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.

Purification and Characterization of Proteases From Oyster (Crassotrea Gigas)

“…The temperatures at which half of the activity of proteases I, I1 and 111 were inactivated under the experimental conditions were 78, 50 and 52"C, respectively. The thermostability of oyster trypsin-like proteases I1 and 111 was similar to that of trypsins from milkfish Chanos chanos (Chen et al 1988), Antarctic krill Euphausia superba (Chen et al 1978) and brine shrimp Artemia salina (Olalla et al 1978) examined under similar conditions. Carboxypeptidase A was more heat stable than trypsinlike I1 and 111.…”

“…7). The optimal temperature for trypsin-like proteases I1 and 111 from oyster (60°C) was close to that from milkfish viscera (Chen et al 1988) and h l l (Seki et al 1975(Seki et al , 1977Chen et al 1978). But it is different from that of deep sea crabs (Geryou affinis) which has a temperature optimum between 45 and 50°C (Galgani and Nagayama 1988).…”

“…5 ) . The molecular weight of protease I is similar to that of Antarctic krill carboxypeptidase A (Chen et al 1978) and the mackerel enzyme (Ooshiro 1962), and different from that of the milkfish (Chen et al 1988), the catfish (Yoshinaka et al 1985a) and the pig enzyme (Folk and Schirmer 1963). The molecular weight of proteases 11 and A, 13,700; 2, chyrnotrypsinogen A, 25,000; 3, ovalburnin, 43,000; and 4, albumin, 67,000 SEPHADEX G-150 GEL FILTRATION 111 were higher than that of the krill (Noguchi et af.…”

“…Carboxypeptidase A was more heat stable than trypsinlike I1 and 111. It is also more stable than the procine carboxypeptidase A with remaining activity of 95% when incubated at 55°C for 30 min (Folk and Schirmer 1963) and that from milk-fish viscera (Chen et al 1988).…”

Catalytic Activities of Crude Enzyme Fractions from Monterey Sardine

“…The sarcoplasmic fluid extract showed a highly hydrolytic enzyme activity at pH 7.6 on both synthetic substrates, hippuryl-L-phenylalanine (HP) and hippuryl-L-arginine (HA) ( Table 1). The HP substrate is commonly used for identification of pancreatic carboxypeptidase A (Chen et al, 1988), and HA for carboxypeptidase B (Tsao and Nagayama, 1991). No evidence of such activity have been reported in fish muscle; nevertheless, several studies have related some fish muscle alkaline proteinase activity with a carboxypeptidase-like enzyme(s).…”