拆分获得(R)-酮基布洛芬酯酶基因的筛选、克隆与表达

以自筛选出的具有一定不对称拆分外消旋酮基布洛芬氯乙酯能力的野生菌Bacillus megaterium NK13为材料,通过构建其基因文库,筛选得到一个阳性克隆重组子pUC18-NK-HYD3。分析测序结果发现外源片段中包含一段完整的741 bp的开放阅读框,其编码的蛋白中含有酯酶的GXSXG保守序列。经在NCBI的BLAST系统中比对,证明该酯酶基因属于首次发现(GenBank Accession Number: GU143552)。将酯酶基因克隆到载体pET21b(+)中,转化E. coli BL21(DE3),经IPTG诱导后在宿主菌得到表达。SDS-PAGE电泳检测证明该酯酶蛋白分子量约为28 kDa。TLC和HPLC检测结果显示,该酯酶优先水解(R)-型底物,在重组菌菌液体系,转化率为15%时,酯酶拆分获得(R)-酮基布洛芬的过量值(e.e.%)最高,达62.74％;改用重组菌湿菌体的PBS体系后,在转化率为10%~50%时,酯酶拆分获得(R)-酮基布洛芬的过量值(e.e.%)一直保持在73%~76%之间。

Screening, Cloning and Expression of Esterase Gene for Enantioselective Resolution of (R)-ketoprofen

It 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 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%.