Lipase from Geobacillus stearothermophilus T6
Enzyme Description
Extremophile
Yes
thermophilic organism
EC Number
Sequence
AAASRANDAPIVLLHGFTGWGREEMFGFKYWGGVRGDIEQWLNDNGYRTYTLAVGPLSSNWDRACEAYAQLVGGTVDYGAAHAAKHGHARFGRTYPGLLPELKRGGRIHIIAHSQGGQTARMLVSLLENGSQEEREYAKAHNVSLSPLFEGGHHFVLSVTTIATPHDGTTLVNMVDFTDRFFDLQKAVLKAAAVASNVPYTSQVYDFKLDQWGLRRQPGESFDQYFERLKRSPVWTSTDTARYDLSVPGAEKLNQWVKASPNTYYLSFATERTYRGALTGNYYPELGMNAFSAVVCAPFLGSYRNATLGIDDRWLENDGIVNTFSMNGPKRGSTDRIVPYDGTIKKGVWNDMGTYNVDHLEVIGVDPNPLFDIRAFYLRLAEQLASLQP
Adi Dror et al. (2014)
β Protein Engineering by Random Mutagenesis and Structure-Guided Consensus of Geobacillus stearothermophilus Lipase T6 for Enhanced Stability in Methanol
Applied and Environmental Microbiology
Β· doi:10.1128/AEM.03371-13 β
Β· Stability - Incubation
▼
Database ID
UniProt: Q93A71 β
Sequence Annotation
Explicit - Provided GenBank Accession Number (29 first residues from UniProt sequence absent from the mature protein)
Protein Source
Recombinant, host bacterium Escherichia coli BL21 (DE3)
Experimental Data (40 measurements)
40 measurements β page 1 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 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| WT | Stability - Incubation | Activity after incubation (1 hour at room temperature), measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement, 405 nm) in aqueous phase | Methanol | 60% | 6.2 | β | 1.8 | 1000 * U/mg | Incubation: 50 mM Tris-HCl, 300 mM NaCl, 1 mM CaCl2, 5% glycerol, Assay: 50 mM Tris-HCl, 1 mM CaCl2, 1% acetonitrile, 9% Isopropanol | Incubation: 7.2, Assay: 8 | Incubation: Room temperature, Assay: 40Β°C | 1 mM p-Nitrophenyl laurate | Lauric Acid , p-Nitrophenol | β | β | Water-incubated control (in 1000 * U/mg), assay in aqueous phase | Clear about assay in aqueous phase ("The incubation was stopped by taking 20-_x0002_l aliquots from the mixture and transferring them into 180 _x0002_l lipase buffer. The residual activity was de- termined in a lipase activity assay as described above.") |
| WT | Stability - Incubation | Activity after incubation (1 hour at room temperature), measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement, 405 nm) in aqueous phase | Ethanol | 80% | 6.2 | β | 0.0 | 1000 * U/mg | Incubation: 50 mM Tris-HCl, 300 mM NaCl, 1 mM CaCl2, 5% glycerol, Assay: 50 mM Tris-HCl, 1 mM CaCl2, 1% acetonitrile, 9% Isopropanol | Incubation: 7.2, Assay: 8 | Incubation: Room temperature, Assay: 40Β°C | 1 mM p-Nitrophenyl laurate | Lauric Acid , p-Nitrophenol | β | β | Water-incubated control (in 1000 * U/mg), assay in aqueous phase | Clear about assay in aqueous phase ("The incubation was stopped by taking 20-_x0002_l aliquots from the mixture and transferring them into 180 _x0002_l lipase buffer. The residual activity was de- termined in a lipase activity assay as described above.") |
| WT | Stability - Incubation | Activity after incubation (1 hour at room temperature), measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement, 405 nm) in aqueous phase | Ethanol | 70% | 6.2 | β | 0.0 | 1000 * U/mg | Incubation: 50 mM Tris-HCl, 300 mM NaCl, 1 mM CaCl2, 5% glycerol, Assay: 50 mM Tris-HCl, 1 mM CaCl2, 1% acetonitrile, 9% Isopropanol | Incubation: 7.2, Assay: 8 | Incubation: Room temperature, Assay: 40Β°C | 1 mM p-Nitrophenyl laurate | Lauric Acid , p-Nitrophenol | β | β | Water-incubated control (in 1000 * U/mg), assay in aqueous phase | Clear about assay in aqueous phase ("The incubation was stopped by taking 20-_x0002_l aliquots from the mixture and transferring them into 180 _x0002_l lipase buffer. The residual activity was de- termined in a lipase activity assay as described above.") |
