Production and stability of protease from Candida buinensis

Cow raw milk from dairy cooperatives was examined for its microbial composition. Among the isolates identified, 17.6% were yeasts. The most frequent genus was Candida, although members belonging to the genera Brettanomyces, Dekkera, and Geotricum were also identified. Although qualitative and quantitative tests for extracellular proteolytic activity were positive for all the species isolated, Candida buinensis showed the highest response (23.5 U/mg); therefore, it was selected for subsequent investigation. The results of fermentations carried out at variable temperature, pH, and soybean flour concentration, according to a 2(3) full factorial design, demonstrated that this yeast ensured the highest production of extracellular proteases (573 U/mL) when cultivated at 35 degrees C, pH 6.5, and using soybean flour concentrations in the range 0.1-0.5% (w/v). The cell-free supernatants showed the highest activity at 25 degrees C and pH 7.0, and satisfactory stability in the ranges 25-30 degrees C and pH 7-9. The first-order rate constants of protease inactivation in the cell-free supernatants were calculated at different temperatures from semi-log plots of the residual activity versus time and then used in Arrhenius and Eyring plots to estimate the main thermodynamic parameters of thermoinactivation (E* = 40.0 kJ/mol; DeltaH* = 37.3 kJ/mol; DeltaS* = -197.5 J/mol K; DeltaG* = 101 kJ/mol).