Information on EC 3.2.1.176 - cellulose 1,4-beta-cellobiosidase (reducing end)

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

EC NUMBER
COMMENTARY hide
3.2.1.176
-
RECOMMENDED NAME
GeneOntology No.
cellulose 1,4-beta-cellobiosidase (reducing end)
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Hydrolysis of (1->4)-beta-D-glucosidic linkages in cellulose and similar substrates, releasing cellobiose from the reducing ends of the chains.
show the reaction diagram
-
-
-
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SYSTEMATIC NAME
IUBMB Comments
4-beta-D-glucan cellobiohydrolase (reducing end)
Some exocellulases, most of which belong to the glycoside hydrolase family 48 (GH48, formerly known as cellulase family L), act at the reducing ends of cellulose and similar substrates. The CelS enzyme from Clostridium thermocellum is the most abundant subunit of the cellulosome formed by the organism. It liberates cellobiose units from the reducing end by hydrolysis of the glycosidic bond, employing an inverting reaction mechanism [2]. Different from EC 3.2.1.91, which attacks cellulose from the non-reducing end.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
the enzyme belongs to the glycoside hydrolase family 7, GH7
malfunction
metabolism
cellulosomal enzyme systems utilize self-assembled scaffolded multimodule enzymes to deconstruct biomass, structure-function relationships governing the action of the large cellulosomal enzyme complex
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(cellobiose)n + H2O
(cellobiose)n-1 + cellobiose
show the reaction diagram
-
release of cellobiose from the reducing end. The enzyme uses a single displacement mechanism resulting in inversion of the anomeric configuration
-
-
?
2,6-diaminopyridine-cellulose + H2O
?
show the reaction diagram
2-chloro-4-nitrophenyl beta-D-cellobioside + H2O
2-chloro-4-nitrophenol + D-cellobiose
show the reaction diagram
4-methylumbelliferyl beta-D-lactoside + H2O
4-methylumbelliferol + D-lactose
show the reaction diagram
4-methylumbelliferyl beta-lactoside + H2O
4-methylumbelliferol + beta-lactose
show the reaction diagram
-
-
-
-
?
4-methylumbelliferyl-beta-D-lactoside + H2O
4-methylumbelliferol + D-lactose
show the reaction diagram
4-nitrophenyl beta-D-cellobioside
4-nitrophenol + D-cellobiose
show the reaction diagram
4-nitrophenyl beta-D-cellobioside + H2O
4-nitrophenol + cellobiose
show the reaction diagram
4-nitrophenyl beta-D-cellobioside + H2O
4-nitrophenyl + D-cellobiose
show the reaction diagram
4-nitrophenyl beta-D-lactopyranoside + H2O
4-nitrophenol + beta-D-lactopyranose
show the reaction diagram
4-nitrophenyl beta-D-lactoside
4-nitrophenol + beta-D-lactose
show the reaction diagram
-
-
-
-
4-nitrophenyl beta-D-lactoside + H2O
4-nitrophenol + beta-D-lactose
show the reaction diagram
-
-
-
-
?
4-nitrophenyl beta-D-lactoside + H2O
4-nitrophenol + D-lactose
show the reaction diagram
4-nitrophenyl beta-D-lactoside + H2O
4-nitrophenol + lactose
show the reaction diagram
4-nitrophenyl beta-D-lactoside + H2O
?
show the reaction diagram
4-nitrophenyl cellotrioside + H2O
cellobiose + 4-nitrophenyl beta-D-glucoside
show the reaction diagram
4-nitrophenyl D-cellobioside + H2O
4-nitrophenol + cellobiose
show the reaction diagram
-
-
-
-
?
4-nitrophenyl lactoside + H2O
4-nitrophenol + lactose
show the reaction diagram
amorphous cellulose + H2O
?
show the reaction diagram
amorphous cellulose + H2O
cellobiose
show the reaction diagram
aqueous ammonia-pretreated corn stover + H2O
?
show the reaction diagram
-
-
-
-
?
avicel + H2O
?
show the reaction diagram
avicel + H2O
cellobiose
show the reaction diagram
avicel + H2O
cellobiose + ?
show the reaction diagram
Avicel PH 101 + H2O
?
show the reaction diagram
cellobiose production is monitored by an amperometric enzyme biosensor based on cellobiose dehydrogenase from Phanerochaete chrysosporium adsorbed onto the surface of a benzoquinone-modified carbon paste electrode
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-
?
