Nanocellulose isolation fromlignocellulose is a tedious and expensive processwith high energy and harsh chemical requirements, primarily due to the recalcitrance of the substrate, which otherwise would have been costeffective due to its abundance. Replacing the chemical steps with biocatalytic processes offers opportunities to solve this bottleneck to a certain extent due to the enzymes substrate specificity and mild reaction chemistry. In this work, we demonstrate the isolation of sulphate-free nanocellulose from organosolv pretreated birch biomass using different glycosyl-hydrolases, along with accessory oxidative enzymes including a lytic polysaccharide monooxygenase (LPMO). The suggested process produced colloidal nanocellulose suspensions (zeta-potential-19.4 mV) with particles of 7-20 nm diameter, high carboxylate content and improved thermostability (T-o= 301 degrees C, T-max= 337 degrees C). Nanocelluloseswere subjected to post-modification using LPMOs of different regioselectivity. The sample from chemical route was the least favorable for LPMO to enhance the carboxylate content, while that from the C1-specific LPMO treatment showed the highest increase in carboxylate content.