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At least 19 recordsLinked to original sources
PubMed1990

Effects of glycosylation on the secretion and enzyme activity of Mucor rennin, an aspartic proteinase of Mucor pusillus, produced by recombinant yeast.

The Mucor rennin gene encoding a prepro form of the fungal aspartic proteinase from Mucor pusillus was expressed under the control of the yeast GAL7 promoter in Saccharomyces cerevisiae. The mature M. pusillus rennin secreted efficiently by yeast was a highly glycosylated protein. Analysis by a combination of site-directed mutagenesis of each of the three possible glycosylation sites and treatment of the secreted M. pusillus rennins with endo-beta-N-acetylglucosaminidase H revealed that the mature yeast M. pusillus rennin contained two asparagine-linked glycosylation sites among the three possible glycosylation sites. A mutation of the 2 glycosylated asparagine residues of M. pusillus rennin resulted in significant decreases in the level of secretion by yeast cells. In addition, the extent of glycosylation of M. pusillus rennin was found to affect the enzyme properties such as milk-clotting and proteolytic activities.

Acetylglucosaminidase↗
PubMed1967

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%.

Amino Acid Sequence↗
PubMed1969

Studies on the preparation of water-insoluble derivatives of rennin and chymotrypsin and their use in the hydrolysis of casein and the clotting of milk.

1. Enzymically active insoluble derivatives of chymotrypsin and rennin were prepared by coupling each enzyme to agarose as described by Porath, Axén & Ernback (1967) and rennin to aminoethylcellulose by the method of Habeeb (1967). 2. Agarose-chymotrypsin was stable over the range pH2-9, but agarose-rennin released active enzyme into solution at above pH2 and aminoethylcellulose-rennin was similarly unstable at certain pH values. 3. Each derivative appeared to catalyse the clotting of milk at 30 degrees , but this was probably entirely due to enzyme released into solution from the carrier. 4. The presence of a competitive inhibitor of chymotrypsin during its coupling to agarose had no effect on the activity or stability of the resulting derivative. 5. The characteristics of agarose and cellulose render them not entirely suitable for use in a continuous system with milk.

Animals↗
PubMed1975

Action of rennin on kappa-casein.

The action of rennin on kappa-casein was studied as a function of time using several methods to measure activity. The first indication of rennin cleavage of kappa-casein is precipitability in .1 M acetate buffer at pH 5.2 and 5 C. A longer exposure to rennin is required to alter kappa-casein so that it forms a precipitate with calcium ions and loses its ability to stabilize alpha s-casein. The least sensitive indication of rennin activity is measurement of nitrogen soluble in 2% trichloroacetic acid. Electrophoresis experiments showed that these methods detect various stages in the conversion of kappa-casein para-kappa-casein.

Calcium Chloride↗
PubMed1977

Further characterization of rennin action on kappa-casein using carboxymethylcellulose: effects of various additives on the enzymatic hydrolysis of kappa-casein.

The effects of various additives on the reaction of rennin with kappa-casein were investigated by using carboxymethylcellulose. Both urea and sodium 1-anilino-8-naphthalenesulfonate effectively inhibited rennin action at concentrations larger than 2 M and 2 mM, respectively. These reagents, however, activated the enzyme action at the lower concentrations. Both alpha S-and beta-caseins had some ranges of concentrations in which the rennin reaction was activated. Calcium chloride had an inhibitory effect on the rennin action. Neither mercaptoethanol nor KCl had any appreciable effect on the enzymatic hydrolysis of kappa-casein. These results are analyzed in terms of the association and dissociation of kappa-casein due to the presence of these additives in the reaction solutions.

Adsorption↗
PubMed1968

Rennin enzyme of Endothia parasitica.

A microbiological screening program was instituted to search for an animal rennet substitute. Among 381 bacteria and 540 fungi tested, only one organism, Endothia parasitica, yielded a suitable enzyme substitute. The fungal rennin enzyme was crystallized and some of its properties were studied. It was found to be water-soluble, nondialyzable, precipitable with (NH(4))(2)SO(4) and organic solvents (e.g., acetone and isopropanol), and destroyed by heating for 5 min at 60 C. It was determined to be most stable in water at pH 4.5 and to have an isoelectric point of pH 5.5. On acid hydrolysis, it yielded: alanine, ammonia, arginine, aspartic acid, cysteic acid, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, phenylalanine, proline, serine, threonine, tyrosine, and valine. No tryptophan was detected after alkaline hydrolysis. Its molecular weight was estimated to be in the range of 34,000 to 39,000. The milk-clotting activities of the fungal and animal rennins proved to be essentially identical in milk containing various concentrations of CaCl(2). Both rennins manifested comparable clotting activities in milk at pH 6.0 to 7.0.

Amino Acids↗
PubMed1983

Secretion and activation of the rennin by the rabbit's stomach.

