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 | 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 | 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 | - |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Aspergillus oryzae | P52956 | - |
- |
Aspergillus oryzae ATCC 42149 | P52956 | - |
- |
Purification (Comment) | Organism |
---|---|
- |
Aspergillus oryzae |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
4-nitrophenyl butyrate + H2O | - |
Aspergillus oryzae | 4-nitrophenol + butyrate | - |
? | |
4-nitrophenyl butyrate + H2O | - |
Aspergillus oryzae ATCC 42149 | 4-nitrophenol + butyrate | - |
? | |
poly(epsilon-caprolactone) + H2O | - |
Aspergillus oryzae | ? | - |
? | |
poly(epsilon-caprolactone) + H2O | - |
Aspergillus oryzae ATCC 42149 | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
cutinase | - |
Aspergillus oryzae |
CutL | - |
Aspergillus oryzae |
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 |