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Scratching the physio-chemical constraints of lytic polysaccharide monooxygenases (LPMOs)

par Magri, Silvia
Président du jury Debaste, Frédéric
Promoteur Cannella, David
Co-Promoteur Doneux, Thomas
Publication Non publié, 2024-08-30

Thèse de doctorat

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Résumé :

Lytic polysaccharide monooxygenases – LPMOs are mono-copper redox enzymes able to oxidatively cleave β-1,4-glycosid bonds characteristic of natural polysaccharides such as cellulose and chitin. Their action depends on the presence of a suitable reducing agent and activated oxygen species (H2O2) and it is a research hot topic in the lignocellulose biorefinery field for their key role as biomass degradation boosters. Today new (bio)technological applications are envisaged for LPMOs like biomaterial and oligosaccharides production and functionalisation, among others, and cumulative knowledge of LPMO’s catalytic constraints is important to foresee future applications that may embrace the design of new products and/or of new production processes.The pushing factor of this thesis was the delineation of the LPMO working limits by testing some extreme conditions not assessed yet. LPMOs are a vast and diversified class of enzymes classified into 8 families showing high variability in substrate scope and affinity, redox partner preferences, and catalytic rates. To harness the full potential of these polyedric enzymes, systematic biochemical screening is required, this implies having a multitude of candidates LPMOs available. To this matter within this thesis, a high throughput expression and purification platform has been developed. Afterward, the array of produced and purified enzymes was tested to assess the role played by the substrate conformation in the interaction with LPMOs. The results here presented showed that the polysaccharide substrate has a dualistic role of being recognised and processed by the LPMO (substrate chemistry level) on one hand and of directing the enzyme orientation thus modulating its catalysis (substrate ultrastructure level) on the other hand. This highlighted new physical constraints posed by the spatial organisation of the substrate in determining the catalysis of the LPMO enzyme. Moreover, we conducted a preliminary and unique investigation of the gravity effect on the enzymatic depolymerisation of cellulose and lignocellulose biomass. Finally, the electrochemical environment of LPMO photobiocatalysis was investigated and framed both in a natural and industrial context. Indeed, the direct interaction between LPMO and lignin, a natural electron sink, has been demonstrated via confocal laser scanning microscopy. The catalytic boosting effect resulting from light-irradiation of LPMO reactions was for the first time successfully implemented in the enzymatic hydrolysis of industrial-relevant biomass. key to this result was the choice of the biomass pretreatment strategy, with the organoslv ligno-first approach being the elected one.