Practical Biotransformations: A Beginner's Guide

✍ Scribed by Gideon Grogan

• Publisher: Wiley-Blackwell
• Year: 2009
• Tongue: English
• Leaves: 353
• Series: Postgraduate Chemistry
• Edition: 1
• Category: Library

✦ Synopsis

Content: Chapter 1: Biotransformations, Microbes and Enzymes. 1.1 Introduction. 1.2 Biotransformations. 1.3 Microorganisms. 1.4 Organism nomenclature. 1.5 Enzymes. 1.6 Types of Enzymatic reactions. 1.7 Enzymatic Cofactors. 1.8 Some Basic Characteristics of Enzyme Catalysis. 1.9 Types of Biocatalyst - Biotransformations by 'whole cells' or isolated enzymes. 1.10. Conclusion. Chapter 2: An overview of biocatalyst sources and web-based information. 2.1 Introduction. 2.2 Microbial culture collections. 2.3 Obtaining organisms from other research groups. 2.4 Selective Enrichments. 2.5 Metagenomics. 2.6 Enzyme Suppliers and Biocatalyst Development Companies. 2.7 Genome mining for biocatalysts. 2.8 Obtaining amino acid and gene sequence information on biocatalysts. 2.9 Obtaining DNA templates for cloning. 2.10 Custom Gene Synthesis. 2.11 Other interesting web resources for biocatalysis. 2.12 Conclusion. Chapter 3: Setting up a laboratory for biotransformations. 3.1 Introduction. 3.2 Microbiological Containment. 3.3 On containment issues and genetically-modified organisms. 3.4 Equipment for handling microorganisms. 3.5 Techniques and terms in microbiology - Sterility, Asepsis and Aseptic Technique. 3.6 Disposal of viable microbial waste and disinfection of reusable equipment. 3.7 Equipment for enzymology and molecular biology. 3.8 General reagents and chemicals in a Biotransformations Laboratory. 3.9 Conclusion. Chapter 4: A beginner's guide to preparative whole-cell microbial biotransformations. 4.1 Introduction. 4.2 Storage, maintenance and growth of microorganisms. 4.3 General Microbiological Methods. 4.4 Examples of Whole-cell Biotransformations using Bacteria. 4.5 Biotransformation by filamentous fungi and yeasts. 4.6 Whole-cell Biotransformations by recombinant strains of E. coli. 4.7 Conclusion. Chapter 5: A beginner's guide to biotransformations by commercially available isolated enzymes. 5.1 Introduction. 5.2 Lipases. 5.3 Hydrolytic Reactions using lipases. 5.4 Using lipases for acylation reactions. 5.5 Other hydrolases. 5.6 Commercially available Coenzyme-dependent Enzymes. 5.7 Carbon-carbon bond forming reactions. 5.8 Conclusion. Chapter 6: A beginner's guide to the isolation and analysis and use of home-grown enzyme biocatalysts. 6.1 Introduction. 6.2 Cell growth and harvesting. 6.3 Cell disruption. 6.4 A typical procedure for making a cell extract from a recombinant strain of E. coli. 6.5 Purification of enzymes - a brief guide. 6.6 Techniques for Protein Purification. 6.7 Isolation of recombinant enzymes using histidine tags. 6.8 Estimation of protein concentration. 6.9 Concentrating protein samples by centrifugation. 6.10 Analysis of protein samples by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). 6.11 Examples of enzyme assays. 6.12 Using home-grown enzymes for biotransformations ? Some recent examples. 6.13 Conclusion. Chapter 7: An introduction to basic gene cloning for the production of designer biocatalysts. 7.1 Introduction. 7.2 Background to gene cloning. 7.3 Gene amplification by polymerase chain reaction (PCR). 7.4 DNA fragment analysis by agarose electrophoresis. 7.5 Gene cloning. 7.6 Analysis by DNA sequencing. 7.7 Troubleshooting the gene amplification and cloning process. 7.8 Ligation-Independent Cloning. 7.9 Gene Expression in E. coli. 7.10 Conclusion. Chapter 8: Engineering Enzymes. 8.1 Introduction. 8.2 Site-directed or targted mutagenesis as a tool for investigating enzyme mechanism or altering catalytic attributes. 8.3 A site-directed mutagenesis experiment. Considerations and practise. 8.4 Engineering using random mutagenesis. Directed Evolution of Enzymes. 8.5 Combining rational and random mutagenesis for biocatalyst improvement. 8.6 Exploiting catalytic promiscuity for creating new enzyme activities. 8.7 Designing enzymes in silico. 8.8 Conclusion. Appendices. 1. Structures of the proteinogenic amino acids. 2. Structures of bases found in nucleic acids. 3. The Genetic Code. 4. Recipes for Microbiological Growth Media. 5. Biological buffers. 6. Ammonium sulphate fractionation table. 7. Restriction enzymes and restriction sites

