Fig. 2.

Prc is a serine endopeptidase. a Prc degrades β-casein, and its endopeptidase activity is dependent on the Ser⁴⁷⁵ and Lys⁵⁰⁰ sites. β-Casein (41.5 μM) was co-incubated with Prc (5 μM) or its recombinant forms (Prc^S475A and PrcK^500A, 10 μM) at 28 °C for the indicated time. Reactions were stopped and analysed by SDS-PAGE together with Coomassie brilliant blue staining. b Quantification of Prc endopeptidase activity via degradation of the substrate azocasein. Azocasein (424 μM) was mixed with Prc (25 μM), and the reaction was carried out at 28 °C for 30 min. The optical absorbance was measured. Error bars represent standard deviations (n = 3). Asterisks indicate significant differences (Student's t-test, P < 0.05). c and d The Michaelis–Menten kinetics of Prc activity for azocasein hydrolysis. The Michaelis–Menten curve c and Lineweaver–Burk plot d were obtained from the specific reaction velocity of the hydrolysation of azocasein by Prc. The maximum specific V_max and K_m values of the Prc activity were determined from the graphic representations. The data were derived from three independent experiments, and the goodness of fit values (R²) is indicated. a–d, the experiment was repeated three times