Kinetic studies on the action of Mucor pusillus, Mucor miehei acid proteases and chymosins A and B on a synthetic chromophoric hexapeptide.

The action of two milk-clotting fungal proteases from Mucos pusillus and Mucor miehei and of chymosins A and B on the hexapeptide, Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe, and on kappa-casein were studied. The effects of pH and temperature on the initial rates of hydrolysis of the hexapeptide were examined. Crystalline chymosin and M. pusillus protease exhibited optimal activities around 49 and 55 degrees C, respectively, whereas the optimum temperature for M. miehei protease is higher than 63 degrees C. The optimum pH was about 4.7 for both fungal proteases whereas chymosin A and chymosin B exhibited optimal activities around 4.2 and 3.7, respectively. Kinetic parameters were then determined under optimal conditions and/or at pH 4.7. Fungal proteases had kcat/Km ratios that were similar to each other and that were significantly greater than the ratios obtained for the chymosins. Nevertheless, chymosins had much greater clotting activities towards kappa-casein relative to their proteolytic activities towards the synthetic peptide.