Information on EC 3.2.1.7 - inulinase

Word Map on EC 3.2.1.7
Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
Specify your search results
Select one or more organisms in this record:
Show additional data
Do not include text mining results
Include (text mining) results (more...)
Include results (AMENDA + additional results, but less precise; more...)


The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
3.2.1.7
-
RECOMMENDED NAME
GeneOntology No.
inulinase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Endohydrolysis of (2->1)-beta-D-fructosidic linkages in inulin
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of O-glycosyl bond
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
1-beta-D-fructan fructanohydrolase
-
CAS REGISTRY NUMBER
COMMENTARY hide
9025-67-6
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain 12
-
-
Manually annotated by BRENDA team
20 OSM
-
-
Manually annotated by BRENDA team
strain NK-126, 12, 817 and TISTR 3570
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain NRRL Y-7571
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain CCMB 300
-
-
Manually annotated by BRENDA team
strain CCMB 300
-
-
Manually annotated by BRENDA team
No.5
-
-
Manually annotated by BRENDA team
OUC2
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1-kestose + H2O
?
show the reaction diagram
-
-
-
-
?
1f-fructofuranosyl nystose + H2O
disaccharides + trisaccharides
show the reaction diagram
-
-
-
?
fructooligosaccharide + H2O
?
show the reaction diagram
-
(fructosylsucrose)4 to (fructosylsucrose)9
-
-
-
inulin + H2O
1-kestose + ?
show the reaction diagram
-
-
-
?
inulin + H2O
?
show the reaction diagram
inulin + H2O
beta-D-fructose + fructooligosaccharides
show the reaction diagram
-
-
-
-
?
inulin + H2O
D-fructose + fructo-oligosaccharides
show the reaction diagram
-
-
-
-
?
inulin + H2O
inulooligofructose
show the reaction diagram
inulin + H2O
inulooligosaccharide
show the reaction diagram
-
-
ranging from DP2 to DP7
-
?
inulin + H2O
inulooligosaccharides
show the reaction diagram
inulin + H2O
inulotetraose + inulotriose
show the reaction diagram
-
-
enzyme produces equal amounts of inulotetraose and inulotriose
-
?
inulin + H2O
inulotriose + inulotetraose + inulopentaose
show the reaction diagram
inulin + H2O
monosaccharide + disaccharide + oligosaccharide
show the reaction diagram
inulin + H2O
oligofructosides
show the reaction diagram
inulin + H2O
oligosaccharides
show the reaction diagram
nystose + H2O
?
show the reaction diagram
-
-
-
-
?
raffinose + H2O
?
show the reaction diagram
-
-
-
-
?
raffinose + H2O
beta-D-fructose + fructooligosaccharides
show the reaction diagram
sucrose + H2O
alpha-D-glucopyranose + beta-D-fructofuranose
show the reaction diagram
sucrose + H2O
beta-D-fructose + fructooligosaccharides
show the reaction diagram
-
-
-
-
?
sucrose + H2O
D-fructose + D-glucose
show the reaction diagram
-
-
-
-
?
sucrose + H2O
D-glucose + D-fructose
show the reaction diagram
-
-
-
-
?
sucrose + H2O
inulooligofructose
show the reaction diagram
additional information
?
