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Information on EC 3.2.1.68 - isoamylase
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3.2.1.68 isoamylaseIUBMB Comments
Also readily hydrolyses amylopectin. Differs from EC 3.2.1.41 (pullulanase) and EC 3.2.1.142 (limit dextrinase) by its inability to hydrolyse pullulan, and by limited action on alpha-limit dextrins. Maltose is the smallest sugar it can release from an alpha-(1->6)-linkage.
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Word Map
- 3.2.1.68
-
salivary
- lipase
- amylopectin
-
hyperamylasemia
- isoenzymes
- alpha-amylase
- pancreas
- phosphorylase
-
exocrine
- pullulanase
- amylose
- endosperm
- abdominal
- dextrin
- glucans
-
salivary-type
- phytoglycogen
- hepatomegaly
-
nonpancreatic
-
macroamylasemia
-
extrapancreatic
-
pancreatic-type
-
dextrinase
-
amylolytic
- beta-amylase
- amylo-1,6-glucosidase
- pseudocysts
-
s-type
- 4-alpha-glucanotransferase
- analysis
- alpha-glucans
-
phadebas
- nutrition
- maltotriose
-
alpha-1,4
-
glycogenolytic
- glycogenin
-
waxy
- isomaltose
-
granule-bound
-
glucanotransferase
- maltooligosaccharides
- detergent
- synthesis
- medicine
- food industry
- degradation
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Reaction Schemes
Synonyms
alkaline isoamylase, alpha 1,6-glucan hydrolase, alpha-1,6-glucosidase, ArDBEp, AtISA1, AtISA1/AtISA2 isoamylase, AtISA2, AtISA3, bacterial isoamylase, CrISA1, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
alkaline isoamylase
AtISA1
AtISA2
AtISA3
bacterial isoamylase
CrISA1
debranching enzyme
glycogen 6-glucanohydrolase
glycogen alpha-1,6-glucanohydrolase
glycogen debranching enzyme
glycogen-6-glucanogydrolase
glycogen-6-glucanohydrolase
IAM
ISA
ISA1
ISA2
ISA3
isoamylase
isoamylase 1
isoamylase 2
isoamylase-type debranching enzyme
isoamylase-type starch debranching enzyme 1
isoamylase-type starch debranching enzyme 2
isoamylase1
starch debranching enzyme
TreX
alkaline isoamylase
-
alkaliphilic Bacillus sp KSM-3309, isolated from soil
alkaline isoamylase
Bacillus sp. (in: Bacteria) KSM-K16
-
alkaliphilic Bacillus sp KSM-3309, isolated from soil
-
debranching enzyme
-
-
-
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hydrolysis of (1->6)-alpha-D-glucosidic branch linkages in glycogen, amylopectin and their beta-limit dextrins
-
-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hydrolysis of O-glycosyl bond
hydrolysis of O-glycosyl bond
-
-
-
-
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PATHWAY SOURCE
PATHWAYS
BRENDA
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SYSTEMATIC NAME
IUBMB Comments
glycogen 6-alpha-D-glucanohydrolase
Also readily hydrolyses amylopectin. Differs from EC 3.2.1.41 (pullulanase) and EC 3.2.1.142 (limit dextrinase) by its inability to hydrolyse pullulan, and by limited action on alpha-limit dextrins. Maltose is the smallest sugar it can release from an alpha-(1->6)-linkage.
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CAS REGISTRY NUMBER
COMMENTARY
9067-73-6
-
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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
alpha-D-glucan + H2O
?
-
cleavage of alpha-D-glucosidic linkages of certain branched alpha-D-glucans
-
-
?
amylopectin phi-dextrin + H2O
maltotetraose
-
substrate on which all 3 types of debranching enzymes act
-
?
amylose-extender amylopectin + H2O
maltose + maltooligosaccharides
O80403
-
-
-
?
beta limit dextrin + H2O
linear maltooligosaccharides
-
-
-
?
beta limit dextrin + H2O
maltose + maltooligosaccharides
O80403
from maize
-
-
?
branched chain oligosaccharides + H2O
maltotriose + higher maltosaccharides
-
-
no detectable maltose released, characteristic action is hydrolysis of alpha 1,6-glucosidic linkages, but alpha 1,6 glucosidic linkages attaching maltose residues to chains of alpha 1,4-linked glucose residues are not readily hydrolyzed
?
D-glucose + H2O
?
-
59% of the activity with dextrin as substrate
-
-
?
di-O-alpha-maltosyl-beta-cyclodextrin + H2O
maltose + beta-cyclodextrin
-
-
-
-
r
ginkgo starch + H2O
ginkgo amylose
-
74.74% yield at pH 5.0 and 52°C after 170 min
-
-
?
maize amylopectin + H2O
?
-
isoamylase hydrolyses both inner and outer branching linkages of amylopectin simultaneously
-
-
?
panose + H2O
D-glucose + maltose
-
acts very slowly, small amounts of product formed after 24 h
-
?
starch + H2O
amylose
-
the enzyme has the ability to attack side chains composed of 1-3 glucose residues
-
-
?
alpha-limit dextrin + H2O
linear maltooligosaccharides
-
-
-
?
alpha-limit dextrin + H2O
linear maltooligosaccharides
-
-
-
?
amylopectin + H2O
?
-
the enzyme can debranch almost all of component chains of amylopectin and shows 9.4% activity compared to phosphorylase a limit dextrin
-
-
?
amylopectin + H2O
?
-
the enzyme shows 0.8% activity compared to phosphorylase a limit dextrin
-
-
?
amylopectin + H2O
?
-
isoform ISA1 removes the wide range of chains with a degree of polymerization (DP) of 6-20 from amylopectin although the long chains of DP higher than 20 are debranched by the prolonged incubation. Isoform ISA1 shows 356% activity compared to phosphorylase a limit dextrin. Isoform ISA3 shows trace activity compared to phosphorylase a limit dextrin
-
-
?
amylopectin + H2O
?
-
the enzyme can debranch almost all of component chains of amylopectin
-
-
?
amylopectin + H2O
amylose + linear maltooligosaccharides
-
-
-
?
amylopectin + H2O
amylose + linear maltooligosaccharides
Bacillus sp. (in: Bacteria) CICIM 304
-
-
-
?
amylopectin + H2O
linear maltooligosaccharides
-
-
-
?
amylopectin + H2O
maltose + maltooligosaccharides
-
specific cleavage of alpha 1,6-glucosidic inter-chain linkages producing essentially linear chains
-
-
?
amylopectin + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins, cleaves the branching points completely in vitro
-
-
?
amylopectin + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins, cleaves the branching points completely in vitro
-
?
amylopectin + H2O
maltose + maltooligosaccharides
-
isoamylase is a direct debranching enzyme with the ability to cleave all the alpha-1,6 linkages of glycogen both inner and outer branching points of soluble amylopectin
-
-
?
amylopectin + H2O
maltose + maltooligosaccharides
-
waxy maize amylopectin, potato amylopectin, amylopectin beta-dextrin
-
?
amylopectin + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins, cleaves the branching points completely in vitro
-
?
amylopectin + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins, cleaves the branching points completely in vitro
-
?
amylopectin + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins, cleaves the branching points completely in vitro
-
?
amylopectin + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins, cleaves the branching points completely in vitro
-
?
