Comparing protein engineering approaches for improvement of lipase stability in non-conventional environments

Improving lipase stability in non-conventional environments, such as organic solvents or extreme pH is desired for different industrial applications, among them biodiesel production from triglycerides and methanol. We have used different protein engineering methods to improve the stability of lipase from Geobacillus stearothermophilus T6 (LipT6) in such conditions. For each approach, activity assays were complemented by biophysical analysis and X-ray structure determination of selected variants.

Directed evolution resulted in one mutation with minor improvement in biodiesel production. Structure guided consensus brought about a variant with 2-fold higher biodiesel production. A semi rational approach of surface charge elimination provided another mutantion which was combined with the previously discovered ones to form a triple mutant with an 87-fold enhanced stability compared to the wild type together with elevated thermostability in buffer and in 50 % methanol. Rational tunnel engineering was performed by incorporating aromatic residues into solvent channels to induce improved hydrophobic packing via π-involving interactions. Computational design of disulfide bonds was another approach that we evaluated and was found successful in generating improved variants. In contrast, computational tools such as the PROSS server or FireProt did not predict superior variants.

In this talk, comparison of the different methods will be discussed and conclusions based on structural work will be presented.