-
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
inulin + H2O
inulooligosaccharides
show the reaction diagram
inulin + H2O
inulotriose + inulotetraose + inulopentaose
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cu2+
-
1 mM, 30% activation
Fe2+
-
1 mM, 35% activation
K+
-
1 mM, 35% activation
Li+
-
1 mM, 35% activation
Mg2+
-
2 mM significantly increases inulinase activity to nearly 114%
Na+
-
1 mM, 35% activation
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
carbodiimide
-
-
Cd2+
-
1 mM, strong inhibition
Co2+
-
1 mM, strong inhibition
inulin
L-Cys
-
1 mM, 6% inactivation
N-bromosuccinimide
Ni2+
-
1 mM, strong inhibition
p-chloromercuribenzoate
-
-
pyridoxal 5'-phosphate
treatment leads to immediate deactivation with reactivation in samples treated at concentrations below 0.25 mM. Treatment results in an increase in alpha-helix content from 13.6% to 17.6% after modification and an increase in melting temperature by 8 degrees
Sodium dodecyl sulfate
-
beyond 0.002% it is inhibitory
sodium dodecylsulfate
-
40 mM, no resiudal activity
Triton X-100
-
-
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
inulin
-
with raw inulin in bioreactor, progressive increase in inulinase production with increase in inulin concentration up to 4.0% with a maximum inulinase activity of 50.2 IU/ml
K+
-
5 mM, 113% of initial activity
Sodium dodecyl sulfate
-
0.001% is most effective for the inulinase activity (36.9 IU/ml)
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.8
(fructosylsucrose)4
-
-
3.8
(fructosylsucrose)5
-
-
0.85
(fructosylsucrose)6
-
-
0.33
(fructosylsucrose)7
-
-
0.24
(fructosylsucrose)9
-
-
8.4
1-kestose
-
-
0.001 - 570
inulin
5.75
raffinose
-
-
0.048 - 184
Sucrose
additional information
inulin
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0367 - 711.7
inulin
0.5
raffinose
Bacillus smithii
-
-
0.035 - 0.1377
Sucrose
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0107
-
60% of nitrogen source for yeast is supplemented as urea
0.0692
-
40% of nitrogen source for yeast is supplemented as urea
0.2031
-
nitrogen source for yeast is ammonium sulfate
0.2416
-
37°C, 15 min, pH 4,5, 20% of nitrogen source for yeast is supplemented as urea
0.5
-
substrate raffinose, pH 5.0, 50°C
8
-
substrate sucrose, pH 5.0, 50°C
23
-
2.5 mM inulin, 50 mM sodium acetate buffer, pH 5.5, 60°C, 1 h
32
-
substrate inulin from chicory, pH 5.0, 50°C
35.22
-
crude extract
1105
-
31.4fold purified enzyme
1407
-
pH 6.0, at 45ºC
1520
-
pH 6.0, 55°C
additional information
-
high specific activity
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
-
immobilized enzyme
4.5 - 5
-
immobilized enzyme
4.7
-
for sucrose
5.5 - 6
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 7
-
pH 4.0: about 75% of maximal activity, pH 7.0: about 60% of maximal activity
4.3 - 6.5
-
pH 4.3: about 40% of maximal activity at pH 4.3 and at pH 6.5
5 - 10
-
pH 5.0: about 55% of maximal activity, pH 10.0: about 40% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
-
both isoforms Endo-I and Endo-II
50 - 60
-
immobilized enzyme
65
-
immobilized enzyme
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 50
-
30°C: about 45% of maximal activity, 50°C: optimum, no activity at 60°C
45 - 65
-
45°C: about 45% of maximal activity, 65°C: about 65% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3
-
isoelctric focusing
3.85
-
chromatofocusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
31000
-
x * 34000, isoform Endo-I, x * 31000, isoform Endo-II, SDS-PAGE
34000
-
x * 34000, isoform Endo-I, x * 31000, isoform Endo-II, SDS-PAGE
49500
-
1 * 49500, SDS-PAGE
53000
-
gel filtration
55000
-
x * 55000, SDS-PAGE
55500
sequence analysis
55800
-
sequence analysis
55900
sequence analysis
57000
-
SDS-PAGE
57600
-
x * 57600, SDS-PAGE
58800
sequence analysis
69000
-
1 * 69000, SDS-PAGE
77000
-
gel filtration on Superose column
87900
sequence analysis
104000
-
SDS-PAGE
139000
-
SDS-PAGE
200000
-
isoform I
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
no modification
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
to 1.5 A resolution. Structural arrangement shows a N-terminal 5fold beta-propeller catalytic domain with four beta-sheets and a C-terminal beta-sandwich domain organized in two beta-sheets with five beta-strands. Comparison with other GH32 enzymes reveals the presence of an extra pocket in the isoform INU2 catalytic site, formed by two loops and the conserved motif W-M(I)-N-D(E)-P-N-G. This cavity explains the endo-activity of the enzyme, the critical role of residue Trp40 and particularly the cleavage at the third unit of the inulin(-like) substrates
-
homology modeling, docking and molecular simulations using structure of Aspergillus awamori as a template. The thermodynamic equilibrium is reached after a few picoseconds, the enzyme toggles between two states during the entire equilibrium phase of the dynamics. The first conformation of the funnel domain corresponds to the closed/inactive state whereas the second conformation corresponds to the open/active conformation
