Chymosin extraction

Three proteases account for almost all sales to the dairy industry, ie, chymosin extracted from calves stomachs, chymosin produced by ... 

Three proteases account for almost all sales to the dairy industry, ie, chymosin extracted from calves stomachs, chymosin produced by fermentation, and substitutes also produced by fermentation. Four different types of enzyme are used in the detergent industry, ie, proteases, amylases, cellulases, and Upases. Cellulases and Upases have only recently been introduced. Table 8 shows a breakdown of estimated woddwide sales consumption by product types. 

The era of modem enzyme technology began in 1874 when the Danish chemist Christian Hansen produced the first industrial batches of chymosin by extracting dried calves stomachs with saline solutions. 

Until about 1950, the predominant method of producing industrial enzymes was by extraction from animal or plant sources by 1993, this accounts for less than 10%. With the exception of trypsin, chymosin, papain [9001 -73-2J, and a few others, industrial enzymes are now produced by microorganisms grown in aqueous suspension in large vessels, ie, by fermentation (qv). A smaH (5%) fraction is obtained by surface culture, ie, soHd-state fermentation, of microorganisms (13). 

The first company based upon applied biocatalysis also dates back to the 19 century. In 1874 Christian Hansen started a company in Copenhagen, Denmark. His company— named Christian Hansen s Laboratory to this day—was the first in the industrial market with a standardized enzyme preparation, rennet, for cheese making. Rennet, a mixture of chymosin (also called rennin) and pepsin, was and still is obtained by salt extraction of the fonrth stomach of suckling calves. 

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. 

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-... 

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). 

Milk Coagulation. The first step in cheese manufacture is the coagulation of milk. Traditionally, this coagulation step is catalyzed by the enzyme rennet. Rennet is a saline extract of the 4th stomach of calves, usually slaughtered before they are 30 days old. The principal protease in rennet is rennin. In an attempt to avoid confusion with the hormone peptide renin, the International Enzyme Nomenclature Committee has assigned the name chymosin to the protease in calf rennet. During the growth of calves, chymosin is replaced by pepsin, the acid protease of the mature stomach. 

Rennet, Calf Aqueous extracts made from the fourth stomach of calves. Produced as a clear, amber to dark brown liquid or a white to tan powder. Major active principle protease (chymosin). Typical application used in the manufacture of cheese. Similar preparations may be made from the fourth stomach of lambs or kids. 

Chymosin. Chymosin is the essential milk-clotting component of rennet, the crude extract of the abomasum of calves. Rennet is used in the production of several hundred cheeses in almost all parts of the world. 

Next, it was essential to establish that the recombinant chymosin preparation was safe. The company showed that, as part of its purification process, recombinant chymosin is extracted from inclusion bodies cell debris, and other impurities, that include nucleic acids. Although the presence of minute amounts of E. coli K-12 cells in the final preparation of chymosin is undesirable,... 

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