Amino acid racemase from Pseudomonas putida DSM 3263
Enzyme Description
Sequence
MQFSRTLLAASLALLITGQAPLYAAPPLSMDNGTTALTAQNSNAWVEISAGALQHNIRTLQAELGGKSKLCAVLKADAYGHGIGLVMPSIIAQGVPCVAVASNEEARVVRASGFTGQLVRVRLASLGEVEDALQYDMEELVGSAEFARQLDAIAERHGKTLRIHMALNSSGMSRNGVEMTTWSGRGEALQITDQKHLQLVALMTHFAVEDKDDVRKGLAAFNEQTDWLIKHAKLDRSKLTLHAANSFATLEVPEAHLDMVRTGGALFGDTVPTRTEYQRVMQFKSHVAAVHSYPAGNTVGYDRTFTLARDSRLANITVGTSDGYRRVFTNKGHVLINGHRVPVVGKVSMNTLMVDVTDFPDVKGGNEVVLFGKQAGREITQAEIEEINGALLADLYTVWGSSNPKILVD
Christian Femmer et al. (2020)
β Semi-rational engineering of an amino acid racemase that is stabilized in aqueous/organic solvent mixtures
Biotechnology and Bioengineering
Β· doi:10.1002/bit.27449 β
Β· Activity - Michaelis-Menten Stability - Half-life
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Database ID
β
Sequence Annotation
Explicit - Provided
Protein Source
Recombinant, host bacterium Escherichia coli GFP JW2786-1
Experimental Data (54 measurements)
54 measurements β page 3 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 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A62R/W182D/K230R/K233R/T262V/Q278R/A308R/N337R/K373R (P3B4) | Activity - Michaelis-Menten | Km (in mM) measured by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 40% | 0.9 | 1.2 | 2.0 | mM | 20 mM Tris-HCl buffer | 7 | 25Β°C | D-ornithine | L-ornithine | 900 rpm | 900 rpm | Classical aqueous control (in mM) |
| K159R/W182D/K230R/K233R/T262V/Q278R/A308R/N337R/K373R (R9F1) | Activity - Michaelis-Menten | kcat (in 1/s) measured by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 40% | 350.0 | 86.0 | 88.0 | 1/s | 20 mM Tris-HCl buffer | 7 | 25Β°C | D-ornithine | L-ornithine | 900 rpm | 900 rpm | Classical aqueous control (in 1/s) |
| A62R/K159R/W182D/K211R/A220S/K230R/K233R/T262V/Q278R/A308R/N337R/K373R (R9) | Stability - Half-life | Half-life (in minutes), measured by activity assay by HPLC (D-ornithine production measurement) in the presence of organic solvent | Acetonitrile | 30% | β | 35.0 | 159.0 | minutes | Incubation: 20 mM Tris-HCl buffer, Assay: 20 mM Tris-HCl | Incubation: 7, Assay: 7 | Incubation: 25Β°C, Assay: 25Β°C | 40 mM D-ornithine | L-ornithine | Incubation: 800 rpm, Assay: 800 rpm | Incubation: 800 rpm, Assay: 800 rpm | Classical aqueous control (in minutes) |
| W182D/T262V/Q278R/A308R/N337R/K373R (P1G4) | Stability - Half-life | Half-life (in minutes), measured by activity assay by HPLC (D-ornithine production measurement) in the presence of organic solvent | Acetonitrile | 30% | β | 35.0 | 243.0 | minutes | Incubation: 20 mM Tris-HCl buffer, Assay: 20 mM Tris-HCl | Incubation: 7, Assay: 7 | Incubation: 25Β°C, Assay: 25Β°C | 40 mM D-ornithine | L-ornithine | Incubation: 800 rpm, Assay: 800 rpm | Incubation: 800 rpm, Assay: 800 rpm | Classical aqueous control (in minutes) |
| K159R/W182D/K230R/K233R/T262V/Q278R/A308R/N337R (P3B8) | Stability - Half-life | Half-life (in minutes), measured by activity assay by HPLC (D-ornithine production measurement) in the presence of organic solvent | Acetonitrile | 30% | β | 35.0 | 298.0 | minutes | Incubation: 20 mM Tris-HCl buffer, Assay: 20 mM Tris-HCl | Incubation: 7, Assay: 7 | Incubation: 25Β°C, Assay: 25Β°C | 40 mM D-ornithine | L-ornithine | Incubation: 800 rpm, Assay: 800 rpm | Incubation: 800 rpm, Assay: 800 rpm | Classical aqueous control (in minutes) |
| A62R/W182D/K230R/K233R/T262V/Q278R/A308R/N337R/K373R (P3B4) | Stability - Half-life | Half-life (in minutes), measured by activity assay by HPLC (D-ornithine production measurement) in the presence of organic solvent | Acetonitrile | 30% | β | 35.0 | 231.0 | minutes | Incubation: 20 mM Tris-HCl buffer, Assay: 20 mM Tris-HCl | Incubation: 7, Assay: 7 | Incubation: 25Β°C, Assay: 25Β°C | 40 mM D-ornithine | L-ornithine | Incubation: 800 rpm, Assay: 800 rpm | Incubation: 800 rpm, Assay: 800 rpm | Classical aqueous control (in minutes) |
| K159R/W182D/K230R/K233R/T262V/Q278R/A308R/N337R/K373R (R9F1) | Stability - Half-life | Half-life (in minutes), measured by activity assay by HPLC (D-ornithine production measurement) in the presence of organic solvent | Acetonitrile | 30% | β | 35.0 | 220.0 | minutes | Incubation: 20 mM Tris-HCl buffer, Assay: 20 mM Tris-HCl | Incubation: 7, Assay: 7 | Incubation: 25Β°C, Assay: 25Β°C | 40 mM D-ornithine | L-ornithine | Incubation: 800 rpm, Assay: 800 rpm | Incubation: 800 rpm, Assay: 800 rpm | Classical aqueous control (in minutes) |
