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Literature summary for 3.2.1.91 extracted from

  • Vuong, T.V.; Wilson, D.B.
    The absence of an identifiable single catalytic base residue in Thermobifida fusca exocellulase Cel6B (2009), FEBS J., 276, 3837-3845.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
expression of wild-type Cel6B and mutant enzymes in Escherichia coli strain BL21(DE3) Thermobifida fusca

Protein Variants

Protein Variants Comment Organism
D226A site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme. The D226A enzyme has a very low activity on insoluble cellulose, not improved by sodium azide Thermobifida fusca
D226A/S232A site-directed mutagenesis, the mutant is almost inactive and shows slightly decreased ligand binding compared to the wild-type enzyme Thermobifida fusca
D274A site-directed mutagenesis, the mutant shows altered and highly reduced substrate specificity compared to the wild-type enzyme Thermobifida fusca
D497A site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme Thermobifida fusca
E495A site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme Thermobifida fusca
S232A site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme. The S232A enzyme retains near wild-type activity on most substrates, but carboxymethylcellulose activity is drastically reduced Thermobifida fusca
Y220A site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme, alomost inactive mutant Thermobifida fusca

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0015
-
2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant mutant D274A Thermobifida fusca
0.0023
-
2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant wild-type enzyme Thermobifida fusca
0.0065
-
2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant mutant D226A Thermobifida fusca
0.044
-
2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant mutant S232A Thermobifida fusca
0.161
-
2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant mutant Y220A Thermobifida fusca
0.214
-
2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant mutant D497A Thermobifida fusca

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
cellulose + H2O Thermobifida fusca
-
cellobiose + ?
-
?

Organism

Organism UniProt Comment Textmining
Thermobifida fusca
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type Cel6B and mutant enzymes from Escherichia coli strain BL21(DE3) by hydrophobic interaction and anion exchange chromatography Thermobifida fusca

Reaction

Reaction Comment Organism Reaction ID
2 cellohexaose + 2 H2O = 2 cellotriose + cellotetraose + cellobiose exocellulase Cel6B acts by an inverting hydrolysis mechanism, that involves a proton-transferring network to carry out the catalytic base function Thermobifida fusca

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
cellulose substrate type specificity of wild-type and mutant enzymes, overview Thermobifida fusca

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2,4-dinitrophenyl cellobiose + H2O
-
Thermobifida fusca 2,4-dinitrophenol + cellobiose
-
?
4-methylumbelliferyl beta-cellobioside + H2O substrate binding analysis of wild-type enzyme and mutant D274A, mutant D274A binds 4-methylumbelliferyl beta-cellobioside to approximately the same extent as the wild-type, overview Thermobifida fusca 4-methylumbelliferol + cellobiose
-
?
carboxymethylcellulose + H2O
-
Thermobifida fusca cellobiose + ?
-
?
cellulose + H2O
-
Thermobifida fusca cellobiose + ?
-
?
cellulose + H2O activity with bacterial microcrystalline cellulose, phosphoric acid-treated swollen cellulose, phosphoric acid-treated cotton, and carboxymethyl cellulose Thermobifida fusca cellobiose + ?
-
?

Subunits

Subunits Comment Organism
More structural model of the Cel6B catalytic domain, structure comparisons to other exocellulases, overview Thermobifida fusca

Synonyms

Synonyms Comment Organism
Cel6B
-
Thermobifida fusca
exocellulase
-
Thermobifida fusca

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
50
-
assay at Thermobifida fusca

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.0001
-
2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant mutant D497A Thermobifida fusca
0.0005
-
2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant mutant S232A Thermobifida fusca
0.0015
-
2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant mutant Y220A Thermobifida fusca
0.0018
-
2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant mutant D226A Thermobifida fusca
0.038
-
2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant mutant D274A Thermobifida fusca
0.057
-
2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant wild-type enzyme Thermobifida fusca

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
5.5
-
assay at Thermobifida fusca

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
additional information
-
additional information
-
Thermobifida fusca