Lytic Polysaccharide Monooxygenases Research Articles

A cellulosomal yeast reaction system of lignin-degrading enzymes for cellulosic ethanol fermentation.

Biofuel production from lignocellulose feedstocks is sustainable and environmentally friendly. However, the lignocellulosic pretreatment could produce fermentation inhibitors causing multiple stresses and low yield. Therefore, the engineering construction of highly resistant microorganisms is greatly significant. In this study, a composite functional chimeric cellulosome equipped with laccase, versatile peroxidase, and lytic polysaccharide monooxygenase was riveted on the surface of Saccharomyces cerevisiae to construct a novel yeast strain YI/LVP for synergistic lignin degradation and cellulosic ethanol production. The assembly of cellulosome was assayed by immunofluorescence microscopy and flow cytometry. During the whole process of fermentation, the maximum ethanol concentration and cellulose conversion of engineering strain YI/LVP reached 8.68g/L and 83.41%, respectively. The results proved the availability of artificial chimeric cellulosome containing lignin-degradation enzymes for cellulosic ethanol production. The purpose of the study was to improve the inhibitor tolerance and fermentation performance of S. cerevisiae through the construction and optimization of a synergistic lignin-degrading enzyme system based on cellulosome.

The rotamer of the second-sphere histidine in AA9 lytic polysaccharide monooxygenase is pH dependent

Lytic polysaccharide monooxygenases (LPMOs) catalyze a reaction that is crucial for the biological decomposition of various biopolymers and for the industrial conversion of plant biomass. Despite the importance of LPMOs, the exact molecular-level nature of the reaction mechanism is still debated today. Here, we investigated the pH-dependent conformation of a second-sphere histidine (His) that we call the stacking histidine, which is conserved in fungal AA9 LPMOs and is speculated to assist catalysis in several of the LPMO reaction pathways.Using constant-pH and accelerated molecular dynamics simulations, we monitored the dynamics of the stacking His in different protonation states for both the resting Cu(II) and active Cu(I) forms of two fungal LPMOs. Consistent with experimental crystallographic and neutron diffraction data, our calculations suggest that the side chain of the protonated and positively charged form is rotated out of the active site toward the solvent. Importantly, only one of the possible neutral states of histidine (HIE state) is observed in the stacking orientation at neutral pH or when bound to cellulose. Our data predict that, in solution, the stacking His may act as a stabilizer (via hydrogen bonding) of the Cu(II)-superoxo complex after the LPMO-Cu(I) has reacted with O2 in solution, which, in fine, leads to H2O2 formation. Also, our data indicate that the HIE-stacking His is a poor acid/base catalyst when bound to the substrate and, in agreement with the literature, may play an important stabilizing role (via hydrogen bonding) during the peroxygenase catalysis.Our study reveals the pH titration midpoint values of the pH-dependent orientation of the stacking His should be considered when modeling and interpreting LPMO reactions, whether it be for classical LPMO kinetics or in industry-oriented enzymatic cocktails, and for understanding LPMO behavior in slightly acidic natural processes such as fungal wood decay.

Diversely regio-oxidative degradation of konjac glucomannan by lytic polysaccharide monooxygenase AA10 and generating antibacterial hydrolysate

Konjac glucomannan (KGM) hydrolysate exhibit various biological activities and health-promoting effects. Lytic polysaccharide monooxygenases (LPMOs) play an important role on enzymatic degradation of recalcitrant polysaccharides to obtain fermentable sugars. It is generally accepted that LPMOs exhibits high substrate specificity and oxidation regioselectivity. Here, a bacteria-derived SmAA10A, with chitin-active with strict C1 oxidation, was used to catalyse KGM degradation. Through ethanol precipitation, two hydrolysed KGM components (4 kDa (KGM-1) and 5 kDa (KGM-2)) were obtained that exhibited antibacterial activity against Staphylococcus aureus. In natural KGM, KGM-1, and KGM-2, the molar ratios of mannose to glucose were 1:2.19, 1:3.05, and 1:2.87, respectively, indicating that SmAA10A preferentially degrades mannose in KGM. Fourier-transform infrared spectroscopy and scanning electron microscopy imaging revealed the breakage of glycosylic bonds during enzymatic catalysis. The regioselectivity of SmAA10A for KGM degradation was determined based on the fragmentation behaviour of the KGM-1 and KGM-2 oligosaccharides and their NaBD4-reduced forms. SmAA10A exhibited diverse oxidation degradation of KGM and generated single C1-, single C4-, and C1/C4-double oxidised oligosaccharide forms. This study provides an alternative method for obtaining KGM degradation components with antibacterial functions and expands the substrate specificity and oxidation regioselectivity of bacterial LPMOs.

