Chymosin

CHYMOSIN (chymase, rennet ferment, rennet), an enzyme causing the coagulation of milk, conditioned by the conversion of milk casein into paracasein, which in the presence of calcium salts precipitates in the form of a curdled mass (cheese). Chymosin is contained in the mucous membrane of the third stomach of ruminants (the abomasum); it is especially abundant in the abomasum of young animals fed on milk (rennet powder for cheesemaking is manufactured from the abomasum of calves). Chymosin is a proteolytic enzyme, and the formation of paracasein is the initial stage of the hydrolytic cleavage of casein under the influence of chymosin (Hammarsten). Chymosin acts at a neutral or weakly acidic reaction (the optimal reaction of the medium lies, according to Michaelis, at pH 6.0–6.4, and according to van Dam, at pH 5), splitting not only casein but also some other proteins (legumin, syntonin) with the formation, albeit insignificant, of free carboxyl and amino groups. Under the influence of alkalis, chymosin is easily destroyed. On the other hand, typical proteases (pepsin, papain, trypsin) at pH 5 also convert casein into paracasein, which with Ca salts precipitates in the form of cheese. The rennet action of the gastric mucosa and preparations made from it usually (but not always) runs parallel to the pepsin content. The separation of both enzymes presents extreme difficulties, which led to the creation of a hypothesis about the identity of chymosin and pepsin, which constitutes the subject of a fierce discussion. In the opinion of Hammarsten, chymosin is contained only in the abomasum of young ruminants (calves, lambs, etc.) and disappears in adults; it is distinct from pepsin, and Hammarsten developed methods for separating both enzymes. The rennet action of the stomachs of adult ruminants and animals of other families depends on the presence in them of 'parachymosin,' which is inseparable from pepsin and is likely identical to the latter. At the pH optimal for 'parachymosin' (pH 5.0), the weak residual proteolytic activity of pepsin manifests itself in the form of rennet action. Chymosin is secreted by the glands of the fundus of the stomach in an inactive form and is activated only upon contact with acidic gastric juice. However, the assumption about the existence of a special 'labzymogen' or 'prochymosin' was refuted by Hedin, who showed that chymosin is secreted in a ready, active form, but is accompanied by a thermostable paralyzer, which is rapidly destroyed by acids. Enzymes of the chymosin type are widely distributed in nature; they have been found in the digestive organs of animals that do not feed on milk (fish, amphibians, birds, invertebrates), and in plant juices (the herb Galium verum, used in cheesemaking), and finally in lower organisms, e.g., in molds (Aspergillus niger). Regarding the synthesizing action of chymosin, see Plastein. Upon immunization of animals with chymosin preparations, a specific immune antibody appears in their blood, inhibiting the action of chymosin; this so-called antilab is one of the most studied immune antienzymes. To obtain active solutions of chymosin, according to Hammarsten, the mucosa of the fundic part of the calf abomasum is washed with water, scraped off, and infused with a 10-fold amount of 0.1% HCl at 0° for 1 day; the extract is filtered and carefully neutralized with soda. To determine the activity of chymosin preparations, increasingly diluted solutions of chymosin are added to a mixture of milk with 1/10 by volume of a 10% CaCl₂ solution, and the greatest dilution at which coagulation still occurs is established. A. Braunstein.