Secreted Expression Of Bacillus Pumilus Lipase Genes In Pichia Pastoris

Lipases have been widely used in the hydrolysis and transesterification of triglycerides and in the enantioselective synthesis of a variety of esters. Bacillus pumilus lipase with a small molecular weight and a strong expressive and secretive character is of great potentiality in industrial application. In this paper, the expression vectors contained Bacillus pumilus lipase gene and its directional evolved gene were constructed, and were electroporated into Pichia pastoris for expression and secretion.Use the Bacillus pumilus YZ02 lipase gene and its directional evolved gene BpL-1369 as the experimental material. Through series of gene cutting / ligation and transforming Escherichia coli as a cloning host, the lipase genes were then inserted into the EcoR I/Not I sites of vector pPIC9K with a HIS4 selectable marker. After verifying the validity of construction by DNA sequencing, the recombinant vectors pPIC9K-BpL and pPIC9K-BpL-1369 were electroporated into Pichia pastoris GS115, and the transformants with high copies of the lipase gene were screened on YPD medium with gradient concentrations of G418.Then, the lipase was induced to over-expressed and secreted with methanol fed-bath inducement, and the relative molecular weight of the lipase was determined to be approximately 25 kDa using SDS-PAGE. Fermentation conditions of the recombinant strains were also optimized with the orthogonal experiment. The results showed that when the optimal fermentation condition was methanol loading 1.0%/24h, the starting yeast concentration 4.0(OD600), inducing time 96h, and the starting pH 7.0, the expression level of GS115/pPIC9K-BpL was increased from 1.60U/mL to 4.95 U/mL; and when the optimal fermentation condition was methanol loading 1.0%/24 h, the starting yeast concentration 4.0(OD600) , inducing time 108h and the starting pH 7.0, the expression level of GS115/pPIC9K-BpL-1369 was increased from 1.85 U/mL to 5.45 U/mL .