Evaluation of cardosin A as a probe for limited proteolysis in non-aqueous environments—complex substrates hydrolysis

Cardosin A is an aspartic proteinase, like pepsin, purified from the cardoon, Cynara cardunculus L. and has been traditionally used in Portugal in the manufacture of traditional cheeses, due to its milk clotting activity. This enzyme is a fine example of a small number of plant aspartic proteinases that have been isolated, purified and characterised both in aqueous and in non-aqueous media. The present work deals with the effect of organic solvents on cardosin A specificity towards complex substrates such as κ-, β- and α-caseins and human type I collagen. At the same time fluorescence studies were carried out to correlate reaction rates with possible structural alterations induced by the organic solvents on the substrates. Results show a strong dependence of the velocity of hydrolysis on the medium composition and additionally, it is shown that none of the organic solvents used in this work, induced alterations of cardosin A specificity. Furthermore, it is shown that cardosin A in the presence of organic solvents can be used as a catalyst in order to obtain reaction intermediary products that are, otherwise, rapidly consumed in aqueous buffer.