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Literature summary 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)

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

Protein Variants

EC Number Protein Variants Comment Organism
3.2.1.91 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
3.2.1.91 D226A/S232A site-directed mutagenesis, the mutant is almost inactive and shows slightly decreased ligand binding compared to the wild-type enzyme Thermobifida fusca
3.2.1.91 D274A site-directed mutagenesis, the mutant shows altered and highly reduced substrate specificity compared to the wild-type enzyme Thermobifida fusca
3.2.1.91 D497A site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme Thermobifida fusca
3.2.1.91 E495A site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme Thermobifida fusca
3.2.1.91 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
3.2.1.91 Y220A site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme, alomost inactive mutant Thermobifida fusca

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.2.1.91 0.0015
-
 2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant mutant D274A Thermobifida fusca
3.2.1.91 0.0023
-
 2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant wild-type enzyme Thermobifida fusca
3.2.1.91 0.0065
-
 2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant mutant D226A Thermobifida fusca
3.2.1.91 0.044
-
 2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant mutant S232A Thermobifida fusca
3.2.1.91 0.161
-
 2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant mutant Y220A Thermobifida fusca
3.2.1.91 0.214
-
 2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant mutant D497A Thermobifida fusca

Natural Substrates/ Products (Substrates)

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

Organism

EC Number Organism UniProt Comment Textmining
3.2.1.91 Thermobifida fusca
-
-
-

Purification (Commentary)

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

Reaction

EC Number Reaction Comment Organism Reaction ID
3.2.1.91 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
a

Specific Activity [micromol/min/mg]

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

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.2.1.91 2,4-dinitrophenyl cellobiose + H2O
-
 Thermobifida fusca 2,4-dinitrophenol + cellobiose
-
 ?
3.2.1.91 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
-
 ?
3.2.1.91 carboxymethylcellulose + H2O
-
 Thermobifida fusca cellobiose + ?
-
 ?
3.2.1.91 cellulose + H2O
-
 Thermobifida fusca cellobiose + ?
-
 ?
3.2.1.91 cellulose + H2O activity with bacterial microcrystalline cellulose, phosphoric acid-treated swollen cellulose, phosphoric acid-treated cotton, and carboxymethyl cellulose Thermobifida fusca cellobiose + ?
-
 ?

Subunits

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

Synonyms

EC Number Synonyms Comment Organism
3.2.1.91 Cel6B
-
 Thermobifida fusca
3.2.1.91 exocellulase
-
 Thermobifida fusca

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.2.1.91 50
-
 assay at Thermobifida fusca

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.2.1.91 0.0001
-
 2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant mutant D497A Thermobifida fusca
3.2.1.91 0.0005
-
 2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant mutant S232A Thermobifida fusca
3.2.1.91 0.0015
-
 2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant mutant Y220A Thermobifida fusca
3.2.1.91 0.0018
-
 2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant mutant D226A Thermobifida fusca
3.2.1.91 0.038
-
 2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant mutant D274A Thermobifida fusca
3.2.1.91 0.057
-
 2,4-dinitrophenyl cellobiose pH 5.5, 50°C, recombinant wild-type enzyme Thermobifida fusca

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.2.1.91 5.5
-
 assay at Thermobifida fusca

kcat/KM [mM/s]

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