Hot-spot mapping of the interactions between chymosin and bovine κ-casein

Chymosin is a commercially important enzyme in the manufacturing of cheese. Chymosin cleaves the milk protein -casein, which initiates the clotting process. Recently, it has been shown that camel chymosin has superior enzymatic properties toward cow’s milk, compared to bovine chymosin. The two enzymes possess a high degree of homology. There are only minor differences in the binding cleft; hence, these must be important for binding the substrate. Models for the binding of a 16 amino acid fragment, consisting of the chymosin-sensitive region of bovine kappa-casein (97-112), to both enzymes have previously been presented. Computational alanine scanning for mutating 39 residues in the substrate and the bovine enzyme are presented herein, and warm- (ΔΔG > 1 kcal/mol) and hot-spot (ΔΔG > 2 kcal/mol) residues in the bovine enzyme are identified. These residues are relevant for site-directed mutagenesis, with the aim of modifying the binding affinity and in turn affecting the catalytic efficacy of the enzyme.