The Expression, Characterization And Application Of A Recombinant Lipase From Bacillus Sp.

A gene coding for Lipase BSL was amplified by PCR from genomic DNA of a Bacillus sp.. The DNA fragments were cloned into vectors pQE-30 and pET22b(+) to construct the expression plasmids, and the two new plasmids were transformed into E. coli Ml5 and E. coli BL21, respectively. After IPTG induction, the recombinant M15/pQE-30-BSL showed the highest hydrolytic activity, which was 17.25-fold higher than that of the wild Bacillus sp. strain. The racemic flurbiprofen ethyl ester was hydrolysed by the recombinant strain to (R)-flurbiprofen with an enantiomeric excess (ee_p) >99%, which was higher than 89.2% of the wild strain. To improve the expression level of the recombinant lipase, the expression conditions were optimized with optimal induction temperature at 37 ℃ and IPTG concentration at 0.1mmol/L. The high density culture of M15/pQE-30-BSL was also studied, final biomass of 55g wet cells weight/L culture broth and activity of 12300U/L culture broth were obtained.After breaking cell wall by supersonic wave, the recombinant lipase was purified by one single step of immobilized metal ion affinity chromatography, yielding specific activity of 30.25U/mg protein as assayed by the hydrolysis of flurbiprofen ethyl ester. The molecular weight of the purified lipase was determined by SDS-PAGE and mass spectrometry to be about 28 kDa, indicating that the mature BSL protein was a monomer. Using flurbiprofen ethyl ester as substrate, characterizations of the purified enzyme revealed that the optimal temperature and pH for the hydrolysis reaction were 55℃ and 9.0, respectively. The enzyme was stable below 55 ℃ and at pH7.0-11.0. The substrate specificity study showed the highest activity was found with p-nitrophenyl butyrate as substrate. The metal ions Ca²⁺ and Mg²⁺ could activate the lipase, whereas Zn²⁺ showed great inhibitory effect on activity. Moreover, except SDS, most surfactants could increase the lipase activity, especially the TritonX-100.The resolution of racemic flurbiprofen ethyl ester catalyzed by the recombinant lipase BSL was studied. The conversion experiment was performed under the optimized conditions at pH 9.0, 50 ℃ and 50mmol/L of initial rac-flurbiprofen ethyl ester. A successful enzymatic chiral resolution was achieved with an enantiomeric excess of 99.5% at 45%-50% conversion, and the enantiomeric ration (E) was 1013.