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Mutagenesis of an Asn156 Residue in a Surface Region of S ‐Selective Hydroxynitrile Lyase from Baliospermum montanum Enhances Catalytic Efficiency and Enantioselectivity
Author(s) -
Kawahara Nobuhiro,
Asano Yasuhisa
Publication year - 2015
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.201500225
Subject(s) - enantiomeric excess , chemistry , enantiomer , enzyme kinetics , mutagenesis , stereochemistry , kinetic resolution , catalysis , enzyme , mutant , substrate (aquarium) , site directed mutagenesis , saturated mutagenesis , biochemistry , active site , enantioselective synthesis , biology , gene , ecology
Abstract The S ‐selective hydroxynitrile lyase from Baliospermum montanum ( Bm HNL) has broad substrate specificity toward aromatic substrates as well as high temperature stability, although with low enantioselectivity and specific activity. To expand the industrial application of this enzyme, we improved its enantioselectivity and specific activity toward ( S )‐mandelonitrile by mutagenesis. The specific activity of the Bm HNL H103C/N156G mutant for ( S )‐mandelonitrile production was raised to 154 U mg −1 (WT Bm HNL: 52 U mg −1 ). The enantiomeric excess was increased to 93 % (WT Bm HNL: 55 %). The kinetic analysis revealed K m for ( R )‐mandelonitrile and k cat for ( S )‐mandelonitrile increased by the mutation at Asn156, thus contributing to the increase in enantiomeric excess. This is the first report on an improvement in catalytic efficiency and enantiomeric excess of Bm HNL for ( S )‐mandelonitrile synthesis by random and site‐directed mutagenesis.