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Natural Diversity to Guide Focused Directed Evolution
Author(s) -
Jochens Helge,
Bornscheuer Uwe T.
Publication year - 2010
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201000284
Subject(s) - diversity (politics) , natural (archaeology) , computational biology , biology , evolutionary biology , data science , computer science , sociology , paleontology , anthropology
Simultaneous multiple site‐saturation mutagenesis was performed at four active‐site positions of an esterase from Pseudomonas fluorescens to improve its ability to convert 3‐phenylbutyric acid esters (3‐PBA) in an enantioselective manner. Based on an appropriate codon choice derived from a structural alignment of 1751 sequences of α/β‐hydrolase fold enzymes, only those amino acids were considered for library creation that appeared frequently in structurally equivalent positions. Thus, the number of mutants to be screened could be substantially reduced while the number of functionally intact variants was increased. Whereas the wild‐type esterase showed only marginal activity and poor enantioselectivity ( E true =3.2) towards 3‐PBA‐ethyl ester, a significant number of hits with improved rates (up to 240‐fold) and enantioselectivities (up to E true =80) were identified in these “smart” libraries.