Characterization of a Dual Cellulolytic/Xylanolytic AA9 Lytic Polysaccharide Monooxygenase from <i>Thermothelomyces thermophilus</i> and its Utilization Toward Nanocellulose Production in a Multi-Step Bioprocess

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are enzymes able to act on a great variety of polysaccharides by an oxidative cleavage mechanism, targeting not only crystalline substrates, such as cellulose and chitin, but also other amorphous structures including xylan, mannan, and pectin. The key role of LPMOs toward the isolation of nanocellulose from natural substrates has been demonstrated since these enzymes promote amorphogenesis of the substrate and facilitate the defibrillation process. In the present study, an AA9 LPMO from the thermophilic fungus Thermothelomyces thermophilus (TtLPMO9G) with C1-regioselectivity and a dual cellulolytic/xylanolytic activity was heterologously produced in Pichia pastoris and biochemically characterized. The enzyme was employed both as a pre- and a post-treatment step alongside with commercially available and in-house produced tailored cocktails of hemicellulases and cellulases in four-step multi-enzymatic processes for the isolation of nanoscale cellulose from OxiOrganosolv pretreated beechwood. Nanostructures obtained from each of these green bio-processes were examined for their morphological features and dimensions, crystallinity, colloidal stability, and the presence of carboxylate groups. The results demonstrate the formation of well-dispersed nanoscale cellulose in the complete absence of any chemical or mechanical treatment step and verify the importance of efficient hemicellulose removal for the isolation of nanocellulose.