Comprehensive characterization of lignin depolymerization products from woody biomass after thermoalkaliphilic laccase treatment

Identifying lignin-derived products is challenging due to their inherent structural complexity from diverse linkage types within lignin. A significant analytical hurdle is the matrix interference from oligosaccharides and lipids in lignin depolymerization products. Therefore, novel identification methods are needed to distinguish lignin-derived compounds from these interferences. In this study, we explored comprehensive strategies to identify lignin-derived products by thermoalkaliphilic laccase (CtLac, V243D) treatment of woody biomass. Our approach combined suspect screening with in silico MS/MS-based identification, integrating targeted and untargeted methods. By applying relative mass defect filtering specific to lignin and elucidating lignin-derived fragment ions, we isolated and characterized chemical features containing phenylpropanoid moieties from plant biomass matrix interferents. Suspect screening using LC-ESI-MS/MS analysis of CtLac-treated woody biomass identified 14 phenolic compounds. An in silico MS/MS database search using MS-FINDER yielded 27 and 18 tentative lignin depolymerization products from six woody biomass and cypress-OSL, respectively. Notably, V243D demonstrated superior performance, producing 5 lignin oligomers at significantly higher levels compared to CtLac. By providing high-coverage identification of degradation products, our approach lays a solid analytical foundation for lignin valorization. Taken together, this methodology advances the characterization of enzymatic lignin depolymerization products and holds potential for broader applications including chemical degradation processes. • Suspect screening + in silico MS/MS identifies lignin monomers and oligomers. • RMD filtering enriches lignin signals by removing aliphatic interferences. • In silico MS/MS search annotated 42 woody lignin depolymerization products. • Mutant laccase V243D yielded 5 unique oligomers vs wild-type CtLac.