Any feedback?
Information on EC 3.4.21.62 - Subtilisin and Organism(s) Bacillus subtilis and UniProt Accession Q84F18
for references in articles please use BRENDA:EC3.4.21.62Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
EC Tree
3.4.21.62 SubtilisinSpecify your search results
Word Map
- 3.4.21.62
- hydrolysates
- chymotrypsin
- prion
- elastase
- papain
- sds
- aeruginosa
- prpsc
- proteinases
- casein
- staphylococcus
- proprotein
- pronase
-
convertase
- alpha-chymotrypsin
- scrapie
- dnase
-
ultrafiltration
-
proteinaceous
-
spongiform
- thermolysin
- pmsf
-
bacteriocins
- licheniformis
- propeptide
-
paraffin-embedded
- gelatin
-
protease-resistant
-
formalin-fixed
- pcsk9
- alpha-amylase
-
cytotoxin
-
boil
- creutzfeldt-jakob
-
phenylmethylsulfonyl
- amyloliquefaciens
- pumilus
- bromelain
-
alkaliphilic
-
shiga
-
i-converting
-
transesterification
- furin
- album
-
defatted
- prodomain
- pancreatin
- p-nitroanilide
-
kazal
- detergent
- industry
- biotechnology
- food industry
- medicine
- synthesis
- degradation
- analysis
The taxonomic range for the selected organisms is: Bacillus subtilis
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
Hydrolysis of proteins with broad specificity for peptide bonds, and a preference for a large uncharged residue in P1. Hydrolyses peptide amides
Synonyms
proteinase k, subtilisin, alkaline protease, alcalase, subtilisin carlsberg, subtilase, subtilisin-like protease, savinase, alkaline serine protease, subtilisin a, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
Alcalase
-
-
-
-
Alcalase 0.6L
-
-
-
-
Alcalase 2.5L
-
-
-
-
ALK-enzyme
-
-
-
-
Alkaline mesentericopeptidase
-
-
-
-
Alkaline protease
-
-
-
-
Bacillopeptidase A
-
-
-
-
Bacillopeptidase B
-
-
-
-
Bacillus subtilis alkaline proteinase Bioprase
-
-
-
-
Bioprase AL 15
-
-
-
-
Bioprase APL 30
-
-
-
-
Colistinase
-
-
-
-
Esperase
-
-
-
-
Genenase I
-
-
-
-
Kazusase
-
-
-
-
Maxatase
-
-
-
-
Nagarse
-
-
-
-
Opticlean
-
-
-
-
Orientase 10B
-
-
-
-
Peptidase, subtilo-, A
-
-
-
-
Protease S
-
-
-
-
Protease VIII
-
-
-
-
Protease XXVII
-
-
-
-
Proteinase, Bacillus subtilis alkaline
-
-
-
-
Protin A 3L
-
-
-
-
Savinase
-
-
-
-
Savinase 16.0L
-
-
-
-
Savinase 32.0 L EX
-
-
-
-
Savinase 4.0T
-
-
-
-
Savinase 8.0L
-
-
-
-
SP 266
-
-
-
-
Subtilisin amylosacchariticus
-
from Bacillus subtilis var. amylosacchariticus
Subtilisin E
Subtilisin GX
-
-
-
-
Subtilisin Novo
-
-
-
-
subtilisin QK
Subtilisin S41
Subtilisin Sendai
-
-
-
-
Subtilopeptidase
-
-
-
-
Superase
-
-
-
-
Thermoase
-
-
-
-
Thermoase PC 10
-
-
-
-
additional information
-
synonyms of subtilisin Carlsberg: subtilisin, subtilisin A, subtilopeptidase A, alcalase Novo, synonyms of subtilisin BPN': nagarse proteinase, Nagarse, BPN', subtilopeptidase C, synonyms of subtilisin Novo: bacterial proteinase Novo, subtilisin B, subtilopeptidase B
Subtilisin E
-
-
-
-
Subtilisin S41
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hydrolysis of peptide bond
-
-
-
-
ester bond hydrolysis
-
-
-
-
transpeptidation
-
-
-
-
transesterification
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
9014-01-1
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
D-Val-Leu-Lys p-nitroanilide + H2O
D-Val-Leu-Lys + p-nitroaniline
-
-
-
?
benzyloxycarbonyl-L-Asp methyl ester + serine amide
benzyloxycarbonyl-L-Asp-L-Ser-NH2 + methanol
-
-
-
-
?
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide + H2O
N-succinyl-Ala-Ala-Pro-Phe + 4-nitroaniline
-
-
-
?
succinyl-Ala-Ala-Pro-Phe 4-nitroanilide + H2O
succinyl-Ala-Ala-Pro-Phe + 4-nitroaniline
-
-
-
-
?
succinyl-L-Ala-L-Ala-L-Pro-L-Phe-4-methyl-coumaryl-7-amide + H2O
succinyl-L-Ala-L-Ala-L-Pro-L-Phe + 7-amino-4-methylcoumarin
-
-
-
?
additional information
?
-
-
random mutagenesis to enhance the activity of subtilisin in organic solvents
-
-
?
additional information
?