The mucosa of the rabbit's stomach has been studied histochemically, electron microscopically and fluorescence immunologically. The main purpose was to find out whether or not this mucosa secrets the enzyme rennin. During the first two weeks after birth, the gastric glands are composed of only undifferentiated cells. The differentiation of these glands into cardiac, fundic and pyloric glands coincides with the final stage of this period. In the course of the period mentioned the P.A.S.-positive material and the fluorescence induced by rennin exhibit a similar location in the apical cytoplasm of the epithelial cells lining the mucosal surface, the gastric pits and the necks of gastric glands. In the light of these findings, the elaboration and activation of the enzyme rennin is being discussed.

Aging↗
PubMed1982

Photo-oxidation of a histidyl residue of milk-clotting acid protease, Mucor rennin.

Mucor rennin, a milk-clotting acid protease produced by a fungus Mucor pusillus, was inactivated by photo-oxidation mediated by methylene blue according to first order kinetics. The pH profile of the inactivation rate showed that a dissociating group with a pK value of 7.6 was involved in the inactivation. Addition of pepstatin A, an inhibitor specific for acid proteases, caused a marked alkaline shift of the pK value. One of two histidyl residues in the enzyme was destroyed by the photo-oxidation, with complete loss of the enzyme activity. Analysis of inhibitor binding activity and chemical modification with diazoacetyl-DL-norleucine suggested that the photo-oxidized enzyme still retained its original conformation. These results indicated that one histidyl residue in addition to the two essential carboxyl groups is involved in the catalytic function of Mucor rennin.

Chymosin↗
PubMed1982

Proton magnetic resonance spectroscopy of an essential histidyl residue in a milk-clotting acid protease, Mucor rennin.

The location and state of an essential histidyl residue in a milk-clotting acid proteases, Mucor rennin, were investigated by NMR spectroscopy. Assignment of the C2H resonance peak of the essential histidyl residue was possible by comparison of the NMR spectrum of the native enzyme with that of the photo-oxidized enzyme. The pH titration curve for the chemical shift of the C2H proton showed two inflections, a major one with pKa = 7.4 and a minor one with pKa = 3.5 at 30 degrees C. The major inflection, corresponding to an intrinsic protonation of the imidazole ring, shifted toward the alkaline side upon addition of acetyl pepstatin, an inhibitor specific for the acid protease. Modification of an essential carboxyl group in the enzyme with diazoacetyl-DL-norleucine caused disappearance of the minor inflection as well as an acidic shift of the major pKa value. Perturbation effects on the C2H resonance of the lanthanide metals, Pr3+, Eu3+, and Gd3+, suggested their selective binding to a carboxyl group and location of the bound metal atom close to the essential histidyl residue. All data suggested that the essential histidyl residue of Mucor rennin is located close to one of the two essential carboxyl groups in the catalytic site of the enzyme.

Animals↗
PubMed1991

The secretion leader of Mucor pusillus rennin which possesses an artificial Lys-Arg sequence directs the secretion of mature human growth hormone by Saccharomyces cerevisiae.

The prepro-peptide of fungal aspartic proteinase, Mucor pusillus rennin, is useful as a secretion leader for efficient secretion of human growth hormone (HGH) from Saccharomyces cerevisiae. For secretion by yeast cells of HGH with the same NH2 terminus as native HGH, an artificial Lys-Arg linker, which is one of the potential KEX2 recognition sequences, was introduced at the junction between the M. pusillus rennin secretion leader and mature HGH. The HGH directed by this construction was the same size as native HGH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino acid sequencing of its NH2 terminus revealed that the secretion leader peptide was removed correctly at the COOH-terminal side of the Lys-Arg linker. On the other hand, when the same plasmid was expressed in a kex2 mutant strain, unprocessed HGH of a higher molecular weight was secreted, indicating that no proteolytic cleavage at the Lys-Arg site occurred. These results clearly showed that the leader peptide with the Lys-Arg linker was recognized and specifically cleaved by the yeast KEX2 protease. The mature HGH purified from yeast culture medium was indistinguishable from native HGH in biological activity, determined by the adipocyte conversion assay, and in secondary structure, determined by circular dichroism spectroscopy.

Amino Acid Sequence↗
PubMed1976

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.

Age Factors↗
PubMed1980

Action of rennin on casein. III. Effect of alpha s- and beta-casein on the secondary phase.

The action of rennin on kappa-casein was studied as a function of time, employing turbidity measurements at 610 nm and the release of nonprotein nitrogen. kappa-Casein was converted to para-kappa-casein by the action of rennin. The para-kappa-casein aggregated to increase turbidity and then precipitated. Turbidity development was enhanced initially and then retarded severely by increasing concentrations of added alpha s- and beta-casein. The addition of selected amino acids and salts had variable effects on increase of turbidity.

Amino Acids↗