Lytic Polysaccharide Monooxygenases Activity Research Articles

Enhancing enzymatic saccharification yields of cellulose at high solid loadings by combining different LPMO activities

BackgroundThe polysaccharides in lignocellulosic biomass hold potential for production of biofuels and biochemicals. However, achieving efficient conversion of this resource into fermentable sugars faces challenges, especially when operating at industrially relevant high solid loadings. While it is clear that combining classical hydrolytic enzymes and lytic polysaccharide monooxygenases (LPMOs) is necessary to achieve high saccharification yields, exactly how these enzymes synergize at high solid loadings remains unclear.ResultsAn LPMO-poor cellulase cocktail, Celluclast 1.5 L, was spiked with one or both of two fungal LPMOs from Thermothielavioides terrestris and Thermoascus aurantiacus, TtAA9E and TaAA9A, respectively, to assess their impact on cellulose saccharification efficiency at high dry matter loading, using Avicel and steam-exploded wheat straw as substrates. The results demonstrate that LPMOs can mitigate the reduction in saccharification efficiency associated with high dry matter contents. The positive effect of LPMO inclusion depends on the type of feedstock and the type of LPMO and increases with the increasing dry matter content and reaction time. Furthermore, our results show that chelating free copper, which may leak out of the active site of inactivated LPMOs during saccharification, with EDTA prevents side reactions with in situ generated H2O2 and the reductant (ascorbic acid).ConclusionsThis study shows that sustaining LPMO activity is vital for efficient cellulose solubilization at high substrate loadings. LPMO cleavage of cellulose at high dry matter loadings results in new chain ends and thus increased water accessibility leading to decrystallization of the substrate, all factors making the substrate more accessible to cellulase action. Additionally, this work highlights the importance of preventing LPMO inactivation and its potential detrimental impact on all enzymes in the reaction.

Carbohydrate-binding modules enhance H2O2 tolerance by promoting lytic polysaccharide monooxygenase active site H2O2 consumption

Lytic polysaccharide monooxygenases (LPMOs) oxidatively depolymerize recalcitrant polysaccharides, which is important for biomass conversion. The catalytic domains of many LPMOs are linked to carbohydrate-binding modules (CBMs) through flexible linkers, but the function of these CBMs in LPMO catalysis is not well understood. In this study, we utilized MtLPMO9L and MtLPMO9G derived from Myceliophthora thermophila to investigate the impact of CBMs on LPMO activity, with particular emphasis on their influence on H2O2 tolerance. Using truncated forms of MtLPMO9G generated by removing the CBM, we found reduced substrate binding affinity and enzymatic activity. Conversely, when the CBM was fused to the C terminus of the single-domain MtLPMO9L to create MtLPMO9L-CBM, we observed a substantial improvement in substrate binding affinity, enzymatic activity, and notably, H2O2 tolerance. Furthermore, molecular dynamics simulations confirmed that the CBM fusion enhances the proximity of the active site to the substrate, thereby promoting multilocal cleavage and impacting the exposure of the copper active site to H2O2. Importantly, the fusion of CBM resulted in more efficient consumption of H2O2 by LPMO, leading to improved enzymatic activity and reduced auto-oxidative damage of the copper active center.

Functional characterization of fungal lytic polysaccharide monooxygenases for cellulose surface oxidation

