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Hyperthermostable Variants of a Highly Thermostable Alpha-Amylase

Bio/Technology volume 10, pages 1579–1583 (1992)Cite this article

Abstract

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.

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  1. Philippe Joyet: Corresponding author.

Authors and Affiliations

  1. Institut National de la Recherche Agronomique, Laboratoire de Génétique des Microorganismes, I.N.R.A./C.N.R.S., 78850, Thiverval-Grignon, France

    Philippe Joyet, Nathalie Declerck & Claude Gaillardin

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Joyet, P., Declerck, N. & Gaillardin, C. Hyperthermostable Variants of a Highly Thermostable Alpha-Amylase. Nat Biotechnol 10, 1579–1583 (1992). https://doi.org/10.1038/nbt1292-1579

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