Information on EC 3.4.23.4 - chymosin

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The expected taxonomic range for this enzyme is: Opisthokonta

EC NUMBER
COMMENTARY hide
3.4.23.4
-
RECOMMENDED NAME
GeneOntology No.
chymosin
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Broad specificity similar to that of pepsin A. Clots milk by cleavage of a single Ser-Phe105-/-Met-Ala bond in kappa-chain of casein
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
-
-
endopeptidase; peptides, endopeptidase
-
CAS REGISTRY NUMBER
COMMENTARY hide
9001-98-3
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
similarar enzyme
-
-
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
similar enzyme
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
similar enzyme
-
-
Manually annotated by BRENDA team
similar enzyme
-
-
Manually annotated by BRENDA team
variant rhizopodiformis
-
-
Manually annotated by BRENDA team
variant rhizopodiformis
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
-
chymosin, an aspartic protease, is the main enzymatic component of calf rennet
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
Abz-A-A-F-F-A-A-N-(2,4-dinitrophenyl)-ethylenediamine + H2O
?
show the reaction diagram
-
low molecular weight, fluorogenic peptide substrate
-
-
?
Abz-A-A-F-F-A-A-p-nitroanilide + H2O
?
show the reaction diagram
-
low molecular weight, fluorogenic peptide substrate
-
-
?
Abz-A-A-F-F-A-N-(2,4-dinitrophenyl)-ethylenediamine + H2O
?
show the reaction diagram
-
low molecular weight, fluorogenic peptide substrate
-
-
?
Abz-A-A-F-F-N-(2,4-dinitrophenyl)-ethylenediamine + H2O
?
show the reaction diagram
-
low molecular weight, fluorogenic peptide substrate
-
-
?
Abz-A-A-F-F-pnA + H2O
?
show the reaction diagram
-
low molecular weight, fluorogenic peptide substrate
-
-
?
Abz-A-F-F-A-A-N-(2,4-dinitrophenyl)-ethylenediamine + H2O
?
show the reaction diagram
-
low molecular weight, fluorogenic peptide substrate
-
-
?
acid denatured hemoglobin + H2O
?
show the reaction diagram
AFPLEFEREL + H2O
AFPLEF + EREL
show the reaction diagram
modified peptide substrate based on residues 165-174 of proopiomelanocortin
-
-
?
AFPLEFFREL + H2O
AFPLEF + FREL
show the reaction diagram
modified peptide substrate based on residues 165-174 of proopiomelanocortin
-
-
?
AFPLEFIREL + H2O
AFPLEF + IREL
show the reaction diagram
modified peptide substrate based on residues 165-174 of proopiomelanocortin
-
-
?
AFPLEFKREL + H2O
AFPLEF + KREL
show the reaction diagram
modified peptide substrate based on residues 165-174 of proopiomelanocortin
-
-
?
alpha-casein + H2O
?
show the reaction diagram
alphaS-casein + H2O
?
show the reaction diagram
alphas1-casein + H2O
?
show the reaction diagram
basic FGF 110-118 + H2O
?
show the reaction diagram
-
parent peptide and Leu115 and Lys115 variants respectively as substrate
-
-
?
beta-casein + H2O
?
show the reaction diagram
beta-chain of proteolytic insulin + H2O
?
show the reaction diagram
-
general proteolytic activity
-
-
?
bovine kappa-casein + H2O
?
show the reaction diagram
-
kappa-casein samples are a mixture of monomers and aggregates at room temperature and pH 7.2, and that heating produces extensive kappa-casein aggregation.The initial polymerization or association state of kappa-casein affects on the aggregation stage after the enzymatic action of chymosin. Sucrose and lactose also affect the aggregation of proteolized particles of kappa-chymosin
-
-
?
bovine kappa-casein + H2O
para-kappa-casein + caseinomacropeptide
show the reaction diagram
bovine kappa-casein residues 97-112 + H2O
?
