Structure and Self-Assembly of Lytic Polysaccharide Monooxygenase-Oxidized Cellulose Nanocrystals

Abstract

Cellulose-derived nanomaterial building blocks, including cellulose nanocrystals (CNCs), have become increasingly important in sustainable materials development. However, the preparation of CNCs requires hazardous chemicals to introduce surface charges that enable liquid crystalline phase behavior, a key parameter for obtaining self-organized, nanostructured materials from CNCs. Lytic polysaccharide monooxygenases (LPMOs), oxidative enzymes that introduce charged carboxyl groups on their cleavage sites in aqueous reaction conditions, offer an environmentally friendly alternative. In this work, two C1-oxidizing LPMOs from fungus Neurospora crassa, one of which contained a carbohydrate-binding module (CBM), were investigated for CNC preparation. The LPMO-oxidized CNCs shared similar features with chemical-derived CNCs, including colloidal stability and a needle-like morphology with typical dimensions of 7 ± 3 nm in width and 142 ± 57 nm in length for CBM-lacking LPMO-oxidized CNCs. The self-organization of the LPMO-oxidized CNCs was characterized in suspensions and solution cast films. Both LPMO-oxidized CNCs showed electrostatically driven self-organization in aqueous colloidal suspension and pseudo-chiral nematic ordering in solid films. The CBM-lacking LPMO generated a higher carboxyl content (0.70 mmol g–1), leading to a more uniform CNC self-organization, favoring LPMOs without CBMs for CNC production. The obtained results demonstrate production of stable colloidal CNCs with self-assembly by C1-oxidizing LPMOs toward a completely green production of advanced, nanostructured cellulose materials.

Carboxylation of sulfated cellulose nanocrystals by family AA9 lytic polysaccharide monooxygenases

“…The approach of producing cellulose nanomaterials using LPMO oxidation of cellulose hierarchies has been applied to prepare carboxylated CNCs from microcrystalline cellulose with C1-oxidizing NcLP-MO9E that has a CBM1 (Koskela et al, 2021). The carboxylated CNC product contained a higher carboxyl group content than in our work, at 0.40 ± 0.07 mmol/g.…”

“…However, proofs of concept for other applications have also emerged. LPMO9s (alone or in combination with cellulases and xylanases) have been demonstrated to produce and oxidize cellulose nanomaterials (Villares et al, 2017;Hu et al, 2018;Moreau et al, 2019;Koskela et al, 2019;Karnaouri et al, 2020;Koskela et al, 2021;Muraleedharan et al, 2021;Rossi et al, 2021;Marjamaa et al, 2022;Magri et al, 2022;Chorozian et al, 2022). In addition, a family AA10 celluloseactive LPMO of bacterial origin has been used to carboxylate cellulose fibers and nanocrystals (Solhi et al, 2022).…”

Oxidative enzyme activation of cellulose substrates for surface modification

“…Here we have optimized a chemo-enzymatic method, based on the unique activity of C1-oxidising LPMOs, to activate cellulose surfaces for further functionalisation. Our study builds upon significant efforts on the kinetic and mechanistic characterisation of LPMOs, 51 recent examples of the use of cellulolytic LPMOs for nanocellulose production, [59][60][61][62][63][64][65] and the development of LPMO assays based on surface carboxylation. 66,92,93 In particular, seminal work by Hsieh and colleagues specifically demonstrated the potential of a chitinolytic LPMO for the chemo-enzymatic functionalisation of insoluble chitin.…”

“…52–58 On the other hand, recent research has demonstrated that the ability of LPMOs to disrupt fibre structure under controlled conditions can be used to generate oxidised nanofibrils for materials applications. 59–65 These latter studies exemplify the use of LPMOs to modify the bulk properties of cellulosic materials. However, the potential of LPMOs to surgically introduce carboxylic acid groups on the cellulose surface for subsequent chemical functionalization is relatively unexplored.…”

Hemicellulose content affects the properties of cellulose nanofibrils produced from softwood pulp fibres by LPMO

“…The released soluble cello-oligosaccharides were quantified as glucose-equivalents with a phenol–sulphuric acid assay according to a previously reported method. 65…”