PURIFICATION AND CHARACTERIZATION OF ALKALINE PROTEINASES FROM THE VISCERA OF ANCHOVY, ENGRAVLIS JAPONICA

Two electrophoretically homogeneous proteinases designated proteinase A and B were isolated from anchovy viscera. Purity was increased 17.7 and 24.6-fold with approximately 1.9 and 1.8% yield for proteinases A and B, respectively. The maximum caseinolytic activity was found to be at pH 9.4 for proteinase A and at pH 9.6 for proteinase B at the optimum temperature of 48°C. The molecular weights of proteinase A and B were determined to be 27, 300 and 25,100 D, respectively, using Sephadex G-100 gel filtration. The amino acid profiles of the enzymes were similar and relative proportion of amino acid residues was comparable to that in bovine pancreatic α-chymotrypsin. Proteinase A and B were identified as α-chymottypsin-like serine proteases by inhibitor and substrate specificity studies. Apparent K_m (K_m′) values of proteinase A and B for benzoyl-L-tyrosine ethyl ester were 4.6 × 10_?4 M and 1.2 × 10_?3 M, respectively.