Mutagenesis of an Asn156 Residue in a Surface Region of S-Selective Hydroxynitrile Lyase from Baliospermum montanum Enhances Catalytic Efficiency and Enantioselectivity

• Published in: Chembiochem : a European journal of chemical biology 2015-09, Vol.16 (13), p.1891-1895
• Main Authors: Kawahara, Nobuhiro, Asano, Yasuhisa
• Format: Article
• Language: English
• Subjects: Bacteriology; cyanohydrins; Efficiency; enzyme catalysis; hydroxynitrile lyase; mandelonitrile; Mutagenesis
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201500225

The S‐selective hydroxynitrile lyase from Baliospermum montanum (BmHNL) 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 BmHNL H103C/N156G mutant for (S)‐mandelonitrile production was raised to 154 U mg−1 (WT BmHNL: 52 U mg−1). The enantiomeric excess was increased to 93 % (WT BmHNL: 55 %). The kinetic analysis revealed Km for (R)‐mandelonitrile and kcat 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 BmHNL for (S)‐mandelonitrile synthesis by random and site‐directed mutagenesis. Hydroxynitrile lyases have commercial potential in the enantioselective synthesis of mandelonitrile. Here we used a combination of random and site‐directed mutagenesis to improve enantioselectivity and specific activity of the enzyme from Baliospermum montanum. A double mutant (H103C/N156G) achieved a threefold improvement in activity with 93 % ee.