(70d) Improving Resistance of Hyperthermophilic Laccase to Ionic Liquids for in Situ Lignin Valorization

Lignin makes up 20-30% of plant biomass and is the most abundant aromatic polymer found in nature. Converting lignin from a waste product to value-added chemicals and precursors can increase the revenue stream of cellulosic biorefineries. Ionic liquids (ILs) have been subjects of numerous studies in recent years for their ability to fractionate biomass during pretreatment. Some ILs that are capable of fractionating biomass are also biocompatible with enzyme activity. Given the unique properties of these ILs, there is a unique opportunity to develop new strategies for lignin valorization via biocatalysis in ILs. Thermophilic enzymes have demonstrated improved resistance to IL inhibition when compared to their mesophilic counterparts. To that end, we screened the biocompatibility of 3 ILs with a hyperthermophilic laccase from Thermus thermophilus. Despite the extreme thermophilicity of this laccase (T_opt = 92 ^oC), it demonstrated sensitivity to low concentrations of cholinium lysinate ([Ch][Lys]) and diethylammonium hydrogensulfate ([DEA][HSO₄]). The IL, 1-ethyl-3-methylimidazolium acetate, [C2C1Im][OAc] was observed to be more biocompatible, with severe inaction only observed in 10% [C2C1Im][OAc]. Docking simulations show that these ILs favor binding to a location near the active site that disrupts substrate binding, leading to the perceived sensitivity to the ILs. Using a rational design approach led to the improved activity of this laccase in aqueous ILs, foreshadowing its capabilities to valorize lignin in ILs after biomass pretreatment. Collectively, these results demonstrate the analysis of laccase-IL interactions and the improvement of laccase activity in ILs via a rational design approach.