Information on EC 4.2.2.18 - inulin fructotransferase (DFA-III-forming)

Word Map on EC 4.2.2.18
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
4.2.2.18
-
RECOMMENDED NAME
GeneOntology No.
inulin fructotransferase (DFA-III-forming)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Produces alpha-D-fructofuranose beta-D-fructofuranose 1,2':2,3'-dianhydride (DFA III) by successively eliminating the diminishing (2->1)-beta-D-fructan (inulin) chain from the terminal D-fructosyl-D-fructosyl disaccharide.
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexosyl group transfer
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
(2->1)-beta-D-fructan lyase (alpha-D-fructofuranose-beta-D-fructofuranose-1,2':2,3'-dianhydride-forming)
This enzyme, like EC 4.2.2.16 [levan fructotransferase (DFA-IV-forming)] and EC 4.2.2.17 [inulin fructotransferase (DFA-I-forming)] eliminates the fructan chain from the terminal disaccharide leaving a difructose dianhydride. These enzymes have long been known as fructotransferases, so this is retained in the accepted name. Since the transfer is intramolecular, the reaction is an elimination and, hence, the enzyme is a lyase, belonging in EC 4.
CAS REGISTRY NUMBER
COMMENTARY hide
50936-42-0
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain C11-1
-
-
Manually annotated by BRENDA team
strain H65-7, gene ift
-
-
Manually annotated by BRENDA team
strain MCI-2493
-
-
Manually annotated by BRENDA team
growth on 4% Cichorium intybus root extract
-
-
Manually annotated by BRENDA team
growth on 4% Cichorium intybus root extract
-
-
Manually annotated by BRENDA team
strain LC-413
-
-
Manually annotated by BRENDA team
strain LC-413
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain OKU17B
-
-
Manually annotated by BRENDA team
strain OKU17B
-
-
Manually annotated by BRENDA team
strain MCI-2524
-
-
Manually annotated by BRENDA team
strain MCI-2524
-
-
Manually annotated by BRENDA team
growth on 4% Cichorium intybus root extract
-
-
Manually annotated by BRENDA team
growth on 4% Cichorium intybus root extract
-
-
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
di-D-fructofuranose 1,2':2,3'-dianhydride + D-glucose
show the reaction diagram
2,1-beta-linked fructose oligosaccharides
?
show the reaction diagram
-
with the exception of inulobiose
-
-
?
beta-D-fructofuranosyl-(2->1)-beta-D-fructofuranosyl-(2->1)-beta-D-fructofuranosyl-(2->1)-alpha-D-glucopyranoside
di-D-fructofuranose 1,2':2,3'-dianhydride + sucrose
show the reaction diagram
inulin
3 di-D-fructofuranose 1,2':2,3' dianhydride + H2O
show the reaction diagram
inulin
?
show the reaction diagram
inulin
di-D-fructofuranose 1,2':2,1' dianhydride
show the reaction diagram
inulin
di-D-fructofuranose 1,2':2,3' dianhydride + ?
show the reaction diagram
nystose
di-D-fructofuranose 1,2':2,3' dianhydride + D-glucose
show the reaction diagram
additional information
?
