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(Received for publication, September 16, 1996, and in revised form, February 10, 1997)
From the Martin-Luther University Halle-Wittenberg, Institute of
Biotechnology, Kurt-Mothes-Strasse 3, D-06120 Halle,
Germany, The thermal inactivation of broad specificity
proteases such as thermolysin and subtilisin is initiated by partial
unfolding processes that render the enzyme susceptible to autolysis.
Previous studies have revealed that a surface-located region in the
N-terminal domain of the thermolysin-like protease produced by
Bacillus stearothermophilus is crucial for thermal
stability. In this region a disulfide bridge between residues 8 and 60 was designed by molecular modelling, and the corresponding single and
double cysteine mutants were constructed. The disulfide bridge was
spontaneously formed in vivo and resulted in a drastic
stabilization of the enzyme. This stabilization presents one of the
very few examples of successful stabilization of a broad specificity
protease by a designed disulfide bond. We propose that the success of
the present stabilization strategy is the result of the localization
and mutation of an area of the molecule involved in the partial
unfolding processes that determine thermal stability.
Volume 272, Number 17,
Issue of April 25, 1997
pp. 11152-11156
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
,
Biocomputing, EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany, the § BIOSON
Research Institute, University of Groningen, Nijenborgh 4, 9747 AG
Groningen, The Netherlands, the ¶ Department of
Genetics, Center for Biological Sciences, University of Groningen,
Kerklaan 30, 9751 NN Haren, The Netherlands, and the
Laboratory of Microbial Gene Technology, Agricultural
University of Norway, P.O. Box 5051, 1432 As, Norway
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