Carbohydrate-binding modules (CBMs) are non-catalytic proteins found appended to carbohydrate-active enzymes. Soil and marine bacteria secrete such enzymes to scavenge nutrition, and they often use CBMs to improve reaction rates and retention of released sugars. Here we present a structural and functional analysis of the recently established CBM family 92. All proteins analysed bind preferentially to β−1,6-glucans. This contrasts with the diversity of predicted substrates among the enzymes attached to CBM92 domains. We present crystal structures for two proteins, and confirm by mutagenesis that tryptophan residues permit ligand binding at three distinct functional binding sites on each protein. Multivalent CBM families are uncommon, so the establishment and structural characterisation of CBM92 enriches the classification database and will facilitate functional prediction in future projects. We propose that CBM92 proteins may cross-link polysaccharides in nature, and might have use in novel strategies for enzyme immobilisation.

Nature has evolved elegant ways to alter the wood cell wall structure through carbohydrate-active enzymes, offering environmentally friendly solutions to tailor the microstructure of wood for high-performance materials. In this work, the cell wall structure of delignified wood is modified under mild reaction conditions using an oxidative enzyme, lytic polysaccharide monooxygenase (LPMO). LPMO oxidation results in nanofibrillation of cellulose microfibril bundles inside the wood cell wall, allowing densification of delignified wood under ambient conditions and low pressure into transparent anisotropic films. The enzymatic nanofibrillation facilitates microfibril fusion and enhances the adhesion between the adjacent wood fiber cells during densification process, thereby significantly improving the mechanical performance of the films in both longitudinal and transverse directions. These results improve the understanding of LPMO-induced microstructural changes in wood and offer an environmentally friendly alternative for harsh chemical treatments and energy-intensive densification processes thus representing a significant advance in sustainable production of high-performance wood-derived materials.

Optically transparent wood has been fabricated by structure-retaining delignification of wood and subsequent infiltration of thermo- or photocurable polymer resins but still limited by the intrinsic low mesopore volume of the delignified wood. Here we report a facile approach to fabricate strong transparent wood composites using the wood xerogel which allows solvent-free infiltration of resin monomers into the wood cell wall under ambient conditions. The wood xerogel with high specific surface area (260 m2 g–1) and high mesopore volume (0.37 cm3 g–1) is prepared by evaporative drying of delignified wood comprising fibrillated cell walls at ambient pressure. The mesoporous wood xerogel is compressible in the transverse direction and provides precise control of the microstructure, wood volume fraction, and mechanical properties for the transparent wood composites without compromising the optical transmittance. Transparent wood composites of large size and high wood volume fraction (50%) are successfully prepared, demonstrating potential scalability of the method.

Lytic polysaccharide monooxygenase (LPMO)-catalysed oxidation of cellulose has emerged as a green alternative to chemical modifications in the production of cellulose nanofibrils (CNFs) from wood pulp fibres. The effect of the hemicellulose content of the starting pulp fibres on the oxidation capabilities of cellulose-active LPMO is important and has not been investigated previously. In this study, the production of LPMO-oxidised CNFs was evaluated on two commercial softwood pulp fibres with different hemicellulose contents. Thin and colloidally stable CNFs were readily obtained from kraft pulp with a hemicellulose content of 16%. The preserved hemicellulose fraction in the kraft pulp enhanced the access of LPMO into the fibre cell wall, enabling the production of homogeneous CNFs with a thin width of 3.7 ± 1.7 nm. By contrast, the LPMO-oxidised dissolving pulp with a lower hemicellulose content of 4% could only be partially disintegrated into thin CNFs, leaving a large amount of cellulose microfibril aggregates with widths of around 50 to 100 nm. CNFs disintegrated from the LPMO-oxidised kraft pulp could be processed into nanopapers with excellent properties including an optical transmittance of 86%, tensile strength of 260 MPa, and Young's modulus of 16.9 GPa. Such CNFs also showed acid-triggered nanofibril gelation owing to the introduced carboxyl groups on cellulose microfibril surfaces. These results indicate that the inherent hemicelluloses present in the wood cell wall are essential for LPMO-mediated CNF production from wood pulp fibres.