| W182D/K230R/K233R/T262V/Q278R/A308R/N337R (R2H4) | Stability - Half-life | Half-life (in minutes), measured by activity assay by HPLC (D-ornithine production measurement) in the presence of organic solvent | Acetonitrile | 30% | β | 35.0 | 206.0 | minutes | Incubation: 20 mM Tris-HCl buffer, Assay: 20 mM Tris-HCl | Incubation: 7, Assay: 7 | Incubation: 25Β°C, Assay: 25Β°C | 40 mM D-ornithine | L-ornithine | Incubation: 800 rpm, Assay: 800 rpm | Incubation: 800 rpm, Assay: 800 rpm | Classical aqueous control (in minutes) |
| A62R/K159R/W182D/K211R/A220S/K230R/K233R/T262V/Q278R/A308R/N337R/K373R (R9) | Stability - Half-life | Half-life (in minutes), measured by activity assay by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 40% | β | 19.0 | 87.0 | minutes | Incubation: 20 mM Tris-HCl buffer, Assay: 20 mM Tris-HCl | Incubation: 7, Assay: 7 | Incubation: 25Β°C, Assay: 25Β°C | 40 mM D-ornithine | L-ornithine | Incubation: 800 rpm, Assay: 800 rpm | Incubation: 800 rpm, Assay: 800 rpm | Classical aqueous control (in minutes) |
| W182D/T262V/Q278R/A308R/N337R/K373R (P1G4) | Stability - Half-life | Half-life (in minutes), measured by activity assay by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 40% | β | 19.0 | 137.0 | minutes | Incubation: 20 mM Tris-HCl buffer, Assay: 20 mM Tris-HCl | Incubation: 7, Assay: 7 | Incubation: 25Β°C, Assay: 25Β°C | 40 mM D-ornithine | L-ornithine | Incubation: 800 rpm, Assay: 800 rpm | Incubation: 800 rpm, Assay: 800 rpm | Classical aqueous control (in minutes) |
| K159R/W182D/K230R/K233R/T262V/Q278R/A308R/N337R (P3B8) | Stability - Half-life | Half-life (in minutes), measured by activity assay by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 40% | β | 19.0 | 157.0 | minutes | Incubation: 20 mM Tris-HCl buffer, Assay: 20 mM Tris-HCl | Incubation: 7, Assay: 7 | Incubation: 25Β°C, Assay: 25Β°C | 40 mM D-ornithine | L-ornithine | Incubation: 800 rpm, Assay: 800 rpm | Incubation: 800 rpm, Assay: 800 rpm | Classical aqueous control (in minutes) |
| A62R/W182D/K230R/K233R/T262V/Q278R/A308R/N337R/K373R (P3B4) | Stability - Half-life | Half-life (in minutes), measured by activity assay by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 40% | β | 19.0 | 140.0 | minutes | Incubation: 20 mM Tris-HCl buffer, Assay: 20 mM Tris-HCl | Incubation: 7, Assay: 7 | Incubation: 25Β°C, Assay: 25Β°C | 40 mM D-ornithine | L-ornithine | Incubation: 800 rpm, Assay: 800 rpm | Incubation: 800 rpm, Assay: 800 rpm | Classical aqueous control (in minutes) |
| K159R/W182D/K230R/K233R/T262V/Q278R/A308R/N337R/K373R (R9F1) | Stability - Half-life | Half-life (in minutes), measured by activity assay by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 40% | β | 19.0 | 142.0 | minutes | Incubation: 20 mM Tris-HCl buffer, Assay: 20 mM Tris-HCl | Incubation: 7, Assay: 7 | Incubation: 25Β°C, Assay: 25Β°C | 40 mM D-ornithine | L-ornithine | Incubation: 800 rpm, Assay: 800 rpm | Incubation: 800 rpm, Assay: 800 rpm | Classical aqueous control (in minutes) |
| W182D/K230R/K233R/T262V/Q278R/A308R/N337R (R2H4) | Stability - Half-life | Half-life (in minutes), measured by activity assay by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 40% | β | 19.0 | 144.0 | minutes | Incubation: 20 mM Tris-HCl buffer, Assay: 20 mM Tris-HCl | Incubation: 7, Assay: 7 | Incubation: 25Β°C, Assay: 25Β°C | 40 mM D-ornithine | L-ornithine | Incubation: 800 rpm, Assay: 800 rpm | Incubation: 800 rpm, Assay: 800 rpm | Classical aqueous control (in minutes) |
Mutations in this dataset (7)
WT
A62R/K159R/W182D/K211R/A220S/K230R/K233R/T262V/Q278R/A308R/N337R/K373R (R9)
A62R/W182D/K230R/K233R/T262V/Q278R/A308R/N337R/K373R (P3B4)
K159R/W182D/K230R/K233R/T262V/Q278R/A308R/N337R (P3B8)
K159R/W182D/K230R/K233R/T262V/Q278R/A308R/N337R/K373R (R9F1)
W182D/K230R/K233R/T262V/Q278R/A308R/N337R (R2H4)
W182D/T262V/Q278R/A308R/N337R/K373R (P1G4)
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.
Visualization : Stability β Half-life
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.