Nanocellulose is one of the most important lignocellulose-derived value added products in the emerging market of biobased polymers. Its isolation upon employment of milder, environmentally friendly processes is particularly attractive. Biocatalysis is a promising approach due to targeted and substrate-specific activity, selectivity, mild and non-toxic chemistry. Endoglucanases are the most exploited enzymes for the production of nanocellulose due to their potential to remove the less ordered amorphous regions from cellulose fibers, leaving intact the more organized, crystalline areas, thus facilitating the nanocellulose isolation without altering the cellulose surface chemistry. Moreover, accessory activities including xylanases and other hemicellulose-acting enzymes hold a key role for the isolation of nanocrystalline cellulose. The newly discovered lytic polysaccharide monooxygenases (LPMOs) are also gaining attention due to their implication in nanocellulose production. Within this context, we report the heterologous expression and production of a novel fungal C1-acting AA9 LPMO from Thermothelomyces thermophila. The enzyme was biochemically characterized for its activity on different polysaccharide substrates, while different electrochemically active compounds were tested as electron donors. The formal potential of the of the Cu(II) center in the active site of the LPMO was determined with the use of large amplitude Fourier Transform alternating current cyclic voltammetry(FTacV). Finally, the application of the enzyme together with other glycoside hydrolases on nanocellulose isolation from beechwood was studied. Starting with an initial mild oxidative organosolv pretreatment, efficient delignification was achieved, leaving behind a cellulose-rich solid fraction. An enzymatic treatment step with LPMO was then applied, followed by different sequential hydrolysis steps with cellulolytic and hemicellulolytic enzymes, thus enabling a fine-tuning of all activities with the aim to shed light on the contribution of different enzymes in each step. Both commercially available enzyme cocktails and a combination of different monoenzymes with specific activities were tested in order to assess their individual effects.