Fluorescein Thiocarbamoyl-Kappa-Casein Assay for the Specific Testing of Milk-Clotting Proteases

Standard plots showing the relationship between fluorescence and the amount of degraded fluorescein thiocarbamoyl (FTC)-casein (Y=59.232X + 13.64; R²=0.993) (□) and FTC-κ-casein (Y=13.464X + 9.0912; R²=0.996) (■). X refers to milligrams of protein per milliliter, and Y refers to fluorescence values. Error bars correspond to standard deviations from triplicate replicas.

Specific activity of milk-clotting proteases from Rhizo-mucor miehei (Y=46.093X + 4.497; R²=0.984) (□), B. licheniformis (Y=2.386X + 5.359; R²=0.984) (■) and the abomasum of Bubalus arnee bubalis (Y=0.190X + 3.079, R²=0.996) (○). X refers to milligrams of protein per milliliter, and Y refers to activity (U/mL). Error bars correspond to standard deviations from triplicate replicas.

Effect of the incubation time and dilution factor on the enzymatic activity of Rhizomucor miehei protease. Dilution factor 1/1,000 (Y=0.071X + 0.574; R²=0.995) (□) and dilution factor 1/10,000 (Y=0.020X – 0.431; R²=0.995) (■). X refers to time in minutes, and Y refers to activity units per milliliter. Error bars correspond to standard deviations from triplicate replicas.

Effect of substrate concentration on the enzymatic activity of samples from; Chymax (■), Rhizomucor miehei (○), Bubalus arnee bubalis (▴), and Cynara cardunculus (♢). Error bars correspond to standard deviations from triplicate replicas.

Lineweaver-Burk plot of enzymatic activity of samples from; Chymax (Y=0.072X + 0.629; R²=0.995) (■), Rhizomucor miehei (Y=0.041X + 0.087; R²=0.996) (○), Bubalus arnee bubalis (Y=0.095X + 0.045; R²=0.997) (▴), and Cynara cardunculus (Y=0.058X + 0.256; R²=0.993) (♢).