Practical Biotransformations: A Beginner's Guide Book

Foreword ix

Preface xi

1 Biotransformations, Microbes and Enzymes 1

1.1 Introduction 1

1.2 Biotransformations 8

1.3 Microorganisms 8

1.4 Organism Nomenclature 14

1.5 Enzymes 15

1.6 Types of Enzymatic Reactions 18

1.7 Enzymatic Cofactors 25

1.8 Some Basic Characteristics of Enzyme Catalysis 32

1.9 Types of Biocatalyst - Biotransformations by 'Whole Cells' or 'Isolated Enzymes' 38

1.10 Conclusion 40

References 41

2 An Overview of Biocatalyst Sources and Web-Based Information 43

2.1 Introduction 43

2.2 Microbial Culture Collections 44

2.3 Obtaining Organisms from Other Research Groups 47

2.4 Selective Enrichments 47

2.5 Metagenomics 48

2.6 Enzyme Suppliers and Biocatalyst Development Companies 50

2.7 Genome Mining for Biocatalysts 54

2.8 Obtaining Amino Acid and Gene Sequence Information on Biocatalysts 55

2.9 Obtaining DNA Templates for Cloning 66

2.10 Custom Gene Synthesis 67

2.11 Other Web Resources for Finding Information on Biocatalysts 68

2.12 Conclusion 69

References 70

3 Setting up a Laboratory for Biotransformations 71

3.1 Introduction 71

3.2 Microbiological Containment 72

3.3 On Containment Issues and Genetically Modified Organisms (GMOs) 75

3.4 Equipment for Handling Microorganisms 77

3.5 Techniques and Terms in Microbiology - Sterility, Asepsis and Aseptic Technique 88

3.6 Disposal of Viable Microbial Waste and Disinfection of Reusable Equipment 89

3.7 Equipment for Enzymology and Molecular Biology 91

3.8 General Reagents and Chemicals in a Biotransformations Laboratory 94

3.9 Conclusion 96

References 97

4 A Beginner's Guide to Preparative Whole-Cell Microbial Transformations 99

4.1 Introduction99

4.2 Storage, Maintenance and Growth of Microorganisms 99

4.3 General Microbiological Methods 103

4.4 Examples of Whole-Cell Biotransformations Using Bacteria 114

4.5 Biotransformation by Filamentous Fungi and Yeasts 125

4.6 Whole-Cell Biotransformation by Recombinant Strains of E. Coli 134

4.7 Conclusion 145

References 145

5 A Beginner's Guide to Biotransformations Catalysed by Commercially Available Isolated Enzymes 147

5.1 Introduction 147

5.2 Lipases 148

5.3 Hydrolytic Reactions Using Lipases 151

5.4 Using Lipases for Acylation Reactions 154

5.5 Other Hydrolases 161

5.6 Commercially Available Coenzyme-Dependent Enzymes 167

5.7 Carbon-Carbon Bond Forming Reactions 178

5.8 Conclusion 182

References 182

6 A Beginner's Guide to the Isolation, Analysis and Use of Home-Grown Enzyme Biocatalysts 185

6.1 Introduction 185

6.2 Cell Growth and Harvesting 186

6.3 Cell Disruption 186

6.4 A Typical Procedure for Making a Cell Extract from a Recombinant Strain of E. Coli 190

6.5 Purification of Enzymes - A Brief Guide 191

6.6 Techniques for Protein Purification 195

6.7 Isolation of Recombinant Enzymes Using Histidine Tags 201

6.8 Estimation of Protein Concentration 203

6.9 Concentrating Protein Samples by Centrifugation 206

6.10 Analysis of Protein Samples by Sodium Dodecylsulfate Polyacylamide Gel Electrophoresis 207

6.11 Examples of Enzyme Assays 212

6.12 Using Home-Grown Enzymes for Biotransformations - Some Recent Examples 224

6.13 Conclusion 232

References 233

7 An Introduction to Basic Gene Cloning for the Production of Designer Biocatalysts 235

7.1 Introduction 235

7.2 Background 238

7.3 Gene Amplification by Polymerase Chain Reaction (PCR) 249

7.4 DNA Fragment Analysis by Agarose Gel Electrophoresis 256

7.5 Gene Cloning 260

7.6 Analysis by DNA Sequencing 266

7.7 Troubleshooting the Gene Amplification and Cloning Process 267

7.8 Ligation-Independent Cloning 268

7.9 Gene Expression 270

7.10 Conclusion 276

References 277

8 Engineering Enzymes 279

8.1 Introduction 279

8.2 Site-Directed or Targeted Mutagenesis as a Tool for Investigating Enzyme Mechanism or Altering Catalytic Attributes 280

8.3 Engineering Using Random Mutagenesis Directed Evolution of Enzymes 291

8.4 Combining Rational and Random Mutagenesis for Biocatalyst Improvement 300

8.5 Exploiting Catalytic Promiscuity for Creating New Enzyme Activities 303

8.6 Designing Enzymes in Silico 305

8.7 Conclusion 305

References 305

Appendices 309

Index 323