-
-
influence of substrate structure of N-protected peptide nitroanilides of the types: benzyloxycarbonyl-A2-A1 4-nitroanilide, benzyloxycarbonyl-A3-A2-A1 4-nitroanilide, benzyloxycarbonyl-A4-A3-A2-A1 4-nitroanilide, subsite S1 is of broad selectivity: preference for hydrophobic amino acid residues (i.e. leucine and phenylalanine) the beta-branched and the basic amino acid residues cannot interact with the S1 subsite and the hydrolysis of the corresponding peptides occurs exclusively at the A2-A1 bond. If S1/A1 interactions are weak (Ala, norvaline, norleucine) the amino acid residue A1 can interact with subsites S1 and S1' resulting in the hydrolysis of two bonds (A1 4-nitroanilide and A2-A1). The subsite S2 reveals a preference for small amino acid residues
-
-
?
additional information
?
-
-
reactions catalyzed: 1. peptide bond hydrolysis, 2. ester bond hydrolysis, 3. transesterification, 4. transpeptidation
-
-
?
additional information
?
-
the enzyme shows activity towards complex substrates, e.g. skimmed milk
-
-
?
additional information
?
-
the enzyme shows activity towards complex substrates, e.g. skimmed milk
-
-
?
additional information
?
-
performance of a thrombolytic activity assay
-
-
?
additional information
?
-
-
the wild-type enzyme performs autoproteolysis, while the enzyme mutant S221C does not. The cleavage patterns of SES7 is determined by hydrolysates peptide profile, it prefers amino acids Q, T, P, or L at P3, P, V and Q at P2, K, L, N, Q, or F at P1, and F, S, or A at P1'
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
Ca2+
importance of calcium in the medium after enzyme induction, both for stability of the proteinase and cell health. The two calcium binding sites have apparent binding constants in the mM range. Binding of calcium to the weaker of those two sites only affects resistance of the enzyme against irreversible thermal inactivation. Kinetics
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1,10-phenanthroline
at 37°C and pH of 7.5, 0.1 mM inhibits prosubtilisin JB1 by 40%
5-Dimethylaminonaphthalene-1-sulfonate
-
circular-dichroism and fluorescence studies of 5-dimethylaminonaphthalene-1-sulfonyl derivative of subtilisin indicate a closely similar structure to that of native subtilisin
antipain
at 37°C and pH of 7.5, 0.1 mM inhibits prosubtilisin JB1 by 75%
Brij 35
at 37°C and pH of 7.5, 0.01% reduces prosubtilisin JB1 relative activity to 30% and 0.05% reduces prosubtilisin JB1 relative activity to 70%
Ca2+
at 37°C and pH of 7.5, 1 mM reduces prosubtilisin JB1 relative activity to 57% and 5 mM reduces prosubtilisin JB1 relative activity to 45%
chymostatin
at 37°C and pH of 7.5, 0.1 mM inhibits prosubtilisin JB1 by 95%
Co2+
at 37°C and pH of 7.5, 1 mM reduces prosubtilisin JB1 relative activity to 14% and 5 mM reduces prosubtilisin JB1 relative activity to 24%
Cu2+
at 37°C and pH of 7.5, with 1 mM results in an almost complete reduction of prosubtilisin JB1 activity, 5 mM reduces prosubtilisin JB1 relative activity to 19%
E-64
at 37°C and pH of 7.5, 0.1 mM inhibits prosubtilisin JB1 by 31%
E-64c
at 37°C and pH of 7.5, 0.1 mM inhibits prosubtilisin JB1 by 25%
EDTA
at 37°C and pH of 7.5, 0.1 mM inhibits prosubtilisin JB1 by 52%
EGTA
at 37°C and pH of 7.5, 0.1 mM inhibits prosubtilisin JB1 by 34%
Hg+
at 37°C and pH of 7.5, 1 mM reduces prosubtilisin JB1 relative activity to 33% and 5 mM reduces prosubtilisin JB1 relative activity to 22%
K+
at 37°C and pH of 7.5, with 1 mM results in an almost complete reduction of prosubtilisin JB1 activity, 5 mM reduces prosubtilisin JB1 relative activity to 53%
Mg2+
at 37°C and pH of 7.5, 1 mM reduces prosubtilisin JB1 relative activity to 79% and 5 mM reduces prosubtilisin JB1 relative activity to 48%
N-ethylmaleimide
at 37°C and pH of 7.5, 0.1 mM inhibits prosubtilisin JB1 by 15%
pepstatin A
at 37°C and pH of 7.5, 0.1 mM inhibits prosubtilisin JB1 by 48%
Sodium dodecyl sulfate
at 37°C and pH of 7.5, 0.01% reduces prosubtilisin JB1 relative activity to 52% and 0.05% reduces prosubtilisin JB1 relative activity to 29%
Triton X-100
at 37°C and pH of 7.5, 0.01% reduces prosubtilisin JB1 relative activity to 92% and 0.05% reduces prosubtilisin JB1 relative activity to 69%
Tween 20
at 37°C and pH of 7.5, 0.01% reduces prosubtilisin JB1 relative activity to 53% and 0.05% reduces prosubtilisin JB1 relative activity to 70%
Inhibitor from Vigna unguiculata subsp. cylindrica
-
properties and kinetics of the inhibitor
-
Inhibitor from Vigna unguiculata subsp. cylindrica
-
inactive with other enzymes
-
phenylmethylsulfonyl fluoride
at 37°C and pH of 7.5, 0.1 mM inhibits prosubtilisin JB1 by 79%
PMSF
-
circular-dichroism and fluorescence studies of PMSF derivative of subtilisin indicate a closely similar structure to that of native subtilisin
Zn2+
