Cloning, Expression And Characterization Of Thermostable Lipase From Chaetomium Thermophilum

Lipase (triacylglycerol acylhydrolases, EC 3.1.1.3), which widely exists in organisms, catalyzes both hydrolysis and synthesis of the ester bonds of triglycerides at oil-water interface. Lipases produced by microorganisms are much more attractive in industrial field because of their various species, easy culturing and great output. Thermalstability lipases show great commercial value because of their stable ability in high temperature, so there has been an increasing interest in them.Chaetomium thermophilum is a thermophilic fungus with high growth temperature, which has been paid more attention with the expectation to produce thermostable enzyme. However the high culture temperature needed by C. thermophilum to produce lipase in a large number is difficult in industrial applications. In recent years, most studies focus on the expression of lipase in different expression systems by gene engineering methods.During growth in an induced medium containing olive oil, C. thermophilum produced lipase. Degenerate primers were designed on the conserved domain of other reported lipases, and the full length cDNA encoding the lipase gene was obtained by Tail-PCR and RT-PCR. We named the lipase gene as lm. DNA sequencing revealed that genomic DNA of the C. thermophilum lipase gene lm was 1180bp and contained four introns. And the cDNA of gene lm we cloned contains an ORF of 870bp encoding 290 amino acids. The DNA and cDNA sequence has been registered in GenBank with accession number GU338249 and GU338248, respectively.Coding region of lm and expression vector pPIC9K were digested with EcoR I and Not I, and ligation was carried out in vitro. The construct plasmid pPIC9K/lm was linearized with restriction enzyme Sac I (insertion at 5'AOX1), transformed into Pichia pastoris GS115 competent cell by electroporation methods, and screened for His+Mut+ transformants on MD and MM plates. Multicopy integrant transformants obtained by PCR analysis and G418 screening, were induced by methanol. These integrants were selected by expression levels of interest protein every 24 hours and we named the engineering strains with high expression level as GS-LM-18. After six days induction, the transformant GS-LM-18 had the highest activity 19.77U/mg and the expression level was 0.93mg/ml.The nature of the expressed lipase was tested and characterized. After subculture for 10 generations on YPD plate, transformants GS-LM-18 kept consistent with the original expression level and the interest gene lm was integrated into P. pastoris genome. The expression level was also kept stable. The expression lipase LM exhibited optimum catalytic activity at pH 10.0 and was thermostable at 60℃. The half-life of the enzyme at 70℃was approximately 40 min. The enzyme was stable in the range of buffer from pH 9.0 to 11.0. Ca²⁺ enhanced the enzyme activity greatly, whereas Ag⁺, Fe⁽2+0, K⁺, Zn²⁺ caused obvious inhibition.The recombinant lipase was secreted into the culture medium by the yeast P. pastoris in an active form, and was a thermostable enzyme. It implied that the expressed lipase could be widely applied in the biological fields and chemical industry.