Sunday, July 26, 2009
P31

Chiral intermediate production by Rhodococcus erythropolis amidase immobilized using a cross-linked enzyme aggregate method

Hyun Joo Park, Ha Ju Park, and Hyung Kwoun Kim. Department of Biotechnology, The Catholic University of Korea, 43-1 Yeokgok 2-dong, Wonmi-gu, Bucheon, South Korea

Rhodococcus erythropolis strain (No. 7) capable of converting 4-chloro-3-hydroxybutyronitrile into 4-chloro-3-hydroxybutyric acid has been isolated. The amidase gene was cloned and expressed in Escherichia coli cells. The Ni column-purified recombinant amidase showed a specific activity of 3.89 U·mg-1 toward isobutyramide. The amidase showed an optimum temperature of 55°C and an optimum pH of 8.5. It was stable at 10-50°C and at pH 7.0-12.0 for 30 min. It hydrolyzed enantioselectively 4-chloro-3-hydroxybutyramide into the corresponding acid; when the conversion yield was 57%, the ee value was 52% toward (R)-form amide substrate. The remaining (S)-form amide substrate could be converted chemically into (S)-acid and then into ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-ECHB). (S)-ECHB is an intermediate for the synthesis of Atorvastatin, a chiral drug for hypercholesterolemia. The amidase was immobilized using the cross-linked enzyme aggregate (CLEA) method. First, the amidase was mixed with the same amount of bovine serum albumin. The mixed proteins were precipitated with ammonium sulfate and the resulting mixed precipitant was cross-linked with glutaraldehyde. The amidase CLEA showed similar optimum temperature and pH with those of the soluble amidase enzyme. It was stable 10-50°C and at pH 5.0-12.0. The CLEA enzyme could be recovered rapidly by centrifugation and it retained 96% activity after 5 repeated cycles of recovery. These results demonstrated that the amidase CLEA was quite stable and could be used repeatedly as a biocatalyst for the production of (S)-ECHB.

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