Subtilisin E from Bacillus subtilis 168
Enzyme Description
Sequence
AQSVPYGISQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVRGGASFVPSETNPYQDGSSHGTHVAGTIAALNNSIGVLGVAPSASLYAVKVLDSTGSGQYSWIINGIEWAISNNMDVINMSLGGPTGSTALKTVVDKAVSSGIVVAAAAGNEGSSGSTSTVGYPAKYPSTIAVGAVNSSNQRASFSSAGSELDVMAPGVSIQSTLPGGTYGAYNGTSMATPHVAGAAALILSKHPTWTNAQVRDRLESTATYLGNSFYYGKGLINVQAAAQ
Keqin Chen et al. (1991)
β Enzyme Engineering for Nonaqueous Solvents. II. Additive Effects of Mutations on the Stability and Activity of Subtilisin E in Polar Organic Media
Biotechnology Progress
Β· doi:10.1021/bp00008a007 β
Β· Activity - Michaelis-Menten Stability - Incubation
▼
Database ID
UniProt: P04189 β
Sequence Annotation
Inferred - from protein name (106 first residues from UniProt sequence absent from the mature protein)
Protein Source
Recombinant, host bacterium Bacillus subtilis DB428
Experimental Data (40 measurements)
40 measurements β page 2 of 2
| Mutation | Property | Assay | Solvent | Solvent Volume | Aqueous Reference | WT Reference | Measured Value | Units | Solution | pH | Temperature | Substrate(s) | Product(s) | Cofactor(s) | Shaking | Comments |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| N218S | Stability - Incubation | Activity after incubation (15 hours at 50Β°C), measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | 100.0 | β | 30.0 | % | Incubation: 0.1 MM Tris-HCl buffer, 2 mM CaCl2, Assay: 0.1 MM Tris-HCl buffer, 2 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | ? mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase ("Enzyme activity was measured by diluting not more than 100FL of the enzyme solution into a standard 1.5-mL reaction volume. In this way the maximum amount of DMF in the assay solution was 2.7%." |
| N218S | Stability - Incubation | Activity after incubation (20 hours at 50Β°C), measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | 100.0 | β | 25.0 | % | Incubation: 0.1 MM Tris-HCl buffer, 2 mM CaCl2, Assay: 0.1 MM Tris-HCl buffer, 2 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | ? mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase ("Enzyme activity was measured by diluting not more than 100FL of the enzyme solution into a standard 1.5-mL reaction volume. In this way the maximum amount of DMF in the assay solution was 2.7%." |
| Q103R/N218S | Stability - Incubation | Activity after incubation (2 hours at 50Β°C), measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | 100.0 | β | 90.0 | % | Incubation: 0.1 MM Tris-HCl buffer, 2 mM CaCl2, Assay: 0.1 MM Tris-HCl buffer, 2 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | ? mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase ("Enzyme activity was measured by diluting not more than 100FL of the enzyme solution into a standard 1.5-mL reaction volume. In this way the maximum amount of DMF in the assay solution was 2.7%." |
| Q103R/N218S | Stability - Incubation | Activity after incubation (4 hours at 50Β°C), measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | 100.0 | β | 75.0 | % | Incubation: 0.1 MM Tris-HCl buffer, 2 mM CaCl2, Assay: 0.1 MM Tris-HCl buffer, 2 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | ? mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase ("Enzyme activity was measured by diluting not more than 100FL of the enzyme solution into a standard 1.5-mL reaction volume. In this way the maximum amount of DMF in the assay solution was 2.7%." |
| Q103R/N218S | Stability - Incubation | Activity after incubation (6 hours at 50Β°C), measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | 100.0 | 40.0 | 70.0 | % | Incubation: 0.1 MM Tris-HCl buffer, 2 mM CaCl2, Assay: 0.1 MM Tris-HCl buffer, 2 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | ? mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase ("Enzyme activity was measured by diluting not more than 100FL of the enzyme solution into a standard 1.5-mL reaction volume. In this way the maximum amount of DMF in the assay solution was 2.7%." |
| Q103R/N218S | Stability - Incubation | Activity after incubation (8 hours at 50Β°C), measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | 100.0 | β | 65.0 | % | Incubation: 0.1 MM Tris-HCl buffer, 2 mM CaCl2, Assay: 0.1 MM Tris-HCl buffer, 2 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | ? mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase ("Enzyme activity was measured by diluting not more than 100FL of the enzyme solution into a standard 1.5-mL reaction volume. In this way the maximum amount of DMF in the assay solution was 2.7%." |
