Para-nitrobenzyl esterase from Bacillus subtilis 168
Enzyme Description
Sequence
MTHQIVTTQYGKVKGTTENGVHKWKGIPYAKPPVGQWRFKAPEPPEVWEDVLDATAYGSICPQPSDLLSLSYTELPRQSEDCLYVNVFAPDTPSKNLPVMVWIHGGAFYLGAGSEPLYDGSKLAAQGEVIVVTLNYRLGPFGFLHLSSFNEAYSDNLGLLDQAAALKWVRENISAFGGDPDNVTVFGESAGGMSIAALLAMPAAKGLFQKAIMESGASRTMTKEQAASTSAAFLQVLGINEGQLDKLHTVSAEDLLKAADQLRIAEKENIFQLFFQPALDPKTLPEEPEKAIAEGAASGIPLLIGTTRDEGYLFFTPDSDVHSQETLDAALEYLLGKPLAEKVADLYPRSLESQIHMMTDLLFWRPAVAYASAQSHYAPVWMYRFDWHPKKPPYNKAFHALELPFVFGNLDGLERMAKAEITDEVKQLSHTIQSAWITFAKTGNPSTEAVNWPAYHEETRETLILDSEITIENDPESEKRQKLFPSKGE
Jeffrey C. Moore et al. (1996)
β Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents
Nature Biotechnology
Β· doi:10.1038/nbt0496-458 β
Β· Activity - Michaelis-Menten
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Database ID
UniProt: P37967 β
Sequence Annotation
Inferred - from protein name
Protein Source
Recombinant, host bacterium Escherichia coli TG1
Experimental Data (14 measurements)
14 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 | kcat/Km (1/(s*M) measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement) in the presence of organic solvent | Dimethylformamide (DMF) | 15% | 5700 | β | 940 | 1/(s*M) | 0.1 M PIPES | 7 | 30Β°C | p-Nitrophenol acetate | Acetic Acid , p-Nitrophenol | β | β | Classical aqueous control (in 1/(s*M)) |
| WT | Activity - Michaelis-Menten | kcat/Km (1/(s*M) measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement) in the presence of organic solvent | Dimethylformamide (DMF) | 30% | 5700 | β | 240 | 1/(s*M) | 0.1 M PIPES | 7 | 30Β°C | p-Nitrophenol acetate | Acetic Acid , p-Nitrophenol | β | β | Classical aqueous control (in 1/(s*M)) |
| H322R/Y370F | Activity - Michaelis-Menten | kcat/Km (1/(s*M) measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement) in the presence of organic solvent | Dimethylformamide (DMF) | 15% | 4100 | 940 | 1100 | 1/(s*M) | 0.1 M PIPES | 7 | 30Β°C | p-Nitrophenol acetate | Acetic Acid , p-Nitrophenol | β | β | Classical aqueous control (in 1/(s*M)) |
| H322R/M358V/Y370F | Activity - Michaelis-Menten | kcat/Km (1/(s*M) measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement) in the presence of organic solvent | Dimethylformamide (DMF) | 15% | 3200 | 940 | 890 | 1/(s*M) | 0.1 M PIPES | 7 | 30Β°C | p-Nitrophenol acetate | Acetic Acid , p-Nitrophenol | β | β | Classical aqueous control (in 1/(s*M)) |
| L144M/H322R/M358V/Y370F | Activity - Michaelis-Menten | kcat/Km (1/(s*M) measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement) in the presence of organic solvent | Dimethylformamide (DMF) | 15% | 2500 | 940 | 860 | 1/(s*M) | 0.1 M PIPES | 7 | 30Β°C | p-Nitrophenol acetate | Acetic Acid , p-Nitrophenol | β | β | Classical aqueous control (in 1/(s*M)) |
| I60V/L144M/H322R/M358V/Y370F | Activity - Michaelis-Menten | kcat/Km (1/(s*M) measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement) in the presence of organic solvent | Dimethylformamide (DMF) | 15% | 3000 | 940 | 1000 | 1/(s*M) | 0.1 M PIPES | 7 | 30Β°C | p-Nitrophenol acetate | Acetic Acid , p-Nitrophenol | β | β | Classical aqueous control (in 1/(s*M)) |
