Calf Chymosin Research Articles

Rennin and pepsin in stomachs of rats (Rattus norvegicus).

The predominant protease in adult rat stomachs has a pH optimum of 2.0 to 2.3 which corresponds almost exactly to that of porcine pepsin. One-day-old rats have a protease with a pH optimum of 3.8 to 4.2 similar to that of calf rennin. The pH optimum of rat stomach proteases decreases with age, and at 28 days it is similar to that of the adult. The pH of stomach contents of rats decreases with age from pH 5.8 to pH 4.6. These data support the contention by other workers that the onset of pepsin secretion is a prerequisite for weaning and that rennin-like enzymes are not unique to ruminant species.

The effect of acid proteinase inhibitors on chicken pepsin.

1. The activity of chicken pepsin was partially inhibited by dimethyl-(2-hydroxy-5-nitrobenzyl)sulphonium bromide, but was unaffected by p-bromophenacyl bromide. 2. In the presence of Cu2+, diazoacetylnorleucine methyl ester completely inactivated chicken pepsin with the incorporation of 1 mol/mol. The mechanism of the reaction was similar to that with pig pepsin. 3. Chicken pepsin was completely inactivated by 2-diazo-4-bromoacetophenone in the presence of Cu2+. 4. Chicken pepsin was almost completely inactivated by 1,2-epoxy-3-(p-nitrophenoxy)propane at 25 degrees C, 3-4mol of inhibitor/mol being incorporated. The reaction at 10 degrees C was investigated briefly. 5. Calf chymosin was inactivated by 1,2-epoxy-3-(p-nitrophenoxy)propane at 10 degrees C, the incorporation of 1 mol/mol being required for complete inhibition. 6. The characteristics of the reactions of chicken pepsin with the above compounds were compared with those of other acid proteinases.

Rhizopus acid proteinases (rhizopus-pepsins): properties and homology with other acid proteinases.

Commercial acid proteinase from Rhizopus chinensis has been further purified by isoelectric focussing. Two forms of the enzyme (I and II) with very similar properties have been obtained. Like other fungal acid proteinases they activate trypsinogen at pH 3.4 and are inhibited by diazoacetyl norleucine methyl ester. Because of the lability of the label it has not been possible to isolate the active site peptide from a peptic digest. N-terminal amino acid sequences were determined for Rhizopus enzyme I (NH2∙Ala∙Gly∙Val∙Gly∙Thr∙Val∙Pro∙Asx∙Thr), for Rhizopus enzyme II (NH2∙Ala∙Gly∙Val∙Gly∙Thr∙Val∙Pro), and for penicillopepsin (NH2∙Ala∙Ala∙Ser∙Gly∙Val∙Ala∙Thr∙Asn∙Thr∙Pro∙Thr). The similarity in enzymic properties and in the sequence suggests that the Rhizopus enzymes are homologous with penicillopepsin and hence also with mammalian pepsins and calf chymosin.

Inactivation of acid proteases from Rhizopus chinensis, Aspergillus saitoi and Mucor pusillus, and calf rennin by diazoactylnorleucine methyl ester.

Journal Article Inactivation of Acid Proteases from Rhizopus chinensis, Aspergillus saitoi and Mucor pusillus, and Calf Rennin by Diazoacetylnorleucine Methyl Ester Get access Kenji TAKAHASHI, Kenji TAKAHASHI The Department of Biophysics and Biochemistry, Faculty of Science, The University of TokyoHongo, Tokyo Search for other works by this author on: Oxford Academic PubMed Google Scholar Fumio MIZOBE, Fumio MIZOBE The Department of Biophysics and Biochemistry, Faculty of Science, The University of TokyoHongo, Tokyo Search for other works by this author on: Oxford Academic PubMed Google Scholar Wen-Jong CHANG Wen-Jong CHANG The Department of Biophysics and Biochemistry, Faculty of Science, The University of TokyoHongo, Tokyo Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 71, Issue 1, January 1972, Pages 161–164, https://doi.org/10.1093/oxfordjournals.jbchem.a129739 Published: 01 January 1972 Article history Received: 01 October 1971 Published: 01 January 1972

PURIFICATION AND PROTEOLYTIC ACTION OF MILK- CLOTTING ENZYME PRODUCED BY PENICILLIUM CITRINUM

Electrophoresis of the partially purified milk-clotting enzyme produced by Penicillium citrinum with 0.02M acetate buffer (pH 3.42) showed four protein components. Fractionation of the enzyme preparation with acetone led to individual isolation of the four electrophoretic components. The course of proteolysis in the first phase of enzymic action of each of the partially purified fungal enzymes and the four pure fractions was conducted and compared with those of trypsin and calf rennin. The fungal enzyme contained two rennin-like fractions constituting most of the milk-clotting activity with limited proteolysis and another two fractions with less clotting activity and more proteolysis. The macroglycopeptide isolated by the action of the fungal rennin-like enzyme fraction comprised 12 amino acids while its carbohydrate moiety consisted of galactose, galactosamine, and N-acetylneuraminic acid. Further studies on the most active rennin-like enzyme fraction showed that calcium chloride enhanced only the primary stage of the enzymic phase but near the end of this phase the rate of NPN release was very slow.

