Clonagem, expressão e caracterização enzimática de lipase recombinante em Pichia pastoris

Abstract

As triacylglycerol lipases, (E.C.3.1.1.3), are called lipases, they catalyze carboxylic ester reactions to triacylglycerol to form free fatty acids, have a characteristic of catalytic activity in aqueous hydrolysis medium. Outside the aqueous medium, esterification, interesterification or transesterification reactions occur. The potential biotechnological, lipolytic enzymes, and their industrial industry for their global leukopithetic lipopruticent, which in the industrial industry, which must be important in the importance of biocatalysis, favoring greater sale in the process. Justifying the study of new molecules with high biotechnological potential to supply new needs of the world market, especially those coming from the Amazon, the region seems richer in biodiversity. In order to enzymatically characterize a recombinant lipase in Pichia pastoris, the lipase coding gene was isolated from a library. The production of clones in the minimum in amino acids and the clones were detected in medium. Selection of recombinant clones was completed in BMMY containing 1% tributary solids in BMMY medium with positive clones grown in 0.5% methanol-induced submerged fermentation for lipase production. The structural region of the lipase gene was enlarged by 1200 bp. Degrading halos were observed around the colonies. The presence of the 50 kDa enzyme in the SDS PAGE gel was verified. Enzymatic characterization was important to verify the biotechnological possibilities of lipase through colorimetric assays, which were positively activated for enzymatic research of 50.80 U / mL with optimum temperature of 50 ° C and pH of 7 being the best time of 5 minutes