Enzymatic machinery of wood-inhabiting fungi that degrade temperate tree species.

Deadwood provides habitat for fungi and serves diverse ecological functions in forests. We already have profound knowledge of fungal assembly processes, physiological and enzymatic activities, and resulting physico-chemical changes during deadwood decay. However, in situ detection and identification methods, fungal origins, and a mechanistic understanding of the main lignocellulolytic enzymes are lacking. This study used metaproteomics to detect the main extracellular lignocellulolytic enzymes in 12 tree species in a temperate forest that have decomposed for 8 ½ years. Mainly white-rot (and few brown-rot) Basidiomycota were identified as the main wood decomposers, with Armillaria as the dominant genus; additionally, several soft-rot xylariaceous Ascomycota were identified. The key enzymes involved in lignocellulolysis included manganese peroxidase, peroxide-producing alcohol oxidases, laccase, diverse glycosyl hydrolases (cellulase, glucosidase, xylanase), esterases and lytic polysaccharide monooxygenases. The fungal community and enzyme composition differed among the 12 tree species. Ascomycota species were more prevalent in angiosperm logs than in gymnosperm logs. Regarding lignocellulolysis as a function, the extracellular enzyme toolbox acted simultaneously and was interrelated (e.g., peroxidases and peroxide-producing enzymes were strongly correlated), highly functionally redundant, and present in all logs. In summary, our in situ study provides comprehensive and detailed insight into the enzymatic machinery of wood-inhabiting fungi in temperate tree species. These findings will allow us to relate changes in environmental factors to lignocellulolysis as an ecosystem function in the future.

The disordered C-terminal tail of fungal LPMOs from phytopathogens mediates protein dimerization and impacts plant penetration

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that oxidatively degrade various polysaccharides, such as cellulose. Despite extensive research on this class of enzymes, the role played by their C-terminal regions predicted to be intrinsically disordered (dCTR) has been overlooked. Here, we investigated the function of the dCTR of an LPMO, called CoAA9A, up-regulated during plant infection by Colletotrichum orbiculare, the causative agent of anthracnose. After recombinant production of the full-length protein, we found that the dCTR mediates CoAA9A dimerization in vitro, via a disulfide bridge, a hitherto-never-reported property that positively affects both binding and activity on cellulose. Using SAXS experiments, we show that the homodimer is in an extended conformation. In vivo, we demonstrate that gene deletion impairs formation of the infection-specialized cell called appressorium and delays penetration of the plant. Using immunochemistry, we show that the protein is a dimer not only in vitro but also in vivo when secreted by the appressorium. As these peculiar LPMOs are also found in other plant pathogens, our findings open up broad avenues for crop protection.

Active roles of lytic polysaccharide monooxygenases in human pathogenicity

Lytic polysaccharide monooxygenases (LPMOs) are redox enzymes widely studied for their involvement in microbial and fungal biomass degradation. The catalytic versatility of these enzymes is demonstrated by the recent discovery of LPMOs in arthropods, viruses, insects and ferns, where they fulfill diverse functions beyond biomass conversion. This mini-review puts a spotlight on a recently recognized aspect of LPMOs: their role in infectious processes in human pathogens. It discusses the occurrence and potential biological mechanisms of LPMOs associated with human pathogens and provides an outlook on future avenues in this emerging and exciting research field.

