RESUMO
Collagenases are proteolytic enzymes capable of degrading both native and denatured collagen, reported to be applied in industrial, medical and biotechnological sectors. Liquid-liquid extraction using aqueous two-phase system (ATPS) is one of the most promising bioseparation techniques, which can substitute difficult solid-liquid separation processes, offering many advantages over conventional methods including low-processing time, low-cost material and low-energy consumption. The collagenase produced by Penicillium sp. UCP 1286 showed a stronger affinity for the bottom salt-rich phase, where the highest levels of collagenolytic activity were observed at the center point runs, using 15.0% (w/w) PEG 3350 g/mol and 12.5% (w/w) phosphate salt at pH 7.0 and concentration. The enzyme was characterized by thermal stability, pH tolerance and effect of inhibitors, showing optimal collagenolytic activity at 37 °C and pH 9.0 and proved to be a serine protease. ATPS showed high efficiency in the collagenase purification, confirmed by a single band in SDS/PAGE, and can in fact be applied as a quick and inexpensive alternative method.
Assuntos
Colagenases/isolamento & purificação , Proteínas Fúngicas/isolamento & purificação , Penicillium/enzimologia , Fosfatos/química , Polietilenoglicóis/química , Colagenases/química , Proteínas Fúngicas/químicaRESUMO
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).