Information on EC 3.2.1.142 - limit dextrinase

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

EC NUMBER
COMMENTARY hide
3.2.1.142
-
RECOMMENDED NAME
GeneOntology No.
limit dextrinase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
hydrolysis of (1->6)-alpha-D-glucosidic linkages in alpha- and beta-limit dextrins of amylopectin and glycogen, and in amylopectin and pullulan
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hyrolysis of O-glycosyl bonds
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
dextrin 6-alpha-glucanohydrolase
Plant enzymes with little or no action on glycogen. Action on amylopectin is incomplete, but action on alpha-limit dextrins is complete. Maltose is the smallest sugar it can release from an alpha-(1->6)-linkage.
CAS REGISTRY NUMBER
COMMENTARY hide
9032-15-9
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
EPZ37738
GenBank
Manually annotated by BRENDA team
-
EPZ37738
GenBank
Manually annotated by BRENDA team
DZ-Cr-387
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-chloro-4-nitrophenyl maltotriosyl-alpha(1->6)-maltotriosyl-beta-D-glucose + H2O
2-chloro-4-nitrophenyl maltotriose + maltotriosyl-beta-D-glucose
show the reaction diagram
-
-
-
-
?
4,6-O-benzylidene-2-chloro-4-nitrophenyl-beta-63-alpha-D-maltotriosyl-maltotrioside + H2O
4,6-O-benzylidene-2-chloro-4-nitrophenyl-beta-63-alpha-D-maltotriose + maltotriose
show the reaction diagram
-
substrate is not susceptible to transglycosylation and may serve as a routine quantitative assay tool
-
-
?
4,6-O-benzylidene-4-methylumbelliferyl-beta-63-alpha-D-maltotriosyl-maltotrioside
4,6-O-benzylidene-4-methylumbelliferyl-beta-63-alpha-D-maltotriose + maltotriose
show the reaction diagram
-
substrate is susceptible to transglycosylation and may serve as a routine quantitative assay tool
-
-
?
amylopectin + H2O
?
show the reaction diagram
-
-
-
-
?
amylopectin + H2O
maltotriose + maltose + maltooligosaccharides
show the reaction diagram
6.0% of the activity with pullulan
-
-
?
beta-limited dextrin + H2O
maltotriose + maltose + maltooligosaccharides
show the reaction diagram
19.7% of the activity with pullulan
-
-
?
glycogen + H2O
maltotriose + maltose + maltooligosaccharides
show the reaction diagram
6.1% of the activity with pullulan
-
-
?
limit dextrin + H2O
?
show the reaction diagram
-
the enzyme hydrolyses alpha-1,6-glucosidic linkages in limit dextrins derived from amylopectin
-
-
?
limit dextrin + H2O
maltotriose + maltotetraose + maltopentaose
show the reaction diagram
limit dextrins + H2O
?
show the reaction diagram
-
limit dextrinase catalyzes the hydrolysis of alpha-1,6 glucosidic linkages in limit dextrins
-
-
?
polysaccharide + H2O
oligosaccharides
show the reaction diagram
pullulan + H2O
?
show the reaction diagram
pullulan + H2O
maltotriose
show the reaction diagram
-
complete conversion to maltotriose
-
?
pullulan + H2O
maltotriose + ?
show the reaction diagram
soluble starch + H2O
maltotriose + maltose + maltooligosaccharides
show the reaction diagram
12.8% of the activity with pullulan
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-
?
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
limit dextrin + H2O
?
show the reaction diagram
-
the enzyme hydrolyses alpha-1,6-glucosidic linkages in limit dextrins derived from amylopectin
-
-
?