amylopectin + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins, cleaves the branching points completely in vitro
-
?
amylopectin + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins, cleaves the branching points completely in vitro
-
-
?
amylopectin + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins, cleaves the branching points completely in vitro
hydrolysis of beta-limit dextrins forming maltotriose as main reaction product
?
amylopectin + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins, cleaves the branching points completely in vitro
hydrolysis of beta-limit dextrins forming maltotriose as main reaction product
?
amylopectin + H2O
maltose + maltooligosaccharides
isoamylase is a direct debranching enzyme with the ability to cleave all the alpha-1,6 linkages of glycogen both inner and outer branching points of soluble amylopectin
-
-
?
amylopectin + H2O
maltose + maltooligosaccharides
isoamylase is a direct debranching enzyme with the ability to cleave all the alpha-1,6 linkages of glycogen both inner and outer branching points of soluble amylopectin
-
-
?
amylopectin + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins, cleaves the branching points completely in vitro
-
-
?
amylopectin + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins, cleaves the branching points completely in vitro
hydrolysis of beta-limit dextrins forming maltotriose as main reaction product
?
amylopectin + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins, cleaves the branching points completely in vitro
-
-
r
beta-amylase limit dextrin + H2O
?
-
the enzyme shows 55% activity compared to phosphorylase a limit dextrin
-
-
?
beta-amylase limit dextrin + H2O
?
-
the enzyme shows 34% activity compared to phosphorylase a limit dextrin
-
-
?
beta-amylase limit dextrin + H2O
?
-
isoform ISA1 shows 71% activity compared to phosphorylase a limit dextrin. Isoform ISA3 shows 48% activity compared to phosphorylase a limit dextrin
-
-
?
beta-limit dextrin + H2O
linear maltooligosaccharides
-
-
-
?
beta-limit dextrin + H2O
linear maltooligosaccharides
-
-
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
cleaves the branching points completely in vitro
-
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins
-
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
cleaves the branching points completely in vitro
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
cleaves the branching points completely in vitro
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
cleaves the branching points completely in vitro
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
cleaves the branching points completely in vitro
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
cleaves the branching points completely in vitro
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
cleaves the branching points completely in vitro
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
cleaves the branching points completely in vitro
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
cleaves the branching points completely in vitro
-
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
oyster glycogen, glycogen beta-dextrin, rabbit liver glycogen
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins
-
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
-
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
cleaves the branching points completely in vitro
-
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins
-
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
cleaves the branching points completely in vitro
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
or the beta-limit dextrins
-
?
glycogen + H2O
maltose + maltooligosaccharides
-
cleaves the branching points completely in vitro
-
-
r
oyster glycogen + H2O
?
-
the enzyme shows 14% activity compared to phosphorylase a limit dextrin
-
-
?
oyster glycogen + H2O
?
-
the enzyme shows 2% activity compared to phosphorylase a limit dextrin
-
-
?
oyster glycogen + H2O
?
-
isoform ISA1 shows 104% activity compared to phosphorylase a limit dextrin. Isoform ISA3 shows 1.5% activity compared to phosphorylase a limit dextrin
-
-
?
phosphorylase a limit dextrin + H2O
?
-
100% activity
-
-
?
phythoglycogen + H2O
?
-
highly branched polysaccharide, complete hydrolysis of the alpha-1,6-glucosidic bonds
-
-
?
phytoglycogen + H2O
?
-
the liberated chains formed two distinct peaks: the first large peak of DP3-4 and the second small peak of DP5-10
-
-
?
phytoglycogen + H2O
?
-
the enzyme removes only exclusively chains of DP3 and DP4 when incubated with phytoglycogen while DP5-7 chains are slightly liberated after the prolonged incubation
-
-
?
phytoglycogen + H2O
?
-
short chains of DP3 and DP4 are most effectively liberated by isoform ISA1 during the short period, and with the elapse of the incubation time intermediate chains with the peak of DP6 are more significantly produced. Almost only short chains of DP3 and DP4 are found after the isoform ISA3 reaction for 5-10 min, then during the course of enzymatic reaction until 60 min a small amount of the DP5-10 chains is found
-
-
?
phytoglycogen + H2O
?
-
the enzyme can debranch almost all of component chains of phytoglycogen
-
-
?
phytoglycogen + H2O
?
-
the liberated chains from phytoglycogen are enriched in short chains of DP3-6, but contained longer chains of DP higer than 7, decreasing in amounts with the increase in chain-length until DP about 15
-
-
?
phytoglycogen + H2O
?
-
the liberated chains from phytoglycogen are enriched in short chains of DP3-6, but contained longer chains of DP higer than 7, decreasing in amounts with the increase in chain-length until DP about 15
-
-
?
starch + H2O
?
hydrolysis of alpha-1,6-glycosidic linkages
-
-
?
starch + H2O
?
specific hydrolysis of alpha-1,6-glucosidic linkages
detection of aminobenzamide-labeled maltooligosaccharide products, overview
-
?
starch + H2O
?
specific hydrolysis of alpha-1,6-glucosidic linkages
detection of aminobenzamide-labeled maltooligosaccharide products, overview
-
?
starch + H2O
?
-
debranching of Cassava starch in concert with pullulanase yielding resistant starch type III. Effects of starch concentration, isoamylase concentration and incubation time on hydrolysis of cassava starch, overview
-
-
?
additional information
?
-
AtISA1 and AtISA2 are required for the production of a functional isoamylase-type of debranching enzyme named Iso1, the major isoamylase found in leaves. The absence of Iso1 leads to an 80% decrease in the starch content and to accumulation of water-soluble polysaccharides whose structure is similar to glycogen
-
-
?
additional information
?
-
AtISA1 and AtISA2 are required for the production of a functional isoamylase-type of debranching enzyme named Iso1, the major isoamylase found in leaves. The absence of Iso1 leads to an 80% decrease in the starch content and to accumulation of water-soluble polysaccharides whose structure is similar to glycogen
-
-
?
additional information
?
-
AtISA1 and AtISA2 are required for the production of a functional isoamylase-type of debranching enzyme named Iso1, the major isoamylase found in leaves. The absence of Iso1 leads to an 80% decrease in the starch content and to accumulation of water-soluble polysaccharides whose structure is similar to glycogen
-
-
?
additional information
?
-
-
AtISA1/AtISA2 isoamylase influences glucan branching pattern
-
-
?
additional information
?
-
-
glucan debranching occurs primarily at the granule surface via ISA3, but in its absence soluble branched glucans are debranched in the stroma via limit dextrinase. Isoamylase acts at the surface of the starch granule. Atisa3 mutants have more leaf starch and a slower rate of starch breakdown than wild-type plants
-
-
?
additional information
?
-
mutant lines carrying a defect in AtISA3 display a strong starch-excess phenotype at the end of both the light and the dark phases accompanied by a small modification of the amylopectin structure
-
-
?
additional information
?
-
mutant lines carrying a defect in AtISA3 display a strong starch-excess phenotype at the end of both the light and the dark phases accompanied by a small modification of the amylopectin structure
-
-
?
additional information
?
-
mutant lines carrying a defect in AtISA3 display a strong starch-excess phenotype at the end of both the light and the dark phases accompanied by a small modification of the amylopectin structure
-
-
?
additional information
?