BackgroundMicrobial lytic polysaccharide monooxygenases (LPMOs) cleave diverse biomass polysaccharides, including cellulose and hemicelluloses, by initial oxidation at C1 or C4 of glycan chains. Within the Carbohydrate-Active Enzymes (CAZy) classification, Auxiliary Activity Family 9 (AA9) comprises the first and largest group of fungal LPMOs, which are often also found in tandem with non-catalytic carbohydrate-binding modules (CBMs). LPMOs originally attracted attention for their ability to potentiate complete biomass deconstruction to monosaccharides. More recently, LPMOs have been applied for selective surface modification of insoluble cellulose and chitin.ResultsTo further explore the catalytic diversity of AA9 LPMOs, over 17,000 sequences were extracted from public databases, filtered, and used to construct a sequence similarity network (SSN) comprising 33 phylogenetically supported clusters. From these, 32 targets were produced successfully in the industrial filamentous fungus Aspergillus niger, 25 of which produced detectable LPMO activity. Detailed biochemical characterization of the eight most highly produced targets revealed individual C1, C4, and mixed C1/C4 regiospecificities of cellulose surface oxidation, different redox co-substrate preferences, and CBM targeting effects. Specifically, the presence of a CBM correlated with increased formation of soluble oxidized products and a more localized pattern of surface oxidation, as indicated by carbonyl-specific fluorescent labeling. On the other hand, LPMOs without native CBMs were associated with minimal release of soluble products and comparatively dispersed oxidation pattern.ConclusionsThis work provides insight into the structural and functional diversity of LPMOs, and highlights the need for further detailed characterization of individual enzymes to identify those best suited for cellulose saccharification versus surface functionalization toward biomaterials applications.

Lytic Polysaccharide Monooxygenase Activity of Tma12 Is Critical for Its Toxicity to Whitefly.

Lytic polysaccharide monooxygenases (LPMOs) are powerful redox enzymes that transform complex carbohydrates through oxidation and make them suitable for saccharification by canonical hydrolases. Due to this property, LPMOs are considered to be a valuable component of enzymatic consortia for industrial biorefineries. Tma12 is a fern entomotoxic protein that kills whitefly and has structural similarities with chitinolytic LPMO. However, its enzymatic activity is poorly understood. Studying the role of the LPMO-like activity in the insecticidal function of Tma12 can be of considerable importance. Our results show that Tma12 preferentially binds and digests β-chitin. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis shows that the digestion of chitin produces chitin oligosaccharides of various lengths (DP2-DP7). The Michaelis constant (km) and catalytic constant (kcat) for hydrocoerulignone are 0.022 mM and 0.044 s-1, respectively. The attenuation of catalytic activity through diethylpyrocarbonate modification abolishes the insecticidal activity of the protein. Our findings reveal that (a) Tma12 is an active LPMO and (b) LPMO activity is indispensable for its function as a bioinsecticide.

Polyphenol Oxidase Products Are Priming Agents For LPMO Peroxygenase Activity.

Polyphenol oxidases catalyze the hydroxylation of monophenols to diphenols, which are reducing agents for lytic polysaccharide monooxygenases (LPMOs) in their degradation of cellulose. In particular, the polyphenol oxidase MtPPO7 from Myceliophthora thermophila convert lignocellulose-derived monophenols, and under the new perspective of the peroxygenase reaction catalyzed by LPMOs, we aim to differentiate the role of the catalytic products of MtPPO7 in priming and fueling of LPMO activity. Exemplified by the activity of MtPPO7 towards guaiacol and by using the benchmark LPMO NcAA9C from Neurospora crassa we show that MtPPO7 catalytic products provide the initial electron for the reduction of Cu(II) to Cu(I) but cannot provide the required reducing power for continuous fueling of the LPMO. We show that the priming reaction occurs with catalytic amounts of MtPPO7 products and that those compounds do not generate substantial amounts of H2O2in situ to fuel the LPMO peroxygenase activity. Reducing agents with a low propensity to generate H2O2 can provide the means for controlling the LPMO catalysis through exogenous H2O2 and thereby minimize any enzyme inactivation.

The Role of the Residue at Position 2 in the Catalytic Activity of AA9 Lytic Polysaccharide Monooxygenases.

