Enzyme Engineering

Selective Catalysts for Applications in Biotechnology, Organic Chemistry, and Life Science
1. Auflage 2023
ISBN: 978-3-527-35033-9
Verlag: Wiley-VCH GmbH

Buch, Englisch, 400 Seiten, Format (B × H): 177 mm x 254 mm, Gewicht: 929 g

Enzyme Engineering
An authoritative and up-to-date discussion of enzyme engineering and its applications
In Enzyme Engineering: Selective Catalysts for Applications in Biotechnology, Organic Chemistry, and Life Science, a team of distinguished researchers deliver a robust treatment of enzyme engineering and its applications in various fields such as biotechnology, life science, and synthesis. The book begins with an introduction to different protein engineering techniques, covers topics like gene mutagenesis methods for directed evolution and rational enzyme design. It includes industrial case studies of enzyme engineering with a focus on selectivity and activity.
The authors also discuss new and innovative areas in the field, involving machine learning and artificial intelligence. It offers several insightful perspectives on the future of this work.
Readers will also find: - A thorough introduction to directed evolution and rational design as protein engineering techniques
- Comprehensive explorations of screening and selection techniques, gene mutagenesis methods in directed evolution, and guidelines for applying gene mutagenesis in organic chemistry, pharmaceutical applications, and biotechnology
- Practical discussions of protein engineering of enzyme robustness relevant to organic and pharmaceutical chemistry
- Treatments of artificial enzymes as promiscuous catalysts
- Various lessons learned from semi-rational and rational directed evolution

A transdisciplinary treatise, Enzyme Engineering: Selective Catalysts for Applications in Biotechnology, Organic Chemistry, and Life Science is perfect for protein engineers, theoreticians, organic, and pharmaceutical chemists as well as transition metal researchers in catalysis and biotechnologists.

Autoren/Hrsg.

Reetz, Manfred T.

Sun, Zhoutong

Qu, Ge

Fachgebiete

• Technische Wissenschaften Verfahrenstechnik | Chemieingenieurwesen | Biotechnologie Biotechnologie
• Naturwissenschaften Chemie Organische Chemie Biochemie

Inhaltsverzeichnis

Introduction to Directed Evolution and Rational Design as Protein Engineering Techniques
-Methods and Aims of Directed Enzyme Evolution
-Short History of Directed Enzyme Evolution
-Methods and Aims of Rational Design of Enzymes
Screening and Selection Techniques
Gene Mutagenesis Methods
Guidelines for Applying Gene Mutagenesis Methods in Organic Chemistry, Pharmaceutical Applications and Biotechnology
Case Studies of Protein Engineering of Activity and Selectivity
-Epoxide Hydrolase
-Transaminase as an Industrial Example with Pharmaceutical Application
-Geranylgeranyl Diphosphate Synthase for Efficient Carotenoid Production
-Cytochrome P450 Monooxygenases for Synthesis of Hydroxylation of Steroids Needed in the Preparation of Pharmaceuticals
-Lipase for Stereocomplementary Production of Organic Compounds with Two Chirality Centers
-Further Examples Using Other Enzyme Types
Protein Engineering of Enzyme Robustness
-Examples of Relevance to Organic and Pharmaceutical Applications
-Examples of Relevance to Biotechnology
Artificial Metallo-Enzymes for Promiscuous Transformations Using Known Organic Reaction Types as a Guide
Learning Lessons from Protein Engineering
Perspectives for Future Work
-In Extending Applications in Organic and Pharmaceutical Chemistry
-In Extending Biotechnological Contributions to Ecology

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

Über Autor(innen)

Manfred T. Reetz is a former Director and presently 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. Reetz has received numerous German and international awards and prizes, and is member of the German National Academy Leopoldina, Academy of the German state of NRW, Royal Dutch Academy of Sciences, European Academy of Sciences, and Distinguished Scientist of the Chinese Academy of Sciences (China).

Zhoutong Sun obtained his Ph.D in microbiology at Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences in 2012, then he moved to Nanyang Technological University in Singapore as a research fellow. One year later, he moved to the MPI für Kohlenforschung and Marburg University for a postdoc with Prof. Manfred T. Reetz. In 2016, he became a full professor at Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences. His research interests are in the discovery, design and engineering of biocatalysts as well as cascade reactions design and metabolic engineering.

Ge Qu received his Ph.D in Bioinformatics from Adam Mickiewicz University in Poland in 2015. Currently, he is working as an associated professor in the Reetz Laboratory at Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences. His major research interests are the discovery and structure-based engineering of enzymes that have potential as industrial biocatalysts.

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.