The flowers of cardoon (Asteraceae) are a rich source of aspartic peptidases which possess milk clotting activity-and are thus used in traditional cheesemaking in the Iberian Peninsula. This study was aimed at characterizing the enzymatic action of the aspartic peptidases present in flowers of Silybum marianum (L.) Gaertn. (Asteraceae), specifically upon degradation of caseins. The proteolytic activities toward Na-caseinates previously prepared from caprine and ovine milks were studied, in a comparative fashion, using urea-PAGE, tricine-SDS-PAGE, densitometry, electroblotting and sequencing. Caprine a s1-and b-caseins were degraded up to 68% and 40%, respectively, during 24 h of incubation. Only one important and well-defined band corresponding to a molecular weight of 14.4 kDa-i.e. a fragment of b-casein, was observed by 12 h of hydrolysis. By 24 h of incubation, ovine a sand b-caseins were degraded up to 76% and 19%, respectively. In what concerns specificity, the major cleavage site in ovine caseinate was Leu99-Arg100 in a s1-casein.

Asclepias fruticosa L. is a small shrub containing latex with proteolytic activity. The crude extract (latex diluted 1:250 and ultracentrifuged) contained 276 microg of protein/mL and the proteolytic activity reached 1.2 caseinolytic U/mL. This enzyme preparation was very stable even after 2 hours at 45 degrees C, but was quickly inactivated after 5 minutes at 80 degrees C. Chromatographic purification was achieved by FPLC using a cation exchanger (SP-Sepharose FF). Thus, a unique proteolitically active fraction could be isolated, being homogeneous by bidimensional electrophoresis and mass spectrometry (Mr = 23,652). The optimum pH range was achieved at 8.5-10.5. The enzyme activity was completely inhibited by specific cysteine peptidases inhibitors. Isoelectric focusing followed by zymogram showed the enzyme had a pI greater than 9.3. The N-terminus sequence (LPDSVDWREKGVVFPIRNQGK) shows a great deal of similarity to those of the other cysteine endopeptidases isolated from latices of Asclepiadaceae even when a high degree of homology could be observed with other plant cysteine endopeptidases.

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