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.

Genomic characterization and expression profiling of the lytic polysaccharide monooxygenases AA9 family in thermophilic fungi Thermothelomyces fergusii in response to carbon source media

Enhancing enzyme activity and stability in biomass degradation can improve substrate saccharification and, increases biorefinery efficiency. For the first time, we identified 20 lytic polysaccharide monooxygenases (LPMOs) AA9 genes in the genome of Thermothelomyces fergusii. Our results showed that TfAA9 was categorized into LPMOs1, LPMOs2, and LPMOs3 subgroups based on protein diversity. Protein- 3D structure analysis showed strong interactions between Myceliophthora thermophila AA9 proteins and 17 TfAA9 proteins. Gene ontology analysis indicated a high enrichment of cellulase activity in TfAA9 genes. KEGG pathways analysis revealed the role of TfAA9 proteins in the endohydrolysis of 1,4-beta-D-glucosidic linkages in cellulose. Numerous TfAA9s gene transcripts were up-regulated on avicel, cellobiose, and glucose, with a higher proportion on avicel. Protein concentration, endoglucanase, and cellulase activity were also boosted on avicel. However, limited fungal biomass was observed on avicel, despite the abundance of AA9 LPMOs in the T. fergusii genome. These findings expand our understanding of fungal AA9 genes and their role in lignocellulolytic degradation. The disparity between biomass and enzymatic activity suggests screening TfAA9 genes for highly active enzymes and redundant genes via heterologous expression. In short, functional characterization of these genes could contribute to improving the saccharification process of industrial raw materials.

Bioconversion of α-Chitin by a Lytic Polysaccharide Monooxygenase OsLPMO10A Coupled with Chitinases and the Synergistic Mechanism Analysis.

The whole enzymatic conversion of chitin is a green and promising alternative to current strategies, which are based on lytic polysaccharide monooxygenases (LPMOs) and chitinases. However, the lack of LPMOs with high activity toward α-chitin limits the efficient bioconversion of α-chitin. Herein, we characterized a high chitin-active LPMO from Oceanobacillus sp. J11TS1 (OsLPMO10A), which could promote the decrystallization of the α-chitin surface. Furthermore, when coupled with OsLPMO10A, the conversion rate of α-chitin to N-acetyl chitobiose [(GlcNAc)2] by three chitinases (Serratia marcescens, ChiA, -B, and -C) reached 30.86%, which was 2.03-folds that without the addition of OsLPMO10A. Moreover, the results of synergistic reactions indicated that OsLPMO10A and chitinases promoted the degradation of α-chitin each other mainly on the surface. To the best of our knowledge, this study achieved the highest yield of N-acetyl chitooligosaccharides (N-acetyl COSs) among reported LPMOs-driven bioconversion systems, which could be regarded as a promising candidate for α-chitin bioconversion.

Structural dissection of two redox proteins from the shipworm symbiont Teredinibacter turnerae.

The discovery of lytic polysaccharide monooxygenases (LPMOs), a family of copper-dependent enzymes that play a major role in polysaccharide degradation, has revealed the importance of oxidoreductases in the biological utilization of biomass. In fungi, a range of redox proteins have been implicated as working in harness with LPMOs to bring about polysaccharide oxidation. In bacteria, less is known about the interplay between redox proteins and LPMOs, or how the interaction between the two contributes to polysaccharide degradation. We therefore set out to characterize two previously unstudied proteins from the shipworm symbiont Teredinibacter turnerae that were initially identified by the presence of carbohydrate binding domains appended to uncharacterized domains with probable redox functions. Here, X-ray crystal structures of several domains from these proteins are presented together with initial efforts to characterize their functions. The analysis suggests that the target proteins are unlikely to function as LPMO electron donors, raising new questions as to the potential redox functions that these large extracellular multi-haem-containing c-type cytochromes may perform in these bacteria.