polysaccharide + H2O
oligosaccharides
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
5 mM, 198% of initial activity
Fe2+
5 mM, 121% of initial activity
K+
EPZ37738
2 mM, 132% of initial activtiy
Na+
EPZ37738
2 mM, 128% of initial activtiy
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
5 mM, 51% residual activity
6,3-alpha-D-glucosylmaltotriose
-
-
6,3-alpha-D-maltosylmaltotetraose
-
-
6,3-alpha-D-maltosylmaltotriose
-
-
6,3-alpha-D-maltotriosylmaltotetraose
-
-
6,3-alpha-D-maltotriosylmaltotriose
-
-
alpha-cyclodextrin
-
competitive inhibitor
Ba2+
EPZ37738
2 mM, 54% of initial activtiy
beta-cyclodextrin
-
competitive inhibitor
Co2+
EPZ37738
2 mM, 15% of initial activtiy
cyclohexaamylose
-
competitive inhibition
K+
-
slight inhibition
maltose
-
-
Mg2+
5 mM, 90% residual activity
Mn2+
EPZ37738
2 mM, 40% of initial activtiy
NH4+
EPZ37738
2 mM, 49% of initial activtiy
Ni2+
EPZ37738
2 mM, 47% of initial activtiy
oligomaltose
-
-
-
SDS
5 mM, 46% residual activity
Sr2+
EPZ37738
2 mM, 73% of initial activtiy
Triton X-100
EPZ37738
1% v/v, 72% of initial activtiy
Urea
EPZ37738
2 mM, 57% of initial activtiy
additional information
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a specific inhibitor of limit dextrinase is found in mature barley kernels, but disappears after several days of germination. Two forms of this proteinaceous inhibitor, identical in amino acid sequence, are isolated and characterized. They differ in attachment of cysteine or glutathione to a sulfhydryl group, possibly that of cysteine residue 59 of the inhibitor. They can form a 1:1 complex with limit dextrinase and are believed to interact specifically with the active site of the enzyme
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
EPZ37738
2 mM, 143% of initial activtiy
Tween-20
EPZ37738
1% v/v, 143% of initial activtiy
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.135 - 0.154
2-chloro-4-nitrophenyl maltotriosyl-alpha(1->6)-maltotriosyl-beta-D-glucose
-
0.192
4,6-O-benzylidene-2-chloro-4-nitrophenyl-beta-63-alpha-D-maltotriosyl-maltotrioside
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pH.0, 40°C
-
0.052
4,6-O-benzylidene-4-methylumbelliferyl-beta-63-alpha-D-maltotriosyl-maltotrioside
-
pH.0, 40°C
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0.2
6-alpha-maltosylmaltotetraose
-
-
0.2
6-alpha-maltosylmaltotriose
-
-
0.16 - 1.5
6-alpha-maltotriosylmaltotetraose
0.2
6-alpha-maltotriosylmaltotriose
-
-
0.6 - 3.24
amylopectin beta-limit dextrin
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26
glycogen beta-limit dextrin
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-
-
0.4
Pullulan
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-
additional information
amylopectin
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3.9 - 15.6
amylopectin
61 - 78
Pullulan
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
amylopectin
607
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1.04
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-
14.2
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pH 5.5, 40°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 5.4
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pullulan as substrate
5.3
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assay at
5.5
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assay at
5.8 - 6.2
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with both pullulan and amylopectin beta-limit dextrin as substrates
6.4 - 6.8
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pullulan and amylopectin as substrates, maximum activity occurred over a relatively broad range
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
-
assay at
60
EPZ37738
-
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 70
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enzyme activity during kilning at different temperatures, first-order rate constants, overview
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
90000
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gel filtration
97000
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x * 97000, SDS-PAGE
97419
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x * 98000, SDS-PAGE, recombinant enzyme, x * 97419, calculated
98000
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x * 98000, SDS-PAGE, recombinant enzyme, x * 97419, calculated
99000
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calculated from amino acid sequence
105000
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SDS-PAGE
180000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
-
binding of alpha-, beta-, and gamma-cyclodextrin binding to limit dextrinase with Kd of 27.2, 0.70, and 34.7 microM, respectively
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
in complex with its endogenous inhibitor LDI, at 2.7 A resolution. A hydrophobic cluster flanked by ionic interactions in the protein-protein interface is vital for the picomolar affinity of LDI to the enzyme
-
