Enzyme Production Optimization Of High Alkalin Lipase Producing Mutant Strain And High-efficient Purification Of Its Alkalin Lipase

Lipase(EC3.1.1.3,triacylglycerol acylhydrolase)is a kind of biocatalysts to hydrolysis long-chain triglycerides to glycerol and lang-chain fatty acids.It can hydrolysis Triglyceride to fatty acid and glycerol in the interface of oil-water.Lipase is widely used in food,medicine,scour,leather and paper industry.In this research work,rejuvenation and enzyme production optimization were used to improve the lipase production of Lp-11-â‘£.Alkalin lipase was purified high efficiently to reduce purification costs.We used plate streaking and coating methods to rejuvenate Lp-11 which was conserved by our laboratory.Finally,the high yield Lp-11-â‘£strain was selected.Stability test demonstrated that the lipase activity remained the same level after five generations.Lipase production condition of Lp-11-â‘£was optimized using seriatim-factorial experiment and orthogonal experiment.The optimum carbon source and the optimum nitrogen source for Lp-11-â‘£screened through seriatim-factorial experiment were glucose and peptone,respectively.By orthogonai experiment we obtained result of the optimum culture medium was:glucose0.5%; peptone5.0%;Na₂HPO₄0.7%;KH₂PO₄0.5%;Tween80 1.0%;olive oil2.0%;pH8.0;inoculation quantity 3.0%(V/V);bacteria age 12h;revolution 250r/min;temperature 30℃.After 30h incubation with the optimum condition,a maximum lipase yield 366.996U/mL was obtained,which was 84.541%higher than that under beginning culture mediums.The alkaline lipase of the supernatant was purified to homogeneity through aqueous two-phase extraction and hydrophobic interaction chromatography on Phenyl-Sepharose Fast Flow column, and the pure alkaline lipase was characterized by SDS-PAGE and IEF electrophoresis.65.15%of the lipase activity was recovered,and purification fold was 26.81.The specific activity was 9901.02 unit/mg.The molecular weight of the lipase is 265.6KD and submits are 64.8KD determined by gel filtration chromatography on Superdex-200 and SDS-PAGE method.Isoelectric point of the pure lipase was 5.43 showed by IEF-PAGE.