AA9 lytic polysaccharide monooxygenases (LPMOs) are copper-dependent metalloenzymes that play a major role in cellulose degradation and plant infection. Understanding the AA9 LPMO mechanism would facilitate the improvement of plant pathogen control and the industrial application of LPMOs. Herein, via point mutation, we investigated the role of glycine 2 residue in cellulose degradation by Thermoascus aurantiacus AA9 LPMOs (TaAA9). A computational simulation showed that increasing the steric properties of this residue by replacing glycine with threonine or tyrosine altered the H-bonding network of the copper center and copper coordination geometry, decreased the surface charge of the catalytic center, weakened the TaAA9-substrate interaction, and enhanced TaAA9-product binding. Compared with wild-type TaAA9, G2T-TaAA9 and G2Y-TaAA9 variants showed attenuated copper affinity, reduced oxidative product diversity and decreased substrate Avicel binding, as determined using ITC, MALDI-TOF/TOF MS and cellulose binding analyses, respectively. Consistently, the enzymatic activity and synergy with cellulase of the G2T-TaAA9 and G2Y-TaAA9 variants were lower than those of TaAA9. Hence, the investigated residue crucially affects the catalytic activity of AA9 LPMOs, and we propose that the electropositivity of copper may correlate with AA9 LPMO activity. Thus, the relationship among the amino acid at position 2, surface charge and catalytic activity may facilitate an understanding of the proteins in AA9 LPMOs.

A fungal lytic polysaccharide monooxygenase is required for cell wall integrity, thermotolerance, and virulence of the fungal human pathogen Cryptococcus neoformans.

Fungi often adapt to environmental stress by altering their size, shape, or rate of cell division. These morphological changes require reorganization of the cell wall, a structural feature external to the cell membrane composed of highly interconnected polysaccharides and glycoproteins. Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that are typically secreted into the extracellular space to catalyze initial oxidative steps in the degradation of complex biopolymers such as chitin and cellulose. However, their roles in modifying endogenous microbial carbohydrates are poorly characterized. The CEL1 gene in the human fungal pathogen Cryptococcus neoformans (Cn) is predicted by sequence homology to encode an LPMO of the AA9 enzyme family. The CEL1 gene is induced by host physiological pH and temperature, and it is primarily localized to the fungal cell wall. Targeted mutation of the CEL1 gene revealed that it is required for the expression of stress response phenotypes, including thermotolerance, cell wall integrity, and efficient cell cycle progression. Accordingly, a cel1Δ deletion mutant was avirulent in two models of C. neoformans infection. Therefore, in contrast to LPMO activity in other microorganisms that primarily targets exogenous polysaccharides, these data suggest that CnCel1 promotes intrinsic fungal cell wall remodeling events required for efficient adaptation to the host environment.

A fast, sensitive and fluorescent LPMO activity assay.

Lytic polysaccharide monooxygenases (LPMOs) are industrially relevant enzymes that utilize a copper co-factor and an oxygen species to break down recalcitrant polysaccharides. These enzymes are secreted by microorganisms and are used in lignocellulosic refineries. As such, they are interesting from both the ecological/biological and industrial perspectives. Here we describe the development of a new fluorescence-based kinetic LPMO activity assay. The assay is based on the enzymatic production of fluorescein from its reduced counterpart. The assay can detect as little as 1 nM LPMO with optimized assay conditions. Furthermore, the reduced fluorescein substrate can also be used to identify peroxidase activity as seen by the formation of fluorescein by horseradish peroxidase. The assay was shown to work well at relatively low H2O2 and dehydroascorbate concentrations. The applicability of the assay was demonstrated.

Comparative studies of two AA10 family lytic polysaccharide monooxygenases from Bacillus thuringiensis.

