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At least 19 recordsLinked to original sources

PubMed1990

Improved production of chymosin in Aspergillus by expression as a glucoamylase-chymosin fusion.

We have extended the work on chymosin production in Aspergillus by constructing an expression vector in which the cDNA encoding bovine prochymosin B was fused in frame immediately following the codon for the last amino acid of the A. awamori glucoamylase (glaA) gene. Transformation of A. awamori with this plasmid led to the secretion of considerably higher amounts of chymosin than obtained with previous chymosin expression vectors. We present evidence that mature chymosin is autocatalytically released from the glucoamylase-chymosin fusion protein after secretion.

Amino Acid Sequence↗

PubMed1988

Structure and mechanism of formation of recombinant-derived chymosin C.

Chymosin C, an autolysis product of chymosin A, is not formed from chymosin B (Foltmann, B. (1966) C. R. Trav. Lab. Carlsberg 35, 143-231) even though chymosins A and B differ in only a single residue (residue 286 is Asp in chymosin A but is Gly in chymosin B). Autolysis of recombinant-derived chymosin A yielded chymosin C for structural analysis. N-terminal sequencing revealed two peptide chains in chymosin C, one of which begins with Gly43, the N terminus of chymosin A; the other begins with Asp289. C-terminal sequencing, peptide mapping, and amino acid analysis showed that chymosin A undergoes autolytic excision of Asp286--Glu287--Phe288 in yielding chymosin C. As predicted from the established disulfide pattern and verified by size-exclusion chromatography under denaturing conditions, no disulfide bond cross-links the two chymosin A fragments which comprise chymosin C. The N-terminal fragment (243 residues) is termed the C-protein, and the C-terminal fragment (77 residues) is termed the C-peptide. Studies with synthetic peptide analogues of relevant regions of the chymosin sequences suggested that Tyr285--Asp286 is the first chymosin A peptide bond hydrolyzed during chymosin C formation and that Tyr285--Gly286 in chymosin B is not cleaved. Cleavage of Phe288--Asp289, a bond which is present in both chymosins A and B, most likely occurs in the A form as a secondary event following nicking at Tyr285--Asp286. This explains why chymosin A gives rise to chymosin C, but chymosin B does not. Examination of the sequence of a prochymosin gene (Hidaka, M., Sasaki, K., Uozumi, T., and Beppu, T. (1986) Gene (Amst.) 43, 197-203) showed that the autolysis-sensitive region of the polypeptide is encoded by genomic DNA located at the end of one exon and at the beginning of the next. Thus, chymosin C illustrates the correlation of protease-sensitive regions of protein sequences with genomic splice junctions.

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↗

PubMed2001

Mathematical modelling of the formation of rennet-induced gels by plant coagulants and chymosin.

Rheological properties of reconstituted skim milk coagulated with plant coagulants Cynara cardunculus L., Cynara humilis L. and chymosin was monitored by dynamic low amplitude oscillation. There are no published reports on the modelling of the gelation behaviour of milk by plant coagulants. Three mathematical models, Scott Blair. Douillard and Carlson, were fitted to the storage modulus (G') as function of time curves. For all coagulants. Scott Blair model was the most efficient in modelling the gelation process, and gave both the smallest residuals and standard error of residuals, Se (P < 0.0001). Douillard model gave the poorest fit and in particular it was not able to predict the initial part of the gelation curves. Carlson model had an intermediate behaviour and, in the case of chymosin, it gave results that were quite comparable to Scott Blair model. The parameters of the Scott Blair model were different for plant coagulants and chymosin. Chymosin had the longest rate constant (tau) and the time shift coefficient (t8) was also different between vegetable coagulants and chymosin (P < 0.0005). These results are in agreement with the overall trends for gelation profiles obtained for vegetable coagulants and chymosin. In the beginning of gelation both plant coagulants produced gels with slightly higher G' values than chymosin, but after longer incubation times chymosin gels had higher G' values. It was concluded that the Scott Blair model was the best equation to follow the gelation of milk induced by both plant coagulants as well as chymosin. Modelling is an important and useful method for comparing the gelation process in gels formed by different types of coagulants.

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↗

PubMed1992

Chymosin: a short review on foetal and neonatal gastric proteases.

All vertebrates in which the age dependent expression of gastric proteases has been investigated show a characteristic developmental pattern. Neonatal proteases which show partial immunochemical identity with calf chymosin have been observed in several species. The amino acid sequence of lamb chymosin shows 94% of identity with that of calf chymosin. Chymosins from pig and cat show about 85% and 75% of identity with calf chymosin. Chicken embryonic pepsinogen appears to be more related to calf chymosin than to other gastric proteases. A pseudo-gene for a chymosin-like protease from man has been identified. But a functional, human chymosin-like neonatal protease has not been identified. The possible physiological significance of chymosin and the clotting of milk are discussed.

Amino Acid Sequence↗

PubMed2001

Mass spectrometric characterisation of proteins in rennet and in chymosin-based milk-clotting preparations.