at 37°C and pH of 7.5, 1 mM reduces prosubtilisin JB1 relative activity to 30%, with 5 mM results in an almost complete reduction of prosubtilisin JB1 activity
additional information
at 37°C and pH of 7.5, 0.1 mM leupeptin does not inhibit prosubtilisin JB1
-
additional information
enzyme destabilization in the presence of EDTA is due to chelation of a tightly bound calcium ion by EDTA, rather than destabilizing effects caused by EDTA on the structure
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
sigma-H is required for full expression of the CSF-processing protease
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.115
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
recombinant enzyme, 5°C, pH 8.6
0.133
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
recombinant enzyme, 25°C, pH 9.4
0.178
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
recombinant enzyme, 25°C, pH 8.6
0.198
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
recombinant enzyme, 25°C, pH 7.4
0.227
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
recombinant enzyme, 45°C, pH 8.6
additional information
additional information
-
the temperature dependence of the kinetics and thermodynamic parameters suggest that the enzyme exists in two, i.e. cold and hot forms, at 22°C the cold form turns into the hot one possibly owing to a conformational change
-
additional information
additional information
Michaelis-Menten kinetics and thermodynamics
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
40.8
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
recombinant enzyme, 5°C, pH 8.6
193.1
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
recombinant enzyme, 25°C, pH 7.4
225.7
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
recombinant enzyme, 25°C, pH 8.6
248.7
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
recombinant enzyme, 25°C, pH 9.4
552.3
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
recombinant enzyme, 45°C, pH 8.6
15
succinyl-L-Ala-L-Ala-L-Pro-L-Phe-4-methyl-coumaryl-7-amide
pH 8.5, 37°C, recombinant wild-type enzyme, in presence of 2 M GdmCl
34
succinyl-L-Ala-L-Ala-L-Pro-L-Phe-4-methyl-coumaryl-7-amide
pH 8.5, 37°C, recombinant mutant S62I/A153V/G166S/I205V, in presence of 2 M GdmCl
35
succinyl-L-Ala-L-Ala-L-Pro-L-Phe-4-methyl-coumaryl-7-amide
pH 8.5, 37°C, recombinant wild-type enzyme, in presence of SDS
42
succinyl-L-Ala-L-Ala-L-Pro-L-Phe-4-methyl-coumaryl-7-amide
pH 8.5, 37°C, recombinant mutant S62I/A153V/G166S/I205V, in absence of GdmCl or SDS
45
succinyl-L-Ala-L-Ala-L-Pro-L-Phe-4-methyl-coumaryl-7-amide
pH 8.5, 37°C, recombinant mutant S62I/A153V/G166S/I205V, in presence of SDS
50
succinyl-L-Ala-L-Ala-L-Pro-L-Phe-4-methyl-coumaryl-7-amide
pH 8.5, 37°C, recombinant wild-type enzyme, in absence of GdmCl or SDS
62
succinyl-L-Ala-L-Ala-L-Pro-L-Phe-4-methyl-coumaryl-7-amide
pH 8.5, 37°C, recombinant mutant G166M, in presence of 2 M GdmCl
65
succinyl-L-Ala-L-Ala-L-Pro-L-Phe-4-methyl-coumaryl-7-amide
pH 8.5, 37°C, recombinant mutant S62I, in presence of SDS
70
succinyl-L-Ala-L-Ala-L-Pro-L-Phe-4-methyl-coumaryl-7-amide
pH 8.5, 37°C, recombinant mutant S62I/G166M, in presence of 2 M GdmCl
81
succinyl-L-Ala-L-Ala-L-Pro-L-Phe-4-methyl-coumaryl-7-amide
pH 8.5, 37°C, recombinant mutant S62I/G166M, in presence of SDS
102
succinyl-L-Ala-L-Ala-L-Pro-L-Phe-4-methyl-coumaryl-7-amide
pH 8.5, 37°C, recombinant mutant S62I, in absence of GdmCl or SDS
104
succinyl-L-Ala-L-Ala-L-Pro-L-Phe-4-methyl-coumaryl-7-amide
pH 8.5, 37°C, recombinant mutant G166M, in presence of SDS
123
succinyl-L-Ala-L-Ala-L-Pro-L-Phe-4-methyl-coumaryl-7-amide
pH 8.5, 37°C, recombinant mutant S62I/G166M, in absence of GdmCl or SDS
141
succinyl-L-Ala-L-Ala-L-Pro-L-Phe-4-methyl-coumaryl-7-amide
pH 8.5, 37°C, recombinant mutant G166M, in absence of GdmCl or SDS
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
354.8
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
recombinant enzyme, 5°C, pH 8.6
975.25
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
recombinant enzyme, 25°C, pH 7.4
1268
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
recombinant enzyme, 25°C, pH 8.6
1869.9
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
recombinant enzyme, 25°C, pH 9.4
2433
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
recombinant enzyme, 45°C, pH 8.6
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
700
guanidinium hydrochloride
Bacillus subtilis
pH 7.5, 37°C, recombinant wild-type enzyme
960
guanidinium hydrochloride
Bacillus subtilis
pH 7.5, 37°C, recombinant mutant N218S
1260
guanidinium hydrochloride
Bacillus subtilis
pH 7.5, 37°C, recombinant mutant T224A
2730
guanidinium hydrochloride
Bacillus subtilis
pH 7.5, 37°C, recombinant mutant S62I/A153V/G166S/I205V
3070
guanidinium hydrochloride
Bacillus subtilis
pH 7.5, 37°C, recombinant mutant S62I/A153V/G166S/T224A/T240S
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.4 - 9.4
