Purification and characterization of exopeptidases from Atlantic short finned squid (Illex illecebrosus) and their application to cheddar cheese ripening

Atlantic short finned squid (Illex illecebrosus) hepatopancreas (HP) aminopeptidases (AP) were partially separated from the endoproteinases, which are predominantly cysteine proteases. Several of the AP identified were metalloproteases and activated by Ca2+, Mn2+, Zn2+ or Mg2+ salts. Squid HP homogenate was held at pH 7 and 0°C for 20 h, incubated with 5 mM ZnSO₄, fractionated by ammonium sulfate (20–80%) and dialyzed against 25 mM ZnSO₄. The procedure resulted in a 3–186 fold increase in the ratio of exopeptidase to endoproteinase activity with different AP substrates. Recovery of AP ranged from 14 to 47%. The partially purified squid HP peptidases had a higher ratio of exopeptidase to endoproteinase activity than did two commercial products with AP activity, Flavourzyme® and Neutrase® .; This squid HP AP fraction, at two levels, was added to Cheddar cheese curd and its influence on ripening indices was determined for up to 3 months storage at 11°C. Neutrase® and Flavourzyme® were similarly tested. Cheese with the higher level of squid AP contained more soluble N, amounts of amino acids and Cheddar flavor after 1 month, but it developed defects in bitterness and texture as ripening progressed. Cheese with less squid AP did not differ from the control with respect to all ripening indices over 3 months storage. Ripening Cheddar contains a cysteine protease inhibitor that inhibit low levels of squid AP, but not Neutrase® or Flavourzyme®.; Carboxypeptidase (CP) activities were detected in a squid HP extract using 33 N-CBZ-dipeptides. A 25 kDa CP was purified to homogeneity by DEAE-cellulose, gel filtration and chromatofocusing chromatography. The purified CP had a pI of 3.4 and was characterized as a metalloprotease, activated by Co2+ and Zn2+. The enzyme had broad specificity towards N-CBZ-dipeptides; however, it preferred substrates with hydrophobic, especially aromatic amino acids at the C-terminus. It was most active with substrate N-CBZ-Ala-Phe (K_m = 0.4mM). The amino acid composition of squid CP-I is similar to CP A from other species. With Ala−Phe and Gly−Phe, its optimum pH was 8 and optimum temperature 70°C. The enzyme has potential for use in hydrolyzing bitter peptides in protein hydrolysates.