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

ABSTRACT: 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 inter

SUBMITTER: Augustyniak W 

PROVIDER: S-EPMC3375749 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature