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Results for “Chymosin enzyme”

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At least 19 recordsLinked to original sources
PubMed2026

Recombinant bovine chymosin expression in microalgae Chlamydomonas reinhardtii chloroplast: A step towards algal biomanufacturing of dairy enzymes.

Chymosin is the major proteolytic enzyme for cheese manufacture, where it plays an important role in the co-precipitation of milk casein. Traditional extraction of chymosin from the abomasum of young ruminants is associated with high limitations, including low yield, high production cost, and ethical issues of animal slaughter. In this study, we report on a recombinant strategy towards the production of bioactive Bos taurus chymosin in the chloroplasts of the microalga Chlamydomonas reinhardtii. The cym gene encoding preprocymosin was inserted into the chloroplast genome by the glass bead-mediated DNA transformation procedure. Successful integration and expression of the transgene were confirmed by spot test analysis, polymerase chain reaction (PCR), western blot, and enzyme-linked immunosorbent assay (ELISA). The functional activities of the recombinant enzyme were checked by the standard milk clotting assay. The engineered microalgal strains produced chymosin with an average concentration of 90 mg/kg fresh weight, i.e., 1.6% of the total soluble protein. These results show that chloroplast-engineered C. reinhardtii is a promising, sustainable, and animal-free platform for the efficient production of the industrially relevant chymosin.

Animals↗
PubMed1979

[Quantitative determination of the activity of acid peptidases of industrial origin].

Reagent ninhydrine-Cd++, reacts with free alpha and epsilon amino groups of proteins. Horse-heart apomyoglobin was subjected to exhaustive succinylation, rendering the product non reactive to ninhydrine. The succinylglobin was submitted to enzyme digestion at pH 2.0, 4.0, 4.7 and 6.0. The commercially available enzymes contain mainly pepsin-like and chymosin-like enzymes. The enzymatic digests of succinyl-globin contain new free alpha-amino groups reacting with ninhydrin. Enzymatic digestion was performed under various condition (ratio E/S, pH). The results were compared to those obtained with synthetic substrate: PRO-HIS-LEU-SER-PHE(NO2)-NLEU-ALA-LEU-OME. The price of the synthetic substrate used, was more than 100 times the cost of succinyl-globin, thus the use of this substrate is a valuable tool for the quantitative estimation of peptidase activity in commercially available (pepsin, chymosin-like) enzymes.

Apoproteins↗
PubMed1979

Characterization of byssochlamyopeptidase A.

The enzyme properties of byssochlamyopeptidase A, a chymosin-like enzyme produced by Byssochlamys fulva were studied. The enzyme was shown to be electrophoretically and immunochemically pure. Most metallic cations had negligible effect, whereas Hg2+ greatly suppressed the enzyme activity. N-Bromosuccinimide and I2 completely inactivated the enzyme. For milk clotting at pH 4.6-6.6, the enzyme was less sensitive to pH than pepsin. The substrate specificity of the enzyme was studied by incubation of the enzyme at 37 degrees C with whole and individual casein fractions at pH 6.6 and pH 3.0, respectively. The proteolysis by the enzyme was found to be most extensive for alphas-, less for kappa-, and least for beta-casein. Studies with different synthetic dipeptides and tripeptides revealed that byssochlamyopeptidase A exhibits specificity for Phe-Tyr and Gly-Phe-Phe, but the enzyme did not hydrolyze Ac-Phe-Tyr(I2).

Ascomycota↗
PubMed1979

Investigations on the activation of bovine prochymosin.

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.

Amino Acid Sequence↗
PubMed1977

The complete amino acid sequence of prochymosin.

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.

Amino Acid Sequence↗
PubMed1977

The first step in the activation of chicken pepsinogen is similar to that of prochymosin.

Chicken pepsinogen was incubated at pH2.5 with pepstatin. The zymogen activated itself by a sequential mechanism and an intact peptide derived from residues 1-26 in the protein was released in the first step. This peptide was found to inhibit the milk-clotting activities of pig and chicken pepsins and calf chymosin but to different extents.

Amino Acid Sequence↗
PubMed2024

The combined effect of the gene copy number and chaperone overexpression on the recombinant bovine chymosin production in Pichia pastoris, with mutant ADH2 promoter.

Chymosin is an enzyme used to coagulate milk, in the cheese industry. This study aimed to increase recombinant production of the chymosin in Pichia pastoris by determining the optimum copy number and overproduction of a Protein Disulfide Isomerase (PpPDI) chaperon protein. Bos taurus chymosin was expressed under the control of a mutant ADH2 promoter. The clones containing 1-4 gene copy numbers of the chymosin were constructed using the in vitro cloning method, and the effect of chaperone protein on chymosin secretion was investigated. The enzyme production levels are 4, 6.3, 4.5, and 3 IMCU/mL for 1, 2, 3, and 4-copy clones. The secreted chymosin levels increased up to two copies, and increasing the number of copies decreased the secretion level. Therefore, PpPDI was over-expressed in the clones regulated with the ADH2 promoter. The over-expression of PDI gene increased chymosin secretion in clones compared to the counterpart host. However, the highest chymosin level was obtained with C2 (2-copy chymosin containing clone; 6.3 IMCU/mL) and C2P2 (2-copy chymosin/2-copy PDI containing clone; 8.2 IMCU/mL). The maximum production was 39 IMCU/mL with the clone C2P2 in the fermenter scale production. The enzyme activity increased approximately 2-fold by adding two copies of the chaperone protein. The combined effect of gene copy number and chaperone overexpression on chymosin production was investigated. Two copies of the chymosin and PpPDI genes were the optimum among the tested clones.