Cellulose is the main structural polymer in wood, and its potential in the form of nanomaterial building blocks, nanocelluloses, has now been recognized. Nanocelluloses, including cellulose nanofibers (CNFs) and cellulose nanocrystals (CNCs), have become increasingly important in development of modern sustainable materials. Nanocelluloses are typically produced from wood pulp fibers by chemical pre-treatments that deposit charged functional groups onto cellulose microfibril surfaces, thereby promoting disintegration of the fiber cell wall during mechanical fibrillation. Due to environmental risks related to the use of harsh chemical treatments, it is crucial to develop greener, nature-inspired alternatives. As renowned decomposers of wood, fungi secrete cellulose-active enzymes that work in aqueous reaction conditions. Of these, lytic polysaccharide monooxygenases (LPMOs) have piqued a special interest in green production of nanocellulose owing to their ability to introduce charged carboxyl groups onto cellulose surfaces. However, little is known about the properties of LPMO-oxidized nanocelluloses, their mechanical performance in bulk materials, and the mechanism how LPMOs facilitate fibrillation of the wood fiber cells.

This PhD thesis aimed to dissect the potential of C1-oxidizing LPMOs in the production of nanocelluloses and to clarify the mechanism of LPMO oxidation that facilitates the disintegration of wood cell wall. LPMOs with and without attached carbohydrate-binding modules (CBMs) were recombinantly produced in Pichia pastoris and studied for the production of CNFs and CNCs, which were further processed into bulk materials. The morphology and properties of the nanocelluloses, and the optical and mechanical properties of the bulk materials were characterized. In addition, delignified wood with a preserved cellular structure was used as a model substrate for LPMO oxidation, and the LPMO-induced changes in the wood cell wall structure were investigated using advanced scattering techniques.

The results on CNF production showed that LPMO-oxidized wood pulp fibers can be transformed into discrete and colloidal CNFs by mild mechanical disintegration, analogous to chemical pre-treatments such as 2,2,6,6-tetramethylpyperidine-1-oxy radical (TEMPO)-mediated oxidation. Importantly, these CNFs were well individualized with an average width of 4 nm, resembling that of cellulose microfibrils in wood. Such CNFs were obtained from softwood holocellulose- and kraft pulp fibers with a hemicellulose content of 16–19%, but not from dissolving pulp with a lower hemicellulose content of 4%. Nanopapers prepared from the LPMO-oxidized CNFs were transparent and they demonstrated tensile strengths of ca. 260 MPa and Young’s moduli of ca. 17 GPa. The water suspensions of LPMO-oxidized CNFs also exhibited acid-triggered gelation behavior due to the enzymatically introduced carboxyl groups.

LPMO oxidation was also found applicable in the preparation of CNCs from microcrystalline cellulose. The LPMO-oxidized CNCs had a needle-like morphology and they formed stable colloidal suspensions in water that demonstrated flow-induced birefringence. Solution cast films showed that the CNCs bearing C1 carboxyl groups possessed the pivotal ability to undergo self-assembly into an anisotropic phase. As some LPMOs are appended to a non-catalytic CBM, the effect of this module on nanocellulose production was also determined. CBM was found to increase the release of carboxyl groups from cellulose microfibril surfaces in the form of soluble cello-oligosaccharides. By contrast, a non-modular LPMO introduced more carboxyl groups to the cellulose surfaces, up to 0.53 mmol g^-1 on CNFs, and 0.70 mmol g^-1 on CNCs. Indeed, a non-modular LPMO was found advantageous in production of both CNFs and CNCs.

Despite the important role of LPMOs for natural and biotechnological degradation of wood biomass, the LPMO-induced changes in the wood cell wall structure have remained unknown. In this work, these changes were characterized for the first time. It was shown that a C1-oxiding LPMO can modulate cellulose microfibrils and disrupt the wood cell wall ultrastructure by modifying cellulose surface chemistry. After the LPMO oxidation, the average distance between cellulose microfibril centers increased from 4.1 nm to 10.7 nm, signifying the separation of microfibrils in a microfibril bundle. This result revealed a previously unidentified role for C1-oxidizing LPMOs in degradation of cellulose at the nanoscale. Remarkably, LPMO-treated wood veneers could be further compressed into anisotropic, transparent films with an ultrahigh tensile strength of 824 MPa.

In summary, this PhD thesis clarified the potential of C1-oxidizing LPMOs in green production of nanocelluloses and showed that LPMO oxidation is a suitable method to obtain high-performing isotropic and anisotropic bulk materials from wood. On the basis of the obtained findings, a new model was also proposed which elucidates the mechanism of cellulose degradation at the nanoscale. This study broadened the understanding of LPMOs including their biological- and biotechnological significance and provided new insights into the use of LPMOs for the preparation of cellulose-based nanomaterials.

