Information on EC 2.4.1.20 - cellobiose phosphorylase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.4.1.20
-
RECOMMENDED NAME
GeneOntology No.
cellobiose phosphorylase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
cellobiose + phosphate = alpha-D-glucose 1-phosphate + D-glucose
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexosyl group transfer
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Starch and sucrose metabolism
-
-
Metabolic pathways
-
-
SYSTEMATIC NAME
IUBMB Comments
cellobiose:phosphate alpha-D-glucosyltransferase
-
CAS REGISTRY NUMBER
COMMENTARY hide
9030-20-0
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
Clostridium stercorarium
strain NCIB 11754
SwissProt
Manually annotated by BRENDA team
strain NCIB 11754
SwissProt
Manually annotated by BRENDA team
Fomes annosus
-
-
-
Manually annotated by BRENDA team
strain MSB8
-
-
Manually annotated by BRENDA team
-
O52504
SwissProt
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1,5-anhydroglucitol + glucose-1-phosphate
?
show the reaction diagram
-
-
-
-
?
alpha-D -glucose 1-fluoride + D-glucose
cellobiose + fluoride
show the reaction diagram
-
-
-
-
ir
alpha-D-glucose 1-phosphate + 1,5-anhydro-D-glucitol
? + phosphate
show the reaction diagram
alpha-D-glucose 1-phosphate + 2-deoxy-2-fluoro-D-glucose
?
show the reaction diagram
-
-
-
-
?
alpha-D-glucose 1-phosphate + 2-deoxy-D-glucose
?
show the reaction diagram
alpha-D-glucose 1-phosphate + 2-deoxy-D-glucose
? + phosphate
show the reaction diagram
alpha-D-glucose 1-phosphate + 5a-carba-alpha-D,L-glucopyranose
4-O-beta-D-glucopyranosyl-5a-carba-beta-D-glucopyranose + phosphate
show the reaction diagram
-
very poor substrate
-
-
?
alpha-D-glucose 1-phosphate + 5a-carba-beta-D,L-glucopyranose
?
show the reaction diagram
-
much better substrate than alpha-anomer, 22% of activity with D-glucose
-
-
?
alpha-D-glucose 1-phosphate + 6-deoxy-6-fluoro-D-glucose
?
show the reaction diagram
-
-
-
-
?
alpha-D-glucose 1-phosphate + 6-deoxy-D-glucose
?
show the reaction diagram
alpha-D-glucose 1-phosphate + 6-deoxy-D-glucose
? + phosphate
show the reaction diagram
alpha-D-glucose 1-phosphate + butyl beta-D-glucoside
? + phosphate
show the reaction diagram
-
no substrate for wild-type
-
-
r
alpha-D-glucose 1-phosphate + cellobiose
? + phosphate
show the reaction diagram
-
no substrate for wilalpha-D-type
-
-
r
alpha-D-glucose 1-phosphate + D-altrose
?
show the reaction diagram
alpha-D-glucose 1-phosphate + D-arabinose
?
show the reaction diagram
alpha-D-glucose 1-phosphate + D-gentobiose
? + phosphate
show the reaction diagram
-
-
-
-
r
alpha-D-glucose 1-phosphate + D-glucosamine
4-O-beta-D-glucopyranosyl-D-glucosamine
show the reaction diagram
-
-
-
-
?
alpha-D-glucose 1-phosphate + D-glucosamine
?
show the reaction diagram
alpha-D-glucose 1-phosphate + D-glucosamine
? + phosphate
show the reaction diagram
-
-
-
-
r
alpha-D-glucose 1-phosphate + D-glucose
? + phosphate
show the reaction diagram
-
-
-
-
r
alpha-D-glucose 1-phosphate + D-glucose
cellobiose + phosphate
show the reaction diagram
alpha-D-glucose 1-phosphate + D-mannose
4-O-beta-D-glucopyranosyl-D-mannose
show the reaction diagram
-
-
-
-
?
