title: Fluorescein thiocarbamoyl-kappa-casein assay for the specific testing of milk-clotting proteases.
abstract: 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 kappa-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.
authors.affiliation: Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Santiago de Compostela, Santiago de Compostela, Spain.
authors.forename: J M
authors.initials: JM
authors.lastname: Ageitos
authors.forename: J A
authors.initials: JA
authors.lastname: Vallejo
authors.forename: M
authors.initials: M
authors.lastname: Poza
authors.forename: T G
authors.initials: TG
authors.lastname: Villa
mesh_terms: D000018:Abomasum / Q000201:enzymology; D000818:Animals; D001407:Bacillus / Q000201:enzymology; D002020:Buffaloes / Q000502:physiology; D002364:Caseins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D031942:Cynara / Q000201:enzymology; D005452:Fluoresceins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D005453:Fluorescence; D005511:Food Handling / Q000379:methods*; D007700:Kinetics; D008892:Milk / Q000201:enzymology*; D009090:Mucorales / Q000201:enzymology; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism
chemical_list: D002364:Caseins; D005452:Fluoresceins; D011480:Protease Inhibitors; C044337:fluorescein thiocarbamoyl casein; D010447:Peptide Hydrolases; D014357:Trypsin