Enzyme Engineering

Preface IX

About the Authors XI

1 Introduction to Directed Evolution and Rational Design as Protein Engineering Techniques 1

1.1 Methods and Aims of Directed Enzyme Evolution 1

1.2 History of Directed Enzyme Evolution 4

1.3 Methods and Aims of Rational Design of Enzymes 19

2 Screening and Selection Techniques 29

2.1 Introductory Remarks 29

2.2 Screening Methods 29

2.3 Selection Methods 38

2.4 Conclusions and Perspectives 52

3 Gene Mutagenesis Methods in Directed Evolution and Rational Enzyme Design 59

3.1 Introductory Remarks 59

3.2 Directed Evolution Approaches 59

3.3 Diverse Approaches to Rational Enzyme Design 112

3.4 Merging Semi-rational Directed Evolution and Rational Enzyme Design by Focused Rational Iterative Site-Specific Mutagenesis (FRISM) 114

3.5 Conclusions and Perspectives 120

4 Guidelines for Applying Gene Mutagenesis Methods in Organic Chemistry, Pharmaceutical Applications, and Biotechnology 141

4.1 Some General Tips 141

4.2 Rare Cases of Comparative Directed Evolution Studies 152

4.3 Choosing the Best Strategy When Applying Saturation Mutagenesis 163

4.4 Techno-economical Analysis of Saturation Mutagenesis Strategies 187

4.5 Generating Mutant Libraries by Combinatorial Solid-Phase Gene Synthesis: The Future of Directed Evolution? 190

4.6 Fusing Directed Evolution and Rational Design: New Examples of Focused Rational Iterative Site-Specific Mutagenesis (FRISM) 192

5 Tables of Selected Examples of Directed Evolution and Rational Design of Enzymes with Emphasis on Stereo- and Regio-selectivity, Substrate Scope and/or Activity 203

5.1 Introductory Explanations 203

6 Protein Engineering of Enzyme Robustness Relevant to Organic and Pharmaceutical Chemistry and Applications in Biotechnology 233

6.1 Introductory Remarks 233

6.2 Rational Design of Enzyme Thermostability and Resistance to Hostile Organic Solvents 234

6.3 Ancestral and Consensus Approaches and Their Structure-Guided Extensions 241

6.4 Further Computationally Guided Methods for Protein Thermostabilization 242

6.5 Directed Evolution of Enzyme Thermostability and Resistance to Hostile Organic Solvents 253

6.6 Application of epPCR and DNA Shuffling 255

6.7 Saturation Mutagenesis in the B-FIT Approach 258

6.8 Iterative Saturation Mutagenesis (ISM) at Protein-Protein Interfacial Sites for Multimeric Enzymes 263

6.9 Conclusions and Perspectives 265

7 Artificial Enzymes as Promiscuous Catalysts in Organic and Pharmaceutical Chemistry 279

7.1 Introductory Background Information 279

7.2 Applying Protein Engineering for Tuning the Catalytic Profile of Promiscuous Enzymes 285

7.3 Applying Protein Engineering to P450 Monooxygenases for Manipulating Activity and Stereoselectivity of Promiscuous Transformations 299

7.4 Conclusions and Perspectives 307

8 Learning Lessons from Protein Engineering 317

8.1 Introductory Remarks 317

8.2 Additive Versus Nonadditive Mutational Effects in Fitness Landscapes Revealed by Partial or Complete Deconvolution 318

8.3 Unexplored Chiral Fleeting Intermediates and Their Role in Protein Engineering 327

8.4 Case Studies Featuring Mechanistic, Structural, and/or Computational Analyses of the Source of Evolved Stereo- and/or Regioselectivity 329

8.5 Conclusions and Suggestions for Further Theoretical Work 358

9 Perspectives for Future Work 367

9.1 Introductory Remarks 367

9.2 Extending Applications in Organic and Pharmaceutical Chemistry 367

9.3 Extending Applications in Biotechnology 372

9.4 Patent Issues 376

9.5 Final Comments 376

References 377

Index 381