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Information on EC 3.2.1.57 - isopullulanase and Organism(s) Aspergillus niger and UniProt Accession O00105

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EC Tree
IUBMB Comments
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).
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This record set is specific for:
Aspergillus niger
UNIPROT: O00105
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Word Map
The taxonomic range for the selected organisms is: Aspergillus niger
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Reaction Schemes
hydrolysis of pullulan to isopanose (6-alpha-maltosylglucose)
Synonyms
isopullulanase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pullulan 4-glucanohydrolase
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of O-glycosyl bond
-
-
-
-
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
-
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
pullulan + H2O
isopanose + ?
show the reaction diagram
Asp353, Asp372 and Asp373 are the catalytic residues of IPU
-
-
?
4(2)-alpha-isomaltosylisomaltose + H2O
?
show the reaction diagram
-
cleaves the alpha-1,4-glycosidic linkage of the panose motif
-
-
?
4(3)-alpha-panosylpanose + H2O
?
show the reaction diagram
-
cleaves the alpha-1,4-glycosidic linkage of the panose motif
-
-
?
6(2)-alpha-isomaltosylmaltose + H2O
?
show the reaction diagram
-
isomaltotriosylglucose, cleaves the alpha-1,4-glycosidic linkage of the panose motif
-
-
?
6(2)-alpha-maltosylmaltose + H2O
?
show the reaction diagram
-
cleaves the alpha-1,4-glycosidic linkage of the panose motif
-
-
?
6(3)-alpha-glucosylmaltotriose + H2O
?
show the reaction diagram
-
cleaves the alpha-1,4-glycosidic linkage of the panose motif
-
-
?
alpha-glucosylmaltotriose + H2O
isomaltose + maltose
show the reaction diagram
-
-
-
-
?
alpha-maltosylmaltose + H2O
isopanose + glucose
show the reaction diagram
-
-
-
-
?
azidopullulan + H2O
?
show the reaction diagram
-
-
-
-
?
panose + H2O
?
show the reaction diagram
-
-
-
-
?
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 + ?
show the reaction diagram
-
-
-
-
?
additional information
?
-
-
recombinant IPU hydrolyzes various substrates containing the structure of panose indicating a strict subsite recognition of the panose motif, cleaves the alpha-1,4-glycosidic linkage
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
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
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
N-bromosuccinimide
-
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
39.2 - 920
panose
0.88
pullulan
-
40°C, recombinant IPU
additional information
additional information
-
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
-
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
25.2
-
pH 3.5, 40°C, recombinant IPU expressed in Pichia pastoris
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35 - 40
-
deglycosylated recombinant IPU, native IPU has a similar optimum temperature
40 - 45
-
recombinant enzyme
45
-
F1 at pH 3.5
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
ATCC9642
Uniprot
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
IPUA_ASPNG
564
0
61449
Swiss-Prot
Secretory Pathway (Reliability: 2)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
59000
69000
-
F2, SDS-PAGE
71000
-
F1, SDS-PAGE
74000
-
gel filtration
91000
-
recombinant enzyme, SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
-
x * 59000, deglycosylated recombinant IPU, SDS-PAGE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D353N
-
enzyme shows no activity with pullulan and panose as substrate
D372N
-
enzyme shows no activity with pullulan and panose as substrate
D373N
-
enzyme shows no activity with pullulan and panose as substrate
E273N
-
45% of wild-type activity with pullulan as substrate, 74% of wild-type activity with panose as substrate
E356Q
-
38% of wild-type activity with pullulan as substrate, 50% of wild-type activity with panose as substrate
W240F
-
130% of wild-type activity with pullulan as substrate, 160% of wild-type activity with panose as substrate
W31F
-
38% of wild-type activity with pullulan as substrate, 140% of wild-type activity with panose as substrate
W32F
-
90% of wild-type activity with pullulan as substrate, 120% of wild-type activity with panose as substrate
W402F
-
0.4% of wild-type activity with pullulan as substrate, 0.1% of wild-type activity with panose as substrate
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2 - 7
-
at 4°C 12 h
136638
2 - 8
-
5°C, 24 h
136633
2.4 - 7
-
recombinant IPU expressed in Pichia pastoris, at 4°C, retains pullulan-hydrolyzing activity for 12 hours
655036
3 - 7
4 - 7
-
-
136636
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35
-
F2, below, 30 min at pH 3.5
40
-
deglycosylated recombinant IPU, stable up to 40°C, native IPU has a similar thermal stability
45
-
F1, below, 30 min at pH 3.5
50
-
30 min at pH 3.7-4.5
55
-
F2, loss of activity
60
-
F1, loss of activity
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant IPU expressed in Pichia pastoris
-
wild-type and mutant enzymes
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant IPU produced by Pichia pastoris
ipuA gene, expression in Aspergillus oryzae M-2-3
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ipuA gene, expression in Pichia pastoris GS115
-
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Sakano, Y.; Higuchi, M.; Kobayashi, T.
Pullulan 4-glucanohydrolase from Aspergillus niger
Arch. Biochem. Biophys.
153
180-187
1972
Aspergillus niger
Manually annotated by BRENDA team
Sakano.Y.; Masuda, N.; Kobayashi, T.
Hydrolysis of pullulan by a novel enzyme from Aspergillus niger
Agric. Biol. Chem.
35
971-973
1972
Aspergillus niger
-
Manually annotated by BRENDA team
Aoki, H.; Yopi; Padmajanti, A.; Sakano, Y.
Two components of cell-bound isopullulanase from Aspergillus niger ATCC 9642 - Their purification and enzymatic properties
Biosci. Biotechnol. Biochem.
60
1795-1798
1996
Aspergillus niger
Manually annotated by BRENDA team
Aoki, H.; Yopi; Sakano, Y.
Molecular cloning and heterologous expression of the isopullulanase gene from Aspergillus niger A.T.C.C. 9642
Biochem. J.
323
757-764
1997
Aspergillus niger
Manually annotated by BRENDA team
Ball, D.H.; Wiley, B.J.; Reese, E.T.
Effect of substitution at C-6 on the susceptibility of pullulan to pullulanases. Enzymatic degradation of modified pullulans
Can. J. Microbiol.
38
324-327
1992
Aspergillus niger
Manually annotated by BRENDA team
Akeboshi, H.; Kashiwagi, Y.; Aoki, H.; Tonozuka, T.; Nishikawa, A.; Sakano, Y.
Construction of an efficient expression system for Aspergillus isopullulanase in Pichia pastoris, and a simple purification method
Biosci. Biotechnol. Biochem.
67
1149-1153
2003
Aspergillus niger
Manually annotated by BRENDA team
Padmajanti, A.; Tonozuka, T.; Sakano, Y.
Deglycosylated isopullulanase retains enzymatic activity
J. Appl. Glycosci.
47
287-292
2000
Aspergillus niger
-
Manually annotated by BRENDA team
Akeboshi, H.; Tonozuka, T.
Insights into the reaction mechanism of glycosol hydrolase family 49. Site-directed mutagenesis and substrate preference of isopullulanase
Eur. J. Biochem.
271
4420-4427
2004
Aspergillus niger
Manually annotated by BRENDA team
Mizuno, M.; Koide, A.; Yamamura, A.; Akeboshi, H.; Yoshida, H.; Kamitori, S.; Sakano, Y.; Nishikawa, A.; Tonozuka, T.
Crystal structure of Aspergillus niger isopullulanase, a member of glycoside hydrolase family 49
J. Mol. Biol.
376
210-220
2008
Aspergillus niger (O00105), Aspergillus niger, Aspergillus niger ATCC 9642 (O00105)
Manually annotated by BRENDA team