Information on EC 4.2.2.16 - levan fructotransferase (DFA-IV-forming)

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
4.2.2.16
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RECOMMENDED NAME
GeneOntology No.
levan fructotransferase (DFA-IV-forming)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
produces di-beta-D-fructofuranose 2,6':2',6-dianhydride (DFA IV) by successively eliminating the diminishing (2->6)-beta-D-fructan (levan) chain from the terminal D-fructosyl-D-fructosyl disaccharide
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SYSTEMATIC NAME
IUBMB Comments
(2->6)-beta-D-fructan lyase (di-beta-D-fructofuranose-2,6':2',6-dianhydride-forming)
This enzyme, like EC 4.2.2.17 [inulin fructotransferase (DFA-I-forming)] and EC 4.2.2.18 [inulin fructotransferase (DFA-III-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
88593-15-1
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
NCIMB 11871
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Manually annotated by BRENDA team
NCIMB 11871
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Manually annotated by BRENDA team
; strain J17-21
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Manually annotated by BRENDA team
strain J17-21
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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
ascorbic acid + levan
ascorbic acid 2-fructoside + ?
show the reaction diagram
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transfructosylation. When 35% of ascorbic acid and 2% of levan are incubated with LFTase of 0.5 unit/g levan at 37°C for 85 h, a maximum 52 g/l of ascorbic acid 2-fructoside is produced
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-
?
levan
5 di-D-fructose-2,6':2',6 dianhydride + H2O
show the reaction diagram
levan
di-D-fructofuranose-2,6':2',6 dianhydride
show the reaction diagram
levan
di-D-fructose-2,6':2',6 dianhydride + fructose + limited levan
show the reaction diagram
phlein
di-D-fructose-2,6':2',6 dianhydride
show the reaction diagram
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while it degrades Phleum pratense phlein more than 90%, formation of 6% reducing sugars as D-fructose
and two or three minor fructooligosaccharides
?
raffinose + D-galactopyranose
?
show the reaction diagram
stachyose + D-galactopyranose
?
show the reaction diagram
sucrose + D-galactopyranose
?
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
levan
5 di-D-fructose-2,6':2',6 dianhydride + H2O
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
MgCl2
-
10 mM, activation to 127% of control
NaCl
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1 mM, activation to 125% of control. 10 mM, activation to 142% of control
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
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10 mM, 81% inhibition
Ag2SO4
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1 mM, complete inhibition
AgNO3
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10 mM, 62% inhibition
CoCl2
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1 mM, 16% inhibition
CuSO4
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1 mM, 36% inhibition
EDTA
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10 mM, 17% inhibition
FeCl2
FeSO4
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1 mM, 99% inhibition
HgCl2
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1 mM, complete inhibition
HgSO4
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1 mM, 84% inhibition
KCl
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1 mM, 11% inhibition
KMnO4
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10 mM, 52% inhibition
LiCl
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1 mM, 8% inhibition
MgCl2
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1 mM, 37% inhibition
MnCl2
NaCl
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1 mM, 10% inhibition
NH4Cl
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1 mM, 54% inhibition
NiCl2
SDS
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10 mM, 68% inhibition
ZnCl2
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1 mM, 76% inhibition
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
Km for levan: 2 mg/ml at pH 7.0 and 40°C
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 5.5
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enzyme immobilized on Chitopearl BCW2501 beads
4
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enzyme immobilized to Diaion WA30
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 9
about 50% of maximal activity at pH 4.5 and pH 9.0
5 - 7.5
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pH 5.0: about 35% of maximal activity, pH 7.5: about 40% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
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assay at
45
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30 min, enzyme retains 87% of initial activity
60
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enzyme immobilized on Chitopearl BCW2501
additional information
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 50
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30°C: about 35% of maximal activity, 50°C: about 80% of maximal activity, 60°C: about 15% of maximal activity
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
46000
