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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. Author summary: Fungi need to adapt quickly to environmental stresses to thrive. The fungal cell wall, which supplies support and integrity to the cell, is an essential compartment to react and interact with the surrounding environment. Rapid changes within the carbohydrate composition and architecture occur in response to environmental stresses. Lytic polysaccharide monooxygenases (LPMOs) are mononuclear copper-enzymes, secreted by microbes to assist in the first steps of remodeling and degradation of complex and recalcitrant carbohydrates. In this study we explore the role of the putative AA9 family LPMO CnCel1 for growth and virulence of the fungal human pathogen Cryptococcus neoformans. The CEL1 gene is highly up-regulated in presence of host stresses and a cel1Δ mutant strain is avirulent in a murine model of infection. Downstream analysis of virulence-associated phenotypes identified the CEL1 gene to be required for thermotolerance as well as cell wall integrity, and efficient cell cycle progression in presence of host-mimicking stresses. Based upon those findings, we propose that CnCel1 likely promotes intrinsic fungal cell wall remodeling events essential for adaptation to the host environment. [ABSTRACT FROM AUTHOR]

Abstract Background The recent development of improved enzymes and pentose-using yeast for cellulosic ethanol processes calls for new attention to the lignocellulose pretreatment step. This study assessed the influence of pretreatment pH, temperature, and time, and their interactions on the enzymatic glucose and xylose yields from mildly pretreated wheat straw in multivariate experimental designs of acid and alkaline pretreatments. Results The pretreatment pH was the most significant factor affecting both the enzymatic glucose and xylose yields after mild thermal pretreatments at maximum 140°C for 10 min. The maximal enzymatic glucose and xylose yields from the solid, pretreated wheat straw fraction were obtained after pretreatments at the most extreme pH values (pH 1 or pH 13) at the maximum pretreatment temperature of 140°C. Surface response models revealed significantly correlating interactions of the pretreatment pH and temperature on the enzymatic liberation of both glucose and xylose from pretreated, solid wheat straw. The influence of temperature was most pronounced with the acidic pretreatments, but the highest enzymatic monosaccharide yields were obtained after alkaline pretreatments. Alkaline pretreatments also solubilized most of the lignin. Conclusions Pretreatment pH exerted significant effects and factor interactions on the enzymatic glucose and xylose releases. Quite extreme pH values were necessary with mild thermal pretreatment strategies (T ≤ 140°C, time ≤ 10 min). Alkaline pretreatments generally induced higher enzymatic glucose and xylose release and did so at lower pretreatment temperatures than required with acidic pretreatments.

The production of regenerated cellulosic fibres, such as viscose, modal and lyocell, is based mainly on the use of dissolving wood pulp as raw material. Enzymatic processes are an excellent alternative to conventional chemical routes in the production of dissolving pulp, in terms of energy efficiency, reagent consumption and pulp yield. The two main characteristics of a dissolving pulp are the cellulose purity and the molecular weight, both of which can be controlled with the aid of enzymes. A purification process for paper-grade kraft pulp has been proposed, based on the use of xylanases in combination with hot and cold caustic extraction, without the conventional pre-hydrolysis step before kraft pulping. This enzyme aided purification allowed the production of a dissolving pulp that met the specifications for the manufacture of viscose, < 3% xylan, > 92% ISO brightness and 70% Fock's reactivity. Endoglucanases (EGs) can efficiently reduce the average molecular weight of the cellulose while simultaneously increasing the pulp reactivity for viscose production. It is shown in this study that lytic polysaccharide monooxygenases act synergistically with EGs in the modification of bleached dissolving pulp. [ABSTRACT FROM AUTHOR]

