INVESTIGADORES
CASTELLI maria Eugenia
congresos y reuniones científicas
Título:
Recovery of toxic protein from inclusion bodies
Autor/es:
HAILS, GUILLERMO; LACAVA FRANCO; ALL JUAN DIEGO; CERCHI MAURO; ANSELMI PABLO; CASTELLI, MARÍA E.; PEIRÚ, SALVADOR; AGUIRRE, ANDRES; MENZELLA HUGO
Reunión:
Congreso; LV ANNUAL SAIB- XIV PABMB 2019; 2019
Resumen:
In the last decades, the world demand for oils to be used as food and for the production of fuels has been in constant increase. In order to produce edible oils, crude oils need to be refined in a process that involves several steps. The first of these steps is the phospholipid removal or oil degumming. Several methods such as water degumming and acid degumming have been used for this purpose. More recently, enzymatic degumming has been implemented showing several advantages over traditional methods, including the reduction of gums volume and an extra-yield of oil produced.For enzymatic degumming, phospholipases are employed. The most commonly used are phospholipases C (PLC) and phospholipases A (PLA). Also, glycerophospholipid:cholesterol acyl tranferases (GCAT) enzymes were described to be suitable for this purpose. GCAT enzymes attack acyl groups from phospholipids just like PLA enzymes, but they transfer the acyl group to a free sterol instead of water, reducing the amount of free fatty acids in treated oil.Our group has recently developed an enzymatic oil degumming process using a GCAT from Aeromonas enteropelogenes (GCATae). We have demonstrated that this enzyme remains active even in the harsh conditions such as the currently used in industry, and that it hydrolyzes all the phospholipids present both in crude oils or water degummed oils. However, the development of a manufacturing process for the efficient production of this enzyme was not simple. GCATae has shown to be toxic to microbial hosts like E. coli and P. pastoris since it recognizes phospholipids present in the host membrane, leading to cell lysis and foam production. As a consequence, heterologous production of soluble GCATae in a fed-batch fermentation process is poor and makes its application non-viable. In order to increase the amount of GCAT produced, we used E. coli thioredoxin (TRX) as a fusion partner. This strategy has been widely used to increase the expression level and the solubility of heterologous proteins synthesized in the E. coli cytoplasm. Surprisingly, the TRX-GCATae fusion protein constructed was expressed as inclusion bodies (IB). This partially folded and inactive form of the protein was not toxic and allowed the host cells to produce up to 17 g of IB per liter of culture. Refolded fusion protein TRX-GCATae showed good activity and was suitable for enzymatic degumming. After several optimizations of the IB refolding protocol, GCAT activity per liter of culture obtained was 2.3 folds higher than the activity reached in the soluble GCATae expression system. Such increase turns TRX-GCATae a cost effective enzyme for its use in industrial enzymatic degumming.