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 kappa-casein, over a 3-h period at pH 6.4, was investigated. Rp-HPLC of the 3% trichloroacetic acid (TCA)-soluble fraction of the kappa-casein digestion mixture showed three peptide peaks, which were identified by amino acid analysis and N-terminal analysis 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 the whole digestion mixture showed no additional peptides. The kinetics of hydrolysis of the Phe105-Met106 bond was studied by spectrofluorometry, using fluorescein isothiocyanate-labeled kappa-casein (FTC-kappa-casein). The values obtained for kcat, km, and kcat/km were 1.04s-1, 0.16 micromolars, and 6.5 micromolars-1s-1,respectively. The proteolytic coefficient is of the same order of magnitude as those obtained for other milk-clotting enzymes, but the km is significantly lower, which reflects the higher affinity of Cynara protease to kappa-casein