the catalytic efficiency (kcat/Km) increases with pH from 7.4 to 9.4 when measured at 25°C, both as a result of increased kcat and lowered Km
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
cold adapted, negatively charged residues on the surface may be responsible for cold adaptation
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
physiological function
-
significantly inhibits carrageenan-induced mouse tail thrombus formation in vivo. Inhibition activity increases along with the injection amount of subtilisin QK, which presents linearity relationship. When the amount of subtilisin QK reaches 12000 IU, thrombus nearly disappears in mouse tail
additional information
evolution
-
enzyme subtilisin E-S7 (SES7) has 3 amino acids different from subtilisin E (1SCJ): S85A, T130S, and T162S
evolution
subtilisin QK is highly homologous to nattokinase (NK, Q548F3)
additional information
structure molecular modeling using the crystal structure of the pro-subtilisin E complex, PDB ID 1SCJ, the active site Cys221 is replaced with the catalytic serine, molecular dynamics simulation, overview
additional information
the salt-bridge triad Gln125-Gln377-Gln381 in subtilisin E contributes to thermostability and activity. The Q125 residue is important for catalytic activity
additional information
-
the salt-bridge triad Gln125-Gln377-Gln381 in subtilisin E contributes to thermostability and activity. The Q125 residue is important for catalytic activity
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
?
-
x * 28800, recombinant mature enzyme, SDS-PAGE, x * 37400, proenzyme, SDS-PAGE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
proteolytic modification
proteolytic modification
-
propeptide is absolutely required to fold into a kinetically trapped conformer. Comparison of folding kinetics and intermediates with intracellular serine proteases
proteolytic modification
-
the enzyme propeptide is cleaved to the mature form
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified recombinant His-tagged enzyme, mixing of 30 mg/ml protein in 30 mM Tris-HCl, pH 7.4, and 150 mM NaCl, with 0.15 M ammonium sulfate, 15% w/v PEG 4000, 0.1 M Tris-HCl, pH 8.0, X-ray diffraction structure determination and analysis at 1.83-1.90 A, molecular replacement and modeling
-
subtilisin S41 surface is rich in hydrophilic residues, particularly Asp, which may contribute to its adaptation to psychrophilic conditions
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
A153V
site-directed mutagenesis, the mutant shows increased activity without detergents compared to the wild-type, but does not show improved properties in chaotropic conditions
G166M
site-directed mutagenesis, the mutant shows increased activity without detergents compared to the wild-type, and improved properties in chaotropic conditions
G166S
site-directed mutagenesis, the mutant shows increased activity without detergents compared to the wild-type, and improved properties in chaotropic conditions
I205V
site-directed mutagenesis, the mutant shows increased activity without detergents compared to the wild-type, but does not show improved properties in chaotropic conditions
K211P/R212A
mutant is more thermostable compared to wild-type, half-life at 60°C 10times longer compared to wild-type. Molecular dynamics simulation at 10°C and 90°C reveal that the average global flexibility of both variants is slightly higher than wild-type
K211P/R212A/S145I/S175T/K221E/N291I/S295T
mutant is more thermostable compared to wild-type, half-life at 60°C 500times longer compared to wild-type. Molecular dynamics simulation at 10°C and 90°C reveal that the average global flexibility of both variants is slightly higher than wild-type
N218S
site-directed mutagenesis, the mutant shows increased activity and stability in the presence of GdmCl or SDS compared to the wild-type enzyme
Q125/Q377E/Q381R
site-directed mutagenesis, the mutant shows increased thermotolerance compared to the wild-type enzyme, but is 60% less active than the wild-type
Q125R
site-directed mutagenesis, the mutant does not show increased thermotolerance compared to the wild-type enzyme, it is 60% less active than the wild-type
Q377E
site-directed mutagenesis, the mutant shows increased thermotolerance and an increase in protease activity of 46.5% compared to the wild-type enzyme
Q381R
site-directed mutagenesis, the mutant shows increased thermotolerance and an increase in protease activity of 46.5% compared to the wild-type enzyme
S221C
-
site-directed mutagenesis, mutation of the critical catalytic residue Ser221 of SES7 to a cysteine to avoid protease self-digestion
S62I
site-directed mutagenesis, the mutant shows increased activity without detergents compared to the wild-type, and slightly improved properties in chaotropic conditions
S62I/A153V/G166S/I205V
