Identification and Characterization of D ‐Succinylase, and a Proposed Enzymatic Method for D ‐Amino Acid Synthesis

Chiral amino acids are important intermediates for the pharmaceutical industry. We have developed a novel one‐pot enzymatic method for D ‐amino acid synthesis by the dynamic kinetic resolution of N ‐succinyl‐ dl ‐amino acids using D ‐succinylase (DSA) and N ‐succinylamino acid racemase (NSAR, EC 4.2.1.113). The DSA from Cupriavidus sp. P4‐10‐C, which hydrolyzes N ‐succinyl‐ D ‐amino acids enantioselectively to their corresponding D ‐amino acids, was identified for the first time by screening soil microorganisms. Subsequently, the DSA gene was cloned and overexpressed in Escherichia coli . DSA was shown to comprise two subunits with molecular masses of 26 kDa and 60 kDa. Additionally, the NSAR gene from Geobacillus stearothermphilus NCA1503, which racemizes N ‐succinylamino acids, was also cloned and overexpressed in E. coli . The highly purified DSA and NSAR prepared from each recombinant E. coli were characterized and used for D ‐amino acid synthesis. A one‐pot enzymatic method converted 100 mM N ‐succinyl‐ dl ‐phenylalanine to D ‐phenylalanine in 91.1% conversion with 86.7% ee . This novel enzymatic method may be useful for the industrial production of many D ‐amino acids.