Bacillus thuringiensis, known to be one of the most important biocontrol microorganisms, contains three AA10 family lytic polysaccharide monooxygenases (LPMOs) in its genome. In previous reports, two of them, BtLPMO10A and BtLPMO10B, have been preliminarily characterized. However, some important biochemical features and substrate preference, as well as their potential applications in chitin degradation, still deserve further investigation. Results from present study showed that both BtLPMO10A and BtLPMO10B exhibit similar catalytic domains as well as highly conserved substrate-binding planes. However, unlike BtLPMO10A, which has comparable binding ability to both crystalline and amorphous form of chitins, BtLPMO10B exhibited much stronger binding ability to colloidal chitin, which mainly attribute to its carbohydrate-binding module-5 (CBM5). Interestingly, the relative high binding ability of BtLPMO10B to colloidal chitin does not lead to high catalytic activity of the enzyme. In contrast, the enzyme exhibited higher activity on β-chitin. Further experiments showed that the binding of BtLPMO10B to colloidal chitin was mainly non-productive, indicating a complicated role for CBM5 in LPMO activity. Furthermore, synergistic experiments demonstrated that both LPMOs boosted the activity of the chitinase, and the higher efficiency of BtLPMO10A can be overridden by BtLPMO10B.

Enzymatic debranching is a key determinant of the xylan-degrading activity of family AA9 lytic polysaccharide monooxygenases

BackgroundPrevious studies have revealed that some Auxiliary Activity family 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) oxidize and degrade certain types of xylans when incubated with mixtures of xylan and cellulose. Here, we demonstrate that the xylanolytic activities of two xylan-active LPMOs, TtLPMO9E and TtLPMO9G from Thermothielavioides terrestris, strongly depend on the presence of xylan substitutions.ResultsUsing mixtures of phosphoric acid-swollen cellulose (PASC) and wheat arabinoxylan (WAX), we show that removal of arabinosyl substitutions with a GH62 arabinofuranosidase resulted in better adsorption of xylan to cellulose, and enabled LPMO-catalyzed cleavage of this xylan. Furthermore, experiments with mixtures of PASC and arabinoglucuronoxylan from spruce showed that debranching of xylan with the GH62 arabinofuranosidase and a GH115 glucuronidase promoted LPMO activity. Analyses of mixtures with PASC and (non-arabinosylated) beechwood glucuronoxylan showed that GH115 action promoted LPMO activity also on this xylan. Remarkably, when WAX was incubated with Avicel instead of PASC in the presence of the GH62, both xylan and cellulose degradation by the LPMO9 were impaired, showing that the formation of cellulose–xylan complexes and their susceptibility to LPMO action also depend on the properties of the cellulose. These debranching effects not only relate to modulation of the cellulose–xylan interaction, which influences the conformation and rigidity of the xylan, but likely also affect the LPMO–xylan interaction, because debranching changes the architecture of the xylan surface.ConclusionsOur results shed new light on xylanolytic LPMO9 activity and on the functional interplay and possible synergies between the members of complex lignocellulolytic enzyme cocktails. These findings will be relevant for the development of future lignocellulolytic cocktails and biomaterials.

H2 O2 feeding enables LPMO-assisted cellulose saccharification during simultaneous fermentative production of lactic acid.

Simultaneous saccharification and fermentation (SSF) is a well-known strategy for valorization of lignocellulosic biomass. Because the fermentation process typically is anaerobic, oxidative enzymes found in modern commercial cellulase cocktails, such as lytic polysaccharide monooxygenases (LPMOs), may be inhibited, limiting the overall efficiency of the enzymatic saccharification. Recent discoveries, however, have shown that LPMOs are active under anoxic conditions if they are provided with H2 O2 at low concentrations. In this study, we build on this concept and investigate the potential of using externally added H2 O2 to sustain oxidative cellulose depolymerization by LPMOs during an SSF process for lactic acid production. The results of bioreactor experiments with 100 g/L cellulose clearly show that continuous addition of small amounts of H2 O2 (at a rate of 80 µM/h) during SSF enables LPMO activity and improves lactic acid production. While further process optimization is needed, the present proof-of-concept results show that modern LPMO-containing cellulase cocktails such as Cellic CTec2 can be used in SSF setups, without sacrificing the LPMO activity in these cocktails.

