Information on EC 3.2.1.57 - isopullulanase

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

EC NUMBER
COMMENTARY hide
3.2.1.57
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RECOMMENDED NAME
GeneOntology No.
isopullulanase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
hydrolysis of pullulan to isopanose (6-alpha-maltosylglucose)
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of O-glycosyl bond
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SYSTEMATIC NAME
IUBMB Comments
pullulan 4-glucanohydrolase (isopanose-forming)
The enzyme has practically no action on starch. Panose (4-alpha-isomaltosylglucose) is hydrolysed to isomaltose and glucose. cf. EC 3.2.1.41 (pullulanase) and EC 3.2.1.135 (neopullulanase).
CAS REGISTRY NUMBER
COMMENTARY hide
37288-43-0
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
formerly Aspergillus niger ATCC 9642
UniProt
Manually annotated by BRENDA team
formerly Aspergillus niger ATCC 9642
UniProt
Manually annotated by BRENDA team
ATCC9642
Uniprot
Manually annotated by BRENDA team
strain US149, likely belonging to Bacillus naganoensis
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Manually annotated by BRENDA team
strain US149, likely belonging to Bacillus naganoensis
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
4(2)-alpha-isomaltosylisomaltose + H2O
?
show the reaction diagram
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cleaves the alpha-1,4-glycosidic linkage of the panose motif
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-
?
4(3)-alpha-panosylpanose + H2O
?
show the reaction diagram
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cleaves the alpha-1,4-glycosidic linkage of the panose motif
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-
?
6(2)-alpha-isomaltosylmaltose + H2O
?
show the reaction diagram
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isomaltotriosylglucose, cleaves the alpha-1,4-glycosidic linkage of the panose motif
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-
?
6(2)-alpha-maltosylmaltose + H2O
?
show the reaction diagram
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cleaves the alpha-1,4-glycosidic linkage of the panose motif
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-
?
6(3)-alpha-glucosylmaltotriose + H2O
?
show the reaction diagram
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cleaves the alpha-1,4-glycosidic linkage of the panose motif
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-
?
alpha-glucosylmaltotriose + H2O
isomaltose + maltose
show the reaction diagram
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-
-
-
?
alpha-maltosylmaltose + H2O
isopanose + glucose
show the reaction diagram
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-
-
-
?
azidopullulan + H2O
?
show the reaction diagram
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-
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?
panose + H2O
?
show the reaction diagram
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-
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-
?
panose + H2O
isomaltose + glucose
show the reaction diagram
pullulan + H2O
isopanose
show the reaction diagram
pullulan + H2O
isopanose + (Glc)4
show the reaction diagram
pullulan + H2O
isopanose + (Glc)6
show the reaction diagram
pullulan + H2O
isopanose + ?
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
pullulan + H2O
isopanose + (Glc)4
show the reaction diagram
pullulan + H2O
isopanose + (Glc)6
show the reaction diagram
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ag+
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5 mM, partial inhibition
Hg2+
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5 mM complete inactivation
KMnO4
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5 mM complete inactivation
N-bromosuccinimide
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
39.2 - 920
panose
0.88
Pullulan
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40C, recombinant IPU
additional information
additional information
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kinetic parameters for deglycosylated recombinant IPU with a sugar content of 13.8%, 6.8% and 2.1%, respectively, deglycosylation decreases the kinetic parameters, except Km for pullulan
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.36 - 180
panose
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
9.6
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pH 5, 60C
25.2
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pH 3.5, 40C, recombinant IPU expressed in Pichia pastoris
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 5.6
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active in the range
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35 - 40
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deglycosylated recombinant IPU, native IPU has a similar optimum temperature
40 - 45
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recombinant enzyme
45
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F1 at pH 3.5
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
69000
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F2, SDS-PAGE
71000
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F1, SDS-PAGE
74000
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gel filtration
91000
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recombinant enzyme, SDS-PAGE
200000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 59000, deglycosylated recombinant IPU, SDS-PAGE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified deglycosylated recombinant enzyme mutant N448A complexed with substrate isopanose, hanging drop vapour diffusion method, 20C, mixing of 0.001 ml protein in 10 mM sodium acetate, pH 3.5, with 0.001 ml of reservoir solution containing 10-13% w/w PEG 8000, and 50 mM sodium acetate, pH 4.5-5.0, cryoprotection with 20% v/v glycerol in reservoir solution, X-ray diffraction structure determination and analysis at 2.2 A resolution, molecular replacement method, the crystal belongs to the P21 space group and contains four molecules in the asymmetric unit, substrate binding structure, overview
