Oxidative Degradation Of Cellulose Research Articles

The influence of photosynthetic pigment chlorophyllin in light-driven LPMO system on the hydrolytic action of cellulases

It has been demonstrated that LPMO reactions can be driven by light, using the photosynthetic pigment chlorophyllin to achieve efficient oxidative degradation of cellulose. However, the effect of chlorophyllin on cellulases remains unclear. This study discovered that chlorophyllin does not affect the hydrolytic activity of cellulases under dark conditions. However, under light exposure, chlorophyllin-derived reactive oxygen species (ROS) exhibit a strong inhibitory effect on cellulases. These ROS primarily inhibit the hydrolytic action of endoglucanase II (Cel5A) and cellobiohydrolase II (Cel6A), while the action of cellobiohydrolase I and β-glucosidase remains unaffected. Scavenger studies revealed that singlet oxygen (1O₂) is the key inhibitory ROS responsible for the inhibition of Cel5A and Cel6A. The removal of 1O₂ by sodium azide effectively mitigates this inhibition, increasing the conversion yield of cellulose to glucose by 25.9 % when using the light-driven LPMO system in conjunction with cellulases. This study provides new insights into the role of chlorophyllin-derived 1O₂ in hindering hydrolytic action of cellulases and demonstrates the successful mitigation of this inhibition by sodium azide, thereby enhancing the cooperative degradation of cellulose to glucose by the light-driven LPMO system and cellulases.

Eco-friendly, efficient and durable flame retardant lyocell fabrics prepared via a feasible grafting of taurine

Inspired by alkaline oxidative degradation of cellulose, a novel eco-friendly scenario for preparation of flame retardant lyocell fabrics was developed by the chemical grafting of biomass taurine. FTIR and XPS results verified the establishment of a covalent bond between taurine and lyocell fabric. Thermogravimetric analysis and vertical combustion test proved the improved char-forming ability and flame resistance of the grafted fabric. The THR and pHRR of the finished fabric were successfully reduced by 67.3 % and 89.9 % respectively after modification. In addition, after 50 wash cycles, the LOI value of the grafted fabric was maintained at 29.5 %. The gas-phase and condensed-phase flame retardant mechanisms were proposed from the analyses of pyrolysis gaseous products and char residue of the modified fabrics. The modification process is simple to operate and provides a feasible green flame retardant strategy for flammable lyocell fabrics.

Functional study of a lytic polysaccharide monooxygenase MsLPMO3 from Morchella sextelata in the oxidative degradation of cellulose

Lytic polysaccharide monooxygenases (LPMOs) can improve the effectiveness with which agricultural waste is utilized. This study described the potent AA9 family protein MsLPMO3, derived from Morchella sextelata. It exhibited strong binding to phosphoric acid swollen cellulose (PASC), and had the considerable binding ability to Cu2+ with a Kd value of 2.70 μM by isothermal titration calorimetry (ITC). MsLPMO3 could also act on PASC at the C1 carbon via MALDI-TOF-MS results. Moreover, MsLPMO3 could boost the hydrolysis efficiency of corncob and wheat bran in combination with glycoside hydrolases. MsLPMO3 also exhibited strong oxidizing ability for 2,6-dimethoxyphenol (2,6-DMP), achieving the best Vmax value of 443.36 U·g−1 for pH 7.4 with a H2O2 concentration of 300 µM. The structure of MsLPMO3 was obtained using AlphaFold2, and the molecular docking results elucidated the specific interactions and key residues involved in the recognition process between MsLPMO3 and cellulose. Altogether, this study expands the knowledge of AA9 family proteins in cellulose degradation, providing valuable insights into the mechanisms of synergistic degradation of lignocellulose with cellulases.

