Cloning and expression of a lipase gene in Bacillus subtilis

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.

Key words:Keywords: lipase; inducible expression; enantioselectivity; (S)-ketoprofen