Proteases in oyster (Crassotrea gigas) were extracted with 10 mM Tris‐HCl buffer solution and purified by ammonium sulfate fractionation, Sephadex G‐150 gel filtration, repeated DEAE‐Sephadex A‐50 and CM‐Sepharose CL‐6B chromatography. Three fractions with caseinolytic activity, named I, II and III, were obtained from CM‐Sepharose CL‐6B and DEAE‐Sephadex A‐50 chromatography. The three proteases were purified to electrophoretic homogeneity. Substrate specificity studies indicated that protease I was a carboxypeptidase A‐like enzyme; II and III were trypsin‐like enzymes.
The optimal pH of protease I for hydrolysis of hippuryl‐L‐phenylalanine was 9.0, II and III for hydrolysis of p‐toluenesulfonyl‐L‐arginine methyl ester (TAME) was 8.0. The temperatures which inactivated 50% of enzymes were 78°C for protease I in 30 min; 50 and 52°C for protease II and III, respectively, in 5 min. The molecular weights of proteases I, II and III were 23,000, 34, 400 and 31, 000, respectively.