| 1-amino cyclopropanecarboxylic acid (ACCA)and its derivatives had been found widespread application in drug design. Derivatives of 1-amino-2-vinyl cyclopropane carboxylic acid ethyl ester is an important drug intermediate of HCV NS3A inhibitor drugs. At present, the chiral separation of derivatives of 1-amino-2-vinyl cyclopropane carboxylic acid ethyl ester are using chemical methods, efficient enzyme from microotganism method is scarce.In this study, we chose racemic 1-amino-2-vinyl cyclopropane carboxylic acid ethyl ester as the single carbon source,378 microorganisms were isolated from 100 soil samples. Through the HPLC test,54 strains can selective hydrolysis of 1S,2R-amino-2-vinyl cyclopropane carboxylic acid ethyl ester, and 15 strains can selective hydrolysis of 1R,2S-amino-2-vinyl cyclopropane carboxylic acid ethyl ester. After subculture and identification, CY-2 show the highest enantioselective activity, and was identified as Sphingomonas aquatilis JSS7.Optimize the reaction conditions of CY-2 chiral separation to derivatives of 1-amino-2-vinyl cyclopropane carboxylic acid ethyl ester. The optimum condition for biotransformation of substrate is at 37℃ for 72 h with 350 mg of wet cell and 1 g·L-1 substrate. Under this condition, substrate e.e was 88.23% and conversion was 62.44%.We do research about CY-2 chiral separation substrates. We prepared derivatives of 1-amino-2-vinyl cyclopropane carboxylic acid ethyl ester and other compounds containing ester and amino substituent. In the 11 different compounds, CY-2 can stereoselectively hydrolysis 7 of them. And especially stereoselectively hydrolysis (1S,2R)-amino-2-vinyl cyclopropane carboxylic acid ethyl ester hydrochloride e.e was 99.6%.Use gene mining to the activity strains SQR-21. Compare SQR-21 with activity enzyme Alcalase 2.4L, use recombinant e.coli bacteria, cloning expressed the four pieces of hydrolytic enzyme protein. And test the stereoselectively hydrolysis substrate activity. Although the results did not achieve ideal value, but still provides a new direction. |
