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Information on EC 3.2.1.57 - isopullulanase
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EC Tree
3.2.1.57 isopullulanaseIUBMB 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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Word Map
- 3.2.1.57
- pullulans
- aspergillus
- niger
- ipu
- glucoamylase
- oligosaccharides
-
subsite
- minioluteum
- dextranases
-
penicillium
-
deglycosylation
- neopullulanase
-
glucosidic
-
hydrolyse
- dextran
-
flask
- oryzae
- polysaccharide
- melanogenum
-
endoglycosidase
-
aureobasidium
-
peptide-n-glycosidase
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Synonyms
isopullulanase, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
IPU
pullulan 4-glucanohydrolase
additional information
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hydrolysis of pullulan to isopanose (6-alpha-maltosylglucose)
-
-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
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).
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CAS REGISTRY NUMBER
COMMENTARY
37288-43-0
-
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
4(2)-alpha-isomaltosylisomaltose + H2O
?
-
cleaves the alpha-1,4-glycosidic linkage of the panose motif
-
-
?
4(3)-alpha-panosylpanose + H2O
?
-
cleaves the alpha-1,4-glycosidic linkage of the panose motif
-
-
?
6(2)-alpha-isomaltosylmaltose + H2O
?
-
isomaltotriosylglucose, cleaves the alpha-1,4-glycosidic linkage of the panose motif
-
-
?
6(2)-alpha-maltosylmaltose + H2O
?
-
cleaves the alpha-1,4-glycosidic linkage of the panose motif
-
-
?
6(3)-alpha-glucosylmaltotriose + H2O
?
-
cleaves the alpha-1,4-glycosidic linkage of the panose motif
-
-
?
panose + H2O
isomaltose + glucose
-
cleaves the alpha-1,4-glycosidic linkage
-
-
?
pullulan + H2O
isopanose
the enzyme cleaves the alpha(1,4) glucosidic linkage using an inverting mechanism and liberating D-glucose
trisaccharide [Glc-alpha(1,4)-Glc-alpha(1,6)-Glc]
-
?
pullulan + H2O
isopanose
the enzyme cleaves the alpha(1,4) glucosidic linkage using an inverting mechanism and liberating D-glucose
trisaccharide [Glc-alpha(1,4)-Glc-alpha(1,6)-Glc]
-
?
pullulan + H2O
isopanose
-
very strict substrate specificity, preferred over panose
-
-
?
pullulan + H2O
isopanose
-
IPU degrades alpha-1,4-glycosidic linkage from the reducing end adjacent to alpha-1,6-glycosidic linkage in pullulan
-
-
?
pullulan + H2O
isopanose
-
IPU degrades alpha-1,4-glycosidic linkage from the reducing end adjacent to alpha-1,6-glycosidic linkage in pullulan
-
-
?
pullulan + H2O
isopanose + ?
Asp353, Asp372 and Asp373 are the catalytic residues of IPU
-
-
?
pullulan + H2O
isopanose + ?
Asp353, Asp372 and Asp373 are the catalytic residues of IPU
-
-
?
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
-
-
?
additional information
?
-
-
not: amylose, maltoheptaose, starch, amylopectin
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-
?
additional information
?
