Understanding Laccase-Ionic Liquid Interactions toward Biocatalytic Lignin Conversion in Aqueous Ionic Liquids

Lignin is a crucial component of plant matter; however, it is also largely responsible for the recalcitrance of lignocellulosic biomass when subjected to pretreatment processes. Lignin is generated in large volumes as a waste product from paper and pulping industry as well as cellulosic biorefineries. As a result, lignin valorization is critical to the successful implementation of cellulosic biofuels. To this end, we investigated interactions between three ionic liquids (ILs) and the lignolytic enzyme laccase toward biocatalytic lignin conversion to aromatic monomers. Laccase exhibited minimal loss of activity in 10% diethylamine hydrogensulfate ([DEA][HSO₄]). Changes in V_max and K_m of laccase with respect to IL concentration indicate that [DEA][HSO₄] is a noncompetitive inhibitor, whereas cholinium lysinate ([Ch][Lys]) and [C₂C₁Im][OAc] are mixed inhibitors. Docking simulations suggested that [Ch][Lys] and [C₂C₁Im][OAc] disrupt residues leading to the active site. Experiments with a β-O-4′ linked model dimer revealed that laccase in [C₂C₁Im][OAc] and [Ch][Lys] requires the presence of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) to oxidize the β-O-4′ linkage, leading to polymerization of the model dimer. Alkaline lignin treated with laccase, ABTS, and the aqueous ILs (AILs) showed few structural changes, although the lignin was partially solubilized and converted to degradation products. The major products obtained from alkaline lignin were vanillin, acetosyringone, syringaldehyde, and acetovanillone. The results of this study provide, for the first time, an in-depth explanation of the interactions between laccase and AILs for the purpose of lignin valorization.