Expansion of the Substrate Specificity of Porcine Kidney D‐Amino Acid Oxidase for <i>S</i>‐Stereoselective Oxidation of 4‐Cl‐Benzhydrylamine

Abstract

Abstract Discovery and development of enzymes for the synthesis of chiral amines have been a hot topic for basic and applied aspects of biocatalysts. Based on our X‐ray crystallographic analyses of porcine kidney D‐amino acid oxidase (pkDAO) and its variants, we rationally designed a new variant that catalyzed the oxidation of ( S )‐4‐Cl‐benzhydrylamine (CBHA) from pkDAO and obtained it by functional high‐throughput screening with colorimetric assay. The variant I230A/R283G was constructed from the variant R283G which had completely lost the activity for D‐amino acids, further gaining new activity toward ( S )‐chiral amines with the bulky substituents. The variant enzyme (I230A/R283G) was characterized to have a catalytic efficiency of 1.85 s −1 for ( S )‐CBHA, while that for ( R )‐1‐phenylethylamine was diminished 10‐fold as compared with the Y228L/R283G variant. The variant was efficiently used for the synthesis of ( R )‐CBHA in 96 % ee from racemic CBHA by the deracemization reaction in the presence of reducing agent such as NaBH 4 in water. Furthermore, X‐ray crystallographic analysis of the new variant complexed with ( S )‐CBHA, together with modelling study clearly showed the basis of understanding the structure‐activity relationship of pkDAO.