-
synthesis of amylopectin and phytoglycogen by associated and separated ISA1 and ISA2, respectively, overview
-
-
?
additional information
?
-
synthesis of amylopectin and phytoglycogen by associated and separated ISA1 and ISA2, respectively, overview
-
-
?
additional information
?
-
-
isoamylase catalyzes the hydrolysis of alpha-1,6-glucosidic linkage specifically in amylopectin, glycogen and derived oligosaccharides
-
-
?
additional information
?
-
-
isoamylase catalyzes the hydrolysis of alpha-1,6-glucosidic linkage specifically in amylopectin, glycogen and derived oligosaccharides
-
-
?
additional information
?
-
pullulan is a poor substrate
-
-
?
additional information
?
-
-
no substrate: pullulan, glycogen
-
-
?
additional information
?
-
pullulan is a poor substrate
-
-
?
additional information
?
-
pullulan is a poor substrate
-
-
?
additional information
?
-
pullulan is a poor substrate
-
-
?
additional information
?
-
pullulan is a poor substrate
-
-
?
additional information
?
-
-
ISA1 activity plays a critical role in the stochastic process in starch synthesis in rice endosperm
-
-
?
additional information
?
-
-
recombinant isoamylase 3 does not readily hydrolyze red pullulan and amylose
-
-
?
additional information
?
-
-
isoform Psisa2 most likely does not have catalytic activity
-
-
?
additional information
?
-
-
direct debranching enzyme, attacks unmodified glycogen and/or amylopectin with hydrolysis of the 1,6-bond, smallest substrate is not known
-
-
?
additional information
?
-
-
direct debranching enzyme, attacks unmodified glycogen and/or amylopectin with hydrolysis of the 1,6-bond, smallest substrate is not known
-
-
?
additional information
?
-
-
distinguished from alpha-dextrin endo-1,6-alpha-glucosidase, EC 3.2.1.41, by the inability of isoamylase to attack pullulan, and by limited action on alpha-limit dextrins, action on glycogen however is complete in contrast to limited action by alpha-dextrin glucanohydrolase, 1,6-linkage hydrolysed only at branch point
-
-
?
additional information
?
-
-
isoamylase is one of the starch-debranching enzymes which catalyzes the hydrolysis of alpha-1,6-glucosidic linkages specific in alpha-glucans including amylopectin, branched starch, and glycogen
-
-
?
additional information
?
-
no activity towards pullulan
-
-
?
additional information
?
-
no activity towards pullulan
-
-
?
additional information
?
-
-
isoamylase is one of the starch-debranching enzymes which catalyzes the hydrolysis of alpha-1,6-glucosidic linkages specific in alpha-glucans including amylopectin, branched starch, and glycogen
-
-
?
additional information
?
-
-
TreX from Sulfolobus solfataricus shows dual activities for alpha-1,4-transferase, EC 2.4.1.25 and alpha-1,6-glucosidase, EC 3.2.1.68, bifunctional mechanism, substrate glycogen, overview. TreX exhibits two different active-site configurations depending on its oligomeric state
-
-
?
additional information
?
-
-
direct debranching enzyme, attacks unmodified glycogen and/or amylopectin with hydrolysis of the 1,6-bond, smallest substrate is not known
-
-
?
additional information
?
-
-
distinguished from alpha-dextrin endo-1,6-alpha-glucosidase, EC 3.2.1.41, by the inability of isoamylase to attack pullulan, and by limited action on alpha-limit dextrins, action on glycogen however is complete in contrast to limited action by alpha-dextrin glucanohydrolase, 1,6-linkage hydrolysed only at branch point
-
-
?
additional information
?
-
-
involved in synthesis of reserve and leaf starches
-
-
?
additional information
?
-
-
isoamylase1 is directly involved in the synthesis of amylopectin
-
-
?
additional information
?
-
-
Zea mays subunit ISA 2 alone is catalytically inactive
-
-
?
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
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
starch + H2O
amylose
-
the enzyme has the ability to attack side chains composed of 1-3 glucose residues
-
-
?
amylopectin + H2O
amylose + linear maltooligosaccharides
-
-
-
?
amylopectin + H2O
amylose + linear maltooligosaccharides
Bacillus sp. (in: Bacteria) CICIM 304
-
-
-
?
starch + H2O
?
hydrolysis of alpha-1,6-glycosidic linkages
-
-
?
additional information
?
-
AtISA1 and AtISA2 are required for the production of a functional isoamylase-type of debranching enzyme named Iso1, the major isoamylase found in leaves. The absence of Iso1 leads to an 80% decrease in the starch content and to accumulation of water-soluble polysaccharides whose structure is similar to glycogen
-
-
?
additional information
?
-
AtISA1 and AtISA2 are required for the production of a functional isoamylase-type of debranching enzyme named Iso1, the major isoamylase found in leaves. The absence of Iso1 leads to an 80% decrease in the starch content and to accumulation of water-soluble polysaccharides whose structure is similar to glycogen
-
-
?
additional information
?
-
AtISA1 and AtISA2 are required for the production of a functional isoamylase-type of debranching enzyme named Iso1, the major isoamylase found in leaves. The absence of Iso1 leads to an 80% decrease in the starch content and to accumulation of water-soluble polysaccharides whose structure is similar to glycogen
-
-
?
additional information
?
-
-
AtISA1/AtISA2 isoamylase influences glucan branching pattern
-
-
?
additional information
?
-
-
glucan debranching occurs primarily at the granule surface via ISA3, but in its absence soluble branched glucans are debranched in the stroma via limit dextrinase. Isoamylase acts at the surface of the starch granule. Atisa3 mutants have more leaf starch and a slower rate of starch breakdown than wild-type plants
-
-
?
additional information
?
-
mutant lines carrying a defect in AtISA3 display a strong starch-excess phenotype at the end of both the light and the dark phases accompanied by a small modification of the amylopectin structure
-
-
?
additional information
?
-
mutant lines carrying a defect in AtISA3 display a strong starch-excess phenotype at the end of both the light and the dark phases accompanied by a small modification of the amylopectin structure
-
-
?
additional information
?
-
mutant lines carrying a defect in AtISA3 display a strong starch-excess phenotype at the end of both the light and the dark phases accompanied by a small modification of the amylopectin structure
-
-
?
additional information
?
-
synthesis of amylopectin and phytoglycogen by associated and separated ISA1 and ISA2, respectively, overview
-
-
?
additional information
?
-
synthesis of amylopectin and phytoglycogen by associated and separated ISA1 and ISA2, respectively, overview
-
-
?
additional information
?
-
-
isoamylase catalyzes the hydrolysis of alpha-1,6-glucosidic linkage specifically in amylopectin, glycogen and derived oligosaccharides
-
-
?
additional information
?
-
-
isoamylase catalyzes the hydrolysis of alpha-1,6-glucosidic linkage specifically in amylopectin, glycogen and derived oligosaccharides
-
-
?
additional information
?
-
-
ISA1 activity plays a critical role in the stochastic process in starch synthesis in rice endosperm
-
-
?
additional information
?
-
-
isoamylase is one of the starch-debranching enzymes which catalyzes the hydrolysis of alpha-1,6-glucosidic linkages specific in alpha-glucans including amylopectin, branched starch, and glycogen
-
-
?
additional information
?