alpha-D-glucose 1-phosphate + D-mannose
?
show the reaction diagram
alpha-D-glucose 1-phosphate + D-mannose
? + phosphate
show the reaction diagram
-
-
-
-
r
alpha-D-glucose 1-phosphate + D-xylose
?
show the reaction diagram
alpha-D-glucose 1-phosphate + D-xylose
? + phosphate
show the reaction diagram
-
-
-
-
r
alpha-D-glucose 1-phosphate + ethyl beta-D-glucoside
? + phosphate
show the reaction diagram
-
no substrate for wild-type
-
-
r
alpha-D-glucose 1-phosphate + L-fucose
?
show the reaction diagram
alpha-D-glucose 1-phosphate + L-galactose
?
show the reaction diagram
alpha-D-glucose 1-phosphate + methyl alpha-D-glucoside
? + phosphate
show the reaction diagram
-
no substrate for wild-type
-
-
r
alpha-D-glucose 1-phosphate + methyl beta-D-glucoside
? + phosphate
show the reaction diagram
-
no substrate for wild-type
-
-
r
alpha-D-glucose 1-phosphate + N-acetyl-D-glucosamine
4-O-beta-D-glucopyranosyl-N-acetyl-D-glucosamine
show the reaction diagram
-
substrate for mutant Y648V only
-
-
?
alpha-D-glucose 1-phosphate + phenyl beta-D-glucoside
? + phosphate
show the reaction diagram
-
no substrate for wild-type
-
-
r
beta-D-glucose 1-phosphate + alpha-D-glucopyranosyl fluoride
beta-cellobiose + HF
show the reaction diagram
-
beta-D-glucopyranosyl fluoride is not ulilized
-
?
cellobiose + arsenate
D-glucose + alpha-D-glucose 1-arsenate
show the reaction diagram
-
-
alpha-D-glucose 1-arsenate hydrolyses spontaneously to form D-glucose and arsenate
ir
cellobiose + phosphate
alpha-D-glucose 1 phosphate + D-glucose
show the reaction diagram
-
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
show the reaction diagram
cellobiose + phosphate
D-glucose 1-phosphate + D-glucose
show the reaction diagram
-
-
-
-
?
D-glucal + 2-deoxy-2-(hydroxymethyl)-beta-D-xylopyranose
2-deoxy-3-O-(2-deoxy-beta-D-arabino-hexopyranosyl)-2-(hydroxymethyl)-beta-D-xylopyranose + ?
show the reaction diagram
-
-
-
-
r
D-glucal + D-glucose
2-deoxy-beta-D-arabino-hexopyranosyl-(1->4)-D-glucose + ?
show the reaction diagram
-
-
-
-
r
D-glucal + D-mannose
2-deoxy-beta-D-arabino-hexopyranosyl-(1->4)-D-mannose + ?
show the reaction diagram
-
-
-
-
r
D-glucal + D-xylose
2-deoxy-beta-D-arabino-hexopyranosyl-(1->4)-D-xylose + ?
show the reaction diagram
-
-
-
-
r
D-glucal + phosphate
2-deoxy-alpha-D-glucose 1-phosphate
show the reaction diagram
-
D-glucal is a slow alternative donor substrate for stereospecific glycosyl transfer to phosphate. Reaction follows a stereochemical course where substrate becomes protonated from below its six-membered ring through stereoselective re side attack at C-2. The proposed catalytic mechanism involves direct protonation of D-glucal by the enzyme-bound phosphate, which then performs nucleophilic attack on the reactive C-1 of donor substrate. When offered D-glucose next to D-glucal and phosphate, the enzyme produces 2-deoxy-beta-D-glucosyl-(1->4)-D-glucose and 2-deoxy-alpha-D-glucose 1-phosphate in a ratio governed by mass action of the two acceptor substrates present. Enzymatic synthesis of 2-deoxy-beta-D-glucosyl-(1->4)-D-glucose is effectively promoted by catalytic concentrations of phosphate, suggesting that catalytic reaction proceeds through a quaternary complex of enzyme, D-glucal, phosphate,and D-glucose
-
-
?