bacterial cellulose + H2O
?
show the reaction diagram
bacterial cellulose + H2O
cellobiose + ?
show the reaction diagram
-
-
-
?
bacterial cellulose + H2O
cellobiose + cellotriose
show the reaction diagram
-
-
no release of D-.glucose is observed
-
?
beta-glucan + H2O
?
show the reaction diagram
-
-
-
-
?
carboxymethyl cellulose + H2O
?
show the reaction diagram
-
-
-
-
?
carboxymethyl cellulose + H2O
cellobiose + ?
show the reaction diagram
cellooligosaccharide + H2O
cellobiose
show the reaction diagram
cellopentaose + H2O
cellobiose + cellotriose
show the reaction diagram
-
-
-
?
cellopentaose + H2O
cellotriose + cellobiose
show the reaction diagram
cellotetraose + H2O
2 cellobiose
show the reaction diagram
cellulose + H2O
?
show the reaction diagram
cellulose + H2O
alpha-cellobiose + beta-cellobiose
show the reaction diagram
the enzyme hydrolyzes the beta-1,4 linkages of a cellulose chain from its reducing end via a retaining mechanism liberating the product, which consists of roughly 63% beta-cellobiose and 37% alpha-cellobiose
-
-
?
cellulose + H2O
cellobiose
show the reaction diagram
cellulose + H2O
cellobiose + ?
show the reaction diagram
cellulose Ialpha + H2O
cellobiose + ?
show the reaction diagram
cellulose IIII + H2O
cellobiose + ?
show the reaction diagram
corn stover + H2O
cellobiose + ?
show the reaction diagram
-
-
-
?
crystalline cellulose + H2O
?
show the reaction diagram
filter paper + H2O
?
show the reaction diagram
21% activity compared to phosphoric acid-swollen cellulose
-
-
?
lichenan + H2O
cellobiose + ?
show the reaction diagram
-
-
-
?
lignocellulose + H2O
cellobiose
show the reaction diagram
microcrystalline cellulose + H2O
?
show the reaction diagram
-
-
-
?
microcrystalline cellulose + H2O
cellobiose
show the reaction diagram
microcrystalline cellulose + H2O
cellobiose + ?
show the reaction diagram
-
-
-
?
milled aspen wood + H2O
?
show the reaction diagram
-
-
-
-
?
phosphoric acid swollen cellulose + H2O
cellobiose + ?
show the reaction diagram
-
-
-
?
phosphoric acid-swollen cellulose + H2O
?
show the reaction diagram
phosphoric acid-swollen cellulose + H2O
cellobiose + cellotriose
show the reaction diagram
-
-
no release of D-.glucose is observed
-
?
pretreated corn stover + H2O
?
show the reaction diagram
reduced cellulose + H2O
?
show the reaction diagram
sigmacell + H2O
?
show the reaction diagram
2% activity compared to phosphoric acid-swollen cellulose
-
-
?
spruce + H2O
?
show the reaction diagram
-
-
-
-
?
additional information
?
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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
2,6-diaminopyridine-cellulose + H2O
?
show the reaction diagram
amorphous cellulose + H2O
?
show the reaction diagram
Avicel PH 101 + H2O
?
show the reaction diagram
G0RVK1
cellobiose production is monitored by an amperometric enzyme biosensor based on cellobiose dehydrogenase from Phanerochaete chrysosporium adsorbed onto the surface of a benzoquinone-modified carbon paste electrode
-
-
?
bacterial cellulose + H2O
?
show the reaction diagram
cellulose + H2O
?
show the reaction diagram
G0RVK1
cellobiohydrolases are exo-active glycosyl hydrolases that processively convert cellulose to soluble sugars, typically cellobiose. Occuring opposite effects on binding and activity by the enzyme can be reconciled if the rate-limiting step is after the catalysis (i.e. in the dissociation process), analysis of the rate-limiting step for cellobiohydrolases, overview
-
-
?
cellulose + H2O
alpha-cellobiose + beta-cellobiose
show the reaction diagram
P62694
the enzyme hydrolyzes the beta-1,4 linkages of a cellulose chain from its reducing end via a retaining mechanism liberating the product, which consists of roughly 63% beta-cellobiose and 37% alpha-cellobiose
-
-
?
cellulose + H2O
cellobiose
show the reaction diagram
reduced cellulose + H2O
?