S62I/A153V/G166S/I205V/N218S/T224A
site-directed mutagenesis, the mutant shows high activity and stability in the presence of GdmCl or SDS compared to the wild-type enzyme
S62I/A153V/G166S/T224A/T240S
S62I/G166M
site-directed mutagenesis, the mutant shows increased activity without detergents compared to the wild-type, and highly improved properties in chaotropic conditions
T224A
site-directed mutagenesis, the mutant shows increased activity and stability in the presence of GdmCl or SDS compared to the wild-type enzyme
additional information
S62I/A153V/G166S/I205V
site-directed mutagenesis, the mutant shows increased activity and stability in the presence of GdmCl or SDS compared to the wild-type enzyme
S62I/A153V/G166S/I205V
site-directed mutagenesis, the mutant shows increased activity without detergents compared to the wild-type, and highly improved properties in chaotropic conditions
S62I/A153V/G166S/T224A/T240S
site-directed mutagenesis, the mutant shows high activity and stability in the presence of GdmCl or SDS compared to the wild-type enzyme
S62I/A153V/G166S/T224A/T240S
site-directed mutagenesis, the mutant shows increased activity and stability in the presence of GdmCl or SDS compared to the wild-type enzyme
additional information
-
mutant lacking three secreted serine proteases regulated by sigma-H, subtilisin, Epr and Vpr, produce less CSF and has less proCSF-processing activity
additional information
directed evolution of subtilisin E into a highly active and guanidinium chloride- and sodium dodecylsulfate-tolerant protease, subtilisin E is engineered into a chaotolerent protease, the enzyme gains a protease stability improved in a chaotropic salt (GdmCl) or a detergent (SDS), nultiple screening, overview
additional information
CRISPR-Cas9 in situ engineering of subtilisin E in Bacillus subtilis by usage of a flexible, co-transformation approach where the single guide RNA is inserted in a plasmid for Cas9 co-expression, and the donor DNA is supplied as a linear PCR product observing an editing efficiency of 76%. The method allows multiple, rapid rounds of in situ editing of the subtilisin E gene to incorporate a salt bridge triad present in the Bacillus clausii thermotolerant homologue, M-protease. Method, overview. The enzyme mutant obtained shows increased thermotolerance and activity
additional information
-
CRISPR-Cas9 in situ engineering of subtilisin E in Bacillus subtilis by usage of a flexible, co-transformation approach where the single guide RNA is inserted in a plasmid for Cas9 co-expression, and the donor DNA is supplied as a linear PCR product observing an editing efficiency of 76%. The method allows multiple, rapid rounds of in situ editing of the subtilisin E gene to incorporate a salt bridge triad present in the Bacillus clausii thermotolerant homologue, M-protease. Method, overview. The enzyme mutant obtained shows increased thermotolerance and activity
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
50
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
stability of wild-type an dmutant enzymes in the presence of GdmCl or SDS, overview
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
2-methyl-2-propanol
-
effect on synthesis of benzyloxycarbonyl-L-Asp-L-Ser-NH2, yield of dipeptide is 56.9% at 85% 2-methyl-2-propanol
acetonitrile
-
effect on synthesis of benzyloxycarbonyl-L-Asp-L-Ser-NH2, yield of dipeptide is 75.5% at 85% acetonitrile
chloroform
-
effect on synthesis of benzyloxycarbonyl-L-Asp-L-Ser-NH2, yield of dipeptide is 51.3% at 85% chloroform
dimethylformamide
-
effect on synthesis of benzyloxycarbonyl-L-Asp-L-Ser-NH2, yield of dipeptide is 33.55% at 85% dimethylformamide
Ethanol
-
effect on synthesis of benzyloxycarbonyl-L-Asp-L-Ser-NH2, yield of dipeptide is 49.4% at 85% ethanol
Ethyl acetate
-
effect on synthesis of benzyloxycarbonyl-L-Asp-L-Ser-NH2, yield of dipeptide is 43.7% at 85% ethyl acetate
Methanol
Methanol
-
effect on synthesis of benzyloxycarbonyl-L-Asp-L-Ser-NH2, yield of dipeptide is 16.3% at 85% methanol
Methanol
-
effect on synthesis of benzyloxycarbonyl-L-Asp-L-Ser-NH2, yield of dipeptide is 33.55% at 85% methanol
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
prosubtilisin purified by ammonium sulfate precipitation and gel filtration
purification of pro-subtilisin E wild-type and mutant (Asp32 to Asn) and intramolecular autoprocessing to active subtilisin E in vitro
-
purified to 23.5fold by a combination of cation and anion exchange chromatography, ethanol precipitation followed by reverse-phase HPLC
-
recombinant extracellular enzyme 7.8fold by ammonium sulfate fractionation, N-carbobenzoxy-D-phenylalanyl-triethylenetetramine-Sepharose (z-D-Phe-TETA) affinity chromatography, hydrophobic interaction chromatography, and anion exchange chrmatography, all in presence of 10 mM Ca2+