Structural and functional study of a novel lytic polysaccharide monooxygenase cPMO2 from compost sample in the oxidative degradation of cellulose

Lytic polysaccharide monooxygenases (LPMOs) are the major groups of oxidizing the recalcitrant polysaccharides. Here, a novel AA9 family protein cPMO2 originated from composting was characterized, and it had a better binding capacity to phosphoric acid swollen cellulose (PASC) and Avicel, as well the isothermal titration calorimetry (ITC) analysis showed that it owned a considerable capacity of binding Cu2+ with the Kd value of 1.58 ± 0.78 μM. Interestingly, the kinetics of cPMO2 by using 2, 6-dimethoxyphenol (2, 6-DMP) as substrate indicated that cPMO2 was a novel member of LPMOs family, with the Vmax of 21.30 ± 0.83 U·g−1 at pH 7.5. In addition, cPMO2 owned a broad substrate specificity, which could act on PASC and xylan at the C1 site based on the MALDI-TOF-MS analysis results. Simultaneously, cPMO2 could also act on Avicel directly resulting the decrease of crystallinity by 4% and further to synergistic degradation of corncob and rice straw with glycoside hydrolases. The structure of the functional region of cPMO2 was obtained by using single-crystal X-ray diffraction, and the enzymatic properties and molecular docking analysis results showed that His1 and Tyr175 were the critical active sites of the cPMO2. Altogether, this study will be a supplement for the AA9 LPMOs in cellulose degradation.

Cellulose Depolymerization with LPMO‐inspired Cu Complexes

AbstractBis(benzimidazole)amine‐based copper complexes, with structural similarities to the active sites of Lytic Polysaccharide Monooxygenase enzymes (LPMOs), were tested for the oxidative degradation of cellulose. Spectroscopic characterization of the complexes, as well as structural authentication of one of them, confirm a tetragonal coordination environment with 3 nitrogen donors, as well as a thioether in axial positions. Aqueous oxidative degradation of cellulose was achieved with the CuII complexes and H2O2 as oxidant through putative cupric‐hydroperoxo intermediates. Conversion of cellulose was achieved in up to 67 % yield of soluble oligosaccharide derivatives at ambient temperature and pressure. The products were analyzed in aqueous solution by HPLC‐MS, confirming oxidative depolymerization of cellulose under ambient conditions, in an analogous fashion to LPMOs.

Selective hydrothermal degradation of cellulose to formic acid in alkaline solutions

Herein, one-step conversion of cellulose dissolved in 1.75 mol L−1 NaOH/0.074 mol L−1 ZnO aqueous solution to organic acids was realized through a hydrothermal degradation without heterogeneous catalyst. The effects of solvent, the concentrations of NaOH and ZnO, O2 pressure, temperature and the reaction time on the yield of organic acids were investigated and discussed. The components of the degraded products were characterized with total organic carbon and high performance liquid chromatography. The results indicated that cellulose was depolymerized successfully to organic acids. Formic acid as the predominant species with 80.0% yield was obtained at the optimal conditions (150°C, 2 h, 3.0 MPa O2), which was much higher than those reported in literatures. The small amount of ZnO in the solvent played an important role not only to promote the cellulose dissolution, but also to accelerate its oxidative degradation. The main features of this one-step degradation of cellulose were the green solvent, pre-dissolution of catalyst, and high selectivity. This work opened up a new avenue for the conversion of cellulose to organic acids under mild conditions. The oxidative degradation of cellulose dissolved in NaOH/ZnO aqueous solution into organic acids was conducted under pressed O2 without heterogeneous catalyst. The yield of FA which was the main product reached up to 80.0%.

Cellobiose dehydrogenase: An essential enzyme for lignocellulose degradation in nature – A review / Cellobiosedehydrogenase: Ein essentielles Enzym für den Lignozelluloseabbau in der Natur – Eine Übersicht

Summary The flavin and heme cofactor containing enzyme cellobiose dehydrogenase (CDH) is ubiquitously distributed in wood-degrading fungi. Current research provides compelling evidence that CDH is an activator for cellulolytic monooxygenases, which enhance the accessibility of crystalline cellulose surfaces for hydrolases. Such oxidative cellulose degradation contributes to the overall cellulolytic capabilities of wood decaying fungi to a large extent, and holds great potential to improve the efficiency of commercial enzyme mixtures for biomass processing and biofuel production. This review summarizes current literature with regard to the distribution, structure and physiological role of CDH in the light of recent findings.

