Biochemical and structural insights of a recombinant AA16 LPMO from the marine and sponge-symbiont Peniophora sp.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidize polysaccharides, leading to their cleavage. LPMOs are classified into eight CAZy families (AA9-11, AA13-17), with the functionality of AA16 being poorly characterized. This study presents biochemical and structural data for an AA16 LPMO (PnAA16) from the marine sponge symbiont Peniophora sp. Phylogenetic analysis revealed that PnAA16 clusters separately from previously characterized AA16s. However, the structural modelling of PnAA16 showed the characteristic immunoglobulin-like fold of LPMOs, with a conserved his-brace motif coordinating a copper ion. The copper-bound PnAA16 showed greater thermal stability than its apo-form, highlighting copper's role in enzyme stability. Functionally, PnAA16 demonstrated oxidase activity, producing 5 µM H2O2 after 30 min, but showed 20 times lower peroxidase activity (0.27 U/g) compared to a fungal AA9. Specific activity assays indicated that PnAA16 acts only on cellohexaose, generating native celloligosaccharides (C3 to C5) and oxidized products with regioselective oxidation at C1 and C4 positions. Finally, PnAA16 boosted the activity of a cellulolytic cocktail for cellulose saccharification in the presence of ascorbic acid, hydrogen peroxide, or both. In conclusion, the present work provides insights into the AA16 family, expanding the understanding of their structural and functional relationships and biotechnological potential.

Transcriptional and secretome analysis of Rasamsonia emersonii lytic polysaccharide mono-oxygenases.

The current study is the first to describe the temporal and differential transcriptional expression of two lytic polysaccharide monooxygenase (LPMO) genes of Rasamsonia emersonii in response to various carbon sources. The mass spectrometry based secretome analysis of carbohydrate active enzymes (CAZymes) expression in response to different carbon sources showed varying levels of LPMOs (AA9), AA3, AA7, catalase, and superoxide dismutase enzymes pointing toward the redox-interplay between the LPMOs and auxiliary enzymes. Moreover, it was observed that cello-oligosaccharides have a negative impact on the expression of LPMOs, which has not been highlighted in previous reports. The LPMO1 (30 kDa) and LPMO2 (47 kDa), cloned and expressed in Pichia pastoris, were catalytically active with (kcat/Km) of 6.6×10-2 mg-1 ml min-1 and 1.8×10-2 mg-1 ml min-1 against Avicel, respectively. The mass spectrometry of hydrolysis products of Avicel/carboxy methyl cellulose (CMC) showed presence of C1/C4 oxidized oligosaccharides indicating them to be Type 3 LPMOs. The 3D structural analysis of LPMO1 and LPMO2 revealed distinct arrangements of conserved catalytic residues at their active site. The developed enzyme cocktails consisting of cellulase from R. emersonii mutant M36 supplemented with recombinant LPMO1/LPMO2 resulted in significantly enhanced saccharification of steam/acid pretreated unwashed rice straw slurry from PRAJ industries (Pune, India). The current work indicates that LPMO1 and LPMO2 are catalytically efficient and have a high degree of thermostability, emphasizing their usefulness in improving benchmark enzyme cocktail performance. KEY POINTS: • Mass spectrometry depicts subtle interactions between LPMOs and auxiliary enzymes. • Cello-oligosaccharides strongly downregulated the LPMO1 expression. • Developed LPMO cocktails showed superior hydrolysis in comparison to CellicCTec3.

The impact of the carbohydrate-binding module on how a lytic polysaccharide monooxygenase modifies cellulose fibers.

BACKGROUND: In recent years, lytic polysaccharide monooxygenases (LPMOs) that oxidatively cleave cellulose have gained increasing attention in cellulose fiber modification. LPMOs are relatively small copper-dependent redox enzymes that occur as single domain proteins but may also contain an appended carbohydrate-binding module (CBM). Previous studies have indicated that the CBM "immobilizes" the LPMO on the substrate and thus leads to more localized oxidation of the fiber surface. Still, our understanding of how LPMOs and their CBMs modify cellulose fibers remains limited. RESULTS: Here, we studied the impact of the CBM on the fiber-modifying properties of NcAA9C, a two-domain family AA9 LPMO from Neurospora crassa, using both biochemical methods as well as newly developed multistep fiber dissolution methods that allow mapping LPMO action across the fiber, from the fiber surface to the fiber core. The presence of the CBM in NcAA9C improved binding towards amorphous (PASC), natural (Cell I), and alkali-treated (Cell II) cellulose, and the CBM was essential for significant binding of the non-reduced LPMO to Cell I and Cell II. Substrate binding of the catalytic domain was promoted by reduction, allowing the truncated CBM-free NcAA9C to degrade Cell I and Cell II, albeit less efficiently and with more autocatalytic enzyme degradation compared to the full-length enzyme. The sequential dissolution analyses showed that cuts by the CBM-free enzyme are more evenly spread through the fiber compared to the CBM-containing full-length enzyme and showed that the truncated enzyme can penetrate deeper into the fiber, thus giving relatively more oxidation and cleavage in the fiber core. CONCLUSIONS: These results demonstrate the capability of LPMOs to modify cellulose fibers from surface to core and reveal how variation in enzyme modularity can be used to generate varying cellulose-based materials. While the implications of these findings for LPMO-based cellulose fiber engineering remain to be explored, it is clear that the presence of a CBM is an important determinant of the three-dimensional distribution of oxidation sites in the fiber.

