Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retentionAugustyniak, 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.
Research output: Contribution to journal › Article › Academic › peer-review
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.
|Number of pages||11|
|Publication status||Published - Apr-2012|
- 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