Three-Step Enzymatic Remodeling of Chitin into Bioactive Chitooligomers.

Here we describe a complex enzymatic approach to the efficient transformation of abundant waste chitin, a byproduct of the food industry, into valuable chitooligomers with a degree of polymerization (DP) ranging from 6 to 11. This method involves a three-step process: initial hydrolysis of chitin using engineered variants of a novel fungal chitinase from Talaromyces flavus to generate low-DP chitooligomers, followed by an extension to the desired DP using the high-yielding Y445N variant of ß-N-acetylhexosaminidase from Aspergillus oryzae, achieving yields of up to 57%. Subsequently, enzymatic deacetylation of chitooligomers with DP 6 and 7 was accomplished using peptidoglycan deacetylase from Bacillus subtilis BsPdaC. The innovative enzymatic procedure demonstrates a sustainable and feasible route for converting waste chitin into unavailable bioactive chitooligomers potentially applicable as natural pesticides in ecological and sustainable agriculture.

Chitinase-functionalized UiO-66 framework nanoparticles active against multidrug-resistant Candida Auris.

Candida auris (C. auris) is a yeast that has caused several outbreaks in the last decade. Cell wall chitin plays a primary role in the antifungal resistance of C. auris. Herein, we investigated the potential of chitinase immobilized with UiO-66 to act as a potent antifungal agent against C. auris. Chitinase was produced from Talaromyces varians SSW3 in a yield of 8.97 U/g dry substrate (ds). The yield was statistically enhanced to 120.41 U/g ds by using Plackett-Burman and Box-Behnken design. We synthesized a UiO-66 framework that was characterized by SEM, TEM, XRD, FTIR, a particle size analyzer, and a zeta sizer. The produced framework had a size of 70.42 ± 8.43 nm with a uniform cubic shape and smooth surface. The produced chitinase was immobilized on UiO-66 with an immobilization yield of 65% achieved after a 6 h loading period. The immobilization of UiO-66 increased the enzyme activity and stability, as indicated by the obtained Kd and T1/2 values. Furthermore, the hydrolytic activity of chitinase was enhanced after immobilization on UiO-66, with an increase in the Vmax and a decrease in the Km of 2- and 38-fold, respectively. Interestingly, the antifungal activity of the produced chitinase was boosted against C. auris by loading the enzyme on UiO-66, with an MIC50 of 0.89 ± 0.056 U/mL, compared to 5.582 ± 0.57 U/mL for the free enzyme. This study offers a novel promising alternative approach to combat the new emerging pathogen C. auris.

Metagenome-derived SusD-homologs affiliated with Bacteroidota bind to synthetic polymers.

Starch utilization system (Sus)D-homologs are well known for their carbohydrate-binding capabilities and are part of the sus operon in microorganisms affiliated with the phylum Bacteroidota. Until now, SusD-like proteins have been characterized regarding their affinity toward natural polymers. In this study, three metagenomic SusD homologs (designated SusD1, SusD38489, and SusD70111) were identified and tested with respect to binding to natural and non-natural polymers. SusD1 and SusD38489 are cellulose-binding modules, while SusD70111 preferentially binds chitin. Employing translational fusion proteins with superfolder GFP (sfGFP), pull-down assays, and surface plasmon resonance (SPR) has provided evidence for binding to polyethylene terephthalate (PET) and other synthetic polymers. Structural analysis suggested that a Trp triad might be involved in protein adsorption. Mutation of these residues to Ala resulted in an impaired adsorption to microcrystalline cellulose (MC), but not so to PET and other synthetic polymers. We believe that the characterized SusDs, alongside the methods and considerations presented in this work, will aid further research regarding bioremediation of plastics. IMPORTANCE: SusD1 and SusD38489 can be considered for further applications regarding their putative adsorption toward fossil-fuel based polymers. This is the first time that SusD homologs from the polysaccharide utilization loci (PUL), largely described for the phylum Bacteroidota, are characterized as synthetic polymer-binding proteins.

