Chymosin activation

Emmons (1970) experienced significant inactivation when commercial pepsin and pepsin-calf rennet mixtures were diluted with high-pH, hard water 10 min before adding them to the cheese vat. Mickelsen and Ernstrom (1972) reported that mixtures of porcine pepsin and calf rennet were stable between pH 5.0 and 6.0, but that pepsin activity was lost from the mixture aboire pH 6.0. This loss was shown to be entirely due to pepsin instability. Below pH 6.0 chymosin activity was destroyed by pepsin. 

Matheson, A. R. 1981. The immunological determination of chymosin activity in cheese. 

The carboxyl proteases are so called because they have two catalytically essential aspartate residues. They were formerly called acid proteases because most of them are active at low pH. The best-known member of the family is pepsin, which has the distinction of being the first enzyme to be named (in 1825, by T. Schwann). Other members are chymosin (rennin) cathepsin D Rhizopus-pepsin (from Rhizopus chinensis) penicillinopepsin (from Penicillium janthinel-lum) the enzyme from Endothia parasitica and renin, which is involved in the regulation of blood pressure. These constitute a homologous family, and all have an Mr of about 35 000. The aspartyl proteases have been thrown into prominence by the discovery of a retroviral subfamily, including one from HIV that is the target of therapy for AIDS. These are homodimers of subunits of about 100 residues.156,157 All the aspartyl proteases contain the two essential aspartyl residues. Their reaction mechanism is the most obscure of all the proteases, and there are no simple chemical models for guidance. 

Cathepsin D (EC3.4.23.5). It has been known for more than 20 years that milk also contains an acid proteinase, (optimum pH ss 4.0) which is now known to be cathepsin D, a lysozomal enzyme. It is relatively heat labile (inactivated by 70°C x 10 min). Its activity in milk has not been studied extensively and its significance is unknown. At least some of the indigenous acid proteinase is incorporated into cheese curd its specificity on asl- and / -caseins is quite similar to that of chymosin but it has very poor milk-clotting activity (McSweeney, Fox and Olson, 1995). It may contribute to proteolysis in cheese but its activity is probably normally overshadowed by chymosin, which is present at a much higher level. 

Rennets. The traditional rennets used to coagulate milk for most cheese varieties are prepared from the stomachs of young calves, lambs or kids by extraction with NaCl (c. 15%) brines. The principal proteinase in such rennets is chymosin about 10% of the milk-clotting activity of calf rennet is due to pepsin. As the animal ages, the secretion of chymosin declines while that of pepsin increases in addition to pepsin, cattle appear to secrete a chymosin-like enzyme throughout life. 

Although in vitro, the cell wall-associated proteinase of the Lactococcus starters is quite active on 8-casein (and that from some strains on asl-casein also), in cheese, they appear to act mainly on casein-derived peptides, produced by chymosin from asl-casein or by plasmin from / -casein. 

Procedures for extraction of chymosin from veils were described by Ernstrom and Wong (1974). Crude rennet extract contains active chymosin and an inactive precursor (prochymosin). Addition of acid to the extract facilitates conversion of prochymosin to chymosin and allows the extract to reach maximum activity. Even though activation at lower pH is faster, poor stability of chymosin below pH 5.0 in the pres-... 

Activation of prochymosin involves the splitting of peptides from the N-terminal end of prochymosin with simultaneous reduction in molecular weight from about 36,000 to 31,000. The rate of conversion increases markedly with decreasing pH below 5.0 (Rand and Emstrom 1964). At pH 5.0, NaCl concentrations up to 2M increase the rate of activation. Milk-clotting activity plotted against activation time at pH 5.0 shows the course of activation (Fig. 12.1) to be autocatalytic. If activation is carried out in the presence of preformed chymosin, the S-shape disappears and the initial rate of the activation process increases with increasing concentration of preformed chymosin. Folt-... 

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). 

M. pusillus var. Lindt protease has given satisfactory results as a chymosin substitute in the manufacture of a number of cheese varieties, but not all varieties of M. pusillus var. Lindt are capable of producing acceptable cheese (Babel and Somkuti 1968). The clotting activity of M. pusillus var. Lindt protease is more sensitive to pH changes between 6.4 and 6.8 than chymosin, but is much less sensitive than that of porcine pepsin (Richardson et al 1967). The same authors reported that CaCL added to milk affected the clotting activity of M. pusillus var. Lindt rennet more than it did that of chymosin rennet. They also reported that this rennet was more stable than chymosin between pH 4.75 and 6.25. M. pusillus var. Lindt rennet is not destroyed during the manufacture of Cheddar cheese, although less than 2% of the enzyme added to the milk remains in the curd. Nearly all of it is found in the whey (Holmes et al. 1977). Mickelsen and Fish (1970) found M. pusillus var. Lindt rennet to be much less proteolytic than E. parasitica rennet but more proteolytic than chymosin rennet on whole casein, a8-casein and /3-casein at pH 6.65. 

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... 

Cheese is made by coagulating milk by the addition of rennet to produce curds. The curds are separated from the liquid whey and then processed and matured to produce a wide variety of cheeses. The active ingredient of rennet is the enzyme chymosin. Until 1990, most rennet was produced from the stomach of slaughtered newly born calves. These days, at a cost one tenth of that before 1990, chymosin is produced by genetically engineered bacteria into which the gene for this enzyme has been inserted, and is used for making cheese in the United States, Europe, and other parts of the world. 

Aspartic proteases include the gastric proteases pepsin A, pepsin B, gastricsin and chymosin that are secreted as inactive zymogens and are activated through removal of autoinhibitory domains in the low pH conditions of the stomach [7-9]. Oesophagitis is caused by undue... 

The stomach environment is acidic as a result of HC1 secretion by the parietal cells. The acidic pH serves to denature many proteins, thus making them susceptible to proteolysis. The chief cells of the stomach produce pepsinogen, which is activated to pepsin by the HC1 (see Table 20.3). The optimum pH of peptic activity is around 2, and pepsin is inactivated at neutrality. Another stomach enzyme is rennin or chymosin, which is present in infants but not in adults. It removes a glycopeptide from milk-K-casein, disrupting the casein micelle and promoting milk protein coagulation and digestion. 

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