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Preliminary investigations by N-terminal sequence analysis showed that pig and calf chymosin possessed 80% amino acid sequence identity but showed considerable differences in their enzymatic properties. A comparison of their structures may therefore contribute to an understanding of the significance of the amino acid residues responsible for the differences in these properties. Pig chymosis was extracted from the stomachs of pigs of less than 3 weeks of age, and was purified by ion exchange chromatography. Half of the primary structure was determined by amino acid sequencing and the complete structure was deduced from a cloned chymosin cDNA. Results showed that the zymogen showed 81% sequence identity with calf prochymosin and 57% identity with pig pepsinogen A. The size of the propart and location of the residue which becomes the N-terminus in the active molecule were the same in the prochymosins. The maximum general proteolytic activity at pH 3.5 of pig chymosin was 2–3% of that of the activity of pig pepsin A at pH 2, whereas the milk clotting activity relative to the general proteolytic activity of pig chymosin was much higher than that of calf chymosin. Agar gel electrophoresis at pH 5.3 of stomach extracts of individual pigs showed the existence of two predominant genetic variants of zymogen and enzyme. The two variants could not be distinguished by amino acid composition or N-terminal sequencing, and no differences in the enzymatic properties of the genetic variants were observed. It was concluded that of the residues that participate in the substrate binding, calf and pig chymosin differ in the following positions (pig pepsin numbering, subsites in parentheses): Ser 12 Thr (S4), Leu 30 Val (S1/S3), His 74 Gln (S2′), Val 111 Ile (S1/S3), Lys 220 Met (S4). With regard to the low general proteolytic activity of pig chymosin, the substitution Asp 303 Val relative to calf chymosin may contribute to an explanation of this.

Development of the porcine gastric proteases (chymosin, pepsin A, B and C) has been studied in the fetal pig in the last third of gestation (term 115 days). The possibility that the prepartum rise in circulating cortisol is involved in gastric maturation was investigated by infusing immature fetuses with cortisol (osmotic minipumps implanted at 82-90 days of gestation). Concentrations of prochymosin in fundic tissue and stomach contents increased before term, correlated positively with logio plasma cortisol values (r = 0.68-0.76, p < 0.001), and were stimulated by cortisol infusion (p < 0.001). The pH of stomach contents decreased (from pH 7 to 3), correlated negatively with logio plasma cortisol values (r = -0.69, p < 0.001), and was reduced by cortisol infusion (p < 0.05). Only trace amounts of pepsinogens could be detected in fetal pigs. By immunohistochemistry, it was shown that cortisol increased the number and distribution of prochymosin-containing cells in the fundic gland. Stimulating effects were also observed for the small populations of pepsinogen-reactive cells present in some of the fetal pigs. The results suggest that endogenous cortisol stimulates the rise in prochymosin synthesis and secretion together with increased gastric acidity in the prenatal period of the pig.

Cheesemaking and fermentation represent the first examples of applied biochemistry and biology. Whereas living microorganisms are used in fermentation processes, the clotting of milk for cheesemaking has always required soluble enzymes. The milk-clotting enzyme from the fourth stomach of the calf was one of the first enzymes of which purification was attempted, and Deschamps1 suggested the name chymosin, derived from the Greek word for gastric liquid ’chyme’. This designation was later used in continental European languages, whereas in English the name rennin, derived from rennet, was used.2 Misunderstandings often occurred between rennin and renin from the kidneys, and therefore the designation chymosin was recently adopted in English3 and it is now used in the recommended international enzyme nomenclature.4

Extracts of cat gastric mucosa contain a zymogen that after activation shows partial immunochemical identity with chymosin (EC 3.4.23.4) from calf. Cat prochymosin has been purified by column chromatography and gel filtration, and car chymosin was obtained after acid activafion of the zymogen. The enzyme showed the optimum of ganeral proteolytic activity at pH 2.5. The amino acid compositions of cat prochymosin and chymosin were similar to those of the corresponding proteins from calf. The first 27 residues of both cat prochymosin and chymosin have been sequenced. Among these 54 positions only 13 differences have been observed between the proteins from cat and calf. The results support the hypothesis that the chymosins form a group of neonatal gastric proteases with high milk-clotting activity, but with such weak general proteolytic activity that postnatal uptake of IgG is not hindered.

1. 1. Calculated as active enzymes the gastric mucosa of piglets contains 1–4 mg of chymosin (EC 3.4.23.4) per g of mucosa during the first week of life. No chymosin is present in pigs older than 2 months. 2. 2. The production of pepsin (EC 3.4.23.1) is very low during the first week of life; a rapid increase occurs after about 3 weeks. 3. 3. Pig chymosin and pig pepsin have higher milk clotting activity against porcine milk than against bovine milk. 4. 4. Neonatal proteinases with high milk clotting and low general proteolytic activities are probably important for postnatal uptake of immunoglobulins.

