Information on EC 5.1.3.11 - cellobiose epimerase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
5.1.3.11
-
RECOMMENDED NAME
GeneOntology No.
cellobiose epimerase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
cellobiose = 4-O-beta-D-glucopyranosyl-D-mannose
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
epimerization
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
beta-(1,4)-mannan degradation
-
-
SYSTEMATIC NAME
IUBMB Comments
cellobiose 2-epimerase
The enzyme catalyses the interconversion between D-glucose and D-mannose residues at the reducing end of beta-1,4-linked disaccharides by epimerizing the hydroxyl group at the C-2 position of the glucose moiety.
CAS REGISTRY NUMBER
COMMENTARY hide
37318-37-9
-
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
metabolism
the enzyme is responsible for conversion of beta-1,4-mannobiose to 4-O-beta-D-mannosyl-D-glucose in mannan metabolism
additional information
determination of residues involved in substrate recognition, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
4-O-beta-D-glucosyl-D-mannose
4-O-beta-D-galactosyl-D-mannose
show the reaction diagram
4-O-beta-D-mannopyranosyl-D-mannose
4-O-beta-D-glucopyranosyl-D-mannose
show the reaction diagram
-
-
-
r
beta-mannobiose
4-O-beta-D-mannopyranosyl-D-glucose
show the reaction diagram
-
putative product
-
r
cellobiose
4-O-beta-D-glucopyranosyl-D-mannose
show the reaction diagram
cellobiose
D-glucosyl-D-mannose
show the reaction diagram
cellotetraose
4-O-beta-D-glucopyranosyl-(1-4)-O-beta-D-glucopyranosyl-(1-4)-O-beta-D-glucopyranosyl-(1-4)-D-mannose
show the reaction diagram
the enzyme is found to 2-epimerize the reducing terminal glucose moieties of cellotriose and cellotetraose in addition to cellobiose
-
-
?
cellotriose
4-O-beta-D-glucopyranosyl-(1-4)-O-beta-D-glucopyranosyl-(1-4)-D-mannose
show the reaction diagram
the enzyme is found to 2-epimerize the reducing terminal glucose moieties of cellotriose and cellotetraose in addition to cellobiose
-
-
?
cellotriose
4-O-beta-D-glucopyranosyl-4-O-beta-D-glucopyranosyl-D-mannose
show the reaction diagram
-
-
-
-
?
D-fructose
D-glucose + D-mannose
show the reaction diagram
-
-
-
-
?
D-glucose
D-mannose
show the reaction diagram
D-lyxose
D-xylose
show the reaction diagram
-
-
-
-
?
D-mannose
D-glucose
show the reaction diagram
-
-
-
-
?
D-xylose
D-lyxose
show the reaction diagram
-
-
-
-
?
D-xylulose
D-xylose + D-lyxose
show the reaction diagram
-
low activity
-
-
-
globotriose
O-alpha-D-galactopyranosyl-(1,4)-O-beta-D-galactopyranosyl-(1,4)-D-mannose
show the reaction diagram
-
putative product
-
r
L-allose
L-altrose
show the reaction diagram
-
low activity
-
-
?
L-altrose
L-allose
show the reaction diagram
-
-
-
-
?
L-arabinose
L-ribose
show the reaction diagram
-
low activity
-
-
?
L-gulose
L-idose
show the reaction diagram
-
low activity
-
-
?
L-idose
L-gulose
show the reaction diagram
-
-
-
-
?
L-psicose
L-allose + L-altrose
show the reaction diagram
-
low activity
-
-
?
L-ribulose
L-arabinose + L-ribose
show the reaction diagram
-
low activity
-
-
?
L-sorbose
L-gulose + L-idose
show the reaction diagram
-
low activity
-
-
?
lactose
4-O-beta-D-galactopyranosyl-D-mannose
show the reaction diagram
lactose
epilactose
show the reaction diagram
lactose
lactulose
show the reaction diagram
maltose
4-O-alpha-D-glucopyranosyl-D-mannose
show the reaction diagram
-
-
-
-
?
maltotriose
4-O-alpha-D-glucopyranosyl-4-O-alpha-D-glucopyranosyl-D-mannose
show the reaction diagram
-
-
-
-
?
mannose
D-glucose + D-fructose
show the reaction diagram
-
the enzyme exhibits the highest isomerization activity for mannose among monosaccharides
-
-
?
