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

Wojciech Augustyniak, Agnieszka A. Brzezinska, Tjaard Pijning, Hans Wienk, Rolf Boelens, Bauke W. Dijkstra, Manfred T. Reetz*

*Bijbehorende auteur voor dit werk

OnderzoeksoutputAcademicpeer review

41 Citaten (Scopus)
161 Downloads (Pure)


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 degrees 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.

Originele taal-2English
Pagina's (van-tot)487-497
Aantal pagina's11
TijdschriftProtein Science
Nummer van het tijdschrift4
StatusPublished - apr-2012

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