Tailoring a Stabilized Variant of Hydroxynitrile Lyase from Arabidopsis thaliana

The R‐selective hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) cannot be applied for stereoselective cyanohydrin syntheses in aqueous media because of its limited stability at pH<5, which is required in order to suppress the uncatalyzed racemic cyanohydrin formation. To stabilize AtHNL we designed a surface‐modified variant incorporating 11 changes in the amino acids on the protein surface. Comparative characterization of the variant and the wild‐type enzyme showed a broadened pH optimum towards the acidic range, along with enhancement of activity by up to twofold and significantly increased pH‐ and thermostabilities. The effect can most probably be explained by a shift of the isoelectic point from pH 5.1 to 4.8. Application of the variant for the synthesis of (R)‐cyanohydrins in an aqueous/organic two‐phase system at pH 4.5 demonstrated the high stereoselectivity and robustness of the variant relative to the wild‐type enzyme, which is immediately inactivated under these conditions.

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