Comparison of the Hydrolysis of Bovine κ-Casein by Camel and Bovine Chymosin: A Kinetic and Specificity Study Kirsten Kastberg Møller,* ,†,‡ Fergal P. Rattray, ‡ Jens Christian Sørensen, † and Ylva Ardö † † Faculty of Science, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark ‡ Chr. Hansen A/S, Bøge Allé 10-12, DK-2970 Hørsholm, Denmark * S Supporting Information 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. KEYWORDS: camel chymosin, bovine chymosin, κ-casein, Michaelis-Menten kinetics, enzyme specificity ■ INTRODUCTION The transformation of liquid milk into a gel is a key step of the cheese-making process. Milk coagulation is typically achieved by enzymatic hydrolysis of κ-casein (CN) at the surface of CN micelles. κ-CN covers the predominantly hydrophobic core of the micelles by its polar, net negatively charged C-terminal, thus preventing micellar aggregation by steric hindrance and charge repulsion. Milk-clotting enzymes specifically remove this part of the protein by cleavage at the Phe105-Met106 bond in bovine κ-CN, which leaves the CN micelles deprived of colloidal properties, thus leading to aggregation. While the hydrophobic para-κ-CN (Glu1-Phe105) remains in the cheese curd, the hydrophilic caseinomacropeptide (CMP) (Met106- Val169) dissolves in the whey (all κ-CN residues are typed in italics). 1 Chymosin (EC 3.4.23.4) of bovine origin (Bos taurus) represents the principal milk-clotting enzyme used in cheese making. 2 It belongs to the aspartic proteinase family and is as such structured mainly in β-sheets that form the N- (Gly1- Tyr175) and C-terminal (Tyr176-Ile323) barrel domains. These domains are divided by a deep substrate binding cleft, at the base of which the two catalytic aspartates (Asp34 and Asp216) reside. 3 Bovine chymosin (pI ≈ 4.7) has a net negative charge at the pH of milk (∼6.6), just like its natural substrate κ- CN (pI ≈ 5.6). 1 The ratio between milk-clotting activity (C) expressed in International Milk-Clotting Units (IMCU) and general proteolytic activity (P) represents a useful measure of the combined activity and specificity of the coagulant and, hence, of its performance during cheese making. 4 Generally, enzymes combining a high C with a low P are desirable to minimize the coagulation time and loss of CN-derived peptides to the cheese whey. In this respect, bovine chymosin is superior to most other aspartic proteinases in use for bovine milk coagulation. 5 Porcine chymosin, for example, has been examined in detail, as it has a higher C/P ratio than bovine chymosin. 6 However, because of a low C, it is not an economic alternative to bovine chymosin and, hence, not commercially available. On the basis of available structural data, numerous kinetic studies have sought to unveil the mechanism behind the highly efficient and specific action of bovine chymosin using as substrate various whole κ-CN fractions, 7,8 peptides of various sizes corresponding in amino acid sequence to the extended chymosin-sensitive region of κ-CN, 9-11 or peptide mutants. 12 The finding that His98-Lys112 corresponding to the peptide joining the N- and C-terminal domains of κ-CN provides kinetic parameters (K M and k cat ) similar to those for whole κ- CN represents an important observation of previous work. 11 Visser et al. 12 proposed a model for the enzyme-substrate (ES) complex in which Leu103-Ile108 is accommodated within the active-site cleft of the enzyme and kept in position through hydrophobic and hydrogen binding to the enzyme. Enzymatic preference to the Phe105-Met106 bond was further enhanced via electrostatic binding of positively charged residues delineating the extended chymosin-sensitive region (His98- His102 and Lys111), whereas Pro99,101,109,110 residues acted as steric stabilizers of a particular constrained substrate conformation in the ES complex. 12 Advances in molecular modeling supported that the substrate binds to bovine Received: February 9, 2012 Revised: May 6, 2012 Accepted: May 7, 2012 Published: May 7, 2012 Article pubs.acs.org/JAFC © 2012 American Chemical Society 5454 dx.doi.org/10.1021/jf300557d | J. Agric. Food Chem. 2012, 60, 5454-5460