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
-
4°C, 24 h, complete loss of activity
136823
4 - 8
-
both isoforms Endo-I and Endo-II
708551
4 - 7
-
stable
679965
4.4
-
highest stability, isoform obtained by submerged fermentation using agroindustrial residues as substrate
707203
4.5 - 8.5
-
30°C, 24 h, stable
136816
4.8
-
highest stability, isoform obtained by submerged fermentation using agroindustrial residues as substrate
707203
5
-
4°C, 24 h, about 10% loss of activity
136823
5 - 7.5
-
4°C, 24 h, stable
136822
5 - 7
-
24 h, stable
136817
6 - 10.5
-
4°C, 24 h, stable
136823
6 - 7
-
-
710444
6 - 9
-
at 20ºC
657202
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
-
pH 7.5, 10 min, stable
64.1
melting temperature, native enzyme
72.2
melting temperature, pyridoxal 5'-phosphate-treated enzyme
additional information
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, immobilized endo-inulinase, stable for at least 1 year
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation
-
ammonium sulfate precipitation, gel filtration on Sephadex G-100
-
by ammonium sulfate precipitation, DEAE-Sephacel column and phenyl-Sepharose column, 28.7fold
-
by DEAE-Trisacryl Plus column and Superose column
-
by metal-affinity chromatography
-
endoinulinase I, II and III
-
from the extracellular extract, by gel filtration, 31.4fold purified
-
immobilized on Ca-alginate beads
-
immobilized onto Amberlite IRC 50
-
recombinant enzyme
-
recombinant protein
-
wild-type and mutant M-30
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expresion in Penicillium canescens
-
expressed on the cell surface of Saccharomyces cerevisiae EBY100 cells
-
expression in Escherichia coli
-
expression in Pichia pastoris
INU2 gene encoding endoinulinase expressed in SUC2-deleted Saccharomyces cerevisiae
-
inulinase gene without the signal sequence subcloned into pPICZaA expression vector and expressed in Pichia pastoris X-33
-
wild-type and mutant M-30 PCR products ligated into pGEM-T easy vector and transformed into the competent cells of Escherichia coli JM109
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
presence of raw inulin extract from Dahlia tubers serves as carbon source for growth and inducer for enzyme expression
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D460A
-
is totally inactive
D460E
-
active to some extent. pKa of the acid/base catalyst decreases from 9.2 for the wild-type enzyme to 7.0 for the mutant
D460N
-
active to some extent
E323A
-
enzyme efficiency (kcat/Km) is significantly lower than that of the wild-type due to a substantial decrease in kcat, but not due to variations in Km consistent with its putative role as nucleophile catalyst
E519A
-
enzyme efficiency (kcat/Km) is significantly lower than that of the wild-type due to a substantial decrease in kcat, but not due to variations in Km consistent with its putative role as acid/base catalyst
D298A
-
mutation within the enlarged cavity formed by the conserved motif W-M(I)-N-D(E)-P-N-G, the so-called loop 1 and the loop 4. 59.7% of wild-typ activity
E43D
-
mutation within the enlarged cavity formed by the conserved motif W-M(I)-N-D(E)-P-N-G, the so-called loop 1 and the loop 4. 5.4% of wild-typ activity
F99A
-
mutation within the enlarged cavity formed by the conserved motif W-M(I)-N-D(E)-P-N-G, the so-called loop 1 and the loop 4. 6.1% of wild-typ activity
I70A
-
mutation within the enlarged cavity formed by the conserved motif W-M(I)-N-D(E)-P-N-G, the so-called loop 1 and the loop 4. 38.9% of wild-typ activity
M41A
-
mutation within the enlarged cavity formed by the conserved motif W-M(I)-N-D(E)-P-N-G, the so-called loop 1 and the loop 4. 56.2% of wild-typ activity
N265A
-
mutation within the enlarged cavity formed by the conserved motif W-M(I)-N-D(E)-P-N-G, the so-called loop 1 and the loop 4. 72.9% of wild-typ activity
N42G
-
mutation within the enlarged cavity formed by the conserved motif W-M(I)-N-D(E)-P-N-G, the so-called loop 1 and the loop 4. 7.1% of wild-typ activity
P62G
-
mutation within the enlarged cavity formed by the conserved motif W-M(I)-N-D(E)-P-N-G, the so-called loop 1 and the loop 4. 0.2% of wild-typ activity
Q59A
-
mutation within the enlarged cavity formed by the conserved motif W-M(I)-N-D(E)-P-N-G, the so-called loop 1 and the loop 4. 2.2% of wild-typ activity
R175A
-
mutation within the enlarged cavity formed by the conserved motif W-M(I)-N-D(E)-P-N-G, the so-called loop 1 and the loop 4. 0.1% of wild-typ activity
R295A
-
mutation within the enlarged cavity formed by the conserved motif W-M(I)-N-D(E)-P-N-G, the so-called loop 1 and the loop 4. 84.6% of wild-typ activity
W67A
-
mutation within the enlarged cavity formed by the conserved motif W-M(I)-N-D(E)-P-N-G, the so-called loop 1 and the loop 4. 2.4% of wild-typ activity
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
industry
nutrition
synthesis
additional information