Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulose surface is lacking. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is also unknown. We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO (from Neurospora crassa) introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed on the surface are degraded into shorter and thinner insoluble fragments. Also using atomic force microscopy, we show that prior action of LPMO enables cellulases to attack otherwise highly resistant crystalline substrate areas and that it promotes an overall faster and more complete surface degradation. Overall, this study reveals key characteristics of LPMO action on the cellulose surface and suggests the effects of substrate morphology on the synergy between LPMO and hydrolytic enzymes in cellulose depolymerization.

The hydrolysis of Whatman no. 1 filter paper by purified cellulolytic components from Trichoderma reesei and the synergistic action of binary combinations of these enzymes on the same substrate were investigated. At 20 milligrams filter paper, enzyme concentrations needed to obtain half-maximal hydrolysis rates (KE values) were in the 3-4 microM range for the cellobiohydrolases (CBHs) and 0.05-0.10 microM for the endoglucanases (EGs). Catalytic-core proteins of CBH I and EG III, lacking the cellulose-binding domain, exhibit KE values 2.3 and 5.1 times higher than those of the intact enzymes. In synergistic combinations of two cellulases, the KE value of at least one enzyme was 3-10-fold reduced. CBH I/CBH II and CBH I/EG III combinations showed the most powerful synergism, and optimal ratios were a function of the total protein concentration. Results obtained in activity and adsorption assays using filter paper pretreated with one component, followed by inactivation and subsequent hydrolysis with the same or another cellulase component, point to a sequential enzymic attack of the cellulose and seems consistent with the mathematical model presented.