Genome sequence of thermophilic cellulolytic actinobacterium Thermobifida fusca strain UPMC 901 producing thermostable cellulases.

Thermophilic cellulolytic bacteria producing thermostable cellulase have multiple potential industrial and biotechnological applications. In this study, we sequenced the genome of Thermobifida fusca UPMC 901 by using nanopore sequencing technology with 100 % genome coverage. The represented genome is 3.76 Mb with a G + C content of 67.4 %. The genome is composed of 1 scaffold with 3207 protein-coding genes. The results indicated that the genome of the Thermobifida fusca gene encodes at least 112 genes for carbohydrate-active enzymes. Of these genes, 40 glycoside hydrolases (GHs), 36 glycosyl transferases (GTs), 22 carbohydrate-binding modules (CBMs), 9 carbohydrate esterases (CEs), 3 polysaccharide lyases (PL) and 2 auxiliary activity (AAs) which refer to as lytic polysaccharide monooxygenase (LPMO), gene encoding the copper-dependent enzymes that catalyse oxidative cleavage of recalcitrant crystalline cellulose, were identified. In addition, two new functional genes, amylo-α-1,6 glucosidase and endo-α-1,4-mannosidase were also detected in the T. fusca genome. This study represented the ninth genome of the T. fusca and the cellulase genes identified will facilitate the exploration of potential sources for industrial and agricultural applications.

Assessing the role of redox partners in TthLPMO9G and its mutants: focus on H2O2 production and interaction with cellulose

BackgroundThe field of enzymology has been profoundly transformed by the discovery of lytic polysaccharide monooxygenases (LPMOs). LPMOs hold a unique role in the natural breakdown of recalcitrant polymers like cellulose and chitin. They are characterized by a “histidine brace” in their active site, known to operate via an O2/H2O2 mechanism and require an electron source for catalytic activity. Although significant research has been conducted in the field, the relationship between these enzymes, their electron donors, and H2O2 production remains complex and multifaceted.ResultsThis study examines TthLPMO9G activity, focusing on its interactions with various electron donors, H2O2, and cellulose substrate interactions. Moreover, the introduction of catalase effectively eliminates H2O2 interference, enabling an accurate evaluation of each donor’s efficacy based on electron delivery to the LPMO active site. The introduction of catalase enhances TthLPMO9G’s catalytic efficiency, leading to increased cellulose oxidation. The current study provides deeper insights into specific point mutations, illuminating the crucial role of the second coordination sphere histidine at position 140. Significantly, the H140A mutation not only impacted the enzyme’s ability to oxidize cellulose, but also altered its interaction with H2O2. This change was manifested in the observed decrease in both oxidase and peroxidase activities. Furthermore, the S28A substitution, selected for potential engagement within the His1–electron donor–cellulose interaction triad, displayed electron donor-dependent alterations in cellulose product patterns.ConclusionThe interaction of an LPMO with H2O2, electron donors, and cellulose substrate, alongside the impact of catalase, offers deep insights into the intricate interactions occurring at the molecular level within the enzyme. Through rational alterations and substitutions that affect both the first and second coordination spheres of the active site, this study illuminates the enzyme’s function. These insights enhance our understanding of the enzyme’s mechanisms, providing valuable guidance for future research and potential applications in enzymology and biochemistry.

