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PubMed1987

Separation of chymosin and pepsin in calf rennet by dye-ligand affinity chromatography.

When calf rennet containing approximately 15% pepsin was applied to a Cibacron Blue agarose column at pH 5.5 in a low salt medium, pepsin passed through unadsorbed while chymosin was bound to the gel in the column. After washing the column, the bound chymosin was eluted with 1.7 M NaCl or 50% (v/v) aqueous ethylene glycol. The salt eluate was analyzed and found to contain greater than 97% pure chymosin. The fraction that passed through unadsorbed was found to contain greater than 96% pure pepsin. Thus a complete separation of chymosin and pepsin was effected by this technique without having to destroy either enzyme. Both enzymes are highly negatively charged at pH 5.5 but the separation does not arise from anion exchange since the gel functions as a cation exchanger. The separation appears to result from a combination of hydrophobic and electrostatic interactions of chymosin with Blue agarose. It is suggested that the enhanced affinity of chymosin to the Blue gel over pepsin may arise from topographically specified interaction between chymosin and the blue chromophore. Differential surface hydrophobicity may also play a key role, since in the presence of 0.7 M Na2SO4 the same behavior as at low ionic strength is observed.

Adsorption↗
PubMed2003

Expression of a synthetic copy of the bovine chymosin gene in Aspergillus awamori from constitutive and pH-regulated promoters and secretion using two different pre-pro sequences.

A copy of the bovine chymosin gene (chy) with a codon usage optimized for its expression in Aspergillus awamori was constructed starting from synthetic oligonucleotides. To study the ability of this filamentous fungus to secrete bovine prochymosin, two plasmids were constructed in which the transcriptional, translational, and secretory control regions of the A. nidulans gpdA gene and pepB genes were coupled to either preprochymosin or prochymosin genes. Secretion of a protein enzymatically and immunologically indistinguishable from bovine chymosin was achieved in A. awamori transformants with each of these constructions. In all cases, the primary translation product (40.5 kDa) was self-processed to a mature chymosin polypeptide having a molecular weight of 35.6 kDa. Immunological assays indicated that most of the chymosin was secreted to the extracellular medium. Hybridization analysis of genomic DNA from chymosin transformants showed chromosomal integration of prochymosin sequences and, in some transformants, multiple copies of the expression cassettes were observed. Expression from the gpdA promoter was constitutive, whereas expression from the pepB promoter was strongly influenced by pH. A very high expression from the pepB promoter was observed during the growth phase. The A. awamori pepB gene terminator was more favorable for chymosin production than the S. cerevisiae CYC1 terminator.

Amino Acid Sequence↗
PubMed1993

Secretion of calf chymosin from the filamentous fungus Aspergillus oryzae.

Active calf chymosin was secreted from Aspergillus oryzae transformants when the chymosin cDNA was expressed under the control of glucoamylase gene (glaA) promoter. Secreted prochymosin was autocatalytically activated to the chymosin (0.07-0.16 mg/l). Western blot analysis showed that a secreted protein immunoreactive with an anti-chymosin antibody was of similar size to authentic chymosin. Northern blot analysis revealed that mRNA of the chymosin cDNA was expressed at as high level as that of the glaA gene. The size and the level of the transcript were different among transformants, due to the integration position of the plasmid on the chromosome.

Animals↗
PubMed1980

Kinetic studies on the action of Mucor pusillus, Mucor miehei acid proteases and chymosins A and B on a synthetic chromophoric hexapeptide.