Structural and Functional Characterization of New Lipid Transfer Proteins with Chitin-Binding Properties: Insights from Protein Structure Prediction, Molecular Docking, and Antifungal Activity.

Faced with the emergence of multiresistant microorganisms that affect human health, microbial agents have become a serious global threat, affecting human health and plant crops. Antimicrobial peptides have attracted significant attention in research for the development of new microbial control agents. This work's goal was the structural characterization and analysis of antifungal activity of chitin-binding peptides from Capsicum baccatum and Capsicum frutescens seeds on the growth of Candida and Fusarium species. Proteins were initially submitted to extraction in phosphate buffer pH 5.4 and subjected to chitin column chromatography. Posteriorly, two fractions were obtained for each species, Cb-F1 and Cf-F1 and Cb-F2 and Cf-F2, respectively. The Cb-F1 (C. baccatum) and Cf-F1 (C. frutescens) fractions did not bind to the chitin column. The electrophoresis results obtained after chromatography showed two major protein bands between 3.4 and 14.2 kDa for Cb-F2. For Cf-F2, three major bands were identified between 6.5 and 14.2 kDa. One band from each species was subjected to mass spectrometry, and both bands showed similarity to nonspecific lipid transfer protein. Candida albicans and Candida tropicalis had their growth inhibited by Cb-F2. Cf-F2 inhibited the development of C. albicans but did not inhibit the growth of C. tropicalis. Both fractions were unable to inhibit the growth of Fusarium species. The toxicity of the fractions was tested in vivo on Galleria mellonella larvae, and both showed a low toxicity rate at high concentrations. As a result, the fractions have enormous promise for the creation of novel antifungal compounds.

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.

Extraction of chitin-glucan complex from shiitake (Lentinula edodes) fruiting bodies using natural deep eutectic solvents and its prebiotic potential.

Chitin-glucan complex (CGC) is an emerging novel prebiotic with numerous physiological activities in amelioration of clinical manifestations. In the present work, natural deep eutectic solvent (NADES), ultrasonication, and submerged fermentation using probiotic microorganisms were deployed for the extraction of CGC from Shiitake fruiting bodies. CGC obtained through non-ultrasonication assisted fermentation employing Lactiplantibacillus plantarum exhibited maximum polysaccharide yield (27.86 ± 0.82 % w/w). However, based on antioxidant potential, NADES combination of urea: glycerol (1:1 M ratio) was selected for further characterization. The rheological behavior of CGC under optimized conditions showed shear thinning property in both 0.1 M NaCl and salt-free solution. FTIR, 1H-(1D), and 2D 1H1H Homonuclear NMR spectra displayed distinctive patterns associated with ß-glycosidic linkage and ß-d-glucopyranose sugar moiety. XRD profiles of CGC exhibited characteristic peaks at 2θ = 23°, 25°, and 28° with corresponding hkl values of (220), (101), and (130) lattice planes, respectively. Enhanced radical scavenging activities were noticed due to the triple helical structure and anionic nature of CGC. CGC exhibited potential prebiotic activity (prebiotic score 118-134 %) and short chain fatty acids liberation (maximum 9.99 ± 0.41 mM by Lactobacillus delbrueckii). Simulated static in-vitro digestion demonstrated that CGC withstands acidic environment of gastric phase, which indicated its suitability for use as a prebiotic in nutraceutical-enriched food products.

Spectroscopic investigation, dielectric and antimicrobial properties of chitin-cellulose@ZnO/CuO conductive nanocomposites.

