Oxidized reducing ends in celluloses: Quantitative profiling relative to molar mass distribution by fluorescence labeling.

Fluorescence labeling with N-(1-naphthyl)ethylenediamine is highly effective for quantifying oxidized reducing end groups (REGs) in cellulosic materials. When combined with size exclusion chromatography in DMAc/LiCl, along with fluorescence / multiple-angle laser light scattering / refractive index detection, a detailed profile of C1-oxidized REGs relative to the molecular weight distribution of the cellulosic material can be obtained. In this work, the derivatization process was extensively optimized, to be carried out heterogeneously in the solvent N-methyl-2-pyrrolidone. Furthermore, we show that to achieve high selectivity for carboxyl groups at the C1 position, keto and aldehyde groups need to be selectively reduced (e.g., by NaBH4), and carboxyl groups other than at C1 need to be blocked (e.g., by methylation with (trimethylsilyl)diazomethane) prior to fluorescence labeling of carboxyl groups at C1 position. Finally, we demonstrate the practical value of the analytical method by measuring the content of the C1-oxidized REGs in cellulose samples after chemical (by Pinnick oxidation) or enzymatic (by treatment with C1-oxidizing LPMO enzymes) oxidation of various pulp samples.

Oxidation of cellulose fibers using LPMOs with varying allomorphic substrate preferences, oxidative regioselectivities, and domain structures.

Lytic polysaccharide monooxygenases (LPMOs) are excellent candidates for enzymatic functionalization of natural polysaccharides, such as cellulose or chitin, and are gaining relevance in the search for renewable biomaterials. Here, we assessed the cellulose fiber modification potential and catalytic performance of eleven cellulose-active fungal AA9-type LPMOs, including C1-, C4-, and C1/C4-oxidizing LPMOs with and without CBM1 carbohydrate-binding modules, on cellulosic substrates with different degrees of crystallinity and polymer chain arrangement, namely, Cellulose I, Cellulose II, and amorphous cellulose. The potential of LPMOs for cellulose fiber modification varied among the LPMOs and depended primarily on operational stability and substrate binding, and, to some extent, also on regioselectivity and domain structure. While all tested LPMOs were active on natural Cellulose I-type fibers, activity on the Cellulose II allomorph was almost exclusively detected for LPMOs containing a CBM1 and LPMOs with activity on soluble hemicelluloses and cello-oligosaccharides, for example NcAA9C from Neurospora crassa. The single-domain variant of NcAA9C oxidized the cellulose fibers to a higher extent than its CBM-containing natural variant and released less soluble products, indicating a more dispersed oxidation pattern without a CBM. Our findings reveal great functional variation among cellulose-active LPMOs, laying the groundwork for further LPMO-based cellulose engineering.