Cloning and Expression of Esterase Gene to Enantioselective Resolution of (S)-Ketoprofen in NK13 / 中国生物工程杂志

ABSTRACT

A strain NK13 was screened for a certain extent asymmetric hydrolyze the rac-ketoprofen chloroethyl ester and identified as Bacillus megaterium. For the preparation of gene libraries, a positive clones was obtained from the tributyrin flat. The sequence of this esterase gene had been analysised, and contained the whole ORF of an esterase gene with the length of 933bp. The esterase gene of NK13 was compared with the esterase genes of GenBank and the result showed that the esterase gene of NK13 was a novel gene(GenBank accession nember DQ196347).The new esterase gene was inserted into the plasmid pET21b+, then the recombinant plasmid transformed E.coli BL21. After being induced by IPTG, it was expressed in the host strain. SDS-PAGE analysis showed that the relative molecular mass of the esterase was about 34kDa. The result of TLC and HPLC showed that the recombinant strain had higher conversion ration than templet strain. 47.4%of the rac-ketoprofen Chloroethl ester was hydrolyzed to ketoprofen by the recombinant strain in 45min. The (S)- ketoprofen enantiomeric excess, in the later,was 55.46%, which indicated that the esterase could hydrolyze (S)-ketoprofen chloroethyl ester firstly.