Information on EC 3.4.24.28 - bacillolysin

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The expected taxonomic range for this enzyme is: Firmicutes

EC NUMBER
COMMENTARY hide
3.4.24.28
-
RECOMMENDED NAME
GeneOntology No.
bacillolysin
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
similar, but not identical, to that of thermolysin
show the reaction diagram
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-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
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-
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CAS REGISTRY NUMBER
COMMENTARY hide
76774-43-1
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
isolated from soil
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
gene mprBi
UniProt
Manually annotated by BRENDA team
A9542
-
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Manually annotated by BRENDA team
strain 76
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Manually annotated by BRENDA team
strain 76
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
gene bmp1
UniProt
Manually annotated by BRENDA team
gene bmp1
UniProt
Manually annotated by BRENDA team
Clostridium histolyticum
-
-
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-aminobenzoyl-AGLA-4-nitrobenzylamide + H2O
?
show the reaction diagram
-
-
-
-
?
2-aminobenzoyl-Ala-Gly-Leu-Ala-4-nitrobenzylamide + H2O
?
show the reaction diagram
-
-
-
-
?
2-Furanacryloyl-Gly-OLeu-NH2 + H2O
?
show the reaction diagram
-
-
-
-
-
3-(2-furylacryloyl)-glycyl-L-leucinamide + H2O
?
show the reaction diagram
-
-
-
-
?
3-(2-furylacryloyl)-glycyl-L-leucine amide + H2O
?
show the reaction diagram
3-(2-furylacryloyl)-L-alanyl-L-phenylalanine amide + H2O
?
show the reaction diagram
-
-
-
?
azocasein + H2O
?
show the reaction diagram
Benzoyl-Gly-OLeu-Ala + H2O
?
show the reaction diagram
-
-
-
-
-
Benzoyl-Gly-OPhe-Ala + H2O
?
show the reaction diagram
-
-
-
-
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beta-casein + H2O
hydrolyzed casein
show the reaction diagram
-
-
-
?
Bovine serum albumin + H2O
?
show the reaction diagram
casein + H2O
?
show the reaction diagram
coagulation factor X + H2O
?
show the reaction diagram
Collagen + H2O
?
show the reaction diagram
Denatured casein + H2O
?
show the reaction diagram
-
-
-
-
?
denatured collagen + H2O
?
show the reaction diagram
-
11.5% of the activity with denatured casein
-
-
?
Dnp-AALR-NH2 + H2O
?
show the reaction diagram
-
-
-
?
Dnp-AAVR + H2O
?
show the reaction diagram
-
-
-
?
Dnp-GGFR + H2O
?
show the reaction diagram
-
-
-
?
Dnp-GGIR + H2O
?
show the reaction diagram
-
-
-
?
Dnp-GGK + H2O
?
show the reaction diagram
-
-
-
?
Dnp-GGLR + H2O
?
show the reaction diagram
-
-
-
?
fibrinogen + H2O
fibrin + ?
show the reaction diagram
-
-
-
-
?
fluorescein isothyocyanate-conjugated bovine serum albumin + H2O
?
show the reaction diagram
Furylacryloyl-Gly-Ile-NH2 + H2O
?
show the reaction diagram
-
-
-
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furylacryloyl-Gly-Leu methyl ester + H2O
?
show the reaction diagram
-
-
-
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furylacryloyl-Gly-Leu-NH2 + H2O
?
show the reaction diagram
Furylacryloyl-Leu-Leu-NH2 + H2O
?
show the reaction diagram
-
-
-
-
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Furylacryloyl-Phe-Phe-NH2 + H2O
?
show the reaction diagram
-
-
-
-
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Furylacryloyl-Thr-Leu-NH2 + H2O
?
show the reaction diagram
-
-
-
-
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Gelatin + H2O
?
show the reaction diagram
Gly-Leu-NH2 + H2O
?
show the reaction diagram
-
-
-
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insulin B chain + H2O
?
show the reaction diagram
-
-
-
?
N-(3-[2-furyl]acryloyl)-Gly-Leu amide + H2O
?
show the reaction diagram
-
-
-
?
nematode cuticle + H2O
?
show the reaction diagram
-
2.8% of the activity with denatured casein
-
-
?
Oxidized insulin B-chain + H2O
?
show the reaction diagram
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major cleavages at the peptide bonds of His5-Leu6, His10-Leu11, Ala14-Leu15, Tyr16-Leu17, Gly23-Phe24 and Phe24-Phe25
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plasminogen + H2O
angiostatin + mini-plasminogen
show the reaction diagram
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cleavage mainly at S441-V442
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?
protein C + H2O
?
show the reaction diagram
prothrombin + H2O
thrombin + thrombin propeptide
show the reaction diagram
-
-
-
-
?
prourokinase + H2O
urokinase + urokinase propeptide
show the reaction diagram
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?
skimmed milk + H2O
?
show the reaction diagram
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60% of the activity with denatured casein
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?
additional information
?
