Optimization of Pichia pastoris fermentation process for PLC production

CERMINATI, SEBASTIAN; MARCHISIO, FIORELA; VAL, DIEGO; HAILS, GUILLERMO; PAOLETTI, LUCIANA; AGUIRRE, ANDRES; PEIRÚ SALVADOR; MENZELLA HUGO; CASTELLI, MARÍA E.

Congreso; LV ANNUAL SAIB- XIV PABMB 2019; 2019

Crude vegetable oils are a complex mix of triglycerides, phospholipids, sterols, glycolipids, free fatty acids, metallic traces and other minor compounds. Oil degumming (or phospholipid removal) is the first step in the industrial process of oil refining and is the one having the major associated losses. Traditionally, physical and chemical methods have been used. More recently, enzymatic degumming has been adopted by many industries, with several advantages over the formers. Enzymatic degumming reduces the gums volume and generates products that are more easily removed by centrifugation, increasing the overall yield, with a significant economic benefit for industry.Our group has developed an enzymatic mixture of two phospholipases C (PC-PLC, PI-PLC) and a lisoacyltransferase GCAT (L-GCAT) that can be employed in the conditions currently used in industry for the treatment of crude oil, efficiently hydrolyzing phospholipids in oil. Each of these enzymes is obtained by heterologous production in fed-batch fermentation processes using different microbial hosts, E coli for Pi-PLC and L-GCAT and Pichia pastoris for PC-PLC. In this work, the optimization of the Pichia pastoris fermentation process to obtain PC-PLC is described. We optimized the culture medium composition, the adaptation phase length and the feeding strategy. We finally established that a continuous strategy using a protease deficient strain is the best option, with remarkably high productivity. This optimized fermentation strategy together with an efficient downstream processing provides a manufacturing alternative for the cost effective production of the enzyme.