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration
-
recombinant wild-type and mutant enzymes from Bacillus subtilis strain WB600 culture supernatant by anion and cation exchange chromatography and ultrafilatration
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
subtilisin gene from Bacillus subtilis PTCC 1023 is cloned into the vector pET-15b and expressed in Escherichia coli strain BL21
gene aprE, construction of error-prone PCR libraries, recombinant expression of wild-type and mutant enzymes in Bacillus subtilis strain WB600 and secretion to the culture medium
gene aprE, genetic structure, recombinant expression of wild-type and mutant enzymes in Bacillus subtilis strain 168
gene aprE, recombinant expression of wild-type and mutant enzymes in Bacillus subtilis strain WB600 and secretion to the culture medium
gene vpr, recombinant expression of extracellular enzyme in Escherichia coli strain XL10-Gold from pET 11a vector, high-level expression of the cold adapted subtilase, VPR, utilizing the rhamnose titratable T7 system of Lemo21 in presence of Ca2+ ions (best at about 100 mM) and rhamnose (best at about 0.076 mM) resulting in a dramatic increase of soluble protein compared to the other systems. Method evaluation, overview. In the absence of rhamnose, toxic effects of the proteinase are clearly observed, as cultures grow slower and end up at lower cell density. Addition of Ca2+ increases the yield of soluble protein. The calcium present in the growth medium is not only aiding in correct folding and stabilization of VPR but is also increasing the resistance of Lemo21 against the toxic effects caused by enzyme VPR
prosubtilisin JB1 cloned into the GST fusion protein expression vector pGEX-4T-1 and transformed into Escherichia coli DH5alphaMCR
recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
-
recombinant expression of the codon-optimized gene encoding enzyme subtilisin QK in Pichia pastoris strain GS115, the thrombolytic activity of QK reaches 112000 IU (urokinase unit) per ml and the specific activity is 14,679 IU/mg under optimal culture and fermentation conditions, method optimization, detailed overview
replacement of the Bacillus subtilis subtilisin structural gene with an in vitro-derived deletion mutant
-
RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
denatured subtilisin E cannot be refolded to form active subtilisin E under the conditions used for processing (of pro-subtilisin to yield active subtilisin)
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
analysis
-
due to high pI-value of enzyme, it does not migrate in the electrophoretic field in the Laemmli buffer system. Fast and simple identification of enzyme by over-running electrophoretic technique with a miniscale culture supernatant
degradation
-
subtilisin releases Phr signalling peptides derived from the C-terminus of their precursor proteins, but does not release Phr peptides derived from an internal portion of its precursor proteins
industry
subtilisin JB1 may serve as a potential source material for use in industrial applications of proteolytic enzymes and microorganisms for fishery waste degradation and fish by-product processing
synthesis
-
synthesis of benzyloxycarbonyl-L-Asp-L-Ser-NH2 with benzyloxycarbonyl-L-Asp methyl ester as acyl donor and serine amide as the nucleophile. Optimum conditions are pH 10.0, 35°C, in acetonitrile/Na2CO3-NaHCO3 buffer system, 85:15, with a dipeptide yield of 75.5%
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Ottesen, M.; Svendsen, I.
The subtilisins
Methods Enzymol.
19
199-215
1970
Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus sp. (in: Bacteria)
-
Nedkov, P.; Oberthur, W.; Braunitzer, G.
Determination of the complete amino-acid sequence of subtilisin DY and its comparison with the primary structures of the subtilisins BPN, Carlsberg and amylosacchariticus
Biol. Chem. Hoppe-Seyler
366
421-430
1985
Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus sp. (in: Bacteria), Bacillus subtilis DY
Kamal, M.; Hg, J.O.; Kaiser, R.; Shafqat, J.; Razzaki, T.; Zaidi, Z.H.; Jrnvall, H.
Isolation, characterization and structure of subtilisin from a thermostable Bacillus subtilis isolate
FEBS Lett.
374
363-366
1995
Bacillus subtilis, Bacillus subtilis RT-5
Stahl, M.L.; Ferrari, E.
Replacement of the Bacillus subtilis subtilisin structural gene with an In vitro-derived deletion mutation
J. Bacteriol.
158
411-418
1984
Bacillus subtilis, Bacillus sp. (in: Bacteria), Bacillus subtilis 1168, Bacillus sp. (in: Bacteria) 1168
Eder, J.; Rheinnecker, M.; Fersht, A.R.
Folding of subtilisin BPN: characterization of a folding intermediate
Biochemistry
32
18-26
1993
Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus sp. (in: Bacteria), Bacillus subtilis DB104
Betzel, C.; Visanji, M.; Eschenburg, S.; Wilson, K.S.; Peters, K.; Fittkau, S.; Singh, T.P.; Genov, N.