Animals↗
PubMed1976

Synthetic peptides for chymosin and pepsin assays: pH effect and pepsin independent-determination in mixtures.

Peptide I [H-Phe-Gly-His-Phe(NO2)-Phe-Ala-Phe-OMe] hydrolyzed by chymosin with kcat=.3+/-.3 s-1 and KM=7+/-3 mM (pH 4.7) inhibited competitively peptide II [H-Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe] hydrolysis by chymosin with KI=.23 +/- .12 mM at pH 4.7. In reference conditions (.4 mM peptide, .01 M acetate buffer pH 4.7), the specific activities of porcine pepsin and chymosin on peptide I were 470 +/- 70 nM S-1 and .8 nM S-1 per mg of enzyme. This difference in specific activity for peptide I allowed development of a chymosin-independent pepsin assay for mixtures of these enzymes. In addition, peptide II with a specific activity of 2400 +/- 300 nM S-1 and 154 +/- 20 nM S-1 per mg of porcine pepsin and chymosin provides an alternative to measurement of milk clotting for measurement of chymosin- and pepsin-like activities in commercial rennets. Hydrolysis products of peptide II by chymosin exhibited one ionized group of apparent pK of 3.5 +/- .2 and a molar absorption coefficient change of 1000 +/- 100 at pH 4.7 and at 310 nm. From measurements of the kinetic constants, kcat and KM, from pH 2.5 to 7 with peptide II, chymosin activity depends on the protonation of one group of apparent pK 5.3 +/- .2 in the free enzyme. Rennet powder proved to be fairly stable after a 17-month storage at 4 C. Within the same period, a crystalline chymosin solution kept at --18 C lost 30 to 50% of its activity.

Animals↗
PubMed1975

Amino-acid sequence of the peptide segment liberated during activation of prochymosin (prorennin).

By conversion of prochymosin into active chymosin and 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.

Amino Acid Sequence↗
PubMed1977

Activation studies of the multiple forms of prochymosin (prorennin).

Activation of the four separate components of prochymosin (prorennin) at pH 5.0 demonstrated that each zymogen was the precursor to an electrophoretically distinct chymosin (rennin). When the increase in milk-clotting activity with time was analysed, the mechanism of activation of unfractionated prochymosin, individual prochymosin components, and a mixture of the prochymosin fractions at pH 5.0 was shown to follow essentially autocatalytic kinetics. The activation of prochymosin C was completed in 70 h, whereas the other three fractions each required more than 110 h for complete activation under the same conditions. Intact prochymosin, the mixture of four components and prochymosin C were activated at similar rates. Interaction of the individual fractions during activation is suggested to explain the increased rate of the activation for the mixture. Comparison of autocatalytic activation of unfractionated prochymosin purified chromatographically at pH 6.7 and 5.7 demonstrated an increased rate of reaction of the zymogen prepared at the lower pH value. The possibility that prochymosin became susceptible to activation during preparation at pH values slightly below 6.0, as a result of changes in the proportion of the components or a conformational change and exposure of the active site, is discussed.

Chymosin↗
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↗
PubMed1977

Influence of age, dietary protein and weaning on calf abomasal enzymic secretion.

Using fistulated calves, the influence of the age, type of dietary protein and weaning on the secretion of chymosin and pepsin by the abomasum were studied. The abomasum secreted both chymosin and pepsin when the animals were fed milk. Chymosin secretion appeared to be independent of the age of the animals whereas a slow increase in pepsin secretion was observed as the calves aged. Several preruminant animals were fed either a skim-milk diet or a milk substitute in which proteins were provided by fish, soya or whey concentrates. Each of these 3 milk substitutes led to a decrease in chymosin secretion without modification of pepsin secretion. Chymosin secretion was partly restored when the claves were again given a skim-milk diet. At weaning, chymosin secretion dropped abruptly, but the pepsin level was not affected. These results indicate that milk (most probably its casein fraction) is responsible for the activation of chymosin secretion.

Abomasum↗
PubMed1977

Gel diffusion--a simple and sensitive technique for the assay of proteinase inhibitors and its use for the determination of the ratio of proteinases in mixtures.

In casein-containing agarose gels, pepsin and chymosin form radial diffusion zones; the diameters of these zones show rectilinear correlations with the logarithm of the enzyme concentration at constant time. The sensitivity for both enzymes is below 1 microgram. Addition of the inhibitor pepstatin A to these enzymes causes a reduction of the diameters of the diffusion zones, with large differences for both the enzymes. With this procedure, the pepsin/chymosin ratio in rennet preparations was assayed with an accuracy of +/- 5%. Identification of the inhibitors allows the determination of amounts in the namomole range. This method is a simple technique for the evaluation of proteinases and their inhibitors in screening systems.

Animals↗
PubMed1977

Differentiation of rennet from other milk-clotting enzymes by polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis was used to differentiate animal rennet and other milk-clotting enzymes. After electrophoresis, the separated components were visualized by staining with aniline blue-black. Two prominent proteins were found in calf and bovine rennet, while only 1 major protein was observed in pepsin and enzymes of microbial origin. These patterns provided a basis for distinguishing animal rennet and the other enzymes as well as a means of identifying each type of enzyme by the characteristic pattern shown.

Animals↗
PubMed1979

The primary structure of calf chymosin.

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.

Amino Acid Sequence↗