| Q103R/N218S | Stability - Incubation | Activity after incubation (10 hours at 50Β°C), measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | 100.0 | 25.0 | 50.0 | % | Incubation: 0.1 MM Tris-HCl buffer, 2 mM CaCl2, Assay: 0.1 MM Tris-HCl buffer, 2 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | ? mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase ("Enzyme activity was measured by diluting not more than 100FL of the enzyme solution into a standard 1.5-mL reaction volume. In this way the maximum amount of DMF in the assay solution was 2.7%." |
| Q103R/N218S | Stability - Incubation | Activity after incubation (15 hours at 50Β°C), measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | 100.0 | β | 30.0 | % | Incubation: 0.1 MM Tris-HCl buffer, 2 mM CaCl2, Assay: 0.1 MM Tris-HCl buffer, 2 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | ? mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase ("Enzyme activity was measured by diluting not more than 100FL of the enzyme solution into a standard 1.5-mL reaction volume. In this way the maximum amount of DMF in the assay solution was 2.7%." |
| Q103R/N218S | Stability - Incubation | Activity after incubation (20 hours at 50Β°C), measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | 100.0 | β | 25.0 | % | Incubation: 0.1 MM Tris-HCl buffer, 2 mM CaCl2, Assay: 0.1 MM Tris-HCl buffer, 2 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | ? mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase ("Enzyme activity was measured by diluting not more than 100FL of the enzyme solution into a standard 1.5-mL reaction volume. In this way the maximum amount of DMF in the assay solution was 2.7%." |
| N218S | Stability - Incubation | Activity after incubation (8 hours at 50Β°C), measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | 100.0 | β | 65.0 | % | Incubation: 0.1 MM Tris-HCl buffer, 2 mM CaCl2, Assay: 0.1 MM Tris-HCl buffer, 2 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | ? mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase ("Enzyme activity was measured by diluting not more than 100FL of the enzyme solution into a standard 1.5-mL reaction volume. In this way the maximum amount of DMF in the assay solution was 2.7%." |
| N218S | Stability - Incubation | Activity after incubation (6 hours at 50Β°C), measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | 100.0 | 40.0 | 70.0 | % | Incubation: 0.1 MM Tris-HCl buffer, 2 mM CaCl2, Assay: 0.1 MM Tris-HCl buffer, 2 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | ? mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase ("Enzyme activity was measured by diluting not more than 100FL of the enzyme solution into a standard 1.5-mL reaction volume. In this way the maximum amount of DMF in the assay solution was 2.7%." |
| N218S | Stability - Incubation | Activity after incubation (4 hours at 50Β°C), measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | 100.0 | β | 75.0 | % | Incubation: 0.1 MM Tris-HCl buffer, 2 mM CaCl2, Assay: 0.1 MM Tris-HCl buffer, 2 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | ? mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase ("Enzyme activity was measured by diluting not more than 100FL of the enzyme solution into a standard 1.5-mL reaction volume. In this way the maximum amount of DMF in the assay solution was 2.7%." |
| N218S | Stability - Incubation | Activity after incubation (2 hours at 50Β°C), measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | 100.0 | β | 90.0 | % | Incubation: 0.1 MM Tris-HCl buffer, 2 mM CaCl2, Assay: 0.1 MM Tris-HCl buffer, 2 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | ? mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase ("Enzyme activity was measured by diluting not more than 100FL of the enzyme solution into a standard 1.5-mL reaction volume. In this way the maximum amount of DMF in the assay solution was 2.7%." |
| Q103R | Stability - Incubation | Activity after incubation (20 hours at 50Β°C), measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | 100.0 | β | 7.0 | % | Incubation: 0.1 MM Tris-HCl buffer, 2 mM CaCl2, Assay: 0.1 MM Tris-HCl buffer, 2 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | ? mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase ("Enzyme activity was measured by diluting not more than 100FL of the enzyme solution into a standard 1.5-mL reaction volume. In this way the maximum amount of DMF in the assay solution was 2.7%." |