The crystallography of calf rennin (chymosin).

The growth of crystals of calf rennin (chymosin, † EC 3.4.4.3) and the control of nucleation to produce crystals of desired size, are described. Only one stable species, orthorhombic rennin I, has been found, but a metastable monoclinic species, rennin II, appeared on one occasion in a solution nearly saturated with glycine. The morphology, optical characteristics and unit cells of these species are recorded. In rennin I, small differences in the properties of sectors built by deposition on the three types of crystal faces are attributed to differences in the proportions of either degradation products or of the slightly different isoenzymes known to be present in calf rennin. Rennin I crystals have been obtained only within the pH range of greatest stability of the protein, 5. 0 to 6. 2; but the crystals tolerate subsequent changes of pH down to 2. 0. The stability of crystals appears to be much greater than that of salt-free protein solutions. Rennin I may be crosslinked with glutaraldehyde with no loss of order, giving crystals stable over a wide range of solution conditions, including pH 2.0 to 10.0 and salt-free solutions. The remarkable swelling behaviour when the pH is raised beyond the stable range is described. Rennin II, though monoclinic in symmetry, shows in its X -ray diffraction patterns strong evidence of a pseudo-orthorhombic arrangement of molecules.

Crystalline Milk-Clotting Protease from Mucor Miehei and Some of its Properties

A milk-clotting protease has been separated in crystalline form from filtrates of Mucor miehei. It is the major proteolytic component produced by the microorganism, and has a molecular weight of 34,000 to 39,000. Determination of amino acid composition indicates: Asp42, Thr18, Ser25, Glu15, Ala22, Val16, ½(Cys)4, Met5, Ileu11, Leu14, Tyr13, Phen14, Orn1, Lys8, His2, Arg4, Try3 (NH3)39. The pH optima for proteolysis of hemoglobin, casein and bovine serum albumin classify the enzyme as an acid protease.The enzyme is most stable at acid pH. The effects of inhibitors indicate that this clotting enzyme is not metal dependent and does not have serine or SH active groups. Aluminum sulfate has a peculiar depressing action on clotting activity, and study of the mechanism has pointed out similarities between this M. miehei enzyme and calf rennin.

Rennin Detection in Cheese with the Passive Indirect Hemagglutination Test

A specific and sensitive method for detecting rennin in cheese has been developed. The method utilizes antiserum and coated erythrocytes as reagents. High titer specific reagents have been produced for calf rennin and the milk coagulating enymes of Mucor pusilus and Endothia parasitica, but not for pepsin.

The N-terminal and C-terminal amino acid sequence of calf rennin

The enzyme rennin (EC 3 . 4 . 4 . 3) is well known as the milk clotting enzyme from the fourth stomach of the calf. It appears, however, that the enzyme may be regarded as a suitable model enzyme for studies of the structure and function of gastric proteases. The general properties of the enzyme have recently been reviewed (Foltmann 1966), and the amino acid sequences of some soluble tryptic peptides and the disulphide bridges have been investigated (Foltmann & Hartley 1967). In the latter paper the only soluble tryptic peptides that were investigated were basic or neutral. Subsequent experiments have shown that two acidic peptides, which only appeared with a modest ninhydrin reaction in the first experiments are in fact significant fractions of the tryptic digest. One of these peptides represents the N-terminal amino acid sequence, while the other is a part of the C-terminal amino acid sequence.

The disulphide bridges and soluble tryptic peptides of calf rennin.

1. Cysteic acid peptides from various digests of calf rennin were purified by diagonal paper electrophoresis. 2. The amino acid sequence of these peptides accounts for 38 amino acids around three unique disulphide bridges in rennin. 3. One bridge connects two acidic regions of the chain, one forms a loop of five residues and the other a loop of six residues. 4. These bridges are homologous with those of hog pepsin. 5. Tryptic peptides from the C-terminus of rennin account for 22 residues, 17 of which are homologous with the C-terminus of pepsin. 6. Altogether, sequences accounting for 94 of the 270 residues in rennin are described and the degree of homology with pepsin approximates to 70%.