-
-
isoamylase is one of the starch-debranching enzymes which catalyzes the hydrolysis of alpha-1,6-glucosidic linkages specific in alpha-glucans including amylopectin, branched starch, and glycogen
-
-
?
additional information
?
-
-
involved in synthesis of reserve and leaf starches
-
-
?
additional information
?
-
-
isoamylase1 is directly involved in the synthesis of amylopectin
-
-
?
additional information
?
-
-
Zea mays subunit ISA 2 alone is catalytically inactive
-
-
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
activates 10% at 0.5 mM, and 25% at 5 mM, Ca2+ highly stabilizes the enzyme at 80°C
additional information
additional information
poor effects by Na+, K+, Mg2+, Li+, and EDTA at 0.5-5 mM. Mn2+ and Mg2+ do not stabilize the enzyme at 70°C and 80°C
additional information
-
poor effects by Na+, K+, Mg2+, Li+, and EDTA at 0.5-5 mM. Mn2+ and Mg2+ do not stabilize the enzyme at 70°C and 80°C
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
maltose
-
10 mM, inhibition slightly, with 0.4% amylose relative activity 90%
N-bromosuccinimide
-
0.01 mg abolishes enzyme activity almost completely
p-chloromercuribenzoate
-
1 mM 34% inhibition; 1 mM, complete inhibition
additional information
no inhibition by EDTA, citrate, maltose, and alpha-cyclodextrin
-
additional information
-
no inhibition by EDTA, citrate, maltose, and alpha-cyclodextrin
-
additional information
-
inhibitory effect of pressure and agitation speed on isoamylase activity in supercritical CO2, overview
-
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
polyethyleneglycol 2000
-
maximal activation of 58% at 0.04% PEG 2000 Da
Triton X-100
-
reagent in the preincubation mixture at concentrations 0.05-1% activates the enzyme by 45-72%
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
-
amylopectin 7.2 mg/ml-1, phytoglycogen 11.9 mg/ml-1, beta-limit dextrin 13.1 mg/ml-1, glycogen 21.8 mg/ml-1
-
additional information
additional information
-
potatoamylopectin 0.59 mg/ml-1, oyster glycogen 0.45 mg/ml-1
-
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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
additional information
additional information
6535 U/mg, measuring 0.01 increase in absorbance at 600 nm per h at 50°C, pH 6.0, per mg protein
additional information
-
6535 U/mg, measuring 0.01 increase in absorbance at 600 nm per h at 50°C, pH 6.0, per mg protein
additional information
-
1130 units/mg, mutant enzyme R505E, at pH 6.0 and 50°C. One unit of enzyme activity is defined as the amount of enzyme capable of resulting 0.01 increase in absorbance at 600 nm per h
additional information
-
1326 units/mg, mutant enzyme G608V, at pH 6.0 and 50°C. One unit of enzyme activity is defined as the amount of enzyme capable of resulting 0.01 increase in absorbance at 600 nm per h
additional information
-
232 units/mg, mutant enzyme N513V, at pH 6.0 and 50°C. One unit of enzyme activity is defined as the amount of enzyme capable of resulting 0.01 increase in absorbance at 600 nm per h
additional information
-
475 units/mg, mutant enzyme G608K, at pH 6.0 and 50°C. One unit of enzyme activity is defined as the amount of enzyme capable of resulting 0.01 increase in absorbance at 600 nm per h
additional information
-
487 units/mg, mutant enzyme N513K, at pH 6.0 and 50°C. One unit of enzyme activity is defined as the amount of enzyme capable of resulting 0.01 increase in absorbance at 600 nm per h
additional information
-
614 units/mg, mutant enzyme N513A, at pH 6.0 and 50°C. One unit of enzyme activity is defined as the amount of enzyme capable of resulting 0.01 increase in absorbance at 600 nm per h
additional information
-
642 units/mg, mutant enzyme R505A, at pH 6.0 and 50°C. One unit of enzyme activity is defined as the amount of enzyme capable of resulting 0.01 increase in absorbance at 600 nm per h
additional information
-
677 units/mg, mutant enzyme G608A, at pH 6.0 and 50°C. One unit of enzyme activity is defined as the amount of enzyme capable of resulting 0.01 increase in absorbance at 600 nm per h
additional information
-
801 units/mg, mutant enzyme R505P, at pH 6.0 and 50°C. One unit of enzyme activity is defined as the amount of enzyme capable of resulting 0.01 increase in absorbance at 600 nm per h
additional information
-
995 units/mg, wild type enzyme, at pH 6.0 and 50°C. One unit of enzyme activity is defined as the amount of enzyme capable of resulting 0.01 increase in absorbance at 600 nm per h
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
3 - 4
5.5
3 - 4
-
starch, 0.1 M acetate-HCl buffer, pH 3.5, 0.1 M phosphate-citric acid buffer, pH 4
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
5.5 - 9
over 90% of maximal activity within this range, profile overview
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
40
52
70
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Bacillus sp. (in: Bacteria) CICIM 304
isolated by screening a soil sample collected from a hot spring of Tengchong volcano in Yunnan, China
UniProt
brenda
isoamylase hyperproducing mutant derived from JD-210
-
-
brenda
isolated by screening a soil sample collected from a hot spring of Tengchong volcano in Yunnan, China
UniProt
brenda
mutants sugary and shrunken, contain reduced activities of isoamylase1
-
-
brenda
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
additional information
the ISA1 homomer does not provide the full physiological function of ISA activity in maize leaves. This is in contrast to the endosperm, where loss of ISA2, and thus the ISA1/ISA2 heteromeric enzyme, can be tolerated without major defects
brenda
three ISA activity forms are observed in leaves, two ISA1/ISA2 heteromultimers and one ISA1 homomultimer. ISA1 homomultimer activity exists in mutants lacking ISA2. Mutants without ISA2 differ in leaf starch content, granule morphology, and amylopectin structure compared with nonmutants or lines lacking both ISA1 and ISA2. The data imply that both the ISA1 homomultimer and ISA1/ISA2 heteromultimer function in the maize leaf. The ISA1 homomer does not provide the full physiological function of ISA activity in maize leaves. This is in contrast to the endosperm, where loss of ISA2, and thus the ISA1/ISA2 heteromeric enzyme, can be tolerated without major defects
brenda
additional information
not detected in the stem, petiole, or root at the four-leaf stage, while at the six-leaf stage the enzyme's mRNA is present in all of the non-storage tissues, quantitative expression analysis, overview
brenda
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
TreX may be located at the cell surface due to the proline-rich region
brenda
-
TreX may be located at the cell surface due to the proline-rich region
-
brenda
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
malfunction
physiological function
additional information
evolution
subunit ISA1 is a family 13 glycoside hydrolase, which has activity for hydrolyzing alpha-1,6-glucosidic linkages corresponding to branch points of growing amylopectin molecules
evolution
subunit ISA2 is a family 13 glycoside hydrolase, but its putative catalytic residues are altered, rendering it enzymatically inactive. Despite its inactivity, ISA2 is evolutionarily conserved in plants, and has been suggested to play a role as a regulatory subunit for ISA1
evolution
the enzyme belongs to the glycosyl hydrolase family 13 GH13, and harbors a carbohydrate-binding module family 48 (CBM48) domain
evolution
the enzyme belongs to the glycosyl hydrolase family 13 GH13, and harbors a carbohydrate-binding module family 48 (CBM48) domain
evolution
the enzyme harbors a carbohydrate-binding module family 48 (CBM48) domain
evolution
-
subunit ISA2 is a family 13 glycoside hydrolase, but its putative catalytic residues are altered, rendering it enzymatically inactive. Despite its inactivity, ISA2 is evolutionarily conserved in plants, and has been suggested to play a role as a regulatory subunit for ISA1
-
evolution
-