show the reaction diagram
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
107% activity at 1 mM
Co2+
127% activity at 1 mM
dithiothreitol
108% activity at 1 mM
K+
115% activity at 1 mM
Li+
110% activity at 1 mM
Mg2+
115% activity at 1 mM
Mn2+
115% activity at 1 mM
Triton X-100
110% activity at 1% (v/v)
Tween 20
116% activity at 1% (v/v)
additional information
not influenced by Ni2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,3-dimethylimidazolium dimethylphosphate
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complete inhibition at 30% v/v, ionic liquids inactivate cellulases, the enzyme is sensitive against 1,3-dimethylimidazolium dimethylphosphate and 1-ethyl-3-methylimidazolium acetate, effects on cellulase substrate binding, overview
1-ethyl-3-methylimidazolium acetate
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complete inhibition at 30% v/v, ionic liquids inactivate cellulases, the enzyme is sensitive against 1,3-dimethylimidazolium dimethylphosphate and 1-ethyl-3-methylimidazolium acetate, effects on cellulase substrate binding, overview
arbutin
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20 mg/ml, 30% inhibition
birchwood xylan
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mixed inhibition
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cellobiose
Cu2+
53% residual activity at 1 mM
ethanol
41% residual activity at 1 mM
Fe2+
93% residual activity at 1 mM; % residual activity at 1 mM
lactose
-
inhibits recombinant CelS activity on cellopentaose, 81% inhibition at 20 mg/ml lactose
mannobiose
competitive
Mannotetraose
competitive
Mannotriose
competitive
methanol
68% residual activity at 1 mM
SDS
complete inhibition at 0.1% (w/v)
xylobiose
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mixed inhibition
xylopentaose
-
mixed inhibition
xylotriose
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mixed inhibition
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Tween 80
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specific activator, 20% increase of activity at 1 mM
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.84
2-chloro-4-nitrophenyl beta-D-cellobioside
catalyticdomain, pH 5.0, 37°C
0.293 - 2.1
4-methylumbelliferyl beta-D-lactoside
0.57 - 1.02
4-nitrophenyl beta-D-cellobioside
3.4
4-nitrophenyl beta-D-lactopyranoside
0.7 - 3.4
4-nitrophenyl beta-D-lactoside
0.44 - 0.81
4-nitrophenyl D-cellobioside
0.0016
4-nitrophenyl lactoside
-
pH 5.0, 37°C
1.4
Cellopentaose
-
pH 5.7, temperature not specified in the publication
additional information
Avicel
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.11
2-chloro-4-nitrophenyl beta-D-cellobioside
catalyticdomain, pH 5.0, 37°C
0.055 - 1.35
4-methylumbelliferyl beta-D-lactoside
0.1 - 0.112
4-nitrophenyl beta-D-cellobioside
0.27 - 0.6
4-nitrophenyl beta-D-lactopyranoside
0.19 - 0.51
4-nitrophenyl beta-D-lactoside
1.8 - 2
amorphous cellulose
-
2.6 - 2.8
bacterial cellulose
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.131
2-chloro-4-nitrophenyl beta-D-cellobioside
catalyticdomain, pH 5.0, 37°C
0.063 - 1.37
4-methylumbelliferyl beta-D-lactoside
0.118 - 0.196
4-nitrophenyl beta-D-cellobioside
0.178
4-nitrophenyl beta-D-lactoside
catalyticdomain, pH 5.0, 37°C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0187 - 0.295
cellobiose
0.0097 - 0.067
xylobiose
0.014 - 0.026
xylopentaose
0.029 - 0.056
xylotriose
additional information
birchwood xylan
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Kic 0.093 g/l, Kiu 0.18 g/l, pH 5.0, 37°C
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.48
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pH 5.0, 22°C, wild-type
0.51
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pH 5.0, 22°C, fusion protein with CBM1 from Hypocrea jecorina Cel7A
0.52
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pH 5.0, 22°C, fusion protein with CBM3 derived from Clostridium thermocellum cellulosomal-scaffolding protein CipA
0.53
-
pH 5.0, 22°C, fusion protein with CBM1 from Hypocrea jecorina Cel7A containing an additional S-S bridge in the catalytic module
0.56
-
pH 5.0, 22°C, fusion protein with CBM2 from Cellulomonas fimi xylanase 10A
0.57
-
pH 5.0, 22°C, fusion protein with CBM3 derived from Clostridium thermocellum cellulosomal-scaffolding protein CipA containing an additional S-S bridge in the catalytic module
1860
substrate carboxymethyl cellulose, pH 5.0, 37°C
1910
substrate lichenan, pH 5.0, 37°C
2150
substrate avicel, pH 5.0, 37°C
40000
substrate bacterial cellulose, pH 5.0, 37°C
275000
substrate phosphoric acid swollen cellulose, pH 5.0, 37°C
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 6
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activity of recombinant CelS on amorphous cellulose
5 - 6
assay at
6 - 6.5
hydrolysis of swollen cellulose
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 6.5
more than 85% of maximum activity
4 - 6
more than 60% activity between pH 4.0 and 6.0
5 - 9
pH 5: about 45% of maximal activity, pH 9.0: about 45% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50 - 75
more than 45% activity between 50 and 75°C
50 - 65
-
the activity is highest at 53°C and slightly lower at 65°C, whereas the enzyme is practically inactive at 75°C
additional information
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
isoelectric focusing
4.2
calculated from sequence