for crystallization, the enzyme is treated with endoglycosidase Hf, by hanging drop vapor-diffusion method, unliganded and isopanose-complexed forms of IPU, both solved at 1.7 A resolution. Unliganded IPU belongs to space group P212121, which contains two molecules and 1273 water molecules in an asymmetric unit. IPU is composed of domains N and C joined by a short linker, with electron density maps for 11 or 12 N-acetylglucosamine residues per molecule. Domain N consists of 13 beta-strands and forms a beta-sandwich. Domain C, where the active site is located, forms a right-handed beta-helix. Overall conformation of IPU in complex with isopanose is essentially identical with that of unliganded IPU
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2 - 7
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at 4C 12 h
136638
2 - 8
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5C, 24 h
136633
2.4 - 7
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recombinant IPU expressed in Pichia pastoris, at 4C, retains pullulan-hydrolyzing activity for 12 hours
655036
4 - 7
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136636
5
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retains activity below pH 5
655376
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35
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F2, below, 30 min at pH 3.5
40
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deglycosylated recombinant IPU, stable up to 40C, native IPU has a similar thermal stability
45
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F1, below, 30 min at pH 3.5
46.6
50% activity remaining for deglycosylated mutant N448A and for mutant Y440A
49.3
50% activity remaining for mutant N448A
52
50% activity remaining for deglycosylated mutant S450A
53
50% activity remaining for the deglycosylated wild-type enzyme
54
50% activity remaining for mutant S450A
90
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pH 5, half-life: 20 min
additional information
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calcium does not increase the thermostability
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
photooxidation in presence of Rose Bengal
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136635
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, no major loss of activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant IPU expressed in Pichia pastoris
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recombinnat wild-type and mutant enzymes from Pichia pastoris strain GS115 by hydrophobic interaction chromatography
wild-type and mutant enzymes
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression of wild-type and mutant enzymes in Pichia pastoris strain GS115
ipuA gene, expression in Aspergillus oryzae M-2-3
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ipuA gene, expression in Pichia pastoris GS115
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recombinant IPU produced by Pichia pastoris
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
N448A
site-directed mutagenesis, glycan-deficient variant, Asn448 is not N-glycosylated, the mutant shows reduced thermal stability compared to the wild-type enzyme
S450A
site-directed mutagenesis, glycan-deficient variant, by replacing Ser450 with Ala, Asn448 is not N-glycosylated, but the Asn448 side chain remains intact, unlike the Asn448Ala mutation, the mutant shows reduced thermal stability compared to the wild-type enzyme
Y440A
site-directed mutagenesis, the mutant shows reduced thermal stability compared to the wild-type enzyme
N448A
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site-directed mutagenesis, glycan-deficient variant, Asn448 is not N-glycosylated, the mutant shows reduced thermal stability compared to the wild-type enzyme
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S450A
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site-directed mutagenesis, glycan-deficient variant, by replacing Ser450 with Ala, Asn448 is not N-glycosylated, but the Asn448 side chain remains intact, unlike the Asn448Ala mutation, the mutant shows reduced thermal stability compared to the wild-type enzyme
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Y440A
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site-directed mutagenesis, the mutant shows reduced thermal stability compared to the wild-type enzyme
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D353N
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enzyme shows no activity with pullulan and panose as substrate
D372N
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enzyme shows no activity with pullulan and panose as substrate
D373N
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enzyme shows no activity with pullulan and panose as substrate
E273N
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45% of wild-type activity with pullulan as substrate, 74% of wild-type activity with panose as substrate
E356Q
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38% of wild-type activity with pullulan as substrate, 50% of wild-type activity with panose as substrate
W240F
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130% of wild-type activity with pullulan as substrate, 160% of wild-type activity with panose as substrate
W31F
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38% of wild-type activity with pullulan as substrate, 140% of wild-type activity with panose as substrate
W32F
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90% of wild-type activity with pullulan as substrate, 120% of wild-type activity with panose as substrate
W402F
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0.4% of wild-type activity with pullulan as substrate, 0.1% of wild-type activity with panose as substrate