Investigating lytic polysaccharide monooxygenase-assisted wood cell wall degradation with microsensors

The study addresses whether hydrogen peroxide (H₂O₂) is the physiologically relevant cosubstrate for lytic polysaccharide monooxygenases (LPMOs) during degradation of plant cell walls and how LPMO activity integrates with cellulases on native wood. LPMOs are copper-dependent enzymes that oxidatively cleave glycosidic bonds in cellulose and other polysaccharides and enhance biomass saccharification. While historically O₂ was considered the cosubstrate, accumulating evidence indicates H₂O₂ drives faster catalysis and higher catalytic efficiencies. However, direct measurements on intact plant cell walls and under conditions mimicking fungal degradation have been lacking, and the functional interplay between LPMOs and cellobiohydrolases on native substrates remains debated. The purpose of this work is to monitor in real time and locally the formation and consumption of H₂O₂ near poplar wood cell walls during enzymatic degradation, to determine whether LPMO consumes H₂O₂ over O₂ when bound to wood, to localize enzyme binding on the cell wall, and to quantify how LPMO activity affects cellobiohydrolase performance.

Prior research established that LPMOs require an initial electron donor (e.g., cellobiose dehydrogenase, CDH) to reduce the active-site copper and then utilize an oxygen species to hydroxylate polysaccharide carbons (C1 or C4), leading to chain scission. Kinetic studies show H₂O₂-driven LPMO catalysis is orders of magnitude faster than O₂-driven catalysis, with reported kcat/KM for H₂O₂ around 10⁶ M⁻¹ s⁻¹ versus ~10³ M⁻¹ s⁻¹ for O₂. Industrial saccharification benefits from supplemental H₂O₂, but the mechanism has not been verified at the microstructural level of wood cell walls. The literature also reports variable synergy between LPMOs and cellobiohydrolases depending on enzyme pairs and substrates; some LPMOs enhance, while others impede cellobiohydrolase activity. Existing LPMO assays (turbidimetric, photometric, O₂ consumption) rely on dispersed or purified substrates and cannot report localized activity on intact plant cell walls, motivating development of microscale, real-time detection methods.

A scanning electrochemical microscopy (SECM) platform with shear-force based positioning was used to place micro(bio)sensors within ~25 µm of poplar wood cell walls in cross-sectional ultramicrotome slices. H₂O₂ microsensors were fabricated by embedding 1–2 µm Pt ultramicroelectrodes in quartz capillaries (outer diameter ~20 ± 5 µm tip), followed by electrodeposition of Prussian blue to catalyze H₂O₂ reduction with high selectivity over O₂. Sensors were calibrated amperometrically (0.0 V vs Ag/AgCl) across 25–200 µM H₂O₂; sensitivity was 0.078 pA µM⁻¹ µm⁻² initially, decreasing to 0.066 pA µM⁻¹ µm⁻² after ~4 h. A glucose microbiosensor was prepared by immobilizing glucose oxidase (GOX) on Pt ultramicroelectrodes using chitosan electrodeposition and polyurethane overcoating to enable localized glucose detection during hydrolysis assays. Positioning relied on measuring resonance frequency attenuation (ca. 340–416 kHz) to approach within ~300 nm and retract to a fixed distance for measurements. Poplar (Populus alba) transverse slices (25 µm) were prepared from debarked branches and soaked in water. Enzymatic systems were applied onto the slice surface by microsyringe under video guidance. For localized H₂O₂ generation, Trichoderma reesei cellobiohydrolases converted cellulose to cellobiose, which was oxidized by an engineered Crassicarpon hotsonii CDH (ChCDH) with increased oxygen turnover, producing H₂O₂ in situ. Control H₂O₂ generation used GOX with β-glucosidase. LPMO activity was probed using Neurospora crassa LPMO9C with its native reductant N. crassa CDHIIA (NcCDH) to reduce and activate LPMO; NcCDH and LPMO possess carbohydrate-binding modules to target cell walls. Time-resolved local H₂O₂ was recorded before and after LPMO addition; dissolved O₂ was monitored with an oxygen sensor positioned near the surface. LPMO dose-response was evaluated (0.5–3 µM). In separate experiments, LPMO and NcCDH were co-applied at the experiment start to assess whether H₂O₂ accumulates or is scavenged. Enzyme localization on cell walls was visualized by fluorescence microscopy using DyLight-labeled LPMO (D633) and NcCDH (D550), assessing co-localization with lignin autofluorescence and identifying binding within S3 secondary walls, middle lamella, and cell corners. To test synergy with cellobiohydrolases, wood slices were pretreated with LPMO9C (with reductant and H₂O₂ cosubstrate), then incubated with TrCel6A (nonreducing-end) or TrCel7A (reducing-end) plus β-glucosidase; glucose formation was monitored locally with the glucose microbiosensor. All reactions were performed in appropriate buffers (e.g., 50 mM sodium acetate pH 5.5 for H₂O₂ measurements, 50 mM potassium phosphate pH 6.0 for glucose assays) at ~20 °C. Data were collected at 2 s⁻¹ and background-corrected; sensors were accepted based on retention of ≥75% sensitivity post-experiment.