gentiobiose + alpha-D-glucose 1-phosphate
?
show the reaction diagram
-
-
-
-
?
isomaltose + alpha-D-glucose 1-phosphate
?
show the reaction diagram
-
-
-
-
?
lactose + phosphate
alpha-D-galactose 1-phosphate + D-glucose
show the reaction diagram
-
-
-
r
melibiose + alpha-D-glucose 1-phosphate
?
show the reaction diagram
-
-
-
-
?
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
alpha-D-glucose 1-phosphate + D-glucose
cellobiose + phosphate
show the reaction diagram
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
no influence of metal ions on reaction
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4-deoxy-D-glucose
-
competitive with D-glucose, dead end inhibitor
4-thio-cellobiose
-
weak inhibition in both directions of reaction
6-deoxy-D-glucose
-
substrate inhibition
alpha-oxogluconate
-
-
cellobiose
D-cellobiose
-
-
D-glucosamine
-
substrate inhibition
D-glucose
D-glucose 1-phosphate
glucono-delta-lactone
-
very weak
N-ethylmaleimide
Nojirimycin
p-chloromercuribenzoate
-
complete inhibition at 0.02 mM
xylose
-
mixed-type inhibitor
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
9.5
1,5-Anhydro-D-glucitol
-
pH 6.0, 37C
8 - 9
1,5-anhydroglucitol
-
enzyme assay at 37C in 50 mM Tris/HCl (pH 7.0)
17.2
2-deoxy-2-fluoro-D-glucose
-
-
24.3 - 250
2-deoxy-D-glucose
7.4
6-deoxy-6-fluoro-D-glucose
-
-
4.1 - 24
6-deoxy-D-glucose
1.46 - 30.7
alpha-D-glucose 1-phosphate
33.7
butyl beta-D-glucoside
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37C
0.29 - 31.4
cellobiose
2.6
D-cellobiose
-
-
10.8
D-gentobiose
-
pH 6.0, 37C
5.7 - 58.1
D-glucosamine
0.69 - 28.9
D-glucose
10.8
D-glucoseamine
-
-
-
11.8 - 200
D-mannose
14 - 84
D-xylose
138 - 155
ethyl beta-D-glucoside
-
83
gentiobiose
-
enzyme assay at 37C in 50 mM Tris/HCl (pH 7.0)
71
isomaltose
-
enzyme assay at 37C in 50 mM Tris/HCl (pH 7.0)
228 - 330
lactose
93
melibiose
-
enzyme assay at 37C in 50 mM Tris/HCl (pH 7.0)
1009
methyl alpha-D-glucoside
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37C
56.6 - 79.8
methyl beta-D-glucoside
17.4
N-acetyl-D-glucosamine
-
mutant enzyme Y648V, at pH 6.0 and 37C
31.2
phenyl beta-D-glucoside
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37C
0.15 - 2.9
phosphate
additional information
additional information
-
simulation of molecular dynamics of the enzyme, the 1S3 pre-transition state conformer is highly stable compared with other conformers, and a conformational change from 4C1 to 1,4B occurs
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.8
1,5-Anhydro-D-glucitol
Ruminococcus albus
-
pH 6.0, 37C
3.5
1,5-anhydroglucitol
Cellulomonas gilvus
-
enzyme assay at 37C in 50 mM Tris/HCl (pH 7.0)
8
2-deoxy-2-fluoro-D-glucose
Cellulomonas uda
-
-
7.83 - 119
2-deoxy-D-glucose
44.6
6-deoxy-6-fluoro-D-glucose
Cellulomonas uda
-
-
17 - 124
6-deoxy-D-glucose
1.3 - 96.5
alpha-D-glucose 1-phosphate
9
butyl beta-D-glucoside
Cellulomonas uda
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37C
1.1 - 88.9
cellobiose
4
D-gentobiose
Ruminococcus albus
-
pH 6.0, 37C
5.2 - 35.4
D-glucosamine
5.3 - 92.3
D-glucose
5.6
D-glucoseamine
Cellulomonas uda
-
-
-
0.626 - 58.3
D-mannose
8.7 - 44.3
D-xylose
5.1 - 8.7
ethyl beta-D-glucoside
-
7.2
gentiobiose
Cellulomonas gilvus
-
enzyme assay at 37C in 50 mM Tris/HCl (pH 7.0)
1.9
isomaltose
Cellulomonas gilvus
-
enzyme assay at 37C in 50 mM Tris/HCl (pH 7.0)
0.109 - 1.11
lactose
2.3
melibiose
Cellulomonas gilvus
-
enzyme assay at 37C in 50 mM Tris/HCl (pH 7.0)
6
methyl alpha-D-glucoside
Cellulomonas uda
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37C
6.8 - 7.9
