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Literature summary for 3.1.1.74 extracted from

  • Herrero Acero, E.; Ribitsch, D.; Dellacher, A.; Zitzenbacher, S.; Marold, A.; Steinkellner, G.; Gruber, K.; Schwab, H.; Guebitz, G.M.
    Surface engineering of a cutinase from Thermobifida cellulosilytica for improved polyester hydrolysis (2013), Biotechnol. Bioeng., 110, 2581-2590.
    View publication on PubMed
Search for more literature for 3.1.1.74

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression in Escherichia coli strain BL21-Gold(DE3) Thermobifida cellulosilytica

Protein Variants

Protein Variants Comment Organism
A30V site-directed mutagenesis, mutation of isozyme Cut2 to the analogue residue of isozyme Cut1, the mutant shows increased catalytic efficiency with polyethyleneterephthalate and higher kcat/KM values on soluble substrates compared to the wild-type enzyme Thermobifida cellulosilytica
L183A site-directed mutagenesis, mutation of isozyme Cut2 to the analogue residue of isozyme Cut1, the mutant shows slightly increased catalytic efficiency with 4-nitrophenyl ester substrates compared to the wild-type enzyme Thermobifida cellulosilytica
additional information exchange of the positively charged arginine (Arg19 and Arg29) located on the enzyme surface to the non-charged amino acids serine and asparagine strongly increased the hydrolysis activity for bis(benzoyloxyethyl)terephthalate and polyethyleneterephthalate. In contrast, exchange of the uncharged glutamine (Glu65) by the negatively charged glutamic acid lead to a complete loss of hydrolysis activity on polyethyleneterephthalate films Thermobifida cellulosilytica
Q65E site-directed mutagenesis, mutation of isozyme Cut2 to the analogue residue of isozyme Cut1, the mutant shows slightly reduced catalytic efficiency with 4-nitrophenyl ester substrates compared to the wild-type enzyme Thermobifida cellulosilytica
R187K site-directed mutagenesis, mutation of isozyme Cut2 to the analogue residue of isozyme Cut1, the mutant shows increased catalytic efficiency with 4-nitrophenyl ester substrates compared to the wild-type enzyme Thermobifida cellulosilytica
R19S site-directed mutagenesis, mutation of isozyme Cut2 to the analogue residue of isozyme Cut1 Thermobifida cellulosilytica
R19S/R29N/A30V site-directed mutagenesis, mutation of isozyme Cut2 to the analogue residue of isozyme Cut1, the mutant shows increased catalytic efficiency with 4-nitrophenyl ester substrates compared to the wild-type enzyme Thermobifida cellulosilytica
R29N site-directed mutagenesis, mutation of isozyme Cut2 to the analogue residue of isozyme Cut1, the mutant shows increased catalytic efficiency with polyethyleneterephthalate and higher kcat/KM values on soluble substrates compared to the wild-type enzyme Thermobifida cellulosilytica
R29N/A30V site-directed mutagenesis, mutation of isozyme Cut2 to the analogue residue of isozyme Cut1, the mutant shows increased catalytic efficiency with polyethyleneterephthalate and higher kcat/KM values on soluble substrates compared to the wild-type enzyme Thermobifida cellulosilytica

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.8
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant isozyme Thermobifida cellulosilytica
0.8
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant R187K Thermobifida cellulosilytica
1
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant mutant R29N/A30V Thermobifida cellulosilytica
1.1
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant mutant A30V Thermobifida cellulosilytica
1.2
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant R19S Thermobifida cellulosilytica
1.2
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant R19S/R29N/A30V Thermobifida cellulosilytica
1.3
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant R29N Thermobifida cellulosilytica
1.3
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant R29N/A30V Thermobifida cellulosilytica
1.4
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant mutant R19S/R29N/A30V Thermobifida cellulosilytica
1.5
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant isozyme Thermobifida cellulosilytica
1.5
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant Q65E Thermobifida cellulosilytica
1.7
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant A30V Thermobifida cellulosilytica
1.9
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant L183A Thermobifida cellulosilytica
1.9
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant wild-type isozyme Thermobifida cellulosilytica
2
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant mutant R29N Thermobifida cellulosilytica
2.1
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant mutant L183A Thermobifida cellulosilytica
2.2
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant mutant R19S Thermobifida cellulosilytica
2.6
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant mutant Q65E Thermobifida cellulosilytica
3.4
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant wild-type isozyme Thermobifida cellulosilytica
4
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant mutant R187K Thermobifida cellulosilytica

Organism

Organism UniProt Comment Textmining
Thermobifida cellulosilytica E9LVH8 Cut1
-
Thermobifida cellulosilytica E9LVH9 Cut2
-
Thermobifida cellulosilytica DSM 44535 E9LVH8 Cut1
-
Thermobifida cellulosilytica DSM 44535 E9LVH9 Cut2
-

Purification (Commentary)

Purification (Comment) Organism
recombinant enzyme from Escherichia coli strain BL21-Gold(DE3) Thermobifida cellulosilytica

