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An Esterase with Superior Activity and Enantioselectivity towards 1,2‐ O ‐Isopropylideneglycerol Esters Obtained by Protein Design
Author(s) -
Godinho Luis F.,
Reis Carlos R.,
van Merkerk Ronald,
Poelarends Gerrit J.,
Quax Wim J.
Publication year - 2012
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/adsc.201200211
Subject(s) - chemistry , kinetic resolution , active site , enantiomer , esterase , docking (animal) , directed evolution , stereochemistry , enantioselective synthesis , combinatorial chemistry , catalysis , enzyme , mutant , organic chemistry , biochemistry , medicine , nursing , gene
The Escherichia coli esterase YbfF displays high activity towards 1,2‐ O ‐isopropylideneglycerol (IPG) butyrate and IPG caprylate, and prefers the R ‐enantiomer of these substrates, producing the S ‐enantiomer of the IPG product in excess. To improve the potential of the enzyme for the kinetic resolution of racemic esters of IPG, an enhancement of the activity and enantioselectivity would be highly desirable. Molecular docking of the R ‐enantiomer of both IPG esters into the active site of YbfF allowed the identification of proximal YbfF active site residues. Four residues (25, 124, 185 and 235) were selected as targets for mutagenesis, in order to enhance YbfF activity and enantioselectivity towards IPG esters. Random mutagenesis at positions 25, 124, 185 and 235 yielded several best YbfF variants with enhanced activity and enantioselectivity towards IPG esters. The best YbfF mutant, W235I, exhibited a 2‐fold higher enantioselectivity than wild‐type YbfF, with an E =38 for IPG butyrate and an E =77 for IPG caprylate. Molecular docking experiments further support the enhanced enantioselectivity shown experimentally and the structural effects of this amino acid substitution on the active site of YbfF are provided. The engineered W235I mutant is an attractive catalyst for practical applications in the kinetic resolution of IPG esters.