The action of Cynara cardunculus L. protease on whole bovine K-casein, over a 3-h period at pH 6.4, was investigated. RpHPLC of the 3 % trichloroacetic acid (TCA)-soluble fraction of the K-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 PhelO5-Met106 bond was studied by spectrofluorometry, using fluorescein isothiocyanate-labeled K-casein (FTC-K-casein). The values obtained for kat, k,, and ka&, were 1.04 s-l, 0.16 pM, and 6.5 pM-l s-l, respectively.The proteolytic coefficient is of the same order of magnitude as those obtained for other milk-clotting enzymes, but the k, is significantly lower, which reflects the higher affinity of Cynara protease to K-casein. INTRODUCTIONThe coagulation of milk is the basic step in the manufacture of all cheeses. Milk-clotting enzymes suitable for cheese-making must have a high, specific cleavage capacity towards K-casein a t the pH of milk. Calf rennet, which contains chymosin as the main enzyme component, has been the most widely used milk-clotting enzyme preparation, but, in the past few years, increasingly high prices have led to a systematic investigation of suitable substitutes, from either animal, bacterial, or plant origin. From this last class, ficin, from the fig tree, papain, from papaya, and bromelain, from pineapple, have been tried, but they all proved to be unsuitable, since they produce extremely bitter cheeses. To our knowledge, there is only one exception to this general unacceptability of plant clotting enzymes-the protease present in the flowers of the cardoon (Cynara cardunculus L.), which is used traditionally in Portugal in the manufacture of the highly appreciated sheep-milk Serra cheese. This aspartic protease has been recently isolated, purified, and partly characterized by Heimgartner et al. (1990) andFaro (1991).Twenty years ago, Sb and Barbosa (1972) studied the total protein breakdown and the rheological behavior of curds prepared with this enzyme and different types of 0021-8561/93/~447-7537$04.00/~ milk. In their study they concluded that, despite its high proteolytic activity, cardoon extract could satisfactorily replace rennet in the manufacture of soft cheeses; for other cheeses, however, lower yields were observed. These authors suggested that cleavage sites of bovine K-casein by chymosin and the cardoon protease might be different.To get more information on the action of the cardoon protease on caseins, the cleavage of whole bovine K-casein by the protease was investigated in this study. The kinetic parameters kat and k, for the cleavage of the PhelO5-Met106 bond were determined and compared to those published for other milk-clotting enzymes. MATERIALS AND METHODSMaterials. K-Casein, phenyl isothio...
The action of cardosin on bovine R s -and -casein at 30°C in 50 mM citrate buffer (pH 6.2) was studied. Peptides were isolated by reversed-phase HPLC on C 18 columns and identified from their amino acid composition and N-terminal amino acid sequence. ), which was less attacked by chymosin in various experimental conditions. The active site cleft of cardosin accommodates sequences as bulky as Trp-Tyr-Tyr in different subsites (S 1 to S′ 2 , S 2 to S′ 1 , and probably S 3 to S 1 ). Several bitter peptides were identified in the digests.
Bovine β-casein was dephosphorylated by the catalytic sub-unit of a type 2A protein phosphatase (PP2Ac) purified from the yeast Yarrowia lipolytica. Complete dephosphorylation was obtained after 24 to 30 h in Tris buffer (pH 7.5). On the contrary, the activity of PP2Ac was largely inhibited (80%) in the presence of 13 mmol⋅L-1 sodium citrate (pH 6.8). After dephosphorylation of β-casein, plasminolysis was increased and more 3% TCA-soluble peptides were obtained (30% increase). Dephosphorylation led to the disappearance of the f 29-105/107 peptide and to the appearance of a new non-phosphorylated 8 200 g. mol-1 peptide, suggested to be f 33-105/107 originated from the middle part of the β-casein sequence and obtained upon plasmin cleavage of the Lys 32-Phe 33 peptide bond. β-casein / dephosphorylation / protein phosphatase / Yarrowia lipolytica / plasmin Résumé-Hydrolyse par la plasmine de la caséine β bovine déphosphorylée par une protéine phosphatase de type 2A purifiée chez Yarrowia lipolytica. La caséine β peut être déphosphorylée par la sous-unité catalytique d'une protéine phosphatase de type 2A purifiée chez la levure Yarrowia lipolytica. La déphosphorylation complète est obtenue en 24 à 30 h en tampon Tris (pH 7.5). À l'inverse, elle est largement inhibée par le citrate de sodium 13 mmol. L-1 à pH 6.8 (80 % d'inhibition). Après déphosphorylation, la plasminolyse de la caséine β est augmentée puisque l'on observe une production supplémentaire de 30 % de peptides solubles dans le TCA à 3 %. La déphosphorylation se traduit par la disparition du peptide f 29-105/107 et l'apparition d'un nouveau peptide non phosphorylé de 8 200 g. mol-1. Il pourrait s'agir du peptide f 33-105/107 provenant de la partie centrale de la séquence protéique de la caséine β et obtenu par attaque de la liaison peptidique Lys 32-Phe 33. caséine β / déphosphorylation / protéine phosphatase / Yarrowia lipolytica / plasmine
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