Crystallization and preliminary X-ray analysis of subtilisin DY, a natural mutant of subtilisin Carlsberg
Arch. Biochem. Biophys.
302
499-502
1993
Bacillus subtilis, Bacillus sp. (in: Bacteria), Bacillus subtilis DY
Economou, C.; Chen, K.; Arnold, F.H.
Random mutagenesis to enhance the activity of subtilisin in organic solvents: characterization of Q103R subtilisin E
Biotechnol. Bioeng.
39
658-662
1992
Bacillus subtilis, Bacillus sp. (in: Bacteria), Bacillus subtilis DB428
Ohta, Y.; Inouye, M.
Pro-subtilisin E: purification and characterization of its autoprocessing to active subtilisin E in vitro
Mol. Microbiol.
4
295-304
1990
Bacillus subtilis, Bacillus sp. (in: Bacteria)
Gololobov,M.Y.; Morozova, I.P.; Vojushina, T.L.; Timokhina, E.A.; Stepanov, V.M.
Subtilisin from Bacillus subtilis strain 72. The influence of substrate structure, temperature and pH on catalytic properties
Biochim. Biophys. Acta
1118
267-276
1992
Bacillus subtilis, Bacillus subtilis 72
Chu, N.M.; Chao, Y.; Bi, R.C.
The 2 A crystal structure of subtilisin E with PMSF inhibitor
Protein Eng.
8
211-215
1995
Bacillus subtilis, Bacillus sp. (in: Bacteria)
Genov, N.; Shopova, M.; Boteva, R.; Jori, G.; Ricchelli, F.
Chemical, photochemical and spectroscopic characterization of an alkaline proteinase from Bacillus subtilis variant DY
Biochem. J.
207
193-200
1982
Bacillus subtilis, Bacillus sp. (in: Bacteria), Bacillus subtilis DY
Ricchelli, F.; Jori, G.; Filippi, B.; Boteva, R.; Shopova, M.; Genov, N.
Effects of pH and urea on the conformational properties of subtilisin DY
Biochem. J.
207
201-205
1982
Bacillus subtilis, Bacillus sp. (in: Bacteria), Bacillus subtilis DY
Vartak, H.G.; Bodhe, A.M.; Paranjpe, S.V.; Jagannathan, V.
Proteinase inhibitors from Vigna unguiculata subsp. cylindrica. II. Inhibitors of subtilisin and trypsin
Arch. Biochem. Biophys.
204
129-133
1980
Bacillus subtilis
Vartak, H.G.; Rele, M.V.; Jagannathan, V.
Proteinase inhibitors from Vigna unguiculata subsp. cylindrica. III. Properties and kinetics of inhibitors of papain, subtilisin, and trypsin
Arch. Biochem. Biophys.
204
134-140
1980
Bacillus subtilis
Dahlen, J.R; Foster, D.C; Kisiel, W.
Human proteinase inhibitor 9 (PI9) is a potent inhibitor of subtilisin A
Biochem. Biophys. Res. Commun.
238
329-333
1997
Bacillus subtilis
Jan, J.; Valle, F.; Bolivar, F.; Merino, E.
Characterization of the 5' subtilisin (aprE) regulatory region from Bacillus subtilis
FEMS Microbiol. Lett.
183
9-14
2000
Bacillus subtilis
Subbian, E.; Yabuta, Y.; Shinde, U.
Positive selection dictates the choice between kinetic and thermodynamic protein folding and stability in subtilases
Biochemistry
43
14348-14360
2004
Bacillus subtilis
Glowacka, A.E.; Podstawka, E.; Szczesna-Antczak, M.H.; Kalinowska, H.; Antczak, T.
Kinetic and molecular properties of Bacillus subtilis IBTC-3 subtilisin
Comp. Biochem. Physiol. B
140
321-331
2005
Bacillus subtilis, Bacillus subtilis IBTC-3
Choi, N.; Ju, S.; Lee, T.Y.; Yoon, K.; Chang, K.; Maeng, P.J.; Kim, S.
Miniscale identification and characterization of subtilisins from Bacillus sp. strains
J. Microbiol. Biotechnol.
15
537-543
2005
Bacillus subtilis, Bacillus sp. (in: Bacteria), Bacillus sp. (in: Bacteria) DJ-1
-
Hou, R.Z.; Yang, Y.; Huang, Y.B.; Wang, H.; Zhang, N.; Liu, Y.J.; Zhang, X.Z.; Li, G.
Alcalase-catalyzed, kinetically controlled synthesis of a precursor dipeptide of RGDS in organic solvents
Prep. Biochem. Biotechnol.
36
93-105
2006
Bacillus subtilis
Uchida, H.; Kondo, D.; Yamashita, S.; Tanaka, T.; Tran, L.H.; Nagano, H.; Uwajima, T.
Purification and properties of a protease produced by Bacillus subtilis CN2 isolated from a Vietnamese fish sauce
World J. Microbiol. Biotechnol.
20
579-582
2004
Bacillus subtilis, Bacillus subtilis CN2
-
Almog, O.; Kogan, A.; de Leeuw, M.; Gdalevsky, G.Y.; Cohen-Luria, R.; Parola, A.H.