Sequence and structure analyses of lytic polysaccharide monooxygenases mined from metagenomic DNA of humus samples around white-rot fungi in Cuc Phuong tropical forest, Vietnam.

Background: White-rot fungi and bacteria communities are unique ecosystems with different types of symbiotic interactions occurring during wood decomposition, such as cooperation, mutualism, nutritional competition, and antagonism. The role of chitin-active lytic polysaccharide monooxygenases (LPMOs) in these symbiotic interactions is the subject of this study. Method: In this study, bioinformatics tools were used to analyze the sequence and structure of putative LPMOs mined by hidden Markov model (HMM) profiles from the bacterial metagenomic DNA database of collected humus samples around white-rot fungi in Cuc Phuong primary forest, Vietnam. Two genes encoding putative LPMOs were expressed in E. coli and purified for enzyme activity assay. Result: Thirty-one full-length proteins annotated as putative LPMOs according to HMM profiles were confirmed by amino acid sequence comparison. The comparison results showed that although the amino acid sequences of the proteins were very different, they shared nine conserved amino acids, including two histidine and one phenylalanine that characterize the H1-Hx-Yz motif of the active site of bacterial LPMOs. Structural analysis of these proteins revealed that they are multidomain proteins with different functions. Prediction of the catalytic domain 3-D structure of these putative LPMOs using Alphafold2 showed that their spatial structures were very similar in shape, although their protein sequences were very different. The results of testing the activity of proteins GL0247266 and GL0183513 show that they are chitin-active LPMOs. Prediction of the 3-D structures of these two LPMOs using Alphafold2 showed that GL0247266 had five functional domains, while GL0183513 had four functional domains, two of which that were similar to the GbpA_2 and GbpA_3 domains of protein GbpA of Vibrio cholerae bacteria. The GbpA_2 - GbpA_3 complex was also detected in 11 other proteins. Based on the structural characteristics of functional domains, it is possible to hypothesize the role of chitin-active GbpA-like LPMOs in the relationship between fungal and bacterial communities coexisting on decomposing trees in primary forests.

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.

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 ß-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.

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 ß-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.

Current insights of factors interfering the stability of lytic polysaccharide monooxygenases.

Cellulose and chitin are two of the most abundant biopolymers in nature, but they cannot be effectively utilized in industry due to their recalcitrance. This limitation was overcome by the advent of lytic polysaccharide monooxygenases (LPMOs), which promote the disruption of biopolymers through oxidative mechanism and provide a breakthrough in the action of hydrolytic enzymes. In the application of LPMOs to biomass degradation, the key to consistent and effective functioning lies in their stability. The efficient transformation of biomass resources using LPMOs depends on factors that interfere with their stability. This review discussed three aspects that affect LPMO stability: general external factors, structural factors, and factors in the enzyme-substrate reaction. It explains how these factors impact LPMO stability, discusses the resulting effects, and finally presents relevant measures and considerations, including potential resolutions. The review also provides suggestions for the application of LPMOs in polysaccharide degradation.

Oxidative Machinery of basidiomycetes as potential enhancers in lignocellulosic biorefineries: A lytic polysaccharide monooxygenases approach.

Basidiomycetes are renowned as highly effective decomposers of plant materials, due to their extensive array of oxidative enzymes, which enable them to efficiently break down complex lignocellulosic biomass structures. Among the oxidative machinery of industrially relevant basidiomycetes, the role of lytic polysaccharide monooxygenases (LPMO) in lignocellulosic biomass deconstruction is highlighted. So far, only a limited number of basidiomycetes LPMOs have been identified and heterologously expressed. These LPMOs have presented activity on cellulose and hemicellulose, as well as participation in the deconstruction of lignin. Expanding on this, the current review proposes both enzymatic and non-enzymatic mechanisms of LPMOs for biomass conversion, considering the significance of the Carbohydrate-Binding Modules and other C-terminal regions domains associated with their structure, which is involved in the deconstruction of lignocellulosic biomass.

