Protein Engineering, Vol. 16, No. 4, 287-293,
April 2003
© 2003 Oxford University Press
Hyperthermostabilization of Bacillus licheniformis 
-amylase and modulation of its stability over a 50°C temperature range
1 Génétique Moléculaire et Cellulaire, CNRS–URA1925, INRA–UMR216, F-78850 Thiverval-Grignon, France, 3 University of Texas Southwestern Medical Center, Dallas, TX 75390, USA and 4 Max-Planck-Institut für Biochemie,D-85152 Planegg-Martinsried, Germany
2 To whom correspondence should be addressed. Present address: Centre de Biochimie Structurale, CNRS-5048, INSERM-554, 29 rue de Navacelles, F-34090 Montpellier, France. E-mail: nathalie{at}cbs.cnrs.fr
Bacillus licheniformis 
-amylase (BLA) is a highly thermostable starch-degrading enzyme that has been extensively studied in both academic and industrial laboratories. For over a decade, we have investigated BLA thermal properties and identified amino acid substitutions that significantly increase or decrease the thermostability. This paper describes the cumulative effect of some of the most beneficial point mutations identified in BLA. Remarkably, the Q264S–N265Y double mutation led to a rather limited gain in stability but significantly improved the amylolytic function. The most hyperthermostable variants combined seven amino acid substitutions and inactivated over 100 times more slowly and at temperatures up to 23°C higher than the wild-type enzyme. In addition, two highly destabilizing mutations were introduced in the metal binding site and resulted in a decrease of 25°C in the half-inactivation temperature of the double mutant enzyme compared with wild-type. These mutational effects were analysed by protein modelling based on the recently determined crystal structure of a hyperthermostable BLA variant. Our engineering work on BLA shows that the thermostability of an already naturally highly thermostable enzyme can be substantially improved and modulated over a temperature range of 50°C through a few point mutations.
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