The plant Baliospermum montanum was found to produce hydroxynitrile lyases (HNL) during a screen for the enzyme among fruits in markets and plant specimens obtained from a botanical garden. The enzymatic properties of the S-stereoselective HNL have been characterized using purified enzyme from the leaves of B. montanum and an Escherichia coli transformant expressing the enzyme. We determined the structure of BmHNL by X-ray crystallography and successfully engineered BmHNL to have H103C and N156G double mutants on the basis of its structure, molecular dynamics simulation with the substrate, and speculation on the reaction mechanism. The specific activity of the BmHNL H103C/N156G mutant was improved to 154 U/mg from 52 U/mg as compared with BmHNL wild type for (S)-mandelonitrile production, and the enantiomeric excess of (S)-mandelonitrile produced by the double mutant was increased to 93% from 55% compared withthe BmHNL wild type. The double mutant is suitable for (S)-mandelonitrile synthesis, improving its enantioselectivity and specific activity.
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