show the reaction diagram
-
substrate binds in an extended conformation with charged residues on either side of the scissile bond playing an important role in stabilizing the binding pose. Substrate residues Lys111 and Lys112 bind to the N-terminal domain of chymosin displacing a conserved water molecule. A cluster of histidine and proline residues, His98-Pro99-His100-Pro101-His102, in kappa-casein binds to the C-terminal domain of the protein, where neighboring conserved arginine residue Arg97 is important for stabilizing the binding pose. The catalytic site including the catalytic water molecule is stable in the starting conformation of the general acid/base catalytic mechanism for 18 ns of molecular dynamics simulations
-
-
?
casein + H2O
?
show the reaction diagram
-
-
-
-
?
dynorphin A 1-7e + H2O
?
show the reaction diagram
-
Ala3Phe7 and ILe3Lys5Phe7 variants respectively as substrate
-
-
?
fluorescein thiocarbamoyl-kappa-casein + H2O
?
show the reaction diagram
-
-
-
?
His-Pro-His-Pro-His-Leu-Ser-Phe-Met-Ala-Ile-Pro-NH2 + H2O
His-Pro-His-Pro-His-Leu-Ser-Phe + Met-Ala-Ile-Pro + NH3
show the reaction diagram
His-Pro-His-Pro-His-Leu-Ser-Phe-Phe(NO2)-Ala-Ile-Pro-Pro-Lys-Lys + H2O
?
show the reaction diagram
-
-
-
-
?
HPHPHLSFMAIPPKK + H2O
?
show the reaction diagram
-
-
-
-
?
kappa-casein + H2O
?
show the reaction diagram
kappa-casein + H2O
casein macropeptide + para-kappa-casein
show the reaction diagram
kappa-casein + H2O
caseinmacropeptide + para-kappa-casein
show the reaction diagram
kappa-casein + H2O
p-kappa-casein + glycomacropeptide
show the reaction diagram
kappa-casein + H2O
para-kappa-casein + glycopeptide
show the reaction diagram
cleavage between Phe105-Met106
-
-
?
kappa-casein + H2O
para-kappa-casein + macropeptide
show the reaction diagram
-
kappa-casein is the primary substrate for the chymosin action
-
-
?
L-leucine-4-nitroanilide + H2O
L-leucine + 4-nitroaniline
show the reaction diagram
-
-
-
-
?
L-S-F-M-A-I-P-NH2 + H2O
?
show the reaction diagram
-
hepapeptide, a fragment of the native chymosin substrate kappa-casein, is most efficiently cleaved by native calf chymosin and less efficiently by transgenic chymosin and recombinant chymosin
-
-
?
Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe + H2O
?
show the reaction diagram
-
-
-
-
?
Leu-Ser-Phe-Met-Ala-Ile-O-methyl ester + H2O
Leu-Ser-Phe + Met-Ala-Ile-O-methyl ester
show the reaction diagram
-
part of the bovine kappa-casein sequence
-
?
Leu-Ser-Phe-Met-Ala-Ile-Pro-NH2 + H2O
Leu-Ser-Phe + Met-Ala-Ile-Pro + NH3
show the reaction diagram
Leu-Ser-Phe-Met-Ala-O-methyl ester + H2O
Leu-Ser-Phe + Met-Ala-O-methyl ester
show the reaction diagram
-
part of the bovine kappa-casein sequence
-
?
Lys-Pro-Ala-Glu-Phe-Phe(NO2)-Ala-Leu-OH + H2O
Lys-Pro-Ala-Glu-Phe + Phe(NO2)-Ala-Leu
show the reaction diagram
Lys-Pro-Leu-Glu-Phe-Phe(NO2)-Arg-Leu + H2O
Lys-Pro-Leu-Glu-Phe + Phe(NO2)-Arg-Leu
show the reaction diagram
-
-
-
?
NT/NMN 142-151 + H2O
?
show the reaction diagram
-
parent peptide, Glu148 and Phe148 variants respectively as substrate
-
-
?
o-aminobenzoyl-Ala-Ala-Phe-Phe-Ala-Ala-NH-C6H4NO2 + H2O
o-aminobenzoyl-Ala-Ala-Phe + Phe-Ala-Ala-NHC6H4NO2
show the reaction diagram
-
-
-
?
o-aminobenzoyl-Ala-Ala-Phe-Phe-NH-C6H4-NO2 + H2O
o-aminobenzoyl-Ala-Ala-Phe + Phe-NH-C6H4-NO2
show the reaction diagram
-
-
-
?
