Information on EC 3.2.1.154 - fructan beta-(2,6)-fructosidase

for references in articles please use BRENDA:EC3.2.1.154
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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
3.2.1.154
-
RECOMMENDED NAME
GeneOntology No.
fructan beta-(2,6)-fructosidase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Hydrolysis of terminal, non-reducing (2->6)-linked beta-D-fructofuranose residues in fructans
show the reaction diagram
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-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis
-
hydrolysis of O-glycosyl bond
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-
SYSTEMATIC NAME
IUBMB Comments
(2->6)-beta-D-fructan fructohydrolase
Possesses one of the activities of EC 3.2.1.80, fructan beta-fructosidase. While the best substrates are the levan-type fructans such as 6-kestotriose [beta-D-fructofuranosyl-(2->6)-beta-D-fructofuranosyl alpha-D-glucopyranoside] and 6,6-kestotetraose [beta-D-fructofuranosyl-(2->6)-beta-D-fructofuranosyl-(2->6)-beta-D-fructofuranosyl alpha-D-glucopyranoside], some (but not all) inulin-type fructans can also be hydrolysed, but more slowly [cf. EC 3.2.1.153, fructan beta-(2,1)-fructosidase]. Sucrose, while being a very poor substrate, can substantially inhibit enzyme activity in some cases.
CAS REGISTRY NUMBER
COMMENTARY hide
1000597-62-5
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37288-56-5
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
ATCC 19246
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-
Manually annotated by BRENDA team
cv. Fulghum
-
-
Manually annotated by BRENDA team
ssp. paracasei P 4134
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-
Manually annotated by BRENDA team
B235
-
-
Manually annotated by BRENDA team
enzyme contains three activities that hydrolyze beta-2,6-glycosidic linkages faster than beta-2,1-glycosidic linkages and two activities hydrolyze beta-2,1-glycosidic linkages faster than beta-2,6-glycosidic linkages
-
-
Manually annotated by BRENDA team
Japanese spurge
UniProt
Manually annotated by BRENDA team
cultivar Hokushu
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
DOPQE8;
the enzyme's the catalytic triad is conserved among glycoside hydrolase family 32, GH32, members
metabolism
DOPQE8;
6-FEHs are key enzymes associated with perennity of forage species
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
1,1,1-kestopentaose + H2O
?
show the reaction diagram
18% activity compared to levan
-
-
?
1,1,1-kestose + H2O
?
show the reaction diagram
-
45% of the activity with levan
-
-
?
1,1-kestose + H2O
?
show the reaction diagram
-
39% of the activity with levan
-
-
?
1,1-kestotetraose + H2O
?
show the reaction diagram
1,6G-kestotetraose + H2O
1-kestotriose + fructose
show the reaction diagram
-
-
-
-
?
1-kestose + H2O
?
show the reaction diagram
1-kestotriose + H2O
?
show the reaction diagram
6,6,6-kestopentaose + H2O
?
show the reaction diagram
-
-
-
-
?
6,6-kestotetraose + H2O
?
show the reaction diagram
6-kestose + H2O
?
show the reaction diagram
6-kestose + H2O
sucrose + D-fructose
show the reaction diagram
as active as levan with native enzyme, 85% of the activity with levan with the heterologous enzyme
-
-
?
6-kestotriose + H2O
?
show the reaction diagram
6G,1,6-kestopentaose + H2O
fructose + 6G-kestotriose + ?
show the reaction diagram
-
-
-
-
?
6G,6-kestotetraoase + H2O
fructose + 6G-kestotriose
show the reaction diagram
-
-
-
-
?
6G-kestotriose + H2O
?
show the reaction diagram
DOPQE8;
-
-
-
?
6G-kestotriose + H2O
sucrose + fructose
show the reaction diagram
-
-
-
-
?
bacterial levan + H2O
?
show the reaction diagram
-
-
-
?
fructan + H2O
?
show the reaction diagram
DOPQE8;
-
-
-
?
garlic fructan + H2O
?
show the reaction diagram
-
60% of the activity with levan
-
-
?
graminan + H2O
?
show the reaction diagram
-
-
-
-
?
inulin + H2O
?
show the reaction diagram
inulin + H2O
fructose
show the reaction diagram
levan + H2O
?
show the reaction diagram
levan + H2O
fructose
show the reaction diagram
levanbiose + H2O
beta-D-fructose
show the reaction diagram
levansucrase + H2O
?
show the reaction diagram
Bacillus subtilis
-
-
?
neokestin + H2O
?
show the reaction diagram
neokestose + H2O
?
show the reaction diagram
-
native enzyme shows 93% of the activity with levan
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-
?
phlein + H2O
?
show the reaction diagram
raffinose + H2O
?
show the reaction diagram
sucrose + H2O
D-fructose + D-glucose
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
6,6-kestotetraose + H2O
?
show the reaction diagram
DOPQE8
-
-
-
?
