| Resumo: | The purpose of this research work was to study the proteolytic activity of aqueous crude extracts of flowers of the plant Cynara cardunculus on the major whey proteins, namely, â-lactoglobulin (â-Lg) and R-lactalbumin (R-La). These extracts, containing a mixture of cardosins A and B (i.e., two distinct aspartic proteases), have been employed for many years in traditional cheese-making in Portugal and Spain. Cow’s milk sweet whey was incubated for up to 24 h at various ratios of addition of crude enzyme extract, under controlled pH (5.2 and 6.0) and temperature (55 °C). The samples collected were assayed by gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A mechanistic model was proposed for the kinetics of the hydrolysis process, which is basically a double-substrate, double-enzyme Michaelis-Menten rate expression; the kinetic parameters were estimated by multiresponse, nonlinear regression analysis. The best estimates obtained for the specificity ratio (i.e., kcat/Km) of each cardosin within the mixture toward each whey protein indicated that said aspartic proteases possess a higher catalytic efficiency for R-La (0.42- 4.2 mM-1âs-1) than for â-Lg (0-0.064 mM-1âs-1), at least under the experimental conditions used. These ratios are below those previously reported for caseins and a synthetic hexapeptide. Cardosins are more active at pH 5.2 than at pH 6.0 and (as expected) at higher enzyme-to-substrate ratios.
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