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
▼
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 2 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/K159R/W182D/K211R/A220S/K230R/K233R/T262V/Q278R/A308R/N337R/K373R (R9) | Activity - Michaelis-Menten | kcat (in 1/s) measured by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 40% | 367.0 | 86.0 | 92.0 | 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) | Activity - Michaelis-Menten | Km (in mM) measured by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 10% | 1.0 | 3.3 | 0.7 | mM | 20 mM Tris-HCl buffer | 7 | 25Β°C | D-ornithine | L-ornithine | 900 rpm | 900 rpm | Classical aqueous control (in mM) |
| A62R/K159R/W182D/K211R/A220S/K230R/K233R/T262V/Q278R/A308R/N337R/K373R (R9) | Activity - Michaelis-Menten | Km (in mM) measured by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 20% | 1.0 | 1.1 | 4.2 | mM | 20 mM Tris-HCl buffer | 7 | 25Β°C | D-ornithine | L-ornithine | 900 rpm | 900 rpm | Classical aqueous control (in mM) |
| A62R/K159R/W182D/K211R/A220S/K230R/K233R/T262V/Q278R/A308R/N337R/K373R (R9) | Activity - Michaelis-Menten | Km (in mM) measured by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 40% | 1.0 | 1.2 | 1.4 | mM | 20 mM Tris-HCl buffer | 7 | 25Β°C | D-ornithine | L-ornithine | 900 rpm | 900 rpm | Classical aqueous control (in mM) |
| 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 | 20% | 0.9 | 1.1 | 2.4 | mM | 20 mM Tris-HCl buffer | 7 | 25Β°C | D-ornithine | L-ornithine | 900 rpm | 900 rpm | Classical aqueous control (in mM) |
| W182D/K230R/K233R/T262V/Q278R/A308R/N337R (R2H4) | Activity - Michaelis-Menten | kcat (in 1/s) measured by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 10% | 345.0 | 217.0 | 249.0 | 1/s | 20 mM Tris-HCl buffer | 7 | 25Β°C | D-ornithine | L-ornithine | 900 rpm | 900 rpm | Classical aqueous control (in 1/s) |
| W182D/K230R/K233R/T262V/Q278R/A308R/N337R (R2H4) | Activity - Michaelis-Menten | kcat (in 1/s) measured by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 20% | 345.0 | 170.0 | 216.0 | 1/s | 20 mM Tris-HCl buffer | 7 | 25Β°C | D-ornithine | L-ornithine | 900 rpm | 900 rpm | Classical aqueous control (in 1/s) |
| W182D/K230R/K233R/T262V/Q278R/A308R/N337R (R2H4) | Activity - Michaelis-Menten | kcat (in 1/s) measured by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 40% | 345.0 | 86.0 | 93.0 | 1/s | 20 mM Tris-HCl buffer | 7 | 25Β°C | D-ornithine | L-ornithine | 900 rpm | 900 rpm | Classical aqueous control (in 1/s) |
| W182D/K230R/K233R/T262V/Q278R/A308R/N337R (R2H4) | Activity - Michaelis-Menten | Km (in mM) measured by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 10% | 0.9 | 3.3 | 0.6 | mM | 20 mM Tris-HCl buffer | 7 | 25Β°C | D-ornithine | L-ornithine | 900 rpm | 900 rpm | Classical aqueous control (in mM) |
| W182D/K230R/K233R/T262V/Q278R/A308R/N337R (R2H4) | Activity - Michaelis-Menten | Km (in mM) measured by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 20% | 0.9 | 1.1 | 1.8 | mM | 20 mM Tris-HCl buffer | 7 | 25Β°C | D-ornithine | L-ornithine | 900 rpm | 900 rpm | Classical aqueous control (in mM) |
| W182D/K230R/K233R/T262V/Q278R/A308R/N337R (R2H4) | 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 | 0.6 | 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 | 10% | 350.0 | 217.0 | 242.0 | 1/s | 20 mM Tris-HCl buffer | 7 | 25Β°C | D-ornithine | L-ornithine | 900 rpm | 900 rpm | Classical aqueous control (in 1/s) |
| 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 | 20% | 350.0 | 170.0 | 194.0 | 1/s | 20 mM Tris-HCl buffer | 7 | 25Β°C | D-ornithine | L-ornithine | 900 rpm | 900 rpm | Classical aqueous control (in 1/s) |
| K159R/W182D/K230R/K233R/T262V/Q278R/A308R/N337R/K373R (R9F1) | Activity - Michaelis-Menten | Km (in mM) measured by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 10% | 0.9 | 3.3 | 0.6 | 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 | Km (in mM) measured by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 20% | 0.9 | 1.1 | 1.1 | 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 | Km (in mM) measured by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 40% | 0.9 | 1.2 | 0.6 | mM | 20 mM Tris-HCl buffer | 7 | 25Β°C | D-ornithine | L-ornithine | 900 rpm | 900 rpm | Classical aqueous control (in mM) |
| A62R/W182D/K230R/K233R/T262V/Q278R/A308R/N337R/K373R (P3B4) | Activity - Michaelis-Menten | kcat (in 1/s) measured by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 10% | 362.0 | 217.0 | 247.0 | 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/W182D/K230R/K233R/T262V/Q278R/A308R/N337R/K373R (P3B4) | Activity - Michaelis-Menten | kcat (in 1/s) measured by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 20% | 362.0 | 170.0 | 205.0 | 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/W182D/K230R/K233R/T262V/Q278R/A308R/N337R/K373R (P3B4) | Activity - Michaelis-Menten | kcat (in 1/s) measured by HPLC (D-ornithine production measurement) in the presence of organic solvent | Methanol | 40% | 362.0 | 86.0 | 90.0 | 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/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 | 10% | 0.9 | 3.3 | 0.4 | mM | 20 mM Tris-HCl buffer | 7 | 25Β°C | D-ornithine | L-ornithine | 900 rpm | 900 rpm | Classical aqueous control (in mM) |
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.