Cloning, Expression Of Lipase Genes From Different Microorganisms

Lipase is a kind of important enzyme hydrolyzing esters bond of triglyceride. In this paper, We cloned, expressed and characterized a novel lipase gene lipB from Serratia marcescens ECU1010, and study on the fermentation of the recombinant lipase. In addition, we cloned seven lipase genes from Aspergillus Clavatus, and achieved their high-level expression in E. coli.LipB from S.marcescens ECU1010 was cloned and overexpressed in E.coli(Genbank Accession:HM440338).The lipB gene is 1845bp long, encoding 614 amino acids.The solubility of the expressed protein was improved by the addition of Ca2+ during IPTG induction.The maximum lipase activity,153720 U/L,was obtained when l.OmM of IPTG was used as an inducer at 15℃overnight,in the presence of 5mM of Ca2+.Based on shake flask cultures,the lipase fermentation was carried out in a 5-L fermenter,containing 10g/L trptone,10g/L yeast extract,10 g/L NaCl,2 g/L NH4Cl,8 g/L K2HPO4,4 g/L KH2PO4,1 g/L MgCl2,pH 7.0.30% of dissolved oxygen (DO) concentration contributed to the enhancements of lipase yield during the whole process, obtaining cell with 245g wet weight.By ammonium sulfate precipitation and Sephadex G-75 gel filtration purification methods,LipB was well purified. Subsequently,the charaterization of the crude enzyme was studied.Effects of temperature, pH value,stability of pH, metal ions on the activity of lipases from S.marcescens ECU1010 were investigated.The results indicated that LipB was sensitive to the changes of temperature and concentration of metal ions. The optimal temperature and pH was 40℃and 8.5.The enzyme was stable in pH range 5 to 7.Its activity was found to increase in the presence of metal ions such as Ca2+.Compared with reported LipA from S.marcescens ECU1010,effect of various organic solvents on the lipB stability was obviously different.To enrich the lipase gene resources,different kinds of extraction methods(modified urea,CTAB)were applied to extract the genomic DNA of Aspergillus Clavatus. We cloned seven lipase genes according to the reports of lipase genes on Genebank and expressed them in E.coli,which laid the foundation for study on structure and function of Aspergillus lipases and development of new uses.