A kinetic model for hydrolysis of whey proteins by cardosin a extracted from Cy...

• Author(s): Barros, Rui M. ; Malcata, F. Xavier
• Date: 2004
• Persistent ID: http://hdl.handle.net/10400.14/3473
• Origin: Veritati - Repositório Institucional da Universidade Católica Portuguesa
• Subject(s): Protease; Enzyme; Dairy foods; Reaction rate
• Document Type: Article
• Language: English

Description

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 1) 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.