Substrate-Dependent Cellulose Saccharification Efficiency and LPMO Activity of Cellic CTec2 and a Cellulolytic Secretome from Thermoascus aurantiacus and the Impact of H2O2-Producing Glucose Oxidase

Understanding and improving the efficiency of enzymatic saccharification of lignocellulosic biomass will promote the use of this renewable material. Here, we have studied several process parameters (reaction temperature, type of enzyme blend, type of substrate, type of reductant, and in situ supply of hydrogen peroxide) to better understand how saccharification could be optimized, focusing on the role of lytic polysaccharide monooxygenases (LPMOs). Comparison of a simple, LPMO-rich cellulolytic secretome from the thermophilic fungus Thermoascus aurantiacus with the commercial cellulase preparation Cellic CTec2 showed that saccharification of (lignin-poor) sulfite-pulped spruce at 60 °C with the secretome was as efficient as saccharification with Cellic CTec2 at 50 °C. Quantification of LPMO products showed that while LPMO activity contributed to saccharification efficiency, high levels of LPMO activity were not necessarily beneficial. Reactions with steam-exploded birch, rich in redox-active lignin, highlighted a strong impact of the feedstock on enzyme performance. In this case, the reaction with Cellic CTec2 at 50 °C was clearly most efficient. At 60 °C, enzyme inactivation became apparent for both enzyme blends, likely due to detrimental redox processes. Addition of H2O2-generating glucose oxidase to reactions with Cellic CTec2 at 50 °C led to strongly increased LPMO activity and, only for reactions with the lignin-poor substrate, improved saccharification yields. These results underpin the potential of the T. aurantiacus secretome for hydrolysis of lignin-poor substrates, and the usefulness of glucose oxidase for optimizing their saccharification. They also show that the efficiency of LPMO-containing cellulase preparations is highly dependent on the nature of the reductant and the substrate.

Natural photoredox catalysts promote light-driven lytic polysaccharide monooxygenase reactions and enzymatic turnover of biomass

Lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative cleavage of crystalline polysaccharides such as cellulose and chitin and are important for biomass conversion in the biosphere as well as in biorefineries. The target polysaccharides of LPMOs naturally occur in copolymeric structures such as plant cell walls and insect cuticles that are rich in phenolic compounds, which contribute rigidity and stiffness to these materials. Since these phenolics may be photoactive and since LPMO action depends on reducing equivalents, we hypothesized that LPMOs may enable light-driven biomass conversion. Here, we show that redox compounds naturally present in shed insect exoskeletons enable harvesting of light energy to drive LPMO reactions and thus biomass conversion. The primary underlying mechanism is that irradiation of exoskeletons with visible light leads to the generation of H2O2, which fuels LPMO peroxygenase reactions. Experiments with a cellulose model substrate show that the impact of light depends on both light and exoskeleton dosage and that light-driven LPMO activity is inhibited by a competing H2O2-consuming enzyme. Degradation experiments with the chitin-rich exoskeletons themselves show that solubilization of chitin by a chitin-active LPMO is promoted by light. The fact that LPMO reactions, and likely reactions catalyzed by other biomass-converting redox enzymes, are fueled by light-driven abiotic reactions in nature provides an enzyme-based explanation for the known impact of visible light on biomass conversion.

Inhibition of the Peroxygenase Lytic Polysaccharide Monooxygenase by Carboxylic Acids and Amino Acids.

Lytic polysaccharide monooxygenases (LPMOs) are widely distributed in fungi, and catalyze the oxidative degradation of polysaccharides such as cellulose. Despite their name, LPMOs possess a dominant peroxygenase activity that is reflected in high turnover numbers but also causes deactivation. We report on the influence of small molecules and ions on the activity and stability of LPMO during catalysis. Turbidimetric and photometric assays were used to identify LPMO inhibitors and measure their inhibitory effect. Selected inhibitors were employed to study LPMO activity and stability during cellulose depolymerization by HPLC and turbidimetry. It was found that the fungal metabolic products oxalic acid and citric acid strongly reduce LPMO activity, but also protect the enzyme from deactivation. QM calculations showed that the copper atom in the catalytic site could be ligated by bi- or tridentate chelating compounds, which replace two water molecules. MD simulations and QM calculations show that the most likely inhibition pattern is the competition between the inhibitor and reducing agent in the oxidized Cu(II) state. A correlation between the complexation energy and the IC50 values demonstrates that small, bidentate molecules interact strongest with the catalytic site copper and could be used by the fungus as physiological effectors to regulate LPMO activity.

