Selection Of One Rhizopus Sp. Mutant Strain High Producing Thermostable Lipase

Lipase （Triacylglycerol hydrolase, E.C.3.1.1.3） also called three acyl glycerin acylhydrolase, refers to a special class of ester hydrolase which can hydrolyze the triglycerides togenerate corresponding fatty acid and glycerol. The lipases which can be applied in theextreme environment were mostly studied at present. Microorganism was one of the importantsources of lipase. Lipases produced by Rhizopus sp. would be applied widely as it showed ahigh thermal stability and catalytic activity.Strain SFE-L01was preserved in our laboratory and mutated by ultraviolet and diethylsulfate. A mutant strain UD-23was selected finally by preliminary screening and secondaryscreening, whose enzyme activity was2.12amount of the initial.The culture medium and the culture condition of thermostable lipase production by mutantstrain UD-23were optimized. The optimal culture medium for lipase production was asfollows （g/L）: soybean flour30, corn powder20, ammonium sulfate10, K₂HPO₄3, MgSO₄0.5, olive oil5, sodium citric acid1, FeSO₄.H₂O1. The optimal fermentation conditions forlipase production were as follows （g/L）: temperature40℃, the initial pH value7.0, andinoculation quantity5%, volume shake flask35mL/250mL, the enzyme activity of mutantstrain UD-23was33000.00U/mL with the optimal culture medium and optimal fermentationconditions, with enzyme activity enhanced by37.5%.Studies on enzymatic properties of the lipase: the lipase produced by the mutant strainUD-23showed well stability at4℃, the enzyme activity remained85%of maximal activity at4℃after10weeks, lipase activity was optimal at60℃, and had a well thermal stability. Incitric acid-sodium citrate buffer system, enzyme activity was stable, the optimum pH valueis7.0, had a better stability in the pH6.5-9.0. The enzyme activity was strongly activated byFe³⁺, Fe²⁺and Zn²⁺, and inhibited by Cu²⁺、Ba²⁺and Ca²⁺. The results suggested that thelipase produced by the mutant strain UD-23had well thermal stability and pH value stability.