Table I.
Thermostability and Equilibrium Unfolding Parameters of Lipase Mutants
|
t(1/2) (min)b |
|||||||||
|---|---|---|---|---|---|---|---|---|---|
| Mutant | Mutationa | 55 °C | 75 °C | 85 °C | T(50)c (°C) | Tm(app)d (°C) | ΔG(H2O)e (kcal/mol) | mf (kcal/mol) | ΔG/m (M) |
| wild-type | — | 2.8 | — | — | 53.3 | 56.0 | 11.39 ± 0.72 | 2.61±0.16 | 2.05 |
| 4D3 | — | — | 4.4 | 15.2 | 68.0 | 71.2 | 13.75 ± 0.40 | 4.74 ± 0.14 | 2.90 |
| 5-A | M134E | — | 38.8 | 46.9 | 93.0 | 72.9 | 14.41 ± 0.35 | 5.04 ± 0.12 | 2.86 |
| 5-B | M137P | — | 101.2 | 54.3 | 93.0 | 74.1 | 14.73 ± 0.37 | 4.71 ± 0.37 | 3.13 |
| 5-D | S163P | — | 22.2 | 49.9 | 72.0 | 72.2 | 13.50 ± 0.43 | 4.55 ± 0.15 | 2.96 |
a
Given mutations are in background of 4D3, which has nine mutations compared to wild-type protein.24
b
Half-life of thermal inactivation.
c
Temperature at which enzyme looses 50% activity upon incubation for 20 min.
d
Mid-point of thermal unfolding transition as calculated by circular dichroism spectroscopy.
e
ΔG relates to the difference beyween the free energies of folded and unfolded states of protein.
f
m indicates the extent of change in free energy as a function of denaturant (GdmCl) concentration.