Improving the Enzymatic Activity and Stability of a Lytic Polysaccharide Monooxygenase.

Lytic Polysaccharide Monooxygenases (LPMOs) are copper-dependent enzymes that play a pivotal role in the enzymatic conversion of the most recalcitrant polysaccharides, such as cellulose and chitin. Hence, protein engineering is highly required to enhance their catalytic efficiencies. To this effect, we optimized the protein sequence encoding for an LPMO from Bacillus amyloliquefaciens (BaLPMO10A) using the sequence consensus method. Enzyme activity was determined using the chromogenic substrate 2,6-Dimethoxyphenol (2,6-DMP). Compared with the wild type (WT), the variants exhibit up to a 93.7% increase in activity against 2,6-DMP. We also showed that BaLPMO10A can hydrolyze p-nitrophenyl-ß-D-cellobioside (PNPC), carboxymethylcellulose (CMC), and phosphoric acid-swollen cellulose (PASC). In addition to this, we investigated the degradation potential of BaLPMO10A against various substrates such as PASC, filter paper (FP), and Avicel, in synergy with the commercial cellulase, and it showed up to 2.7-, 2.0- and 1.9-fold increases in production with the substrates PASC, FP, and Avicel, respectively, compared to cellulase alone. Moreover, we examined the thermostability of BaLPMO10A. The mutants exhibited enhanced thermostability with an apparent melting temperature increase of up to 7.5 °C compared to the WT. The engineered BaLPMO10A with higher activity and thermal stability provides a better tool for cellulose depolymerization.

The histidine brace: nature's copper alternative to haem?

The copper histidine brace is a structural unit in metalloproteins (Proc Natl Acad Sci USA 2011, 108, 15079). It consists of a copper ion chelated by the NH2 and π-N atom of an N-terminal histidine, and the τ-N atom of a further histidine, in an overall T-shaped coordination geometry (Nat Catal 2018, 1, 571). Like haem-containing proteins, histidine-brace-containing proteins have peroxygenase and/or oxygenase activity, where the substrates are notable for resistance to oxidation, for example, lytic polysaccharide monooxygenases (LPMOs). Moreover, the histidine brace is an invariant unit around which different protein structures exert different activities. Given the similarities in the diversity of function of proteins that contain either the copper histidine brace or haem, the question arises as to whether the functions of histidine brace-containing proteins duplicate those containing haem groups.

The crystal structure of CbpD clarifies substrate-specificity motifs in chitin-active lytic polysaccharide monooxygenases.

Pseudomonas aeruginosa secretes diverse proteins via its type 2 secretion system, including a 39 kDa chitin-binding protein, CbpD. CbpD has recently been shown to be a lytic polysaccharide monooxygenase active on chitin and to contribute substantially to virulence. To date, no structure of this virulence factor has been reported. Its first two domains are homologous to those found in the crystal structure of Vibrio cholerae GbpA, while the third domain is homologous to the NMR structure of the CBM73 domain of Cellvibrio japonicus CjLPMO10A. Here, the 3.0 Šresolution crystal structure of CbpD solved by molecular replacement is reported, which required ab initio models of each CbpD domain generated by the artificial intelligence deep-learning structure-prediction algorithm RoseTTAFold. The structure of CbpD confirms some previously reported substrate-specificity motifs among LPMOAA10s, while challenging the predictive power of others. Additionally, the structure of CbpD shows that post-translational modifications occur on the chitin-binding surface. Moreover, the structure raises interesting possibilities about how type 2 secretion-system substrates may interact with the secretion machinery and demonstrates the utility of new artificial intelligence protein structure-prediction algorithms in making challenging structural targets tractable.

Extending the diversity of Myceliophthora thermophila LPMOs: Two different xyloglucan cleavage profiles.

Lytic polysaccharide monooxygenases (LPMOs) play a key role in enzymatic conversion of plant cell wall polysaccharides. Continuous discovery and functional characterization of LPMOs highly contribute to the tailor-made design and improvement of hydrolytic-activity based enzyme cocktails. In this context, a new MtLPMO9F was characterized for its substrate (xyloglucan) specificity, and MtLPMO9H was further delineated. Aided by sodium borodeuteride reduction and hydrophilic interaction chromatography coupled to mass spectrometric analysis, we found that both MtLPMOs released predominately C4-oxidized, and C4/C6-double oxidized xylogluco-oligosaccharides. Further characterization showed that MtLPMO9F, having a short active site segment 1 and a long active site segment 2 (-Seg1+Seg2), followed a "substitution-intolerant" xyloglucan cleavage profile, while for MtLPMO9H (+Seg1-Seg2) a "substitution-tolerant" profile was found. The here characterized xyloglucan specificity and substitution (in)tolerance of MtLPMO9F and MtLPMO9H were as predicted according to our previously published phylogenetic grouping of AA9 LPMOs based on structural active site segment configurations.