Structural insights into cold inactivation of tryptophanase and cold adaptation degrees of subtilisin S41: Minireview
Biopolymers
89
354-359
2008
Bacillus subtilis, Bacillus licheniformis, Lysinibacillus sphaericus, Bacillus amyloliquefaciens (P00782), Vibrio sp. (Q8GB52), Bacillus subtilis TA41
Lanigan-Gerdes, S.; Dooley, A.N.; Faull, K.F.; Lazazzera, B.A.
Identification of subtilisin, Epr and Vpr as enzymes that produce CSF, an extracellular signalling peptide of Bacillus subtilis
Mol. Microbiol.
65
1321-1333
2007
Bacillus subtilis, Bacillus licheniformis
Anikina, O.M.; Semashko, T.A.; Oksenoit, E.S.; Lysogorskaya, E.N.; Filippova, I.Y.
Native subtilisin Karlsberg and modified subtilisin 72 as effective catalysts of peptide bond formation in organic media
Russ. J. Bioorg. Chem.
32
116-121
2006
Bacillus subtilis, Bacillus licheniformis, Bacillus subtilis 72
-
Sung, J.H.; Ahn, S.J.; Kim, N.Y.; Jeong, S.K.; Kim, J.K.; Chung, J.K.; Lee, H.H.
Purification, molecular cloning, and biochemical characterization of subtilisin JB1 from a newly isolated Bacillus subtilis JB1
Appl. Biochem. Biotechnol.
162
900-911
2010
Bacillus subtilis (B6VFQ8), Bacillus subtilis JB1 (B6VFQ8), Bacillus subtilis JB1
Yan, F.; Yan, J.; Sun, W.; Yao, L.; Wang, J.; Qi, Y.; Xu, H.
Thrombolytic effect of Subtilisin QK on carrageenan induced thrombosis model in mice
J. Thromb. Thrombolysis
28
444-448
2009
Bacillus subtilis, Bacillus subtilis QK02
Rai, S.; Mukherjee, A.
Statistical optimization of production, purification and industrial application of a laundry detergent and organic solvent-stable subtilisin-like serine protease (Alzwiprase) from Bacillus subtilis DM-04
Biochem. Eng. J.
48
173-180
2010
Bacillus subtilis
-
Ghasemi, Y.; Dabbagh, F.; Ghasemian, A.
Cloning of a fibrinolytic enzyme (Subtilisin) gene from Bacillus subtilis in Escherichia coli
Mol. Biotechnol.
52
1-7
2012
Bacillus subtilis (Q84F18), Bacillus subtilis
Martinez, R.; Schwaneberg, U.; Roccatano, D.
Temperature effects on structure and dynamics of the psychrophilic protease subtilisin S41 and its thermostable mutants in solution
Protein Eng. Des. Sel.
24
533-544
2011
Bacillus subtilis (Q45681)
Li, Z.; Roccatano, D.; Lorenz, M.; Schwaneberg, U.
Directed evolution of subtilisin E into a highly active and guanidinium chloride- and sodium dodecylsulfate-tolerant protease
ChemBioChem
13
691-699
2012
Bacillus subtilis (P04189)
Li, Z.; Roccatano, D.; Lorenz, M.; Martinez, R.; Schwaneberg, U.
Insights on activity and stability of subtilisin E towards guanidinium chloride and sodium dodecylsulfate
J. Biotechnol.
169
87-94
2014
Bacillus subtilis (P04189)
Tang, H.; Zhang, J.; Shi, K.; Aihara, H.; Du, G.
Insight into subtilisin E-S7 cleavage pattern based on crystal structure and hydrolysates peptide analysis
Biochem. Biophys. Res. Commun.
512
623-628
2019
Bacillus subtilis, Bacillus subtilis JN-S7
Oskarsson, K.R.; Kristjansson, M.M.
Improved expression, purification and characterization of VPR, a cold active subtilisin-like serine proteinase and the effects of calcium on expression and stability
Biochim. Biophys. Acta
1867
152-162
2019
Bacillus subtilis (P29141), Bacillus subtilis 168 (P29141)
Price, M.A.; Cruz, R.; Baxter, S.; Escalettes, F.; Rosser, S.J.
CRISPR-Cas9 in situ engineering of subtilisin E in Bacillus subtilis
PLoS ONE
14
e0210121
2019
Bacillus subtilis (P04189), Bacillus subtilis, Bacillus subtilis 168 (P04189)
Zhou, K.; Dong, Y.; Zheng, H.; Chen, B.; Mao, R.; Zhou, L.; Wang, Y.
Expression, fermentation, purification and lyophilisation of recombinant subtilisin QK in Pichia pastoris
Process Biochem.
54
1-8
2017
Bacillus subtilis (Q7WVA6), Bacillus subtilis QK02 (Q7WVA6)
-
EXTERNAL LINKS (specific for EC-Number 3.4.21.62)
Use of this online version of BRENDA is free under the CC BY 4.0 license. See terms of use for full details.
Release 2023.1 (January 2023)
Legal
Curated at
Member of
