Enzymes ability to catalyze reactions in non-natural environments of organic solvents has opened new opportunities for enzyme-based industrial processes. An example for such a process is the enzymatic production of biodiesel by transesterification of oil and an alcohol which involves lipases as catalysts in a non-aqueous environment. Methanol is utilized frequently as the alcohol in the reaction and the limited stability of lipases in high methanol concentrations hinders industrial implementation of the process.
In this research we applied random and rational protein engineering approaches to evolve a lipase for enhanced stability in methanol in order to improve its performance in biodiesel production. We have chosen to work with an unexplored lipase from a thermophilic bacterium, Geobacillus stearothermophilus T6 because a positive correlation between enzyme thermostability and stability in organic solvents was reported in the literature.
A high throughput colorimetric screening assay in 96-well plates was developed to evaluate lipase activity after an incubation period in high methanol concentrations. We found three single mutations which significantly enhanced the lipase stability in 70% methanol compare to the wild type enzyme. The combination of the mutations to a triple variant, H86Y/A269T/R374W, resulted in an additive stabilizing effect in methanol, elevated thermostability and improved methanolysis activity of both soybean and waste oils. Insights from crystal structures of the methanol-stable variants and the wild type enzyme revealed that all three mutations were situated on the surface and enabled the formation of new hydrogen bonds to other residues and to structural water molecules.
