Analytical strategies for the investigation of enzymatic and microbial ligninolytic activities

Lignocelluloses provide a carbon source for bacteria and fungi species equipped with enzymatic systems able to degrade plant cell-wall components. Ligninolytic enzymes play a key role in these systems through delignification and subsequent increased accessibility of hydrolases to carbohydrates. Studying their action mechanism on lignin substrate is needed not only to understand biodegradation but also to design biocatalysts and biorefinery pathways converting lignocelluloses into valuable bio-based molecules. The choice of an analytical strategy depends on the state and composition of the matrix where the reaction takes place, along with the objective of the study. Solid state analyses including Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), surface mass spectrometry or pyrolysis-gas chromatography-mass spectrometry (pyr-GC-MS) enable rapid detection of changes in lignin overall structural profile in complex matrix like wood, lignocellulosic fibers or insect guts. Accurate investigation of the enzyme reaction mechanisms requires strategies using isolated lignins and lignin-model compounds as substrates. These strategies involve the extraction of phenolic fractions, their kinetic analysis through size-exclusion chromatography (SEC) and liquid chromatography-MS (LC-MS), and in-depth structural analysis by NMR. Though progress have been made in identifying new ligninolytic enzymes and controlling their activity, the chemical heterogeneity and poor solubility of lignins, along with the propensity of their depolymerization products to undergo re-condensation, are limits to address. This chapter illustrates strategies implemented so far to investigate lignin microbial or enzymatic depolymerization, with particular focus on recent high-throughput approaches. It encompasses detailed protocols and leads to recommendations to avoid artefacts or erroneous interpretations of analytical results.