POm C165-174 + H2O
?
show the reaction diagram
-
parent peptide and Glu171 variant respectively as substrate
-
-
?
Pro-His-Leu-Ser-Phe-Met-Ala-Ile-O-methyl ester + H2O
Pro-His-Leu-Ser-Phe + Met-Ala-Ile-O-methyl ester
show the reaction diagram
-
part of the bovine kappa-casein sequence
-
?
Ser-Phe-Met-Ala-Ile-O-methyl ester + H2O
Ser-Phe + Met-Ala-Ile-O-methyl ester
show the reaction diagram
-
part of the bovine kappa-casein sequence
-
?
skim milk + H2O
?
show the reaction diagram
Substance P + H2O
?
show the reaction diagram
-
parent peptide and Lys8 variant respectively as substrate
-
-
?
undecapeptide analogue to chymosin sensitive region of bovine kappa casein
?
show the reaction diagram
-
synthetic substrate
-
-
?
undecapeptide analogue to chymosin sensitive region of bovine kappa-casein
?
show the reaction diagram
-
synthetic substrate
-
-
?
undecapeptide analogue to chymosin sensitive region of camel kappa casein
?
show the reaction diagram
-
synthetic substrate
-
-
?
undecapeptide analogue to chymosin sensitive region of camel kappa-casein
?
show the reaction diagram
-
synthetic substrate
-
-
?
YGISSKFCE + H2O
YGISSKF + L-Cys-L-Glu
show the reaction diagram
-
modified peptide based on prochymosin sequence
100% cleavage at enzyme to substrate ratio of 1:1, pH 6.2, 16 h
-
?
YGISSKFfE + H2O
YGISSKF + FE
show the reaction diagram
-
modified peptide based on prochymosin sequence
100% cleavage at enzyme to substrate ratio of 1:1, pH 6.2, 16 h
-
?
YGISSKFHE + H2O
YGISSKF + His-Glu
show the reaction diagram
-
modified peptide based on prochymosin sequence
51% cleavage at enzyme to substrate ratio of 1:1, pH 6.2, 16 h
-
?
YGISSKFIE + H2O
YGISSKF + Ile-Glu
show the reaction diagram
-
modified peptide based on prochymosin sequence
54% cleavage at enzyme to substrate ratio of 1:1, pH 6.2, 16 h
-
?
YGISSKFKE + H2O
YGISSKF + Lys-Glu
show the reaction diagram
-
modified peptide based on prochymosin sequence
33% cleavage at enzyme to substrate ratio of 1:1, pH 6.2, 16 h
-
?
YGISSKFLE + H2O
YGISSKF + L-Leu-L-Glu
show the reaction diagram
-
modified peptide based on prochymosin sequence
42% cleavage at enzyme to substrate ratio of 1:1, pH 6.2, 16 h
-
?
YGISSKFME + H2O
YGISSKF + L-Met-L-Glu
show the reaction diagram
-
modified peptide based on prochymosin sequence
52% cleavage at enzyme to substrate ratio of 1:1, pH 6.2, 16 h
-
?
YGISSKFNE + H2O
YGISSKF + L-Asn-L-Glu
show the reaction diagram
-
modified peptide based on prochymosin sequence
33% cleavage at enzyme to substrate ratio of 1:1, pH 6.2, 16 h
-
?
YGISSKFRE + H2O
YGISSKF + Arg-Glu
show the reaction diagram
-
modified peptide based on prochymosin sequence
59% cleavage at enzyme to substrate ratio of 1:1, pH 6.2, 16 h
-
?
YGISSKFVE + H2O
YGISSKF + Val-Glu
show the reaction diagram
-
modified peptide based on prochymosin sequence
36% cleavage at enzyme to substrate ratio of 1:1, pH 6.2, 16 h
-
?
YGISSKFWE + H2O
YGISSKF + Trp-Glu
show the reaction diagram
-
modified peptide based on prochymosin sequence
100% cleavage at enzyme to substrate ratio of 1:1, pH 6.2, 16 h
-
?