The histidine brace (His‐brace) is a copper‐binding motif that is associated with both oxidative enzymes and proteinaceous copper chaperones. Here, we used biochemical and structural methods to characterize mutants of a His‐brace‐containing copper chaperone from Pseudomonas fluorescens (PfCopC). A total of 15 amino acid variants in primary and second‐sphere residues were produced and characterized in terms of their copper binding and redox properties. PfCopC has a very high affinity for Cu(II) and also binds Cu(I). A high reorganization barrier likely prevents redox cycling and, thus, catalysis. In contrast, mutations in the conserved second‐sphere Glu27 enable slow oxidation of ascorbate. The crystal structure of the variant E27A confirmed copper binding at the His‐brace. Unexpectedly, Asp83 at the equatorial position was shown to be indispensable for Cu(II) binding in the His‐brace of PfCopC. A PfCopC mutant that was designed to mimic the His‐brace from lytic polysaccharide monooxygenase‐like family X325 did not bind Cu(II), but was still able to bind Cu(I). These results highlight the importance of the proteinaceous environment around the copper His‐brace for reactivity and, thus, the difference between enzyme and chaperone. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) are mononuclear copper enzymes that catalyse the oxidative cleavage of glycosidic bonds. They are characterised by two histidine residues that coordinate copper in a configuration termed the Cu-histidine brace. Although first identified in bacteria and fungi, LPMOs have since been found in all biological kingdoms. LPMOs are now included in commercial enzyme cocktails used in industrial biorefineries. This has led to increased process yield due to the synergistic action of LPMOs with glycoside hydrolases. However, the introduction of LPMOs makes control of the enzymatic step in industrial stirred-tank reactors more challenging, and the operational stability of the enzymes is reduced. It is clear that much is still to be learned about the interaction between LPMOs and their complex natural and industrial environments, and fundamental scientific studies are required towards this end. Several atomic-resolution structures have been solved providing detailed information on the Cu-coordination sphere and the interaction with the polysaccharide substrate. However, the molecular mechanisms of LPMOs are still the subject of intense investigation; the key question being how the proteinaceous environment controls the copper cofactor towards the activation of the O-O bond in O2 and cleavage of the glycosidic bonds in polysaccharides. The need for biochemical characterisation of each putative LPMO is discussed based on recent reports showing that not all proteins with a Cu-histidine brace are enzymes. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) are copper enzymes discovered within the last 10 years. By degrading recalcitrant substrates oxidatively, these enzymes are major contributors to the recycling of carbon in nature and are being used in the biorefinery industry. Recently, two new families of LPMOs have been defined and structurally characterized, AA14 and AA15, sharing many of previously found structural features. However, unlike most LPMOs to date, AA14 degrades xylan in the context of complex substrates, while AA15 is particularly interesting because they expand the presence of LPMOs from the predominantly microbial to the animal kingdom. The first two neutron crystallography structures have been determined, which, together with high-resolution room temperature X-ray structures, have putatively identified oxygen species at or near the active site of LPMOs. Many recent computational and experimental studies have also investigated the mechanism of action and substrate-binding mode of LPMOs. Perhaps, the most significant recent advance is the increasing structural and biochemical evidence, suggesting that LPMOs follow different mechanistic pathways with different substrates, co-substrates and reductants, by behaving as monooxygenases or peroxygenases with molecular oxygen or hydrogen peroxide as a co-substrate, respectively. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent enzymes discovered within the last ten years. They oxidatively cleave polysaccharides (chitin, lignocellulose, hemicellulose and starch-derived), presumably making recalcitrant substrates accessible to glycoside hydrolases. Recently, the first crystal structure of an LPMO-substrate complex was reported, giving insights into the interaction of LPMOs with β-linked substrates (Frandsen et al., 2016). The LPMOs acting on α-linked glycosidic bonds (family AA13) display binding surfaces that are quite different from those of LPMOs that act on β-linked glycosidic bonds (families AA9-AA11), as revealed from the first determined structure (Lo Leggio et al., 2015), and thus presumably the AA13s interact with their substrate in a distinct fashion. Here, several new structures of the same AA13 enzyme, Aspergillus oryzae AA13, are presented. Crystals obtained in the presence of high zinc-ion concentrations were used, as they can be obtained more reproducibly than those used to refine the deposited copper-containing structure. One structure with an ordered zinc-bound active site was solved at 1.65 Å resolution, and three structures from crystals soaked with maltooligosaccharides in solutions devoid of zinc ions were solved at resolutions of up to 1.10 Å. Despite similar unit-cell parameters, small rearrangements in the crystal packing occur when the crystals are depleted of zinc ions, resulting in a more occluded substrate-binding surface. In two of the three structures maltooligosaccharide ligands are bound, but not at the active site. Two of the structures presented show a His-ligand conformation that is incompatible with metal-ion binding. In one of these structures this conformation is the principal one (80% occupancy), giving a rare atomic resolution view of a substantially misfolded enzyme that is presumably rendered inactive. [ABSTRACT FROM AUTHOR]