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
outer membrane
Manually annotated by BRENDA team
PDB
SCOP
CATH
UNIPROT
ORGANISM
Clostridium thermocellum;
Q55FE6
Dictyostelium discoideum;
F0ZJZ1
Dictyostelium purpureum;
P15828
Humicola grisea var. thermoidea;
G2Q665
Myceliophthora thermophila (strain ATCC 42464 / BCRC 31852 / DSM 1799);
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
46000
x * 75000, full-length enzyme, x * 46000, catalytic domain, SDS-PAGE
75000
x * 75000, full-length enzyme, x * 46000, catalytic domain, SDS-PAGE
75244
x * 75244, calculated from sequence
80670
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x * 80670, calculated from sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
three-dimensional structure of the catalytic domain of TrCel7A cellobiohydrolase, overview
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
proteolytic modification
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structures in complex with cellobiose, cellotetraose and triethylene glycol molecules. The product cellobiose occupies subsites +1 and +2 in the open active-site cleft of the enzyme-cellotetraose complex structure, and three triethylene glycol molecules and one pentaethylene glycol molecule are located at active-site subsites -2 to -6 in the structure of the ExgS-triethylene glycol complex. Glu50 acts as a proton donor and Asp222 plays a nucleophilic role
sitting drop vapor diffusion method, using 12.5% (w/v) polyethylene glycol 1000, 12.5% (w/v) PEG 3350, 12.5% (v/v) 2-methyl-2,4-pentanediol, 0.1 M sodium HEPES-MOPS pH 7.5, 0.03 M MgCl2, and 0.03 M CaCl2
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to 2.1 A resolution. Protein natively consists of a catalytic domain and does not exhibit a carbohydrate-binding module. Strucutre is homologous to those of other GH7 CBHs with an enclosed active-site tunnel
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sitting drop vapor diffusion method, using 0.1 M sodium malonate, pH 7.0 and 12% (w/v) PEG 3350
to 1.8 A resolution. Protein natively consists of a catalytic domain and does not exhibit a carbohydrate-binding module. Structure is homologous to those of other GH7 CBHs with an enclosed active-site tunnel
structures, with and without bound thio-oligosaccharides, show conformational diversity of tunnel-enclosing loops, including a form with partial tunnel collapse at subsite -4
protein lacking the C-terminal linker-CBM1 part, yielding crystals of space group P212121 with two protein molecules, chains A and B, in the asymmetric unit, to 1.8 A resolution. The catalytic triad consisting of Glu213 (nucleophile), Asp215 and Glu218 (acid/base), and the tryptophan platforms at subsites -7, -4, -2 and +1 are highly conserved; structure of catalytic module, 1.8 A resolution. The crystal structure of the enzyme reveals considerable flexibility of the active-site-defining loop regions
crystals of truncated CelS in complex with cellobiose are obtained by ammonium sulfate precipitation, crystal structure of the catalytic domain of Clostridium thermocellum CelS in complex with oligosaccharides
purified recombinant enzyme, sitting drop vapor diffusion method, mixing of 0.001 ml each of 80 mg/ml protein, in 20 mM Tris, pH 7.0, with 100 mM NaCl, and 1.9 M sodium malonate, pH 6.0, or with 1.9 M sodium malonate, pH 6.5, 20% v/v ethylene glycol, 50 mM KI. In a second method, 15 mg/ml protein in 20 mM acetic acid buffer, pH 5.0, with 100 mM NaCl is mixed with 0.2 M ammonium iodide, 20% w/v PEG 3350, pH 6.2, X-ray diffraction structure determination and analysis at 1.7 A resolution, single isomorphous replacement with anomalous signal from iodine and molecular-replacement
3D structural modeling of the Cel7A catalytic module with N-linked glucans attached to Asn45, Asn194, and Asn388 glycosylation sites. Residue Asn45 is located near the entrance to the enzyme active site tunnel, while the two other N-glycosylation sites are located on the loops forming the tunnel, Asn194 being closer to the entrance and Asn388 being closer to the exit of the tunnel
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mutant D224N, to 1.6 A resolution. There is one molecule in the asymmetric unit in complex with a cellobiose and a cellohexaose molecule. The active site tunnel entrance is enriched in aromatic residues. Only the aromatic residues located around the tunnel entrance appear to be important for the ability of Cel48A to access individual cellulose chains on bacterial cellulose
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hanging drop vapor diffusion method, using 25% (w/v) PEG 8K and 2.5 mM CaCl2
sitting drop vapor diffusion method, using 25.5% (w/v) PEG 4000, 0.17 M ammonium sulfate, and 15% (v/v) glycerol
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structures in complex with xylotriose, xylotetraose and xylopentaose reveal a predominant binding mode at the entrance of the substrate-binding tunnel of the enzyme, in which each xylose residue is shifted about 2.4 A towards the catalytic center compared with binding of cellooligosaccharides. Partial occupancy of two consecutive xylose residues at subsites -2 and -1 suggests an alternative binding mode for xylooligosaccharides in the vicinity of the catalytic center
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 7
24 h, 37°C, stable
736095
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50 - 65
