A kinetic model for hydrolysis of whey proteins by cardosin A extracted from Cynara cardunculus

The enzymatic hydrolysis of the major whey proteins, namely b-lactoglobulin (b-Lg) and a-lactalbumin (a-La), was experimentally studied using whey as substrate; an aspartic protease (cardosin A), previously extracted from the flowers of Cynara cardunculus and purified by gel filtration and ion exchange chromatographies, was used for this purpose. Sweet whey was incubated for 24 h at various enzyme:substrate ratios, at controlled pH (5.2 and 6.0) and temperature (55 C); the hydrolyzates were assayed by gel permeation chromatography and electrophoresis. A mechanistic model was proposed for the kinetics, which basically leads to a double-substrate, single-enzyme Michaelis–Menten rate expression containing four adjustable parameters; these parameters were estimated by applying multiresponse, nonlinear regression analysis to the experimental data, so that the model would yield good fits. The best estimates obtained for Km were markedly lower for a-La than for b-Lg, so cardosin A shows a higher affinity for a-La than for b-Lg. The experimental results also suggest that b-Lg is rather resistant to enzyme-mediated hydrolysis under all experimental conditions tested. The highest activity (measured by kcat) of cardosin A was recorded toward a-La (i.e. 0.013 s ) at pH 5.2. Furthermore, the specificity ratio (kcat=Km), obtained toward each whey protein, indicated that cardosin A possesses a higher catalytic efficiency for hydrolysis of a-La than of b-Lg; the highest value for this ratio was recorded for a-La at pH 5.2, and was close to that reported elsewhere for cardosin A acting on caseins and casein-like substrates. Aspartic proteases (EC 3.4.23), also known as acid proteases or aspartyl proteinases, are a widely distributed subfamily of proteolytic enzymes, which belong to the endopeptidase family and have been found in vertebrates, plants, viruses and retroviruses. Recent studies (Faro, 1991; Faro, Ver ıssimo, Lin, Tang, & Pires, 1995; Heimgartner et al., 1990; Ver ıssimo et al., 1996) have shown that it is possible to extract and purify two aspartic proteases, namely cardosins A and B, from the flowers of the thistle, Cynara cardunculus; these enzymes are characterized by somewhat related amino acid sequences – to a degree of 73% similarity (Vieira et al., 2001). Crude aqueous extracts of the flowers of C. carIntroduction * Corresponding author. Tel.: +351-22-5580004; fax: 351-22-