Prokaryotic Expression And Application Of A New Lipase Gene Liphy From Propionibacterium Acnes

Gene cloning, expression, characterization and degradation of lipase from Propionibacterium acnes were explored in this paper.The liphy gene cloned from Propionibacterium acnes genome was inserted into pUCm-T to construct the recombinant cloning vector pUCm-T-liphy. After the DNA sequence was determined, the liphy was subcloned into expression vector pET-15b to construct the recombinant expression vector pET-15b-liphy that would be transformed into the host strain E.coli BL21(DE3) PlysS. Upon the induction of IPTG, the result of SDS-PAGE showed a band with apparent molecular weight of37kDa.However, most part of the protein was expressed in the form of inclusion body. The best expression condition with culture pH6.5, temperature30℃,inoculum-size1:60,IPTG concentration0.4mM,and culture time24h was decided-Expression product was purified by Ni column. The SDS-PAGE analysis showed a single band of purified enzyme of which purity was more than95%and recovery was37.25%. Characterization studies have shown that the specific activity of the enzyme was91.43U/ml, the optimum pH was9.0,the optimum temperature was30℃and it had a thermal stability. In addition, Mg2+and Ca2+have positive effects on the enzyme.However, Co2+, Fe3+, Cu2+, Zn2+, Tween20, Tween80, Triton-100, SDS and EDTA-Na2have negative effects. Km value of the recombinant lipase is36.084uM with the Nitrophenyl palmit as a substrate. Toxicological experiment of the enzyme on the SKOV3ovarian cancer cells and human diploid cells showed a result that the IC50value of them were150μg/mL and300μg/mL. The result of degradation experiments told people that the enzyme could degradate olive oil and cooking oil.This paper increase the likelihood of use of lipase in the detergent industry and environmental governance.