Biologia, Bratislava, 57/Suppl. 11: 203-211, 2002.
ISSN 0006-3088 (Biologia).
Engineering the thermostability of Bacillus licheniformis a-amylase.
Nathalie Declerck1,2*, Mischa Machius3,4, Philippe Joyet1, Georg Wiegand3, Robert Huber3 & Claude Gaillardin1
1 Laboratoire de Genetique Moleculaire et Cellulaire, INRA, CNRS-1925, F-78850 Thiverval-Grignon, France
2 Centre de Biochimie Structurale, CNRS-5048, INSERM-554, 29 rue de Navacelles, F-34090, Montpellier, France; e-mail: email@example.com
3 Max Planck-Institut fur Biochemie, D-85152 Planegg-Martinsried, Germany
4 Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA
* corresponding author
Received: November 26, 2001 / Accepted: March 07, 2002
Bacillus licheniformis a-amylase (BLA) is a highly thermostable enzyme which
is widely used in biotechnological processes. Although it is produced by a
it remains active for several hours at temperatures over 90 °C under conditions
of industrial starch hydrolysis. It is also far more thermostable than the a-amylases
from B. stearothermophilus and B. amyloliquefaciens despite the strong
sequence similarities between these three proteins. BLA provides therefore an
interesting model for protein engineers investigating
thermostability and thermostabilization. Over the last decade, we have
performed an extensive mutational and structural analysis on BLA in order to
elucidate the origin of its unusual thermal properties and, if possible,
increase its thermostability even further. Before the three-dimensional
structure was known, we had used “blind” mutagenesis and identified two
critical positions where aminoacid substitutions could either increase or
decrease significantly the rate of irreversible thermoinactivation. Once a
detailed X-ray structure of BLA was solved, structure-based mutagenesis was
used to probe the role of residues involved in salt-bridges, calcium-binding or
potential deamidation processes. Our results revealed the key role of domain B
and its interface with domain A in determining the overall thermostability of
BLA. Most of the mutations we introduced in this region modify the stability in
one way or another by influencing the network of electrostatic interactions
entrapping a Ca-Na-Ca metal triad at the domain A/B interface. In the course of
this mutational study we have constructed over 500 BLA variants bearing single
or multiple mutations, among which many were found to be either highly detrimental
or slightly beneficial to the stability. The cumulative effect of the mutations
modulate the enzyme stability over a 50 °C temperature range without perturbing
significantly the amylolytic function. Although a full understanding of the origin
of BLA natural thermoresistance has not yet been reached, our study
demonstrated that it is not optimized and that it can be increased or decreased
artificially by several means.
Key words: alpha-amylase, thermostable enzyme, protein engineering, mutagenesis, X-ray structure, calcium binding.