The protein composition of natural rennet and of chromatographic and crystalline chymosin preparations has been defined by on-line reverse-phase high performance liquid chromatography/electrospray ionisation mass spectrometry (RP-HPLC/ESI-MS) and by tandem mass spectrometry (MS/MS). Natural rennet was found to consist of six chymosin species, corresponding to chymosin A and B genetic variants, each of which comprised a mixture of two other forms differing at theN-terminal end, with one being three residues longer, and the other two residues shorter, than the mature chymosin. Two main tissue proteins were also identified as lysozyme (isozyme 2 plus a novel isozyme labelled 4) and bovine serum albumin. In addition to the proteins, chymosin fragments 247-323 and 288-323 were consistently present in natural rennet. Conversely, chromatographic and crystalline chymosin preparations lacked bovine serum albumin and/or lysozyme, although they contained the same six chymosin species as natural rennet. Since these tissue-specific contaminating proteins each possess specific functions in terms of stabilising enzyme solutions and protecting proteins from proteolytic enzymes, oxidising agents and bacterial proliferation, the rennet may be considered as a functional enzyme preparation that is effectively and naturally adapted to the purposes of cheesemaking. In practice, the highly complex protein composition inherent to natural rennet provided the possibility to differentiate the natural product from other bovine chymosin-based milk-clotting preparations examined in this work.

Amino Acid Sequence↗

PubMed1991

Functional implications of the three-dimensional structure of bovine chymosin.

Many aspects of the structure of chymosin are quite unique even though structure comparisons indicate a high degree of structural homology with other eukaryotic aspartic proteinases. The structural homology is shown to be directly related to the sequence homology which varies from 30 to 60%. The recent structures of pepsin (Abad-Zapatero et al., 1990; Sielecki et al., 1990; Cooper et al., 1990) have allowed the first preliminary comparisons of two different gastric enzymes. These structures are quite similar, even more so than the structures of the fungal proteinases. However, unlike chymosin, the position of Tyr77 in the flap of pepsin is similar to that found in the fungal aspartic proteinases despite the fact that pepsin is more similar in the flap sequence and the S1 binding site to chymosin than to the fungal proteinases. Attempts at obtaining crystals complexed with substrate analogs which are suitable for diffraction studies have been unsuccessful. Therefore, substrate binding has been examined by model building substrates and substrate analogs into the active site cleft of the structure determined from X-ray studies. The model complexes have been compared with the structures of inhibitor-aspartic proteinase complexes have been previously reported. The results reported here indicate that there are valid reasons why the natural substrate, kappa-casein, binds and is cleaved between positions 105-106. The positively charged histidine residues (98, 100, and 102) of kappa-casein, which are located prior to the cleavage site, appear to be able to interact with negatively charged residues of chymosin which are quite distant from the active site. These residues include Glu288, Asp279, and Glu280 of chymosin. The latter two residues are approximately 20 and 25 A from the center of the active site. These studies also suggest that the difference in activities of the A and B isozymes of chymosin may be due to the increased binding affinity of the substrate as a result of strong electrostatic interactions with Asp244 of chymosin and positively charged His102 of the substrate. An examination of the charged amino acid residues of the chymosin structure has produced two interesting observations. First, there is an asymmetric distribution of charged residues; the N-terminal domain has a smaller net negative charge than the C-terminal domain. This is due to a patch of positive charges on the surface located in the region from residues 48 to 62. Electrostatic calculations in which overall dipole moments were estimated for each of the eukaryotic aspartic proteinases have been performed.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1996

The primary structure and enzymic properties of porcine prochymosin and chymosin.

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 (S'2), 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.

Amino Acid Sequence↗

PubMed1994

Proteolysis of bovine alpha s2-casein by chymosin.

Proteolysis of bovine alpha s2-casein by chymosin (E. C. 3.4.23.4) in solution in 100 mM Na phosphate buffer, pH 6.5, at 30 degrees C was studied by reversed-phase (RP)-HPLC and urea-polyacrylamide gel electrophoresis (PAGE). Chymosin hydrolyzed alpha s2-casein in solution to eight peptides detectable by urea-PAGE. Peptides soluble in acetate buffer, pH 4.6, were isolated by RP-HPLC on a C18 column using an acetonitrile/water gradient and identified from their N-terminal amino acid sequence. The chymosin cleavage sites were at the bonds Phe88-Tyr89, Tyr95-Leu96, Gln97-Tyr98, Tyr98-Leu99, Phe163-Leu164, Phe174-Ala175 and Tyr179-Leu180. Chymosin cleavage sites were restricted to the hydrophobic regions of the molecule. The bond-type in alpha s2-casein cleaved by chymosin was in agreement with that found to be susceptible to chymosin in other caseins. The primary site of chymosin action on alpha s2-casein appeared to be at Phe88-Tyr89.

Amino Acid Sequence↗

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↗

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↗

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↗