Enhanced in situ H2O2 production explains synergy between an LPMO with a cellulose-binding domain and a single-domain LPMO

Lytic polysaccharide monooxygenases (LPMOs) are mono-copper enzymes that catalyze oxidative depolymerization of recalcitrant substrates such as chitin or cellulose. Recent work has shown that LPMOs catalyze fast peroxygenase reactions and that, under commonly used reaction set-ups, access to in situ generated H2O2 likely limits catalysis. Based on a hypothesis that the impact of a cellulose-binding module (CBM) on LPMO activity could relate to changes in in situ H2O2 production, we have assessed the interplay between CBM-containing ScLPMO10C and its truncated form comprising the catalytic domain only (ScLPMO10CTR). The results show that truncation of the linker and CBM leads to elevated H2O2 production and decreased enzyme stability. Most interestingly, combining the two enzyme forms yields strong synergistic effects, which are due to the combination of high H2O2 generation by ScLPMO10CTR and efficient productive use of H2O2 by the full-length enzyme. Thus, cellulose degradation becomes faster, while enzyme inactivation due to off-pathway reactions with excess H2O2 is reduced. These results underpin the complexity of ascorbic acid-driven LPMO reactions and reveal a potential mechanism for how LPMOs may interact synergistically during cellulose degradation.

Inhibition of LPMOs by Fermented Persimmon Juice.

Fermented persimmon juice, Kakishibu, has traditionally been used for wood and paper protection. This protective effect stems at least partially from inhibition of microbial cellulose degrading enzymes. The inhibitory effect of Kakishibu on lytic polysaccharide monooxygenases (LPMOs) and on a cocktail of cellulose hydrolases was studied, using three different cellulosic substrates. Dose dependent inhibition of LPMO activity by a commercial Kakishibu product was assessed for the well-characterized LPMO from Thermoascus aurantiacus TaAA9A, and the inhibitory effect was confirmed on five additional microbial LPMOs. The model tannin compound, tannic acid exhibited a similar inhibitory effect on TaAA9A as Kakishibu. It was further shown that both polyethylene glycol and tannase can alleviate the inhibitory effect of Kakishibu and tannic acid, indicating a likely mechanism of inhibition caused by unspecific tannin–protein interactions.

The impact of reductants on the catalytic efficiency of a lytic polysaccharide monooxygenase and the special role of dehydroascorbic acid.

Monocopper lytic polysaccharide monooxygenases (LPMOs) catalyse oxidative cleavage of glycosidic bonds in a reductant-dependent reaction. Recent studies indicate that LPMOs, rather than being O2 -dependent monooxygenases, are H2 O2 -dependent peroxygenases. Here, we describe SscLPMO10B, a novel LPMO from the phytopathogenic bacterium Streptomyces scabies and address links between this enzyme's catalytic rate and in situ hydrogen peroxide production in the presence of ascorbic acid, gallic acid and l-cysteine. Studies of Avicel degradation showed a clear correlation between the catalytic rate of SscLPMO10B and the rate of H2 O2 generation in the reaction mixture. We also assessed the impact of oxidised ascorbic acid, dehydroascorbic acid (DHA), on LPMO activity, since DHA, which is not considered a reductant, was recently reported to drive LPMO reactions. Kinetic studies, combined with NMR analysis, showed that DHA is unstable and converts into multiple derivatives, some of which are redox active and can fuel the LPMO reaction by reducing the active site copper and promoting H2 O2 production. These results show that the apparent monooxygenase activity observed in SscLPMO10B reactions without exogenously added H2 O2 reflects a peroxygenase reaction.

