Full Description
Natural selection created optimal catalysts. However, optimal performance of enzyme catalysis does not necessarily refer to maximum reaction rate. Rather, it may be a compromise between specificity, rate, stability, and other chemical constraints that makes enzymes capable of catalyzing reactions under mild conditions and with high substrate specificity, accompanied by high regio- and enantioselectivity.
The book presented here focuses on the directed evolution of proteins, which has established itself as a powerful method for designing enzymes showing new substrate specificities. It includes a comprehensive repertoire of techniques for producing combinatorial enzyme libraries, while the functional gene expression in a suitable host helps in selecting the appropriate structure, making fast screening a necessity. This book illustrates both the theoretical background as well as the potential of this interesting method in practice - which is becoming ever more important even in classical organic synthesis!
Contents
List of Contributors xi
1 Introduction 1
2 Evolutionary Biotechnology - From Ideas and Concepts to Experiments and Computer Simulations 5
2.1 Evolution in vivo - From Natural Selection to Population Genetics 5
2.2 Evolution in vitro - From Kinetic Equations to Magic Molecules 8
2.3 Evolution in silico - From Neutral Networks to Multi-stable Molecules 16
2.4 Sequence Structure Mappings of Proteins 25
2.5 Concluding Remarks 26
3 Using Evolutionary Strategies to Investigate the Structure and Function of Chorismate Mutases 29
3.1 Introduction 29
3.2 Selection versus Screening 30
3.3 Genetic Selection of Novel Chorismate Mutases 33
3.4 Summary and General Perspectives 57
4 Construction of Environmental Libraries for Functional Screening of Enzyme Activity 63
4.1 Sample Collection and DNA Isolation from Environmental Samples 65
4.2 Construction of Environmental Libraries 68
4.3 Screening of Environmental Libraries 71
4.4 Conclusions 76
5 Investigation of Phage Display for the Directed Evolution of Enzymes 79
5.1 Introduction 79
5.2 The Phage Display 79
5.3 Phage Display of Enzymes 81
5.4 Creating Libraries of Mutants 87
5.5 Selection of Phage-enzymes 89
5.6 Conclusions 108
6 Directed Evolution of Binding Proteins by Cell Surface Display: Analysis of the Screening Process 111
6.1 Introduction 111
6.2 Library Construction 113
6.3 Mutant Isolation 115
6.4 Summary 124
7 Yeast n-Hybrid Systems for Molecular Evolution 127
7.1 Introduction 127
7.2 Technical Considerations 130
7.3 Applications 147
7.4 Conclusion 155
8 Advanced Screening Strategies for Biocatalyst Discovery 159
8.1 Introduction 159
8.2 Semi-quantitative Screening in Agar-plate Formats 161
8.3 Solution-based Screening in Microplate Formats 164
8.4 Robotics and Automation 169
9 Engineering Protein Evolution 177
9.1 Introduction 177
9.2 Mechanisms of Protein Evolution in Nature 178
9.3 Engineering Genes and Gene Fragments 187
9.4 Gene Fusion ± From Bi- to Multifunctional Enzymes 203
9.5 Perspectives 208
10 Exploring the Diversity of Heme Enzymes through Directed Evolution 215
10.1 Introduction 215
10.2 Heme Proteins 216
10.3 Cytochromes P450 218
10.4 Peroxidases 223
10.5 Comparison of P450s and Peroxidases 227
10.6 Chloroperoxidase 228
10.7 Mutagenesis Studies 229
10.8 Directed Evolution of Heme Enzymes 233
10.9 Conclusions 238
11 Directed Evolution as a Means to Create Enantioselective Enzymes for Use in Organic Chemistry 245
11.1 Introduction 245
11.2 Mutagenesis Methods 247
11.3 Overexpression of Genes and Secretion of Enzymes 248
11.4 High-Throughput Screening Systems for Enantioselectivity 250
11.5 Examples of Directed Evolution of Enantioselective Enzymes 257
11.6 Conclusions 273
12 Applied Molecular Evolution of Enzymes Involved in Synthesis and Repair of DNA 281
12.1 Introduction 281
12.3 Directed Evolution of DNA polymerases 289
12.4 Directed Evolution of Thymidine Kinase 295
12.5 Directed Evolution of Thymidylate Synthase 297
12.6 O 6 -Alkylguanine-DNA Alkyltransferase 300
12.7 Discussion 302
13 Evolutionary Generation versus Rational Design of Restriction Endonucleases with Novel Specificity 309
13.1 Introduction 309
13.2 Design of Restriction Endonucleases with New Specificities 313
13.3 Summary and Outlook 324
14 Evolutionary Generation of Enzymes with Novel Substrate Specificities 329
14.1 Introduction 329
14.2 General Considerations 331
14.3 Examples 333
14.4 Conclusions 339
Index 343
