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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).
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Reaction Schemes
hydrolysis of pullulan to isopanose (6-alpha-maltosylglucose)
Synonyms
isopullulanase,
more
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pullulan 4-glucanohydrolase
IPU
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pullulan 4-glucanohydrolase
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pullulan 4-glucanohydrolase
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-
additional information
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classified under pullulanase type II
additional information
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classified under pullulanase type II
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hydrolysis of pullulan to isopanose (6-alpha-maltosylglucose)
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-
-
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hydrolysis of O-glycosyl bond
-
-
-
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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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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
?
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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
?
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cleaves the alpha-1,4-glycosidic linkage of the panose motif
-
-
?
alpha-glucosylmaltotriose + H2O
isomaltose + maltose
-
-
-
-
?
alpha-maltosylmaltose + H2O
isopanose + glucose
-
-
-
-
?
azidopullulan + H2O
?
-
-
-
-
?
panose + H2O
isomaltose + glucose
pullulan + H2O
isopanose + (Glc)4
pullulan + H2O
isopanose + (Glc)6
pullulan + H2O
isopanose + ?
additional information
?
-
panose + H2O
isomaltose + glucose
-
-
-
-
?
panose + H2O
isomaltose + glucose
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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
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cleaves the alpha-1,4-glycosidic linkage
-
-
?
pullulan + H2O
isopanose
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very strict substrate specificity, preferred over panose
-
-
?
pullulan + H2O
isopanose
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IPU degrades alpha-1,4-glycosidic linkage from the reducing end adjacent to alpha-1,6-glycosidic linkage in pullulan
-
-
?
pullulan + H2O
isopanose
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IPU degrades alpha-1,4-glycosidic linkage from the reducing end adjacent to alpha-1,6-glycosidic linkage in pullulan
-
-
?
pullulan + H2O
isopanose + (Glc)4
-
-
-
?
pullulan + H2O
isopanose + (Glc)4
-
-
-
?
pullulan + H2O
isopanose + (Glc)4
-
-
-
?
pullulan + H2O
isopanose + (Glc)4
-
-
small amount
?
pullulan + H2O
isopanose + (Glc)6
-
-
-
?
pullulan + H2O
isopanose + (Glc)6
-
-
-
?
pullulan + H2O
isopanose + (Glc)6
-
-
-
?
pullulan + H2O
isopanose + ?
-
-
-
-
?
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
-
-
?
pullulan + H2O
isopanose + ?
-
-
-
?
pullulan + H2O
isopanose + ?
-
-
-
?
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
?
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not: amylose, maltoheptaose, starch, amylopectin
-
-
?
additional information
?
-
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not: amylose, maltoheptaose, starch, amylopectin
-
-
?
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pullulan + H2O
isopanose + (Glc)4
pullulan + H2O
isopanose + (Glc)6
pullulan + H2O
isopanose + ?
pullulan + H2O
isopanose + (Glc)4
-
-
-
?
pullulan + H2O
isopanose + (Glc)4
-
-
-
?
pullulan + H2O
isopanose + (Glc)4
-
-
-
?
pullulan + H2O
isopanose + (Glc)4
-
-
small amount
?
pullulan + H2O
isopanose + (Glc)6
-
-
-
?
pullulan + H2O
isopanose + (Glc)6
-
-
-
?
pullulan + H2O
isopanose + (Glc)6
-
-
-
?
pullulan + H2O
isopanose + ?
-
-
-
?
pullulan + H2O
isopanose + ?
-
-
-
?
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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
Fe3+
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5 mM completeinactivation
N-bromosuccinimide
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5 mM complete inactivation
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0.88
pullulan
-
40°C, 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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39.2
panose
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40°C, recombinant IPU
160
panose
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pH 3.5, 40°C, wild-type enzyme
920
panose
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pH 3.5, 40°C, mutant enzyme W402F
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0.36
panose
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mutant enzyme W402F
180
panose
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wild-type enzyme
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25.2
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pH 3.5, 40°C, recombinant IPU expressed in Pichia pastoris
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3.5
assay at
3.5
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deglycosylated recombinant IPU, native IPU has a similar pH-optimum
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4.5 - 5.6
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active in the range
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35 - 40
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deglycosylated recombinant IPU, native IPU has a similar optimum temperature
40 - 45
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recombinant enzyme
40
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assay at
60
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4.2
calculated from amino acid sequence
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formerly Aspergillus niger ATCC 9642
UniProt
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formerly Aspergillus niger ATCC 9642
UniProt
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ATCC9642
Uniprot
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UniProt
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UniProt
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likely belonging to Bacillus naganoensis
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likely belonging to Bacillus naganoensis
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brenda
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T6, single enzyme with G2-dextranase EC 3.2.1.94 and isopullanase activity
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brenda
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ATCC 9642, IPU F1 and IPU F2
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ATCC9642
Uniprot
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strain ATCC 9642
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brenda
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evolution
the enzyme belongs to the glycoside hydrolase family 49 (GH49)
evolution
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the enzyme belongs to the glycoside hydrolase family 49 (GH49)
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IPUA_ASPNG
564
0
61449
Swiss-Prot
Secretory Pathway (Reliability: 2 )
A0A345BDZ2_9PEZI
610
0
66864
TrEMBL
Secretory Pathway (Reliability: 2 )
A0A1C9HIY7_9PEZI
380
0
41369
TrEMBL
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91000
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recombinant enzyme, SDS-PAGE
95000
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2 * 95000, SDS-PAGE
59000
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59000
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F1 and F2 deglycosylated
59000
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x * 59000, deglycosylated recombinant IPU, SDS-PAGE
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?
-
x * 59000, deglycosylated recombinant IPU, SDS-PAGE
?
x * 66864, calculated from amino acid sequence
?
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x * 66864, calculated from amino acid sequence
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dimer
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2 * 95000, SDS-PAGE
dimer
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2 * 95000, SDS-PAGE
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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
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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
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glycoprotein
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N-glycosylated
glycoprotein
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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
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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
glycoprotein
the enzyme has 7 glycosylation sites
glycoprotein
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the enzyme has 7 glycosylation sites
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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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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
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2 - 7
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at 4°C 12 h
136638
2.4 - 7
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recombinant IPU expressed in Pichia pastoris, at 4°C, retains pullulan-hydrolyzing activity for 12 hours
655036
5
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retains activity below pH 5
655376
3 - 7
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recombinant enzyme, at 4°C 12 h
136639
3 - 7
-
deglycosylated recombinant IPU, stable
655805
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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
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
-
pH 5, half-life: 20 min
additional information
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calcium does not increase the thermostability
50
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below completely stable
50
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30 min at pH 3.7-4.5
55
50% activity remaining for the wild-type enzyme
55
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F2, loss of activity
60
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30 min, stable up to
60
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F1, loss of activity
60
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60 min, 16% loss of activity
70
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10 min, 80% loss of activity
70
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60 min, 30% loss of activity
80
-
inactivated
80
-
pH 5, half-life: 48 min
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photooxidation in presence of Rose Bengal
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136635
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4°C, no major loss of activity
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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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expressed in Escherichia coli DH5alpha cells
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
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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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