in complex with the competitive inhibitor beta-cyclodextrin, hanging drop vapor diffusion method, with streak seeding in a reservoir solution of 22% (w/v) PEG 3350, 5% (v/v) Gol, and 0.3 M NaI, or without seeding in a reservoir solution of 30% (w/v) PEG 3350, 10% (v/v) Gol, and 0.3 M NaI. In complex with the competitive inhibitor alpha-cyclodextrin, hanging drop vapor diffusion method, without seeding in a reservoir solution of 30% (w/v) PEG 3350 and 0.3 M NaI
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purified free enzyme with a glycerol molecule in the active site or purified enzyme in complex with competitive inhibitors alpha- and beta-cyclodextrin, hanging drop vapour diffusion method, streak-seeding, mixing of 10 mg/mlprotein in 50 mM MES buffer pH 6.6, 250 mM NaCl, 0.5 mM CaCl2, 0.67mM maltotriose, with a reservoir solution consisting of 30% w/v PEG 3350, 5% glycerol, 0.3 M NaI, cysteine is added to the crystallization drops to a final concentration of 5-7 mM, one week, 20°C, X-ray diffraction structure determination and analysis at 1.9-2.5 A resolution, molecular replacement and modelling
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structures of barley limit dextrinase and active-site mutants in complex with natural substrates, products and substrate analogues. Avoidance of alpha-1,4-hydrolytic activity and strong preference for alpha-1,6-hydrolytic activity seems to be based on the strong interaction to Trp512 and Phe553 on each flank of the catalytic cleft, which will keep non-branched polysaccharides out of the reach of the catalytic nucleophile and acid-base catalyst
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 10
30 min, more than 93% residual activity
738951
5 - 6
EPZ37738
-
737518
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 60
EPZ37738
20 min, stable
30 - 70
-
enzyme activity during kilning at different temperatures, first-order rate constants, overview. the enzyme is stable up to 50°C for 40 h, increases in activity at 30°C for 30 h, and loses activity at 70°C
50
pH 5-8, 10 min, more than 70% of maximum activity
70
-
malted enzyme, strong decrease in activity within 1-2 days during mashing
additional information
-
the level of limit dextrinase thermostability is inversely correlated with total limit dextrinase activity
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
acetone precipitation, affinity chromatography
-
acetone precipitation, DEAE-cellulose, Sephadex G-150
continuous electrophoresis, starch-celite, Sephadex G-150
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enzyme from malted barley 31.23fold by ammonium sulfate fractionation and column chromatography
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from rice grain
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Pichia pastoris
-
expression in Escherichia coli
expression in Pichia pastoris
-
gene HvLDI, cloning from barley seedling leaves and genotyping of 56 different cultivars
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recombinant epression in Pichia pastoris
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recombinant expression in Pichia pastoris and Escherichia coli
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
the enzyme is induced in the aleurone layer in response to gibberellin produced by the embryo, slight induction by salicylic acid. Antagonistic effcts of abscisic acid and salicylic acid on gibberellic acid function, overview
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A885S
single nucleotide polymorphism associated with thermostability; single nucleotide polymorphism associated with thermostability; single nucleotide polymorphism associated with thermostability
D730R
-
modest change in the association rate constant with endogenous inhibitor LDI. Residue D730 binds in a positively charged pocket on the surface of LDI via a hydrogen bond and a salt bridge to residue Arg34 and a hydrogen bond to Arg84 of LDI. D730 adopts different rotamers in the free enzyme and enzyme bound to LDI
D730W
-
modest change in the association rate constant with endogenous inhibitor LDI. Residue D730 binds in a positively charged pocket on the surface of LDI via a hydrogen bond and a salt bridge to residue Arg34 and a hydrogen bond to Arg84 of LDI. D730 adopts different rotamers in the free enzyme and enzyme bound to LDI
E510A
-
0.0004% residual activity with pullulan
L102R/T233A/S235G/G298A/C415R/A885S/G888C
amino acid substitutions identified by alignment of the limit dextrinase sequences of varieties Galleon and Maud; amino acid substitutions identified by alignment of the limit dextrinase sequences of varieties Galleon and Maud
M440G
-
2.6fold decrease in catalytic efficiency
T233A
single nucleotide polymorphism associated with thermostability; single nucleotide polymorphism associated with thermostability; single nucleotide polymorphism associated with thermostability
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
food industry
synthesis
-
expression of the limit dextrinase encoding gene fragment without signal peptide, with the Saccharomyces cerevisiae alpha-factor secretion signal under control of the alcohol oxidase 1 promoter, in Pichia pastoris leads to highly active barley limit dextrinase secreted during high cell-density fermentation. Optimization of a fedbatch fermentation procedure enables efficient production in a 5-l bioreactor, yielding 34 mg homogenous enzyme with 84% recovery