YGISSKFYE + H2O
YGISSKF + L-Tyr-L-Glu
show the reaction diagram
-
modified peptide based on prochymosin sequence
100% cleavage at enzyme to substrate ratio of 1:1, pH 6.2, 16 h
-
?
additional information
?
-
-
does not cleave alpha-casein and beta-casein
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
alpha-casein + H2O
?
show the reaction diagram
alphas1-casein + H2O
?
show the reaction diagram
beta-casein + H2O
?
show the reaction diagram
bovine kappa-casein + H2O
para-kappa-casein + caseinomacropeptide
show the reaction diagram
kappa-casein + H2O
?
show the reaction diagram
kappa-casein + H2O
casein macropeptide + para-kappa-casein
show the reaction diagram
-
cleaves a single bond between phenylalanine 105 and methionine 106
-
-
?
kappa-casein + H2O
caseinmacropeptide + para-kappa-casein
show the reaction diagram
-
cleaves a single bond between phenylalanine 105 and methionine 106
-
-
?
kappa-casein + H2O
para-kappa-casein + glycopeptide
show the reaction diagram
Q5VK60
cleavage between Phe105-Met106
-
-
?
skim milk + H2O
?
show the reaction diagram
-
-
-
-
?
additional information
?
-
-
does not cleave alpha-casein and beta-casein
-
-
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cr3+
-
1 mM increases the milk clotting activity by 15.1%
Mg2+
-
1 mM increases the milk clotting activity by 20.1%
Zn2+
-
1 mM increases the milk clotting activity by 12%
additional information
-
Cu2+, Mn2+, and Co2+ ions have no effect on the enzyme activity
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1-2-epoxy-3(p-nitrophenoxy)propane
-
-
alpha2-Macroglobulin
Blood serum
-
porcine and particularly equine serum impairs the coagulation of milk by chymosin, the inhibitor(s) may be heat labile, alpha2-macroglobulin may be the inhibitory substance
-
Ca2+
-
milkclotting activity decrease when the concentration of Ca2+ is higher than 40 mM
Diazoacetylnorleucine methyl ester
-
in presence of Cu2+
EDTA
-
10 mM EDTA causes inhibition of 73.8% of the milk-clotting activity
iodoacetamide
-
4 mM iodoacetamide causes inhibition of 7.2% of the milk-clotting activity
Pepstatin
pepstatin A
Sn2+
-
28.8% inhibition at 1 mM
additional information
-
phenylmethylsulfonyl fluoride does not affect the enzyme activity
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
His-Pro-His-Pro-His-NH2
-
stimulation, part of kappa-casein
additional information
-
when subjected to a low pH, recombinant prochymosin is converted into mature and active chymosin
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0056 - 0.007
Abz-A-A-F-F-A-A-N-(2,4-dinitrophenyl)-ethylenediamine
0.004
Abz-A-A-F-F-A-A-p-nitroanilide
-
hydrolysis by calf chymosin
0.0022 - 0.018
Abz-A-A-F-F-A-N-(2,4-dinitrophenyl)-ethylenediamine
0.003
Abz-A-A-F-F-N-(2,4-dinitrophenyl)-ethylenediamine
-
hydrolysis by calf chymosin
0.0018
Abz-A-A-F-F-p-nitroanilide
0.0021 - 0.0078
Abz-A-F-F-A-A-N-(2,4-dinitrophenyl)-ethylenediamine
3.3
AFPLEFEREL
-
pH 4.0, 37C
0.041
AFPLEFFREL
-
pH 4.0, 37C
0.079
AFPLEFIREL
-
pH 4.0, 37C
0.18
AFPLEFKREL
-
pH 4.0, 37C
0.01223 - 0.89
kappa-casein
0.333
Leu-Ser-Phe(NO2)-Nle-Ala-Leu-O-methyl ester
-
point mutant G243D
1.11
Leu-Ser-Phe(NO2)-Nle-Ala-Leu-Ome
-
point mutant A115T
0.85
Leu-Ser-Phe-Met-Ala-Ile-O-methyl ester
-
-
6.9
Leu-Ser-Phe-Met-Ala-O-methyl ester
-
-
0.016 - 0.111
NT/NMN 142-151
0.065 - 0.463
POm C165-174
0.34
Pro-His-Leu-Ser-Phe-Met-Ala-Ile-O-methyl ester
-
-
8.5
Ser-Phe-Met-Ala-Ile-O-methyl ester
-
-
0.077 - 0.165
undecapeptide analogue to chymosin sensitive region of bovine kappa casein
0.056 - 0.134
undecapeptide analogue to chymosin sensitive region of camel kappa casein
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.12 - 1.6
Abz-A-A-F-F-A-A-N-(2,4-dinitrophenyl)-ethylenediamine
1.4
Abz-A-A-F-F-A-A-p-nitroanilide
Bos taurus
-