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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
casein + H2O
?
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Calcium
Co2+
30% activation at 8 mM, 40% inhibition at 10 mM
Li+
-
activates 20% at 10 mM
Mn2+
-
activates 2.90fold at 10 mM
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
94.2% inhibition at 0.5 mM, 100% at 5 mM
2-mercaptoethanol
-
27% inhibition at 50 mM
Ba2+
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88% inhibition at 10 mM
Ca2+
30-40% inhibition at 4-8 mM
dithio-bis-nitrobenzoic acid
-
64% inhibition at 50 mM
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dithiothreitol
Fe2+
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86% inhibition at 10 mM
Fe3+
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98% inhibition at 10 mM
guanidine hydrochloride
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competitive inhibitor
HgCl2
49% inhibition at 0.5 mM, 60% at 5 mM
Mg2+
72% inhibition at 4-8 mM
Mn2+
64% inhibition at 4-8 mM
N-[alpha-L-rhamnopyranosyl-oxyhydroxyphosphinyl]-L-leucyl-L-tryptophan
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Organic solvents
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and related compounds, overview
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PCMB
6% inhibition at 0.5 mM, 98.1% at 5 mM
pepstatin A
slight inhibition
phenylmethylsulfonyl fluoride
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0.1 mM, 77% residual activity
SDS
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37% inhibition at 50 mM
Triton X-100
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slight inhibition at 50 mM
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
CoCl2
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0.3 mM, 1.8fold increase in activity
Tween-20
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1.61fold activation at 50 mM
Tween-80
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1.34fold activation at 50 mM
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.3 - 1.4
3-(2-furylacryloyl)-glycyl-L-leucine amide
0.25 - 0.3
3-(2-furylacryloyl)-L-alanyl-L-phenylalanine amide
0.06
azocasein
recombinant wild-type enzyme, pH 8.0, 37C
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0.003
plasminogen
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pH 7.4, 25C
-
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
80 - 256
3-(2-furylacryloyl)-glycyl-L-leucine amide
64 - 99
3-(2-furylacryloyl)-L-alanyl-L-phenylalanine amide
1210
azocasein
Bacillus intermedius
B3V4Z0
recombinant wild-type enzyme, pH 8.0, 37C
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0.7
plasminogen
Bacillus megaterium
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pH 7.4, 25C
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1000
guanidine hydrochloride
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pH 7.5, 25C
0.0035 - 0.0037
N-[alpha-L-rhamnopyranosyl-oxyhydroxyphosphinyl]-L-leucyl-L-tryptophan
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0057
with substrate Dnp-AAVR, pH 8.0, 37C
0.0145
with substrate Dnp-GGLR, pH 8.0, 37C
0.0264
with substrate Dnp-GGK, pH 8.0, 37C
0.0282
with substrate Dnp-GGIR, pH 8.0, 37C
0.0365
with substrate Dnp-GGFR, pH 8.0, 37C
0.0515
with substrate Dnp-AALR-NH2, pH 8.0, 37C
17.4
with substrate azocasein, recombinant purified wild-type enzyme, pH 8.0, 37C
55.6
-
pH 6.5, 55C
78.5
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Boilysin at 37C with casein as substrate
81.5
-
pseudo wild type at 37C with casein as substrate
81.9
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wild type enzyme at 37C with casein as substrate
82.4
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double mutant G8C/N60C at 37C with casein as substrate
242
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wild type enzyme at temperature optimum 75C
273
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pseudo wild type at temperature optimum 75C
375
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double mutant G8C/N60C at temperature optimum 80C
608
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boilysin at temperature optimum 95C
3556
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purified enzyme, pH 7.5, 37C
11280
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purified native enzyme, pH 7.0, 40C
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 10
activity range, recombinant enzyme
5.5
sharp decrease in activity below
6 - 10
6.5 - 11
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decrease of activity from pH 6.5 to 11
11
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no residual activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 7.2
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-
37 - 65
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assay at
75
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pseudo wild type; wild type enzyme
80
-
double mutant G8C/N60C
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 60
activity range, recombinant enzyme
22 - 70
activity range
30 - 70
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacillus cereus (strain ATCC 14579 / DSM 31 / JCM 2152 / NBRC 15305 / NCIMB 9373 / NRRL B-3711)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
34500
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Bacillus stearothermophilus NCIB 8924
35000
