Xu Lijuan, Zhao Yuhong, Liu Ruien, Zhao Yunying, Zhang Jinhong
College of Life Science of Nankai University, (Tianjin 300071, China.
Sheng Wu Gong Cheng Xue Bao. 2010 Jan;26(1):108-13.
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 Sau3AI partially and recovered the fragment from 2 kb to 6 kb, cleaved the plasmid of pUC18 with BamH I, ligated the 2-6 kb fragment of NK13 genomic DNA into pUC18 plasmid, and then transformed an Escherichia coli strain DH5alpha. 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-beta-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.
我们筛选了一株NK13菌株，其能对消旋酮洛芬氯乙酯进行一定程度的不对称水解生成(S)-酮洛芬。经鉴定，NK13为巨大芽孢杆菌。用Sau3AI对NK13基因组DNA进行部分酶切，回收2 kb至6 kb的片段，用BamH I酶切pUC18质粒，将NK13基因组DNA的2 - 6 kb片段连接到pUC18质粒中，然后转化大肠杆菌DH5α菌株。我们构建了NK13的基因文库，并从三丁酸平板上的文库中获得了一个阳性克隆pUC - NK1。测序结果表明，pUC - NK1质粒中有一个633 bp的脂肪酶基因的完整开放阅读框（ORF）。与GenBank中的基因进行比较，该脂肪酶基因首次被报道（GenBank登录号EU381317）。以pUC - NK1质粒为模板，通过PCR扩增脂肪酶基因，并亚克隆到T7启动子控制下的高表达载体pET21b(+)中。然后将重组质粒pET - NKest1转化到大肠杆菌BL21(DE3)菌株中以生产重组脂肪酶蛋白。用异丙基-β-D-硫代半乳糖苷（IPTG）诱导3小时后，脂肪酶得以表达。SDS - PAGE分析表明，脂肪酶蛋白的相对分子质量约为20 kDa。高效液相色谱（HPLC）结果表明，重组菌株的转化率是野生菌株NK13的50倍。重组菌株的(S)-酮洛芬对映体过量为75.28%，这表明该脂肪酶能优先水解(S)-酮洛芬氯乙酯。如果我们进一步研究该脂肪酶水解消旋酮洛芬氯乙酯的条件，或许可为(S)-酮洛芬的工业化生产提供基础。