| WT | Stability - Incubation | Activity after incubation (1 hour at room temperature), measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement, 405 nm) in aqueous phase | Ethanol | 60% | 6.2 | β | 0.0 | 1000 * U/mg | Incubation: 50 mM Tris-HCl, 300 mM NaCl, 1 mM CaCl2, 5% glycerol, Assay: 50 mM Tris-HCl, 1 mM CaCl2, 1% acetonitrile, 9% Isopropanol | Incubation: 7.2, Assay: 8 | Incubation: Room temperature, Assay: 40Β°C | 1 mM p-Nitrophenyl laurate | Lauric Acid , p-Nitrophenol | β | β | Water-incubated control (in 1000 * U/mg), assay in aqueous phase | Clear about assay in aqueous phase ("The incubation was stopped by taking 20-_x0002_l aliquots from the mixture and transferring them into 180 _x0002_l lipase buffer. The residual activity was de- termined in a lipase activity assay as described above.") |
| WT | Stability - Incubation | Activity after incubation (1 hour at room temperature), measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement, 405 nm) in aqueous phase | Ethanol | 50% | 6.2 | β | 0.7 | 1000 * U/mg | Incubation: 50 mM Tris-HCl, 300 mM NaCl, 1 mM CaCl2, 5% glycerol, Assay: 50 mM Tris-HCl, 1 mM CaCl2, 1% acetonitrile, 9% Isopropanol | Incubation: 7.2, Assay: 8 | Incubation: Room temperature, Assay: 40Β°C | 1 mM p-Nitrophenyl laurate | Lauric Acid , p-Nitrophenol | β | β | Water-incubated control (in 1000 * U/mg), assay in aqueous phase | Clear about assay in aqueous phase ("The incubation was stopped by taking 20-_x0002_l aliquots from the mixture and transferring them into 180 _x0002_l lipase buffer. The residual activity was de- termined in a lipase activity assay as described above.") |
| WT | Stability - Incubation | Activity after incubation (1 hour at room temperature), measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement, 405 nm) in aqueous phase | Methanol | 50% | 6.2 | β | 4.5 | 1000 * U/mg | Incubation: 50 mM Tris-HCl, 300 mM NaCl, 1 mM CaCl2, 5% glycerol, Assay: 50 mM Tris-HCl, 1 mM CaCl2, 1% acetonitrile, 9% Isopropanol | Incubation: 7.2, Assay: 8 | Incubation: Room temperature, Assay: 40Β°C | 1 mM p-Nitrophenyl laurate | Lauric Acid , p-Nitrophenol | β | β | Water-incubated control (in 1000 * U/mg), assay in aqueous phase | Clear about assay in aqueous phase ("The incubation was stopped by taking 20-_x0002_l aliquots from the mixture and transferring them into 180 _x0002_l lipase buffer. The residual activity was de- termined in a lipase activity assay as described above.") |
| WT | Stability - Incubation | Activity after incubation (1 hour at room temperature), measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement, 405 nm) in aqueous phase | Methanol | 70% | 6.2 | β | 0.0 | 1000 * U/mg | Incubation: 50 mM Tris-HCl, 300 mM NaCl, 1 mM CaCl2, 5% glycerol, Assay: 50 mM Tris-HCl, 1 mM CaCl2, 1% acetonitrile, 9% Isopropanol | Incubation: 7.2, Assay: 8 | Incubation: Room temperature, Assay: 40Β°C | 1 mM p-Nitrophenyl laurate | Lauric Acid , p-Nitrophenol | β | β | Water-incubated control (in 1000 * U/mg), assay in aqueous phase | Clear about assay in aqueous phase ("The incubation was stopped by taking 20-_x0002_l aliquots from the mixture and transferring them into 180 _x0002_l lipase buffer. The residual activity was de- termined in a lipase activity assay as described above.") |