hydrolysis by calf chymosin
0.02 - 0.8
Abz-A-A-F-F-A-N-(2,4-dinitrophenyl)-ethylenediamine
0.27
Abz-A-A-F-F-N-(2,4-dinitrophenyl)-ethylenediamine
Bos taurus
-
hydrolysis by calf chymosin
0.48
Abz-A-A-F-F-p-nitroanilide
Bos taurus
-
hydrolysis by calf chymosin
0.06 - 0.14
Abz-A-F-F-A-A-N-(2,4-dinitrophenyl)-ethylenediamine
2600
AFPLEFEREL
Bos taurus
-
pH 4.0, 37C
290
AFPLEFFREL
Bos taurus
-
pH 4.0, 37C
427
AFPLEFIREL
Bos taurus
-
pH 4.0, 37C
29
AFPLEFKREL
Bos taurus
-
pH 4.0, 37C
2 - 191
NT/NMN 142-151
12 - 219
POm C165-174
11.7 - 44.3
undecapeptide analogue to chymosin sensitive region of bovine kappa casein
4.3 - 5.1
undecapeptide analogue to chymosin sensitive region of camel kappa casein
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
790
AFPLEFEREL
Bos taurus
-
pH 4.0, 37C
12112
7100
AFPLEFFREL
Bos taurus
-
pH 4.0, 37C
12114
5400
AFPLEFIREL
Bos taurus
-
pH 4.0, 37C
12113
160
AFPLEFKREL
Bos taurus
-
pH 4.0, 37C
40659
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1.987
-
crude enzyme, in 50 mM acetate buffer (pH 5.6) at 37C
7.272
-
after 33.2fold purification, in 50 mM acetate buffer (pH 5.6) at 37C
additional information
-
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2.5
-
-
3.7
-
isoform chymosin B
4.2
-
isoform chymosin A
4.5
recombinant chymosin
4.9
-
for proteolysis of undecapeptide analogue to chymosin sensitive region of the bovine kappa casein
5.1
-
for proteolysis of the undecapeptide analogue to chymosin sensitive region of bovine kappa casein
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1 - 5
-
acid-denatured hemoglobin, pH 1: about 80% of maximum activity, pH 5: about 30% of maximum activity
2 - 6
-
-
2 - 6.7
3 - 10
-
pH 3: about 80% of maximum activity, pH 10: about 20% of maximum activity
4.5
-
using alpha- and beta-casein as substrate
4.6 - 5.6
-
-
4.85
-
calculated isoelectric point
5.1 - 6.7
-
at pH 5.1, the relative clotting activity decreases to 50% and further declines to 17% and 10% at pH 5.6 and pH 6.7, respectively
5.5 - 8
sharp decline of activity up to pH 5.8, moderate decline at higher pH with complete loss of activity at pH 8
5.6
-
-
5.8
-
using whole casein as substrate
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 50
-
-
45 - 55
-
optimum for milk clotting activity
47
-
for proteolysis of the undecapeptide analogue to chymosin sensitive region of camel kappa casein
50
-
-
55
proteolytic activity
56
-
for proteolysis of the undecapeptide analogue to chymosin sensitive region of camel kappa casein
additional information
the substrate temperature was found to influence milk clotting activity, optimum substrate temperature is 65C
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 52.5
-
-
20 - 60
-
20C: about 25% of maximum activity, 60C: about 50% of maximum activity
30 - 60
milk clotting activity declines moderately up to 55C (80% of maximum activity) and than drops to 40% of activity at 60C proteolytic activity at 30C and 60C approximately 80% of maximum activity
35 - 60
-
relative activity of 50% is observed at temperatures of 35C and 60C. Chymosin activity completely ceases below 20C or above 70C
additional information
the substrate temperature was found to influence milk clotting activity, sharp optimum at 65C with complete loss of activity at 75C, less than 20% of activity at 30C
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.55
-
isoelectric focusing
4.6
-
recombinant chymosin
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
enzyme extracted from abdomasal tissue of 15 days old kid
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
-
rennet from abomasal tissue from a local indigenous breed
-
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30700
-
amino acid composition
33000
-
gel filtration
36000
-
purified chymosin, gel filtration
36300
-
