Cloning And Expression Of Lipase Genes From Thermophilic Fungus Neosartorya Fischeri P1

Lipase is an enzyme that hydrolyzes the breakage of ester bonds of triglycerides to release fatty acids and glycerol at the interface between oil and water. Besides hydrolysis activity, it also has the ability to catalyze ester synthesis and trans-esterification. These multiple functions make lipase tremendously valuable in application in the food, medicine, bioethanol and detergent industries. Thermophilic lipase can meet the demand requirements of harsh industrial process, and thus is attracting much attention for basic research.In this study, the thermophilic fungus Neosartorya fischeri P1 with significant lipase-producing ability was used as the donor strain. After 24 h cultivation at 45℃, strain P1 showed great growth, secreted many lipases, and clarified the olive oil medium to transparency. A total of seven lipase genes were cloned from the genome of N. fischeri P1 based on homology. The gene fragments coding for the mature proteins without the signal peptides were subcloned into pET-22b (+) and expressed in Escherichia coli BL21 (DE3). With IPTG induction, all E. coli cells harboring recombinant plasmids showed lipase activities, which were too low for purification and enzyme characterization.Heterologous expression of the genes described above was also conducted in yeast. The gene fragments were subcloned to expression vector pPIC9 and expressed in Pichia pastoris GS115. Only two lipase genes, Lip24 and LipO9, showed detectable lipase activity. The expression level of LipO9 was higher, reaching 2 g/L in shake flasks. Recombinant Lip24 and LipO9 were also produced by high-density fermentation in 3.7 L fermentor with 96 h methanol induction, and showed the lipase activity of 4.2 U/mL and 1900 U/mL, respectively. Enzyme characterization indicated that purified recombinant Lip24 and LipO9 were acidothermophilic. The optimal pH of LipO9 was 5.0, and the enzyme showed broad pH adaptability (>70% activity at pH 3.5-8.0 and 40% activity at pH 2.0). The optimal temperature of Lip09 was found to be 60℃, and the enzyme remained 37% activity at 70℃ and >80% activity at 50-65℃. Lip09 was highly thermostable, retaining >95% activity after incubation at 50℃ for 30 min and 51% activity at 55℃ for 30 min. Lip24 exhibited maximum activity at 50℃ and pH 7.0, and had broad pH stability (retaining >90% activity after incubation at pH 3.0-7.0 for 1 h at 40℃ and retaining 40% activity at pH 2.0).In summary, an acidothermophilic recomibinant lipase with high activity was obtained through gene cloning and heterologous expression. This study provides not only a good material for the study of acidophilic and thermophilic mechanisms, but also a good candidate enzyme for potential industrial applications.