Table 2. ADHA mutants with highest ΔTm and retained activity.
In parentheses the temperature of the first unfolding event (minor peak) is given. Michaelis-Menten kinetics for wild type and M9 are shown in Figure 3.
| Single mutants |
kobs*
(U/mg) |
Tm app
(°C) |
ΔTm
(°C) |
|---|---|---|---|
| Wild type | 0.6 | 43.0 | - |
| L150F | 0.5 | 57.25 ± 0.25 | 14.0 ± 0.25 |
| G149A | 0.4 | 52.5 ± 0 | 9.5 ± 0 |
| M157K | 0.4 | 51.0 ± 0 | 8.0 ± 0 |
| S197E | 0.5 | 49 ± 1 | 7.5 ± 1 |
| V238L | 0.4 | 50.0 ± 0 | 7.0 ± 0 |
| G159A | 1.2 | 48.5 ± 0 | 5.5 ± 0 |
| N212R | 1.1 | 48.0 ± 0 | 5.0 ± 0 |
| T194V | n.d. | 47.0 ± 0 | 4.0 ± 0 |
| V193L | n.d. | 46.5 ± 0 | 3.5 ± 0 |
| V217P | n.d. | 46.0 ± 0 | 3.0 ± 0 |
| Combination mutants |
kobs*
(U/mg) |
Tm app
(°C) |
ΔTm
(°C) |
| M2 (L150F + M157K) | 0.4 | 64.0 ± 0 | 21.0 ± 0 |
| M3 (M2 + S197E) | 0.1 | 69.0 ± 0 | 26.0 ± 0 |
| M4 (M3 + V238L) | 0.2 | 72 (62) ± 0.25 | 29.0 ± 0.25 |
| M5 (M4 + N212R) | 0.2 | 75.5 (64) ± 0 | 32.5 ± 0 |
| M6 (M5 + G149A) | 0.1 | 81.0 (69) ± 0 | 38.0 ± 0 |
| M7 (M6 + G159A) | 0.1 | 85.0 (74) ± 0 | 42.0 ± 0 |
| M8 (M7 + V193L + T194V) | 0.05 | 90.0 (81) ± 0.25 | 47.0 ± 0.25 |
| M9 (M8 + V217P) | 0.03 | 94.5 (84) ± 0 | 51.5 ± 0 |
| M9* (M9 - S197E) | 0.8 | 88.0 (78.5) ± 0 | 45.0 ± 0 |
*kobs values are averages based on 2–3 replicates and for each average the error was smaller than 5% (between ± 0.0015–0.04 U/mg). Cyclohexanol was used as substrate.