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Bacillus stearothermophilus NRRL B-3880
additional information
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure at 3.0 A resolution
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 5
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purified extracellular enzyme, 50C, loss of 70% activity after 60 min
733130
5 - 9
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37C, 60 min, stable
669317
5 - 8
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stable within this range
667347
6
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purified extracellular enzyme, 50C, no loss of activity after 60 min, stable at
733130
6 - 9
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purified enzyme, 60C, loss of 20-25% activity after 10 min
733469
7
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purified extracellular enzyme, 50C, loss of 20% activity after 60 min
733130
8 - 10
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purified extracellular enzyme, 50C, loss of 40% activity after 60 min
733130
10
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purified enzyme, 60C, loss of 40% activity after 10 min
733469
11.7
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60 min, complete loss of activity
669317
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 60
-
stable
30
-
purified enzyme, 2 h, stable
30 - 60
-
stable for 30 min
40 - 50
58.6
-
T50 value at pH 7.0
59.5
-
T50 value at pH 5.3
92.5
-
G8C/N60C double mutation extremely thermostable, half-life 35.9 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
Ca2+ stabilizes against thermal inactivation
-
MgCl2 stabilizes
-
native wild type and G8C/N60C enzymes are stable toward autoproteolysis and keep their structural integrity for several weeks
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2-propanol
-
48 h, 37C, 50% residual activity with 44.5% (v/v) and 65.5% (v/v) for pseudo wild-type and mutant G8C/N60C respectively
Acetone
-
48 h, 37C, 50% residual activity with 30.0% (v/v) and 50.1% (v/v) for pseudo wild-type and mutant G8C/N60C respectively
acetonitrile
-
48 h, 37C, 50% residual activity with 30.6% (v/v) and 51.6% (v/v) for pseudo wild-type and mutant G8C/N60C respectively
dimethylformamide
-
48 h, 37C, 50% residual activity with 29.8% (v/v) and 45.8% (v/v) for pseudo wild-type and mutant G8C/N60C respectively
dioxane
-
48 h, 37C, 50% residual activity with 32.1% (v/v) and 47.9% (v/v) for pseudo wild-type and mutant G8C/N60C respectively
DMSO
-
48 h, 37C, 50% residual activity with 32.9% (v/v) and 48.7% (v/v) for pseudo wild-type and mutant G8C/N60C respectively
Methanol
-
48 h, 37C, 50% residual activity with 56.6% (v/v) and 86.4% (v/v) for pseudo wild-type and mutant G8C/N60C respectively
additional information
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
after expression in Escherichia coli with N-terminal His-tag
-
extracellular enzyme from culture medium by anion exchange chromatography
-
extracellular enzyme from culture medium by several chromatographic steps including one affinity based step
Clostridium histolyticum
-
native enzyme 13.1fold by ethanol precipitation, gel filtration, and anion exchange chromatography
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native extracellular enzyme 5.6fold from culture supernatant by ammonium sulfate fractionation, anion exchange and hydrophobic interaction chromatography, and gel filtration
-
recombinant enzyme from Bacillus subtilis culture medium by ammonium sulfate fractionation and hydrophobic interaction chromatography to homogeneity
recombinant enzyme from Escherichia coli strain BL21(DE3)
wild-type and mutant enzymes
-
wild-type and mutant TLPs
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis
-
expression in Bacillus subtilis
gene bmp1, recombinant expression in Escherichia coli strain BL21(DE3)
gene contains a large open reading frame between the regions coding for signal sequence and mature protein
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gene mprBi, DNA and amino acid sequence determination and analysis, enzyme expression in Bacillus subtilis and secretion to the culture medium
nucleotide sequence and promoter region
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protease-deficient strain Bacillus subtilis DB117 used as host for plasmids, subcloned in Escherichia coli for site-directed mutagenesis
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
I157D
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site-directed mutagenesis
G8C/N60C
W55F
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higher Ca2+ concentration is needed than for wild-type for occupation of Ca2+-binding site III at amino acids 55-59
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
protease activity can be significantly regained if the completely unfolded enzyme is transferred from 8 M guanidine-HCl into native-like conditions (0.4 M guanidine-HCl) by dilution with buffer
-
renaturation of enzyme from inclusion bodies after solubilization using guanidine hydrochloride
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
food industry
synthesis
-
production of enzyme as an active mature enzyme in Escherichia coli in the absence of its prosequence. Expression of enzyme with N-terminal His-tag, which accumulates as inclusion bodies and renaturation after solubilization using guanidine hydrochloride
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