The recent discovery of copper-dependent lytic polysaccharide mono-oxygenases (LPMOs) has opened up a vast area of research covering several fields of application. The biotech company Novozymes A/S holds patents on the use of these enzymes for the conversion of steam-pre-treated plant residues such as straw to free sugars. These patents predate the correct classification of LPMOs and the striking synergistic effect of fungal LPMOs when combined with canonical cellulases was discovered when fractions of fungal secretomes were evaluated in industrially relevant enzyme performance assays. Today, LPMOs are a central component in the Cellic CTec enzyme products which are used in several large-scale plants for the industrial production of lignocellulosic ethanol. LPMOs are characterized by an N-terminal histidine residue which, together with an internal histidine and a tyrosine residue, co-ordinates a single copper atom in a so-called histidine brace. The mechanism by which oxygen binds to the reduced copper atom has been reported and the general mechanism of copper-oxygen-mediated activation of carbon is being investigated in the light of these discoveries. LPMOs are widespread in both the fungal and the bacterial kingdoms, although the range of action of these enzymes remains to be elucidated. However, based on the high abundance of LPMOs expressed by microbes involved in the decomposition of organic matter, the importance of LPMOs in the natural carbon-cycle is predicted to be significant. In addition, it has been suggested that LPMOs play a role in the pathology of infectious diseases such as cholera and to thus be relevant in the field of medicine. [ABSTRACT FROM AUTHOR]

Mechanical agitation during enzymatic hydrolysis of insoluble plant biomass at high dry matter contents is indispensable for the initial liquefaction step in biorefining. It is known that particle size reduction is an important part of liquefaction, but the mechanisms involved are poorly understood. Here we put forward a simple model based on mechanical principles capable of capturing the result of the interaction between mechanical forces and cell wall weakening via hydrolysis of glucosidic bonds. This study illustrates that basic material science insights are relevant also within biochemistry, particularly when it comes to up-scaling of processes based on insoluble feed stocks. [ABSTRACT FROM AUTHOR]

Background Ensiling may act as a pretreatment of fresh grass biomass and increase the enzymatic conversion of structural carbohydrates to fermentable sugars. However, ensiling does not provide sufficient severity to be a standalone pretreatment method. Here, ensiling of grass is combined with hydrothermal treatment (HTT) with the aim of improving the enzymatic biomass convertibility and decrease the required temperature of the HTT. Results Grass silage (Festulolium Hykor) was hydrothermally treated at temperatures of 170, 180, and 190°C for 10 minutes. Relative to HTT treated dry grass, ensiling increased the solubilization of dry matter (DM) during HTT and gave increased glucan content, but lower lignin in the insoluble fiber fraction. Ensiling improved glucose yields in the enzymatic hydrolysis of the washed solid fiber fraction at the lower HTT temperatures. At 170°C glucose yield improved from 17 to 24 (w/w)% (45 to 57% cellulose convertibility), and at 180°C glucose yield improved from 22 to 29 (w/w)% (54 to 69% cellulose convertibility). Direct HTT of grass at 190°C gave the same high glucose yield as for grass silage (35 (w/w)% (77% cellulose convertibility)) and improved xylan yields (27% xylan convertibility). The effect of ensiling of grass prior to HTT improved the enzymatic conversion of cellulose for HTT at 170 and 180°C, but the increased glucose release did not make up for the loss of water soluble carbohydrates (WSC) during ensiling. Overall, sugar yields (C6 + C5) were similar for HTT of grass and grass silage at both 170 and 180°C, but at 190°C the overall sugar yield was better for HTT of dry grass. Conclusions This study unequivocally establishes that ensiling of grass as a biomass pretreatment method comes with a loss of WSC. The loss of WSC by ensiling is not necessarily compensated for by providing a lower temperature requirement for HTT for high enzymatic monosaccharide release. However, ensiling can be an advantageous storage method prior to grass processing. [ABSTRACT FROM AUTHOR]

Background: Plant cell wall polysaccharide composition varies substantially between species, organs and genotypes. Knowledge of the structure and composition of these polysaccharides, accompanied by a suite of well characterised glycosyl hydrolases will be important for the success of lignocellulosic biofuels. Current methods used to characterise enzymatically released plant oligosaccharides are relatively slow. Results: A method and software was developed allowing the use of a DNA sequencer to profile oligosaccharides derived from plant cell wall polysaccharides (DNA sequencer-Assisted Saccharide analysis in High throughput, DASH). An ABI 3730xl, which can analyse 96 samples simultaneously by capillary electrophoresis, was used to separate fluorophore derivatised reducing mono- and oligo-saccharides from plant cell walls. Using electrophoresis mobility markers, oligosaccharide mobilities were standardised between experiments to enable reproducible oligosaccharide identification. These mobility markers can be flexibly designed to span the mobilities of oligosaccharides under investigation, and they have a fluorescence emission that is distinct from that of the saccharide labelling. Methods for relative and absolute quantitation of oligosaccharides are described. Analysis of a large number of samples is facilitated by the DASHboard software which was developed in parallel. Use of this method was exemplified by comparing xylan structure and content in Arabidopsis thaliana mutants affected in xylan synthesis. The product profiles of specific xylanases were also compared in order to identify enzymes with unusual oligosaccharide products. Conclusions: The DASH method and DASHboard software can be used to carry out large-scale analyses of the compositional variation of plant cell walls and biomass, to compare plants with mutations in plant cell wall synthesis pathways, and to characterise novel carbohydrate active enzymes. [ABSTRACT FROM AUTHOR]

