Chymosin effects

The Daily Industiy. The first step in cheese manufacture is the coagulation of milk. Coagulation can be divided into two distinct phases, enzymatic and the non-enzymatic. In the primary enzymatic phase a proteol ic enzyme such as chymosin (rennet), or less effectively, pepsin, carries out an extremely specific and limited proteolysis, cleaving a phenylalanine-methionine bond of /c-casein, making the casein micelle metastabie. In the second, non-enzymatic phase, the... 

Shalabi, S. I. and Wheelock, J. V. 1977. Effect of sulphydryl blocking agents on the primary phase of chymosin action on heated casein micelles and heated milk. J. Dairy Res. 44, 351-355. 

Reddy, D., Payens, T. A. and Brown, R. J. 1986. Effect of pepstatin on the chymosin-triggered coagulation of casein micelles. J. Dairy Sci. 69 (Suppl. 1), 72. 

Castillo, M., Lucey, J.A., and Payne, F A. (2006). The effect of temperature and inoculum concentration on rheological and light scatter properties of milk coagulated by a combination of bacterial fermentation and chymosin cottage cheese-type gels. Int. Dairy J. 16, 131-146. 

Walstra, P., Bloomfield, V.A., Wei, G.J., and Jenness, R. (1981). Effect of chymosin action on the hydrodynamic diameter of casein micelles. Biochim. Biophys. Acta 669, 258-259. 

One of the major benefits of biotechnology is for the cost effective production of valuable enzymes. In 1987, sales of chymosin alone were a half a billion dollars. Kuraishi (this volume) reports some of the potential applications of transglutaminase, an enzyme recently commercialized by Ajinomoto. The enzyme catalyzes the acyltransfer between the y-carboxyamide group of glutamine and the 8-aminogroup of lysine crosslinking proteins with a e-(y-... 

The residues Phe and Met are not intrinsically essential for chymosin action. Replacement of Phe by Phe (NO2) or cyclohexylamine reduces k J K 3- and 50-fold, respectively (Visser et al, 1977). Oxidation of Metioe reduces kcJKm 10-fold but substitution of norleucine for Met increases this ratio 3-fold. Neither porcine nor human K-casein possesses a Phe-Met bond [both have a Phe-Ile bond at this position (Brignon et al, 1985 Chobert et al, 1976 Fiat et al, 1977)], yet both are readily hydrolyzed by calf chymosin, although more slowly than bovine K-casein in contrast, porcine milk is coagulated more effectively than bovine milk by porcine chymosin (Foltmann, 1987). Thus, the sequence around the Phe-Met bond, rather than the bond itself, contains the important determinants for hydrolysis. The particularly important residues are Ser jo4, the hydrophobic residues Leuio3 and Ileiog, at least one of the three histidines (residues 98, 100, or 102), some or all of the four prolines (residues 99, 101, 109, and 110), and Lysiii. 

Nuflez, M., Medina, M., Gaye, P., Guillen, A. M., and Rodriguez-Martin, M. A. (1992). Effect of recombinant chymosin on ewes milk coagulation and Manchego cheese characteristics. 

Salesse, R., and Gamier, J. (1976). Synthetic peptides for chymosin and pepsin assays pH effect and pepsin independent determination in mixtures. J. Dairy Sci. 1215-1221. 

Thus, K-casein in its native stabilizing role exists, probably as small disulphide linked polymers, bound to the micellar surface (the ill-defined boundary between the hydrophobic interior of the micelle and the aqueous phase). C-terminal polypeptides (61 residues) of the protein project from the surface into the solution. In this position, the macropeptide moiety of the protein is conformationally free (H), constrained only by its interactions with its neighbours (J ), and the bond 105-106 of the protein is held in a particularly advantageous position for attack by enzymes such as chymosin ( ). The importance of this will be apparent when emulsions stabilized by K-casein are being discussed. The enzymic action has a relatively small but detectable effect on the hydrodynamic diameters of the particles, and a large effect on their electrophoretic mobilities, which decrease by between one-third and one-half, depending on the solution conditions ( ). 

Since peptide Al appeared to have been derived from residues 1-16, it was of interest to see whether this peptide, generated in the course of a stopped activation, could inhibit pepsin and the homologous milk-clotting calf protein, chymosin. The results (V. Barkholt Pedersen and P. H. Ward - personal communications) show that Al had no effect on the milk-clotting activity of chymosin (confirming that all of the pepstatin must have been removed) while independently the Danish and American labs produced similar curves for the inactivation of porcine pepsin (Fig.5). Thus, it would seem that the first peptide produced on activation is the inhibitor peptide. This seems to make sense from a control point of view, i.e., the first bond being hydrolyzed on activation releases the pepsin inhibitor so it can exert its effect if required. 

Figure 5. The effect of peptide A1 on the milk-clotting activities of pepsin (— — —) and chymosin ( A pH 5.3. The peptide was premixed with the enzyme before measuring the clotting activity remaining.

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