HIGHLY EFFICIENT PHOSPHOLIPASE C PRODUCTION FOR INDUSTRIAL OIL DEGUMMING USING Pichia pastoris

RAVASI, PABLO; ELENA, CLAUDIA; CASTELLI, MARÍA EUGENIA; CERMINATI, SEBASTIÁN; PEIRÚ, SALVADOR; MENZELLA, HUGO

Rosario

Congreso; 50th Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2014

Crude soybean oil is refined to remove impurities that adversely impact in oil stability, color and flavor. Phospholipids are a major class of impurities removed in conventional processes by oil degumming at significant cost to oil yield. In recent years, enzymatic degumming methods using phospholipase C (PLC) were introduced and are already available on industrial scale with further reduction in yield loss. In this study, Pichia pastoris was used as the expression system for the production of a recombinant PLC enzyme derived from Bacillus cereus. Secreted expression of PLC was driven by the methanol inducible promoter AOX1. In order to optimize the yield of expression, we performed genetic engineering on this strain including generation of multi-copy chromosomal integrations clones, gene codon optimization and the co-expression of several helper factors supporting protein folding, processing and ecretion processes. Based on screening activity tests, the most productive clone was selected. Different approaches were used in order to maximize the yield obtained from fed-batch fermentations reaching secreted protein levels up to 7 g/l. In conclusion, this work set the basis for the development of a scalable model for PLC production. Cost reduction of enzyme production is a basic principle for industrial applications.