Background: Wheat straw used for bioethanol production varies in enzymatic digestibility according to chemical structure and composition of cell walls and tissues. In this work, the two biologically different wheat straw organs, leaves and stems, are described together with the effects of hydrothermal pretreatment on chemical composition, tissue structure, enzyme adhesion and digestion. To highlight the importance of inherent cell wall characteristics and the diverse effects of mechanical disruption and biochemical degradation, separate leaves and stems were pretreated on lab-scale and their tissue structures maintained mostly intact for image analysis. Finally, samples were enzymatically hydrolysed to correlate digestibility to chemical composition, removal of polymers, tissue composition and disruption, particle size and enzyme adhesion as a result of pretreatment and wax removal. For comparison, industrially pretreated wheat straw from Inbicon A/S was included in all the experiments. Results: Within the same range of pretreatment severities, industrial pretreatment resulted in most hemicellulose and epicuticular wax/cutin removal compared to lab-scale pretreated leaves and stems but also in most redeposition of lignin on the surface. Tissues were furthermore degraded from tissues into individual cells while lab-scale pretreated samples were structurally almost intact. In both raw leaves and stems, endoglucanase and exoglucanase adhered most to parenchyma cells; after pretreatment, to epidermal cells in all the samples. Despite heavy tissue disruption, industrially pretreated samples were not as susceptible to enzymatic digestion as lab-scale pretreated leaves while lab-scale pretreated stems were the least digestible. Conclusions: Despite preferential enzyme adhesion to epidermal cells after hydrothermal pretreatment, our results suggest that the single most important factor determining wheat straw digestibility is the fraction of parenchyma cells rather than effective tissue disruption. [ABSTRACT FROM AUTHOR]

Background: The recent development of improved enzymes and pentose-using yeast for cellulosic ethanol processes calls for new attention to the lignocellulose pretreatment step. This study assessed the influence of pretreatment pH, temperature, and time, and their interactions on the enzymatic glucose and xylose yields from mildly pretreated wheat straw in multivariate experimental designs of acid and alkaline pretreatments. Results: The pretreatment pH was the most significant factor affecting both the enzymatic glucose and xylose yields after mild thermal pretreatments at maximum 140°C for 10 min. The maximal enzymatic glucose and xylose yields from the solid, pretreated wheat straw fraction were obtained after pretreatments at the most extreme pH values (pH 1 or pH 13) at the maximum pretreatment temperature of 140°C. Surface response models revealed significantly correlating interactions of the pretreatment pH and temperature on the enzymatic liberation of both glucose and xylose from pretreated, solid wheat straw. The influence of temperature was most pronounced with the acidic pretreatments, but the highest enzymatic monosaccharide yields were obtained after alkaline pretreatments. Alkaline pretreatments also solubilized most of the lignin. Conclusions: Pretreatment pH exerted significant effects and factor interactions on the enzymatic glucose and xylose releases. Quite extreme pH values were necessary with mild thermal pretreatment strategies (T ≥ 140°C, time ≥ 10 min). Alkaline pretreatments generally induced higher enzymatic glucose and xylose release and did so at lower pretreatment temperatures than required with acidic pretreatments. [ABSTRACT FROM AUTHOR]

The nutritional effects of two barley mutant lines with an altered phosphorus (P) profile and phytate content (13 and 43% of total P, respectively) were studied. Because grain quantities were limited, rats served as a model for the pig in a feeding trail. Four groups of five Wistar rats (weighing 65 g) were fed the low-phytate lines, the mother variety and a mixture of barley varieties. All diets were supplemented with vitamins and minerals except for P, calcium, zinc and copper. The apparent digestibility of P was improved by up to 13% in the mutant lines. Although all diets were very similar in their zinc content, only rats fed the mutant lines had a net absorption and a positive zinc balance, indicating the improved availability of this mineral. Rats appear to be a suitable model for P utilization in pigs and can thus provide plant breeders with a mineral bioavailability assay for use at an early stage. [ABSTRACT FROM AUTHOR]