The action of two milk-clotting fungal proteases from Mucos pusillus and Mucor miehei and of chymosins A and B on the hexapeptide, Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe, and on kappa-casein were studied. The effects of pH and temperature on the initial rates of hydrolysis of the hexapeptide were examined. Crystalline chymosin and M. pusillus protease exhibited optimal activities around 49 and 55 degrees C, respectively, whereas the optimum temperature for M. miehei protease is higher than 63 degrees C. The optimum pH was about 4.7 for both fungal proteases whereas chymosin A and chymosin B exhibited optimal activities around 4.2 and 3.7, respectively. Kinetic parameters were then determined under optimal conditions and/or at pH 4.7. Fungal proteases had kcat/Km ratios that were similar to each other and that were significantly greater than the ratios obtained for the chymosins. Nevertheless, chymosins had much greater clotting activities towards kappa-casein relative to their proteolytic activities towards the synthetic peptide.

Caseins↗
PubMed1980

Binding of Streptomyces pepsin inhibitor (acetyl-pepstatin) with chymosin (Rennin).

Chymosin (Rennin) was effectively purified using an AH-Sepharose 4B column. Binding of Streptomyces pepsin inhibitor (acetul-pepstatin) with chymosin was studied spectroscopically. The binding caused ultraviolet difference and CD spectral changes suggesting microenvironmental changes around tryptophan and/or tyrosine residue(s) in chymosin. The fluorescence intensity of a hydrophobic probe, 2-p-toluidinylnaphthalene-6-sulfonate, increased in the presence of chymosin and was further amplified when Streptomyces pepsin inhibitor was added to the chymosin-2-p-toluidinylnaphthalene-6-sulfonate solution. The binding and dissociation-rate constants between chymosin and the inhibitor were determined using 2-p-toluidinylhnaphthalene-6-sulfonate as a probe. The binding constant was determined from the binding and dissociation-rate constants, to be 3.1 . 10(7) M-1 at 25 degrees C, pH 5.5.

Chymosin↗
PubMed1994

Strain improvement of chymosin-producing strains of Aspergillus niger var. awamori using parasexual recombination.

Parasexual recombination was used to obtain improved chymosin-producing strains and to perform genetic analysis on existing strains. Chlorate resistance was used to select for a variety of spontaneous nitrate assimilation pathway mutations in strains previously improved for chymosin production using classical strain improvement methods including mutation and screening, and selection for 2-deoxyglucose resistance (dgr). Diploids of these improved strains were generated via parasexual recombination and were isolated on selective media by complementation of nitrate assimilation mutations. A preliminary genetic analysis of diploid and haploid segregants indicated that the dgr trait, resulting in overexpression of chymosin, was recessive. Also, mutations in two different dgr genes resulted in an increased level of chymosin production. When these mutations were combined via parasexual recombination, the resulting haploid segregants produced about 15% more chymosin than either parental strain. CHEF gel electrophoresis was used to determine the chromosomal location of the integrated chymosin DNA sequences, and to verify diploidy in one case where the chromosome composition of two haploid parents differed.

Animals↗
PubMed2005

Effect of chymosin and salt reduction on the quality of ultrafiltrated white-salted cheese.

This study demonstrated that both chymosin and salt-in-moisture (SM) were important factors for proteolysis in the manufacture of ultrafiltrated white-salted cheese, with significant effects on water-soluble nitrogen and nitrogen soluble in trichloroacetic acid. In contrast, the levels of free amino acids were not significantly affected by chymosin and salt treatments. The cheeses made, using high levels of chymosin with low SM had lower levels of residual alpha(S1)- and beta-casein at the end of ripening. On texture profile analysis, the hardness and fracturability of the cheeses significantly increased with SM and decreased during ripening. Increases in chymosin significantly contributed to the overall weakening of the structure throughout ripening. Bitter flavour was detected after 12 weeks in the cheese made with the higher chymosin level and lower SM, which could be the result of accumulation of gamma-casein fractions. The sensory data indicated that the hedonic responses for low chymosin with low SM cheeses were good and acceptable in flavour, which may be due to the moderate levels of proteolysis products.

Cheese↗
PubMed2006

A comparative study of functional properties of calf chymosin and its recombinant forms.

