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At least 19 recordsLinked to original sources
PubMed2002

Stimulation of chymosin secretion by simultaneous expression with chymosin-binding llama single-domain antibody fragments in yeast.

We studied the effect of coexpression of chymosin and chymosin-binding llama single-domain antibody fragments (VHHs) on the secretion of chymosin by Saccharomyces cerevisiae cells. A VHH expression library containing chymosin-specific VHHs was obtained by immunization of a llama and coexpressed with chymosin in yeast. From this library, we obtained two VHH clones that stimulated chymosin secretion by screening colonies for the level of chymosin secreted. These VHHs bound biotinylated chymosin in an immunoblot procedure but failed to bind chymosin in ELISA, suggesting that their interaction with chymosin was of low affinity. In a second approach, chymosin-specific VHHs were first selected using phage display and then coexpressed with chymosin in yeast cells. Screening yeast cells for higher levels of chymosin secretion resulted in 11 VHHs. Sequence analysis revealed that these 11 VHHs formed four sets of related VHHs that were different from the previously isolated two VHHs. Although binding of VHHs to chymosin could not be demonstrated in ELISA using soluble VHHs, it could be unambiguously demonstrated for clones isolated by phage display, using phage-displayed VHHs. Finally, quantitative Western blot analysis of chymosin amounts demonstrated that coexpression with VHH domains can stimulate the level of secreted chymosin 1.5- to 6-fold.

Animals↗
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↗
PubMed2002

Thermal inactivation of chymosin during cheese manufacture.

The aspartic proteinase, chymosin (EC 3.4.23.4) is the principal milk clotting enzyme used in cheese production and is one of the principal proteolytic agents involved in cheese ripening. Varietal differences in chymosin activity, due to factors such as cheese cooking temperature, fundamentally influence cheese characteristics. Furthermore, much chymosin is lost in whey, and further processing of this by-product may require efficient inactivation of this enzyme, with minimal effects on whey proteins. In the first part of this study, the thermal inactivation kinetics of Maxiren 15 (a recombinant chymosin preparation) were studied in skim milk ultrafiltration permeate, whole milk whey and skim milk whey. Inactivation of chymosin in these systems (at pH 6.64) followed first order kinetics with a D45.5 value of 100 +/- 21 min and a z-value of 5.9 +/- 0.3 degrees C. D-Values increased linearly with decreasing pH from 6.64 to 6.2, while z-values decreased as pH decreased from 6.64 to 6.4, but were similar at pH 6.4 and 6.2. Subsequent determination of chymosin activity during manufacture of Cheddar and Swiss-type cheese showed good correlations between predicted and experimental values for thermal inactivation of chymosin in whey. However, both types of cheese curd exhibited relatively constant residual chymosin activity throughout manufacture, despite the higher cooking temperature applied in the manufacture of Swiss cheese. Electrophoretic analysis of slurries made from Cheddar and Swiss cheese indicated decreased proteolysis due to chymosin activity during storage of the Swiss cheese slurry, but hydrolysis of sodium caseinate by coagulant extracted from both cheese types indicated similar levels of residual chymosin activity. This may suggest that some form of conformational change other than irreversible thermal denaturation of chymisin takes place in cheese curd during cooking, or that some other physico-chemical difference between Swiss and Cheddar cheese controls the activity of chymosin during ripening.

Animals↗
PubMed2006

Characterization of recombinant camel chymosin reveals superior properties for the coagulation of bovine and camel milk.

Enzymatic milk coagulation for cheese manufacturing involves the cleavage of the scissile bond in kappa-casein by an aspartic acid protease. Bovine chymosin is the preferred enzyme, combining a strong clotting activity with a low general proteolytic activity. In the present study, we report expression and enzymatic properties of recombinant camel chymosin expressed in Aspergillus niger. Camel chymosin was shown to have different characteristics than bovine chymosin. Camel chymosin exhibits a 70% higher clotting activity for bovine milk and has only 20% of the unspecific protease activity for bovine chymosin. This results in a sevenfold higher ratio of clotting to general proteolytic activity. The enzyme is more thermostable than bovine chymosin. Kinetic analysis showed that half-saturation is achieved with less than 50% of the substrate required for bovine chymosin and turnover rates are lower. While raw camel milk cannot be clotted with bovine chymosin, a high clotting activity was found with camel chymosin.

Amino Acid Sequence↗
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↗
PubMed2006

Purification and characterization of chymosin and pepsin from kid.

The objective of this work was to study the characteristics of the gastric aspartic proteinases chymosin and pepsin which are constituents of the kid rennet. The two enzymes were extracted from abomasal tissue of one kid from a local indigenous breed, separated from each other by DEAE-cellulose chromatography and then were purified by gel filtration and anion-exchange chromatography. The molecular weights of the purified kid chymosin and pepsin as determined by gel filtration were 36 kDa and 40 kDa respectively. The isoelectric point of kid chymosin was as multiple forms of 3-6 zones at pH 4.6-5.1, while that of kid pepsin was at pH < or =3.0. Kid pepsin contained 0.37 molecules phosphorous per molecule and was totally inhibited by 5 muM pepstatin A, being more sensitive than kid chymosin. Both enzymes were almost equally as proteolytic as calf chymosin on total casein at pH 5.6. Kid pepsin activity was more pH and temperature dependent than kid chymosin activity. In comparison with the calf chymosin temperature sensitivity, the order of increased sensitivity was: calf chymosin <kid chymosin <kid pepsin.

