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

Augustyniak, W., Brzezinska, A. A., Pijning, T., Wienk, H., Boelens, R., Dijkstra, B. W. & Reetz, M. T., Apr-2012, In : Protein Science. 21, 4, p. 487-497 11 p.

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DOI

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

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.

Original languageEnglish
Pages (from-to)487-497
Number of pages11
JournalProtein Science
Volume21
Issue number4
Publication statusPublished - Apr-2012

    Keywords

  • lipase, directed evolution, iterative saturation mutagenesis, thermal inactivation, aggregation, ITERATIVE SATURATION MUTAGENESIS, DIRECTED EVOLUTION, MACROMOLECULAR STRUCTURES, PROTEIN THERMOSTABILITY, ESCHERICHIA-COLI, ENZYME STABILITY, STRUCTURAL BASIS, SELECTION, PH

ID: 5522940