The action of calf chymosin obtained from transgenic sheep milk and the recombinant protein expressed in yeast Kluyveromyces lactis (Maxiren) on fluorogenic peptide substrates, namely Abz-A-A-F-F-A-A-Ded, Abz-A-A-F-F-A-A-pNA, Abz-A-F-F-A-A-Ded, Abz-A-A-F-F-A-Ded, Abz-A-A-F-F-Ded, Abz-A-A-F-F-pNA, and heptapeptide L-S-F-M-A-I-P-NH2, a fragment of kappa-casein (the native chymosin substrate), was investigated. It has been established that transgenic chymosin and recombinant chymosin (Maxiren) differ from the native enzyme in their action on low molecular weight substrates, whereas there was no difference in enzymatic action on protein substrates. Pepstatin, a specific inhibitor of aspartic proteinases, inhibits the recombinant chymosin forms less efficiently than the native enzyme. Perhaps this is associated with local conformational changes in the substrate binding site of recombinant chymosin occurring during the formation of the protein globule.

Amino Acid Sequence↗
PubMed2001

[Site-directed mutagenesis at disulfide bond Cys206-Cys210 of prochymosin (chymosin)].

During the work of site-directed mutagenesis at disulfide bond Cys206-Cys210 of prochymosin, it was found that the corresponding template sequence had the potential to form a loop-stem structure with free energy of -16.1 kcal/mol, which prevent the template from pairing with primer and, in turn, the synthesis of the mutated DNA strand. Rapid annealing can overcome this difficulty. Five expression plasmids of prochymosin muants with deletion of Cys206-Cys210 (C206A, C210A, C206A/C210A, C210S and C206S/C210S) were constructed. Except for C206A they were expressed at high level in E. coli amounting to 50% of the total cellular proteins. Renaturation of the mutant prochymosin indicated that Cys206-Cys210 is dispensable for correct refolding of prochymosin. However, the amino acid residues at Cys206 and/or Cys 210 play a critical role in determining the renaturation. Among the five mutants the reactivation efficiency of C206A/C210A were about 4.5-fold, 20-fold and 30-fold higher than that of C206S/C210S, C210A and C210S respectively. C206A can not correctly refold at all. CD spectra in the far UV region indicate that C206A/C210A and C206S/C210S chymosin analogs have a secondary structure almost identical to that of the wild-type chymosin. Fluorescence spectroscopic analysis revealed that mutant chymosins have the same emission maximum at 333 nm as the wild-type chymosin but their fluorescence intensities at 333 nm are much higher than that of the wild-type chymosin. Considering that the mutants and the wild-type chymosin exhibit almost the same specific activity, it is reasonable to conclude that the mutant proteins assume a native active information with a perturbance around some tryptophan residues.

Chymosin↗
PubMed1991

Hydrolysis of beta-casein by gastric proteases. I. Comparison of proteolytic action of bovine chymosin and pepsin A.

Hydrolysis of beta A2-casein by bovine chymosin and pepsin A was performed in order to compare the hydrolysis of the two enzymes on this protein. Different conditions have been tested: pH 5.5 for 116h and pH 3.5 for 7 h [E/S = 1/100 (w/w)] for chymosin. pH 3.0 for 24 h [E/S = 1/1000 (w/w)] for pepsin A. Under these conditions 17 peptides were obtained after the action of chymosin and 23 after the action of pepsin A. They corresponded respectively to the cleavage of 14 and 15 peptide bonds for chymosin and pepsin A. However, six of the peptide bonds were only hydrolyzed by chymosin and seven other bonds only by pepsin A. Our results showed a preferential splitting at the Leu-X, Ser-X, and Trp-X bonds for chymosin and Leu-X, Met-X, and Thr-X, for pepsin A. Some of the identified peptides contained sequences with possible physiological roles.

Amino Acid Sequence↗
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↗
PubMed1985

Kinetic study of the action of bovine chymosin and pepsin A on bovine kappa-casein.