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
4-O-beta-D-mannopyranosyl-D-mannose
4-O-beta-D-glucopyranosyl-D-mannose
show the reaction diagram
F8WRK9
-
-
-
r
beta-mannobiose
4-O-beta-D-mannopyranosyl-D-glucose
show the reaction diagram
Q5LH66
-
putative product
-
r
cellobiose
4-O-beta-D-glucopyranosyl-D-mannose
show the reaction diagram
cellobiose
D-glucosyl-D-mannose
show the reaction diagram
globotriose
O-alpha-D-galactopyranosyl-(1,4)-O-beta-D-galactopyranosyl-(1,4)-D-mannose
show the reaction diagram
Q5LH66
-
putative product
-
r
lactose
4-O-beta-D-galactopyranosyl-D-mannose
show the reaction diagram
lactose
epilactose
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
-
the activity of the cellobiose 2-epimerase is not affected by the addition of the cofactor ATP, FAD, FMN, NAD+, NADP+, NADH, or NADPH
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
the enzyme is not activated by monovalent or divalent cations
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4-chloromercuribenzoate
-
-
iodoacetate
-
-
N-bromosuccinimide
-
-
additional information
-
the enzyme is not inhibited by EDTA
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
-
the enzyme is not affected by ATP
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3.7 - 146
cellobiose
70.9
D-glucose
-
in 50 mM PIPES buffer (pH 7.5), at 75°C
51.8
D-mannose
-
in 50 mM PIPES buffer (pH 7.5), at 75°C
6.56 - 145
lactose
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4.64 - 215
cellobiose
0.27
D-glucose
Caldicellulosiruptor saccharolyticus
-
in 50 mM PIPES buffer (pH 7.5), at 75°C
0.74
D-mannose
Caldicellulosiruptor saccharolyticus
-
in 50 mM PIPES buffer (pH 7.5), at 75°C
1.79 - 111
lactose
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.037 - 18.1
cellobiose
82
0.0038
D-glucose
Caldicellulosiruptor saccharolyticus
-
in 50 mM PIPES buffer (pH 7.5), at 75°C
35
0.0143
D-mannose
Caldicellulosiruptor saccharolyticus
-
in 50 mM PIPES buffer (pH 7.5), at 75°C
216
0.019 - 12.1
lactose
114
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0095
-
in 50 mM PIPES buffer (pH 7.5) containing 10 mM D-fructose as substrate at 75°C
0.035
-
in 50 mM PIPES buffer (pH 7.5) containing 10 mM D-glucose as substrate at 75°C
0.04
-
in 50 mM PIPES buffer (pH 7.5) containing 10 mM D-xylose as substrate at 75°C
0.06
-
in 50 mM PIPES buffer (pH 7.5) containing 10 mM D-lyxose as substrate at 75°C
0.15
-
in 50 mM PIPES buffer (pH 7.5) containing 10 mM D-mannose as substrate at 75°C
0.44
-
F114A mutant, kinetic value for cellobiose
0.53
-
W303F mutant, kinetic value for cellobiose
1.15
-
F114A mutant, kinetic value for cellobiose
1.4
-
M378T mutant, kinetic value for cellobiose
3
-
the specific activity toward cellobiose is 3–5 micromol/min/mg at a pH optimum 7.8 in 100 mM glycylglycine buffer
3.6
-
in 50 mM PIPES buffer (pH 7.5) containing 10 mM cellobiose as substrate at 75°C
5.6
-
M106T mutant, kinetic value for cellobiose
5.99
-
W303F mutant, kinetic value for cellobiose
6.3
-
M179T mutant, kinetic value for cellobiose
6.35
-
M212T mutant, kinetic value for cellobiose
6.6
-
R377A mutant, kinetic value for cellobiose
8.73
-
M96T mutant, kinetic value for cellobiose
9
-
M270T mutant, kinetic value for cellobiose
10.9
-
M62T mutant, kinetic value for cellobiose
12.1
-
R377A mutant, kinetic value for cellobiose
12.3
-
M95T mutant, kinetic value for cellobiose
14.2
-
purified recombinant His-tagged enzyme, substrate lactose, pH 7.0, 70°C
16.8 - 24.4
-
purified recombinant His-tagged enzyme, substrate cellolbiose, pH 7.0, 70°C
22.7
-
W55F mutant, kinetic value for cellobiose
25.8
-
M380T mutant, kinetic value for cellobiose
29.9
crude extract after expression in Escherichia coli cells
30
-
purified recombinnat His-tagged enzyme, substrate lactose, pH 7.5, 80°C
42
-
W55F mutant, kinetic value for cellobiose
58.3
recombinant enzyme after first purification step on a DEAE Sepharose FF column
61.8
recombinant enzyme after second purification step on a hydroxyapatite column, substrate is cellobiose
79.7
recombinant enzyme after second purification step on a hydroxyapatite column, substrate is lactose
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3 - 9
-
activity range, profile, overview
5 - 5.5
-
no activity
6 - 9.5
-
activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
16
-
maximum activity when reaction mixture is incubated for 25 h
31
-
maximum activity when reaction mixture is incubated for 5.5 h
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50 - 100
-
activity range, profile, overview
50 - 90
-
high activity within this range
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.3
-
sequence calculation
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
41100
determined by SDS-PAGE, the mass discrepancy putatively results from partial hydrolysis at the C-terminus during expression or in the purification steps
43700
-
approximate molecular weight for wild type and mutant proteins, determined by SDS-PAGE
44000
-
gel filtration
45780
calculated by amino acid sequence, confirmed by SDS-PAGE
46700
x * 46963, calculated, x * 46700, SDS-PAGE
46963
x * 46963, calculated, x * 46700, SDS-PAGE
47200
-
1 * 47000, recombinant His-tagged enzyme, SDS-PAGE, 1 * 47200, about, recombinant His-tagged enzyme, sequence calculation
47340
-
1 * 47340, calculated from amino acid sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
-
enzyme structure modeling with bound mannose, three-dimensional structure, overview