In this research, we fabricated a functional conductive nanocomposite with valuable properties through a chitin (CH) and cellulose (CE) polymerization process, incorporating ZnO/(0.1, 0.2, 0.3 mol.%) CuO bioactive nanoparticles. These bioactive nanoparticles, synthesized through sol-gel and polymerization interactions, greatly enhanced the structural, dielectric, and antimicrobial characteristics of CH-CE@ZnO/CuO conductive nanocomposites. The morphological analysis revealed that these nanoparticles, with diameters ranging from 11-25 nm, formed covalent bonds with the membrane matrix, bolstering the conductive nanocomposites ' structural integrity and dielectric performance. The dielectric properties of the conductive nanocomposites were significantly enhanced by the even distribution of ZnO/CuO nanoparticles within the CH-CE composite. Additionally, antimicrobial assessments demonstrated that the CH-CE@ZnO/CuO conductive nanocomposites displayed significant antibacterial properties against the Escherichia coli and Staphylococcus aureus, showcasing their potential as active packaging materials for electronic, biosensors, and sustainable applications.

Extraction of chitin from flammulina velutipes waste: A low-concentration acid pretreatment and aspergillus Niger fermentation approach.

In recent years, with the booming of the edible mushroom industry, chitin production has become increasingly dependent on fungi and other non-traditional sources. Fungal chitin has advantages including superior performance, simpler separation processes, abundant raw materials, and the absence of shellfish allergens. As a kind of edible mushroom, flammulina velutipes (F. velutipes) also has the advantages of wide source and large annual yield. This provided the possibility for the extraction of chitin. Here, a procedure to extract chitin from F. velutipes waste be presented. This method comprises low-concentration acid pretreatment coupled with consolidated bioprocessing with Aspergillus niger. Characterization by SEM, FTIR, XRD, NMR, and TGA confirmed that the extracted chitin was ß-chitin. To achieve optimal fermentation of F. velutipes waste (80 g/L), ammonium sulfate and glucose were selected as nitrogen and carbon sources (5 g/L), with a fermentation time of 5 days. The extracted chitin could be further deacetylated and purified to obtain high-purity chitosan (99.2 % ± 1.07 %). This chitosan exhibited a wide degree of deacetylation (50.0 % ± 1.33 % - 92.1 % ± 0.97 %) and a molecular weight distribution of 92-192 kDa. Notably, the yield of chitosan extracted in this study was increased by 56.3 % ± 0.47 % compared to the traditional chemical extraction method.

α-Chitosan and ß-Oligochitosan Mixtures-Based Formula for In Vitro Assessment of Melanocyte Cells Response.

Chitosan is a natural polymer with numerous biomedical applications. The cellular activity of chitosan has been studied in various types of cancer, including melanoma, and indicates that these molecules can open new perspectives on antiproliferative action and anticancer therapy. This study analyzes how different chitosan conformations, such as α-chitosan (CH) or ß-oligochitosan (CO), with various degrees of deacetylation (DDA) and molar mass (MM), both in different concentrations and in CH-CO mixtures, influence the cellular processes of SK-MEL-28 melanocytes, to estimate the reactivity of these cells to the applied treatments. The in vitro evaluation was carried out, aiming at the cellular metabolism (MTT assay), cellular morphology, and chitinase-like glycoprotein YKL-40 expression. The in vitro effect of the CH-CO mixture application on melanocytes is obvious at low concentrations of α-chitosan/ß-oligochitosan (1:2 ratio), with the cell's response supporting the hypothesis that ß-oligo-chitosan amplifies the effect. This oligochitosan mixture, favored by the ß conformation and its small size, penetrates faster into the cells, being more reactive when interacting with some cellular components. Morphological effects expressed by the loss of cell adhesion and the depletion of YKL-40 synthesis are significant responses of melanocytes. ß-oligochitosan (1.5 kDa) induces an extension of cytophysiological effects and limits the cell viability compared to α-chitosan (400-900 kDa). Statistical analysis using multivariate techniques showed differences between the CH samples and CH-CO mixtures.

Marine Bacillus haynesii chitinase: Purification, characterization and antifungal potential for sustainable chitin bioconversion.