Cellulosa är den huvudsakliga strukturella polymeren i trä, och dess potential i form av nanomaterialbyggstenar, nanocellulosa, har nu erkänts. Nanocellulosa, inklusive cellulosa nanofibrer (CNF) och cellulosa nanokristaller (CNC), har blivit allt viktigare i utvecklingen av moderna hållbara material. Nanocellulosa tillverkas vanligtvis av trämassafibrer genom kemiska förbehandlingar som avsätter laddade funktionella grupper på cellulosamikrofibrillerytor, vilket främjar sönderdelning av fibercellväggen under mekanisk fibrillering. På grund av miljörisker relaterade till användningen av hårda kemiska behandlingar är det avgörande att utveckla grönare, naturinspirerade alternativ. Som välkända nedbrytare av trä utsöndrar svampar cellulosaaktiva enzymer som arbetar under vattenhaltiga reaktionsförhållanden. Av dessa enzymer har lytiska polysackaridmonooxygenaser (LPMO) väckt ett speciellt intresse för grön produktion av nanocellulosa på grund av deras förmåga att introducera laddade karboxylgrupper på cellulosaytor. Lite är dock känt om egenskaperna hos LPMO-oxiderade nanocellulosa, deras mekaniska prestanda i bulkmaterial och mekanismen för hur LPMO underlättar fibrillering av träfibercellerna.

Denna doktorsavhandling syftade till att dissekera potentialen hos C1-oxiderande LPMO vid produktion av nanocellulosa och att klargöra mekanismen för LPMO-oxidation som underlättar sönderfallet av träcellvägg. LPMO:er med och utan bifogade kolhydratbindande moduler (CBM) producerades rekombinant i Pichia pastoris och studerades för produktion av CNF:er och CNC:er, som vidarebearbetades till bulkmaterial. Nanocellulosornas morfologi och egenskaper samt de optiska och mekaniska egenskaperna hos bulkmaterialen karakteriserades. Dessutom användes delignifierat trä med en bevarad cellstruktur som modellsubstrat för LPMO-oxidation, och de LPMO-inducerade förändringarna i träets cellväggsstruktur undersöktes med hjälp av avancerad spridningsteknik.

Resultaten av CNF-produktion visade att LPMO-oxiderade trämassafibrer kan omvandlas till diskreta och kolloidala CNF:er genom mild mekanisk sönderdelning, analogt med kemiska förbehandlingar såsom 2,2,6,6-tetrametylpyperidin-1-oxiradikal (TEMPO)-medierad oxidation. Viktigt är att dessa CNF var väl individualiserade med en genomsnittlig bredd på 4 nm, som liknar den för cellulosamikrofibriller i trä. Sådana CNF erhölls från barrvedsholocellulosa- och kraftmassafibrer med en hemicellulosahalt på 16–19 %, men inte från dissolvingmassa med en lägre hemicellulosahalt på 4 %. Nanopapper framställda från LPMO-oxiderade CNF var transparenta och de visade draghållfastheter på 260 MPa och Youngs moduler på 17 GPa. Vattensuspensionerna av LPMO-oxiderade CNF:er uppvisade också syrautlöst gelningsbeteende på grund av de enzymatiskt införda karboxylgrupperna.

LPMO-oxidation visade sig också vara användbar vid framställning av CNC från mikrokristallin cellulosa. De LPMO-oxiderade CNC:erna hade en nålliknande morfologi och de bildade stabila kolloidala suspensioner i vatten som visade flödesinducerad dubbelbrytning. Lösningsgjutna filmer visade att CNC:er som bär C1-karboxylgrupper hade den avgörande förmågan att genomgå självmontering till en anisotrop fas. Eftersom vissa LPMOs är bifogade till en icke-katalytisk CBM, bestämdes också effekten av denna modul på nanocellulosaproduktionen. CBM visade sig öka frisättningen av karboxylgrupper från cellulosamikrofibrillerytor i form av lösliga cello-oligosackarider. Däremot introducerade en icke-modulär LPMO fler karboxylgrupper på cellulosaytorna, upp till 0,53 mmol g^-1 på CNF och 0,70 mmol g^-1 på CNC. I själva verket befanns en icke-modulär LPMO vara fördelaktig vid produktion av både CNF och CNC.

Trots LPMOs viktiga roll för naturlig och bioteknisk nedbrytning av träbiomassa, har de LPMO-inducerade förändringarna i träcellväggstrukturen förblivit okända. I detta arbete präglades dessa förändringar för första gången. Det visades att en C1-oxiderande LPMO kan modulera cellulosamikrofibriller och störa träcellväggens ultrastruktur genom att modifiera cellulosaytans kemi. Efter LPMO-oxidationen ökade det genomsnittliga avståndet mellan cellulosamikrofibrillerscentra från 4,1 nm till 10,7 nm, vilket indikerar separationen av mikrofibriller i en mikrofibrillbunt. Detta resultat avslöjade en tidigare oidentifierad roll för C1-oxiderande LPMO vid nedbrytning av cellulosa i nanoskala. Anmärkningsvärt nog kunde LPMO-behandlade träfaner komprimeras ytterligare till anisotropa, transparenta filmer med en ultrahög draghållfasthet på 824 MPa.

