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| Screening, Cloning and Expression of Esterase Gene for Enantioselective Resolution of (R)-ketoprofen |
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Abstract The research was carried out on a Bacillus megaterium NK13 strain which to a certain extent could asymmetricly hydrolyzes the rac-ketoprofen chloroethyl ester to (S)-Ketoprofen. By constructing Bacillus megaterium NK13 gene library, one positive clone containing the plasmid pUC18-NK- HYD3 was obtained. The analysis of sequence detection showed that the positive clone included one Open Read Frame of 741 bp nucleotide sequence which contains esterases’ conserved motif —— GXSXG. The results of blast in NCBI database showed that it was was a novel esterase gene. Then the esterase gene was cloned to pET-21b(+) vector and transformed into E.coli BL21(DE3). After being induced by IPTG, SDS-PAGE analysis showed that the relative molecular mass of the esterase was about 28 kDa. The results of TLC and HPLC indicated that the esterase is a (R)-ketoprofen enantioselective enzyme. In the recombinant strain suspension system, The highest enantiomeric yield of (R)-Ketoprofen was 62.74% when conversion rate was 15%. In PBS system, which contained the recombinant strain wet cells, when the conversion rate increased from 10% to 50%, the e.e.% of (R)-Ketoprofen were maintained at 73%~76%.
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Received: 12 June 2010
Published: 19 October 2010
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