Purification Of Cold-adapted Lipase From Pseudomonas Lip35 And Enzyme Characterizations

Cold-adapted lipase is a kind of hydrolase that mainly applied in fields such as food, medicine,environment protecting etc.This research established a set of method which is easy,fast and inexpensive for extracting and purifying the cold-adapted lipase from Pseudomonas Lip35,and then studied the characterizations of purified enzyme systematically.This experiment use a strain of Pseudomonas Lip35 which preserved in the laboratory as the fermentation strain to ferment cold-adapted lipase in the 10L fermentation tank.Cold-adapted lipase was purified by using ultrafiltration,ammonium sulfate fractionation,Sephadex G-25,DEAE-cellulose ion-exchange chromatography and Sephacryl^TMS-200 gel filtration.The result indicated that by controlling the detail conditions of ion-exchange chromatography and molecular sieve chromatography,most miscellaneous protein had been removed.The purified cold-adapted lipase showed a single band in PAGE electrophoresis.The specific activity reached 834.72 U/mg and which was 26.02 folds higher than the crude fermenting liquor and the yield was 15.76%.In the base of obtaining a single component cold-adapted lipase from Pseudomonas Lip35,the characterizations of purified cold-adapted lipase was studied.The molecular weight of the enzyme was estimated to be 39.8 kDa by SDS-PAGE.The optimum temperature of the cold-adapted lipase was 25℃.The activity was almost unchanged between 15～35℃,and was always above 80%.the enzyme was thermal lability,only 10 %of its activity was remained after 10 min incubation at 60℃and higher temperature. The lipase showed high lipolytic activity from pH 8.0 to pH 9.5 and its optimal pH for activity was 8.1,belonged to the alkaline cold-adapted lipase.It has a good pH tolerance, the relative enzyme activity were all above 80%after being kept 24h at room temperature (25℃)in the condition of pH 6～9;Metallic ions had influence on cold-adapted lipase. The lipase activity was inhibited strongly by Al³⁺,Zn²⁺,Fe²⁺and was inhibited a litter by other metallic ions.But was stimulated by Ca²⁺(more than two times of the initial activity was achieved).The EDTA test testified that this lipase was Ca²⁺dependent enzyme.The denaturation engendered by SDS on enzyme protein can severely affect the enzyme activity.For p-NPP the Km is 14.31mmol/min,Vm is 7.93mmol/L which is tested by dual-reciprocal graph.This paper has done some research on the cold-adapted lipase including,purification and the enzyme properties and this will be a foundation for the study of its catalysed mechanism,clone and industrial application.