A study was carried out to determine the Michaelian parameters relative to the action of chymosin and pepsin A on bond Phe105-Met106 of bovine kappa0-casein (carbohydrate-free fraction in micellar state). The reaction was performed in citrate buffer, pH 6.2, at 30 degrees C. The reaction mixture was analysed by reverse phase HPLC. Dosages of peptide 106-169 (caseino macropeptide) at different reaction times from recordings of its absorbance at 220 nm gave the initial rates of reaction at each substrate concentration. From these values the following parameters were determined: kcat = 68.5 s-1, Km = 0.048 mM, kcat/Km = 1,413 mM-1 s-1 for chymosin, and kcat = 45 s-1, Km = 0.018 mM, kcat/Km = 2,439 mM-1 s-1 for pepsin A. For chymosin they are similar to those obtained previously in dimethyl glutarate buffer, pH 6.6, at 30 degrees C, using fragment 98-111 of kappa-casein as substrate. It can thus be concluded that neither the micellar state nor the presence of the whole peptide chain of kappa-casein (our conditions) significantly affect the action of chymosin on fragment 98-111, which seems to contain all information that makes bond 105-106 highly sensitive to chymosin. For pepsin A, only the information contained in fragment 103-108 appears to be required.

Amino Acids↗
PubMed1982

Isolation and partial characterization of prochymosin and chymosin from cat.

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 cat chymosin was obtained after acid activation of the zymogen. The enzyme showed the optimum of general 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.

Amino Acid Sequence↗
PubMed1993

Expression and characterisation of chymosin pH optima mutants produced in Trichoderma reesei.

The production of chymosin mutants designed to have altered pH optima using the cellulolytic filamentous fungus Trichoderma reesei is described. The strong promoter of the gene encoding the major cellulase, cellobiohydrolase I (CBHI) has been used for the expression and secretion of active calf chymosin. Structural analysis of the hydrogen bonding network around the two active site aspartates 32 and 215 in chymosin have suggested that residues Thr 218 and Asp 303 may influence the rate and pH optima for catalysis. The chymosin mutants Thr218Ala and the double mutant Thr218Ala/Asp303Ala have been made by site-directed mutagenesis and expressed in T. reesei. Enzyme kinetics of the active enzyme T218A indicate a pH optimum of 4.2 compared to 3.8 for native chymosin B using a synthetic octa-peptide substrate, confirming the previous analysis undertaken in E. coli. The double mutant T218A/D303A exhibits a similar optimum of 4.4 to that reported for the D303A, indicating that the combination of these changes is not additive. The application of protein engineering in the rational design of specific modifications to tailor the properties of enzymes offers a new approach to the development of industrial processes.

Amino Acid Sequence↗
PubMed1996

Investigating the use of the chymosin-sensitive sequence of kappa-casein as a cleavable linker site in fusion proteins.

The chymosin-sensitive sequence of bovine k-casein A (kappa-CN A) was investigated as a cleavable linker site between the two domains of a streptavidin-chloramphenicol acetyltransferase fusion protein. Two DNA sequences were synthesized which encode the amino acids from 101 to 107 and from 97 to 113 of bovine kappa-CN A. These sequences were separately cloned in-frame to a streptavidin expression vector used for fusion protein construction. The gene for chloramphenicol acetyltransferase (CAT) was then cloned in-frame to a streptavidin-chymosin-sensitive linker vector forming plasmids pStCL1CAT and pStCL2CAT. The fusion protein was expressed in Escherichia coli and SDS-PAGE and Western blot analysis of chymosin-treated cell lysates showed a pH-dependent cleavage of the fusion proteins. Fusion proteins were also bioselectively immobilized onto biotinylated controlled-pore glass beads and treated with chymosin. CAT was specifically released by chymosin treatment and was identified by SDS-PAGE.

Amino Acid Sequence↗
PubMed2005

Characterization and study of a kappa-casein-like chymosin-sensitive linkage.