The development of chitinase tailored for the bioconversion of chitin to chitin oligosaccharides has attracted significant attention due to its potential to alleviate environmental pollution associated with chemical conversion processes. In this present investigation, we purified extracellular chitinase derived from marine Bacillus haynesii to homogeneity and subsequently characterized it. The molecular weight of BhChi was approximately 35 kDa. BhChi displayed its peak catalytic activity at pH 6.0, with an optimal temperature of 37 °C. It exhibited stability across a pH range of 6.0-9.0. In addition, BhChi showed activation in the presence of Mn2+ with the improved activity of 105 U mL-1. Ca2+ and Fe2+ metal ions did not have any significant impact on enzyme activity. Under the optimized enzymatic conditions, there was a notable enhancement in catalytic activity on colloidal chitin with Km of 0.01 mg mL-1 and Vmax of 5.75 mmol min-1. Kcat and catalytic efficiency were measured at 1.91 s-1 and 191 mL mg-1 s-1, respectively. The product profiling of BhChi using thin layer chromatography and Mass spectrometric techniques hinted an exochitinase mode of action with chitobiose and N-Acetyl glucosamine as the products. This study represents the first report on an exochitinase from Bacillus haynesii. Furthermore, the chitinase showcased promising antifungal properties against key pathogens, Fusarium oxysporum and Penicillium chrysogenum, reinforcing its potential as a potent biocontrol agent.

A Peritrophin-44 gene in Litopenaeus vannamei is involved in disease resistance.

Peritrophic membrane (PM) is a pellicle structure present in the midgut of some invertebrates, such as insects and crustaceans. It could isolate harmful components and pathogens in food from intestinal epithelial cells; and it also plays a role in improving digestion and absorption efficiency. So PM is important for survival of its owner. In current study, 44 PM proteins were identified in Litopenaeus vannamei by PM proteome analysis. Among these PM proteins, the Peritrophin-44 homologous protein (LvPT44) was further studied. Chitin-binding assay indicated that LvPT44 could bind to colloidal chitin, and immunoeletron microscopy analysis shown that it was located to PM of L. vannamei. Furthermore, LvPT44 promoter was found to be activated by L. vannamei STAT and c-Jun. Besides, LvPT44 was induced by ER-stress as well as white spot syndrome virus infection. Knocked-down expression of LvPT44 by RNA inference increased the cumulative mortality of shrimp that caused by ER-stress or white spot syndrome virus. These results suggested that LvPT44 has an important role in disease resistance.

Dissecting the manipulation of lufenuron on chitin synthesis in Helicoverpa armigera.

Lufenuron, a benzoylurea chitin synthesis inhibitor, is effective against many insect pests. However, the insecticidal activity of lufenuron has not been completely elucidated, nor has its disturbing effect on chitin synthesis genes. In this study, bioassay results demonstrated an outstanding toxicity of lufenuron against Helicoverpa armigera larvae. The treated larvae died from abortive molting and metamorphosis defects, and severe separation of epidermis and subcutaneous tissues was observed. Treatment of 3rd- and 4th-instar larvae with LC25 lufenuron significantly extended the duration of larval and pupal stage, reduced the rates of pupation and emergence, and adversely affected pupal weight. Besides, lufenuron can severely reduce chitin content in larval integument, and the lufenuron-treated larvae showed reduced trehalose content in their hemolymph. Further analysis using RNA sequencing revealed that five chitin synthesis genes were down-regulated, whereas the expressions of two chitin degradation genes were significantly enhanced. Knockdown of chitin synthase 1 (HaCHS1), uridine diphosphate-N-acetylglucosamine-pyrophosphorylase (HaUAP), phosphoacetyl glucosamine mutase (HaPGM), and glucosamine 6-phosphate N-acetyl-transferase (HaGNPAT) in H. armigera led to significant increase in larval susceptibilities to LC25 lufenuron by 75.48%, 65.00%, 68.42% and 28.00%, respectively. Our findings therefore revealed the adverse effects of sublethal doses of lufenuron on the development of H. armigera larvae, elucidated the perturbations on chitin metabolism, and proved that the combination of RNAi and lufenuron would improve the control effect of this pest.

