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Author:   Susanne Brakmann (University of Leipzig, Germany) ,  Kai Johnsson (Department of Chemistry, Inst. of Organic Chemistry, EPFL - BCH, Switzerland)
Publisher:   Wiley-VCH Verlag GmbH
Imprint:   Blackwell Verlag GmbH
Edition:   illustrated edition
Dimensions:   Width: 17.80cm , Height: 2.30cm , Length: 24.70cm
Weight:   0.794kg
ISBN:  

9783527304233

ISBN 10:   3527304231
Pages:   368
Publication Date:   11 March 2002
Audience:   College/higher education ,  Professional and scholarly ,  Undergraduate ,  Postgraduate, Research & Scholarly
Format:   Hardback
Publisher's Status:   Out of Print
Availability:   Out of stock  

Table of 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

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Author Information

"Susanne Brakmann is head of the junior research group ""Applied Molecular Evolution"" at the University of Leipzig (Germany) and a Member of the Biotechnological-Biomedical Center of Leipzig. She studied Chemistry at the Technical University of Braunschweig where she received her diploma in 1988, moving afterwards to the University of Karlsruhe to work on her thesis under the supervision of Reinhold Tacke (Ph. D. 1991). She was postdoctoral fellow at the Max-Planck-Institute for Biophysical Chemistry in Göttingen where she worked with Manfred Eigen before she moved to Leipzig in 2001. She is interested in directed evolution as a tool for understanding and optimizing enzyme functions, focusing on nucleic acid polymerases and their biotechnological applications. ***** Kai Johnsson is assistant professor for Bioorganic Chemistry at the Swiss Federal Institute for Technology (EPFL) where he heads the laboratory for protein engineering. Prior to joining EPFL, he was a junior group leader at Ruhr-University Bochum in Germany, after spending three and a half years in the laboratory of Prof. Peter G. Schultz (University of California, Berkeley) as a postdoctoral research fellow. Kai Johnsson studied chemistry and did his PhD with Prof. Steven Benner at ETH Zurich. Since the start of his PhD thesis, Kai Johnsson's research interests focus on biological chemistry and in particular enzyme mechanisms and protein chemistry. Prof. Johnsson is the inventor of the Covalys technology."

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