Enzyme Engineering: Methods and Protocols

Part I: Enzyme Libraries Preparation and Screening

1. Preparation of Soil Metagenome Libraries and Screening for Gene Specific Amplicons

            Luke J. Stevenson, David F. Ackerley, and Jeremy G. Owen

2. Ultrahigh-Throughput Screening of Metagenomic Libraries Using Droplet Microfluidics

            Davide Agostino Cecchini, Mercedes Sánchez-Costa, Alejandro H. Orrego, Jesús Fernández-Lucas, and Aurelio Hidalgo

3. Synthetic DNA Libraries for Protein Engineering toward Process Improvement in Drug Synthesis

            Michele Tavanti

Part II: Directed Evolution

4. In Silico Prediction Methods for Site-Saturation Mutagenesis

            Ge Qu and Zhoutong Sun

5. Recombination of Compatible Substitutions by 2GenReP and InSiReP

            Haiyang Cui, Mehdi D. Davari, and Ulrich Schwaneberg

Part III: Semi-Rational and De Novo Design

6. Using the Evolutionary History of Proteins to Engineer Insertion-Deletion Mutants from Robust, Ancestral Templates Using Graphical Representation of Ancestral Sequence Predictions (GRASP)

            Connie M. Ross, Gabriel Foley, Mikael Boden, and Elizabeth M.J. Gillam

7. Resurrecting Enzymes by Ancestral Sequence Reconstruction

            Maria Laura Mascotti

8. Expression and In Vivo Loading of De Novo Proteins with Tetrapyrrole Cofactors

            Paul Curnow and J.L. Ross Anderson

Part IV: Rational Design

9. Rational-Design Engineering to Improve Enzyme Thermostability

            Vinutsada Pongsupasa, Piyanuch Anuwan, Somchart Maenpuen, and Thanyaporn Wongnate

10. Using Molecular Simulation to Guide Protein Engineering for Biocatalysis in Organic Solvents

            Haiyang Cui, Markus Vedder, Ulrich Schwaneberg, and Mehdi D. Davari

11. In Silico Engineering of Enzyme Access Tunnels

            Alfonso Gautieri, Federica Rigoldi, Archimede Torretta, Alberto Redaelli, and Emilio Parisini

12. Quantum-Mechanical/Molecular-Mechanical (QM/MM) Simulations for Understanding Enzyme Dynamics

13. Computational Enzyme Design at Zymvol

            Emanuele Monza, Victor Gil, and Maria Fatima Lucas

Part V: Biocatalytic Process Development

14. Advanced Enzyme Immobilization Technologies: An Eco-Friendly Support, a Polymer-Stabilizing Immobilization Strategy, and an Improved Cofactor Co-Immobilization Technique

            Ana I. Benítez-Mateos and Francesca Paradisi

15. Chemical Reaction Engineering to Understand Applied Kinetics in Free Enzyme Homogeneous Reactors

            Alvaro Lorente-Arevalo, Alberto Garcia-Martin, Miguel Ladero, and Juan M. Bolivar

16. Enzymatic Oxidative Cascade for Oxofunctionalization of Fatty Acids in One-Pot

            Somayyeh Gandomkar and Mélanie Hall

17. CRISPR-Cas9 Editing of the Synthesis of Biodegradable Polyesters Polyhydroxyalkanaotes (PHA) in Pseudomonas putida KT2440

            Si Liu, Tanja Narancic, Chris Davis, and Kevin E. O'Connor