Exposure to teflubenzuron reduces drought tolerance of collembolans.

Chitin synthesis inhibitors (CSIs) are commonly used insecticides compromising cuticle formation and structure in arthropods. Arthropods rely on intact cuticles to maintain water balance and cellular homeostasis to survive in different weather conditions. We hypothesized that physiological impacts of CSIs may make arthropods more vulnerable to harsh environmental conditions, such as extreme heat, cold or drought. The aim of this study was to investigate if pre-exposure to teflubenzuron (a common CSI) would influence Folsomia candida's (Collembola: Isotomidae) sensitivity to natural stressors. Here, we exposed adult collembolans to teflubenzuron through food for two weeks, then survivors were immediately divided into three groups for subsequent acute heat, cold, and drought exposure. After acute exposure to these natural stressors, the collembolans were moved to optimal conditions for a one-week recovery period during which their survival, time to regain reproduction, and egg production were examined. We analyzed the interaction between effects of teflubenzuron and natural stressors using a multiplicative model. No interaction between effects of teflubenzuron and heat was observed in any test endpoints. A synergistic interaction between effects of teflubenzuron and cold was observed in the time to regain reproduction. Both survival and egg production, on the other hand, showed synergistic interaction between effects of teflubenzuron and drought, as well as a tendency for longer reproduction recovery times. Our results suggest that pre-exposure to teflubenzuron reduces drought tolerance in F. candida, while its impact on heat or cold tolerance is minor or absent. This study is among the first to explore the combined effects of CSI and natural stressors on soil arthropods, providing more insight on potential risks posed by such chemicals in the environment.

Low protein digestibility-corrected amino acid score and net nitrogen-to-protein conversion factor value of edible insects.

The amount of chitin, a nitrogen-containing dietary fiber, in edible insects can mislead the exact nitrogen-to-protein conversion factor (NPF) and true protein content. We determined the amino acid score (AAS), protein digestibility-corrected AAS (PDCAAS), chitin content, and net NPF of five edible insects. Additionally, the effect of the amino acid composition of migratory locust on rat growth were investigated. The AAS of the insects were ranged from 63 to 94. The chitin contents were ranged from 1.6 g/100 g to 10.7 g/100 g. The PDCAAS, calculated by AAS and gut-intestinal digestibility, ranged from 44 to 81, which was lower than casein (97). The net NPF ranged from 4.93 to 5.76, which were lower than the conventional value. Dietary migratory locust, whose PDCAAS was the lowest, decreased growth and altered lipid metabolism. Therefore, a lower PDCAAS and overestimation of net NPF of insects can affect the true protein calculations and growth.

Structure information analysis and relative content determination of protein and chitin from yellow mealworm larvae using Raman spectroscopy.

Insect protein extract is one of the high-quality protein sources and is frequently viewed as a potential nutrition alternative. However, a more precise method for protein measurement is still needed due to protein overestimation by the Kjeldahl method due to the presence of a large amount of chitin in insects. Therefore, we demonstrated the monitoring of chitin and protein extracted from yellow mealworm larvae through the information on molecular vibration obtained using Raman spectroscopy and infrared (IR) spectroscopy. The NH vibration at 3475 cm-1 is the characteristic peak of chitin in defatted product observed in the Raman spectra. The nitrogen-to-protein conversion factor in protein extracted from larvae by the Raman method was determined based on the NH vibration and found to be 5.66 ± 0.01. We also compared these experimental data to theoretical Raman and IR spectra and determined the possible reasons for why nitrogen elements in chitin affect the determination of protein content. The method of sequentially removing fat and protein could provide more accurate quantification of protein and chitin. Raman spectroscopy is feasible for various types of insects with high chitin content. Compared with the Kjeldahl method, the Raman method is a faster and more accurate measurement method. Moreover, it provides the content of impurities, purity, and structural information.

Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase.

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high-resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.

