拆分获得(S)-酮基布洛芬脂肪酶基因在枯草芽孢杆菌中的克隆与表达

Cloning and expression of a lipase gene in Bacillus subtilis

摘要:

摘要：【目的】筛选具有不对称拆分消旋酮基布洛芬氯乙酯能力的脂肪酶基因，构建其表达分泌型工程菌，并进一步提高该脂肪酶的立体选择性。【方法】以自筛选出的一株具有不对称拆分消旋酮基布洛芬氯乙酯能力的菌株NK13为材料，通过构建其基因组文库，筛选具有不对称拆分消旋酮基布洛芬氯乙酯能力的脂肪酶基因。通过构建该脂肪酶基因的分泌型诱导表达载体pHY300-plk-sacR-gene，将其转入枯草芽孢杆菌WB600，获得基因重组菌WB600(pHY300-plk-sacR-gene)。用SDS-PAGE检测其表达和转化情况，采用非变性聚丙烯酰胺凝胶电泳的方法纯化脂肪酶；并利用TLC和HPLC检测该酶的立体选择专一性。【结果】得到了具有专一性拆分获得(S)-酮基布洛芬能力、长度为633 bp的脂肪酶基因（GenBank登录号为：EU381317）。该脂肪酶在枯草芽孢杆菌WB600中得到了分泌表达。TLC和HPLC检测结果显示，纯化的脂肪酶对底物转化40 h时转化率为30%， 生成(S)-酮基布洛芬的e.e.%值最高，达60.02%，与未加Tween-80的枯草芽孢杆菌转化子体系相同。而在含Tween-80的环境下，枯草芽孢杆菌表达重组菌对底物转化36 h时转化率约为45%，生成(S)-酮基布洛芬的e.e.%值最高，达93.64%，是野生菌NK13的16倍。【结论】从NK13号菌株中筛选得到的新的脂肪酶具有很高的不对称拆分获得（S）-酮基布洛芬的能力， 实现了NK13菌中633 bp脂肪酶基因在枯草芽孢杆菌中的分泌表达，研究证明Tween-80能提高该脂肪酶的拆分专一性。

Abstract:

Abstract: [Objective] We screened a new lipase gene to asymmetricly hydrolyze the rac- ketoprofen Chloroethyl ester, to construct a secretive expression strain and to improve its enantioselectivity.[Method] Strain NK13 was screened for asymmetricly hydrolyzing the rac-ketoprofen Chloroethyl ester. We prepared NK13 chromosomal DNA library to screen the lipase gene. An inducible secretion vector (pHY300–plk- sacR-gene) was developed based on the sequence that was ligated by the lipase gene and the regulatory region and the signal sequence (sacR) of the SacB gene. Transforming Bacillus subtilis WB600 with this vector, we attained a new recombinant strain. The expression of the lipase gene was analyzed by SDS-PAGE. The lipase was purified through native PAGE. The ability of enantioselected Ketoprofen Chloroethyl ester of the recombinant was identified by thin-layer chromatography (TLC) and HPLC. [Results] A 633bp lipase gene with enantioselectivity was attained (GeneBank accession number: EU381317). SDS-PAGE analysis showed that the expression of the inserted lipase gene could be induced by addition of sucrose into the medium. The results of TLC and HPLC showed that the highest enantiomeric excess of (S)-Ketoprofen was 93.64% at 36 h, and the conversion rate was about 45% of the Bacillus subtilis recombinant strain by adding Tween-80. The enantiomeric excess of (S)-Ketoprofen of was 16 times of that of NK13. For the purifed lipase the highest enantiomeric excess was 60.22% at 40 h and the conversion rate was about 30%, which was same to the recombinant strain without Tween-80. [Conclusion] The lipase screened from NK13 had high ability to hydrolyze the rac-ketoprofen Chloroethyl ester to (S)-Ketoprofen. The 633 bp lipase gene of NK13 was induced to secretively express. By adding Tween-80, the enantioselectivity of the lipase was improved.