methyl beta-D-glucoside
6.54
N-acetyl-D-glucosamine
Ruminococcus albus
-
mutant enzyme Y648V, at pH 6.0 and 37C
4
phenyl beta-D-glucoside
Cellulomonas uda
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37C
15.4
phosphate
Cellulomonas uda
-
pH 6.6, 30C, native enzyme; pH 6.6, 30C, recombinant His6-tagged enzyme
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.29
1,5-Anhydro-D-glucitol
Ruminococcus albus
-
pH 6.0, 37C
2926
0.0198 - 1.43
2-deoxy-D-glucose
531
12.7
6-deoxy-D-glucose
Ruminococcus albus
-
pH 6.0, 37C
1527
0.0829 - 63.2
alpha-D-glucose 1-phosphate
107
0.266
butyl beta-D-glucoside
Cellulomonas uda
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37C
20171
0.0681 - 47.1
cellobiose
82
0.37
D-gentobiose
Ruminococcus albus
-
pH 6.0, 37C
42200
0.61 - 0.72
D-glucosamine
699
0.031 - 63.2
D-glucose
35
0.0263 - 0.931
D-mannose
216
0.65
D-xylose
Ruminococcus albus
-
pH 6.0, 37C
115
0.033 - 0.063
ethyl beta-D-glucoside
19675
0.00048 - 0.0034
lactose
114
0.006
methyl alpha-D-glucoside
Cellulomonas uda
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37C
5684
0.099 - 0.12
methyl beta-D-glucoside
3478
0.0376
N-acetyl-D-glucosamine
Ruminococcus albus
-
mutant enzyme Y648V, at pH 6.0 and 37C
300
0.129
phenyl beta-D-glucoside
Cellulomonas uda
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37C
4858
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4.4
4-deoxy-D-glucose
-
-
13
4-thio-cellobiose
-
in presence of saturating concentrations of phosphate
14.8
cellobiose
-
-
6.8
D-cellobiose
-
-
1.2 - 255
D-glucose
3.2
D-glucose 1-phosphate
-
-
0.045
Nojirimycin
-
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.033
purifed recombinant wild-type enzyme, substrate lactose
0.1
purifed recombinant mutant A397V/T508A/A512T/D557N/N667T/G681S, substrate lactose
0.171
purifed recombinant mutant T508I/N667A, substrate lactose
0.249
purifed recombinant mutant T508A/N667T, substrate lactose
1.35
purifed recombinant mutant A397V/T508A/A512T/D557N/N667T/G681S, substrate cellobiose
1.71
purifed recombinant mutant T508I/N667A, substrate cellobiose
2.61
purifed recombinant mutant T508A/N667T, substrate cellobiose
2.66
-
purified enzyme
4.7
Clostridium stercorarium
purified enzyme
7.78
purifed recombinant wild-type enzyme, substrate cellobiose
14.4
-
pH 6.6, 30C
20
-
purified enzyme
27.4
-
purified enzyme
33.3
-
purified enzyme
40.8
-
purified enzyme
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 7
Clostridium stercorarium
-
6.5
-
with D-xylose and 2-deoxyglucose as acceptor
7.5
-
with D-glucosamine as acceptor
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 8
Clostridium stercorarium
50% activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
38
-
assay at
65
Clostridium stercorarium
-
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 80
-
very low activity at 20 and 80C
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
72000
-
SDS-PAGE
90000
-
gel filtration
92000
-
SDS-PAGE, native PAGE
93760
-
calculated from DNA-sequence
94320
-
calculated from DNA-sequence
150000
180000
-
gel filtration
187000
-
gel filtration
280000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
tetramer
-
4 * 72000, gel filtration, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
at 25C using the sitting-drop vapour diffusion method. Cellobiose phosphorylase-SO4 and cellobiose phosphorylase-PO4 structures are determined at 2.0 and 2.1 A resolution to R factor (Rfree) of 17.6% (21.3%) and 18.0% (22%) respectively. The pH values for crystallization are pH 8.2 for cellobiose phosphorylase-PO4 and pH 6.5 for cellobiose phosphorylase-SO4.
