Characterization of ionic liquid-compatible extremophilic laccases for biological lignin valorization

Abstract

Enzymatic lignin depolymerization, as a biomolecular engineering strategy, offers milder reaction conditions and improved selectivity compared to thermochemical methods; however, the low solubility of lignin in water remains a significant challenge. Certain biocompatible ionic liquids (ILs) with high lignin dissolution show promise in overcoming the solubility limitation and improving enzyme effectiveness. In this study, we evaluated the enzymatic activity and stability of five extremophilic laccases sourced from Streptomyces lavendulae REN-7 (Sl), Thermus thermophilus (Tt), Haloferax volcanii (Hv), Trametes versicolor (Tv) and Myceliophthora thermophila (Mt) in the presence of two selected ILs—ethanolamine acetate ([ETA][OAc]) and triethylammonium hydrogen sulfate ([Et₃NH][HSO₄]). The evaluation was conducted using the 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay and nanostructure-initiator mass spectrometry (NIMS). Results showed that the tested enzymes possess greater compatibility with [Et₃NH][HSO₄] than with [ETA][OAc], as demonstrated by stability tests in 5% (w/w) aqueous solutions. Notably, laccases Mt and Tt exhibit a slight activity increase of 10% and 8%, respectively, in the presence of 10% [Et₃NH][HSO₄] compared to the citrate buffer without IL, indicating strong compatibility with such IL under the tested conditions. Laccase Tt showed good compatibility with the ILs and efficiently transformed β-O-4′ and β-β′ model lignin dimers in NIMS assays, highlighting its potential to act on lignin substrates. This study provides a foundation for integrating enzyme engineering with solvent system design to advance lignin valorization and related applications in biological engineering.