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

Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Santiago de Compostela, Santiago de Compostela, Spain

¹ Corresponding author: mpvilla{at}usc.es

Milk-clotting proteases, which are widely used in the cheese-making industry, are enzymes that use soluble caseins as their preferential substrates. Here, we propose a modification to a method previously described for the specific determination of milk-clotting proteases by using -casein labeled with fluorescein isothiocyanate as substrate. Validation of the modified method was confirmed using natural bacterial, fungal, plant, and animal milk-clotting proteases, as well as a milk-clotting enzyme of recombinant origin. The new modified method described here allowed specific quantification of the activity of milk-clotting proteases in a very sensitive way and permitted determination of the appropriate kinetic parameters of all the enzymes tested, consistent with their origin and degree of purity.

Key Words: milk-clotting protease • enzymatic assay • -casein