Immobilization of Endoproteases from Crude Extract of Cynara cardunculus L. Flowers

The cardoon (Cynara cardunculus L.) is a variety of thistle which grows wild and abundantly in uncultivated areas of many regions of Portugal. Its dried flowers are used as a milk coagulant in the manufacture of traditional cheeses, due to its high content in aspartic proteases, namely cardosins A and B. In general, proteases can be immobilized without significant loss of their catalytic activity and their use as milk coagulant offers several advantages over their soluble counterparts, namely simplified protocols, long term stability of the coagulant and successful reutilization. Since proteases are often expensive, especially at high degrees of purity, immobilization may increase their industrial applicability. In this work, aqueous extracts of cardoon were immobilized on celite by adsorption. The properties of free and immobilized extracts from cardoon (kinetic parameters, pH effects, activation energy, ionic effects, clotting time) were compared. Optima pH and temperature were 5.0 and 60 °C, respectively, for extracts in both forms. Upon immobilization, a 60--70% loss of enzyme activity, as well as a decrease in the specificity constants (up to 90%) were observed. Immobilized biocatalyst was evaluated for bovine and caprine caseins, at a laboratory scale, showing a higher specificity towards the latter.