Sammanfattningsvis klargjorde denna doktorsavhandling potentialen hos C1-oxiderande LPMOs i grön produktion av nanocellulosa och visade att LPMO-oxidation är en lämplig metod för att erhålla högpresterande isotropa och anisotropa bulkmaterial från trä. På basis av de erhållna resultaten föreslogs också en ny modell som belyser mekanismen för cellulosanedbrytning på nanoskala. Denna studie breddade förståelsen av LPMO inklusive deras biologiska och biotekniska betydelse och gav nya insikter om användningen av LPMO för framställning av cellulosabaserade nanomaterial.

Composite polymer hydrogels are of significant interests for high optical transparency and mechanical performance. In this work, a strong and transparent composite hydrogel is developed from a highly mesoporous cellulose network prepared from wood via top-down delignification followed by TEMPO-mediated oxidation and in situ polymerization of PNIPAM. Individualization of cellulose microfibrils inside the wood cell wall is critical for the fabrication of free-standing composite hydrogel with high water content of 94.9 wt% and high optical transmittance of 85.8% with anisotropic light scattering behavior. The composite hydrogel also showed anisotropic mechanical properties with a tensile strength, Young's modulus and toughness of 317 kPa, 5.4 MPa, and 39.2 kJ m- 3 in axial direction, and 152 kPa, 0.31 MPa and 57.1 kJ m- 3 in the transverse direction, respectively. It also showed thermochromic behavior, i.e., reversibly changing between transparent and brightly white by a temperature change between 25 and 40 degrees C, demonstrating great potential for optical applications.

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.

Protein immobilization on a stationary phase, such as nanocelluloses, is widely used in biodiagnostic, biocatalytic, and bioseparation applications. With the top-down approach which utilizes the native hardwood honeycomb structure, mesoporous cellulose scaffolds can be fabricated without the need for energy-consuming production and bottom-up assembly of nanocelluloses. However, this approach is difficult for preparing softwood-based cellulose scaffolds due to the disintegration of wood cells after complete delignification. Herein, for the first time the use of spruce softwood with a homogenous cellular structure of longitudinally positioned and top-to-bottom joined tracheids is explored as a scaffold for protein immobilization. 1,4-butanediol diglycidyl ether is utilized to crosslink cell wall polysaccharides before the delignification step, thus improving the adhesion between tracheids. The native cellular structure of spruce is well preserved after the complete removal of lignin, enabling the successful production of a highly mesoporous and mechanically robust spruce-derived cellulose scaffold with exceptionally high specific surface area (219 m² g⁻¹). Further amination of the cellulose scaffold allows covalent immobilization of functional biomolecules, such as a lectin protein concanavalin A (Con A) and biotin, on the lumen surfaces and inside the porous cell wall. The Con A immobilized scaffold demonstrates native glycoprotein-binding activity and possible glycoprotein separation application.

The production of cellulose nanofibres (CNFs) typically requires harsh chemistry and strong mechanical fibrillation, both of which have negative environmental impacts. A possible solution is offered by lytic polysaccharide monooxygenases (LPMOs), oxidative enzymes that boost cellulose fibrillation. Although the role of LPMOs in oxidative modification of cellulosic substrates is rather well established, their use in the production of cellulose nanomaterials is not fully explored, and the effect of the carbohydrate-binding module (CBM) on nanofibrillation has not yet been reported. Herein, we studied the activity of two LPMOs, one of which was appended to a CBM, on delignified softwood fibres for green and energy-efficient production of CNFs. The CNFs were used to prepare cellulose nanopapers, and the structure and properties of both nanofibres and nanopapers were determined. Both enzymes were able to facilitate nanocellulose fibrillation and increase colloidal stability of the produced CNFs. However, the CBM-lacking LPMO was more efficient in introducing carboxyl groups (0.53 mmol/g) on the cellulose fibre surfaces and releasing CNFs with thinner width (4.3 ± 1.5 nm) from delignified spruce fibres than the modular LPMO (carboxylate content of 0.38 mmol/g and nanofibre width of 6.7± 2.5 nm through LPMO pretreatment followed by mild homogenisation. The prepared nanopapers showed improved mechanical properties (tensile strength of 262 MPa, and modulus of 16.2 GPa) compared to conventional CNFs preparation methods, demonstrating the potential of LPMOs as green alternatives for cellulose nanomaterials preparation.