Single-domain flavoenzymes trigger lytic polysaccharide monooxygenases for oxidative degradation of cellulose.

The enzymatic conversion of plant biomass has been recently revolutionized by the discovery of lytic polysaccharide monooxygenases (LPMOs) that carry out oxidative cleavage of polysaccharides. These very powerful enzymes are abundant in fungal saprotrophs. LPMOs require activation by electrons that can be provided by cellobiose dehydrogenases (CDHs), but as some fungi lack CDH-encoding genes, other recycling enzymes must exist. We investigated the ability of AA3_2 flavoenzymes secreted under lignocellulolytic conditions to trigger oxidative cellulose degradation by AA9 LPMOs. Among the flavoenzymes tested, we show that glucose dehydrogenase and aryl-alcohol quinone oxidoreductases are catalytically efficient electron donors for LPMOs. These single-domain flavoenzymes display redox potentials compatible with electron transfer between partners. Our findings extend the array of enzymes which regulate the oxidative degradation of cellulose by lignocellulolytic fungi.

The effect of oil binders on paper supports via VOC analysis

The effect of the presence of drying oils in paper supports on the rate of cellulose degradation is investigated in a novel manner using Solid Phase Micro-extraction (SPME), which is employed to analyse volatile organic compounds (VOCs), emitted from oiled paper. This technique is applied as a non-destructive means of analysing original works of art on paper, in order to detect volatile cellulose degradation products. It is also applied to artificially aged paper samples with and without oil, in order to investigate the extent to which the presence of drying oil accelerates the degradation of cellulose. Furfural and other volatile cellulose degradation products containing a furan ring are selected as representative cellulose degradation products to be measured for the purpose of the investigation. It is demonstrated, by the finding of increased emissions of the selected compounds, that the presence of drying oils accelerates the thermal and oxidative degradation of cellulose in cotton paper and two types of wood pulp based papers.

Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation.

A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization.

Crystal Structure and Substrate Recognition of Cellobionic Acid Phosphorylase, Which Plays a Key Role in Oxidative Cellulose Degradation by Microbes

The microbial oxidative cellulose degradation system is attracting significant research attention after the recent discovery of lytic polysaccharide mono-oxygenases. A primary product of the oxidative and hydrolytic cellulose degradation system is cellobionic acid (CbA), the aldonic acid form of cellobiose. We previously demonstrated that the intracellular enzyme belonging to glycoside hydrolase family 94 from cellulolytic fungus and bacterium is cellobionic acid phosphorylase (CBAP), which catalyzes reversible phosphorolysis of CbA into glucose 1-phosphate and gluconic acid (GlcA). In this report, we describe the biochemical characterization and the three-dimensional structure of CBAP from the marine cellulolytic bacterium Saccharophagus degradans. Structures of ligand-free and complex forms with CbA, GlcA, and a synthetic disaccharide product from glucuronic acid were determined at resolutions of up to 1.6 Å. The active site is located near the dimer interface. At subsite +1, the carboxylate group of GlcA and CbA is recognized by Arg-609 and Lys-613. Additionally, one residue from the neighboring protomer (Gln-190) is involved in the carboxylate recognition of GlcA. A mutational analysis indicated that these residues are critical for the binding and catalysis of the aldonic and uronic acid acceptors GlcA and glucuronic acid. Structural and sequence comparisons with other glycoside hydrolase family 94 phosphorylases revealed that CBAPs have a unique subsite +1 with a distinct amino acid residue conservation pattern at this site. This study provides molecular insight into the energetically efficient metabolic pathway of oxidized sugars that links the oxidative cellulolytic pathway to the glycolytic and pentose phosphate pathways in cellulolytic microbes.