The present report is dealing with the identification, in various unrelated proteins, of protein fragments sharing local sequence and structure similarities with the chymosin-sensitive linkage surrounding the Phe-Met/Ile bond of kappa-caseins. In all these proteins, this linkage is observed within an exposed beta-strand-like structure, as also predicted for kappa-caseins. The structure of one of these fragments, included in glutamine synthetase, particularly superimposes well with the conformation observed for a chymosin inhibitor (CP-113972) within the complex it forms with chymosin and can be similarly accommodated by specificity pockets within the enzyme substrate binding cleft. The effect of the enzyme activity of chymosin was thus tested on glutamine synthetase. Chymosin cut the latter at the Phe-Met linkage, suggesting that this system may locally resemble the kappa-casein/chymosin complex.

Animals↗
PubMed1996

Comparative study of methods for the isolation and purification of bovine kappa-casein and its hydrolysis by chymosin.

kappa-Casein was purified from a single batch of whole acid casein (kappa-A variant) using different methods in order to compare their merits in producing a purified material with a carbohydrate and phosphate heterogeneity representative of the whole kappa-casein complement in milk. Ion-exchange methods of purification gave products of higher purity than precipitation techniques involving final purification by ethanol fractionation, but all methods resulted in kappa-caseins of apparently similar heterogeneity and chemical composition. The purified kappa-caseins were hydrolysed with chymosin and the derived macropeptides isolated. These were all virtually identical as determined by reversed-phase chromatography and gel electrophoresis. Some observations on chymosin hydrolysis of kappa-casein were made. In addition to formation of the major para-kappa-casein (Glu1-Phe105) and macropeptide (Met106-Val169), chymosin hydrolysis at pH 6.6 also resulted in two minor para-kappa-caseins with N-termini corresponding to Phe18 and Ser33 of kappa-casein. At pH 5.5 and 4.5 para-kappa-casein was rapidly hydrolysed into at least six fragments, one of which had an N-terminus corresponding to Trp76 of kappa-casein. At pH 6.6, 5.5 and 4.5 the kappa-casein macropeptide was stable to chymosin, but at pH 2.3 it was hydrolysed by chymosin into fragments with N-termini corresponding to Met106, Ile125, Ala138, Val139, Thr145 and Glu147 of kappa-casein.

Animals↗
PubMedDate not supplied

Sensitive fluorometric assay for the activity of chymosin.

Fluorogenic substrates for chymosin [Dns-Leu-Ser-Phe-Trp-Ala-Leu-OCH2Py (I), Dns-Leu-Ser-Phe-Met-Trp-Leu-OCH2Py (II), Dns-Leu-Ser-Leu-Trp-Ala-Leu-OCH2Py (III), Dns-Leu-Ala-Phe-Trp-Ala-Leu-OCH2Py (IV), Dns-Leu-Ser-Phe-Leu-Ala-Leu-OCH2Py (V) and Dns-Leu-Ser-Phe-Phe-Ala-Leu-OCH2Py (VI)] were synthesized by a solution method. The obtained substrates I-VI were cleaved specifically (between the Phe and Trp residues for substrates I and IV, the Phe and Met residues for substrate II, the Leu and Trp residues for substrate III, the Phe and Leu residues for substrate V, and the Phe and Phe residues for substrate VI) by chymosin. The fluorescence of substrates I-IV (345 nm) increased with their hydrolysis, and hydrolysis rates were obtained by measuring the increase in fluorescence. The minimum detectable chymosin concentrations for substrates I and IV were about 1 nM; those for substrates II and III were about 4 and 2 nM. This assay method is very sensitive, and it is possible to determine the chymosin activity rapidly and easily. Substrates I and IV-VI were hydrolyzed by chymosin two times faster than substrates II and III. The effect of the amino-acid residues of the substrates on the hydrolysis rate is discussed.

Amino Acid Sequence↗