Optimization Of Lipase Production From Pseudomonas Fluorescens 26-2 And Expression Of The Lipase Gene In Pichia Pastoria

By plates screening with Rhodamine B as indicator and flask fermentation, a strain with the lipase activity higher than 5.0U/mL were obtained, and identified as Pseudomonas fluorescens 26-2 by phenotypic characteristics and partial 16S rRNA sequence.Lipase production condition of P. fluorescens 26-2 was optimized using monofactorial experiment and orthogonal test. The optimum carbon source and nitrogen source for P. fluorescens 26-2 screened through monofactorial experiment were soluble starch and yeast extract, respectively. Four factors, soluble starch, yeast extract, olive and MgSO4 were investigated by orthogonal test to obtain optimal medium composition. The effect of pH and temperature on the production of the lipase was also examined with the optimized medium by monofactorial experiment. The optimized condition was: yeast extract 3%, starch 2.0%, K₂HPO₄ 0.2%, MgSO₄ 0.05%, olive oil 0.2%; the initial pH of fermentation was pH7.0, and the termperature was 30°C; After 60 h, the lipase activity reached the maxium15 U/ml. The optimal pH and temperature of enzymes was 9.0 and 50℃, respectively. The Ca2+ can enhance the activity of the enzyme, while the EDTA can inhibit the activity.The lipase gene of P. fluorescens 26-2 was cloned by PCR. The Open Reading Frame (ORF) of the lipase gene is 1854bp and encodes 617 amino acids. Digested with EcoRI/NotI, the lipase gene was cloned into vector pPIC9K, and transformed into Pichia pastoris GS115 by lithium chloride transformation method. The inducible expression of the lipase in recombinant yeast was checked by Rhodamin B plates and the enzyme activity up to 5.0 U/mL in fermentation liquid.