Biochemical characterization of an isoform of chymotrypsin from the viscera of Monterey sardine ( Sardinops sagax caerulea), and comparison with bovine chymotrypsin

Abstract

Chymotrypsin II from the viscera of Monterey sardine was characterized as an isoform of chymotrypsin I previously characterized from the same source and compared with bovine chymotrypsin. Chymotrypsin II had a molecular weight of 25,500 Da, similar to bovine chymotrypsin. The isoform identity as chymotrypsin was established by its catalytic specificity on the specific substrates succinyl- l-ala-ala-pro- l-phenylalanine- p-nitroanilide and benzoyl- l-tyrosine ethyl ester, showing higher specific activity than bovine chymotrypsin. Both enzymes showed maximal activity at pH 8.0, chymotrypsin II being stable at alkaline pH while bovine chymotrypsin was stable at acid and alkaline pH. Optimum temperature was 45 °C for chymotrypsin II and 55 °C for bovine chymotrypsin. Both enzymes were inhibited by phenylmethylsulfonyl-fluoride and soybean trypsin inhibitor, and partially by N-toluenesulfonyl- l-phenylalanine chloromethyl-ketone. This is valid only in specific conditions of this work. K m and k cat for chymotrypsin II were 0.048 mM and 4.8 s −1, and 0.09 mM and 1.9 s −1 for bovine chymotrypsin. Catalytic efficiency of chymotrypsin II was 4.8-fold higher than bovine chymotrypsin.