Production in Fed-Batch Reactor of Bacillus subtilis LipaseA Immobilized on its own Producer Saccharomyces cerevisiae Cells

Abstract

Lipases (EC 3.1.1.3, triacylglycerol hydrolases), a subclass of the esterases (EC 3.1.1.1, carboxyl ester hydrolases) are one of the most important groups of biocatalysts for biotechnological uses such as synthesis of biopolymers and biodiesel, production of enantiopure pharmaceuticals, detergent formulation or the production of flavour compounds. Lipases can be commercialized as free or immobilized form. Nowadays it is of great interest to obtain immobilized enzymes especially for industrial applications since immobilization confers stability giving the possibility to recover the biocatalyst after its use, simplifying downstream processing. In the present work, Lipase A from Bacillus subtilis has been expressed in different strains of the yeast S. cerevisiae as a fusion protein with the yeast cell-wall mannoprotein Pir4. The corresponding gene fusion was created by inserting all the coding sequence of the lipase A gene in the BglII restriction site of PIR4 gene, leading to the expression of a fusion protein still containing the four cysteine residues responsible for the anchorage of Pir4 to the yeast cell wall. Production of lipase as a naturally immobilized biocatalyst was carried out allowing yeast cells to proliferate in a fed-batch reactor under glucose limitation to promote a fully respiratory metabolism and consequently high biomass yield. The performance of the producer strains was evaluated in terms of cell density and enzyme productivity. A preliminary study of economic feasibility of a single fermentation run has been performed based on the experimental results obtained.