subunit ISA1 is a family 13 glycoside hydrolase, which has activity for hydrolyzing alpha-1,6-glucosidic linkages corresponding to branch points of growing amylopectin molecules
-
evolution
Bacillus sp. (in: Bacteria) CICIM 304
-
the enzyme harbors a carbohydrate-binding module family 48 (CBM48) domain
-
malfunction
-
both overexpression and loss of function of isoamylase 3 in the endosperm generated pleomorphic amyloplasts and starch granules
malfunction
loss of isozyme ISA1 or ISA2 causes phytoglycogen accumulation
malfunction
mutants of the STA8 locus accumulate both phytoglycogen and a reduced amount of high amylose starch
malfunction
mutation of the STA7 locus leads to a very severe reduction of starch content and its replacement by a water-soluble polysaccharide phytoglycogen
malfunction
the ISA1 homomer does not provide the full physiological function of ISA activity in maize leaves. This is in contrast to the endosperm, where loss of ISA2, and thus the ISA1/ISA2 heteromeric enzyme, can be tolerated without major defects. Mutants without ISA2 differ in leaf starch content, granule morphology, and amylopectin structure compared with nonmutants or lines lacking both ISA1 and ISA2, mutant phenotypes, overview
malfunction
the ISA1 homomer does not provide the full physiological function of ISA activity in maize leaves. This is in contrast to the endosperm, where loss of ISA2, and thus the ISA1/ISA2 heteromeric enzyme, can be tolerated without major defects. Mutants without ISA2 differ in leaf starch content, granule morphology, and amylopectin structure compared with nonmutants or lines lacking both ISA1 and ISA2. Plastids from maize leaves lacking ISA2 exhibit a nearly normal appearance with the exception that starch granules appear to be slightly smaller than in wild-type, mutant phenotypes, overview
malfunction
-
seeds of the homozygous mutants, cr-isa1-1 (type 1, with an adenine insertion) and cr-isa1-2 (type 3, with a cytosine deletion) display a shrunken endosperm with significantly lower grain weight. Abnormal starch granules and amyloplasts are found in cr-isa1-1 and cr-isa1-2 endosperm cells. The contents of total starch, amylose and amylopectin in the endosperm of the cr-isa1 mutants are significantly reduced, whereas sugar content and starch gel consistency were observably increased compared to the wild type. The gelatinization temperature and starch chain length distributions of the cr-isa1 mutants are also altered. The transcript levels of most starch synthesis-related genes are significantly lower in cr-isa1 mutants
malfunction
-
mutants of the STA8 locus accumulate both phytoglycogen and a reduced amount of high amylose starch
-
malfunction
-
mutation of the STA7 locus leads to a very severe reduction of starch content and its replacement by a water-soluble polysaccharide phytoglycogen
-
physiological function
-
isoamylase 3 facilitates starch metabolism and affects morphological characteristics of plastids in rice. Both overexpression and loss of function of isoamylase 3 in the endosperm generates pleomorphic amyloplasts and starch granules
physiological function
-
the isoamylase 1 is essential for amylopectin biosynthesis and starch production in rice endosperm. Overexpression of the isoamylase 2 gene brings about a dramatic reduction in kernel size in the dry seed and the transformants contain less than 50% of the starch in the kernel, while the content of soluble sugars, including maltodextrins, malto-oligosaccharides, and simple sugars, increase by about 8fold when compared with the host cultivar Kinmaze
physiological function
the isoamylase ISA1/ISA2 heteromeric complexes II and III are not required in maize endosperm for normal starch content and structure, and the ISA1 homomeric complex is sufficient for most ISA functions. ISA1 is required for the accumulation of ISA2, which is regulated posttranscriptionally, while the absence of ISA2 in isa2-339 mutants has no effect on the accumulation of ISA1 mRNA or protein
physiological function
isoamylase catalyzes hydrolysis of alpha-D-(1,6)-glucosidic branch linkages in amylopectin and glycogen to release amylose and linear maltooligosaccharides
physiological function
starch debranching enzymes isoamylase 1 and 2, ISA1 and ISA2, are known to exist in a large complex and are involved in the biosynthesis and crystallization of starch. The function of the complex is to remove misplaced branches of growing amylopectin molecules, which would otherwise prevent the association and crystallization of adjacent linear chains
physiological function
starch debranching enzymes isoamylase 1 and 2, ISA1 and ISA2, are known to exist in a large complex and are involved in the biosynthesis and crystallization of starch. The function of the complex is to remove misplaced branches of growing amylopectin molecules, which would otherwise prevent the association and crystallization of adjacent linear chains. ISA2 plays a role as a regulatory subunit for ISA1
physiological function
the ISA1 class of DBE is the one primarily associated with amylopectin synthesis
physiological function
-
starch debranching enzymes isoamylase 1 and 2, ISA1 and ISA2, are known to exist in a large complex and are involved in the biosynthesis and crystallization of starch. The function of the complex is to remove misplaced branches of growing amylopectin molecules, which would otherwise prevent the association and crystallization of adjacent linear chains. ISA2 plays a role as a regulatory subunit for ISA1
-
physiological function
-
starch debranching enzymes isoamylase 1 and 2, ISA1 and ISA2, are known to exist in a large complex and are involved in the biosynthesis and crystallization of starch. The function of the complex is to remove misplaced branches of growing amylopectin molecules, which would otherwise prevent the association and crystallization of adjacent linear chains
-
physiological function
Bacillus sp. (in: Bacteria) CICIM 304
-
isoamylase catalyzes hydrolysis of alpha-D-(1,6)-glucosidic branch linkages in amylopectin and glycogen to release amylose and linear maltooligosaccharides
-
additional information
Arabidopsis thaliana subunit ISA1 requires subunit ISA2 as a partner for enzymatic function. Images of purified starch granules of wild-type and mutant lines, overview
additional information
Arabidopsis thaliana subunit ISA1 requires subunit ISA2 as a partner for enzymatic function. Images of purified starch granules of wild-type and mutant lines, overview
additional information
Arabidopsis thaliana subunit ISA1 requires subunit ISA2 as a partner for enzymatic function. Images of purified starch granules of wild-type and mutant lines, overview. Recombinant AtISA1 is capable of enzymatic activity if mixed with recombinant AtISA2 but does not display such function on its own
additional information
Arabidopsis thaliana subunit ISA1 requires subunit ISA2 as a partner for enzymatic function. Images of purified starch granules of wild-type and mutant lines, overview. Recombinant AtISA1 is capable of enzymatic activity if mixed with recombinant AtISA2 but does not display such function on its own
additional information
isozyme ISA1 interacts with its homologue ISA2, but no evidence for interaction with other starch biosynthetic enzymes. ISA1 and ISA2 form a heteromultimeric enzyme with ISA1 being the catalytic subunit and ISA2 subunit not catalytically active
additional information
isozyme ISA1 interacts with its homologue ISA2, but no evidence for interaction with other starch biosynthetic enzymes. ISA1 and ISA2 form a heteromultimeric enzyme with ISA1 being the catalytic subunit and ISA2 subunit not catalytically active
additional information
subunit ISA2 interacts physically with ISA1, presence of both homomeric ISA1 and heteromeric ISA1-ISA2 complexes in vivo
additional information
subunit ISA2 interacts physically with ISA1, presence of both homomeric ISA1 and heteromeric ISA1-ISA2 complexes in vivo