Abomasum↗
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↗
PubMed2004

Role of S'1 loop residues in the substrate specificities of pepsin A and chymosin.

Proteolytic specificities of human pepsin A and monkey chymosin were investigated with a variety of oligopeptides as substrates. Human pepsin A had a strict preference for hydrophobic/aromatic residues at P'1, while monkey chymosin showed a diversified preferences accommodating charged residues as well as hydrophobic/aromatic ones. A comparison of residues forming the S'1 subsite between mammalian pepsins A and chymosins demonstrated the presence of conservative residues including Tyr(189), Ile(213), and Ile(300) and group-specific residues in the 289-299 loop region near the C terminus. The group-specific residues consisted of hydrophobic residues in pepsin A (Met(289), Leu/Ile/Val(291), and Leu(298)) and charged or polar residues in chymosins (Asp/Glu(289) and Gln/His/Lys(298)). Because the residues in the loop appeared to be involved in the unique specificities of respective types of enzymes, site-directed mutagenesis was undertaken to replace pepsin-A-specific residues by chymosin-specific ones and vice versa. A yeast expression vector for glutathione-S-transferase fusion protein was newly developed for expression of mutant proteins. The specificities of pepsin-A mutants could be successfully altered to the chymosin-like preference and those of chymosin mutants, to pepsin-like specificities, confirming residues in the S'1 loop to be essential for unique proteolytic properties of the enzymes. An increase in preference for charged residues at P'1 in pepsin-A mutants might have been due to an increase in the hydrogen-bonding interactions. In chymosin mutants, the reverse is possible. The changes in the catalytic efficiency for peptides having charged residues at P'1 were dominated by k(cat) rather than K(m) values.

Amino Acid Sequence↗
PubMed1999

Bovine chymosin: production by rDNA technology and application in cheese manufacture.

Bovine chymosin, an aspartyl protease extracted from abomasum of suckling calves, is synthesized in vivo as preprochymosin and secreted as prochymosin which is autocatalytically activated to chymosin. Chymosin is bilobular, with Asp 32 and Asp 215 acting as the catalytic residues. Chymosin A and chymosin B have pH optima of 4.2 and 3.8, respectively, and act to initiate milk clotting by cleaving kappa-casein between Phe 105 and Met 106. The gene encoding chymosin has been cloned and expressed in suitable bacteria and yeast hosts under the control of lac, trp, trp-beta, gly A genes, and serine hydroxymethyl-transferase promoters. Protein engineering of chymosin has also been attempted. A number of companies are now producing recombinant chymosin for commercial use in cheese manufacture.

Journal Article↗
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↗
PubMed2003

Isolation, purification and characterization of chymosin from riverine buffalo (Bubalos bubalis).

Chymosin, an aspartyl proteinase, is used for curdling of milk and manufacture of cheese. We report the purification and the physicochemical properties of chymosin isolated from the abomasal tissue of buffalo calves. The enzyme preparation extracted from buffalo abomasal tissues could be purified 29-fold using anion exchange and gel filtration chromatography. The molecular weight of the purified enzyme was 35.6 kDa on SDS-PAGE. Partial N-terminal amino acid sequence of the first eight amino acid sequences of buffalo chymosin was identical to the first eight amino acid sequences of cattle chymosin. Buffalo chymosin exhibited a skewed bell-shaped stability profile as a function of temperature with maximum activity near 55 degrees C. Milk clotting activity decreased gradually as pH increased. The enzyme became completely inactive, however, above pH 7.0. The ratio of milk clotting to proteolytic activity was 3.03. When compared with cattle chymosin, there were subtle differences in the stability and relative proteolytic activity of buffalo chymosin.

Abomasum↗
PubMed2005

Validation of recombinant and bovine chymosin by mass spectrometry.

Mass spectrometry has been used to map chymosin from a fermentative source. The copresence of the two known genetic variants A (Asp(244)) and B (Gly(244)) was ascertained in bovine chymosin. By contrast, either the A or the B genetic variant occurred in the three commercial samples of recombinant calf chymosin (RCC). Specific biomarker proteins were searched to identify the enzyme source, in both bovine chymosin and RCC samples. Analyzing the derived tryptic peptides, evidence was provided that RCC and bovine chymosin are mainly formed by (1-323), (3-323), and (40p-323) (suffix "p" denotes residues in the pro-segment region of chymosin), whereas the minor components, (4-323), (5-323), and (6-323), were only detected in bovine chymosin. Additionally, the three commercial RCC samples contained the protein species (1-323), (38p-323), (39p-323), and (40p-323) and the shorter form (3-323). Differentiation of the natural and bioengineered enzyme is based upon the detection of these unique minor components by mass spectrometry.

Amino Acid Sequence↗