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Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention

MPG-Autoren
http://pubman.mpdl.mpg.de/cone/persons/resource/persons58403

Augustyniak,  Wojciech
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons132994

Brzezinska,  Agnieszka A.
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons58919

Reetz,  Manfred T.
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;
Fachbereich Chemie, Philipps-Universität Marburg;

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Zitation

Augustyniak, W., Brzezinska, A. A., Pijning, T., Wienk, H., Boelens, R., Dijkstra, B. W., et al. (2012). Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention. Protein Science, 21(4), 487-497. doi:10.1002/pro.2031.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-000F-EFFF-9
Zusammenfassung
Previously, Lipase A from Bacillus subtilis was subjected to in vitro directed evolution using iterative saturation mutagenesis, with randomization sites chosen on the basis of the highest B-factors available from the crystal structure of the wild-type (WT) enzyme. This provided mutants that, unlike WT enzyme, retained a large part of their activity after heating above 65°C and cooling down. Here, we subjected the three best mutants along with the WT enzyme to biophysical and biochemical characterization. Combining thermal inactivation profiles, circular dichroism, X-ray structure analyses and NMR experiments revealed that mutations of surface amino acid residues counteract the tendency of Lipase A to undergo precipitation under thermal stress. Reduced precipitation of the unfolding intermediates rather than increased conformational stability of the evolved mutants seems to be responsible for the activity retention.