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gel filtration
48000
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gel filtration
51000
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2 * 51000, SDS-PAGE
52000
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1 * 52000, SDS-PAGE
54000
x * 54000, SDS-PAGE
96000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
in apo form, as well as in complexes with sucrose and levanbiose. Enzyme contains an active site that accommodates a difructosaccharide using the -1 and -2 subsites. Binding is facilitated by small side chain residues in the loop region of a catalytic beta-propeller N-domain. An additional oligosaccharide-binding site is in the beta-sandwich C-domain, supporting its role in carbohydrate recognition
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55
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60 min, 45% loss of activity of enzyme immobilized on Chitopearl BCW2501 beads, 95% loss of activity of native enzyme. Native enzyme loses about 80% of its initial activity after 30 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
enzyme immobilized on Chitopearl BCW2501 beads retains about 60% of its initial activity after being used for 20 cycles
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the enzyme is immobilized on Eupergit C250 L and Trisospor-amino without loss of enzymatic activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
after Ni-NTA chromatogrphy the enzyme solution is further purified on a Hi-Prep 16/60 Sephacryl S-200 high resolution column using fast-performance liquid chromatography
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recombinant enzyme
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Bacillus subtilis strain 168
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expressed in Escherichia coli as a His-tagged fusion protein
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expressed in Escherichia coli BL21(DE3) cells
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expressed with N-terminal fusion of a LacZ-derived secretion motif TMITNSSSVP using lac promoter system in recombinant Escherichia coli JM109[pUDF-A8]
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expression in Escherichia coli
expression in Escherichia coli. An Escherichia coli transformant carrying the plasmid pLFT-BB1 expresses six times as much activity of levan fructotransferase as that of the original strain Arthrobacter nicotinovorans GS-9
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levan fructotransferase gene is cloned from Arthrobacter nicotinovorans GS-9 and expressed in levan producing Bacillus subtilis 168
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overexpression in Escherichia coli
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subcloned into a high-expression vector, pET-29b, and the recombinant enzyme with a tag of six histidine is overexpressed in Escherichia coli
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A264G
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relative hydrolytic activity: 20.4% compared to levanbiohydrolase from Microbacterium laevaniformans. Relative hydrolytic activity of wild-type: 10.7%
N238K
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relative hydrolytic activity: 23.6% compared to levanbiohydrolase from Microbacterium laevaniformans. Relative hydrolytic activity of wild-type: 10.7%
N85S
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relative hydrolytic activity: 34.8% compared to levanbiohydrolase from Microbacterium laevaniformans. Relative hydrolytic activity of wild-type: 10.7%
N85S/A264G
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relative hydrolytic activity: 49.9% compared to levanbiohydrolase from Microbacterium laevaniformans. Relative hydrolytic activity of wild-type: 10.7%
N85S/D268E
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relative hydrolytic activity: 30% compared to levanbiohydrolase from Microbacterium laevaniformans. Relative hydrolytic activity of wild-type: 10.7%
N85S/N224K/Q229E/T253N
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relative hydrolytic activity: 37% compared to levanbiohydrolase from Microbacterium laevaniformans. Relative hydrolytic activity of wild-type: 10.7%
P313R/F518L
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relative hydrolytic activity: 15.8% compared to levanbiohydrolase from Microbacterium laevaniformans. Relative hydrolytic activity of wild-type: 10.7%
D54N
inactive mutant, used for the structural analysis of the complex with sucrose
additional information
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residues that are critical to the intramolecular fructosyl transfer reaction of LFTase are analysed. An error-prone PCR mutagenesis process is carried out, and the mutants that led to a shift in activity from transfructosylation towards hydrolysis of levan are screened by the dinitrosalicylic acid (DNS) method. Selected mutants reveal two major products: one is a di-D-fructose-2,6':6,2'-dianhydride (DFAIV) and the other is a levanbiose. The detected levanbiose corresponds to the reaction product from LFTase lacking transferring activity N85S substitution is responsible for the strong enhancement of the hydrolytic activity
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
medicine
synthesis