| WT | Stability - Incubation | Activity after incubation (1 hour at room temperature), measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement, 405 nm) in aqueous phase | Methanol | 80% | 6.2 | β | 0.0 | 1000 * U/mg | Incubation: 50 mM Tris-HCl, 300 mM NaCl, 1 mM CaCl2, 5% glycerol, Assay: 50 mM Tris-HCl, 1 mM CaCl2, 1% acetonitrile, 9% Isopropanol | Incubation: 7.2, Assay: 8 | Incubation: Room temperature, Assay: 40Β°C | 1 mM p-Nitrophenyl laurate | Lauric Acid , p-Nitrophenol | β | β | Water-incubated control (in 1000 * U/mg), assay in aqueous phase | Clear about assay in aqueous phase ("The incubation was stopped by taking 20-_x0002_l aliquots from the mixture and transferring them into 180 _x0002_l lipase buffer. The residual activity was de- termined in a lipase activity assay as described above.") |
| A269T | Stability - Incubation | Activity after incubation (1 hour at room temperature), measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement, 405 nm) in aqueous phase | Ethanol | 80% | 6.5 | 0.0 | 0.5 | 1000 * U/mg | Incubation: 50 mM Tris-HCl, 300 mM NaCl, 1 mM CaCl2, 5% glycerol, Assay: 50 mM Tris-HCl, 1 mM CaCl2, 1% acetonitrile, 9% Isopropanol | Incubation: 7.2, Assay: 8 | Incubation: Room temperature, Assay: 40Β°C | 1 mM p-Nitrophenyl laurate | Lauric Acid , p-Nitrophenol | β | β | Water-incubated control (in 1000 * U/mg), assay in aqueous phase | Clear about assay in aqueous phase ("The incubation was stopped by taking 20-_x0002_l aliquots from the mixture and transferring them into 180 _x0002_l lipase buffer. The residual activity was de- termined in a lipase activity assay as described above.") |
| Q185L | Stability - Incubation | Activity after incubation (1 hour at room temperature), measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement, 405 nm) in aqueous phase | Ethanol | 60% | 5.4 | 0.0 | 1.2 | 1000 * U/mg | Incubation: 50 mM Tris-HCl, 300 mM NaCl, 1 mM CaCl2, 5% glycerol, Assay: 50 mM Tris-HCl, 1 mM CaCl2, 1% acetonitrile, 9% Isopropanol | Incubation: 7.2, Assay: 8 | Incubation: Room temperature, Assay: 40Β°C | 1 mM p-Nitrophenyl laurate | Lauric Acid , p-Nitrophenol | β | β | Water-incubated control (in 1000 * U/mg), assay in aqueous phase | Clear about assay in aqueous phase ("The incubation was stopped by taking 20-_x0002_l aliquots from the mixture and transferring them into 180 _x0002_l lipase buffer. The residual activity was de- termined in a lipase activity assay as described above.") |
| Q185L | Stability - Incubation | Activity after incubation (1 hour at room temperature), measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement, 405 nm) in aqueous phase | Ethanol | 70% | 5.4 | 0.0 | 1.0 | 1000 * U/mg | Incubation: 50 mM Tris-HCl, 300 mM NaCl, 1 mM CaCl2, 5% glycerol, Assay: 50 mM Tris-HCl, 1 mM CaCl2, 1% acetonitrile, 9% Isopropanol | Incubation: 7.2, Assay: 8 | Incubation: Room temperature, Assay: 40Β°C | 1 mM p-Nitrophenyl laurate | Lauric Acid , p-Nitrophenol | β | β | Water-incubated control (in 1000 * U/mg), assay in aqueous phase | Clear about assay in aqueous phase ("The incubation was stopped by taking 20-_x0002_l aliquots from the mixture and transferring them into 180 _x0002_l lipase buffer. The residual activity was de- termined in a lipase activity assay as described above.") |
| Q185L | Stability - Incubation | Activity after incubation (1 hour at room temperature), measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement, 405 nm) in aqueous phase | Ethanol | 80% | 5.4 | 0.0 | 0.6 | 1000 * U/mg | Incubation: 50 mM Tris-HCl, 300 mM NaCl, 1 mM CaCl2, 5% glycerol, Assay: 50 mM Tris-HCl, 1 mM CaCl2, 1% acetonitrile, 9% Isopropanol | Incubation: 7.2, Assay: 8 | Incubation: Room temperature, Assay: 40Β°C | 1 mM p-Nitrophenyl laurate | Lauric Acid , p-Nitrophenol | β | β | Water-incubated control (in 1000 * U/mg), assay in aqueous phase | Clear about assay in aqueous phase ("The incubation was stopped by taking 20-_x0002_l aliquots from the mixture and transferring them into 180 _x0002_l lipase buffer. The residual activity was de- termined in a lipase activity assay as described above.") |