| Q103R | Stability - Incubation | Activity after incubation (15 hours at 50Β°C), measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | 100.0 | β | 10.0 | % | Incubation: 0.1 MM Tris-HCl buffer, 2 mM CaCl2, Assay: 0.1 MM Tris-HCl buffer, 2 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | ? mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase ("Enzyme activity was measured by diluting not more than 100FL of the enzyme solution into a standard 1.5-mL reaction volume. In this way the maximum amount of DMF in the assay solution was 2.7%." |
| Q103R | Stability - Incubation | Activity after incubation (10 hours at 50Β°C), measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | 100.0 | 25.0 | 20.0 | % | Incubation: 0.1 MM Tris-HCl buffer, 2 mM CaCl2, Assay: 0.1 MM Tris-HCl buffer, 2 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | ? mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase ("Enzyme activity was measured by diluting not more than 100FL of the enzyme solution into a standard 1.5-mL reaction volume. In this way the maximum amount of DMF in the assay solution was 2.7%." |
| Q103R | Stability - Incubation | Activity after incubation (8 hours at 50Β°C), measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | 100.0 | β | 30.0 | % | Incubation: 0.1 MM Tris-HCl buffer, 2 mM CaCl2, Assay: 0.1 MM Tris-HCl buffer, 2 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | ? mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase ("Enzyme activity was measured by diluting not more than 100FL of the enzyme solution into a standard 1.5-mL reaction volume. In this way the maximum amount of DMF in the assay solution was 2.7%." |
| Q103R | Stability - Incubation | Activity after incubation (6 hours at 50Β°C), measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | 100.0 | 40.0 | 40.0 | % | Incubation: 0.1 MM Tris-HCl buffer, 2 mM CaCl2, Assay: 0.1 MM Tris-HCl buffer, 2 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | ? mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase ("Enzyme activity was measured by diluting not more than 100FL of the enzyme solution into a standard 1.5-mL reaction volume. In this way the maximum amount of DMF in the assay solution was 2.7%." |
| Q103R | Stability - Incubation | Activity after incubation (4 hours at 50Β°C), measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | 100.0 | β | 50.0 | % | Incubation: 0.1 MM Tris-HCl buffer, 2 mM CaCl2, Assay: 0.1 MM Tris-HCl buffer, 2 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | ? mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase ("Enzyme activity was measured by diluting not more than 100FL of the enzyme solution into a standard 1.5-mL reaction volume. In this way the maximum amount of DMF in the assay solution was 2.7%." |
| Q103R | Stability - Incubation | Activity after incubation (2 hours at 50Β°C), measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | 100.0 | β | 70.0 | % | Incubation: 0.1 MM Tris-HCl buffer, 2 mM CaCl2, Assay: 0.1 MM Tris-HCl buffer, 2 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | ? mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase ("Enzyme activity was measured by diluting not more than 100FL of the enzyme solution into a standard 1.5-mL reaction volume. In this way the maximum amount of DMF in the assay solution was 2.7%." |
Mutations in this dataset (4) β Keqin Chen et al. (1991)
WT
Q103R
Q103R/N218S
N218S
Visualization : Activity β Michaelis-Menten
Keqin Chen et al. (1991)
One bar per measurement. Colour = solvent, shade = solvent volume. β β β Reference value. Hover for details.
Mutation Effect β Michaelis-Menten
Mutation impact on kinetic parameters in the presence of organic solvent: comparison of wild-type and mutant values in identical conditions.
Visualization : Stability β Incubation
Keqin Chen et al. (1991)
One bar per measurement. Colour = solvent, shade = solvent volume. β β β Reference value. Hover for details.
Mutation Effect
Mutation impact on enzyme stability and function in the presence of organic solvent: comparison of wild-type and mutant values in identical conditions.
