Laboratory Evolved Biocatalysts for Stereoselective Syntheses of Substituted Benzaldehyde Cyanohydrins

Abstract

Random mutagenesis and recombination of PaHNL5 was carried out in Pichia pastoris by employment of an overlap extension PCR-based strategy. New fast and stereoselective enzyme variants were obtained, and these led to improved turnover rates with the nonnatural substrate 2-chlorobenzaldehyde. A new catalyst combining all favorable mutations was generated, and even very small amounts of this variant enabled complete conversion of 2-chlorobenzaldehyde into the (R)-hydroxynitrile with >99 % ee in only 4 h.