Screening And Classification Of Lipase-producing Strains And Optimization Of Their Fermentation Conditions

Ten oil-contaminated soil samples were collected from Haikou city.145 lipolytic bacterial strains were isolated from soil samples rich in oil. The screening method is based on the formation of clearance zones on turbid solid media supplemented with emulsified peanut oil. Two strains with high ability of lipases production were obtained by second flask assay. These strains were characterized according to their conventional morphplogical, physiological, biochenmistrica characteristics and also with phylogenetic methods. The strains C737-11 and C7828-5 were identified as B. cepacia and P. aeruginosa, respectively.The optimal reaction conditions of the lipase produced by C737-11 were 37℃, at a pH of 8.0. The optional medium for the induction or supporting of lipase from C737-11 was preliminarily determined, and the lipase activity reached the maximal level of 10.5 U mL-1.Lipase-induction conditions of P. aeruginosa C7828-5 were optimized using monofactorial experiment and orthogonal test. The optimum carbon source and nitrogen source for P. aeruginosa C7828-5 were peanut oil, sucrose and beaf extract. Fours factors, peanut oil, sugar, beaf extract and (NH4)2SO4 were investigated by orthogonal test to obtain optimal medium composition. The effect of pH,temperature,inoculum and speed of shaker on the production of the lipase was also examined with the optimized medium by monofactorial experiment. The optimized condition was:sugar 5 g L-1, beaf extract 20 g L"1, (NH4)2SO4 1 g L-1, MgSO4·7H2O 0.5 g L-1, CaCl2 0.5 g L-1, emulsified peanut oil 120 mL L-1; the initial pH of fermentation was pH 7.0, and the temperature was 28℃; After 72 h, the alkaline lipase activity reached the maximal level of 8.08 U mL"1.The enzymatic properties of the lipase produced by C737-11 and C7828-5 in the fermentation medium was studied. Result showed that they had the same optimal reaction pH and temperature, which were 8.0 and 37℃, respectively, suggesting that they were warm-adapted alkaline lipases. The lipase produced by C737-11 retained 97%,87.7%, and 62.9% of its activity after incubation at 50,60, and 70℃for 20 min, respectively; and 85.9%,55.3%, and 22.6% of its activity after incubation at 50,60, and 70℃for 40 min, respectively, similar to the lipase produced by C7828-5, which retained 97.8%,97%, and 54.3 % of its activity after incubation at 50,60 and 70℃for 20 min, respectively, and 87.2%, 61.5%, and 6.3% of its activity after incubation at 50,60, and 70℃for 40 min, respectively. The lipase produced by C7828-5 was stable between pH 5 and 10, and remained 45% of its activity after incubation at a pH of 10 for 1 h. However, the lipase produced by C737-11 was stable at a pH range of 6-8, and the activity dropped sharply after incubation at pH>9.0.