-
crystallization of recombinant enzyme by the sitting-drop vapour-diffusion method using 10 mg/ml purified enzyme, 1.5 M ammonium sulfate, 0.1 M MES buffer pH 7.0 and 5 mM glucose. A suitable crystal is obtained after 10 d incubation at 298 K. The crystal belongs to space group P2(1), with unit-cell parameters a = 84.77, b = 98.31, c = 104.04 A, beta = 102.73. X-ray diffraction data to 2.1 A resolution collected at KEK-PF BL-5A
-
in complex with phosphate, to 2.4 A resolution. Enzyme is composed of four distinct domains, an N-terminal domain with residues 1-279, a helical linker with residues 280-314, an (alpha/beta)6-barrel domain with residues 321-734, and a C-terminal beta-sandwich domain with residues 315-320 and 735-811. The side chains of His653, Gln699 and Thr718 and the backbone N-atom of Gly719 coordinate the phosphate bound in the interior of the (alpha/beta)6-barrel domain. Two short helical extensions, residues 158-169, from the N-terminal domain of the adjacent subunit extend into the cleft which runs along the face of the (alpha/beta)6-barrel domain, forming an active-site pocket. An additional loop, residues 488-507, lays on top of the active-site pocket, forming a restrictive active-site pocket that is large enough to bind a disaccharide but not large enough to bind an oligosaccharide
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 9
-
most stable at pH 7, rapid loss of activity at different pH
637863
5.5 - 8.5
-
the enzyme remains stable over the pH range of 5.5 to 8.5
735893
5.5 - 8.8
-
stable within
719128
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37 - 60
-
in contrast to the rapid inactivation at 60C, the enzyme is remarkably stable at 37C. At 50C, the enzyme retains over 58% of its initial activity after 24 h
85
O52504
active for 2 h in presence of substrate, without substrate little activity after 15 min
90
-
25% loss of activity after heating for 30 min at pH 6-8
100
-
complete loss of activity after heating for 30 min at pH 6-8
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
enzyme immobilization with cross-linked enzyme aggregates boosts the half-life at 50C from 34 h to almost 11 days
-
storage of purified enzyme at -20C without thiol reagents causes rapid loss of activity
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ethanol
Ethyl acetate
Methanol
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
His-bind resin column chromatography
-
Ni-chelating column chromatography, and gel filtration
-
Ni-NTA agarose slurry, Superdex 200 column and Mono Q column
-
Ni-NTA column chromatography
-
recombinant enzyme
-
recombinant enzyme from E. coli
recombinant enzyme with a cellobiose-binding intein tag, development of a simple, low-cost, and scalable protein purification method using a biodegradable regenerated amorphous cellulose, affinity adsorption on regenerated amorphous cellulose followed by intein self-cleavage, method evaluation, overview
-
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by immobilized metal ion affinity chromatography
use of thiol reagent necessary during purification to maintain activity
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
CP gene, expression of His-tagged wild-type and mutant enzymes in Escherichia coli
expressed in Escherichia coli
expressed in Escherichia coli BL21 (DE3)
-
expressed in Escherichia coli BL21(DE3) cells
-
expressed in Escherichia coli JW0987 cells
-
expressed in Escherichia coli strain BL21 (DE3)
-
expressed in Escherichia coli, part of oligosaccharide catabolic cluster
O52504
