Résumé
Genetic screening at temperatures between 70–80°C far exceeds the range of growth of most bacteria, and is not applicable to isolate easily thermostable protein variants. We describe a temperature shift protocol and an
in vivo
screening method which allowed us to identify a hyperthermostable variant of the thermostable
α
-amylase from
Bacillus licheniformis
. Our strategy was to select, after hydroxylamine mutagenesis, an intragenic suppressor mutation which overcomes a mutation leading to a thermolabile enzyme. Sequence analysis of the mutated gene revealed only one change in the amino acid sequence, substituting a valine for alanine at position 209. This single amino acid replacement increased the half-life of the protein at 90°C by a factor of two to three relative to the wild-type enzyme. When this substitution was combined with another stabilizing substitution (H133Y) we described previously, the stabilizing effects were additive. The half-life of the new protein was about 12 hours at 90°C, corresponding to a nine to ten-fold increase over the wild-type enzyme and the industrial
Bacillus licheniformis
α
-amylase Termamyl
®
. These mutations are located in a predicted folding domain of the protein which appears crucial in determining thermal stability.