On the expansion of biological functions of lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) constitute an enigmatic class of enzymes, the discovery of which has opened up a new arena of riveting research. LPMOs can oxidatively cleave the glycosidic bonds found in carbohydrate polymers enabling the depolymerisation of recalcitrant biomasses, such as cellulose or chitin. While most studies have so far mainly explored the role of LPMOs in a (plant) biomass conversion context, alternative roles and paradigms begin to emerge. In the present review, we propose a historical perspective of LPMO research providing a succinct overview of the major achievements of LPMO research over the past decade. This journey through LPMOs landscape leads us to dive into the emerging biological functions of LPMOs and LPMO-like proteins. We notably highlight roles in fungal and oomycete plant pathogenesis (e.g. potato late blight), but also in mutualistic/commensalism symbiosis (e.g. ectomycorrhizae). We further present the potential importance of LPMOs in other microbial pathogenesis including diseases caused by bacteria (e.g. pneumonia), fungi (e.g. human meningitis), oomycetes and viruses (e.g. entomopox), as well as in (micro)organism development (including several plant pests). Our assessment of the literature leads to the formulation of outstanding questions, promising for the coming years exciting research and discoveries on these moonlighting proteins.

The periplasmic expression and purification of AA15 lytic polysaccharide monooxygenases from insect species in Escherichia coli.

Lytic polysaccharide monooxygenases (LPMOs) are metalloenzymes that cleave structural polysaccharides through an oxidative mechanism. The enzymatic activity of LPMOs relies on the presence of a Cu2+ histidine-brace motif in their flat catalytic surface. Upon reduction by an external electron donor and in the presence of its co-substrates, O2 or H2O2, LPMOs can generate reactive oxygen species to oxidize the substrates. Fungal and bacterial LPMOs are involved in the catabolism of polysaccharides, such as chitin, cellulose, and hemicelluloses, and virulence mechanisms. Based on the reports on the discovery of LPMOs from the family AA15 in termites, firebrats, and flies, the functional role of the LPMO in the biosphere could expand, as these enzymes may be correlated with chitin remodeling and molting in insects. However, there is limited knowledge of AA15 LPMOs due to difficulties in recombinant expression of soluble proteins and purification protocols. In this study, we describe a protocol for the cloning, expression, and purification of insect AA15 LPMOs from Arthropoda, mainly from termites, followed by the expression and purification of an AA15 LPMO from the silkworm Bombyx mori, which contains a relatively high number of disulfide bonds. We also report the recombinant expression and purification of a protein with homology to AA15 family from the western European honeybee Apis mellifera, an LPMO-like enzyme lacking the canonical histidine brace. Therefore, this work can support future studies concerning the role of LPMOs in the biology of insects and inspire molecular entomologists and insect biochemists in conducting activities in this field.

Lytic polysaccharide monooxygenases as powerful tools in enzymatically assisted preparation of nano-scaled cellulose from lignocellulose: A review.

Nanocellulose, either in the form of fibers or crystals, constitutes a renewable, biobased, biocompatible material with advantageous mechanical properties that can be isolated from lignocellulosic biomass. Enzyme-assisted isolation of nanocellulose is an attractive, environmentally friendly approach that leads to products of higher quality compared to their chemically prepared counterparts. Lytic polysaccharide monooxygenases (LPMOs) are enzymes that oxidatively cleave the ß-1,4-glycosidic bond of polysaccharides upon activation of O2 or H2O2 and presence of an electron donor. Their use for treatment of cellulose fibers towards the preparation of nano-scaled cellulose is related to the ability of LPMOs to create nicking points on the fiber surface, thus facilitating fiber disruption and separation. The aim of this review is to describe the mode of action of LPMOs on cellulose fibers towards the isolation of nanostructures, thus highlighting their great potential for the production of nanocellulose as a novel value added product from lignocellulose.

Structure of a C1/C4-oxidizing AA9 lytic polysaccharide monooxygenase from the thermophilic fungus Malbranchea cinnamomea.

