Isolation Of Cold-adapted Lipase Producing Strain And Characterization Of Lipase

Cold-adapted lipase become the focus of scientific interests and being the one of the hot research fields in the world, due to its apparent optimal activity at lower temperature(0℃～30℃) and higher catalytic activity. Cold-adapted lipase display higher Kcat and physiological efficiency (Kcat/Km). It can save energy and protect environment. On this thesis, the study mainly concerns about cold-adapted lipase screening,species identification,studying characterization of the lipase and Fermentation Conditions. The mostly content as following: About 200 soil samples were collected from some low temperature regions in Xinjiang and screened by olive oil agar containing Rhodamine B. 11strains showed lipolytic activity at lower temperature. A higher cold-adapted lipase activity producing yeast strain GL-1 was isolated. On the basis of physiology,morphology,structural characteristics and phylogenetic analysis by comparing the sequence of 26SrDNA, the strain GL-1 was identified as Trichosporon pullulans.The cold-adapted lipase was purified by using ammonium sulfate precipitation. Its specific activity was improved in saturation range from 20%to 60%. Properties of the cold-adapted lipase showed: The optimum temperature and pH for the enzymatic reaction was from 25℃to 30℃and 6.5 respectively. Under 30℃this enzyme was stable,about 80% of its activity was remained after 80min, but it was rapidly inactivated at 40℃. It was stable in the pH range from 3 to 10, about 80% of its activity was remained .The enzyme activity was stimulated by K+ but was inhibited by Cu2+,Mn2+.The fermentation technology of cold-adapted lipase produced by GL-1 was studied.The optimal cultural conditions in shaking flask were as follows:medium consisted of 1%glucose,2% soybean meal,3% corn steep liquor,0.5% K2HPO4,0.5% NaNO3,0.25%olive oil and 0.1%Tween80. The cultivation was carried out at the conditions of 20℃with pH 7 in initial, culture medium volumes for 50 ml in 250 ml flask , 24 h germ age, 4% inoculum size, 175 r/min agitation speed for 48 h. With these conditions a maximum lipase yield of 7.17 U/ml was obtained. The result of the orthogonal trial showed the corn steep liquor is the important factor that influences lipase production. and the lipase produced was induced by olive oil.