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.

Characterization of Flavin-Based Fluorescent Proteins: An Emerging Class of Fluorescent Reporters

“…High values of B-factor indicate uncertainty in precisely locating the coordinates of amino acid side chains in the crystal structure of a protein, which is usually the case for side chains that are relatively less restrained, such as surface exposed residues. In particular, protein engineering techniques that specifically target high B-factor residues for mutations (relative to lower B-factor amino acids) have been shown to improve stability of enzymes [40], [41]. In the context of EcFbFP and iLOV, a higher B-factor in the iLOV binding site may indicate a binding cavity that is less rigid relative to the chromophore-binding cavity in EcFbFP.…”

Cold active lipases – an update

“…In another study using circular dichroism, X-ray structure analysis and nuclear magnetic resonance spectroscopy on B. subtilis Lip-A, it was observed that mutation of surface residues hinder the tendency of Lip-A to undergo precipitation under thermal stress (Augustyniak et al 2012).…”

The Importance of Additive and Non‐Additive Mutational Effects in Protein Engineering

“…Even greater cooperative effects were encountered in a B‐FIT/ISM‐based study of the heat stabilization of the lipase from Bacillus subtilis 22b. 33 Biophysical characterization of the best mutants, including by protein NMR spectroscopy, uncovered the factors contributing to the increased retention of activity at high temperatures, which point to inhibition of protein–protein interactions leading to undesired aggregation and precipitation upon heating 33a. Cooperative long‐range interactions between point mutations on the surface of the lipase mutants are in line with the dramatic non‐additive effects 33b…”