Chapter 11. Applications of Recombinant DNA Technology: Directed Evolution

  1. Prof. Dr. Andreas Sebastian Bommarius1,
  2. Dr. Bettina R. Riebel2

Published Online: 28 JAN 2005

DOI: 10.1002/3527602364.ch11

Book Title

Biocatalysis

Biocatalysis

Additional Information

How to Cite

Bommarius, A. S. and Riebel, B. R. (2005) Applications of Recombinant DNA Technology: Directed Evolution, in Biocatalysis, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, FRG. doi: 10.1002/3527602364.ch11

Author Information

  1. 1

    School of Chemical and Biomolecular Engineering, Parker H. Petit Biotechnology Institute, Georgia Institute of Technology, 315 Ferst Drive, N. W. Atlanta, GA 30332-0363, USA

  2. 2

    Emory University School of Medicine, Whitehead Research Building, 615 Michael Street, Atlanta, GA 30322, USA

Publication History

  1. Published Online: 28 JAN 2005
  2. Published Print: 28 JAN 2004

ISBN Information

Print ISBN: 9783527303441

Online ISBN: 9783527602360

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Keywords:

  • DNA technology;
  • evolvability of proteins;
  • DNA library;
  • directed evolution;
  • error-prone PCR;
  • subtilisin;
  • DMF;
  • DNA shuffling;
  • p-nitrobenzyl esterase;
  • p-nitrophenyl esterase;
  • monomeric chorismate mutase;
  • enantioselectivity;
  • pseudomonas aeruginosa lipase;
  • hydantoinase;
  • KDPG aldolase;
  • aminotransferases;
  • carotenoids

Summary

  • Background of Evolvability of Proteins

    • Purpose of Directed Evolution

    • Evolution and Probability

    • Evolution: Conservation of Essential Components of Structure

  • Process steps in Directed Evolution: Creating Diversity and Checking for Hits

    • Creation of Diversity in a DNA Library

    • Testing for Positive Hits: Screening or Selection

  • Experimental Protocols for Directed Evolution

    • Creating Diversity: Mutagenesis Methods

    • Creating Diversity: Recombination Methods

    • Checking for Hits: Screening Assays

    • Checking for Hits: Selection Procedures

    • Additional Techniques of Directed Evolution

  • Successful Examples of the Application of Directed Evolution

    • Application of Error-prone PCR: Activation of Subtilisin in DMF

    • Application of DNA Shuffling: Recombination of p-Nitrobenzyl Esterase Genes

    • Enhancement of Thermostability: p-Nitrophenyl Esterase

    • Selection instead of Screening: Creation of a Monomeric Chorismate Mutase

    • Improvement of Enantioselectivity: Pseudomonas aeruginosa Lipase

    • Inversion of Enantioselectivity: Hydantoinase

    • Redesign of an Enzyme's Active Site: KDPG Aldolase

  • Comparison of Directed Evolution Techniques

    • Comparison of Error-Prone PCR and DNA Shuffling: Increased Resistance against Antibiotics

    • Protein Engineering in Comparison with Directed Evolution: Aminotransferases

    • Directed Evolution of a Pathway: Carotenoids

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