CONICET | Buscador de Institutos y Recursos Humanos

Introduction: During fish processing, between 20-60% of starting raw material become solid waste whose final disposal creates environmental pollution issues. It is of interest to valorize those by-products through the use of viscera animal waste, a source of enzymes such as porcine pepsin widely used in food and pharmaceutical industry. Objective: The aim of this study was to isolate a protease with enzymatic activity at low pHs from the stomach of Sábalo (Prochilodus lineatus). Material and methods: Fish were captured from the Paraná-Paraguay basin. Crude acid protease extract was obtained by mechanical and sonic digestion of gastric mucosa with 0.1 M Glycine-HCl buffer pH 2 (buffer A). Only two steps were necessary to obtain the purified enzyme from the extract, a saline precipitation followed by a molecular exclusion chromatography. After treatment with ammonium sulfate (60% saturation), the precipitated proteins were dissolved in buffer A, filtered and lyophilized. Dry powder was suspended in phosphate buffer 20Mm pH 4.5 (buffer B) and applied to a gel filtration column (Sephadex G-75) equilibrated and eluted with the same buffer. Fractions with proteolytic activity on 2% acid hemoglobin substrate and a specific activity greater than 1000 U/mg protein were collected, pooled and concentrated. Protein concentration was determined by Coomassie Blue staining and SDS-PAGE was carried out to determine the purity and molecular weight of the protease. Gels were stained with Coomassie Blue R-250. Results and discussions: The purification factor increased 19-fold in specific activity and the recovery of the target protein was 13%. The enzyme appeared as a single band of approximately 38kDa on SDS-PAGE. Conclusions: In the present study, the use of simple procedures allows the isolation of a protease enzyme such as pepsin from gastric mucosa of Sábalo with potential industrial application.