additional information
-
subunit ISA2 interacts physically with ISA1, presence of both homomeric ISA1 and heteromeric ISA1-ISA2 complexes in vivo
additional information
subunit ISA2 interacts physically with ISA1, presence of both homomeric ISA1 and heteromeric ISA1-ISA2 complexes in vivo. Subunit ISA1 enzyme is also partially functional without subunit ISA2
additional information
subunit ISA2 interacts physically with ISA1, presence of both homomeric ISA1 and heteromeric ISA1-ISA2 complexes in vivo. Subunit ISA1 enzyme is also partially functional without subunit ISA2
additional information
-
subunit ISA2 interacts physically with ISA1, presence of both homomeric ISA1 and heteromeric ISA1-ISA2 complexes in vivo. Subunit ISA1 enzyme is also partially functional without subunit ISA2
additional information
-
Zea mays subunit ISA1 is active by itself and does not require subunit ISA2 for activity, subunit ISA 2 alone is catalytically inactive
additional information
Zea mays subunit ISA1 is active by itself and does not require subunit ISA2 for activity, subunit ISA 2 alone is catalytically inactive
additional information
-
Zea mays subunit ISA1 is active by itself, either in vitro or in transgenic Arabidopsis leaves, and does not require subunit ISA2 for activity
additional information
Zea mays subunit ISA1 is active by itself, either in vitro or in transgenic Arabidopsis leaves, and does not require subunit ISA2 for activity
additional information
-
subunit ISA2 interacts physically with ISA1, presence of both homomeric ISA1 and heteromeric ISA1-ISA2 complexes in vivo
-
additional information
-
subunit ISA2 interacts physically with ISA1, presence of both homomeric ISA1 and heteromeric ISA1-ISA2 complexes in vivo. Subunit ISA1 enzyme is also partially functional without subunit ISA2
-
additional information
-
Arabidopsis thaliana subunit ISA1 requires subunit ISA2 as a partner for enzymatic function. Images of purified starch granules of wild-type and mutant lines, overview. Recombinant AtISA1 is capable of enzymatic activity if mixed with recombinant AtISA2 but does not display such function on its own
-
additional information
-
Arabidopsis thaliana subunit ISA1 requires subunit ISA2 as a partner for enzymatic function. Images of purified starch granules of wild-type and mutant lines, overview
-
Show AA Sequence and Transmembrane Helices (351 entries)
Please use the AA Sequence and Transmembrane Helices Search for a specific query.
Please use the AA Sequence and Transmembrane Helices Search for a specific query.
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PDB
SCOP
CATH
UNIPROT
ORGANISM
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
101155
1 * 100000, SDS-PAGE, 1 * 101155, sequence calculation
150000
x * 150000, ISA1 and ISA2 as stable dimer, SDS-PAGE
180000
subunit ISA1, crystal structure analysis and SDS-PAGE
300000
wild type endosperm contains three high molecular mass ISA complexes resolved by gel filtration and native-PAGE. Two complexes of approximately 400 kDa contain both isoform ISA1 and isoform ISA2, and an approximately 300 kDa complex contains isoform ISA1 but not isoform ISA2
400000
wild type endosperm contains three high molecular mass ISA complexes resolved by gel filtration and native-PAGE. Two complexes of approximately 400 kDa contain both isoform ISA1 and isoform ISA2, and an approximately 300 kDa complex contains isoform ISA1 but not isoform ISA2
510000
O80403
SDS-PAGE, heterooligomer, hetero-hexamer composed of five OsISA1 and one OsISA2
65000
75000
79000
80000
83000
830000
85000
86000 - 94000
87000
93000
94000
95000
additional information
83000
-
SDS-PAGE, co-purified with a second minor band of 75000, probably C-terminal truncation of the native enzyme in vivo or during purification
830000
O80403
homooligomer OsISA1 5 * 830000 and heterooligomer OsISA1-OsISA2 6 * 85000, HPLC gel filtration
85000
O80403
homooligomer OsISA1 5 * 830000 and heterooligomer OsISA1-OsISA2 6 * 85000, HPLC gel filtration
87000
heterotetramer with PvISA1, 2 * 87000 PyISA1 and 2 * 93000 PvISA2, SDS-PAGE
87000
heterotetramer with PvISA2, 2 * 87000 PvISA1 and 2 * 93000 PvISA2, SDS-PAGE
93000
heterotetramer with PvISA1, 2 * 87000 PyISA1 and 2 * 93000 PvISA2, SDS-PAGE
93000
heterotetramer with PvISA2, 2 * 87000 PvISA1 and 2 * 93000 PvISA2, SDS-PAGE
93000
2 * 93000, subunit ISA1, crystal structure analysis and SDS-PAGE
additional information
comparable mobility in the native PAGE of native enzyme recombinnat enzyme complex
additional information
comparable mobility in the native PAGE of native enzyme recombinnat enzyme complex
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
dimer or tetramer
-
the enzyme exists in two oligomeric states in solution, as a dimer and tetramer
homodimer
monomer
oligomer
tetramer
additional information
dimer
-
dimers probably obtained by partial proteolytic degradation
homodimer
2 * 93000, subunit ISA1, crystal structure analysis and SDS-PAGE
homodimer
-
2 * 93000, subunit ISA1, crystal structure analysis and SDS-PAGE
-
monomer
1 * 100000, SDS-PAGE, 1 * 101155, sequence calculation
monomer
Bacillus sp. (in: Bacteria) CICIM 304
-
1 * 100000, SDS-PAGE, 1 * 101155, sequence calculation
-
oligomer
O80403
homooligomer OsISA1 5 * 830000 and heterooligomer OsISA1-OsISA2 6 * 85000, HPLC gel filtration
tetramer
heterotetramer with PvISA1, 2 * 87000 PyISA1 and 2 * 93000 PvISA2, SDS-PAGE
tetramer
heterotetramer with PvISA2, 2 * 87000 PvISA1 and 2 * 93000 PvISA2, SDS-PAGE
additional information
the enzym eis an (alphabeta)8-barrel protein
additional information
-
ISA1 and ISA2 proteins are subunits of the same enzyme
additional information
-
enzymic activity lies within a 88000 Da subunit of a multimeric enzyme complex
additional information
-
isozymes Psisa1, Psisa2, and Psisa3, structure comparisons
additional information
-
the structural lid, amino acids 99-97, at the active site generated by the tetramerization is closely associated with the bifunctionality and in particular with the alpha-1,4-transferase activity
additional information
associated with Stisa1 to form a multimeric complex
additional information
associated with Stisa1 to form a multimeric complex
additional information
associated with Stisa1 to form a multimeric complex
additional information
-
associated with Stisa1 to form a multimeric complex
additional information
associated with Stisa2 to form a multimeric complex
additional information
associated with Stisa2 to form a multimeric complex
additional information
associated with Stisa2 to form a multimeric complex
additional information
-
associated with Stisa2 to form a multimeric complex
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified subunit ISA1 alone and in complex with maltoheptaose, X-ray diffraction structure determination at 2.3 A and 2.4 A, respectively
purified subunit ISA1 alone and in complex with maltoheptaose, X-ray diffrcation structure determination at 2.3 A and 2.4 A, respectively. The subunit structure includes highly conserved catalytic (beta/alpha)8 A-domain (aa 184-750), N-terminal beta-sandwich domain (aa 76-183), and C-terminal beta-sandwich domain (aa 751-875)
crystallized from ammonium sulfate solution, crystals belong to orthorhombic space group P212121
-
TreX in complex with an acarbose ligand, microbatch method under oil at 18°C, dimeric crystal from 16% PEG 8000, 0.2 M NaCl, and 0.1 M CHES buffer, pH 9.5, tetrameric crystal form from 2.2 M ammonium phosphate and 0.1 M Tris-HCl buffer, pH 8.5. For the acarbose intermediate complex crystal, 0.1% acarbose is added to the protein, followed by incubation for 1 h prior to the setup of the crystal in 8% PEG 3000, 0.2M lithium sulfate, and 0.1 M imidazole buffer, pH 8.0, cyroprotection by 20% glycerol in mother liquor, in both crystal forms, the asymmetric unit consists of one dimer, X-ray diffraction structure determination and analysis at 2.8-3.0 A resolution
-
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D367A