Mimicking the Reactivity of LPMOs with a Mononuclear Cu Complex

AbstractLytic polysaccharide monooxygenases (LPMOs) are Cu‐dependent metalloenzymes that catalyze the hydroxylation of strong C−H bonds in polysaccharides using O2 or H2O2 as oxidants (monooxygenase/peroxygenase). In the absence of C−H substrate, LPMOs reduce O2 to H2O2 (oxidase) and H2O2 to H2O (peroxidase) using proton/electron donors. This rich oxidative reactivity is promoted by a mononuclear Cu center in which some of the amino acid residues surrounding the metal might accept and donate protons and/or electrons during O2 and H2O2 reduction. Herein, we utilize a podal ligand containing H‐bond/proton donors (LH2) to analyze the reactivity of mononuclear Cu species towards O2 and H2O2. [(LH2)CuI]1+ (1), [(LH2)CuII]2+ (2), [(LH−)CuII]1+ (3), [(LH2)CuII(OH)]1+ (4), and [(LH2)CuII(OOH)]1+ (5) were synthesized and characterized by structural and spectroscopic means. Complex 1 reacts with O2 to produce 5, which releases H2O2 to generate 3, suggesting that O2 is used by LPMOs to generate H2O2. The reaction of 1 with H2O2 produces 4 and hydroxyl radical, which reacts with C−H substrates in a Fenton‐like fashion. Complex 3, which can generate 1 via a reversible protonation/reduction, binds H2O and H2O2 to produce 4 and 5, respectively, a mechanism that could be used by LPMOs to control oxidative reactivity.

The upsurge of lytic polysaccharide monooxygenases in biomass deconstruction: characteristic functions and sustainable applications.

Lytic polysaccharide monooxygenases (LPMOs) are one of the emerging classes of copper metalloenzymes that have received considerable attention due to their ability to boost the enzymatic conversion of intractable polysaccharides such as plant cell walls and chitin polymers. LPMOs catalyze the oxidative cleavage of β-1,4-glycosidic bonds using molecular O2 or H2 O2 in the presence of an external electron donor. LPMOs have been classified as an auxiliary active (AA) class of enzymes and, further based on substrate specificity, divided into eight families. Until now, multiple LPMOs from AA9 and AA10 families, mostly from microbial sources, have been investigated; the exact mechanism and structure-function are elusive to date, and recently discovered AA families of LPMOs are just scratched. This review highlights the origin and discovery of the enzyme, nomenclature, three-dimensional protein structure, substrate specificity, copper-dependent reaction mechanism, and different techniques used to determine the product formation through analytical and biochemical methods. Moreover, the diverse functions of proteins in various biological activities such as plant-pathogen/pest interactions, cell wall remodeling, antibiotic sensitivity of biofilms, and production of nanocellulose along with certain obstacles in deconstructing the complex polysaccharides have also been summarized, while highlighting the innovative and creative ways to overcome the limitations of LPMOs in hydrolyzing the biomass.

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.

A novel cellulolytic/xylanolytic SbAA14 from Sordaria brevicollis with a branched chain preference and its synergistic effects with glycoside hydrolases on lignocellulose

In this study, the novel auxiliary activity (AA) family 14 lytic polysaccharide monooxygenase (LPMO) SbAA14 from Sordaria brevicollis was successfully characterized. It was active against heteroxylan, xyloglucan and cellulose in β-cellulose and released native oligosaccharides and corresponding C1- and/or C4-oxidized products. SbAA14 showed a branched chain preference, because partial removal of arabinosyl substituents from heteroxylan led to a decrease in activity. SbAA14 had synergistic effects with the debranching enzyme EpABF62C in an enzyme- and ascorbic acid-dependent manner. SbAA14 had synergistic effects with the GH10 endoxylanase EpXYN1, and the degree of synergy was greater with step-by-step addition than with simultaneous addition. SbAA14 could also synergize with Celluclast® 1.5 L on NaOH-pretreated wheat straw and on NaOH-pretreated and hydrogen peroxide-acetic acid (HPAC)-H2SO4-pretreated bamboo substrates. The greatest synergistic effect between SbAA14 and Celluclast® 1.5 L was observed for HPAC-H2SO4–200 mM pretreated bamboo, in which the degree of synergy reached approximately 1.61. The distinctive substrate preference of SbAA14 indicated that it is a novel AA14 LPMO that may act mainly on heteroxylan with numerous arabinosyl substituents between cellulose fibers rather than on recalcitrant xylan tightly associated with cellulose. These findings broaden the understanding of enigmatic AA14 LPMOs and provide new insights into the substrate specificities and biological functionalities of AA14 LPMOs in fungi.

