Optimization Of Burkholderia Mana SYBC Fermentation For Lipase Production And Enzymatic Characterization

Lipase, which catalyses the hydrolysis of long chain triglycerides, played an important role in industrial catalyzation. Cold-active lipase had relative high catalytic activity at low temperature and good prospect in applications. Most cold-active lipase producing microorganisms were psychrophiles. Usually, psychrophiles were difficult to culture. In this paper, a high lipase producing strain had been screened from mesophilic natural soil samples. According to the morphological, physiological, biochemical identification and 16S rDNA sequence analysis, this strain belonged to Burkholderia and named as B. mana SYBC LI-1.The fermentation conditions of B. mana SYBC LI-1 was investigated in shake flask. The optimized fermentation medium contained (g/L): soluble starch 10, yeast extract 15, NaNO3 0.252, olive oil 40, OP 10. While the optimized fermentation condition was: pH 7.5, 30℃, 200 r/min, 30 mL/250 mL, and 10% (V/V) of the inoculum size, for 48 h. Under such conditions, the lipase activity reached 85.23 U/mL, which was 3.63 folds of that before optimization.Three methods were researched in the purification of B. mana SYBC LI-1 lipase, including ammonium sulphate fractional precipitation, ethanol fractional precipitation and aqueous two phase system (ATPS). The optimal system of ATPS was: PEG 2000 (W/W) 10%, K2HPO4 (W/W) 15%, pH 8.0. Under these conditions, the partition coefficiency of the lipase is 3.7, purification fold is 4.1, and lipase yield is 86.5%. This system could be applied to separation of commercial production of lipase.Furthermore, the characteristics of crude lipase was investigated, its optimal pH was 9.5, with relative activities were all above 75% at the rang from pH 3.5 to pH 10. Its optimal temperature was 20℃, and residual activity retained 70% at 0℃, belonged to the cold-active lipase. It was proved to have good temperature stability, more than 75% of the initial activity retained at 60℃for 60 min. The lipase activity was stimulated by Fe2+ and Fe3+, while inhibited by Co2+ and EDTA at the concentration of 1 mmol/L. Moreover, the lipase was resistant to alcohol.