Specificity and kinetics of the milk-clotting enzyme from cardoon (Cynara cardunculus L.) toward bovine .kappa.-casein

The action of Cynara cardunculus L. protease on whole bovine K-casein, over a 3-h period at pH 6.4, investigated. RpHPLC of the 3% trichloroacetic acid (TCA)-solublefraction of the K-casein digestion showed three peptide peaks, which were identified by amino acid analysis and N-terminal as the 106-169 fragment [caseinomacropeptide (CMP)]. Upon selective precipitation with 12% TCA, one glycosylated and two nonglycosylated forms of CMP were distinguished. Analysis of whole digestion mixture showed no additional peptides. The kinetics of hydrolysis of the PhelO5- 106 bond was studied by spectrofluorometry, using fluorescein isothiocyanate-labeled K-casein (FTC- -casein). The values obtained for kat, k, and k were 1.04 s-l, 0.16 pM, and 6.5 pM-l s-l, respectively. proteolytic coefficient is of the same order of magnitude as those obtained for other milk-clotting , but the k, is significantly lower, which reflects the higher affinity of Cynara protease to -casein