Action on bovine alpha(s1)-casein of cardosins A and B, aspartic proteinases from the flowers of the cardoon Cynara cardunculus L

The cleavage of purified bovine alpha(s1)-casein separately by cardosin A and cardosin B, two distinct milk-clotting aspartic proteinases (APs) present in the stigmas of the plant Cynara cardunculus L., was studied. Casein digestion peptides were separated either by SDS-PAGE or by reverse-phase HPLC, and their N-terminal amino-acid sequences were subsequently determined by automated Edman degradation, thus identifying the cleavage sites. Results showed that both enzymes exert a similar but distinct action on bovine alpha(s1)-casein. In common they have the preference for the bond Phe(23)-Phe(24), and the cleavage of Trp(164)-Tyr(165) and Phe(153)-Tyr(154). Cardosin A also cleaves the bond Tyr(165)-Tyr(166), whereas Cardosin B cleaves an extra type of bond, Phe(150)-Arg(151), revealing a slightly broader specificity. A model for the action of both enzymes on bovine alpha(s1)-casein is proposed and discussed. In comparison with the reported action of chymosin on bovine alpha(s1)-casein, both cardosins proved to have a broader specificity towards this particular substrate due to a higher ability to cleave bonds between residues with large hydrophobic side-chains.