• The Prussian blue-modified Pt H₂O₂ microsensor provided selective, stable amperometric detection of H₂O₂ under air-saturated conditions with negligible O₂ reduction, showing linear response from 25–200 µM and sensitivity of 0.078 pA µM⁻¹ µm⁻² (decreasing by 14.3% after ~4 h).
• Localized H₂O₂ generation on poplar cell walls by T. reesei cellobiohydrolases plus engineered ChCDH increased H₂O₂ nearly linearly to 106.1 ± 6.7 µM over 120 min; control without ChCDH showed no H₂O₂ formation. Replacement with GOX + β-glucosidase yielded 208.5 ± 6.2 µM, indicating substrate availability was not limiting for CDH.
• Upon addition of N. crassa LPMO9C with NcCDH, the local H₂O₂ concentration rapidly decreased and plateaued within ~30 min, while dissolved O₂ remained unchanged, demonstrating that wall-bound LPMO consumes H₂O₂ and not O₂ under these conditions.
• LPMO dose-response experiments (0.5–3 µM) showed initial H₂O₂ consumption rates of 3.2–16.7 µM min⁻¹. The lowest estimate of turnover number, assuming all added LPMO was active, was ~0.1 s⁻¹ on wood cell walls, exceeding reported O₂-driven turnovers on amorphous cellulose by ~6-fold.
• High accumulated H₂O₂ ultimately led to LPMO deactivation, consistent with prior observations of H₂O₂-induced inactivation of uncoupled LPMOs; the observed cessation likely reflects a combination of kinetic equilibrium and deactivation.
• When LPMO (1 µM) and NcCDH (0.5 µM) were present from the beginning of hydrolysis, LPMO fully consumed the H₂O₂ generated by the cellobiohydrolase/ChCDH system, preventing accumulation (Fig. 5a), and glucose formation proceeded (Fig. 5b).
• Fluorescence imaging showed that LPMO9C and NcCDH predominantly bind to the S3 secondary cell wall layer rich in cellulose, with some NcCDH binding in middle lamella and cell corners; co-localization suggests frequent interaction and enhanced LPMO affinity upon reduction by CDH.
• LPMO pretreatment of wood slices increased subsequent cellobiohydrolase activity measured by local glucose formation: +40% for TrCel6A and +44% for TrCel7A at 30 min compared to untreated controls, indicating LPMO promotes exoglucanase action on native wood cell walls.
• The microsensor approach provided the first localized, time-resolved measurements of LPMO peroxygenase activity on intact plant cell walls and demonstrated compatibility with complex enzyme mixtures without matrix interference.

The real-time, localized measurements establish that, when bound to native wood cell walls, LPMO utilizes H₂O₂ rather than O₂ as the cosubstrate, supporting the physiological relevance of H₂O₂-driven peroxygenase activity during fungal degradation. The lack of detectable O₂ consumption upon LPMO addition, contrasted with rapid H₂O₂ consumption, resolves uncertainty about cosubstrate usage in situ. Enzyme localization to S3 cell walls confirms access to cellulose-rich domains where oxidative cleavage is most impactful. Although excessive H₂O₂ leads to LPMO deactivation in the experimental setup with high H₂O₂ production, in natural settings extracellular peroxidases and other H₂O₂-consuming enzymes likely maintain low H₂O₂ levels, sustaining LPMO activity longer. The observed enhancement of TrCel6A and TrCel7A activity on LPMO-pretreated wood indicates that LPMO augments cellobiohydrolase performance on native substrates, likely through creating new chain ends and increasing accessibility by modifying hemicellulose and cellulose microfibril interfaces. Collectively, the findings support a synergistic role for LPMOs with hydrolytic enzymes in fungal extracellular catabolism and suggest strategies to optimize industrial saccharification by controlling local H₂O₂ supply.

This work introduces a microsensor-based SECM approach to monitor, with spatial and temporal resolution, H₂O₂ dynamics at wood cell walls and to quantify LPMO peroxygenase activity under near-natural conditions. It demonstrates that N. crassa LPMO9C binds to cellulose-rich wall layers and preferentially consumes H₂O₂ (not O₂), efficiently scavenging H₂O₂ generated in situ by auxiliary oxidoreductases. LPMO activity enhances cellobiohydrolase-catalyzed hydrolysis on native wood, evidencing synergy relevant to fungal physiology and industrial biomass processing. The platform can be extended to study other H₂O₂-consuming/producing enzymes (e.g., peroxidases) and various LPMOs on different solid substrates. Future work should quantify bound-enzyme fractions to refine kinetics, tune H₂O₂ generation to avoid LPMO deactivation, and investigate how cell wall composition and structure modulate LPMO–cellulase synergy.

• Accumulated H₂O₂ concentrations in the experimental setup are higher than typically expected in natura, potentially accelerating LPMO deactivation.
• Exact enzyme and substrate concentrations at the sensor–substrate interfacial region are unknown, preventing precise kinetic parameter determination; turnover estimates assume all added enzyme is active and thus are lower bounds.
• Sensor performance drifts over multi-hour experiments (∼14% sensitivity loss over 4 h); oxygen sensor tip is larger than the H₂O₂ microsensor, potentially affecting spatial resolution.
• Structural complexity and heterogeneity of wood cell walls limit accessibility and may intensify oxidative deactivation of LPMO; findings may vary with different wood species or enzyme cocktails.