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Identification and Characterization of D ‐Succinylase, and a Proposed Enzymatic Method for D ‐Amino Acid Synthesis
Author(s) -
Sumida Yosuke,
Iwai Sachio,
Nishiya Yoshiaki,
Kumagai Shinya,
Yamada Toshihide,
Azuma Masayuki
Publication year - 2016
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/adsc.201600105
Subject(s) - chemistry , amino acid , escherichia coli , enzyme , biochemistry , hydrolysis , phenylalanine , enzymatic hydrolysis , recombinant dna , gene
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.