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

2006/10/01 by José Manuel Ageitos, Juán A. Vallejo, Margarita Poza +1 · 23 citations
Biochemistry, Genetics and Molecular Biology · Agricultural and Biological Sciences · Chemistry · #Enzyme Production and Characterization #Protein Hydrolysis and Bioactive Peptides #Phytase and its Applications #Proteases #Casein #Chemistry #Enzyme #Biochemistry #Clotting time #Fluorescein isothiocyanate #Chymosin #Rennet #Chromatography #Biology #Fluorescence

paper · pdf · doi:10.3168/jds.s0022-0302(06)72418-3

published in Journal of Dairy Science 89(10), 3770-3777 (Elsevier BV)

openalex publication_date 2006/10/01 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/31

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.

Citations

• Structure and Stability of Bovine Casein Micelles
• Partial Identification of Water-Soluble Peptides Released at Early Stages of Proteolysis in Sterilized Ovine Cheese-Like Systems: Influence of Type of Coagulant and Starter
• Casein Micelles as Colloids: Surface Structures and Stabilities
• Production and characterization of the milk-clotting protease of Myxococcus xanthus strain 422
• A Rapid Method for Measuring Protease Activity in Milk Using Radiolabeled Casein
• Effect of pH on the Gelation Properties of Skim Milk Gels Made From Plant Coagulants and Chymosin
• Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes
• Gelation Mechanism of Milk as Influenced by Temperature and pH; Studied by the Use of Transglutaminase Cross-Linked Casein Micelles
• Formation and Physical Properties of Milk Protein Gels
• Invited Review: Perspectives on the Basis of the Rheology and Texture Properties of Cheese
• Specificity of milk-clotting enzymes towards bovine κ-casein
• Studies on Milk Proteins. II. Colorimetric Determination of the Partial Hydrolysis of the Proteins in Milk
• Molecular and Biotechnological Aspects of Microbial Proteases
• Kinetics of milk coagulation: II. Kinetics of the secondary phase: Micelle flocculation
• The voluminosity of bovine casein micelles and some of its implications
• Kinetics of milk coagulation: I. The kinetics of kappa casein hydrolysis in the presence of enzyme deactivation
• Casein Interactions: Casting Light on the Black Boxes, the Structure in Dairy Products
• Systematic risk behavior of financially distressed firms

Cited by

• K-casein [wikipedia]