-
the enzyme is stable at 50°C at least for 3 h. At 60 and 65°C, the enzyme half-life times are in the range of 40-43 min and 11-13 min, respectively
50 - 70
the enzyme has a half-life of 60 min at 70°C and after incubation for 72 h retains more than 80% of enzymatic activity at 60°C and 100% at 50°C, respectively
62
melting temperature
62 - 69
-
the melting temperature is 62.5°C. The wild type enzyme shows half-lives of 19.9, 9.3 and 8.6 min at 62°C, 66°C, and 69°C, respectively
64.2 - 74.7
-
melting tempeartures, recombinant expression in Saccharomyces cerevisiae and in Neurospora crassa. The Cel7A expressed in Neurospora crassa has a by 10°C higher melting temperature and higher specific activity than the Cel7A expressed in Saccharomyces cerevisiae
72
-
melting temperature, fusion protein with CBM2 from Cellulomonas fimi xylanase 10A
72.5
melting temperature
73
-
melting temperature, wild-type
74
-
melting temperature, fusion protein with CBM1 from Hypocrea jecorina Cel7A
75
-
melting temperature, fusion protein with CBM3 derived from Clostridium thermocellum cellulosomal-scaffolding protein CipA
75.5
-
melting temperature, fusion protein with CBM1 from Hypocrea jecorina Cel7A containing an additional S-S bridge in the catalytic module
77
-
melting temperature, fusion protein with CBM3 derived from Clostridium thermocellum cellulosomal-scaffolding protein CipA containing an additional S-S bridge in the catalytic module
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation and phenyl Sepharose resin column chromatography
-
ammonium sulfate precipitation, phenyl Sepharose column chromatography, Source 15Q column chromatography, and Superdex 75 gel filtration
ammonium sulfate precipitation, Source 15Q column chromatography, and Source 15 isopropyl column chromatography
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phenyl Sepharose column chromatography, Source 15Q column chromatography, and Sephadex G-25 gel filtration
-
Q Sepharose column chromatography and DEAE-Sephadex gel filtration
recombinant -type and mutant W40A enzymes from Trichoderma reesei strain ALKO 3413 culture filtrate by gel filtration, anion exchange, hydrophobic interaction, and affinity chromatography
recombinant C-terminally His6-tagged enzyme from Saccharomyces cerevisiae by anion exchange chromaatography and dialysis, recombinant enzyme from Neurospora crassa by ammonium sulfate fractionation, gel filtration, anion exchange chromatography and dialysis
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recombinant His6-tagged enzyme from Escherichia coli (BL21) by cation exchange and hydrophobic interaction chromatography, followed by another step of cation exchange and anion exchange chromatography and gel filtration
the enzyme and catalytic domain alone are each expressed in and purified from Streptomyces lividans
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned into Escherichia coli and Streptomyces lividans
expressed in Aspergillus niger variant awamori AP4
-
expressed in Aspergillus oryzae
-
expressed in Escherichia coli strains BL21, Origami, and Origami B
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expressed in Penicillium canescens strain PCA10
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expressed in Pichia pastoris Pichia Pink strain2
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expressed in Trichoderma reesei
expression in Escherichia coli
-
expression in Penicillium canescens PCA10
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gene cel7A, heterologous expression of wild-type and mutant enzymes in Aspergillus oryzae
gene cel7A, recombinant expression of wild-type and mutant W40A enzymes under the control of its own promoter in Trichoderma reesei strain ALKO 3413 lacking the genes encoding for endogenous Cel7A and Cel7B
gene cel7AF, recombinant expression of extracellular C-terminally FLAG-tagged enzyme with its native signal peptide in Saccharomyces cerevisiae strain CEN.PK102-3, the enzyme is secreted to the culture medium
recombinant expression of C-terminally His6-tagged enzyme in Saccharomyces cerevisiae, and expression in Neurospora crassa using an Neurospora crassa codon bias and Neurospora crassa CBHI signal peptide and cloned in pCSR1:GPD vector, which directs gene integration to the csr-1 locus. Gene expression is promoted by the constitutive Myceliophthora thermophila gpdA promoter. The Cel7A expressed in Neurospora crassa has a higher melting temperature and higher specific activity than the Cel7A expressed in Saccharomyces cerevisiae. The underlying cause of this disparity is the lack of N-terminal glutamine cyclization in the Cel7A expressed in Saccharomyces cerevisiae. Treating the enzyme in vitro with glutaminyl cyclase improves the properties of Cel7A expressed in Saccharomyces cerevisiae to match those of Cel7A expressed in Neurospora crassa
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recombinant expression of His6-tagged enzyme in Escherichia coli (BL21)
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
