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.

Manufacture of dry‐ and brine‐salted soft camel cheeses for the camel dairy industry

“…), probably because of the higher proteolysis activity of camel chymosin (Moller et al . ). Camel SCC‐D and SCC‐B were not distinguished by their saltiness in spite of the different salting procedure (Table ).…”

Advances in enzyme substrate analysis with capillary electrophoresis

“…An additional role for surface coating is to decrease the analyte interaction with the fused silica walls. To facilitate this specialized coatings are applied to the capillary wall in order to suppress the electroosmotic flow, in the case of polyvinyl acrylamide [49] and phospholipids [26,50], or to reverse the electroosmotic flow, in the case of polybrene [15]. The use of coatings may be expanded to analytes that are not anionic by derivatizing the analytes.…”

“…An off-line enzyme incubation is preferred if the working pH range of the enzyme reaction does not support the separation of product from substrate. These reactions typically use reaction volumes of 100 μL or greater [29,38,39,49,53–55], although smaller volumes have been reported [36,37]. Off-line enzyme reactions can be quenched to allow more flexibility in the separation of each sample; for example, when reactions are run in parallel.…”

Determination of the influence of high pressure processing on calf rennet using response surface methodology: Effects on milk coagulation

“…Thus, the search for a substitute for rennet enzymes is growing (Walstra, Wouters, & Geurts, 2006;Møller, Rattray, Sørensen, & Ardo, 2012). These substitutes must mimic the calf rennet characteristics, such as high specificity and good proteolytic activity at the usual pH and temperature of cheese manufacture (Dalgleish, 1992;Fox & Kelly, 2004;Bansal et al, 2009).…”