two-dimensional electrophoresis technique
36500
-
sedimentation, diffusion
40000
-
apparent molecular weight, determined by comparison to a Novex Mark12TM
additional information
-
-
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
additional information
-
insertion of a glycosylation site (S351T), glycosylation has significant inhibitory effect but this can be circumvented by deglycosylation with endoglucosidase H
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
loop exchange mutant
-
modeling of chymosin in complex with residues 97-112 of bovine kappa-casein. Substrate binds in an extended conformation with charged residues on either side of the scissile bond playing an important role in stabilizing the binding pose. Substrate residues Lys111 and Lys112 bind to the N-terminal domain of chymosin displacing a conserved water molecule. A cluster of histidine and proline residues, His98-Pro99-His100-Pro101-His102, in kappa-casein binds to the C-terminal domain of the protein, where neighboring conserved arginine residue Arg97 is important for stabilizing the binding pose. The catalytic site including the catalytic water molecule is stable in the starting conformation of the general acid/base catalytic mechanism for 18 ns of molecular dynamics simulations
-
point mutants
-
prochymosin mutant
-
unglycosylated chymosin, vapor diffusion method, using 100 mM NaH2PO4 pH 5.5, 1.5 M NaCl
-
doubly and singly glycosylated variants of chymosin, vapor diffusion method, using 2 M ammonium sulfate, 100 mM bis-Tris buffer in the pH range 5.1-6.5
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2 - 6
-
under incubation at 37C for 24 and 48 h, loss of activity at pH 3.5 and pH 7.0, trangenic chymosin and recombinant chymosin are inactivated slightly faster than the native enzyme
667763
2 - 6.5
-
crystalline chymosin exhibits optimum stability at pH between 5.3 and 6.3, the enzyme is moderately stable at pH 2.0, but the enzyme is unstable at pH around 3.5 and above 6.5, clotting activity is not observed at pH 7.0
708236
2
-
-
36882
2 - 3.8
-
-
667435
2 - 8
-
the enzyme retains 80% of activity within pH 2.0-8.0
731169
2.5 - 6.5
-
the recombinant enzyme is highly active and stable over a wide pH range (from 2.5 to 6.0) at 20C for 8 h. Relative clotting activity declines when the recombinant chymosin is kept at a pH value above 6.0. No detectable clotting activity at pH 6.5
732866
3.5
-
rapid loss of activity
36882
5.3 - 6.3
-
highest stability
36882
7
-
rapid loss of activity above
36882
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0 - 50
-
after 8 h of incubation, between 0 and 20C the enzyme shows 100% activity, while at 30C and 40C 70% and 35% is retained, respectively. The enzyme is inactive after 8 h at 50C
5
-
very stable below, suspension of crystals
25 - 50
-
transgenic chymosin loses activity slightly faster than abomasal enzyme and recombinant enzyme
25
-
most stable at pH 5.0, denaturation can be fitted to the two-state irreversible model
40 - 55
-
no residual clotting activities at temperatures higher than 55C
40
-
the enzyme is stable until 40C for 30 min while losing 40 and 80% of its activity after incubation at 45 and 50C for 30 min, respectively
45
-
about 25% loss of activity after 60 min
50 - 60
-
chymosin is stable up to 50C and a relative milk-clotting activity of 50% is recorded when the temperature is raised to 60C
50
-
about 80% loss of activity after 30 min
60
-
melting temperature of the singly glycosylated enzyme
65
-
transition midpoint
additional information
-
the enzyme undergoes irreversible, highly scan-rate-dependent thermal denaturation under all the experimental conditions investigated (pH 2-12, 20-50C). Between pH 3.0 and 7.0, only one endotherm characterizes the thermal denaturation of the enzyme. Upon reaching pH 7.5, the denaturation is characterized by two endotherms
