PURIFICATION AND CHARACTERIZATION OF PROTEASES FROM THE VISCERA OF MILKFISH (CHANOS CHANOS)

Abstract

Proteases in acetone powder prepared from milkfish (Chanos chanos) viscera were extracted with deionized water and purified by ammonium sulfate fractionation, Sephadex G-75 gel filtration, repeated DEAE-Sephadex A-50 and CM-Sepharose CL-6B chromatography. Four fractions with caseinolytic activity, named A, B, C and D, were obtained from CM-Sepharose CL-6B and DEAE-Sephadex A-50 chromatography. The four proteases were purified to electrophoretic homogeneity. Substrate specificity studies indicated that proteases A and B were carboxypeptidase A-like and chymotrypsin-like enzymes, respectively; C and D were trypsin-like enzymes. ABSTRACT The optimal temperatures of proteases A, B, C and D for hydrolysis of casein were found to be 60, 60, 55 and 60°C, respectively. The optimal pH of protease A for hydrolysis of hippuryl-L-phenylalanine was 9.0, B for hydrolysis of acetyl-L-tyrosine ethyl ester was 8.0, C and D for hydrolysis of tosyl-L-arginine methyl ester was 8.0. The temperatures which inactivated 50% of enzymes in 5 min were 20°C for protease B; 51°C for protease C; 56°C for protease A; and 61°C for protease D. The molecular weights of proteases A, B, C, and D were 14,800, 16,800, 24,800 and 22,000 daltons, respectively.