The Effect of Iron Ions from Prussian Blue Pigment on the Deterioration of Japanese Paper

AbstractPaper is often damaged by ink or pigments containing transition metal ions. Damage of Kozo paper (a kind of Japanese paper) caused by iron ions from Prussian blue pigments (iron ferrocyanide) or by the acidity of dilute sulfuric acid during artificial ageing was studied. The degradation induced by iron ions is suggested to be a synergistic process consisting of hydrolytic and oxidative reactions. The degradation state of the cellulose polymer in Kozo paper was investigated using fluorescence labeling of carbonyl and carboxyl groups in combination with gel permeation chromatography-multiple-angle laser light scattering. In addition to cellulose, Kozo paper contains a relatively large amount of hemicellulose, which was characterized by determination of uronic acid and neutral carbohydrates. The amount of carboxyl groups in Kozo paper decreased during ageing. A decrease in the amount of uronic acid originating from the hemicellulose in Kozo paper is considered the dominant factor in the observed decrease of total carboxyl contents, even if the oxidative reaction would increase the number of carboxyl groups. The amount of uronic acid and xylose in hemicellulose was influenced by iron ions during ageing, indicating the decomposition of hemicellulose. This result suggested that the hemicellulose in Kozo paper were affected first and prevented the hydrolytic and oxidative degradation of cellulose, which might enable Kozo paper to last longer.

Mechanistic studies of polysaccharide monooxygenases (584.8)

Polysaccharide monooxygenases (PMOs) from filamentous fungi have recently been shown to play a central role in oxidative degradation of cellulose. These enzymes contain a mononuclear copper active site on a flat surface of the protein that would allow for direct interaction with the substrate. The active site copper coordination consists of two histidine ligands. One of these ligands is the unusual N‐terminal Ne‐methyl‐histidine residue that binds in a bidentate mode using its imidazole and amino groups. PMOs activate dioxygen leading to hydroxylation of either C1 or C4 position of the glycosidic linkage, which subsequently results in the cleavage of this bond. Newly developed expression methods have enabled the characterizeation and classification of the PMO family into three major groups based on their selectivity toward C1 and C4. Factors that control the regioselectivity of PMOs are underway, including residues that contribute to orientation of the copper site on the cellulose surface. Several PMOs have been identified that hydroxylate soluble cellodextrins to form similar products as observed for cellulose. Oxygen activation by these PMOs is also underway.

Transcriptional analysis of selected cellulose-acting enzymes encoding genes of the white-rot fungus Dichomitus squalens on spruce wood and microcrystalline cellulose

The recent discovery of oxidative cellulose degradation enhancing enzymes has considerably changed the traditional concept of hydrolytic cellulose degradation. The relative expression levels of ten cellulose-acting enzyme encoding genes of the white-rot fungus Dichomitus squalens were studied on solid-state spruce wood and in microcrystalline Avicel cellulose cultures. From the cellobiohydrolase encoding genes, cel7c was detected at the highest level and showed constitutive expression whereas variable transcript levels were detected for cel7a, cel7b and cel6 in the course of four-week spruce cultivation. The cellulolytic enzyme activities detected in the liquid cultures were consistent with the transcript levels. Interestingly, the selected lytic polysaccharide monooxygenase (LPMO) encoding genes were expressed in both cultures, but showed different transcription patterns on wood compared to those in submerged microcrystalline cellulose cultures. On spruce wood, higher transcript levels were detected for the lpmos carrying cellulose binding module (CBM) than for the lpmos without CBMs. In both cultures, the expression levels of the lpmo genes were generally higher than the levels of cellobiose dehydrogenase (CDH) encoding genes. Based on the results of this work, the oxidative cellulose cleaving enzymes of D. squalens have essential role in cellulose degrading machinery of the fungus.

