Enzyme Engineering

Manfred T. Reetz is Emeritus Professor at the Max-Planck-Institut für Kohlenforschung in Mülheim/Germany. He is a synthetic organic chemist who pioneered the concept of directed evolution of stereo- and regioselective enzymes as a prolific source of catalysts in organic chemistry and biotechnology.Zhoutong Sun, PhD, is Full Professor at Tianjin Institute of Industrial Biotechnology at the Chinese Academy of Sciences. His research interests include enzyme engineering,metabolic engineering and synthetic biology.Ge Qu, PhD, is Associated Professor at the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences/China and member of the Sun group. He specializes in computational enzyme design and biocatalysis.

• Preface IXAbout the Authors XI1 Introduction to Directed Evolution and Rational Design as Protein Engineering Techniques 11.1 Methods and Aims of Directed Enzyme Evolution 11.2 History of Directed Enzyme Evolution 41.3 Methods and Aims of Rational Design of Enzymes 192 Screening and Selection Techniques 292.1 Introductory Remarks 292.2 Screening Methods 292.3 Selection Methods 382.4 Conclusions and Perspectives 523 Gene Mutagenesis Methods in Directed Evolution and Rational Enzyme Design 593.1 Introductory Remarks 593.2 Directed Evolution Approaches 593.3 Diverse Approaches to Rational Enzyme Design 1123.4 Merging Semi-rational Directed Evolution and Rational Enzyme Design by Focused Rational Iterative Site-Specific Mutagenesis (FRISM) 1143.5 Conclusions and Perspectives 1204 Guidelines for Applying Gene Mutagenesis Methods in Organic Chemistry, Pharmaceutical Applications, and Biotechnology 1414.1 Some General Tips 1414.2 Rare Cases of Comparative Directed Evolution Studies 1524.3 Choosing the Best Strategy When Applying Saturation Mutagenesis 1634.4 Techno-economical Analysis of Saturation Mutagenesis Strategies 1874.5 Generating Mutant Libraries by Combinatorial Solid-Phase Gene Synthesis: The Future of Directed Evolution? 1904.6 Fusing Directed Evolution and Rational Design: New Examples of Focused Rational Iterative Site-Specific Mutagenesis (FRISM) 1925 Tables of Selected Examples of Directed Evolution and Rational Design of Enzymes with Emphasis on Stereo- and Regio-selectivity, Substrate Scope and/or Activity 2035.1 Introductory Explanations 2036 Protein Engineering of Enzyme Robustness Relevant to Organic and Pharmaceutical Chemistry and Applications in Biotechnology 2336.1 Introductory Remarks 2336.2 Rational Design of Enzyme Thermostability and Resistance to Hostile Organic Solvents 2346.3 Ancestral and Consensus Approaches and Their Structure-Guided Extensions 2416.4 Further Computationally Guided Methods for Protein Thermostabilization 2426.5 Directed Evolution of Enzyme Thermostability and Resistance to Hostile Organic Solvents 2536.6 Application of epPCR and DNA Shuffling 2556.7 Saturation Mutagenesis in the B-FIT Approach 2586.8 Iterative Saturation Mutagenesis (ISM) at Protein-Protein Interfacial Sites for Multimeric Enzymes 2636.9 Conclusions and Perspectives 2657 Artificial Enzymes as Promiscuous Catalysts in Organic and Pharmaceutical Chemistry 2797.1 Introductory Background Information 2797.2 Applying Protein Engineering for Tuning the Catalytic Profile of Promiscuous Enzymes 2857.3 Applying Protein Engineering to P450 Monooxygenases for Manipulating Activity and Stereoselectivity of Promiscuous Transformations 2997.4 Conclusions and Perspectives 3078 Learning Lessons from Protein Engineering 3178.1 Introductory Remarks 3178.2 Additive Versus Nonadditive Mutational Effects in Fitness Landscapes Revealed by Partial or Complete Deconvolution 3188.3 Unexplored Chiral Fleeting Intermediates and Their Role in Protein Engineering 3278.4 Case Studies Featuring Mechanistic, Structural, and/or Computational Analyses of the Source of Evolved Stereo- and/or Regioselectivity 3298.5 Conclusions and Suggestions for Further Theoretical Work 3589 Perspectives for Future Work 3679.1 Introductory Remarks 3679.2 Extending Applications in Organic and Pharmaceutical Chemistry 3679.3 Extending Applications in Biotechnology 3729.4 Patent Issues 3769.5 Final Comments 376References 377Index 381