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
-
slight stimulation
Mn2+
-
slight stimulation
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ag2+
-
1 mM, almost complete inhibition
BaCl2
-
1 mM, 64% inhibition
Co2+
-
4 mM, 32% inhibition
Fe2+
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4 mM, 94% inhibition
Fe3+
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1 mM, 90% inhibition
FeCl3
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1 mM, 70% inhibition
HgCl2
-
1 mM, complete inhibition
MnCl2
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1 mM, 19% inhibition
PCMB
-
4 mM, complete inhibition
Sucrose
ZnCl2
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1 mM, 93% inhibition
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
77
6-kestose
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-
additional information
additional information
-
the KM-value for neokestin is 2.8% w/v. The Km-value for 6G,6-kestotetraose is 5.6% w/v
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
6-kestose
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
134
Sucrose
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-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
66.4
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enzyme from Streptococcus mutans
204.5
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recombinant enzyme expressed in Escherichia coli
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 5
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hydrolysis of neokestin
4.5
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sucrose degradation
5
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hydrolysis of sucrose
5.1 - 5.6
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-
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.5 - 6.5
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pH 3.5: about 70% of maximal activity, pH 6.5: about 55% of maximal activity, hydrolysis of neokestin
3.5 - 7
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pH 3.5: about 55% of maximal activity, pH 7.0: about 60% of maximal activity
4 - 6
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pH 4.0: about 85% of maximal activity, pH 6.0: about 70% of maximal activity, reaction with sucrose
4 - 7
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pH 4.0: about 40% of maximal activity, pH 7.0: about 50% of maximal activity, reaction with levan or inulin
4.3 - 6.8
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pH 4.3: about 65% of maximal activity, pH 6.8: about 40% of maximal activity
5 - 7.5
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pH 5.0: about 50% of maximal activity, pH 7.5: about 60% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55
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hydrolysis of levan
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 30
the activity of the enzyme remains relatively high even at 4°C, with up to 38% of its activity at 30°C
10 - 40
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10°C: 60% of maximal activity, 40°C: about 50% of maximal activity
18 - 38
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18°C: about 45% of maximal activity, 38°C: about 20% of maximal activity
35 - 50
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35°C: about 50% of maximal activity, 50°C: about 80% of maximal activity
55 - 60
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55°C: maximal activity, 60°C: 61% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5
putative mature protein, calculated from amino acid sequence
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
enzyme is induced after defoliation
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22000
1 * 53000 + 1 * 22000, SDS-PAGE
42000
-
x * 42000, SDS-PAGE
43000
-
x * 43000, SDS-PAGE
53000
1 * 53000 + 1 * 22000, SDS-PAGE
63400
x * 63400, putative mature protein, calculated from amino acid sequence
65000
-
gel filtration
69000
-
1 * 69000, SDS-PAGE
78000
x * 78000, recombinant enzyme, SDS-PAGE
89000
-
x * 89000, SDS-PAGE
125000
-
1 * 125000, SDS-PAGE
126000
-
gel filtration
127000
-
x * 127000, SDS-PAGE
140000
-
x * 140000, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterodimer
1 * 53000 + 1 * 22000, SDS-PAGE
monomer
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
temperature stability is increased in the presence of 6,6-kestotetraose
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation, concanavalin A column chromatography, Mono S (pH 4.5) column chromatography and Mono S (pH 4.0) column chromatography
partial, enzyme contains three activities that hydrolyze beta-2,6-glycosidic linkages faster than beta-2,1-glycosidic linkages and two activities hydrolyze beta-2,1-glycosidic linkages faster than beta-2,6-glycosidic linkages
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cDNA library screening, gene Lp6-FEHa, DNA and amino acid sequence determination and analysis, phylogenetic analysis
DOPQE8;
expressed in Pichia pastoris
expressed in Pichia pastoris strain X-33
expression in Pichia pastoris
fruA is expressed under its own control in Escherichia coli JM83(pFRU1)
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heterologous expression in Pichia pastoris
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heterologous expression in Pichia pastoris, pPICZalphaA vector
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
in the stubble of seedlings and in young haplocorms from adult timothy plants, transcripts of 6-FEH1 are significantly increased (about 7fold) within 3 h of defoliation, followed by an increase in fructan 6-exohydrolase activity
Wfh-sm3 transcripts increase in wheat leaf tissues inoculated with snow mold and incubated under snow cover
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
F233D
introduction of acidic amino acid in vicinity of acid-base catalyst creating a beta-fructofuranosidase type of enzyme with dual activity against sucrose and levan, generally lower activities against levan and 6-kestose but conserving preference for these substrates over sucrose