Augustyniak_2012_Protein.Sci_21_487

Reference

Title : Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: factors contributing to increased activity retention - Augustyniak_2012_Protein.Sci_21_487
Author(s) : Augustyniak W , Brzezinska AA , Pijning T , Wienk H , Boelens R , Dijkstra BW , Reetz MT
Ref : Protein Science , 21 :487 , 2012
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 degC 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.

PubMedSearch : Augustyniak_2012_Protein.Sci_21_487
PubMedID: 22267088
Gene_locus related to this paper: bacsu-lip

Related information

Gene_locus bacsu-lip
Family Lipase_2
Structure 3QZU

Citations formats

Augustyniak W, Brzezinska AA, Pijning T, Wienk H, Boelens R, Dijkstra BW, Reetz MT (2012)
Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: factors contributing to increased activity retention
Protein Science 21 :487

Augustyniak W, Brzezinska AA, Pijning T, Wienk H, Boelens R, Dijkstra BW, Reetz MT (2012)
Protein Science 21 :487