Action of an AA9-type lytic polysaccharide monooxygenase on the cellulose surface and its role for synergy with cellulases

Lytic polysaccharide monooxygenase (LPMO) represents a recently discovered, unique principle of O₂-dependent oxidative degradation of highly resistant insoluble polysaccharides such as chitin and cellulose. When used in combination with hydrolytic enzymes, LPMO appears to constitute a highly important factor of the efficiency of enzymatic biomass depolymerization into soluble sugars. LPMO activity on different cellulose substrates was demonstrated from the relatively slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the action of LPMO on the cellulose surface was not available. Specificity of LPMO for degradation of crystalline and amorphous cellulose material on the surface of the substrate was also unknown. We showed by a new fluorescence dye adsorption assay analyzed with confocal laser scanning microscopy that LPMO from Neurospora crassa (AA9-type, C1'-oxidizing) introduced carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further showed that cellulose nano-fibrils exposed on the surface were degraded into shorter and thinner insoluble fragments. Also using AFM, we showed that action of LPMO enabled cellulases to degraded otherwise highly recalcitrant crystalline substrate regions, thus promoting an overall faster and more complete hydrolytic surface degradation. Overall, key characteristics of LPMO action on the cellulose surface were revealed and effects of substrate morphology on the synergy between LPMO and hydrolytic enzymes in cellulose depolymerization were suggested. The proposed important role of "substrate factors" in determining synergy between LPMO and cellulases was strongly supported.