celS appears to be regulated at the transcriptional level, and its expression is modulated by growth rate both under conditions of cellobiose and nitrogen limitation. The amount of celS mRNA transcripts per cell is about 180 for cells grown under carbon limitation at growth rates of 0.04 to 0.21 per h and 80 and 30 transcripts per cell for batch cultures at growth rates of 0.23 and 0.35 per hour, respectively. Under nitrogen limitation, the corresponding levels were 110, 40, and 30 transcripts/cell for growth rates of 0.07, 0.11, and 0.14 per hour, respectively. Two major transcriptional start sites are detected at positions 140 and 145 bp, upstream of the translational start site of the celS gene. The relative activity of the two promoters remains constant under the conditions studied and is in agreement with the results of the RNase protection assay, in which the observed transcriptional activity is inversely proportional to the growth rate
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A201P
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predicted from structure fold, calculation of the thermostability of the mutant
A329G
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predicted from structure fold, calculation of the thermostability of the mutant
A383Y
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predicted from structure fold, calculation of the thermostability of the mutant, stabilizing mutation
D300K
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predicted from structure fold, calculation of the thermostability of the mutant
D354V
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predicted from structure fold, calculation of the thermostability of the mutant, stabilizing mutation
D52T
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predicted from structure fold, calculation of the thermostability of the mutant
E325P
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predicted from structure fold, calculation of the thermostability of the mutant
H208Y
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predicted from structure fold, calculation of the thermostability of the mutant, stabilizing mutation
H358K
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predicted from structure fold, calculation of the thermostability of the mutant
H358R
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predicted from structure fold, calculation of the thermostability of the mutant
H358V
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predicted from structure fold, calculation of the thermostability of the mutant
L113M
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predicted from structure fold, calculation of the thermostability of the mutant
N126G
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predicted from structure fold, calculation of the thermostability of the mutant
N439G
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predicted from structure fold, calculation of the thermostability of the mutant, the mutation results in loss of expression
N93K
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predicted from structure fold, calculation of the thermostability of the mutant, increased thermotability compared to the wild-type enzyme
P399G
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predicted from structure fold, calculation of the thermostability of the mutant
Q345M
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predicted from structure fold, calculation of the thermostability of the mutant
S130T
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predicted from structure fold, calculation of the thermostability of the mutant
S13P
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predicted from structure fold, calculation of the thermostability of the mutant
S13P/Y60L7S324P/A383Y7Y43
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predicted from structure fold, calculation of the thermostability of the mutant, the mutant is the most thermostable variant
S222K
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predicted from structure fold, calculation of the thermostability of the mutant
S324P
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predicted from structure fold, calculation of the thermostability of the mutant, stabilizing mutation
S57D
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predicted from structure fold, calculation of the thermostability of the mutant
S5T
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predicted from structure fold, calculation of the thermostability of the mutant
T164K
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predicted from structure fold, calculation of the thermostability of the mutant
T257 V
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predicted from structure fold, calculation of the thermostability of the mutant
T257I
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predicted from structure fold, calculation of the thermostability of the mutant
T257K
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predicted from structure fold, calculation of the thermostability of the mutant
T273K
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predicted from structure fold, calculation of the thermostability of the mutant
T273P
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predicted from structure fold, calculation of the thermostability of the mutant
T339P
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predicted from structure fold, calculation of the thermostability of the mutant
T339Q
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predicted from structure fold, calculation of the thermostability of the mutant
T392I
