Study On The Cloning And Expression, Characterization Of Lipase From Paenibacillus Pasadenensis CS0611 And The Synthesis Of Ethyl Palmitate

Lipase?EC 3.1.1.3?can catalyze various reactions including hydrolysis of eater,ester synthesis and transesterification.As biocatalysts,lipase is characterized by substrate richness,mild reaction condition and high selectivity.Due to these features,lipase is widely used in the production of food and daily chemical as well as the filed of pharmaceutical and bio-energy.Microbial lipase is the main source of lipase in industrial application.Searching for lipase with special enzymatic properties is vital for futher expanding utilization of it.In this study,Paenibacillus pasadenensis CS0611 was used as the starting strain and a potential lipase gene was found according to the bioinformatics analysis of the whole gene sketch of it.Finally,the lipase gene lp2252 was successfully cloned and expressed in Escherichia coli and Pichia pastoris respectively.The main results are as follows:The gene lp2252 from Paenibacillus pasadenensis CS0611 was cloned into the expression vector pET-28a,and the recombinant plasmid was introduced into the E.coli BL21?DE3?.The recombinant strain BL21?DE3??pET-28a-lp2252?was obtained successfully.An obvious band at 45 KDa was obtained by SDS-PAGE after ultrasonication,which was consistent with the expected target protein size,indicating that the target gene lp2252 had been successfully expressed in E.coli BL21?DE3?.Lipase Elp2252 was purified by Ni affinity chromatography,and the specific activity was 456.33 U/mg.The enzymatic properties of Elp2252 were studied.The results showed that the optimum pH and temperature of Elp2252 were 7.0 and 50°C respectively.Elp2252 showed high stability under the condition of pH 3.0⁸.0 and temperature of 20⁵0°C.Metal ions such as Ca²⁺and Mg²⁺could strongly activate its activity.Substrate spectrum analysis indicated that Elp2252showed stronger selectivity to pNPC₈.Elp2252 was active on pNPC₈ with an apparent K_m value of 0.12 mmol/L and V_maxax value of 3.33?mol/min.When investigating its organic solvent tolerance,we found that solvents with high polar such as methanol and ethanol could obviously activate its catalytic activity.In order to further improve the expression level and activity of the lipase,the lp2252 gene was also cloned into the expression vector pPICZ?A,and then integrated into the genome of Pichia pastoris host X33.The recombinant strain X33?pPICZ?A-lp2252?was constructed successfully.An obvious band at 51 KDa was found by the SDS-PAGE analysis of the supernatant,which was in accordance with the expected target protein size,indicating that the lp2252 had been successfully expressed in Pichia pastoris X33.The activity of Ylp2252 was472.31 U/mg,which increased by 15.98 U/mg compared with Elp2252 under the same condition.Futhermore,the enzyme properties of Ylp2252 were studied.The results showed that the optimum pH and temperature of Ylp2252 were 7.0 and 50°C respectively.Besides,Ylp2252had a stronger specificity for the pNPC₆.The synthesis of ethyl palmitate catalyzed by Elp2252 was investigated.The optimal molar ratio of palmitic acid to ethanol was 1:2,the optimal reaction temperature and time were 40°C and 6 h,and the optimal amount of water and enzyme were 4%and 0.2g respectively.Under the above condition,the yield of ethyl palmitate reached 50.3%.