site-directed mutagenesis of the catalytic nucleophile Asp-367 in ISA1, the mutation abolishes the catalytic activity of the enzyme complex
G608A
-
the kcat/Km values and the specific activity of the mutant are decreased as compared to the wild type enzyme
G608K
-
the kcat/Km values and the specific activity of the mutant are decreased as compared to the wild type enzyme
G608V
-
the kcat/Km values of the mutant are promoted by 49% and the specific activity increases by 33% as compared to the wild type enzyme
N513A
-
the kcat/Km values of the mutant are slightly decreased and the specific activity is decreased as compared to the wild type enzyme
N513K
-
the kcat/Km values and the specific activity of the mutant are decreased as compared to the wild type enzyme
N513V
-
the kcat/Km values and the specific activity of the mutant are strongly decreased as compared to the wild type enzyme
R505A
-
the kcat/Km values of the mutant are promoted and the specific activity is decreased as compared to the wild type enzyme
R505E
-
the acidic stability is enhanced and the specific activity of the mutant is increased by 13% compared to the wild type enzyme
R505P
-
the thermal stability of the mutant is enhanced compared to the wild type enzyme
G608A
Lederbergia lentus JNU3
-
the kcat/Km values and the specific activity of the mutant are decreased as compared to the wild type enzyme
-
G608V
Lederbergia lentus JNU3
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the kcat/Km values of the mutant are promoted by 49% and the specific activity increases by 33% as compared to the wild type enzyme
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R505A
Lederbergia lentus JNU3
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the kcat/Km values of the mutant are promoted and the specific activity is decreased as compared to the wild type enzyme
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R505E
Lederbergia lentus JNU3
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the acidic stability is enhanced and the specific activity of the mutant is increased by 13% compared to the wild type enzyme
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R505P
Lederbergia lentus JNU3
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the thermal stability of the mutant is enhanced compared to the wild type enzyme
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additional information
additional information
construction of a single mutant line isa2-2 by T-DNA insertion mutagenesis. The isa1-1 isa2-2 double mutant line is obtained by a cross between the two corresponding single mutants and screening of the progeny by PCR amplification of genomic DNA using a T-DNA end primer and gene-specific primers. The phenotype of the isa1-1 isa2-2 double mutant is complemented but the ZmISA1 activity
additional information
construction of a single mutant line isa2-2 by T-DNA insertion mutagenesis. The isa1-1 isa2-2 double mutant line is obtained by a cross between the two corresponding single mutants and screening of the progeny by PCR amplification of genomic DNA using a T-DNA end primer and gene-specific primers. The phenotype of the isa1-1 isa2-2 double mutant is complemented but the ZmISA1 activity
additional information
construction of single mutant line isa1-1 by T-DNA insertion mutagenesis. The isa1-1 isa2-2 double mutant line is obtained by a cross between the two corresponding single mutants and screening of the progeny by PCR amplification of genomic DNA using a T-DNA end primer and gene-specific primers. The phenotype of the isa1-1 isa2-2 double mutant is complemented but the ZmISA1 activity, Zea mays ISA1 subunit is expressed via transfection with Agrobacterium tumefaciens strain GV3101 in Arabidopsis thaliana lacking endogenous ISA1 or lacking both native ISA1 and ISA2. The maize protein functions in Arabidopsis leaves to support nearly normal starch metabolism in the absence of any native ISA1 or ISA2
additional information
construction of single mutant line isa1-1 by T-DNA insertion mutagenesis. The isa1-1 isa2-2 double mutant line is obtained by a cross between the two corresponding single mutants and screening of the progeny by PCR amplification of genomic DNA using a T-DNA end primer and gene-specific primers. The phenotype of the isa1-1 isa2-2 double mutant is complemented but the ZmISA1 activity, Zea mays ISA1 subunit is expressed via transfection with Agrobacterium tumefaciens strain GV3101 in Arabidopsis thaliana lacking endogenous ISA1 or lacking both native ISA1 and ISA2. The maize protein functions in Arabidopsis leaves to support nearly normal starch metabolism in the absence of any native ISA1 or ISA2
additional information
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construction of single mutant line isa1-1 by T-DNA insertion mutagenesis. The isa1-1 isa2-2 double mutant line is obtained by a cross between the two corresponding single mutants and screening of the progeny by PCR amplification of genomic DNA using a T-DNA end primer and gene-specific primers. The phenotype of the isa1-1 isa2-2 double mutant is complemented but the ZmISA1 activity, Zea mays ISA1 subunit is expressed via transfection with Agrobacterium tumefaciens strain GV3101 in Arabidopsis thaliana lacking endogenous ISA1 or lacking both native ISA1 and ISA2. The maize protein functions in Arabidopsis leaves to support nearly normal starch metabolism in the absence of any native ISA1 or ISA2
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additional information
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construction of a single mutant line isa2-2 by T-DNA insertion mutagenesis. The isa1-1 isa2-2 double mutant line is obtained by a cross between the two corresponding single mutants and screening of the progeny by PCR amplification of genomic DNA using a T-DNA end primer and gene-specific primers. The phenotype of the isa1-1 isa2-2 double mutant is complemented but the ZmISA1 activity
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additional information
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mutant sta8, about 35% residual enzymic activity, loss of two out of three activity bands in zymogram gels, loss of about 50% of mass of the multimeric complex
additional information
functional complementation of sta8 mutant strains BafV13 and BafO6 with ISA2 or ISA2-HA, overview
additional information
functional complementation of sta8 mutant strains BafV13 and BafO6 with ISA2 or ISA2-HA, overview
additional information
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functional complementation of sta8 mutant strains BafV13 and BafO6 with ISA2 or ISA2-HA, overview
additional information
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functional complementation of sta8 mutant strains BafV13 and BafO6 with ISA2 or ISA2-HA, overview
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additional information
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Cassava starch is debranched by treatment with isoamylase and pullulanase and the yield of resistant starch type III, method optimization with respect to starch solids concentration, incubation time, and enzyme concentration using central composite rotatable design
additional information
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mutations in the N-terminal region result in a sharp increase in alpha-1,4-transferase activity and a reduced level of alpha-1,6-glucosidase activity, overview
additional information
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transgenic plants expressing antisense RNA for Stisa1 or Stisa2, accumulate small amounts of soluble glucan and large numbers of tiny starch granules
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4.5
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during 10 h incubation at pH 4.5 and 20°C, the activity of wild type enzyme rapidly declines with the increase of time until 35% of its initial activity is retained
