Enzymes chymosin

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. 

This, maybe somewhat peculiar, example demonstrates that the use of digestible catalysts, like the enzyme phytase, further broadens the field of catalysis, i.e. not in a visible production rmit but in the species or product where the catalytic conversion is really needed. Other examples in the food industry are the application of the enzymes chymosin, invertase and amylase for cheese, bonbon and bread manufacturing, respectively. 

In the manufacture of fresh cheeses, e. g. quark, the pasteurised skim milk is inoculated with micro-organisms (Sc. lactis, Sc. cremoris). To accelerate the thickening, the enzym chymosin is added. After ripening - with a pH value of about 4.6 - the coagulated milk must be pumped through a separator, possibly including an ultrafiltration system, in order to separate the sour whey. The ultrafiltration would separate the whey into 2 phases the permeate (water soluble) and the retentate (protein phase). Finally the quark, retentate, cream, (fruit) preparations, flavourings or spices and herbs are added. 

FIGURE 7.9 Heating times needed (t ) for inactivation (reduction of activity to about 1 %) of the enzymes chymosin (Ch), lipoxygenase (LP), acid phosphatase (AP), and plasmin (PI) and for 30% of ovalbumin (OA) and /1-lactoglobulin (LG) to become insoluble. Because of the narrow temperature intervals involved, also plots versus T, rather than fT, can be approximately linear. 

BC. Calves stomachs and the enzyme chymosin were used for cheese making. 

The environmental benefits are (1) cheese makers are no longer dependent upon enzymes recovered from slaughtered calves and lambs for production of rennet needed for most cheese making processes, and (2) based on current demands for chymosin, commercial needs for rennet could not be met from animal sources. The consumer benefits are (1) plentiful, consistently high quality enzyme chymosin is available at low prices that help assure availability of excellent cheeses at a reasonable cost and (2) people who follow kosher and vegetarian eating practices can consume cheese since the enzyme is from a microbe and not a calf. 

Pepsin, pepsin-like enzymes, chymosin, rennin, and other acid proteinases have an activity optimum at pH 2.0-3.5 papain, trypsin, chymotrypsin, and similar enzymes are most active at neutral pH (pH 6-8). Subtilisin BPN, pancreatic elastase, leucine... 

Such processes have existed for many thousands of years in the manufacture of cheese from milk and in the brewing of beer from barley. In the former process the enzyme chymosin catalyses the hydrolysis of one peptide bond in casein, causing the milk to clot, and in the latter the amylases from malted barley (the diastases of Peyen and Persoz) catalyse the hydrolysis of starch. While these remain important processes the application of enzymes such as these to industrial chemistry is quite recent, dating back only to about 1960. 

Bulk Enzymes. Enzymes such as proteases, amylases, glucose isomerases, and rennin are used in food processing. Similarly proteases and Hpases are used in detergents. CeUulases and xylanases are used in the paper pulp industry. The genes for most of the enzymes used in the various commercial processes have been cloned and overexpressed. Rennin (chymosin) produced from E. coli and A. nigerhas been approved by FDA for use in the dairy industry. 

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. 

Yeast. Several yeast species, including Saccharomjces cerevisiae (baker s yeast) and Klujveromjces lactis are good candidates for the production of certain industrial enzymes, although their abiUty to secrete is much inferior to Bacilli 2in.d Yispergilli. The best-known example of K. lactis is used for commercial production of chymosin [9001-98-3]. 

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). 

Milk from cows contains 3.2% protein, about 80% of which is casein. Casein is isolated by a precipitation process from milk, involving heating, rinsing to remove whey, and drying to a powder. The yield is about 3 kg/ 100 kg skim milk. Rennet casein is obtained when the casein is precipitated by chymosin enzyme, also known as rennet, and acid casein is produced when precipitation is accomplished by acidification. Acid casein is usually found in the form of sodium caseinate or calcium caseinate, which are water-soluble salts. Caseinates are made by reacting NaOH or CaOH with a slurry of casein curd or powder and then spray drying (Southward, 2010). 

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. 

The most abundant milk protein is casein, of which there are several different kinds, usually designated a-, (1-, and K-casein. The different caseins relate to small differences in their amino acid sequences. Casein micelles in milk have diameters less than 300 nm. Disruption of the casein micelles occurs during the preparation of cheese. Lactic acid increases the acidity of the milk until the micelles crosslink and a curd develops. The liquid portion, known as whey, containing water, lactose and some protein, is removed. Addition of the enzyme rennet (chymosin) speeds up the process by hydrolysing a specific peptide bond in K-casein. This opens up the casein and encourages further cross-linking. 

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... 

Microbial coagulants are now useful and are responsible for about one third of all the cheese produced worldwide, but suffer from the disadvantage of being too stable and so are threatened commercially by improved methods of produdng chymosin by recombinant DNA technology. The use of thermally destabilized microbial rennets results in residual enzyme levels in the milk product similar to or below those encountered when calf rennet is use (55). An unexpected benefit has been an increase on some occasions of the specificity of the microbial enzyme, making it virtually indistinguishable from the action of calf rennet. Also some microbial rennets help impart a flavor that is popular with consumers. 

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. 

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. 

Recent reviews (Green 1977 Phelan 1977 Visser 1981) have dealt with the role of milk-clotting enzymes in cheese manufacture, while Foltmann (1981) has provided an excellent discussion of the structure of chymosin and its enzymic properties. 

Fungal proteases have been investigated extensively in search of suitable milk clotting enzymes. Patents have been issued for production of rennets from E. parasitica, M. Pusillus var. Lindt and M. miehei var. Cooney et Emerson. These have been approved in the United States as secondary direct food additives (FDA. 1984B) and have experienced considerable commercial success in the United States as milk-clotting enzymes for cheese manufacture. Many other fungal sources have also been tried in the effort to find an inexpensive replacement for chymosin. 

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. 

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