Screening Of Cold-adapted Lipase Producing Bacterium、Gene Clone And Expression In Escherichia Coli

Lipases(triacylglycerol acylhydrolases EC 3.1.1.3) are a class of hydrolase which catalyze the hydrolysis of triglycerides to glycerol and free fatty acids over an oil–water interface. In addition, lipases catalyze the hydrolysis and transesterification of other esters as well as the synthesis esters and exhibit enantioselective properties. The ability of lipases to perform very specific chemical transformation( biotransformation) has make them increasingly popular in the food, detergent, cosmetic, organic synthesis, and pharmaceutical industries.A total of 55 lipase producing bacteria strains were isolated from the different samples by the Rhodamine B agar plate method. Strain LHY-1 grew fastwith high enzyme production at low temperature.The strain was then choosed for the research of enzyme production and the characteristic. The sequence similarity of 16 S rDNA between LHY-1 and Serratia nematodiphila, Serratia marcescens, Serratia rubidaea, Serratia ficaria were 99%, 99%, 98% and 98% respectively. The strain LHY-1 was preliminarily identified as Serratia sp. by morphological characteristics, physiological and biochemical, and 16 S rDNA sequence analysis.The conditions of growth and enzyme production for strains LHY-1 were optimized. The growth curve of the bacterium was obtained in LB liquid medium with 0 to 10 h of logarithmic phase, with the best culture time for the lipase production at 16 h. The optimum carbon source and nitrogen source for cell growth and enzyme production of LHY-1 were beef extract, yeast powder, respectively. The optimum temperature and pH for cell growth were 10-40℃ and 4.0-10.0, respectively. The optimum temperature and pH for the lipase production were 20℃ and 7.0, respectively. The bacterium grew with salinity of 9%, but the salinity for the best enzyme production is 0.The lipase gene of LHY-1 was cloned and expressed. The expressed lipase was purified by Ni-NTA metal affinity chromatography. The recombinant protein was 36 kDa on SDS-PAGE. The optimal temperature for the enzyme activity was 30℃. The enzyme was stable at temperatures of 0℃-30℃ and pH7.0-11.0. Addition of Co2+, Zn2+, SDS, PMSF, EDTA, EGTA inhibited the enzyme activity, but addition of Mg2+and Ca2+ increased its activity.The lipase of LHY-1 has transesterification activities, which catalyzeed soybean oil to synthesize biodiesel with a transesterification rate of 5.60%.