| S94G/L144M/H322R/L334V/M358V/Y370F | Activity - Michaelis-Menten | kcat/Km (1/(s*M) measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement) in the presence of organic solvent | Dimethylformamide (DMF) | 15% | 2400 | 940 | 810 | 1/(s*M) | 0.1 M PIPES | 7 | 30Β°C | p-Nitrophenol acetate | Acetic Acid , p-Nitrophenol | β | β | Classical aqueous control (in 1/(s*M)) |
| L144M/K267R/H322R/L334S/M358V/Y370F | Activity - Michaelis-Menten | kcat/Km (1/(s*M) measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement) in the presence of organic solvent | Dimethylformamide (DMF) | 15% | 2600 | 940 | 640 | 1/(s*M) | 0.1 M PIPES | 7 | 30Β°C | p-Nitrophenol acetate | Acetic Acid , p-Nitrophenol | β | β | Classical aqueous control (in 1/(s*M)) |
| H322R/Y370F | Activity - Michaelis-Menten | kcat/Km (1/(s*M) measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement) in the presence of organic solvent | Dimethylformamide (DMF) | 30% | 4100 | 240 | 320 | 1/(s*M) | 0.1 M PIPES | 7 | 30Β°C | p-Nitrophenol acetate | Acetic Acid , p-Nitrophenol | β | β | Classical aqueous control (in 1/(s*M)) |
| H322R/M358V/Y370F | Activity - Michaelis-Menten | kcat/Km (1/(s*M) measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement) in the presence of organic solvent | Dimethylformamide (DMF) | 30% | 3200 | 240 | 280 | 1/(s*M) | 0.1 M PIPES | 7 | 30Β°C | p-Nitrophenol acetate | Acetic Acid , p-Nitrophenol | β | β | Classical aqueous control (in 1/(s*M)) |
| L144M/H322R/M358V/Y370F | Activity - Michaelis-Menten | kcat/Km (1/(s*M) measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement) in the presence of organic solvent | Dimethylformamide (DMF) | 30% | 2500 | 240 | 330 | 1/(s*M) | 0.1 M PIPES | 7 | 30Β°C | p-Nitrophenol acetate | Acetic Acid , p-Nitrophenol | β | β | Classical aqueous control (in 1/(s*M)) |
| I60V/L144M/H322R/M358V/Y370F | Activity - Michaelis-Menten | kcat/Km (1/(s*M) measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement) in the presence of organic solvent | Dimethylformamide (DMF) | 30% | 3000 | 240 | 360 | 1/(s*M) | 0.1 M PIPES | 7 | 30Β°C | p-Nitrophenol acetate | Acetic Acid , p-Nitrophenol | β | β | Classical aqueous control (in 1/(s*M)) |
| S94G/L144M/H322R/L334V/M358V/Y370F | Activity - Michaelis-Menten | kcat/Km (1/(s*M) measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement) in the presence of organic solvent | Dimethylformamide (DMF) | 30% | 2400 | 240 | 290 | 1/(s*M) | 0.1 M PIPES | 7 | 30Β°C | p-Nitrophenol acetate | Acetic Acid , p-Nitrophenol | β | β | Classical aqueous control (in 1/(s*M)) |
| L144M/K267R/H322R/L334S/M358V/Y370F | Activity - Michaelis-Menten | kcat/Km (1/(s*M) measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement) in the presence of organic solvent | Dimethylformamide (DMF) | 30% | 2600 | 240 | 270 | 1/(s*M) | 0.1 M PIPES | 7 | 30Β°C | p-Nitrophenol acetate | Acetic Acid , p-Nitrophenol | β | β | Classical aqueous control (in 1/(s*M)) |
Mutations in this dataset (7)
WT
I60V/L144M/H322R/M358V/Y370F
S94G/L144M/H322R/L334V/M358V/Y370F
L144M/H322R/M358V/Y370F
L144M/K267R/H322R/L334S/M358V/Y370F
H322R/M358V/Y370F
H322R/Y370F
Visualization : Activity β Michaelis-Menten
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.