Pascal Martinez et al. (1992)
β Stabilization of substilisin E in organic solvents by site-directed mutagenesis
Biotechnology and Bioengineering
Β· doi:10.1002/bit.260390204 β
Β· Stability - Incubation Stability - Half-life
▼
Database ID
UniProt: P04189 β
Sequence Annotation
Inferred - from protein name (106 first residues from UniProt sequence absent from the mature protein)
Protein Source
Recombinant, host bacterium Bacillus subtilis DB428
Experimental Data (52 measurements)
52 measurements β page 1 of 3
| Mutation | Property | Assay | Solvent | Solvent Volume | Aqueous Reference | WT Reference | Measured Value | Units | Solution | pH | Temperature | Substrate(s) | Product(s) | Cofactor(s) | Shaking | Comments |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| WT | Stability - Half-life | Half-life in organic solvent at 30Β°C, with residual activity measured with absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 80% | β | β | 5.7 | hours | Incubation: 0.1 M tris-HCl, 10 mM CaCl2, Assay: 0.1 M tris-HCl, 10 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 30Β°C, Assay: 37Β°C | 0.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Classical aqueous control (in hours) | Clear about assay in aqueous phase |
| WT | Stability - Half-life | Half-life in organic solvent at 50Β°C, with residual activity measured with absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | β | β | 4.7 | hours | Incubation: 0.1 M tris-HCl, 10 mM CaCl2, Assay: 0.1 M tris-HCl, 10 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | 0.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Classical aqueous control (in hours) | Clear about assay in aqueous phase |
| WT | Stability - Incubation | Activity after incubation (2 hours at 50Β°C), measured with absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 80% | 100.0 | β | 69.6 | % | Incubation: 0.1 M tris-HCl, 10 mM CaCl2, Assay: 0.1 M tris-HCl, 10 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | 0.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase |
| WT | Stability - Incubation | Activity after incubation (4 hours at 50Β°C), measured with absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 80% | 100.0 | β | 52.1 | % | Incubation: 0.1 M tris-HCl, 10 mM CaCl2, Assay: 0.1 M tris-HCl, 10 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | 0.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase |
| WT | Stability - Incubation | Activity after incubation (6 hours at 50Β°C), measured with absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 80% | 100.0 | β | 39.3 | % | Incubation: 0.1 M tris-HCl, 10 mM CaCl2, Assay: 0.1 M tris-HCl, 10 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | 0.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase |
| WT | Stability - Incubation | Activity after incubation (8 hours at 50Β°C), measured with absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 80% | 100.0 | β | 31.4 | % | Incubation: 0.1 M tris-HCl, 10 mM CaCl2, Assay: 0.1 M tris-HCl, 10 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | 0.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase |
| WT | Stability - Incubation | Activity after incubation (12 hours at 30Β°C), measured with absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 80% | 100.0 | β | 22.5 | % | Incubation: 0.1 M tris-HCl, 10 mM CaCl2, Assay: 0.1 M tris-HCl, 10 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 30Β°C, Assay: 37Β°C | 0.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase |
| WT | Stability - Incubation | Activity after incubation (10 hours at 30Β°C), measured with absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 80% | 100.0 | β | 29.6 | % | Incubation: 0.1 M tris-HCl, 10 mM CaCl2, Assay: 0.1 M tris-HCl, 10 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 30Β°C, Assay: 37Β°C | 0.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase |
| WT | Stability - Incubation | Activity after incubation (8 hours at 30Β°C), measured with absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 80% | 100.0 | β | 38.0 | % | Incubation: 0.1 M tris-HCl, 10 mM CaCl2, Assay: 0.1 M tris-HCl, 10 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 30Β°C, Assay: 37Β°C | 0.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase |
| WT | Stability - Incubation | Activity after incubation (6 hours at 30Β°C), measured with absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 80% | 100.0 | β | 50.3 | % | Incubation: 0.1 M tris-HCl, 10 mM CaCl2, Assay: 0.1 M tris-HCl, 10 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 30Β°C, Assay: 37Β°C | 0.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase |
