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- A kinetic model for hydrolysis of whey proteins by cardosin a extracted from Cynara CardunculusPublication . Barros, Rui M.; Malcata, F. XavierThe 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.
- Quantitative studies on the enzymatic hydrolysis of milk proteins brought about by cardosins precipitated by ammonium sulfatePublication . Barros, Rui M.; Ferreira, Carla A. Ferreira; Silva, Sofia V. Silva; Malcata, F. XavierHydrolysis of whey proteins may produce peptide mixtures with better functional properties than the original protein mixture, viz. higher solubilites and lower allergenic effects. Cynara cardunculus is a wild plant that possesses (aspartic) proteases in its flower cells; those enzymes exhibit general proteolytic and specific milk clotting activities, which are rather useful in traditional cheesemaking. This study was thus aimed at characterizing the enzymatic action of crude extracts of said plant after preliminary purification by salting out with ammonium sulfate at two different concentration levels, viz. 30% and 70% saturation. The coagulant activity on milk, and the proteolytic activity using casein and azocasein as substrates, of the crude extract and of each precipitated fraction were measured at 37°C and pH 5.2. The profile of hydrolysis of the major whey proteins, i.e. α-lactalbumin (α-La), β-lactoglobulin (β-Lg) and bovine serum albumin (BSA) was characterized by gel permeation chromatography and polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate. The 30% and 70% saturation fractions exhibited lower coagulant and proteolytic specific activities than the crude extract. However, the relative ratio of coagulant to proteolytic activity, which is a useful indicator of appropriateness for cheesemaking, was higher for the partially purified fractions. The extents of hydrolysis of whey proteins brought about by the partially purified extracts were above those by their crude counterpart, but qualitative hydrolysis patterns were essentially identical to each other; by 24 h, α-La was substantially depleted, whereas β-Lg was very poorly hydrolyzed and BSA was only slightly hydrolyzed. The native proteins were converted to lower and lower molecular weight peptides.
- Hydrolysis of α-lactalbumin by cardosin A immobilized on highly activated supportsPublication . Barros, Rui M.; Extremina, Clara I.; Gonçalves, Inês C.; Braga, Beatriz O.; Balcão, Victor M.; Malcata, F. XavierIn the present research effort, production of derivatives of cardosin A (a plant protease) encompassing full stabilization of its dimeric structure has been achieved, via covalent, multi-subunit immobilization onto highly activated agarose-glutaraldehyde supports. Boiling such enzyme derivatives in the presence of sodium dodecyl sulfate and β-mercaptoethanol did not lead to leaching of enzyme, thus providing evidence for the effectiveness of the attachment procedure. Furthermore, the cardosin A derivatives prepared under optimal conditions presented ca. half the specific activity of the enzyme in soluble form, and were successfully employed at laboratory-scale trials to perform (selective) hydrolysis of α-lactalbumin (α-La), one of the major proteins in bovine whey. Hydrolysates of α-La were assayed for by the OPA method, as well as by FPLC, SDS–PAGE and HPLC. Thermal inactivation of the immobilized cardosin A was also assessed at 40, 50 and 55 °C; at these temperatures, no thermal denaturation took place during incubation for 48 h. The highest degree of hydrolysis was attained by 5 h reaction, at 55 °C and pH 5.2. SDS–PAGE of α-La hydrolysates displayed bands corresponding to low molecular weight peptides. Our results suggest that cardosin A in immobilized form is a good candidate to bring about proteolysis in the dairy industry, namely in whey processing.