751254
4.5 - 9
purified native enzyme, 50°C, 1 h, over 70% activity remaining
732278
5 - 11.5
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more than 50% of the original activity detectable between pH 6.6-10.5
136809
6.5 - 7
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assayed at 22°C enzyme displays broader activity and stability optima of pH 5.0 -7.5
136813
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
21
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recombinant wild-type and His-tagged enzyme are more stable at room temperature than at 4°C
30 - 70
purified native enzyme, pH 6.0, 1 h, over 70% activity remaining
4
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recombinant wild-type and His-tagged enzyme are more stable at room temperature than at 4°C
40
45
50
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amylopectin, incubated in the absence of substrate enzyme stable only up to 45°C
75
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2 h, recombinant wild-type enzyme and His-tagged enzyme are sstable
80
40
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stable up to 40°C, at temperature below 30°C and above 65°C enzyme is almost inactive
80
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the wild type enzyme loses 50% of its initial activity after 1 h at 80°C
80
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2 h, recombinant wild-type enzyme and His-tagged enzyme are sstable
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
Pseudomonas isoamylase immobilized physically by raw starch adsorption results in an increase in pH and temperature stability and a retention of about 75% of activity even after 75 days
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C for 5 months 85% enzyme activity retained, preservation with 0.1% potassium sorbate isoamylase activity was not reduced until 2 months of incubation
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation, DEAE cellulose column chromatography, CM-cellulose column chromatography, and DEAE-Sephadex gel filtration
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ammonium sulfate precipitation, Ni-NTA column chromatography, and Superdex 200 gel filtration
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copurification of native isozymes ISA1 and ISA2 partially from leaves by gel filtration, copurification of TAP-tagged ISA1 and ISA2 from Escherichia coli strain BL21(DE3) by tandem-affinity chromatography on TEV and calmodulin, followed by gel filtration
HitrapQ column chromatography and TSKgel DEAE-5PW column chromatography
native extracellular enzyme 369fold from culture supernatant by ultrafiltration, ammonium sulfate fractionation, gel filtration, two different steps of anion exchange chromatography, and dialysis, to homogeneity
native subunit ISA2 from leaves by anion exchange chromatography, ultrafiltration, and gel filtration, separation from subunit ISA1
native subunit ISAI from leaves by anion exchange chromatography, ultrafiltration, and gel filtration, separation from subunit ISA2
recombinant C-terminally His8-tagged single subunit ISA1 or ISA1 in complex with His8-tagged ISA2 from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
recombinant C-terminally His8-tagged single subunit ISA2 or His8-tagged ISA2 in complex with ISA1 from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
recombinant His-tagged subunit ISA1 from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
recombinant wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography
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wild type enzyme by HitrapQ HP and TSKgel G3000SWXL column chromatography, recombinant OsISA2 by HitrapQ HP column chromatography
O80403
HitrapQ column chromatography and TSKgel DEAE-5PW column chromatography
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HitrapQ column chromatography and TSKgel DEAE-5PW column chromatography
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HitrapQ column chromatography and TSKgel DEAE-5PW column chromatography
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recombinant C-terminally His8-tagged single subunit ISA1 or ISA1 in complex with His8-tagged ISA2 from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
recombinant C-terminally His8-tagged single subunit ISA1 or ISA1 in complex with His8-tagged ISA2 from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
recombinant C-terminally His8-tagged single subunit ISA2 or His8-tagged ISA2 in complex with ISA1 from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
recombinant C-terminally His8-tagged single subunit ISA2 or His8-tagged ISA2 in complex with ISA1 from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
cloned to an expression vector with a T7lac promoter. Both wild-type and His-tagged isoamylases are expressed in Escherichia coli
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cloning of cDNAs of different isoforms, Psisa1-Psisa3, DNA and amino acid sequence determination and analysis, phylogenetic analysis and sequence comparisons, overview
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coexpression of wild-type His-tagged ISA1 and ISA2, and of mutant D367A ISA1 with wild-type ISA2, in Escherichia coli strain BL21(DE3)
DNA and amino acid sequence determination and analysis, phylogenetic tree
expressed in Escherichia coli BL21 cells
expressed in Escherichia coli BL21(DE3) cells
expression of wild-type and mutant enzymes in Escherichia coli, sequence comparison
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gene ArDBE, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis
gene isa1, single expression of C-terminally His8-tagged subunit in Escherichia coli strain BL21(DE3), co-expression of untagged ISA1 subunit with C-terminally His8-tagged subunit ISA2 in Escherichia coli
gene isa2, single expression of C-terminally His8-tagged subunit in Escherichia coli strain BL21(DE3) or co-expression with untagged ISA1 subunit in Escherichia coli
gene su-1, single expression of C-terminally His8-tagged subunit ISA1 in Escherichia coli strain BL21(DE3), co-expression of untagged ISA1 subunit with C-terminally His8-tagged subunit ISA2 in Escherichia coli. Zea mays ISA1 subunit is heterologously expressed in Arabidopsis thaliana lacking endogenous ISA1 or lacking both native ISA1 and ISA2 via transfection with Agrobacterium tumefaciens strain GV3101. The maize protein functions in Arabidopsis leaves to support nearly normal starch metabolism in the absence of any native ISA1 or ISA2
gene treX, component of a treXYZ operon, DNA and amino acid sequence determination and analysis, sequence comparison, genetic organization in the treXYZ operon, overview
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iam gene cloned and expressed in Escherichia coli JM101, pMON17481, production of a nonsecreted signal peptide-less isoamylase
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into Agrobacterium tumefaciens strain GV310, GFP/GUS promoter fusion constructs for AtISA1, AtISA2, AtISA3 into Arabidopsis thaliana using the floral dip method
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introduction of ISA1 gene from Triticum aestivum (TaISA1 from the D genome donor Aegilops tauschii) into a rice sugary-1 mutant line EM914 in which starch is completely replaced by phytoglycogen in the endosperm
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plasmid pIAM275, pUC9 carrying iam gene, Escherichia coli, recominant plasmid does not direct the synthesis of isoamylase in Escherichia coli , sequenced
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recombinant GST-ISA2 fusion protein is expressed in Escherichia coli Rosetta DE3-pLysS cells
the STA7 locus encodes ISA1, recombinant expression of His-tagged subunit ISA1 in Escherichia coli strain BL21(DE3)
the STA8 locus encodes ISA2, recombinant expression of HA-tagged ISA2 in strain BafV13 and BafO6