expressed in Saccharomyces cerevisiae strain D452-2
expressed in Saccharomyces cerevisiae strain D452-2 and Escherichia coli BL21(DE3)
-
expression in Escherichia coli
expression of a functional recombinant enzyme containing an N-terminal metal affinity fusion peptide in Escherichia coli. Catalytic properties of the natural enzyme are retained completely in the recombinant cellobiose phosphorylase
-
expression of intein-tagged enzyme in Escherichia coli strain BL21, subcloning in Escherichia coli strain DH5alpha
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A397V
random, site-saturation, and site-directed mutagenesis, the mutant enzyme shows reduced activity with lactose compared to the wild-type enzyme
A397V/T508A/A512T/D557N/N667T/G681S
random, site-saturation, and site-directed mutagenesis, the mutant enzyme shows 3fold increased activity with lactose and 6fold decreased activity with cellobiose compared to the wild-type enzyme
A512T
random, site-saturation, and site-directed mutagenesis, the mutant enzyme shows similar activity with lactose compared to the wild-type enzyme
D557N
random, site-saturation, and site-directed mutagenesis, the mutant enzyme shows similar activity with lactose compared to the wild-type enzyme
E649C
-
mutant accepts methyl beta-D-glucoside and ethyl beta-D-glucoside as substrates
G681S
random, site-saturation, and site-directed mutagenesis, the mutant enzyme shows reduced activity with lactose compared to the wild-type enzyme
N156D/N163D/T508I/E649G/N667A
-
mutant with very broad acceptor specificity
T508A/N667T
random, site-saturation, and site-directed mutagenesis, the mutant enzyme shows increased activity with lactose compared to the wild-type enzyme
T508I/E649C/N667A
-
triple mutation broadens acceptor specificity
T508I/N667A
A423S
-
mutation introduced to increase thermostability, mutant displays a half-life of 11.3 min at 70C
A781K
-
mutation introduced to increase thermostability, mutant displays a half-life of 15.3 min at 70C
Q130H/K131Y/S411G/A423S/A781K
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mutations introduced to increase thermostability, mutant displays a half-life of 17.7 min at 70C
R48R/Q130H/K131Y/K142R/S411G/A423S/V526A/A781K
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mutations introduced to increase thermostability, mutant displays a half-life of 24.6 min at 70C
S411G
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mutation introduced to increase thermostability, mutant displays a half-life of 13.6 min at 70C
C485A
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the mutant shows 43% of wild type activity with cellobiose. The mutant shows higher preference for D-glucosamine than the wild type enzyme
E654A
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inactive
E654D
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inactive
Y648A
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inactive
Y648F
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The mutant shows 23% of wild type activity with cellobiose. Apparent kcat/Km values of the mutant for D-mannose and 2-deoxy-D-glucose are 8.2 and 4.0fold higher than those of the wild type enzyme, respectively
Y648V
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the mutant shows 0.888% of wild type activity with cellobiose and has synthetic activity toward N-acetyl-D-glucosamine
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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quantitative determination of cellobiose in presence of glucose or glucose-1-phosphate
synthesis