DESIGN AND OPTIMIZATION OF THE PRODUCTION OF PHOSPHOLIPASE IN Corynebacterium glutamicum

LACAVA, FRANCO EMANUEL; PONCE DE LEÓN, CAMILA; CERMINATI, SEBASTIÁN; MENZELLA, HUGO GABRIEL; AGUIRRE, ANDRÉS

Congreso; XVIII Congreso de la Sociedad Argentina de Microbiología General; 2023

Enzymes are used in numerous modern industries because of a variety of advantages.Argentina is the world's leading exporter of soybean oil. A refining process is necessary toproduce edible oils. The first step of this process is the removal of phospholipids (PLs),called “degumming”. There are several methods for this, including aqueous degumming,acid degumming, and enzymatic degumming. In recent years, enzymatic degumming hasshown several advantages over other methods, including the reduction of gums generatedand an additional yield of the oil produced. The main enzymes used in this process arephospholipases. The most commonly used are type C phospholipases (PLCs) and type Aphospholipases (PLAs). Within type C phospholipases, there are those that are specific forphosphatidylcholine (PC) and phosphoethanolamine (PE), called PC-PLCs, and those thatare specific for phosphatidylinositol (PI), called PI-PLCs. PC, PE and PI are the main PLSpresent in crude soybean oil. Since PC-PLCs hydrolyze both PC and PE, a cocktailcontaining both PI and PC-PLCs would result in a 91 % reduction of the phospholipidspresent in soybean oil. Our group has focused on the development of an enzymaticproduction platform that uses Corynebacterium glutamicum as a host microorganism, as ithas certain important characteristics that support its use at an industrial level.In order to achieve this, we have used: molecular biology techniques to construct C.glutamicum strains, high-density cell fermentations in a 3L agitated bioreactor, micro- andultrafiltration techniques to purify the target protein, fluorometric and colorimetric techniquesand 31P RMN to measure phospholipase activity,.In this work it was possible to obtain a recombinant PI-PLC. First, a defined medium forC. glutamicum growth was designed, optimized and evaluated in a batch fermentation. Forthis, several vitamins, trace elements, and siderophores were evaluated. In a second stage,the optimized medium was evaluated in a fed-batch fermentation, reaching high celldensities and improving the yield of the protein of interest. In the third stage, the downstreamprocess was carried out by optimizing parameters such as cross-flow, LMH andtransmembrane pressure. Finally, the minimum amount of enzyme required for thecomplete removal of PI in soybean oil was determined, and the reaction conditions wereoptimized. Also, the activity was evaluated in the presence of a PC-PLC, obtaining thecomplete removal of PC, PE, and PI, the major phospholipids present in soybean oil.In conclusion, in order to be competitive in the production of industrial enzymes, whichare marketed as commodities, it is necessary to reduce manufacturing costs, optimize thedesign of fermentations and obtain high yields of the protein of interest. The design andoptimization of a protein production process using C. glutamicum as a host is a significantand attractive undertaking at both the scientific and industrial levels.