Isolation and characterization of proteases from the viscera of Labeo rohita

A preliminary study on the isolation and characterization of proteases from the crude extract of visceral organs of Labeo rohita were carried out. In order to estimate the number of proteases and their mass, the crude protease extract were separated by SDS-PAGE and then the activity was revealed by Zymogram. The crude enzyme extract showed two clear bands on casein and gelatin Zymogram, which indicated the presence of two major proteases. The optimal pH and temperature of the proteases obtained in crude extract were 8.0 and 400 C respectively, using casein as a substrate. Furthermore, the crude enzymes were characterized by protease inhibitors namely phenylmethylsulphonyl fluride (PMSF), EDTA and β mercaptoethanol, in which the protease enzymes were strongly inhibited by PMSF (serine protease inhibitor). In addition, the enzymes were found to be highly active in the presence of activators like Mg2+, Ca2+, Mn2+ and Na+. The protease kinetic constants, Km and Vmax of the crude enzymes for casein, were 0.65 mM and 2.5 U/ml/min respectively. With respect to properties of the enzyme and its capacity for degradation of different protein sources, these proteases finds potential application for waste treatment used in detergent and leather industry.