Combining pieces: a thorough analysis of light activation boosting power and co-substrate preferences for the catalytic efficiency of lytic polysaccharide monooxygenase MtLPMO9A

Cost-efficient plant biomass conversion using biochemical and/or chemical routes is essential for transitioning to sustainable chemical technologies and renewable biofuels. Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that make part of modern hydrolytic cocktails destined for plant biomass degradation. Here, we characterized MtLPMO9A from Thermothelomyces thermophilus M77 (formerly Myceliophthora thermophila) and demonstrated that it could be efficiently driven by chlorophyllin excited by light in the presence of a reductant agent. However, in the absence of chemical reductant, chlorophyllin and light alone do not lead to a significant release of the reaction products by the LPMO, indicating a low capacity of MtLPMO9A reduction (either via direct electron transfer or via superoxide ion, O2•-). We showed that photocatalysis could significantly increase the LPMO activity against highly crystalline and recalcitrant cellulosic substrates, which are poorly degraded in the absence of chlorophyllin and light. We also evaluated the use of co-substrates by MtLPMO9A, revealing that the enzyme can use both hydrogen peroxide (H2O2) and molecular oxygen (O2) as co-substrates for cellulose catalytic oxidation.

Oxidative Power: Tools for Assessing LPMO Activity on Cellulose.

Lytic polysaccharide monooxygenases (LPMOs) have sparked a lot of research regarding their fascinating mode-of-action. Particularly, their boosting effect on top of the well-known cellulolytic enzymes in lignocellulosic hydrolysis makes them industrially relevant targets. As more characteristics of LPMO and its key role have been elucidated, the need for fast and reliable methods to assess its activity have become clear. Several aspects such as its co-substrates, electron donors, inhibiting factors, and the inhomogeneity of lignocellulose had to be considered during experimental design and data interpretation, as they can impact and often hamper outcomes. This review provides an overview of the currently available methods to measure LPMO activity, including their potential and limitations, and it is illustrated with practical examples.

In situ measurements of oxidation\u2013reduction potential and hydrogen peroxide concentration as tools for revealing LPMO inactivation during enzymatic saccharification of cellulose

BackgroundBiochemical conversion of lignocellulosic biomass to simple sugars at commercial scale is hampered by the high cost of saccharifying enzymes. Lytic polysaccharide monooxygenases (LPMOs) may hold the key to overcome economic barriers. Recent studies have shown that controlled activation of LPMOs by a continuous H2O2 supply can boost saccharification yields, while overdosing H2O2 may lead to enzyme inactivation and reduce overall sugar yields. While following LPMO action by ex situ analysis of LPMO products confirms enzyme inactivation, currently no preventive measures are available to intervene before complete inactivation.ResultsHere, we carried out enzymatic saccharification of the model cellulose Avicel with an LPMO-containing enzyme preparation (Cellic CTec3) and H2O2 feed at 1 L bioreactor scale and followed the oxidation–reduction potential and H2O2 concentration in situ with corresponding electrode probes. The rate of oxidation of the reductant as well as the estimation of the amount of H2O2 consumed by LPMOs indicate that, in addition to oxidative depolymerization of cellulose, LPMOs consume H2O2 in a futile non-catalytic cycle, and that inactivation of LPMOs happens gradually and starts long before the accumulation of LPMO-generated oxidative products comes to a halt.ConclusionOur results indicate that, in this model system, the collapse of the LPMO-catalyzed reaction may be predicted by the rate of oxidation of the reductant, the accumulation of H2O2 in the reactor or, indirectly, by a clear increase in the oxidation–reduction potential. Being able to monitor the state of the LPMO activity in situ may help maximizing the benefit of LPMO action during saccharification. Overcoming enzyme inactivation could allow improving overall saccharification yields beyond the state of the art while lowering LPMO and, potentially, cellulase loads, both of which would have beneficial consequences on process economics.