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

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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
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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
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-
Manually annotated by BRENDA team
ssp. paracasei P 4134
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-
Manually annotated by BRENDA team
B235
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-
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
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-
?
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
?
show the reaction diagram
-
native enzyme shows 63% of the activity with levan
-
-
?
levanbiose + H2O
D-fructose
show the reaction diagram
i.e. O-beta-D-fructofuranosyl-(2-6)-beta-D-fructofuranoside. 63% of the activity with levan with the native enzyme, 57% of the activity with levan with the heterologous enzyme
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-
?
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
?
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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
6,6-kestotetraose + H2O
?
show the reaction diagram
DOPQE8
-
-
-
?
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
-
slight stimulation
Mn2+
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slight stimulation
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ag2+
-
1 mM, almost complete inhibition
BaCl2
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1 mM, 64% inhibition
Co2+
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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
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1 mM, complete inhibition
MnCl2
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1 mM, 19% inhibition
PCMB
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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
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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 4C, with up to 38% of its activity at 30C
10 - 40
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10C: 60% of maximal activity, 40C: about 50% of maximal activity
18 - 38
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18C: about 45% of maximal activity, 38C: about 20% of maximal activity
35 - 50
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35C: about 50% of maximal activity, 50C: about 80% of maximal activity
55 - 60
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55C: maximal activity, 60C: 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