulteress or the maiden who had dishonoured her
father’s house was compelled to hang herself, was burned, and her
paramour hung over the blazing pile; or she was scourged or cut to
pieces with knives by all the women of the village till she was
dead.97.2 Among the Slav peoples of the Balkan
The Southern
Slavs.
peninsula women convicted of immoral conduct
used to be stoned to death. About the year 1770 a
young betrothed couple were thus executed near Cattaro in
Dalmatia, because the girl was found to be with child. The youth
offered to marry her, and the priest begged that the sentence of
death might be commuted to perpetual banishment; but the people
declared that they would not have a bastard born among them; and
the two fathers of the luckless couple threw the first stones at them.
When Miss M. Edith Durham related this case to some Montenegrin
peasantry, they all said that in the old days stoning was the proper
punishment for unchaste women; the male paramours were shot by
the relations of the girls whom they had seduced. When “that
modern Messalina,” Queen Draga of Servia, was murdered, a
decent peasant woman remarked that “she ought to be under the
cursed stone heap” (pod prokletu gomilu). The country-folk of
Montenegro, who heard the news of the murder from Miss Durham,
“looked on it as a cleansing—a casting out of abominations—and
genuinely believed that Europe would commend the deed, and that
the removal of this sinful woman would bring prosperity to the
land.”97.3 Even down to the second half of the nineteenth century in
cases of seduction among the Southern Slavs the people proposed
to stone both the culprits to death.98.1 This happened, for example, in
Herzegovina in the year 1859, when a young man named Milutin
seduced or (to be more exact) was seduced by three unmarried girls
and got them all with child. The people sat in judgment upon the
sinners, and, though an elder proposed to stone them all, the court
passed a milder sentence. The young man was to marry one of the
girls, to rear the infants of the other two as his legitimate children,
and next time there was a fight with the Turks he was to prove his
manhood by rushing unarmed upon the enemy and wresting their
weapons from them, alive or dead. The sentence was fulfilled to the
letter, though many years passed before the culprit could carry out
the last part of it. However, his time came in 1875, when
Herzegovina revolted against the Turks. Then Milutin ran unarmed
upon a regiment of the enemy and found among the Turkish
bayonets a hero’s death.98.2 Even now the Old Catholics among the
South Slavs believe that a village in which a seducer is not
compelled to marry his victim will be punished with hail and
excessive rain. For this article of faith, however, they are ridiculed by
their enlightened Catholic neighbours, who hold the far more
probable view that thunder and lightning are caused by the village
priest to revenge himself for unreasonable delays in the payment of
his salary. A heavy hail-storm has been known to prove almost fatal
to the local incumbent, who was beaten within an inch of his life by
his enraged parishioners.98.3
It is difficult to believe that in these and similar
Inference from the cases the community would inflict such severe
severe punishments
inflicted for sexual punishment for sexual offences if it did not believe
offences. that its own safety, and not merely the interest of a
few individuals, was imperilled thereby.
If now we ask why illicit relations between the
Why should illicit sexes should be supposed to disturb the balance
relations between
the sexes be of nature and particularly to blast the fruits of the
thought to disturb earth, a partial answer may be conjecturally
the balance of suggested. It is not enough to say that such
nature?
relations are displeasing to the gods, who punish
indiscriminately the whole community for the sins of a few. For we
must always bear in mind that the gods are creations of man’s fancy;
he fashions them in human likeness, and endows them with tastes
and opinions which are merely vast cloudy projections of his own. To
affirm, therefore, that something is a sin because the gods will it so,
is only to push the enquiry one stage farther back and to raise the
further question, Why are the gods supposed to dislike and punish
these particular acts? In the case with which we
The reason why the are here concerned, the reason why so many
gods of savages
are supposed to savage gods prohibit adultery, fornication, and
punish sexual incest under pain of their severe displeasure may
crimes so severely
may perhaps be perhaps be found in the analogy which many
found in a mistaken savage men trace between the reproduction of the
belief that human species and the reproduction of animals
irregularities of the
human sexes and plants. The analogy is not purely fanciful, on
prevent the the contrary it is real and vital; but primitive
reproduction of peoples have given it a false extension in a vain
edible animals and
plants and thereby attempt to apply it practically to increasing the food
supply. They have imagined, in fact, that by
strike a fatal blow at
the food supply. performing or abstaining from certain sexual acts
they thereby directly promoted the reproduction of
animals and the multiplication of plants.99.1 All such acts and
abstinences, it is obvious, are purely superstitious and wholly fail to
effect the desired result. They are not religious but magical; that is,
they compass their end, not by an appeal to the gods, but by
manipulating natural forces in accordance with certain false ideas of
physical causation. In the present case the principle on which
savages seek to propagate animals and plants is that of magical
sympathy or imitation: they fancy that they assist the reproductive
process in nature by mimicking or performing it among themselves.
Now in the evolution of society such efforts to control the course of
nature directly by means of magical rites appear to have preceded
the efforts to control it indirectly by appealing to the vanity and
cupidity, the good-nature and pity of the gods; in short, magic seems
to be older than religion.100.1 In most races, it is true, the epoch of
unadulterated magic, of magic untinged by religion, belongs to such
a remote past that its existence, like that of our ape-like ancestors,
can be a matter of inference only; almost everywhere in history and
the world we find magic and religion side by side, at one time allies,
at another enemies, now playing into each other’s hands, now
cursing, objurgating, and vainly attempting to exterminate one
another. On the whole the lower intelligences cling closely, though
secretly, to magic, while the higher intelligences have discerned the
vanity of its pretensions and turned to religion instead. The result has
been that beliefs and rites which were purely magical in origin often
contract in course of time a religious character; they are modified in
accordance with the advance of thought, they are translated into
terms of gods and spirits, whether good and beneficent, or evil and
malignant. We may surmise, though we cannot prove, that a change
of this sort has come over the minds of many races with regard to
sexual morality. At some former time, perhaps, straining a real
analogy too far, they believed that those relations of the human
sexes which for any reason they regarded as right and natural had a
tendency to promote sympathetically the propagation of animals and
plants and thereby to ensure a supply of food for the community;
while on the contrary they may have imagined that those relations of
the human sexes which for any reason they deemed wrong and
unnatural had a tendency to thwart and impede the propagation of
animals and plants and thereby to diminish the common supply of
food.
Such a belief, it is obvious, would furnish a
Such a belief would sufficient motive for the strict prohibition of what
account both for the
horror with which were deemed improper relations between men
many savages and women; and it would explain the deep horror
regard such crimes, and detestation with which sexual irregularities are
and for the severity
with which they viewed by many, though certainly not by all,
punish them. savage tribes. For if improper relations between
the human sexes prevent animals and plants from
multiplying, they strike a fatal blow at the existence of the tribe by
cutting off its supply of food at the roots. No wonder, therefore, that
wherever such superstitions have prevailed the whole community,
believing its very existence to be put in jeopardy by sexual
immorality, should turn savagely on the culprits, and beat, burn,
drown or otherwise exterminate them in order to rid itself of so
dangerous a pollution. And when with the advance of knowledge
men began to perceive the mistake they had made in imagining that
the commerce of the human sexes could affect the propagation of
animals and plants, they would still through long habit be so inured
to the idea of the wickedness of certain sexual relations that they
could not dismiss it from their minds, even when they discerned the
fallacious nature of the reasoning by which they had arrived at it. The
old practice would therefore stand, though the old theory had fallen:
the old rules of sexual morality would continue to be observed, but if
they were to retain the respect of the community, it was necessary to
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