(S)-酮基布洛芬拆分用脂肪酶基因的克隆及表达

Cloning and expression of lipase gene to enantioselective resolution of (S)-ketoprofen

摘要:

本实验室筛选出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，从中筛选得到阳性克隆重组子pUC-NK1。测序分析表明，该重组子质粒中包含一长度为633 bp的脂肪酶基因的完整开放阅读框，核苷酸同源性对比证明该脂肪酶基因属首次发现 (GenBank Accession No. EU381317)，将此基因克隆到原核表达载体pET21b(+) 中构建重组表达质粒pET-NKest1，转化Escherichia coli BL21，经Isopropyl-β-D-Thiogalactoside (IPTG) 诱导在宿主菌中得到表达，经SDS-PAGE电泳检测证明该脂肪酶成熟蛋白分子量约为20 kDa。薄层层析与HPLC检测结果显示，表达菌株转化外消旋酮基布洛芬氯乙酯得到(S)-酮基布洛芬过量 (e.e.％)，由野生菌NK13的5.84％提高到75.28％，提高约15倍，说明该脂肪酶具有优先拆分得到(S)-酮基布洛芬的特性。

Abstract:

We screened a strain NK13 for a certain extent asymmetric hydrolysis the rac-ketoprofen Chloroethyl ester to (S)-Ketoprofen. As identified, NK13 was Bacillus megaterium. Digested NK13 genomic DNA with Sau3AΙ partially and recovered the fragment from 2 kb to 6 kb, cleaved the plasmid of pUC18 with BamH Ι, ligated the 2?6 kb fragment of NK13 genomic DNA into pUC18 plasmid, and then transformed an Escherichia coli strain DH5α. We created the gene library of NK13 and obtained a positive clone, pUC-NK1 in the library from the tributyrin flat. The result of sequencing showed that there was a whole open read frame (ORF) of 633 bp lipase gene in the plasmid of pUC-NK1. To compare with the genes of GenBank, this lipase gene was reported firstly (GenBank Accession No. EU381317). The lipase gene was amplified by PCR, using pUC-NK1 plasmid as template, and subcloned into the high expression vector pET21b(+) under the control of T7 promoter. The recombinant plasmid, pET-NKest1, was then transformed into an Escherichia coli strain BL21 (DE3) for the production of recombinant lipase protein. After 3 hours of induction by isopropyl-β-D-thiogalactoside (IPTG), lipase was expressed. SDS-PAGE analysis showed that the relative molecular mass of the lipase protein was about 20 kDa. The result of high performance liquid chromatography (HPLC) showed that the conversion rate of the recombinant strain was fifty times than the wild strain NK13’s. The (S)-Ketoprofen enantiomeric excess of the recombinant strain was 75.28%, which indicated that the lipase could hydrolyze (S)-Ketoprofen Chloroethyl ester firstly. If we research the conditions of the hydrolysis rac-ketoprofen Chloroethyl ester of this lipase further, maybe it could offer a foundation to product (S)-Ketoprofen industrially.