| Lipase, a special ester hydrolase, can catalyze hydrolysis of triglyceride ester to generate diglyceride, monoglyceride, glycerol and fatty acids. Lipase has been widely used in oil processing, food industry, medical and pharmaceuticals and fine chemical synthesis due to its function of catalyze hydrolysis, transesterification and esterification reaction. It is one of the most important groups of the industrial enzymes. Low-temperature alkaline lipase has a good application prospect in the detergent industry due to its high activity in low and room temperature. In this study, the lipase production of Acinetobacter johnsonii G23was optimized which favors to the further purification and sequence analysis and purified by optimizing the condition of Hitrap Q HP ion exchange chromatography and characteristics of it was studied. The lipase activity of the strain was improved through N+ion implantation.The Optimized culture medium containing(w/v):soluble starch1%, soy peptone1.5%, MgSO40.04%, CaCl20.05%and olive oil1%(v/v).The lipase was purified to homogeneity by centrifugation, leaching, ammonium sulfate precipitation, dialysis, microsepcenyrifugal device (10kD cut off) and Hitrap Q HP ion exchange chromatography. The enzyme was purified about29.59fold with a final yield of13%and the relative molecular mass of the enzyme was determined to be40kDa by SDS-PAGE.The characterization of the purified enzyme exhibited maximum activity at25℃and the optimum pH was8.0.The enzyme shows very strong heat and alkali resistance characteristics, but the organic resistance is not obvious.For detergent process stability, the enzyme was highly stable in the presence of various oxidizing agents, some commercial detergent and protease.The strain A. johnsonii G23was mutated by N+ion implantation and several positive mutations were obtained in the end. Lipase activity of a new strain H-21which had exhibited good genetic stability and the lipase activity can reached to20.92U/ml. |
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