Expression And Characterization Of A Lipase From Proteus Sp.SW1 In Pichia Pastoris

Microbial lipases have been widely used in industry.In this study,the lipA gene was cloned into plasmid pHBM905BDM to generate the recombinant plasmid pHBM905BDM-LipA.Subsequently,pHBM905BDM-LipA was transformed into Pichia pastoris GS115 and positive transformtants were identified by plate screening and verified by PCR.The positive transformtant with the highest ratio of the diameter of a transformtant to its hydrolysis circle was chosen to induce LipA expression in shake flasks.SDS-PAGE analysis of the culture supernatant showed that the the highest expression level reached 294 mg/L at 168 h induction.The purified lipase preferentially hydrolysed triacylglycerols with acyl chain lengths of 8 and 12 carbon atoms.It exhibited optimal activity at pH8.0 and 40?.In addition,it was stable in the range of 25-45? with 70%residual activity and under alkaline conditions(pH 7.5-9.0)retaining 40%activity,respectively.The presence of Mn2+and Mg2+ ions could significantly enhance the activity of the lipase.SDS and Tween20 had obviously inhibitory effects on the activity of the lipase,but 0.1%and 0.05%TrtitonX-100 had remarkably stimulative effects.The enzyme also displayed good stability in several organic solvents indicating its potential application in biodiesel preparation.