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant enzyme in apoform and complexed with D-glucose-D-mannose, epilactose, and reaction intermediate analogue cellobiitol, sitting drop vapor diffusion method, mixing of 500 nl of 5 mg/ml protein in 30 mM Tris-HCl, pH 8.5, and 60 mM NaCl, with 500 nl reservoir solution containing 0.1 M sodium acetate, pH 4.5, and 1.2 M ammonium hydrogen phosphate, 5 days, 20°C, method optimization, X-ray diffraction structure determination and analysis at 1.74-2.19 A resolution
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.2 - 10.8
-
purified recombinant enzyme, stable at
727191
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
23
-
unstable
38
-
rapid loss of activity
60 - 80
-
the half-lives of the enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C are 142, 71, 35, 18, and 4.6 h, respectively
65 - 85
-
purified recombinant His-tagged enzyme, thermal inactivation of the cellobiose 2-epimerase follows first-order kinetics and the half-lives of the enzyme at 65°C, 70°C, 75°C, 80°C, and 85°C are 79 h, 55 h, 35 h, 20 h, and 4.8 h, respectively
80
-
purified recombinant enzyme, stable up to
additional information
-
the enzyme displays first-order kinetics for thermal inactivation, and the half-lives (inactivation rate constants) at 65°C, 70°C, 75°C, and 80°C are 74 h, 34 h, 16h , and 5 h, respectively
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by anion exchange chromatography, enzyme is further purified on a hydroxyapatite column
His-Trap HP column chromatography, gel filtration
-
protein is purified by loading the extract directly onto coupled columns (15 x 150 mm each) of CM Sepharose Fast Flow and DEAE Sepharose CL-6B
-
purification of wild-type and mutant proteins by successive chromatography on columns of Q-Sepharose FF and hydroxyapatite
-
recombinant enzyme
recombinant enzyme from Escherichia coli
-
recombinant enzyme from Escherichia coli by ammonium sulfate fractionation, ultrafiltration, and gel filtration
-
recombinant enzyme from Escherichia coli by heat treatment, anion exchange chromatography, and dialysis
recombinant His-tagged enzyme 10fold from Escherichia coli by heat treatment and metal affinity chromatography
-
recombinant His-tagged enzyme 10fold from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
-
expressed in Escherichia coli ER2566 cells
-
expression in Escherichia coli
expression of His-tagged enzyme in Escherichia coli
-
expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
-
gene RmCE, DNA and amino acid sequence determination and analysis, recombinant expression in Escherichia coli
-
gene RmCE, recombinant expression in Escherichia coli
-
overexpression in Escherichia coli
-
recombinant expression in Escherichia coli
recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
-
recombinant wild type and mutants are expressed in BL21(DE3) Escherichia coli cells
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E246A
-
production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose
E246Q
-
production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose
E308A
-
production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose
E308D
-
production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose
E308Q
-
production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose
F114A
-
production by site-directed mutagenesis, mutant has a very low specific activity toward cellobiose and lactose, residue protrudes into the active-site cleft in the model core barrel structure
H184A
-
site-directed mutagenesis
H184N
-
site-directed mutagenesis
H243A
-
production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose
H243K
-
production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose
H243R
-
production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose
H374A
-
production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose
H374K
-
production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose
H374R
-
production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose
M106T
-
mutant is active
M179T
-
mutant is active
M212T
-
mutant is active
M251T
-
mutant is inactive
M270T
-
mutant is active
M378T
-
mutant is inactive
M380T
-
mutant is active
M62T
-
mutant is active
M95T
-
mutant is active
M96T
-
mutant is active
R377A
-
production by site-directed mutagenesis, mutant retains 43% of the activity toward cellobiose and 23% activity toward lactose, compaired to the wild type
R377H
-
production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose
R52A
-
production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose
R52H
-
production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose
R52K
-
production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose
W249F
-
mutant completely loses acitivity toward cellobiose and lactose
W303F
-
11% and 1.9% activity toward cellobiose and lactose, compared to the wild type, contributes to catalysis, residue protrudes into the active-site cleft in the model core barrel structure
W304F
-
mutant completely loses acitivity toward cellobiose and lactose
W369A
-
site-directed mutagenesis
W55F
-
mutant is active, the specific activities toward cellobiose and lactose of the mutant retains more than 80% of those of the wild type enzyme although their Km values are increase greatly
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
nutrition
-
cellobiose 2-epimerase is an attractive enzyme to produce epilactose (4-O-beta-Dgalactosyl-D-mannose) from lactose. Epilactose is a non-digestible oligosaccharide, and enhances proliferation of bifidobacilli and lactobacilli in the human gut
synthesis
-
cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus is used in the presence of borate to increase the production of lactulose from lactose, method optimization, incubation at pH 7.5 and 80°C for 3 h, with a conversion yield of 88% and a productivity of 205 g/l/h, overview
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