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Information on EC 3.2.1.176 - cellulose 1,4-beta-cellobiosidase (reducing end) and Organism(s) Acetivibrio thermocellus and UniProt Accession A3DCH2
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3.2.1.176 cellulose 1,4-beta-cellobiosidase (reducing end)IUBMB Comments
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 . Different from EC 3.2.1.91, which attacks cellulose from the non-reducing end.
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- 3.2.1.176
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crew
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subsystem
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mission
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hydroponic
- cellulosome
- thermocellum
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bioregenerative
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life-support
- cellulases
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lunar
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long-duration
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inedible
- degradation
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breadboard
- avicel
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dockerin
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moon
- synthesis
- biotechnology
- biofuel production
- analysis
- paper production
The taxonomic range for the selected organisms is: Acetivibrio thermocellus
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Reaction Schemes
2
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2
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Hydrolysis of (1->4)-beta-D-glucosidic linkages in cellulose and similar substrates, releasing cellobiose from the reducing ends of the chains.
Synonyms
celss, cel48s, cbh-1, cbhi.1, cel48c, gh7 cbh, cbh7b, cellobiohydrolase cels, 1,4-beta-d-glucan cellobiohydrolase i, 1,4-beta-d-glucan-cellobiohydrolase i, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
Cel48A
cellobiohydrolase CelS
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Cellulase SS
celS
CelSS
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endoglucanase SS
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Cel48A
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Cellulase SS
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celS
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Hydrolysis of (1->4)-beta-D-glucosidic linkages in cellulose and cellotetraose, releasing cellobiose from the non-reducing ends of the chains
mechanism
PATHWAY SOURCE
PATHWAYS
BRENDA
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.
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(cellobiose)n + H2O
(cellobiose)n-1 + cellobiose
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release of cellobiose from the reducing end. The enzyme uses a single displacement mechanism resulting in inversion of the anomeric configuration
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-
?
4-nitrophenyl beta-D-cellotetraoside + H2O
?
enzyme activity starts from the reducing end in a processive mode after making random cuts
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-
?
4-nitrophenyl cellotrioside + H2O
cellobiose + 4-nitrophenyl beta-D-glucoside
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-
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?
carboxymethylcellulose + H2O
glucose + cellobiose + cellutetraose
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-
-
-
?
phosphoric acid-swollen cellulose + H2O
?
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about 30% activity compared to Avicel
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-
?
cellulose + H2O
cellobiose + ?
the recombinant gene product is active on cellodextrins, barley beta-glucan, carboxymethylcellulose and insoluble cellulose. Cellobiose is the only product released from amorphic and crystalline cellulose, cellotetraose and higher cellooligosaccharides. The cleavage pattern of p-nitrophenyl beta-D-cellotetraoside, blockage of the hydrolysis of NaBH4-reduced cellopentaose and the reduction in substrate viscosity suggests activity from the reducing end in a processive mode after making random cuts. Binding to insoluble, i.e. amorphous, and crystalline cellulose is mediated by the carbohydrate-binding module CBM3b, with a preference for the crystalline substrate
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-
?
additional information
?
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role in the cellusome, an active cellulase system
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?
additional information
?
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role in the cellusome, an active cellulase system
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?
additional information
?
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enzyme is exclusively an exocellulase
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?
additional information
?
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rCelO completely lacks chromogenic activity on the p-nitrophenyl-cellodextrins from p-nitrophenyl glucoside to 4-nitrophenyl cellopentaoside
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?
additional information
?
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rCelO completely lacks chromogenic activity on the p-nitrophenyl-cellodextrins from p-nitrophenyl glucoside to 4-nitrophenyl cellopentaoside
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?
additional information
?
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no activity with pectin, xylan, cellobiose and cellotriose
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-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
additional information
?
-
-
role in the cellusome, an active cellulase system
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-
?
additional information
?
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role in the cellusome, an active cellulase system
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-
?
