Cold Resistant Pseudomonas Pseudomonas Sp. The G6 Lipase Production Optimization And Enzymology Properties Research

Lipase could hydrolyze triglycercide to glycerol and fatty acid which is widelyapplied to industrial production such as food, fats,medicine and chemical.The presentresearch on lipase is mainly focused on medium-temperature lipase, whose optimaltemperature of hydrolyzation is around40℃，and has no catalytic activity at lowtemperature. The cold-adapted lipase has the feature of have a high catalytic activity atlow temperature, and still have catalytic activity at0℃.This makes cold-adapted lipasetake advantages of applying to fat processes, environmental restoration anddetergents,it also arouses widely attentions in recent yearsIn this paper,4strains of psychrotrophic bacteria were identified aslipase-producing bacteria with high capability of producing cold-adapted lipase,separatedly named G6，H8，Z4and W7by using the method of Rhodamine B. Thestrain G6was proved to be with the highest lipase activity, and further identified as astrain of Pseudomonas sp. based on morphological，physiological characteristics andphylogenetic analysis of16SrDNA. The strain G6was named as Pseudomonas sp. G6.In order to improve the lipase yield produced by the strain of Pseudomonas sp.G6the fermentation conditions were optimized by single factors experiments,including carbon soure, nitrogen source, inorganic salt,cultural temperature and soon.The optimal fermentation nutritions was determined as glucose5.0g·L^-1,（NH₄）2SO₄1.0g·L^-1,peptone10g·L^-1,K2HPO41.0g·L^-1,Tween-8010mL·L^-1,MgSO₄2.0g·L^-1,NaCL2.0g·L^-1. By seeding2%bacteria liquid into the medium with a volume of20%ofincubator, the initial pH7.5, then incubated at20℃with rotation speed of100rpm, theyield of the lipase reached to65.66U·mL^-1.Base on the results of the single factor experiment, response surface methodologywas applied for further optimization. Finally, the optimized culturing conditions wereglucose5.0g·L^-1,（NH₄）2SO₄1.0g·L^-1,peptone12g·L^-1,K2HPO41.0g·L^-1,MgSO₄2.0g·L^-1, NaCl1.2g·L^-1Tween8010mL·L^-1,cultural temperature20℃, the initial pH7.5, theseeding volumes2%, medium volume20%of the incubator and Speed110rpm. Afterthe optimization, the yield of the lipase increased significantly and reached to107.03U·mL^-1.The lipase was purified by a subsequent procedure of homogeneity, acetoneprecipition, and high-speed countercurrent chromatography.SDS-PAGE analysisindicated that the molecular mass of the purified-lipase was about65KDa.The purifiedlipase characteristic analysis revealed that the optimal reaction temperature was at30℃, and30%activity remained when placed to a temperature as low as5℃.Thelipase was thermal lability, only20%of its activity was remained after30minincubation50℃. The lipase activity was stable at pH range from7to8, its optimal pHwas7.5.It’s catalyse activity was inhabited by Cu²⁺、Hg²⁺、Fe³⁺、Zn²⁺,but promoted byMn²⁺.The detergents of EDTA、Tween80、SDS and Tritonx^-100could inhabit itsactivity obviously.