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show all sequences of 1.1.99.B3

Direct comparison of gluco-oligosaccharide oxidase variants and glucose oxidase substrate range and H2O2 stability

Vuong, T.V.; Foumani, M.; MacCormick, B.; Kwan, R.; Master, E.R.; Sci. Rep. 6, 37356 (2016)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
recombinant expression of His-tagged mutant enzyme in Pichia pastoris strain KM71H
Sarocladium strictum
Engineering
Amino acid exchange
Commentary
Organism
Y300A
the mutant variant of gluco-oligosaccharide oxidase (GOOX) from Sarocladium strictum shows a broader substrate range and higher H2O2 stability compared to the wild-type enzyme. The mutant Y300A exhibits up to 40times higher activity on all tested sugars except glucose, compared to wild-type. Fusion of the Y300A variant to a family 22 carbohydrate binding module from Clostridium thermocellum (CtCBM22A) nearly doubles its catalytic efficiency on glucose, while retaining significant activity on oligosaccharides. In the presence of 200 mM of H2O2, the recombinant CtCBM22A_Y300A retains 80% of activity on glucose and 100% of activity on cellobiose, the preferred substrate for this enzyme, while the wild-type enzyme retains 60% activity on D-glucose under the same conditions. GOOX variants appear to undergo a different mechanism of inactivation, as a loss of histidine instead of methionine is observed after H2O2 incubation. The addition of CtCBM22A also promotes functional binding of the fusion enzyme to xylan, facilitating its simultaneous purification and immobilization using edible oat spelt xylan, which might benefit the usage of this enzyme preparation in food and baking applications. The presence of CtCBM22A also permits enzyme binding to oat spelt xylans, facilitating simultaneous purification and immobilization of the enzyme, and ancillary fibre enrichment in potential food applications
Sarocladium strictum
Inhibitors
Inhibitors
Commentary
Organism
Structure
additional information
mutants Y300D and CtCBM22A_Y300A GOOX variants show no H2O2 inactivation on cellobiose
Sarocladium strictum
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.21
-
cellotriose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
0.25
-
cellobiose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A; pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
0.25
-
cellotriose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
0.3
-
Cellopentaose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
0.31
-
cellohexaose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
3
-
xylohexaose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
3.15
-
xylotriose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
4.49
-
xylopentaose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
4.57
-
xylotriose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
5.11
-
xylobiose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
5.4
-
D-glucose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
5.84
-
xylobiose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
8.1
-
D-glucose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
37
-
D-xylose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
51.8
-
D-xylose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Sarocladium strictum
Q6PW77
-
-
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
cellobiose + O2
preferred substrate
743824
Sarocladium strictum
? + H2O2
-
-
-
?
cellohexaose + O2
-
743824
Sarocladium strictum
Glc-beta-(1->4)-Glc-beta-(1->4)-Glc-beta-(1->4)-Glc-beta-(1->4)-Glc-beta-(1->4)-D-glucono-1,5-lactone + H2O2
-
-
-
?
cellopentaose + O2
-
743824
Sarocladium strictum
Glc-beta-(1->4)-Glc-beta-(1->4)-Glc-beta-(1->4)-Glc-beta-(1->4)-D-glucono-1,5-lactone + H2O2
-
-
-
?
cellotetraose + O2
-
743824
Sarocladium strictum
Glc-beta-(1->4)-Glc-beta-(1->4)-Glc-beta-(1->4)-D-glucono-1,5-lactone + H2O2
-
-
-
?
cellotriose + O2
-
743824
Sarocladium strictum
Glc-beta-(1->4)-Glc-beta-(1->4)-D-glucono-1,5-lactone + H2O2
-
-
-
?
D-glucose + O2
-
743824
Sarocladium strictum
D-glucono-1,5-lactone + H2O2
-
-
-
?
D-xylose + O2
-
743824
Sarocladium strictum
D-xylono-1,5-lactone + H2O2
-
-
-
?
maltose + O2
-
743824
Sarocladium strictum
4-O-alpha-D-glucopyranosyl-D-glucono-1,5-lactone + H2O2
-
-
-
?
additional information
enzyme GOOX oxidizes the anomeric carbon of carbohydrate substrates through an FAD reduction mechanism. The enzyme displays a comparatively broad substrate profile, ranging from D-xylose, and galactose, to malto-, cello- and xylooligosaccharides. GOOX oxidizes oligosaccharides more effectively than monosaccharides
743824
Sarocladium strictum
?
