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Many Pathways in Laboratory Evolution Can Lead to Improved Enzymes: How to Escape from Local Minima.
MedLine Citation:
PMID:  22522601     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
Directed evolution is a method to tune the properties of enzymes for use in organic chemistry and biotechnology, to study enzyme mechanisms, and to shed light on Darwinian evolution in nature. In order to enhance its efficacy, iterative saturation mutagenesis (ISM) was implemented. This involves: 1) randomized mutation of appropriate sites of one or more residues; 2) screening of the initial mutant libraries for properties such as enzymatic rate, stereoselectivity, or thermal robustness; 3) use of the best hit in a given library as a template for saturation mutagenesis at the other sites; and 4) continuation of the process until the desired degree of enzyme improvement has been reached. Despite the success of a number of ISM-based studies, the question of the optimal choice of the many different possible pathways remains unanswered. Here we considered a complete 4-site ISM scheme. All 24 pathways were systematically explored, with the epoxide hydrolase from Aspergillus niger as the catalyst in the stereoselective hydrolytic kinetic resolution of a chiral epoxide. All 24 pathways were found to provide improved mutants with notably enhanced stereoselectivity. When a library failed to contain any hits, non-improved or even inferior mutants were used as templates in the continuation of the evolutionary pathway, thereby escaping from the local minimum. These observations have ramifications for directed evolution in general and for evolutionary biological studies in which protein engineering techniques are applied.
Authors:
Yosephine Gumulya; Joaquin Sanchis; Manfred T Reetz
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-4-20
Journal Detail:
Title:  Chembiochem : a European journal of chemical biology     Volume:  -     ISSN:  1439-7633     ISO Abbreviation:  -     Publication Date:  2012 Apr 
Date Detail:
Created Date:  2012-4-23     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  100937360     Medline TA:  Chembiochem     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Affiliation:
Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr (Germany).
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