LPMO-Catalyzed\nOxidation of Cellulosic Fibers with\nControlled Addition of a Reductant and H<sub>2</sub>O<sub>2</sub>

Cellulose-derived\nbiomaterials offer a sustainable and\nversatile\nplatform for various applications. Enzymatic engineering of these\nfibers, particularly using lytic polysaccharide monooxygenases (LPMOs),\nshows promise due to the ability to introduce functional groups onto\ncellulose surfaces, potentially enabling further functionalization.\nHowever, harnessing LPMOs for fiber engineering remains challenging,\npartly because controlling the enzymatic reaction is difficult and\npartly because limited information is available about how LPMOs modify\nthe fibers. In this study, we explored controlling LPMO-mediated fiber\noxidation by sequentially adding a reductant (gallic acid, GA) and\nH<sub>2</sub>O<sub>2</sub>, using three different carbohydrate-binding\nmodule (CBM)-containing LPMOs. An in-depth analysis of the soluble\nproducts and the <i>M</i><sub>n</sub>, <i>M</i><sub>w</sub>, and carbonyl content in the fiber fraction indicates\nthat fiber oxidation can indeed be controlled by adjusting the amount\nof GA and H<sub>2</sub>O<sub>2</sub> added to the reaction. In particular,\nat lower overall dosages of GA and H<sub>2</sub>O<sub>2</sub>, corresponding\nto low oxidation levels, fiber oxidation occurs rapidly with almost\nno release of soluble oxidized products. Conversely, at higher dosages,\nfiber oxidation levels off, while oxidized oligosaccharides continue\nto be released and the fibers are eroded. Importantly, next to demonstrating\ncontrolled fiber oxidation, this study shows that different cellulose-active\nLPMOs modify the fibers in different manners.