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predicted from structure fold, calculation of the thermostability of the mutant, stabilizing mutation
T395P
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predicted from structure fold, calculation of the thermostability of the mutant, the mutation results in loss of expression
T408D
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predicted from structure fold, calculation of the thermostability of the mutant
T41V
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predicted from structure fold, calculation of the thermostability of the mutant
V110L
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predicted from structure fold, calculation of the thermostability of the mutant
V217I
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predicted from structure fold, calculation of the thermostability of the mutant
V227L
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predicted from structure fold, calculation of the thermostability of the mutant
V331M
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predicted from structure fold, calculation of the thermostability of the mutant
V404A
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predicted from structure fold, calculation of the thermostability of the mutant
Y430F
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predicted from structure fold, calculation of the thermostability of the mutant, increased thermotability compared to the wild-type enzyme
Y60I
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predicted from structure fold, calculation of the thermostability of the mutant
Y60L
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predicted from structure fold, calculation of the thermostability of the mutant, increased thermotability compared to the wild-type enzyme
N430A
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expression of N430A mutant could not be achieved
F195A
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mutagenesis of active site tunnel residues, moderate reduction in activity
W313A
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residue is important for processivity on bacterial cellulose
W315A
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residue is important for processivity on bacterial cellulose
Y213A
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mutagenesis of active site tunnel residues, moderate reduction in activity
Y213A/S311A
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mutagenesis of active site tunnel residues, moderate reduction in activity
Y213A/W313A
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mutagenesis of active site tunnel residues, moderate reduction in activity
Y97A
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mutagenesis of active site tunnel residues, moderate reduction in activity
D214N
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the mutant shows slightly reduced activity compared to the wild type enzyme
E212Q
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the mutant loses most of its hydrolysis capability
G22D/N49S/A68T/P227L/S278P/T296P
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the mutant shows an increase in melting temperature and a greater half-life compared with the wild type enzyme
N45A
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the mutant shows 114% of wild type activity
N45A/N64A
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the mutant shows 143% of wild type activity
N64A
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the mutant shows 106% of wild type activity
S128C
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mutant generated for single-molecule fluorescence imaging analysis, shows hydrolysis activity comparable with those of the wild-type against cellulose Ialpha and IIII and similar dependence on the enzyme concentration
S8P/G22D/T41I/N49S/A68T/S113N/P227L /D249K/S278P/T296P/N301R
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the mutant shows an increase in melting temperature and a greater half-life compared with the wild type enzyme
S8P/T41I/N49S/A68T/N89D/S92T/S113N /S196T/P227L/D249K/T255P/S278P/E295K /T296P/T332Y/V403D/S411F/T462I
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the mutant, which is active up to 75°C, shows a 10.4°C increase in melting temperature and a 44fold greater half-life compared with the wild type enzyme
W40A
mutation of Trp40 at the entrance of the catalytic tunnel drastically decreases the ability to degrade crystalline cellulose. Comparison of activities of the wild-type and mutant W40A enzymes (with and without the cellulose-binding domain) for various substrates, overview
W38A
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the mutant shows an increased Km value for Avicel compared to the wild type enzyme
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S128C
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mutant generated for single-molecule fluorescence imaging analysis, shows hydrolysis activity comparable with those of the wild-type against cellulose Ialpha and IIII and similar dependence on the enzyme concentration
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additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
biotechnology
degradation
synthesis
Show AA Sequence (118 entries)
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