Screening Of Alkaline Lipase Producing Bacteria And Studying On Gene Cloning

Oily soil samples were colleted. After repeating enrichment culture for several times, the culture fluid was spread on the agar plate, in which tributyrin served as substrate and Victoria blue as pH indicator. About 300 strains producing lipase or esterase were isolated. The existence of lipases has to be verified by using olive oil agar containing Rhodamine B. Five strains were isolated as the alkaline lipase producers. Among them an isolated bacterial strain secreting a large amount of alkaline lipases was selected. By testing morphologic and physiological-biochemical characteristics the strain was identified as Pseudomonas sp.. We called it Lzl.Studying on the effects of nitrogen sources, carbon sources and other influence factors has optimized the lipase fermentation conditions for the strain. The optimal medium composition for the lipase production is(%) : yeast extract 1. 5, soybean meal 1, NaCl 0.2, K2HP04 0.2, NaH2P04 0.2, MgSO₄7H20 0.01. The optimal conditions for lipase producing are inoculation for 10%, seeds age for 12h, culture medium volumes for lOmL in lOOmL flask, initial pH8.5, cultivated aerobically at 30℃ with an aeration rate of 150r/min for 46 hours. Then a maximum yield of 19. lu/mL was obtained.By studying on the supernatant of the culture fluids, the enzymatic characteristics were concluded. The optimum temperature and pH for it were 40℃ and 8.5. It was stable in the pH range from 7. 5¹0. 0 and had good thermal stabilities梐bove 80% enzyme activity remained after incubated in the temperature range from 30⁵5℃ for 3Omin. The Lipase activity was stimulated in the presence of metal ions such as Li+, Na+, K+, but was inhibited by Ni+ s Co2+ Zn2+ . In addition, it had good detergents stabilities.To improve the strain' s lipase yield, it was treated by means of UV mutagenesis. The mutant Lzl-10 was selected and produced lipase activity 10% higher than that of parent strain.The mutant Lzl-10 was used as the donor for the isolation of alkaline lipase gene. Chromosomal DNA from Pseudomonas sp. Lzl-10 was partially digested with the restriction enzyme. The plasmid DNA was cut and dephosphorylated. The linearized plasmid DNA was ligated to the Pseudomonas sp. DNA fragments and resulting recombinant plasmids were used to transform Escherichia coli XLl-blue. Then Pseudomonas sp genomic library was constructed.