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-degrees-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 alpha-chymotrypsin. Proteinase A and B were identified as alpha-chymotrypsin-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 x 10(-4) M and 1.2 x 10(-3) M, respectively.