Characterization of the Two Neurospora crassa Cellobiose Dehydrogenases and Their Connection to Oxidative Cellulose Degradation

The genome of Neurospora crassa encodes two different cellobiose dehydrogenases (CDHs) with a sequence identity of only 53%. So far, only CDH IIA, which is induced during growth on cellulose and features a C-terminal carbohydrate binding module (CBM), was detected in the secretome of N. crassa and preliminarily characterized. CDH IIB is not significantly upregulated during growth on cellulosic material and lacks a CBM. Since CDH IIB could not be identified in the secretome, both CDHs were recombinantly produced in Pichia pastoris. With the cytochrome domain-dependent one-electron acceptor cytochrome c, CDH IIA has a narrower and more acidic pH optimum than CDH IIB. Interestingly, the catalytic efficiencies of both CDHs for carbohydrates are rather similar, but CDH IIA exhibits 4- to 5-times-higher apparent catalytic constants (k(cat) and K(m) values) than CDH IIB for most tested carbohydrates. A third major difference is the 65-mV-lower redox potential of the heme b cofactor in the cytochrome domain of CDH IIA than CDH IIB. To study the interaction with a member of the glycoside hydrolase 61 family, the copper-dependent polysaccharide monooxygenase GH61-3 (NCU02916) from N. crassa was expressed in P. pastoris. A pH-dependent electron transfer from both CDHs via their cytochrome domains to GH61-3 was observed. The different properties of CDH IIA and CDH IIB and their effect on interactions with GH61-3 are discussed in regard to the proposed in vivo function of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.

Cellobiose Dehydrogenase and a Copper-Dependent Polysaccharide Monooxygenase Potentiate Cellulose Degradation by Neurospora crassa

The high cost of enzymes for saccharification of lignocellulosic biomass is a major barrier to the production of second generation biofuels. Using a combination of genetic and biochemical techniques, we report that filamentous fungi use oxidative enzymes to cleave glycosidic bonds in cellulose. Deletion of cdh-1, the gene encoding the major cellobiose dehydrogenase of Neurospora crassa, reduced cellulase activity substantially, and addition of purified cellobiose dehydrogenases from M. thermophila to the Δcdh-1 strain resulted in a 1.6- to 2.0-fold stimulation in cellulase activity. Addition of cellobiose dehydrogenase to a mixture of purified cellulases showed no stimulatory effect. We show that cellobiose dehydrogenase enhances cellulose degradation by coupling the oxidation of cellobiose to the reductive activation of copper-dependent polysaccharide monooxygenases (PMOs) that catalyze the insertion of oxygen into C-H bonds adjacent to the glycosidic linkage. Three of these PMOs were characterized and shown to have different regiospecifities resulting in oxidized products modified at either the reducing or nonreducing end of a glucan chain. In contrast to previous models where oxidative enzymes were thought to produce reactive oxygen species that randomly attacked the substrate, the data here support a direct, enzyme-catalyzed oxidation of cellulose. Cellobiose dehydrogenases and proteins related to the polysaccharide monooxygenases described here are found throughout both ascomycete and basidiomycete fungi, suggesting that this model for oxidative cellulose degradation may be widespread throughout the fungal kingdom. When added to mixtures of cellulases, these proteins enhance cellulose saccharification, suggesting that they could be used to reduce the cost of biofuel production.

