Laccase catalytic reaction chemistry in relation to enzymatic lignin modicatio

Lignocellulosic biomass is a natural and abundant resource for energy and materials in biorefineries. Lignocellulose is composed typically of 15-30% of lignin, which is a hydrophobic biopolymer built of phenylpropanoid units that act as a waterproof, protective shield in plant cells. Lignin is the least exploited biomass component due to a high degree of polymerization and a complex structure, which makes its degradation difficult.

Typically, lignin is burned for energy production in bioprocessing. However considering its complex structure and the fact that it is the only biopolymer exclusively composed of aromatic units, development of an efficient enzymatic processes should be exploited to produce other value-added compounds and thus contribute to valorization of lignin.

Laccases have received lots of attention because they are thought to be able to degrade lignin due to their ability to oxidize phenolic compound using atmospheric oxygen as electron acceptor. Laccase modification of lignin has been studied for decades but the mechanisms remain unclear. The core hypothesis behind this PhD work was to enhance the knowledge of the laccase reaction mechanism and develop new methods to understand the reaction chemistry on small soluble lignin subunits to then progress to laccase reaction on lignin itself.

Ganoderma lucidum laccase was shown in previous study to be able to enhance sugar release during lignocellulose degradation which makes Ganoderma lucidum laccase a good candidate for further characterization studies. The development of both an optimization of the recombinant production and of the purication were studied to yield a highly pure enzyme.

Two methods were developed to measure laccase activity and kinetics on small phenolic compounds related to lignin subunits. The methods used LC-MS analysis and Fourier Transform Infrared (FTIR) spectroscopy coupled to Parallel Factor (PARAFAC) analysis. LC-MS was used to measure kinetics on hydroxycinnamic acids and on a dimeric compound to study, first, the specificity of laccase towards the different lignin subunits and, second, the product profiles generated after laccase activation of the substrates.

The FTIR-PARAFAC coupled method was used to assess potential differences in the reaction profile of laccases of different origins duringoxidation of different lignin subunits. Both methodologies enabled the development of activity assays on small soluble phenolic compounds and an understanding of the preferred laccase reaction mechanism on theses soluble compounds.

The kinetics of organosolv lignin was also studied by measuring the first product after laccase oxidation of lignin, namely radicals, using Electron Paramagnetic Resonance (EPR) spectroscopy. Laccase kinetic parameters on lignin were compared to the one of the soluble hydroxycinnametes to examine laccase specificity toward lignin.

This method also made it possible to study the radical disappearance rate; during laccase oxidation of lignin, radicals were formed by laccase action but at the same time these radicals were quenched due to the highly unstable nature of radicals. The fate of the radicals was also studied. The activation of oxygen from these radicals was studied by measuring the concentration of hydrogen peroxide during laccase oxidation of organosolv lignin and raw birch wood.

The biological role of the laccase-induced hydrogen peroxide production was tested through activation of two lytic polysaccharide monooxygenases (LPMO) reactions, one of which was active on chitin and the other active on cellulose. Both LPMOs could be activated by the levels of hydrogen peroxide produced after laccase oxidation of lignin.

The results obtained in this work showed that a correlation between laccase activation of small soluble compounds cannot be directly translated into laccase action on lignin. Laccase reaction towards soluble substrates was found to be relatively fast and a clear laccase preference towards highly methoxylated compounds, i.e. sinapic acid, was found.

Moreover the product profile after laccase oxidation of these soluble substrates was characterized by the presence of oligomeric compounds derived from the starting substrate. Reactions fingerprints, accounting for both substrate depletion and products formation during laccase oxidation of soluble phenolic compounds, were studied with FTIR and they appeared to be dependent on the laccase origin.

Laccase kinetics on lignin were determined by measuring the semiquinone radicals formed during laccase oxidation. The radical propagation in lignin after laccase oxidation was slower than the one observed for the hydroxycinnamic acids and therefore possible to be measured. The observed radical formation was a sum of two reaction taking place at the same time, namely the radical formation after laccase oxidation and the spontaneous radical quenching.

It appeared that hydrogen peroxide was one possible route for radical propagation of the radicals formed on lignin after laccase oxidation. Hydrogen peroxide formation was induced by laccases and the concentrations produced was high enough to activate lytic polysaccharide monooxygenases reactions, hence suggesting that one role of laccases in lignocellulose degradation could be the controlled formation of hydrogen peroxide in order to activate hydrogen peroxide-dependent enzymes like LPMOs or lignin peroxidases during the initial phase of degradation.