| WT | Stability - Incubation | Activity after incubation (4 hours at 30Β°C), measured with absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 80% | 100.0 | β | 63.7 | % | Incubation: 0.1 M tris-HCl, 10 mM CaCl2, Assay: 0.1 M tris-HCl, 10 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 30Β°C, Assay: 37Β°C | 0.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase |
| WT | Stability - Incubation | Activity after incubation (2 hours at 30Β°C), measured with absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 80% | 100.0 | β | 80.4 | % | Incubation: 0.1 M tris-HCl, 10 mM CaCl2, Assay: 0.1 M tris-HCl, 10 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 30Β°C, Assay: 37Β°C | 0.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Non-incubated control (in %), assay in aqueous phase | Clear about assay in aqueous phase |
| D248N | Stability - Half-life | Half-life in organic solvent at 30Β°C, with residual activity measured with absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 80% | β | 5.7 | 10.2 | hours | Incubation: 0.1 M tris-HCl, 10 mM CaCl2, Assay: 0.1 M tris-HCl, 10 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 30Β°C, Assay: 37Β°C | 0.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Classical aqueous control (in hours) | Clear about assay in aqueous phase |
| N218S | Stability - Half-life | Half-life in organic solvent at 50Β°C, with residual activity measured with absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | β | 4.7 | 10.0 | hours | Incubation: 0.1 M tris-HCl, 10 mM CaCl2, Assay: 0.1 M tris-HCl, 10 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | 0.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Classical aqueous control (in hours) | Clear about assay in aqueous phase |
| D248L | Stability - Half-life | Half-life in organic solvent at 50Β°C, with residual activity measured with absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | β | 4.7 | 4.2 | hours | Incubation: 0.1 M tris-HCl, 10 mM CaCl2, Assay: 0.1 M tris-HCl, 10 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | 0.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Classical aqueous control (in hours) | Clear about assay in aqueous phase |
| D248A | Stability - Half-life | Half-life in organic solvent at 50Β°C, with residual activity measured with absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | β | 4.7 | 3.6 | hours | Incubation: 0.1 M tris-HCl, 10 mM CaCl2, Assay: 0.1 M tris-HCl, 10 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | 0.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Classical aqueous control (in hours) | Clear about assay in aqueous phase |
| D248N | Stability - Half-life | Half-life in organic solvent at 50Β°C, with residual activity measured with absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | β | 4.7 | 3.7 | hours | Incubation: 0.1 M tris-HCl, 10 mM CaCl2, Assay: 0.1 M tris-HCl, 10 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | 0.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Classical aqueous control (in hours) | Clear about assay in aqueous phase |
| D248N/N218S | Stability - Half-life | Half-life in organic solvent at 30Β°C, with residual activity measured with absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 80% | β | 5.7 | 19.2 | hours | Incubation: 0.1 M tris-HCl, 10 mM CaCl2, Assay: 0.1 M tris-HCl, 10 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 30Β°C, Assay: 37Β°C | 0.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Classical aqueous control (in hours) | Clear about assay in aqueous phase |
| D248N/N218S | Stability - Half-life | Half-life in organic solvent at 50Β°C, with residual activity measured with absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 40% | β | 4.7 | 8.0 | hours | Incubation: 0.1 M tris-HCl, 10 mM CaCl2, Assay: 0.1 M tris-HCl, 10 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 50Β°C, Assay: 37Β°C | 0.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Classical aqueous control (in hours) | Clear about assay in aqueous phase |
| D248A | Stability - Half-life | Half-life in organic solvent at 30Β°C, with residual activity measured with absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in aqueous phase | Dimethylformamide (DMF) | 80% | β | 5.7 | 10.3 | hours | Incubation: 0.1 M tris-HCl, 10 mM CaCl2, Assay: 0.1 M tris-HCl, 10 mM CaCl2 | Incubation: 8, Assay: 8 | Incubation: 30Β°C, Assay: 37Β°C | 0.5 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Classical aqueous control (in hours) | Clear about assay in aqueous phase |
Mutations in this dataset (7) β Pascal Martinez et al. (1992)
WT
N218S
N218S/D248N
D248A
D248L
D248N
D248N/N218S
Visualization : Stability β Incubation
Pascal Martinez et al. (1992)
One bar per measurement. Colour = solvent, shade = solvent volume. β β β Reference value. Hover for details.