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
cellobiose
inhibits recombinant CelS activity on cellopentaose. Complete inhibition at 10 mg/ml cellobiose. Cellobiose appeared to inhibit the CelS activity through a competitive mechanism. The inhibition is relieved when beta-glucosidase is added, presumably because of the conversion of cellobiose into glucose
lactose
inhibits recombinant CelS activity on cellopentaose, 81% inhibition at 20 mg/ml lactose
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.4
cellopentaose
pH 5.7, temperature not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
5.7
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4.5 - 7
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more than 85% activity between pH 4.5 and 6.5, about 75% activity at pH 7.0
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
60
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
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the amount of CelS in the supernatant fluids of cellobiose-grown cultures is lower than that of cellulose-grown cultures
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
malfunction
physiological function
additional information
crystal structures of the two X1 modules from the enzyme CbhA
malfunction
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CelS knockout strains show reduced growth rate and cell yield on cellulose
malfunction
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family 48 glycoside hydrolases are not necessary for growth of Clostridium thermocellum on crystalline cellulose. Creation of knockout mutants for Cel48S, the most abundant cellulosome subunit. The cellulose hydrolysis rate of the S mutant strain is 60% lower than the parent strain, with the S mutant strain also exhibiting a 40% reduction in cell yield. Although most cellulolytic bacteria have one family 48 cellulase, Clostridium thermocellum has two, Cel48S and Cel48Y. Cellulose solubilization by a Cel48S and Cel48Y double knockout is essentially the same as that of the Cel48S single knockout. Solubilization of crystalline cellulose by Clostridium thermocellum can proceed to completion without expression of a family 48 cellulase
physiological function
CelS is the exoglucanase component of the Clostridium thermocellum cellulosome multicomponent cellulase complex
physiological function
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CelS is the major enzymatic component of the Clostridium thermocellum cellulosome
physiological function
the enzyme was a component of the cellulosome complex
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
?
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x * 74268, His12-tagged GH domain, calculated from amino acid sequence
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
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
Ig-GH9 CbhA: immunoglobulin-like module and the catalytic module, and Ig-GH9 CbhA-(E795Q) mutant in complex with cellotetraose
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
Ni2+ Sepharose column chromatography and Superdex S200 gel filtration
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
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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RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
biotechnology
recombination of the catalytic domains of three glycoside hydrolase family 48 bacterial cellulases (Cel48), i.e. Clostridium cellulolyticum CelF, Clostridium stercorarium CelY, and Clostridium thermocellum CelS, to create a diverse library of Cel48 enzymes with an average of 106 mutations from the closest native enzyme. The library is based on the Clostridium thermocellum CelS architecture, which consists of a 70-kDa catalytic domain connected to the organism's respective dockerin domain. Two of the most stabilizing blocks are predicted to be from the parent CelS at blocks located in the C-terminus of the catalytic domain, close to where the dockerin attaches. Two of the highly stable chimeras also hydrolyze more cellulose than the most active parental enzyme
synthesis
heterologous expression in Bacillus subtilis combined with customized signal peptides for secretion from a random libraries with 173 different signal peptides originating from the Bacillus subtilis genome. The customized signal peptide does not affect enzyme performance when assayed on carboxymethyl cellulose, phosphoric acid swollen cellulose, and microcrystalline cellulose
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Kruus, K.; Andreacchi, A.; Wang, W.K.; Wu, J.H.D.
Product inhibition of the recombinant CelS, and exoglucanase component of the Clostridium thermocellum cellulosome
Appl. Microbiol. Biotechnol.
44
399-404
1995
Acetivibrio thermocellus, Acetivibrio thermocellus (A3DH67)
Morag, E.; Halevy, I.; Bayer, E.A.; Lamed, R.
Isolation and properties of a major cellobiolhydrolase from the cellulosome of Clostridium thermocellum
J. Bacteriol.
173
4155-4162
1991
Acetivibrio thermocellus
Schubot, F.D.; Kataeva, I.A.; Chang, J.; Shah, A.K.; Ljungdahl, L.G.; Rose, J.P.; Wang, B.C.
Structural Basis for the exocellulase activity of the cellobiohydrolase CbhA from Clostridium thermocellum
Biochemistry
43
1163-1170
2004
Acetivibrio thermocellus
Guimaraes, B.G.; Souchon, H.; Lytle, B.L.; David Wu, J.H.; Alzari, P.M.