-
-
not: amylose, maltoheptaose, starch, amylopectin
-
-
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
3.5
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
35 - 40
-
deglycosylated recombinant IPU, native IPU has a similar optimum temperature
40
60
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
formerly Aspergillus niger ATCC 9642
UniProt
brenda
T6, single enzyme with G2-dextranase EC 3.2.1.94 and isopullanase activity
-
-
brenda
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
evolution
the enzyme belongs to the glycoside hydrolase family 49 (GH49)
evolution
-
the enzyme belongs to the glycoside hydrolase family 49 (GH49)
-
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UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). IPUA_ASPNG
564
0
61449
Swiss-Prot
Secretory Pathway (Reliability: 2)
A0A345BDZ2_9PEZI
610
0
66864
TrEMBL
Secretory Pathway (Reliability: 2)
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PDB
SCOP
CATH
UNIPROT
ORGANISM
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
59000
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
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POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
glycoprotein
glycoprotein
highly N-glycosylated enzyme with 15 potential N-glycosylation sites (Asn-X-Ser/Thr), the side chain of a glycosylated asparagine residue is important for the stability of the enzyme, enzyme deglycosylation results in a decrease in thermostability. Enzyme deglycosylated with peptide-N-glycosidase F shows virtually no enzymatic activity, while a single GlcNAc residue remains following digestion with Endo H, and the activity of the Endo H-treated wild-type enzyme is 65% compared to intact, non-deglycosylated, wild-type enzyme
glycoprotein
-
highly N-glycosylated enzyme with 15 potential N-glycosylation sites (Asn-X-Ser/Thr), the side chain of a glycosylated asparagine residue is important for the stability of the enzyme, enzyme deglycosylation results in a decrease in thermostability. Enzyme deglycosylated with peptide-N-glycosidase F shows virtually no enzymatic activity, while a single GlcNAc residue remains following digestion with Endo H, and the activity of the Endo H-treated wild-type enzyme is 65% compared to intact, non-deglycosylated, wild-type enzyme
-
glycoprotein
-
recombinant IPU expressed in Pichia pastoris GS115 has a higher carbohydrate content, 41%, than native IPU, 12-15%, or recombinant IPU expressed in Aspergillus oryzae M-2-3, 34%
glycoprotein
-
the sugar content of native IPU is 12.4-15.3%, that of recombinant IPU is 33.8%, deglycosylated enzyme by endoglycosidase H with a sugar content of 2.1% retains 65% of its original activity, kinetics of deglycosylated IPU, hybrid- and/or high-mannose types
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified deglycosylated recombinant enzyme mutant N448A complexed with substrate isopanose, hanging drop vapour diffusion method, 20°C, 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
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
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
-
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
-
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
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
2.4 - 7
-
recombinant IPU expressed in Pichia pastoris, at 4°C, retains pullulan-hydrolyzing activity for 12 hours
655036
3 - 7
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
40
-
deglycosylated recombinant IPU, stable up to 40°C, native IPU has a similar thermal stability
46.6
50% activity remaining for deglycosylated mutant N448A and for mutant Y440A
50
53
50% activity remaining for the deglycosylated wild-type enzyme
55
60
70
80
additional information
-
calcium does not increase the thermostability
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinnat wild-type and mutant enzymes from Pichia pastoris strain GS115 by hydrophobic interaction chromatography
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of wild-type and mutant enzymes in Pichia pastoris strain GS115
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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
brenda
Okada, G.; Takayanagi, T.; Miyahara, S.; Sawai, T.
An isomalto-dextranase accompanied by isopullulanase activity from Arthrobacter globiformis T6
Agric. Biol. Chem.
52
829-836
1988
Arthrobacter globiformis
-
brenda
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
-
brenda
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
brenda
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
brenda
Sakano.Y.; Masuda, N.; Kobayashi, T.
Isopullanase from Arthrobacter globiformis T6
Agric. Biol. Chem.
41
909-910
1977
Arthrobacter globiformis
-
brenda
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
brenda
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
brenda
Roy, A.; Ben Messaoud, E.; Bejar, S.
Isolation and purification of an acidic pullulanase type II from newly isolated Bacillus sp. US149
Enzyme Microb. Technol.
33
720-724
2003
Bacillus sp. (in: Bacteria), Bacillus sp. (in: Bacteria) US149
-
brenda
Padmajanti, A.; Tonozuka, T.; Sakano, Y.
Deglycosylated isopullulanase retains enzymatic activity
J. Appl. Glycosci.
47
287-292
2000
Aspergillus niger
-
brenda
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
brenda
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)
brenda
Miyazaki, T.; Yashiro, H.; Nishikawa, A.; Tonozuka, T.
The side chain of a glycosylated asparagine residue is important for the stability of isopullulanase
J. Biochem.
157
225-234
2015
Aspergillus brasiliensis (O00105), Aspergillus brasiliensis ATCC 9642 (O00105)
brenda
Liu, N.N.; Chi, Z.; Liu, G.L.; Chen, T.J.; Jiang, H.; Hu, Z.; Chi, Z.M.
alpha-Amylase, glucoamylase and isopullulanase determine molecular weight of pullulan produced by Aureobasidium melanogenum P16
Int. J. Biol. Macromol.
117
727-734
2018
Aureobasidium melanogenum (A0A1C9HIY7), Aureobasidium melanogenum, Aureobasidium melanogenum P16 (A0A1C9HIY7)
brenda
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