title: 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 intermediates rather than increased conformational stability of the evolved mutants seems to be responsible for the activity retention.
authors.affiliation: Max-Planck-Institut fur Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mulheim an der Ruhr, Germany.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Augustyniak
authors.forename: Agnieszka A
authors.initials: AA
authors.lastname: Brzezinska
authors.forename: Tjaard
authors.initials: T
authors.lastname: Pijning
authors.forename: Hans
authors.initials: H
authors.lastname: Wienk
authors.forename: Rolf
authors.initials: R
authors.lastname: Boelens
authors.forename: Bauke W
authors.initials: BW
authors.lastname: Dijkstra
authors.forename: Manfred T
authors.initials: MT
authors.lastname: Reetz
mesh_terms: D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D055592:Biophysical Phenomena; D011232:Chemical Precipitation; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D019143:Evolution, Molecular; D006358:Hot Temperature; D008049:Lipase / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011489:Protein Denaturation; D013312:Stress, Physiological
chemical_list: D001426:Bacterial Proteins; D008049:Lipase