The crystal structure and catalytic mechanism of cellobiohydrolase CelS, the major enzymatic component of the Clostridium thermocellum Cellulosome
J. Mol. Biol.
320
587-596
2002
Acetivibrio thermocellus, Acetivibrio thermocellus (P0C2S5)
Zverlov, V.V.; Velikodvorskaya, G.A.; Schwarz, W.H.
A newly described cellulosomal cellobiohydrolase, CelO, from Clostridium thermocellum: investigation of the exo-mode of hydrolysis, and binding capacity to crystalline cellulose
Microbiology
148
247-255
2002
Acetivibrio thermocellus, Acetivibrio thermocellus (Q9L3J2), Acetivibrio thermocellus F7, Acetivibrio thermocellus F7 (Q9L3J2)
Wang, W.K.; Kruus, K.; Wu, J.H.
Cloning and expression of the Clostridium thermocellum celS gene in Escherichia coli
Appl. Microbiol. Biotechnol.
42
346-352
1994
Acetivibrio thermocellus (A3DH67), Acetivibrio thermocellus ATCC 27405 (A3DH67)
Wang, W.K.; Kruus, K.; Wu, J.H.
Cloning and DNA sequence of the gene coding for Clostridium thermocellum cellulase Ss (CelS), a major cellulosome component
J. Bacteriol.
175
1293-1302
1993
Acetivibrio thermocellus (A3DH67)
Dror, T.W.; Morag, E.; Rolider, A.; Bayer, E.A.; Lamed, R.; Shoham, Y.
Regulation of the cellulosomal CelS (cel48A) gene of Clostridium thermocellum is growth rate dependent
J. Bacteriol.
185
3042-3048
2003
Acetivibrio thermocellus
Saharay, M.; Guo, H.; Smith, J.C.
Catalytic mechanism of cellulose degradation by a cellobiohydrolase, CelS
PLoS One
5
e1294
2010
Acetivibrio thermocellus
Olson, D.G.; Tripathi, S.A.; Giannone, R.J.; Lo, J.; Caiazza, N.C.; Hogsett, D.A.; Hettich, R.L.; Guss, A.M.; Dubrovsky, G.; Lynd, L.R.
Deletion of the Cel48S cellulase from Clostridium thermocellum
Proc. Natl. Acad. Sci. USA
107
17727-17732
2010
Acetivibrio thermocellus
Wilson, D.B.
Demonstration of the importance for cellulose hydrolysis of CelS, the most abundant cellulosomal cellulase in Clostridium thermocellum
Proc. Natl. Acad. Sci. USA
107
17855-17856
2010
Acetivibrio thermocellus
Brunecky, R.; Alahuhta, M.; Bomble, Y.J.; Xu, Q.; Baker, J.O.; Ding, S.Y.; Himmel, M.E.; Lunin, V.V.
Structure and function of the Clostridium thermocellum cellobiohydrolase A X1-module repeat: enhancement through stabilization of the CbhA complex
Acta Crystallogr. Sect. D
68
292-299
2012
Acetivibrio thermocellus (A3DCH2)
Smith, M.A.; Rentmeister, A.; Snow, C.D.; Wu, T.; Farrow, M.F.; Mingardon, F.; Arnold, F.H.
A diverse set of family 48 bacterial glycoside hydrolase cellulases created by structure-guided recombination
FEBS J.
279
4453-4465
2012
Acetivibrio thermocellus (A3DH67), Thermoclostridium stercorarium (P50900), Acetivibrio thermocellus DSM 1237 (A3DH67), Thermoclostridium stercorarium DSM 8532 (P50900)
Lan Thanh Bien, T.; Tsuji, S.; Tanaka, K.; Takenaka, S.; Yoshida, K.
Secretion of heterologous thermostable cellulases in Bacillus subtilis
J. Gen. Appl. Microbiol.
60
175-182
2014
Acetivibrio thermocellus (A3DH67), Acetivibrio thermocellus DSM 1237 (A3DH67)
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