Chymosin bovine

Today, active bovine chymosin has been successfully produced from several filamentous fungi and they are indistinguishable from the natural enzyme obtained from calves stomachs. 

To study its mode of inhibition, we prepared several derivatives and measured their kinetics of inhibition. Both N-acetyl-statine and N-acetyl-alanyl-statine are competitive inhibitors for pepsin with values of 1.2 X lO M and 5.65 x 10 M, respectively. The value for N-acetyl-valyl-statine is 4.8 x 10 M. These statyl derivatives, therefore, are very strong inhibitors. The value for N-acetyl-statine is 600-fold smaller than that of its structural analog N-acetyl-leucine. The derivative which contains two statyl residues in a tetrapeptide exhibits inhibitory properties which approach those of pepstatin itself. Other acid proteases, human pepsin, human gastricsin, renin, cathepsin D, the acid protease from R. chinensis and bovine chymosin, also are inhibited by pepstatin and its derivatives. We suggest that the statyl residue is responsible for the unusual inhibitory capability of pepstatin and that statine is an analog of the previously proposed transition state for catalysis by pepsin and other acid proteases. 

Figure 4.12 Amino acid sequence of bovine k-casein, showing the amino acid substitutions in genetic polymorphs A and B and the chymosin cleavage site, Sites of post-translational phosphorylation or glycosylation are italicized (from Swaisgood, 1992).

Visser, S., van Rooijen, P.J., Schattenkerk, C., and Kerling, K.E.T. (1976) Peptide substrates for chymosin (rennin). Kinetic studies with peptides of different chain length including parts of the sequence 101-112 of bovine K-casein. Biochim. Biophys. Acta, 438, 265-72. 

Several proteases from animal organs have been investigated for their milk-clotting potential, but only chymosin, porcine pepsin, and bovine pepsin are of interest to the cheese industry. 

Linklater (1961) reported that bovine pepsin accounted for only 0 to 6% of the milk-clotting activity of commercial rennet extracts. He used porcine pepsin as a reference standard. Bovine pepsin has increased in use as a coagulant because of the practice of extracting the stomach from older calves and adult cattle. More recently, Sellers (1982) reported that 85 to 95% of the proteolytic activity of calf rennet is due to chymosin and the remainder is from bovine pepsin. Adult bovine rennets preparations may contain 55 to 60% bovine pepsin. Mixtures of calf rennet and porcine pepsin may contain 40 to 45% chymosin, 5 to 10% bovine pepsin, and 50% porcine pepsin. Mixtures of adult bovine rennet and porcine pepsin typically contain 20 to 25% chymosin, 40 to 45% bovine pepsin, and 30 to 40% porcine pepsin activity (McMahon and Brown 1985). 

Proteolysis of casein begins with the addition of rennet to the milk and the formation of a coagulum. Calf rennet is actually 80% chymosin and 20% bovine pepsin A (Grappin et al 1985). Rennet can remain active in Cheddar and Camembert cheeses for up to three months, but... 

Chymosin, bovine Cathepsin D, spleen Cathepsin E, bone marrow Thyroid acid protease... 

Bovine pepsin Porcine pepsin Chymosin Penicillopepsin ... 

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. 

Chicken pepsin is the least suitable of these and is used widely only in Israel. Bovine pepsin is probably the most satisfactory and many commercial calf rennets contain up to 50% bovine pepsin its proteolytic specificity is generally similar to that of calf chymosin. The proteolytic specificities of the three principal fungal rennets are considerably different from that of calf chymosin but the acceptability of most cheese varieties made using fungal rennets is fairly good. Microbial rennets are widely used in the United States but to only a limited extent in Europe. The extensive literature on rennet substitutes has been reviewed by Sardinas (1972), Emstrom and Wong (1974), Nelson (1975), Green (1977), and Phelan (1985). 

Calf rennet contains about 10% bovine pepsin (EC 3.4.23.1, Rothe et ai, 1977). The proteolytic products produced from Na-caseinate by bovine pepsin are similar to those produced by chymosin (Fox, 1%9), although as far as we are aware the specificity of bovine or porcine pepsins on bovine caseins has not been rigorously determined. 

Carles, C., and Ribadeau-Dumas, B. (1984). Kinetics of action of chymosin (rennin) on some peptide bonds of bovine /3-casein. Biochemistry 23, 6839-6843. 

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