Cloning and Expression of a Lipase Gene from Indigenous Bacillus pumilus in Pichia pastoris Print

Lipases (Triacylglycerol acylhydrolases; EC 3.1.1.3) catalyze the hydrolysis of triacylglycerol to glycerol and fatty acids at the interface between water and oil. These enzymes are versatile biocatalysts with a wide range of application in food, dairy, leather, paper, and pharmaceutical and detergent industries. In this study, mesophilic lipase gene from an indigenous Bacillus pumilus F3 was cloned and expressed in methylotrophic yeast Pichia pastoris. The lipase gene of 648bp with natural signal peptid sequence from B. pumilus F3 was amplified by PCR and inserted into the PGEM5Zf vector. The lipase gene was excised from the recombinant plasmid with BamHI and EcoRI enzymes and ligated to the pPIC9 linearized with the same enzymes. The recombinant plasmid was confirmed by the PCR and restriction enzyme digestion. The Bgl II linearized Ppic9 recombinant plasmid was introduced into the yeast P. pastoris GS115 by electroporation and confirmed by PCR. One of the lipase expressing P. pastoris transformants was cultivated in expression medium. The expression of the lipase gene was confirmed by p-nitrophenyl palmitate test and SDS-PAGE.