✦ Table of Contents

Practical Biotransformations......Page 3
Contents......Page 7
Foreword......Page 11
Preface......Page 13
1.1 Introduction......Page 15
1.3 Microorganisms......Page 22
1.4 Organism Nomenclature......Page 28
1.5 Enzymes......Page 29
1.6 Types of Enzymatic Reactions......Page 32
1.7 Enzymatic Cofactors......Page 39
1.8 Some Basic Characteristics of Enzyme Catalysis......Page 46
1.9 Types of Biocatalyst - Biotransformations by ‘Whole Cells’ or ‘Isolated Enzymes’......Page 52
1.10 Conclusion......Page 54
References......Page 55
2.1 Introduction......Page 57
2.2 Microbial Culture Collections......Page 58
2.4 Selective Enrichments......Page 61
2.5 Metagenomics......Page 62
2.6 Enzyme Suppliers and Biocatalyst Development Companies......Page 64
2.7 Genome Mining for Biocatalysts......Page 68
2.8 Obtaining Amino Acid and Gene Sequence Information on Biocatalysts......Page 69
2.9 Obtaining DNA Templates for Cloning......Page 80
2.10 Custom Gene Synthesis......Page 81
2.11 Other Web Resources for Finding Information on Biocatalysts......Page 82
2.12 Conclusion......Page 83
References......Page 84
3.1 Introduction......Page 85
3.2 Microbiological Containment......Page 86
3.3 On Containment Issues and Genetically Modified Organisms (GMOs)......Page 89
3.4 Equipment for Handling Microorganisms......Page 91
3.5 Techniques and Terms in Microbiology - Sterility, Asepsis and Aseptic Technique......Page 102
3.6 Disposal of Viable Microbial Waste and Disinfection of Reusable Equipment......Page 103
3.7 Equipment for Enzymology and Molecular Biology......Page 105
3.8 General Reagents and Chemicals in a Biotransformations Laboratory......Page 108
3.9 Conclusion......Page 110
References......Page 111
4.2 Storage, Maintenance and Growth of Microorganisms......Page 113
4.3 General Microbiological Methods......Page 117
4.4 Examples of Whole-Cell Biotransformations Using Bacteria......Page 128
4.5 Biotransformation by Filamentous Fungi and Yeasts......Page 139
4.6 Whole-Cell Biotransformations by Recombinant Strains of E. Coli......Page 148
References......Page 159
5.1 Introduction......Page 161
5.2 Lipases......Page 162
5.3 Hydrolytic Reactions Using Lipases......Page 165
5.4 Using Lipases for Acylation Reactions......Page 168
5.5 Other Hydrolases......Page 175
5.6 Commercially Available Coenzyme-Dependent Enzymes......Page 181
5.7 Carbon-Carbon Bond Forming Reactions......Page 192
References......Page 196
6.1 Introduction......Page 199
6.3 Cell Disruption......Page 200
6.4 A Typical Procedure for Making a Cell Extract from a Recombinant Strain of E. Coli......Page 204
6.5 Puri.cation of Enzymes – A Brief Guide......Page 205
6.6 Techniques for Protein Purification......Page 209
6.7 Isolation of Recombinant Enzymes Using Histidine Tags......Page 215
6.8 Estimation of Protein Concentration......Page 217
6.9 Concentrating Protein Samples by Centrifugation......Page 220
6.10 Analysis of Protein Samples by Sodium Dodecylsulfate Polyacylamide Gel Electrophoresis......Page 221
6.11 Examples of Enzyme Assays......Page 226
6.12 Using Home-Grown Enzymes for Biotransformations – Some Recent Examples......Page 238
6.13 Conclusion......Page 246
References......Page 247
7.1 Introduction......Page 249
7.2 Background......Page 252
7.3 Gene Amplification by Polymerase Chain Reaction (PCR)......Page 263
7.4 DNA Fragment Analysis by Agarose Gel Electrophoresis......Page 270
7.5 Gene Cloning......Page 274
7.6 Analysis by DNA Sequencing......Page 280
7.7 Troubleshooting the Gene Amplification and Cloning Process......Page 281
7.8 Ligation-Independent Cloning......Page 282
7.9 Gene Expression......Page 284
7.10 Conclusion......Page 290
References......Page 291
8.1 Introduction......Page 293
8.2 Site-Directed or Targeted Mutagenesis as a Tool for Investigating Enzyme Mechanism or Altering Catalytic Attributes......Page 294
8.3 Engineering Using Random Mutagenesis Directed Evolution of Enzymes......Page 305
8.4 Combining Rational and Random Mutagenesis for Biocatalyst Improvement......Page 314
8.5 Exploiting Catalytic Promiscuity for Creating New Enzyme Activities......Page 317
8.6 Designing Enzymes in Silico......Page 318
References......Page 319
Appendices......Page 323
Index......Page 337