(216f) Towards Biocatalytic Lignin Valorization in Aqueous Ionic Liquids Using Thermophilic Laccases

Lignin accounts for 20-30% of terrestrial biomass and is the most abundant aromatic polymer in nature. Converting lignin into high-value chemicals adds revenue for a biorefinery, improving the economic viability of biofuel production. Recently, ionic liquids (ILs) have received increased interest due to their ability to fractionate lignocellulosic biomass during pretreatment. Given the unique properties of ILs for lignin solubility and enzyme biocompatibility, we foresee a great opportunity to develop new strategies for lignin valorization via biocatalysis in aqueous ILs. Indeed, thermophilic enzymes, which exhibited an increased resistance to IL inhibition relative to their mesophilic counterparts, have shown promising results for lignin oxidation in ionic liquids. In this study, the inhibitory effects were further examined using molecular modelling to investigate protein–ligand docking mechanisms. To that end, the stability and activity of thermophilic laccases were evaluated in different concentration of ILs. In the presence of 1-ethyl-3-methylimidazolium acetate ([C₂C₁Im][OAc]), the activity of a thermophilic laccase increased. To date, this activity trend has never been observed with any enzyme-alkylimidazolium IL pair. Extending this system to native lignin, we pretreated biomass with aqueous ILs and characterized the extracted lignin with GPC and 2D HSQC NMR. Subsequently, we introduced a thermophilic laccase into the lignin-containing liquid fraction after aqueous IL pretreatment. Changes to lignin before and after introduction of the laccase were characterized to determine plausible lignolytic oxidation mechanisms of laccase on lignin in ILs. Immobilization on iron nanoparticles further improved the laccase resistance to IL inhibition, resulting in higher lignin conversion and yield of phenolic compounds as quantified by GC-MS. Collectively, these results demonstrate in situ lignin valorization with a thermophilic laccase in aqueous ILs.