ITRAQ-based quantitative secretome analysis of Phanerochaete chrysosporium

The basidiomycete fungi such as Phanerochaete chrysosporium secrete large amount of hydrolytic and oxidative enzymes and degrade lignocellulosic biomass. The lignin depolymerizing proteins were extensively studied, but cellulose, hemicellulose and pectin hydrolyzing enzymes were poorly explored. In this study P. chrysosporium was grown in cellulose, lignin and mixture of cellulose and lignin, and secretory proteins were quantified by isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics using liquid chromatography tandem mass spectrometry (LC-MS/MS). An iTRAQ quantified 117 enzymes comprising cellulose hydrolyzing endoglucanases, exoglucanases, beta-glucosidases; hemicelluloses hydrolyzing xylanases, acetylxylan esterases, mannosidases, mannanases; pectin-degrading enzymes polygalacturonase, rhamnogalacturonase, arabinose and lignin degrading protein belonging to oxidoreductase family. Under cellulose and cellulose with lignin culture conditions, enzymes such as endoglucanases, exoglucanases, β-glucosidases and cellobiose dehydrogenase were significantly upregulated and iTRAQ data suggested hydrolytic and oxidative cellulose degradation. When lignin was used as a major carbon source, enzymes such as copper radical oxidase, isoamyl oxidase, glutathione S-transferase, thioredoxin peroxidase, quinone oxidoreductase, aryl alcohol oxidase, pyranose 2-oxidase, aldehyde dehydrogenase, and alcohol dehydrogenase were expressed and significantly regulated. This study explored cellulose, hemicellulose, pectin and lignin degrading enzymes of P. chrysosporium that are valuable for lignocellulosic bioenergy.

Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components

The enzymatic degradation of recalcitrant plant biomass is one of the key industrial challenges of the 21st century. Accordingly, there is a continuing drive to discover new routes to promote polysaccharide degradation. Perhaps the most promising approach involves the application of "cellulase-enhancing factors," such as those from the glycoside hydrolase (CAZy) GH61 family. Here we show that GH61 enzymes are a unique family of copper-dependent oxidases. We demonstrate that copper is needed for GH61 maximal activity and that the formation of cellodextrin and oxidized cellodextrin products by GH61 is enhanced in the presence of small molecule redox-active cofactors such as ascorbate and gallate. By using electron paramagnetic resonance spectroscopy and single-crystal X-ray diffraction, the active site of GH61 is revealed to contain a type II copper and, uniquely, a methylated histidine in the copper's coordination sphere, thus providing an innovative paradigm in bioinorganic enzymatic catalysis.

Evolution of organic matter during composting of different organic wastes assessed by CPMAS 13C NMR spectroscopy

In this paper, the evolution of organic matter (OM) during composting of different mixtures of various organic wastes was assessed by means of chemical analyses and CPMAS 13C NMR spectroscopy measured during composting. The trends of temperatures and C/N ratios supported the correct evolution of the processes. The CPMAS 13C NMR spectra of all composting substrates indicated a reduction in carbohydrates and an increase in aromatic, phenolic, carboxylic and carbonylic C which suggested a preference by microorganisms for easily degradable C molecules. The presence of hardly degradable pine needles in one of the substrates accounted for the lowest increase in alkyl C and the lowest reduction in carbohydrates and carboxyl C as opposite to another substrate characterized by the presence of a highly degradable material such as spent yeast from beer production, which showed the highest increase of the alkyl C/O-alkyl C ratio. The highest increase of COOH deriving by the oxidative degradation of cellulose was shown by a substrate composed by about 50% of plant residues. The smallest increases in alkyl C/O-alkyl C ratio and in polysaccharides were associated to the degradation of proteins and lipids which are major components of sewage sludge. Results obtained were related to the different composition of fresh organic substrates and provided evidence of different OM evolution patterns as a function of the initial substrate composition.

The role of transition metals in oxidative degradation of cellulose

The role of transition metals in oxidative degradation of cellulose has been studied. Degradation experiments with model papers and studies of hydroxyl radical production in solution have been performed with Fe, Cu, Mn, Co, Cr, Ni, and Zn. Rates of production of hydroxyl radicals in solution have been estimated using the radical scavenger N, N′-(5-nitro-1,3-phenylene)bisglutaramide in the pH interval 7–9. Hydroxyl radical production during degradation of Cu-containing cellulose has been studied. To gain a better insight into chemistry behind degradation processes, chemiluminometric experiments were also performed. The experiments provide strong evidence that the role of transition metals during the oxidative degradation of cellulose is catalytic. A correlation between the behaviour of transition metals in solution and in paper was established at low contents of transition metal in paper.