-
-
-
-
xylobiose + O2
-
743824
Sarocladium strictum
xyl-beta-(1->4)-D-xylono-1,5-lactone + H2O2
-
-
-
?
xylohexaose + O2
-
743824
Sarocladium strictum
xyl-beta-(1->4)-xyl-beta-(1->4)-xyl-beta-(1->4)-xyl-beta-(1->4)-xyl-beta-(1->4)-D-xylono-1,5-lactone + H2O2
-
-
-
?
xylooligosaccharide + O2
-
743824
Sarocladium strictum
? + H2O2
-
-
-
?
xylopentaose + O2
-
743824
Sarocladium strictum
xyl-beta-(1->4)-xyl-beta-(1->4)-xyl-beta-(1->4)-xyl-beta-(1->4)-D-xylono-1,5-lactone + H2O2
-
-
-
?
xylotriose + O2
-
743824
Sarocladium strictum
xyl-beta-(1->4)-xyl-beta-(1->4)-D-xylono-1,5-lactone + H2O2
-
-
-
?
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
37
-
assay at
Sarocladium strictum
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
11.17
-
cellotriose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
11.33
-
D-xylose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
13.22
-
D-glucose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
13.28
-
xylobiose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
13.72
-
cellobiose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
13.87
-
xylotriose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
15.68
-
D-glucose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
16.25
-
D-xylose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
16.33
-
cellobiose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
16.5
-
cellohexaose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
17.43
-
xylohexaose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
18.33
-
Cellopentaose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
18.67
-
cellotriose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
19.67
-
xylotriose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
19.72
-
xylobiose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
26.17
-
xylopentaose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
8
-
assay at
Sarocladium strictum
Cofactor
Cofactor
Commentary
Organism
Structure
FAD
the FAD cofactor is bi-covalently linked through cysteine and histidine residues
Sarocladium strictum
Cloned(Commentary) (protein specific)
Commentary
Organism
recombinant expression of His-tagged mutant enzyme in Pichia pastoris strain KM71H
Sarocladium strictum
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
FAD
the FAD cofactor is bi-covalently linked through cysteine and histidine residues
Sarocladium strictum
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
Y300A
the mutant variant of gluco-oligosaccharide oxidase (GOOX) from Sarocladium strictum shows a broader substrate range and higher H2O2 stability compared to the wild-type enzyme. The mutant Y300A exhibits up to 40times higher activity on all tested sugars except glucose, compared to wild-type. Fusion of the Y300A variant to a family 22 carbohydrate binding module from Clostridium thermocellum (CtCBM22A) nearly doubles its catalytic efficiency on glucose, while retaining significant activity on oligosaccharides. In the presence of 200 mM of H2O2, the recombinant CtCBM22A_Y300A retains 80% of activity on glucose and 100% of activity on cellobiose, the preferred substrate for this enzyme, while the wild-type enzyme retains 60% activity on D-glucose under the same conditions. GOOX variants appear to undergo a different mechanism of inactivation, as a loss of histidine instead of methionine is observed after H2O2 incubation. The addition of CtCBM22A also promotes functional binding of the fusion enzyme to xylan, facilitating its simultaneous purification and immobilization using edible oat spelt xylan, which might benefit the usage of this enzyme preparation in food and baking applications. The presence of CtCBM22A also permits enzyme binding to oat spelt xylans, facilitating simultaneous purification and immobilization of the enzyme, and ancillary fibre enrichment in potential food applications
Sarocladium strictum
Inhibitors (protein specific)
Inhibitors
Commentary
Organism
Structure
additional information
mutants Y300D and CtCBM22A_Y300A GOOX variants show no H2O2 inactivation on cellobiose
Sarocladium strictum
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.21
-
cellotriose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
0.25
-
cellobiose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A; pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
0.25
-
cellotriose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
0.3
-
Cellopentaose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
0.31
-
cellohexaose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
3
-
xylohexaose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
3.15
-
xylotriose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
4.49
-
xylopentaose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
4.57
-
xylotriose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
5.11
-
xylobiose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
5.4
-
D-glucose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
5.84
-
xylobiose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
8.1
-
D-glucose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
37
-
D-xylose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
51.8
-
D-xylose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
cellobiose + O2
preferred substrate
743824
Sarocladium strictum
? + H2O2
-
-
-
?
cellohexaose + O2
-
743824
Sarocladium strictum
Glc-beta-(1->4)-Glc-beta-(1->4)-Glc-beta-(1->4)-Glc-beta-(1->4)-Glc-beta-(1->4)-D-glucono-1,5-lactone + H2O2
-
-
-
?
cellopentaose + O2
-
743824
Sarocladium strictum
Glc-beta-(1->4)-Glc-beta-(1->4)-Glc-beta-(1->4)-Glc-beta-(1->4)-D-glucono-1,5-lactone + H2O2
-
-
-
?
cellotetraose + O2
-
743824
Sarocladium strictum
Glc-beta-(1->4)-Glc-beta-(1->4)-Glc-beta-(1->4)-D-glucono-1,5-lactone + H2O2
-
-
-
?
cellotriose + O2
-
743824
Sarocladium strictum
Glc-beta-(1->4)-Glc-beta-(1->4)-D-glucono-1,5-lactone + H2O2
-
-
-
?
