Laccase is essential for the biodepolymerization of lignin, but the challenge is that the reaction mechanism has not been fully elucidated. The laccase (Lacc) inactivated mutant of Bacillus ligniniphilus L1 had a sharp decline in its ability to degrade lignin, which proved its indispensable role in lignin depolymerization. The purified Lacc from recombinant Escherichia coli BL21 and its mediator system (LMS) displayed significant lignin degradation capacities as well as remarkable thermotolerance and solvent resistance. The chemical oxygen demand removal rates of LMS for alkaline and milled wood lignin have reached 67.0% and 80.9%, respectively. Comprehensive analyses, including Fourier-transform infrared spectrometry, gas chromatography–mass spectrometry, 2D-HSQC-NMR, and time-of-flight secondary ion mass spectrometry, unveiled that Lacc- and LMS-oxidized lignin include at least 10 or more catalytic reactions. Lacc can effectively degrade G-lignin even without a mediator, and the removal rate of G-lignin is higher than that of S-lignin. In addition, the supplementation of the mediator increased the removal rate of H-lignin by Lacc and the cleavage of interunit linkages such as β-O-4, β-5, β-β, 4-O-5, and 5-5. Moreover, we found that Lacc cannot polymerize some aromatic monomers into dimers or polymers, which is different from fungal and plant laccases. It is by far the most detailed study describing the reaction mechanism of lignin oxidation by bacterial laccase. These results provide new insights into the catalytic mechanism of bacterial laccase and lay the foundation for the application of laccase in lignin valorization.

Symbiotic bacteria in the termite gut system may play an important role in lignin degradation that can assist the subsequent saccharification process. Pseudocitrobacter anthropi MP-4, which is capable of degrading lignin components and rapidly growing on various lignin analogue dyes, was successfully screened from the gut of a wood-feeding termite Microtermes pakistanicus. Further decolorization tests with this strain showed that the strain MP-4 potentially produced some relevant extracellular enzymes to participate in lignin degradation. The removal rate of chemical oxygen demand by this strain was recorded as high as 52.1% when it was incubated in a mineral-salt medium with lignin as the sole carbon source. For the degrading process of MP-4 on lignin, it was proposed through a series of evaluations by field emission scanning electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis, and pyrolysis gas chromatography/mass spectroscopy, that the lignin degradation mechanism of the strain MP-4 would primarily include the cleavage of various chemical linkages and the demethylation reactions. This resulted in a change in the S/G ratio and the disappearance of the biphenyl structure in the lignin components. Thus, these findings suggested that the strain MP-4 uniquely presented an attractive capability to deconstruct lignin components from biomass, which may be potentially valuable for a future industrial exploration.