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4-nitrophenyl acetate + H2O
-
 Thermobifida cellulosilytica 4-nitrophenol + acetate
-
 ?
4-nitrophenyl acetate + H2O
-
 Thermobifida cellulosilytica DSM 44535 4-nitrophenol + acetate
-
 ?
4-nitrophenyl butyrate + H2O
-
 Thermobifida cellulosilytica 4-nitrophenol + butyrate
-
 ?
4-nitrophenyl butyrate + H2O
-
 Thermobifida cellulosilytica DSM 44535 4-nitrophenol + butyrate
-
 ?
bis(benzoyloxyethyl) terephthalate + H2O
-
 Thermobifida cellulosilytica terephthalate + benzoic acid + 2-hydroxyethylbenzoate + mono-(2-hydroxyethyl) terephthalate + bis-(2-hydroxyethyl) terephthalate
-
 ?
bis(benzoyloxyethyl) terephthalate + H2O
-
 Thermobifida cellulosilytica terephthalate + benzoic acid + 2-hydroxyethylbenzoate + mono-(2-hydroxyethyl) terephthalate + bis-(2-hydroxyethyl) terephthalate product ratios of wild-type and mutant enzymes, overview ?
bis(benzoyloxyethyl) terephthalate + H2O
-
 Thermobifida cellulosilytica DSM 44535 terephthalate + benzoic acid + 2-hydroxyethylbenzoate + mono-(2-hydroxyethyl) terephthalate + bis-(2-hydroxyethyl) terephthalate
-
 ?
bis(benzoyloxyethyl) terephthalate + H2O
-
 Thermobifida cellulosilytica DSM 44535 terephthalate + benzoic acid + 2-hydroxyethylbenzoate + mono-(2-hydroxyethyl) terephthalate + bis-(2-hydroxyethyl) terephthalate product ratios of wild-type and mutant enzymes, overview ?
polyethyleneterephthalate + H2O
-
 Thermobifida cellulosilytica terephthalate + benzoic acid + 2-hydroxyethylbenzoate + mono-(2-hydroxyethyl)terephthalate + bis-(2-hydroxyethyl)terephthalate
-
 ?
polyethyleneterephthalate + H2O
-
 Thermobifida cellulosilytica terephthalate + benzoic acid + 2-hydroxyethylbenzoate + mono-(2-hydroxyethyl)terephthalate + bis-(2-hydroxyethyl)terephthalate product ratios of wild-type and mutant enzymes, overview ?
polyethyleneterephthalate + H2O
-
 Thermobifida cellulosilytica DSM 44535 terephthalate + benzoic acid + 2-hydroxyethylbenzoate + mono-(2-hydroxyethyl)terephthalate + bis-(2-hydroxyethyl)terephthalate
-
 ?
polyethyleneterephthalate + H2O
-
 Thermobifida cellulosilytica DSM 44535 terephthalate + benzoic acid + 2-hydroxyethylbenzoate + mono-(2-hydroxyethyl)terephthalate + bis-(2-hydroxyethyl)terephthalate product ratios of wild-type and mutant enzymes, overview ?

Synonyms

Synonyms Comment Organism
Thc_Cut1
-
 Thermobifida cellulosilytica
Thc_Cut2
-
 Thermobifida cellulosilytica

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at Thermobifida cellulosilytica

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3 6 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant R19S Thermobifida cellulosilytica
7
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant Q65E Thermobifida cellulosilytica
15
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant mutant Q65E Thermobifida cellulosilytica
16
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant wild-type isozyme Thermobifida cellulosilytica
17
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant wild-type isozyme Thermobifida cellulosilytica
25
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant L183A Thermobifida cellulosilytica
32
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant mutant L183A Thermobifida cellulosilytica
35
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant R187K Thermobifida cellulosilytica
76
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant R19S/R29N/A30V Thermobifida cellulosilytica
79
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant mutant R187K Thermobifida cellulosilytica
80
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant mutant R19S Thermobifida cellulosilytica
100
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant R29N/A30V Thermobifida cellulosilytica
136
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant mutant R19S/R29N/A30V Thermobifida cellulosilytica
173
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant mutant R29N/A30V Thermobifida cellulosilytica
183
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant mutant A30V Thermobifida cellulosilytica
191
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant mutant R29N Thermobifida cellulosilytica
207
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant R29N Thermobifida cellulosilytica
260
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant A30V Thermobifida cellulosilytica
327
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant isozyme Thermobifida cellulosilytica
436
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant isozyme Thermobifida cellulosilytica

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
 assay at Thermobifida cellulosilytica

General Information

General Information Comment Organism
evolution modeling and comparison of the structures of the two closely related cutinases Thc_Cut1 and Thc_Cut2 from Thermobifida cellulosilytica DSM44535 reveal that dissimilarities in their electrostatic and hydrophobic surface properties in the vicinity to the active site might be responsible for pronounced differences in hydrolysis efficiencies of polyester (i.e., PET, polyethyleneterephthalate), isozyme Thc_Cut2 hydrolyzes PET much less efficiently than Thc_Cut1 Thermobifida cellulosilytica
evolution modeling and comparison of the structures of the two closely related cutinases Thc_Cut1 and Thc_Cut2 from Thermobifida cellulosilytica DSM44535 reveal that dissimilarities in their electrostatic and hydrophobic surface properties in the vicinity to the active site might be responsible for pronounced differences in hydrolysis efficiencies of polyester (i.e., PET, polyethyleneterephthalate), Thc_Cut2 hydrolyzes PET much less efficiently than Thc_Cut1 Thermobifida cellulosilytica

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
5
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant Q65E Thermobifida cellulosilytica
5
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant wild-type isozyme Thermobifida cellulosilytica
9
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant wild-type isozyme Thermobifida cellulosilytica
13
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant L183A Thermobifida cellulosilytica
30
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant R19S Thermobifida cellulosilytica
44
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant R187K Thermobifida cellulosilytica
65
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant R19S/R29N/A30V Thermobifida cellulosilytica
76
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant R29N/A30V Thermobifida cellulosilytica
153
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant A30V Thermobifida cellulosilytica
159
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant mutant R29N Thermobifida cellulosilytica
291
-
 4-nitrophenyl acetate pH 7.0, 25°C, recombinant isozyme Thermobifida cellulosilytica
425
-
 4-nitrophenyl butyrate pH 7.0, 25°C, recombinant isozyme Thermobifida cellulosilytica