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Literature summary for 3.1.1.74 extracted from

  • Shirke, A.N.; Basore, D.; Butterfoss, G.L.; Bonneau, R.; Bystroff, C.; Gross, R.A.
    Toward rational thermostabilization of Aspergillus oryzae cutinase Insights into catalytic and structural stability (2016), Proteins, 84, 60-72 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.1.1.74

Cloned(Commentary)

Cloned (Comment) Organism
heterologously expressed in Pichia pastoris using the strong methanol-induced AOX I promoter. The protein is expressed with the Saccharomyces cerevisiae alpha factor tag on the N-terminal for extracellular secretion and the six histidine tag on the C-terminus for ease of purification using affinity chromatography Aspergillus oryzae

Protein Variants

Protein Variants Comment Organism
A102D/Q105R/G106E pH-optima for activity and stability are identical to wild-type enzym. Improvement in Tm-value of 3.4°C. Increased half-life at 6°C relative to the wild-type enzyme of approximately 3fold Aspergillus oryzae
A102D/Q105R/G106E/N133A/S140P/E161T/A166P large improvement of stability at 60°C Aspergillus oryzae
A102D/Q105R/G106E/N133A/S140P/E161T/A166P/K137E large improvement of stability at 60°C Aspergillus oryzae
A102D/Q105R/G106E/Q98N/A99D/E109Q thermodynamically most stable variant, improving on wild-type enzyme by 6.7 kJ/mol Aspergillus oryzae
A178P/V179P loss of stability and activity Aspergillus oryzae
K174R/Y176F/A178E/D200R/G202E/D203E/D206R mutant enzyme shows an increased kinetic stability Aspergillus oryzae
L26D/G28E/D30R/K67R improvement in Tm-value of 0.7°C Aspergillus oryzae
additional information mutational analysis toward the thermostabilization of the enzyme. Mutants with increased thermal unfolding temperature and increase in the half-life of the enzyme activity at 60°C do not display improved rate or temperature optimum of enzyme activity. Surface salt bridge optimization produces enthalpic stabilization. Mutations to proline reduces the entropy loss upon folding. The lack of a correlative increase in the temperature optimum of catalytic activity with thermodynamic stability suggests that the active site is locally denatured at a temperature below the thermal unfolding temperature of the global structure Aspergillus oryzae
N133A/S140P/E161T/A166P proline mutations contribute to themostabilization by decreasing the entropy lost upon folding. Improvement in Tm of 1.7°C. Increased half-life at 6°C relative to the wild-type enzyme of approximately 2fold Aspergillus oryzae
Q110W/K114W the mutant enzyme is retained in the endoplasmic reticulum whereas wild-type enzyme is secreted Aspergillus oryzae
R46P the Tm-value is 3°C below that of wild-type enzyme Aspergillus oryzae
T84R/D86L/A99E/A100S decrease in thermostability relative to the wild-type enzyme. Large losses in 4-nitrophenyl butyrate (about 70%) and poly(epsilon-caprolactone) (about 90%) activities Aspergillus oryzae
V150I/I136V mutation do not provide any improvement in stability Aspergillus oryzae

Localization

Localization Comment Organism GeneOntology No. Textmining
endoplasmic reticulum the mutant enzyme Q110W/K114W is retained in the endoplasmic reticulum whereas wild-type enzyme is secreted Aspergillus oryzae 5783
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Organism

Organism UniProt Comment Textmining
Aspergillus oryzae P52956
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-
Aspergillus oryzae ATCC 42149 P52956
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-

Purification (Commentary)

Purification (Comment) Organism
-
 Aspergillus oryzae

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4-nitrophenyl butyrate + H2O
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 Aspergillus oryzae 4-nitrophenol + butyrate
-
 ?
4-nitrophenyl butyrate + H2O
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 Aspergillus oryzae ATCC 42149 4-nitrophenol + butyrate
-
 ?
poly(epsilon-caprolactone) + H2O
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 Aspergillus oryzae ?
-
 ?
poly(epsilon-caprolactone) + H2O
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 Aspergillus oryzae ATCC 42149 ?
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 ?

Synonyms

Synonyms Comment Organism
cutinase
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 Aspergillus oryzae
CutL
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 Aspergillus oryzae

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
60
-
 t1/2-value: 4.5 h (wild-type enzyme), 40 h (mutant enzymes A102D/Q105R/G106E/N133A/S140P/E161T/A166P and 102D/Q105R/G106E/N133A/S140P/E161T/A166P/K137E), 11.2 h (mutant enzyme A102D/Q105R/G106E/Q98N/A99D/E109Q) Aspergillus oryzae
65
-
 t1/2-value: 10 min (wild-type enzyme), 4 h (mutant enzyme A102D/Q105R/G106E/N133A/S140P/E161T/A166P), 5 h (mutant enzyme A102D/Q105R/G106E/N133A/S140P/E161T/A166P/K137E) Aspergillus oryzae