Immobilized chitinase as effective biocatalytic platform for producing bioactive di-N-acetyl chitobiose from recycled chitin food waste.

Described is chitinase immobilization on magnetic nanoparticles (MNPs) as biocompatible support for enzymatic production of di-N-acetyl chitobiose from chitin waste. Chitinase immobilization was feasible with an immobilization yield of 88.9 ± 1.6 % with 97.8 ± 1.0 % retention of activity and compared to free enzyme work, immobilization conferred better thermal and storage stability. As practical benefit the attachment to magnetic nanocarriers enabled easy enzyme recovery after repeated application runs and thus sustainable reuse. In fixed state chitinase retained a remarkable 39.7 ± 2.6 % of the starting activity after 16 reaction cycles. Furthermore, immobilized chitinase showed higher catalytic activity than free chitinase in converting shrimp shells and squid-pens chitins into di-N-acetyl chitobiose in a single-step reaction. The final yield of purified compound was 37.0 ± 1.2 % from shrimp shells and 61.1 ± 0.5 % from squid-pens chitin. In conclusion, an efficient MNP-based chitinase immobilization system with the potential for large-scale production was developed.

Heterologous Expression and Characterization of a pH-Stable Chitinase from Micromonospora aurantiaca with a Potential Application in Chitin Degradation.

To promote the bioconversion of marine chitin waste into value-added products, we expressed a novel pH-stable Micromonospora aurantiaca-derived chitinase, MaChi1, in Escherichia coli and subsequently purified, characterized, and evaluated it for its chitin-converting capacity. Our results indicated that MaChi1 is of the glycoside hydrolase (GH) family 18 with a molecular weight of approximately 57 kDa, consisting of a GH18 catalytic domain and a cellulose-binding domain. We recorded its optimal activity at pH 5.0 and 55 °C. It exhibited excellent stability in a wide pH range of 3.0-10.0. Mg2+ (5 mM), and dithiothreitol (10 mM) significantly promoted MaChi1 activity. MaChi1 exhibited broad substrate specificity and hydrolyzed chitin, chitosan, cellulose, soluble starch, and N-acetyl chitooligosaccharides with polymerization degrees ranging from three to six. Moreover, MaChi1 exhibited an endo-type cleavage pattern, and it could efficiently convert colloidal chitin into N-acetyl-D-glucosamine (GlcNAc) and (GlcNAc)2 with yields of 227.2 and 505.9 mg/g chitin, respectively. Its high chitin-degrading capacity and exceptional pH tolerance makes it a promising tool with potential applications in chitin waste treatment and bioactive oligosaccharide production.

Hierarchical structure and chemical composition of complementary segments of the fruiting bodies of Fomes fomentarius fungi fine-tune the compressive properties.

Humanity is often fascinated by structures and materials developed by Nature. While structural materials such as wood have been widely studied, the structural and mechanical properties of fungi are still largely unknown. One of the structurally interesting fungi is the polypore Fomes fomentarius. The present study deals with the investigation of the light but robust fruiting body of F. fomentarius. The four segments of the fruiting body (crust, trama, hymenium, and mycelial core) were examined. The comprehensive analysis included structural, chemical, and mechanical characterization with particular attention to cell wall composition, such as chitin/chitosan and glucan content, degree of deacetylation, and distribution of trace elements. The hymenium exhibited the best mechanical properties even though having the highest porosity. Our results suggest that this outstanding strength is due to the high proportion of skeletal hyphae and the highest chitin/chitosan content in the cell wall, next to its honeycomb structure. In addition, an increased calcium content was found in the hymenium and crust, and the presence of calcium oxalate crystals was confirmed by SEM-EDX. Interestingly, layers with different densities as well as layers of varying calcium and potassium depletion were found in the crust. Our results show the importance of considering the different structural and compositional characteristics of the segments when developing fungal-inspired materials and products. Moreover, the porous yet robust structure of hymenium is a promising blueprint for the development of advanced smart materials.