The complete amino acid sequence of calf chymosin (rennin) (EC 3.4.23.4) has been determined. The sequence consists of a single peptide chain of 323 amino acid residues. The primary structure of the precursor part of calf prochymosin was published previously (Pedersen, V.B., and Foltmann, B. (1975) Eur. J. Biochem. 55, 95-103), thus we are now able to account for the total 365 amino acid residues of calf prochymosin. Comparison of the sequence of calf prochymosin with that of pig pepsinogen A (EC 3.4.23.1) shows extensive homology. In the precursor part of the sequence, 15 residues are located at identical positions, as compared to 189 identical residues in the respective enzymes. Furthermore comparison to Penicillium janthinellum acid proteinase (penicillopepsin) (EC 3.4.23.7) shows that 76 residues are common to this enzyme and to the two gastric proteinases. These homologies in sequence further suggest that the folding of the peptide chain in chymosin is very similar to that of other acid proteinases.

Activation of prochymosin at pH below 2.5 results in formation of the active enzyme pseudochymosin by proteolytic cleavage of the bond 27--28. Pseudochymosin is 15 amino acid residues longer than chymosin. It is the final activation product at low pH, whereas chymosin is formed by activation between pH 4 and 5. Pseudochymosin is converted to chymosin when it is brought to pH 5.5. Our present knowledge does not allow quantitative evaluation of the possible reactions involved in formation of pseudochymosin, but the course of activation at pH 2 is in accordance with an intermolecular reaction between two zymogen molecules as the predominant reaction. We find indications of an intramolecular reaction when intermolecular reactions are prevented by immobilization of the zymogen.

Chymosin (EC 3.4.4.3) is the predominant protease in the fourth stomach of the young calf. For many years the term rennin has been used in English literature, but since mistakes often occur between rennin from the calf stomach and renin (EC 3.4.4.15) from the kidneys, we return to the older term chymosin, which previously has been used in European continental literature. Like other proteases from the digestive tract chymosin is secreted as an inactive precursor, which is transformed into active enzyme by a limited proteolysis. The chemistry of the enzyme and the kinetics of the activation process have been reviewed a few years ago [ 1 ] . This communication describes isolation and analyses of some peptides obtained after tryptic digestion of prochymosin in which the amino groups were blocked by reaction with maleic anhydride, and it is shown that these peptides constitute the N-terminal and the C-terminal amino acid sequences of the zymogen.

By conversion of prochymosin into active chymosin an N-terminal segment of 42 amino acid residues is liberated. In one activation experiment this segment was recovered in two peptides; in a second experiment the activation segment was cleaved into three peptides. The primary structures of the peptides have been determined. Overlaps between these peptides and between the activation segment and the active enzyme have been obtained from peptides produced by tryptic digestion of denatured prochymosin. Comparison of the amino acid sequences of the activation segments from bovine prochymosin, bovine pepsinogen and porcine pepsinogen shows considerable homology.

The total sequence of 365 amino acid residues in bovine prochymosin is presented. Alignment with the amino acid sequence of porcine pepsinogen shows that 204 amino acid residues are common to the two zymogens. Further comparison and alignment with the amino acid sequence of penicillopepsin shows that 66 residues are located at identical positions in all three proteases. The three enzymes belong to a large group of proteases with two aspartate residues in the active center. This group forms a family derived from one common ancestor.

1. 1. Prochymosin (EC 3.4.23.4) and pepsinogen A (EC 3.4.23.1) from Mongolian lamb (Ovis platyurea) were purified to homogeneity by salt precipitation, gel filtration and ion-exchange chromatography. 2. 2. Immunoelectrophoresis shows partial immunochemical identity between chymosins and pepsins from lamb and cattle, respectively. 3. 3. Activity determinations, N-terminal amino acid sequences and amino acid compositions also show a close relationship between the proteinases from lamb and cattle. 4. 4. Lamb prochymosin and pepsinogen are both glycosylated.

Limited proteolysis of bovine kappa-casein has been investigated with porcine pepsin A and C, and with the 2 microbial proteinases Mucor miehei proteinase and Endothia parasitica proteinase. The liberated C-terminal glycomacropeptide of kappa-casein was isolated after precipitation in 3% trichloroacetic acid followed by high-performance gel-permeation chromatography on a TSK G3000 SW column. From amino acid analyses and N-terminal sequencing of the liberated peptide it is concluded that porcine pepsin A, C and Mucor miehei proteinase cleave the same bond as chymosin: Phe-105-Met-106 whereas Endothia parasitica proteinase cleaves the bond Ser-104-Phe-105.

Proteinases with a pH-optimum of general proteolytic activity at pH below 6 are called acidic proteinases. Such enzymes were at first recognized as the proteinases of the vertebrate gastric juice, and for many years porcine pepsin and calf chymosin have been the most well-known members of this group.