| A269T | Stability - Incubation | Activity after incubation (1 hour at room temperature), measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement, 405 nm) in aqueous phase | Ethanol | 50% | 6.5 | 0.7 | 4.0 | 1000 * U/mg | Incubation: 50 mM Tris-HCl, 300 mM NaCl, 1 mM CaCl2, 5% glycerol, Assay: 50 mM Tris-HCl, 1 mM CaCl2, 1% acetonitrile, 9% Isopropanol | Incubation: 7.2, Assay: 8 | Incubation: Room temperature, Assay: 40Β°C | 1 mM p-Nitrophenyl laurate | Lauric Acid , p-Nitrophenol | β | β | Water-incubated control (in 1000 * U/mg), assay in aqueous phase | Clear about assay in aqueous phase ("The incubation was stopped by taking 20-_x0002_l aliquots from the mixture and transferring them into 180 _x0002_l lipase buffer. The residual activity was de- termined in a lipase activity assay as described above.") |
| A269T | Stability - Incubation | Activity after incubation (1 hour at room temperature), measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement, 405 nm) in aqueous phase | Ethanol | 60% | 6.5 | 0.0 | 1.3 | 1000 * U/mg | Incubation: 50 mM Tris-HCl, 300 mM NaCl, 1 mM CaCl2, 5% glycerol, Assay: 50 mM Tris-HCl, 1 mM CaCl2, 1% acetonitrile, 9% Isopropanol | Incubation: 7.2, Assay: 8 | Incubation: Room temperature, Assay: 40Β°C | 1 mM p-Nitrophenyl laurate | Lauric Acid , p-Nitrophenol | β | β | Water-incubated control (in 1000 * U/mg), assay in aqueous phase | Clear about assay in aqueous phase ("The incubation was stopped by taking 20-_x0002_l aliquots from the mixture and transferring them into 180 _x0002_l lipase buffer. The residual activity was de- termined in a lipase activity assay as described above.") |
| A269T | Stability - Incubation | Activity after incubation (1 hour at room temperature), measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement, 405 nm) in aqueous phase | Ethanol | 70% | 6.5 | 0.0 | 0.7 | 1000 * U/mg | Incubation: 50 mM Tris-HCl, 300 mM NaCl, 1 mM CaCl2, 5% glycerol, Assay: 50 mM Tris-HCl, 1 mM CaCl2, 1% acetonitrile, 9% Isopropanol | Incubation: 7.2, Assay: 8 | Incubation: Room temperature, Assay: 40Β°C | 1 mM p-Nitrophenyl laurate | Lauric Acid , p-Nitrophenol | β | β | Water-incubated control (in 1000 * U/mg), assay in aqueous phase | Clear about assay in aqueous phase ("The incubation was stopped by taking 20-_x0002_l aliquots from the mixture and transferring them into 180 _x0002_l lipase buffer. The residual activity was de- termined in a lipase activity assay as described above.") |
| H86Y/A269T | Stability - Incubation | Activity after incubation (1 hour at room temperature), measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement, 405 nm) in aqueous phase | Ethanol | 80% | 6.4 | 0.0 | 2.2 | 1000 * U/mg | Incubation: 50 mM Tris-HCl, 300 mM NaCl, 1 mM CaCl2, 5% glycerol, Assay: 50 mM Tris-HCl, 1 mM CaCl2, 1% acetonitrile, 9% Isopropanol | Incubation: 7.2, Assay: 8 | Incubation: Room temperature, Assay: 40Β°C | 1 mM p-Nitrophenyl laurate | Lauric Acid , p-Nitrophenol | β | β | Water-incubated control (in 1000 * U/mg), assay in aqueous phase | Clear about assay in aqueous phase ("The incubation was stopped by taking 20-_x0002_l aliquots from the mixture and transferring them into 180 _x0002_l lipase buffer. The residual activity was de- termined in a lipase activity assay as described above.") |
