NK13中(S)- 酮基布洛芬拆分用酯酶基因的克隆及表达

以本实验室筛选出的一株具有不对称拆分消旋酮基布洛芬氯乙酯的菌株NK13为材料，经初步鉴定为巨大芽孢杆菌(Bacillus megaterium)，通过构建其基因文库，从中筛选得到一阳性克隆重组子pUC-NK。测序分析表明，该重组子质粒中包含一长度为933bp的酯酶基因的完整开放阅读框，核苷酸同源性对比证明该酯酶基因属首次发现（GenBank登录号为DQ196347），将此基因克隆到原核表达载体pET21b+中构建重组表达质粒pET-NKest，转化E.coli BL21，经IPTG诱导在宿主菌中得到表达，经SDS-PAGE电泳检测证明该酯酶蛋白分子量约为34KDa。薄层层析与HPLC检测结果显示，表达菌株的转化效率较原始菌有明显提高，由表达菌45min就能转化酮基布洛芬氯乙酯47.4％，而得到的(S)-酮基布洛芬过量（e.e.%）由野生菌NK13的5.84％提高到55.46％,提高将近10倍，说明该酯酶具有优先拆分得到(S)-酮基布洛芬的特性。

Cloning and expression of esterase gene to enantioselective resolution of (S)-ketoprofen in NK13

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 este firstly.