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
for prochymosins activation, the supernatant is adjusted to pH 2.0 and incubated for 2 h at 25C, then adjusted to pH 6.3 and left again at 25C for 1 h
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
urea
-
chymosin loses 50% of its activity after incubation in 4.6 M urea at 37C for 30 min
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, Tris-HCl buffer, pH 8.0, glycerol
-
0-20C, at pH 5.5, 8 h, no loss of activity
-
40C, at pH 5.5, 8 h, 37.5% residual activity
-
4C, at pH 3.0 and 6.2, 3 days, maximum stability
-
50C, at pH 5.5, complete loss of activity
-
very stable below 5C, suspension of crystals
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation, Q Sepharose column chromatography, and Sephadex S-100 gel filtration
-
by affinity chromatography MIMO1300 matrix
-
by affinity chromatography on a bacitracin-Sepharose column
-
DEAE-cellulose column chromatography, gel filtration
-
extraction by application of ultrasound
-
gel filtration and anion-exchange chromatography
-
metal affinity chromatography
phenyl Superose column chromatography
purification by partitioning between aqueous two-phase systems
-
resolves into three major peaks in DEAE chromatography
similar enzyme
-
ultrafiltration and Superdex 75 gel filtration
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed as inclusion bodies in Escherichia coli
-
expressed in Escherichia coli and in Saccharomyces cerevisiae
-
expressed in Escherichia coli BL21(DE3) cells
expressed in Pichia pastoris strain GS115
expression in Aspergillus niger
-
expression in Aspergillus niger var. awamori dgr246pyrG
-
expression in Kluyveromyces lactis
-
expression in Pichia pastoris
-
expression in Saccharomyces cerevisiae strain INVSC1
-
loop exchange mutant expressed in Trichoderma reesei
-
point mutants expressed in Escherichia coli and Trichoderma reesei
-
prochymosin cDNA, expressed as a NusA fusin protein in Escherichia coli, low level of milk clotting activity after activation at acidic pH
-
prochymosin fusion product expressed in Escherichia coli
-
S351T mutant of prochymosin as fusion protein with the Aspergillus niger enzyme glucoamylase in Aspergillus niger var. awamori, insertion of a short peptide linker between the proteins with the sequence TDNST
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A115T
-
reduction of Km compared to wild-type
D304A
-
pH optima shift
D304A/T218A
G243D
-
increase of Km compared to wild-type
G244D
-
pH optima shift
Q288K
-
-
S351T
-
insertion of a glycosylation site in the linker region between chymosin and glucoamylase, strongly improves enzyme secretion
T218A
-
pH optima shift
D289M/Q298L
-
decreased hydrolytic activity
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
oxidative refolding of solubilized enzyme obtained from inclusion bodies at pH 2 to obtain autoconversion of prochymosin to active chymosin
-
using 8 M urea, at 25C
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
food industry
synthesis
-
use of chymosin to cleave a pro-chymosin derived fusion tag releasing native target proteins. After modification of the pro-chymosin fusion tag chymosin can remove this tag at more neutral pH 6.2, less prone to compromise the integrity of target proteins. Chymosin produces intact native target protein both at the level of small and large-scale preparations
additional information
-
under typical cheese-making conditions (pH 6.6, 0-2 mM CaCl2) the clotting activity of camel chymosin is ca. 80% higher than the activity of bovine chymosin (average clotting activity of camel chymosin is 70% higher than the activity of bovine chymosin), camel chymosin is more thermostable than bovine chymosin