Electrochemical Monitoring of Heterogeneous Peroxygenase Reactions Unravels LPMO Kinetics.

Biological conversion of plant biomass depends on peroxygenases and peroxidases acting on insoluble polysaccharides and lignin. Among these are cellulose- and hemicellulose-degrading lytic polysaccharide monooxygenases (LPMOs), which have revolutionized our concept of biomass degradation. Major obstacles limiting mechanistic and functional understanding of these unique peroxygenases are their complex and insoluble substrates and the hard-to-measure H2O2 consumption, resulting in the lack of suitable kinetic assays. We report a versatile and robust electrochemical method for real-time monitoring and kinetic characterization of LPMOs and other H2O2-dependent interfacial enzymes based on a rotating disc electrode for the sensitive and selective quantitation of H2O2 at biologically relevant concentrations. The H2O2 sensor works in suspensions of insoluble substrates as well as in homogeneous solutions. Our characterization of multiple LPMOs provides unprecedented insights into the substrate specificity, kinetics, and stability of these enzymes. High turnover and total turnover numbers demonstrate that LPMOs are fast and durable biocatalysts.

A novel starch-active lytic polysaccharide monooxygenase discovered with bioinformatics screening and its application in textile desizing

BackgroundLytic polysaccharide monooxygenases (LPMOs) catalyzing the oxidative cleavage of different types of polysaccharides have potential to be used in various industries. However, AA13 family LPMOs which specifically catalyze starch substrates have relatively less members than AA9 and AA10 families to limit their application range. Amylase has been used in enzymatic desizing treatment of cotton fabric for semicentury which urgently need for new assistant enzymes to improve reaction efficiency and reduce cost so as to promote their application in the textile industry.ResultsA total of 380 unannotated new genes which probably encode AA13 family LPMOs were discovered by the Hidden Markov model scanning in this study. Ten of them have been successfully heterologous overexpressed. AlLPMO13 with the highest activity has been purified and determined its optimum pH and temperature as pH 5.0 and 50 °C. It also showed various oxidative activities on different substrates (modified corn starch > amylose > amylopectin > corn starch). The results of enzymatic textile desizing application showed that the best combination of amylase (5 g/L), AlLPMO13 (5 mg/L), and H2O2 (3 g/L) made the desizing level and the capillary effects increased by 3 grades and more than 20%, respectively, compared with the results treated by only amylase.ConclusionThe Hidden Markov model constructed basing on 34 AA13 family LPMOs was proved to be a valid bioinformatics tool for discovering novel starch-active LPMOs. The novel enzyme AlLPMO13 has strong development potential in the enzymatic textile industry both concerning on economy and on application effect.

A frontier-orbital view of the initial steps of lytic polysaccharide monooxygenase reactions.

Lytic polysaccharide monooxygenases (LPMOs) are copper enzymes that oxidatively cleave the strong C-H bonds in recalcitrant polysaccharide substrates, thereby playing a crucial role in biomass degradation. Recently, LPMOs have also been shown to be important for several pathogens. It is well established that the Cu(II) resting state of LPMOs is inactive, and the electronic structure of the active site needs to be altered to transform the enzyme into an active form. Whether this transformation occurs due to substrate binding or due to a unique priming reduction has remained speculative. Starting from four different crystal structures of the LPMO LsAA9A with well-defined oxidation states, we use a frontier molecular orbital approach to elucidate the initial steps of the LPMO reaction. We give an explanation for the requirement of the unique priming reduction and analyse electronic structure changes upon substrate binding. We further investigate how the presence of the substrate could facilitate an electron transfer from the copper active site to an H2O2 co-substrate. Our findings could help to control experimental LPMO reactions.

Production of lignin containing cellulose nanofibrils (CNF) after enzymatic treatment of curl-induced, unbleached kraft pulps

Curling treatment of unbleached Kraft pulp fibers creates disordered cellulose, enhancing enzyme accessibility for fiber modification. This facilitates micro/nano-fibrillation, improving mechanical performance for potential functional applications.