-
-
2,6-beta-fructans are no substrates
-
-
-
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
3 di-D-fructofuranose 1,2':2,3' dianhydride + H2O
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
-
1 mM, 86% residual activity
Ba2+
-
slight inhibition
Mg2+
-
1 mM, 78% residual activity
N-bromosuccinimide
-
1 mM, 29% residual activity
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
-
no effect of EDTA and cysteine
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.52 - 10
inulin
additional information
additional information
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
16.4
-
lyophilized enzyme, pH 5.5, 60C
45.8
-
purified enzyme
81.5
-
purified enzyme
100.9
-
pH 6.0, 440C
254.9
-
pH 6.0, 440C
256
-
purified enzyme
294
-
purified enzyme
384
-
purified enzyme
604
-
purified enzyme
644
-
di-D-fructofuranose 1,2':2,3' dianhydride produced
740
purified recombinant enzyme
853
-
purified enzyme
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 9
-
-
4 - 7
-
pH 4.0: about 20% of maximal activity, pH 7.0: about 60% of maximal activity
4.5 - 7.5
-
more than 50% of maximum activity within
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 70
30
-
80% of maximal activity at 30C and 60C
35 - 65
-
more than 50% of maximum activity within
40
-
about 50% of activity maximum at 40C and 75C
40 - 75
-
40C: about 45% of maximal activity, 75C: about 25% of maximal activity
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27000
-
2 * 27000, SDS-PAGE
35000
-
gel filtration
36000
-
2 * 36000, SDS-PAGE
43700
x * 43700, calculated
44000
-
2 * 44000, SDS-PAGE
45000
-
SDS-PAGE
46000
-
gel filtration
46500
x * 46500, SDS-PAGE
58000
2 * 58000, SDS-PAGE
62000
-
x * 62000, SDS-PAGE
70000
-
gel filtration
72000
-
x * 72000, SDS-PAGE
73000
-
gel filtration
74000
-
gel filtration
78000
-
x * 78000, SDS-PAGE
88000
gel filtration
100000
-
gel filtration
145000
-
gel filtration
additional information
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
trimer
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystals are obtained at 20C by hanging drop vapour-diffusion method using 0.1 M Na HEPES pH 7.5 buffer containing 1.5 M lithium sulfate as a precipitant. Crystals of the recombinant wild-type enzyme diffract to better than 1.5 A at -173C. The crystals belong to space group R32, with unit-cell parameters a = b = 90.02 A, c = 229.82 A in the hexagonal axes. Selenomethionine-derivative crystals belong to a different space group, C2, with unit-cell parameters a = 159.32, cb = 91.92, c = 92.58 A, beta = 125.06. The C2 selenomethionine-derived crystal contains three molecules per asymmetric unit
-
hanging drop vapor diffusion method
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 10
4C, 24, stable
681352
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
65
-
stable up to
75
-
pH 5.0, 20 min, stable
85
-
1 h, pH 5.5, about 90% loss of activity
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-18C, lyophilized enzyme, 120 days, 3% loss of activity
-
20C, lyophilized enzyme, 120 days, 5% loss of activity
-
4C, 1 year, 96% residual activity
-
4C, lyophilized enzyme, 120 days, 4% loss of activity
-
refrigerator, pH 6.0, under toluene, stable for a few months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
enzyme from strain A-6 recombinant in Escherichia coli
from strain H65-7
-
from strain MCI-2493
-
large scale, flocculation from fermenter broth after disruption of recombinant Escherichia coli cells, expressing the enzyme intracellularly
-
strain A-6
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli as a functionally active, His-tagged protein
expression in Escherichia coli
functional expression of gene ift, strain H65-7, in Escherichia coli strain JM109, DNA sequence determination and analysis, most of the enzyme activity is intracellular
-
overexpression in Escherichia coli
-
overexpression in Escherichia coli XL-1 blue
-
strain A-6, functional expression in Escherichia coli DH5alpha/pDFE
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
30 and 40 g/l difructose anhydride III intensely inhibit cell growth and enzymic activity
highest production levels of inulinase at 50C, growth on yeast extract and peptone as nitrogen sources
IFTase expression can be significantly promoted by the supplement of inulin at 5-50 g/l and difructose ahhydride III at 5-20 g/l.
inulin at high initial concentrations gives no indication of catabolite repression
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
G221R
-
point-mutation introduced via error-prone PCR, increase in activity of 35%
D233N
-
inactive mutant enzyme
E244Q
-
inactive mutant enzyme
D233N
-
inactive mutant enzyme
-
E244Q
-
inactive mutant enzyme
-
additional information
-
genetic engineering of the enzyme for enlarged thermotolerance in large scale production of DFA III, recombinant expression in Escherichia coli, elimination of sequence coding for a signal-peptide: 18% secretion, 82% intracellular localisation of the enzyme in Escherichia coli; immobilization of enzyme on alginate beads lowered the activity
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
synthesis