Mutation Effect
Mutation impact on enzyme stability and function in the presence of organic solvent: comparison of wild-type and mutant values in identical conditions.
Visualization : Stability β Half-life
Pascal Martinez et al. (1992)
One bar per measurement. Colour = solvent, shade = solvent volume. β β β Reference value. Hover for details.
Mutation Effect
Mutation impact on enzyme stability and function in the presence of organic solvent: comparison of wild-type and mutant values in identical conditions.
Chariklia Economou et al. (1992)
β Random mutagenesis to enhance the activity of subtilisin in organic solvents: Characterization of Q103R subtilisin E
Biotechnology and Bioengineering
Β· doi:10.1002/bit.260390610 β
Β· Activity - Michaelis-Menten
▼
Database ID
UniProt: P04189 β
Sequence Annotation
Inferred - from protein name (106 first residues from UniProt sequence absent from the mature protein)
Protein Source
Recombinant, host bacterium Bacillus subtilis DB428
Experimental Data (6 measurements)
6 measurements
| Mutation | Property | Assay | Solvent | Solvent Volume | Aqueous Reference | WT Reference | Measured Value | Units | Solution | pH | Temperature | Substrate(s) | Product(s) | Cofactor(s) | Shaking | Comments |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| WT | Activity - Michaelis-Menten | Km (mM) measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 10% | 0.51 | β | 2.9 | mM | 0.1 M tris-HCl, 10 mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Classical aqueous control (in mM) | Clear about assay in aqueous phase |
| WT | Activity - Michaelis-Menten | kcat (/s) measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 10% | 21.0 | β | 20.0 | 1/s | 0.1 M tris-HCl, 10 mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Classical aqueous control (in 1/s) | Clear about assay in aqueous phase |
| WT | Activity - Michaelis-Menten | kcat/Km (M^-1 * s^-1) * 10^-3 measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 10% | 41.0 | β | 7.0 | 10^-3 * M^-1 * s^-1 | 0.1 M tris-HCl, 10 mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Classical aqueous control (in 10^-3 * M^-1 * s^-1) | Clear about assay in aqueous phase |
| Q103R | Activity - Michaelis-Menten | Km (mM) measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 10% | 0.27 | 2.9 | 1.3 | mM | 0.1 M tris-HCl, 10 mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Classical aqueous control (in mM) | Clear about assay in aqueous phase |
| Q103R | Activity - Michaelis-Menten | kcat (/s) measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 10% | 34.0 | 20.0 | 34.0 | 1/s | 0.1 M tris-HCl, 10 mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Classical aqueous control (in 1/s) | Clear about assay in aqueous phase |
| Q103R | Activity - Michaelis-Menten | kcat/Km (M^-1 * s^-1) * 10^-3 measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 10% | 126.0 | 7.0 | 26.0 | 10^-3 * M^-1 * s^-1 | 0.1 M tris-HCl, 10 mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinyl-Ala-Ala-Pro-Phe , p-Nitroaniline | β | β | Classical aqueous control (in 10^-3 * M^-1 * s^-1) | Clear about assay in aqueous phase |
Mutations in this dataset (2) β Chariklia Economou et al. (1992)
WT
Q103R
Visualization : Activity β Michaelis-Menten
Chariklia Economou et al. (1992)
One bar per measurement. Colour = solvent, shade = solvent volume. β β β Reference value. Hover for details.
Mutation Effect β Michaelis-Menten
Mutation impact on kinetic parameters in the presence of organic solvent: comparison of wild-type and mutant values in identical conditions.
K Chen et al. (1993)
β Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide.
