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.