The thermophilic fungus Malbranchea cinnamomea contains a host of enzymes that enable its ability as an efficient degrader of plant biomass and that could be mined for industrial applications. This thermophilic fungus has been studied and found to encode eight lytic polysaccharide monooxygenases (LPMOs) from auxiliary activity family 9 (AA9), which collectively possess different substrate specificities for a range of plant cell-wall-related polysaccharides and oligosaccharides. To gain greater insight into the molecular determinants defining the different specificities, structural studies were pursued and the structure of McAA9F was determined. The enzyme contains the immunoglobulin-like fold typical of previously solved AA9 LPMO structures, but contains prominent differences in the loop regions found on the surface of the substrate-binding site. Most significantly, McAA9F has a broad substrate specificity, with activity on both crystalline and soluble polysaccharides. Moreover, it contains a small loop in a region where a large loop has been proposed to govern specificity towards oligosaccharides. The presence of the small loop leads to a considerably flatter and more open surface that is likely to enable the broad specificity of the enzyme. The enzyme contains a succinimide residue substitution, arising from intramolecular cyclization of Asp10, at a position where several homologous members contain an equivalent residue but cyclization has not previously been observed. This first structure of an AA9 LPMO from M. cinnamomea aids both the understanding of this family of enzymes and the exploration of the repertoire of industrially relevant lignocellulolytic enzymes from this fungus.

Comparison of C4-oxidizing and C1/C4-oxidizing AA9 LPMOs in substrate adsorption, H2O2-driven activity and synergy with cellulase on celluloses of different crystallinity.

Two C1/C4-oxidizing AA9 lytic polysaccharide monooxygenases (AA9 LPMOs), AoLPMO9A and AoLPMO9B, and one C4-oxidizing AoLPMO9C from Aspergillus oryzae, were characterized and compared with the well-studied C4-oxidizing NcLPMO9C. NcLPMO9C and AoLPMO9C harboring carbohydrate-binding module 1 (CBM1) exhibited much stronger adsorption capacity than AoLPMO9A and B without CBM1. The binding affinity is crucial for the efficacy of H2O2 as cosubstrate and oxidative activity of AA9 LPMOs on crystalline cellulose. C4-oxidizing AA9 LPMOs had a striking boosting effect on cellobiohydrolase I (CBHI), while C1/C4-oxidizing AA9 LPMOs boosted CBHII and endoglucanase I (EGI) activity. Our results indicated that two types of AA9 LPMOs with different modularities and regioselectivities varied in cellulose adsorption, H2O2-driven activity and synergy with cellulase on celluloses of different crystallinity which could complement each other in lignocellulose degradation. C4-oxidizing AA9 LPMOs with CBM1 were particularly essential in cellulase cocktail due to high H2O2-driven activity and a striking boosting effect on CBHI.

Enhanced Fenton Reaction for Xenobiotic Compounds and Lignin Degradation Fueled by Quinone Redox Cycling by Lytic Polysaccharide Monooxygenases.

The Fenton reaction is considered to be of great significance in the initial attack of lignocellulose in wood-decaying fungi. Quinone redox cycling is the main way to induce the Fenton reaction in fungi. We show that lytic polysaccharide monooxygenases (LPMOs), through LPMO-catalyzed oxidation of hydroquinone, can efficiently cooperate with glucose dehydrogenase (GDH) to achieve quinone redox cycling. The LPMO/GDH system can enhance Fe3+-reducing activity, H2O2 production, and hydroxyl radical generation, resulting in a fueled Fenton reaction. The system-generated hydroxyl radicals exhibited a strong capacity to decolorize different synthetic dyes and degrade lignin. Our results reveal a potentially critical connection between LPMOs and the Fenton reaction, suggesting that LPMOs could be involved in xenobiotic compound and lignin degradation in fungi. This new role of LPMOs may be exploited for application in biorefineries.

Role and significance of lytic polysaccharide monooxygenases (LPMOs) in lignocellulose deconstruction.

Lytic polysaccharide monooxygenases (LPMOs) emerged a decade ago and have been described as biomass deconstruction boosters as they play an extremely important role in unravelling the enzymatic biomass hydrolysis scheme. These are oxidative enzymes requiring partners to donate electrons during catalytic action on cellulose backbone. Commercial cellulase preparations are mostly from the robust fungal sources, hence LPMOs from fungi (AA9) have been discussed. Characterisation of LPMOs suffers due to multiple complications which has been discussed and challenges in detection of LPMOs in secretomes has also been highlighted. This review focuses on the significance of LPMOs on biomass hydrolysis due to which it has become a key component of cellulolytic cocktail available commercially for biomass deconstruction and its routine analysis challenge has also been discussed. It has also outlined a few key points that help in expressing catalytic active recombinant AA9 LPMOs.