Proceedings of the National Academy of Sciences
Β· doi:10.1073/pnas.90.12.5618 β
Β· Activity - Michaelis-Menten Stability - Half-life
▼
Database ID
UniProt: P04189 β
Sequence Annotation
Inferred - from protein name (106 first residues from UniProt sequence absent from the mature protein)
Protein Source
Recombinant, host bacterium Bacillus subtilis DB428
Experimental Data (65 measurements)
65 measurements β page 1 of 4
| Mutation | Property | Assay | Solvent | Solvent Volume | Aqueous Reference | WT Reference | Measured Value | Units | Solution | pH | Temperature | Substrate(s) | Product(s) | Cofactor(s) | Shaking | Comments |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| WT | Activity - Michaelis-Menten | Km (mM) measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 20% | 0.56 | β | 12.2 | mM | 0.1 M Tris-HCl, 10mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinylβAlaβAlaβProβPhe , p-Nitroaniline | β | β | Classical aqueous control (in mM) |
| WT | Activity - Michaelis-Menten | kcat/Km (M^-1 * s^-1) * 10^-3 measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 20% | 38.0 | β | 1.4 | 10^-3 * M^-1 * s^-1 | 0.1 M Tris-HCl, 10mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinylβAlaβAlaβProβPhe , p-Nitroaniline | β | β | Classical aqueous control (in 10^-3 * M^-1 * s^-1) |
| WT | Activity - Michaelis-Menten | kcat (/s) measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 40% | 21.0 | β | 3.3 | 1/s | 0.1 M Tris-HCl, 10mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinylβAlaβAlaβProβPhe , p-Nitroaniline | β | β | Classical aqueous control (in 1/s) |
| WT | Activity - Michaelis-Menten | Km (mM) measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 40% | 0.56 | β | 20.9 | mM | 0.1 M Tris-HCl, 10mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinylβAlaβAlaβProβPhe , p-Nitroaniline | β | β | Classical aqueous control (in mM) |
| WT | Activity - Michaelis-Menten | kcat/Km (M^-1 * s^-1) * 10^-3 measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 40% | 38.0 | β | 0.16 | 10^-3 * M^-1 * s^-1 | 0.1 M Tris-HCl, 10mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinylβAlaβAlaβProβPhe , p-Nitroaniline | β | β | Classical aqueous control (in 10^-3 * M^-1 * s^-1) |
| WT | Activity - Michaelis-Menten | kcat (/s) measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 20% | 21.0 | β | 17.0 | 1/s | 0.1 M Tris-HCl, 10mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinylβAlaβAlaβProβPhe , p-Nitroaniline | β | β | Classical aqueous control (in 1/s) |
| WT | Stability - Half-life | Half-life in organic solvent, measured by residual activity with absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) | Dimethylformamide (DMF) | 40% | β | β | 6.0 | hours | 0.1 M Tris-HCl, 10mM CaCl2 | 8 | 50Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinylβAlaβAlaβProβPhe , p-Nitroaniline | β | β | Classical aqueous control (in hours) |
| N181S | Activity - Michaelis-Menten | kcat/Km (M^-1 * s^-1) * 10^-3 measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 20% | 120.0 | 1.4 | 4.4 | 10^-3 * M^-1 * s^-1 | 0.1 M Tris-HCl, 10mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinylβAlaβAlaβProβPhe , p-Nitroaniline | β | β | Classical aqueous control (in 10^-3 * M^-1 * s^-1) |
| Q103R | Activity - Michaelis-Menten | Km (mM) measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 20% | 0.25 | 12.0 | 2.7 | mM | 0.1 M Tris-HCl, 10mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinylβAlaβAlaβProβPhe , p-Nitroaniline | β | β | Classical aqueous control (in mM) |
| N181S | Activity - Michaelis-Menten | Km (mM) measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 20% | 0.23 | 12.0 | 3.8 | mM | 0.1 M Tris-HCl, 10mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinylβAlaβAlaβProβPhe , p-Nitroaniline | β | β | Classical aqueous control (in mM) |
