Hyperthermostable variants of a highly thermostable alpha-amylase.
Genetic screening at temperatures between 70-80 degrees 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 alpha-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 degrees 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 degrees C, corresponding to a nine to ten-fold increase over the wild-type enzyme and the industrial Bacillus licheniformis alpha-amylase Termamyl. These mutations are located in a predicted folding domain of the protein which appears crucial in determining thermal stability.
Institut National de la Recherche Agronomique, Laboratoire de Génétique des Microorganismes, I.N.R.A./C.N.R.S., Thiverval-Grignon, France.
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Joyet
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Declerck
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Gaillardin
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006358:Hot Temperature; D019811:Hydroxylamine; D006898:Hydroxylamines; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D014633:Valine / Q000235:genetics; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D006898:Hydroxylamines; D019811:Hydroxylamine; D000516:alpha-Amylases; D014633:Valine; D000409:Alanine; D002118:Calcium