title: Comparison of the hydrolysis of bovine κ-casein by camel and bovine chymosin: a kinetic and specificity study.
abstract: Bovine chymosin constitutes a traditional ingredient for enzymatic milk coagulation in cheese making, providing a strong clotting capacity and low general proteolytic activity. Recently, these properties were surpassed by camel chymosin, but the mechanistic difference behind their action is not yet clear. We used capillary electrophoresis and reversed-phase liquid chromatography-mass spectrometry to compare the first site of hydrolysis of camel and bovine chymosin on bovine κ-casein (CN) and to determine the kinetic parameters of this reaction (pH 6.5; 32 °C). The enzymes showed identical specificities, cleaving the Phe105-Met106 bond of κ-CN to produce para-κ-CN and caseinomacropeptide. Initial formation rates of both products validated Michaelis-Menten modeling of the kinetic properties of both enzymes. Camel chymosin bound κ-CN with ∼30% lower affinity (K(M)) and exhibited a 60% higher turnover rate (k(cat)), resulting in ∼15% higher catalytic efficiency (k(cat)/K(M)) as compared to bovine chymosin. A local, less dense negatively charged cluster on the surface of camel chymosin may weaken electrostatic binding to the His-Pro cluster of κ-CN to simultaneously impart reduced substrate affinity and accelerated enzyme-substrate dissociation as compared to bovine chymosin.
authors.affiliation: Faculty of Science, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark. dkkkm@chr-hansen.com
authors.forename: Kirsten Kastberg
authors.initials: KK
authors.lastname: Møller
authors.forename: Fergal P
authors.initials: FP
authors.lastname: Rattray
authors.forename: Jens Christian
authors.initials: JC
authors.lastname: Sørensen
authors.forename: Ylva
authors.initials: Y
authors.lastname: Ardö
mesh_terms: D000818:Animals; D002162:Camelus; D002364:Caseins / Q000737:chemistry*; D002417:Cattle; D012085:Chymosin / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D013045:Species Specificity
chemical_list: D002364:Caseins; D012085:Chymosin