| N181S | Activity - Michaelis-Menten | kcat (/s) measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 20% | 27.0 | 17.0 | 17.0 | 1/s | 0.1 M Tris-HCl, 10mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinylβAlaβAlaβProβPhe , p-Nitroaniline | β | β | Classical aqueous control (in 1/s) |
| E156G | Activity - Michaelis-Menten | kcat/Km (M^-1 * s^-1) * 10^-3 measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 20% | 39.0 | 1.4 | 1.5 | 10^-3 * M^-1 * s^-1 | 0.1 M Tris-HCl, 10mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinylβAlaβAlaβProβPhe , p-Nitroaniline | β | β | Classical aqueous control (in 10^-3 * M^-1 * s^-1) |
| E156G | Activity - Michaelis-Menten | Km (mM) measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 20% | 0.25 | 12.0 | 7.8 | mM | 0.1 M Tris-HCl, 10mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinylβAlaβAlaβProβPhe , p-Nitroaniline | β | β | Classical aqueous control (in mM) |
| E156G | Activity - Michaelis-Menten | kcat (/s) measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 20% | 10.0 | 17.0 | 12.0 | 1/s | 0.1 M Tris-HCl, 10mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinylβAlaβAlaβProβPhe , p-Nitroaniline | β | β | Classical aqueous control (in 1/s) |
| Q131D | Activity - Michaelis-Menten | kcat/Km (M^-1 * s^-1) * 10^-3 measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 20% | 33.0 | 1.4 | 2.8 | 10^-3 * M^-1 * s^-1 | 0.1 M Tris-HCl, 10mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinylβAlaβAlaβProβPhe , p-Nitroaniline | β | β | Classical aqueous control (in 10^-3 * M^-1 * s^-1) |
| Q131D | Activity - Michaelis-Menten | Km (mM) measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 20% | 1.15 | 12.0 | 10.0 | mM | 0.1 M Tris-HCl, 10mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinylβAlaβAlaβProβPhe , p-Nitroaniline | β | β | Classical aqueous control (in mM) |
| Q131D | Activity - Michaelis-Menten | kcat (/s) measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 20% | 39.0 | 17.0 | 29.0 | 1/s | 0.1 M Tris-HCl, 10mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinylβAlaβAlaβProβPhe , p-Nitroaniline | β | β | Classical aqueous control (in 1/s) |
| Q103R | Activity - Michaelis-Menten | kcat/Km (M^-1 * s^-1) * 10^-3 measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 20% | 124.0 | 1.4 | 6.8 | 10^-3 * M^-1 * s^-1 | 0.1 M Tris-HCl, 10mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinylβAlaβAlaβProβPhe , p-Nitroaniline | β | β | Classical aqueous control (in 10^-3 * M^-1 * s^-1) |
| S182G | Activity - Michaelis-Menten | kcat (/s) measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 20% | 18.0 | 17.0 | 11.0 | 1/s | 0.1 M Tris-HCl, 10mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinylβAlaβAlaβProβPhe , p-Nitroaniline | β | β | Classical aqueous control (in 1/s) |
| S182G | Activity - Michaelis-Menten | Km (mM) measured by absorbance spectrophotometry (p-nitroaniline absorbance measurement, 410 nm) in the presence of organic solvent | Dimethylformamide (DMF) | 20% | 0.47 | 12.0 | 8.0 | mM | 0.1 M Tris-HCl, 10mM CaCl2 | 8 | 37Β°C | N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide | N-succinylβAlaβAlaβProβPhe , p-Nitroaniline | β | β | Classical aqueous control (in mM) |
Mutations in this dataset (14) β K Chen et al. (1993)
WT
D60N
D60N/D97G/Q103R/G131D/E156G/N181S/S182G/S188P/N218S/T255A
D60N/D97G/Q103R/N218S
D60N/Q103R/N218S
D97G
Q103R
Q131D
E156G
N181S
S182G
S188P
N218S
T255A
Visualization : Activity β Michaelis-Menten
K Chen et al. (1993)
One bar per measurement. Colour = solvent, shade = solvent volume. β β β Reference value. Hover for details.
Mutation Effect β Michaelis-Menten
Mutation impact on kinetic parameters in the presence of organic solvent: comparison of wild-type and mutant values in identical conditions.
Visualization : Stability β Half-life
K Chen et al. (1993)
One bar per measurement. Colour = solvent, shade = solvent volume. β β β Reference value. Hover for details.
Mutation Effect
Mutation impact on enzyme stability and function in the presence of organic solvent: comparison of wild-type and mutant values in identical conditions.