Functional expression of Candida Antarctica Lipase in Recombinant Pichia Pastoris

Abstract

The nature s only catalysts are considered to be the enzymes. Enzymes offer the advantages of mild conditions of reactions, specificity, and reduced wastage of chemicals. It is required in 0.1 % - 1.0 % of the substrate, minimal waste treatment cost and contribution to the BOD, and the industries utilizing enzymes in their processes require less cost to set up the facility. Lipases are the principal enzymes that are at the forefront of this development and are being used to resolve the racemic mixtures, synthesis of chiral building blocks for pharmaceuticals, agrochemicals and pesticides. The Candida antarctica lipase B gene was amplified from the genomic DNA isolated from Candida antarctica and cloned in pPICZand#945;B, pGAPZand#945;B, pPIC9K vectors, and electroporated into Pichia pastor is strains GS115 and KM71 for over expression of a 33kDa CALB protein. The expression studies on fermenter level were carried out by a three phase process in defined medium. An initial batch phase on 4 % glycerol, followed by fed batch cultivation on glycerol, to further increase the biomass and the final induction phase was with methanol to induce lipase secretion and biomass build up. The three stage fermentation process gave a productivity of around 160 u / mL of CALB. To further increase the yield high copy number clones were produced and fermentation parameters standardized The high copy number integrations of the lipase B gene was done by increasing the concentration of the pPICZand#945;B lipase B plasmid DNA during electroporation of the Pichia pastoris GS115 strain and selection of the positive clones at higher concentrations of 1 mg / ml of zeocin. The positive colonies were taken for fermentation studies and yielded 600 u / ml in 125 hours. Also we had attempted partial purification of CALB from the fermentation broth. Among the various techniques tried simple ultrafiltration with lyophilisation gave higher percentage recovery. newline