D-glucose + O2
-
743824
Sarocladium strictum
D-glucono-1,5-lactone + H2O2
-
-
-
?
D-xylose + O2
-
743824
Sarocladium strictum
D-xylono-1,5-lactone + H2O2
-
-
-
?
maltose + O2
-
743824
Sarocladium strictum
4-O-alpha-D-glucopyranosyl-D-glucono-1,5-lactone + H2O2
-
-
-
?
additional information
enzyme GOOX oxidizes the anomeric carbon of carbohydrate substrates through an FAD reduction mechanism. The enzyme displays a comparatively broad substrate profile, ranging from D-xylose, and galactose, to malto-, cello- and xylooligosaccharides. GOOX oxidizes oligosaccharides more effectively than monosaccharides
743824
Sarocladium strictum
?
-
-
-
-
xylobiose + O2
-
743824
Sarocladium strictum
xyl-beta-(1->4)-D-xylono-1,5-lactone + H2O2
-
-
-
?
xylohexaose + O2
-
743824
Sarocladium strictum
xyl-beta-(1->4)-xyl-beta-(1->4)-xyl-beta-(1->4)-xyl-beta-(1->4)-xyl-beta-(1->4)-D-xylono-1,5-lactone + H2O2
-
-
-
?
xylooligosaccharide + O2
-
743824
Sarocladium strictum
? + H2O2
-
-
-
?
xylopentaose + O2
-
743824
Sarocladium strictum
xyl-beta-(1->4)-xyl-beta-(1->4)-xyl-beta-(1->4)-xyl-beta-(1->4)-D-xylono-1,5-lactone + H2O2
-
-
-
?
xylotriose + O2
-
743824
Sarocladium strictum
xyl-beta-(1->4)-xyl-beta-(1->4)-D-xylono-1,5-lactone + H2O2
-
-
-
?
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
37
-
assay at
Sarocladium strictum
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
11.17
-
cellotriose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
11.33
-
D-xylose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
13.22
-
D-glucose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
13.28
-
xylobiose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
13.72
-
cellobiose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
13.87
-
xylotriose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
15.68
-
D-glucose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
16.25
-
D-xylose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
16.33
-
cellobiose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
16.5
-
cellohexaose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
17.43
-
xylohexaose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
18.33
-
Cellopentaose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
18.67
-
cellotriose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
19.67
-
xylotriose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
19.72
-
xylobiose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
26.17
-
xylopentaose
pH 8.0, 37°C, recombinant mutant CtCBM22A_Y300A
Sarocladium strictum
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
8
-
assay at
Sarocladium strictum
General Information
General Information
Commentary
Organism
malfunction
comparison of the substrate profile and H2O2 inactivation of the wild-type glucose oxidase, EC 1.1.3.4, and the Y300A mutant variant of GOOX, the mutant shows a comparatively broad substrate range along with reduced substrate inhibition compared to glucose oxidase, overview
Sarocladium strictum
additional information
open active site of GOOX compared to glucose oxidase
Sarocladium strictum
General Information (protein specific)
General Information
Commentary
Organism
malfunction
comparison of the substrate profile and H2O2 inactivation of the wild-type glucose oxidase, EC 1.1.3.4, and the Y300A mutant variant of GOOX, the mutant shows a comparatively broad substrate range along with reduced substrate inhibition compared to glucose oxidase, overview
Sarocladium strictum
additional information
open active site of GOOX compared to glucose oxidase
Sarocladium strictum
KCat/KM [mM/s]
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
0.25
-
cellobiose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
0.25
-
cellotriose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
0.45
-
D-xylose
pH 8.0, 37°C, recombinant enzyme mutant
Sarocladium strictum
2.883
-
D-glucose
pH 8.0, 37°C, recombinant enzyme mutant
Sarocladium strictum
3.15
-
xylotriose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
5.11
-
xylobiose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
8.1
-
D-glucose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
51.8
-
D-xylose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
KCat/KM [mM/s] (protein specific)
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
0.25
-
cellobiose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
0.25
-
cellotriose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
0.45
-
D-xylose
pH 8.0, 37°C, recombinant enzyme mutant
Sarocladium strictum
2.883
-
D-glucose
pH 8.0, 37°C, recombinant enzyme mutant
Sarocladium strictum
3.15
-
xylotriose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
5.11
-
xylobiose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
8.1
-
D-glucose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
51.8
-
D-xylose
pH 8.0, 37°C, recombinant mutant Y300A
Sarocladium strictum
Other publictions for EC 1.1.99.B3
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
741429
Vuong
Direct comparison of gluco-oli ...
Sarocladium strictum
Sci. Rep.
6
37356
2016
-
-
1
-
1
-
1
6
-
-
-
-
-
1
-
-
1
-
-
-
-
-
11
-
-
-
-
6
-
-
-
-
-
-
-
-
-
1
-
-
1
-
-
1
-
6
-
-
-
-
-
-
-
1
-
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