Screening Of Lipase Producing Strain And Study On Its Culture Conditions And Enzymatic Characteristic In Gansu Pastureland

Lipase, the abbreviations of TG hydrolase, is an important class of ester bond hydrolase,which can be hydrolyzed triglycerides to fatty acid and glycerol in the oil-water interface,also can catalyze the synthesis of lipids transesterification. Therefore, lipase have wide application in food, pharmaceutical, leather production, detergents, paper, environmental protection and many other industries areas. In recent years, microbial lipase become the focus of attention for its unique advantages and has been more and more widely in industrial applications.In this study, lipase-producing microorganisms were screened from yaks butter, Qula and soil of yak grazing plot in the Tianzhu and Gannan pasturing area of Gansu province. Isolated and identified the microorganism which had high lipase activity after secondary screened and studied the condition of lipase production and purification of lipase and the characteristics of lipase.The main contents of the study were as following:1. Screening and identification of lipase producing microorganism.More than 10 lipase-producing strains were isolated from the Tianzhu and Gannan pasturing area of Gansu province, in which, the high lipase-producing strain S122 was isolated by repeated screening and its lipase activity is 39U/mL.Through the morphological identification, S122 was branches, forming single or chains meristem arthrospores and the arthrospores were square or cylinder shaped and oval or round shaped,and 26s rDNA sequence and phylogenetic analysis,the strain S122 was finally ascertained to be Geotrichum sp.S122.2. Take the Geotrichum sp.S122 fermentation conditions:culture temperature,the initiation pH of the culture medium,inoculation volume,rotation speed,liquid volume in flask and the culture time for single factor experiment;and then Plackett-Burman and CCD design were employed for optimization of fermentation medium components for lipase production.The result showd that the culture temperature was 28℃,the initiation pH of the culture medium was 8.0, inoculation volume was 4%,rotationa speed was 200r/min, liquid volume in flask was 60mL/250mL,the culture time was 96 h, sucrose was 35.54g/L, peptone was 30.00g/L, urea was 5.00g/L, magnesium sulfate was 1.01g/L, zinc sulfate was 0.50g/L, ferrous sulfate was 0.50g/L,manganese sulfate was 1.02g/L, Calcium chloride was 1.00g/L and dipotassium hydrogen phosphate was 5.00g/L.3. Isolation and purification lipase.The lipase of Geotrichum sp.S122 was purified by aqueous two-phase(ATP) systems extraction.It was discovered that PEG 10%,(NH4)2SO4 10%, NaCI 2%,pH 5.0 was the best conditions of ATP extraction, and the recovery rate of lipase was 78%.4. The effect of optimum temperature and pH, heating and pH stability, different metal ions, the concentration of Mg²⁺ were studied on lpase purified from Geotrichum sp.S122.The result showed: the optimum temperature and pH of lipase was respectively 30℃and 10.0;the variation amplitude of lipase was not obvious when keeping the temperature of heat preservation at 30 and 40℃respectively, but at 50 and 60℃the variation amplitude was obvious;the pH stability of lipase was 8～10; so the results indicated the lipase was moderate cold-adapted and alkaline lipase. The lipase activity was inhibited by K⁺,Na+,Zn²⁺,Ca²⁺,Fe²⁺ and Mn²⁺,and the Zn²⁺ had strongly inhibitory effect;Mg²⁺ had activation effect on lipase activity.