| Q185L/A269T | Stability - Incubation | Activity after incubation (1 hour at room temperature), measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement, 405 nm) in aqueous phase | Ethanol | 50% | 6.4 | 0.7 | 3.9 | 1000 * U/mg | Incubation: 50 mM Tris-HCl, 300 mM NaCl, 1 mM CaCl2, 5% glycerol, Assay: 50 mM Tris-HCl, 1 mM CaCl2, 1% acetonitrile, 9% Isopropanol | Incubation: 7.2, Assay: 8 | Incubation: Room temperature, Assay: 40Β°C | 1 mM p-Nitrophenyl laurate | Lauric Acid , p-Nitrophenol | β | β | Water-incubated control (in 1000 * U/mg), assay in aqueous phase | Clear about assay in aqueous phase ("The incubation was stopped by taking 20-_x0002_l aliquots from the mixture and transferring them into 180 _x0002_l lipase buffer. The residual activity was de- termined in a lipase activity assay as described above.") |
| Q185L/A269T | Stability - Incubation | Activity after incubation (1 hour at room temperature), measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement, 405 nm) in aqueous phase | Ethanol | 60% | 6.4 | 0.0 | 2.8 | 1000 * U/mg | Incubation: 50 mM Tris-HCl, 300 mM NaCl, 1 mM CaCl2, 5% glycerol, Assay: 50 mM Tris-HCl, 1 mM CaCl2, 1% acetonitrile, 9% Isopropanol | Incubation: 7.2, Assay: 8 | Incubation: Room temperature, Assay: 40Β°C | 1 mM p-Nitrophenyl laurate | Lauric Acid , p-Nitrophenol | β | β | Water-incubated control (in 1000 * U/mg), assay in aqueous phase | Clear about assay in aqueous phase ("The incubation was stopped by taking 20-_x0002_l aliquots from the mixture and transferring them into 180 _x0002_l lipase buffer. The residual activity was de- termined in a lipase activity assay as described above.") |
| Q185L/A269T | Stability - Incubation | Activity after incubation (1 hour at room temperature), measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement, 405 nm) in aqueous phase | Ethanol | 70% | 6.4 | 0.0 | 1.7 | 1000 * U/mg | Incubation: 50 mM Tris-HCl, 300 mM NaCl, 1 mM CaCl2, 5% glycerol, Assay: 50 mM Tris-HCl, 1 mM CaCl2, 1% acetonitrile, 9% Isopropanol | Incubation: 7.2, Assay: 8 | Incubation: Room temperature, Assay: 40Β°C | 1 mM p-Nitrophenyl laurate | Lauric Acid , p-Nitrophenol | β | β | Water-incubated control (in 1000 * U/mg), assay in aqueous phase | Clear about assay in aqueous phase ("The incubation was stopped by taking 20-_x0002_l aliquots from the mixture and transferring them into 180 _x0002_l lipase buffer. The residual activity was de- termined in a lipase activity assay as described above.") |
| Q185L/A269T | Stability - Incubation | Activity after incubation (1 hour at room temperature), measured by absorbance spectrophotometry (p-nitrophenol absorbance measurement, 405 nm) in aqueous phase | Ethanol | 80% | 6.4 | 0.0 | 1.5 | 1000 * U/mg | Incubation: 50 mM Tris-HCl, 300 mM NaCl, 1 mM CaCl2, 5% glycerol, Assay: 50 mM Tris-HCl, 1 mM CaCl2, 1% acetonitrile, 9% Isopropanol | Incubation: 7.2, Assay: 8 | Incubation: Room temperature, Assay: 40Β°C | 1 mM p-Nitrophenyl laurate | Lauric Acid , p-Nitrophenol | β | β | Water-incubated control (in 1000 * U/mg), assay in aqueous phase | Clear about assay in aqueous phase ("The incubation was stopped by taking 20-_x0002_l aliquots from the mixture and transferring them into 180 _x0002_l lipase buffer. The residual activity was de- termined in a lipase activity assay as described above.") |
Mutations in this dataset (5)
WT
H86Y/A269T
Q185L
Q185L/A269T
A269T
Visualization : Stability β Incubation
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.