Arabinoxylan (Wheat Flour; Medium Viscosity)

endo-1,4-β-Xylanase (EC 3.2.1.8) is employed across a broad range of industries including animal feed, brewing, baking, biofuels, detergents and pulp (paper). Despite its importance, a rapid, reliable, reproducible, automatable assay for this enzyme that is based on the use of a chemically defined substrate has not been described to date. Reported herein is a new enzyme coupled assay procedure, termed the XylX6 assay, that employs a novel substrate, namely 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-4⁵-O-glucosyl-xylopentaoside. The development of the substrate and associated assay is discussed here and the relationship between the activity values obtained with the XylX6 assay versus traditional reducing sugar assays and its specificity and reproducibility were thoroughly investigated.

A range of α-L-arabinofuranosyl-(1-4)-β-D-xylo-oligosaccharides (AXOS) were produced by hydrolysis of wheat flour arabinoxylan (WAX) and acid debranched arabinoxylan (ADWAX), in the presence and absence of an AXH-d3 α-L-arabinofuranosidase, by several GH10 and GH11 β-xylanases. The structures of the oligosaccharides were characterised by GC-MS and NMR and by hydrolysis by a range of α-L-arabinofuranosidases and β-xylosidase. The AXOS were purified and used to characterise the action patterns of the specific α-L-arabinofuranosidases. These enzymes, in combination with either Cellvibrio mixtus or Neocallimastix patriciarum β -xylanase, were used to produce elevated levels of specific AXOS on hydrolysis of WAX, such as 3²-α-L-Araf-(1-4)-β-D-xylobiose (A³X), 2³-α-L-Araf-(1-4)-β-D-xylotriose (A²XX), 3³-α-L-Araf-(1-4)-β-D-xylotriose (A³XX), 2²-α-L-Araf-(1-4)-β-D-xylotriose (XA²X), 3²-α-L-Araf (1-4)-β-D-xylotriose (XA³X), 2³-α-L-Araf-(1-4)-β-D-xylotetraose (XA²XX), 3³-α-L-Araf-(1-4)-β-D-xylotetraose (XA³XX), 2³ ,3³-di-α-L-Araf-(1-4)-β-D-xylotriose (A²⁺³XX), 2³,3³-di-α-L-Araf-(1-4)-β-D-xylotetraose (XA²⁺³XX), 2⁴,3⁴-di-α-L-Araf-(1-4)-β-D-xylopentaose (XA²⁺³XXX) and 3³,3⁴-di-α-L-Araf-(1-4)-β-D-xylopentaose (XA³A³XX), many of which have not previously been produced in sufficient quantities to allow their use as substrates in further enzymic studies. For A^2,3XX, yields of approximately 16% of the starting material (wheat arabinoxylan) have been achieved. Mixtures of the α-L-arabinofuranosidases, with specific action on AXOS, have been combined with β-xylosidase and β-xylanase to obtain an optimal mixture for hydrolysis of arabinoxylan to L-arabinose and D-xylose.

To achieve sustainable environmental development, various biodegradable films have been designed to replace plastic films. The cell walls of the wheat bran aleurone layers primarily comprise alternately overlapping arabinoxylan (AX), β-glucan (BG) and small quantities of cellulose and ferulic acid. The differences in their composition can affect their mechanical strength and hydration. Therefore, multilayer films of AX, BG, cellulose nanofibers (CNFs), and free ferulic acid were prepared to simulate the cell walls of wheat aleurone layers, and the effect of different CNF contents on cell walls characteristics was investigated. The microstructure, physical properties, and hydration characteristics of multilayer films were studied using scanning electron microscopy, time-domain nuclear magnetic resonance, and thermogravimetric analysis. The AX/BG/8%CNF multilayer films possessed excellent thermal stability and mechanical properties along with slow moisture diffusion and flowability. When the CNF content within the films was 8%, the polymer composite structure hindered the diffusion of moisture. Inspired by the biological effects of wheat grain cell walls, cellulose multilayer films were used for blueberries preservation, and the decay rate, weight loss index, and respiration rate of blueberries were found to decrease significantly.

Carbohydrate-binding module (CBM) family 91 is a novel module primarily associated with glycoside hydrolase (GH) family 43 enzymes. However, our current understanding of its function remains limited. PphXyl43B is a β-xylosidase/α-L-arabinofuranosidase bifunctional enzyme from physcomitrellae patens XB belonging to the GH43_11 subfamily and containing CBM91 at its C terminus. To fully elucidate the contributions of the CBM91 module, the truncated proteins consisting only the GH43_11 catalytic module (rPphXyl43B-dCBM91) and only the CBM91 module (rCBM91) of PphXyl43B were constructed, respectively. The result showed that rPphXyl43B-dCBM91 completely lost hydrolysis activity against both p-nitrophenyl-β-D-xylopyranoside and p-nitrophenyl-α-L-arabinofuranoside; it also exhibited significantly reduced activity towards xylobiose, xylotriose, oat spelt xylan and corncob xylan compared to the control. Thus, the CBM91 module is crucial for the β-xylosidase/α-L-arabinofuranosidase activities in PphXyl43B. However, rCBM91 did not exhibit any binding capability towards corncob xylan. Structural analysis indicated that CBM91 of PphXyl43B might adopt a loop conformation (residues 496–511: ILSDDYVVQSYGGFFT) to actively contribute to the catalytic pocket formation rather than substrate binding capability. This study provides important insights into understanding the function of CBM91 and can be used as a reference for analyzing the action mechanism of GH43_11 enzymes and their application in biomass energy conversion.

Wood-degrading white-rot fungi can efficiently degrade all plant biomass components, but the molecular mechanisms behind the degradation of plant polysaccharides remain poorly understood. For example, the gene sets and expression levels induced by the plant polysaccharide-derived monosaccharides in white-rot fungi do not reflect those induced by crude plant biomass substrates. To explore the molecular response of the white-rot fungus Dichomitus squalens to plant-derived oligo- and polysaccharides, we investigated the transcriptomes from mono- and dikaryotic strains of the fungus on 10 substrates and compared the expression of carbohydrate-active enzyme-encoding genes to that previously reported for different monosaccharides and cellobiose. Our results revealed that in D. squalens, a robust response to cellulose leads to its effective depolymerization, with an orthologue of the ascomycete Trichoderma reesei ACE3 likely acting as a central transcriptional regulator. The conserved response between cellulose and cellobiose further confirms cellobiose as the main cellulase inducer in D. squalens. Surprisingly, despite low abundance of pectin in the natural wood substrate of D. squalens, we identified polygalacturonic acid as a major inducer of a broad-targeted pectinolytic response including pectinase, pectin-related sugar transporter and catabolism genes, and four fungal specific transcription factors. This indicates that D. squalens has not only maintained its ability to degrade minor polysaccharide components in its biotope, but also a regulatory system spanning from extracellular degradation to metabolic conversion. Our study contributes to a deeper understanding of the molecular mechanisms behind white-rot fungal plant polysaccharide degradation and provides leads for functional studies of potential transcriptional regulators in basidiomycetes.

Lignocellulosic biomass holds promise as a renewable feedstock for various applications, but its efficient conversion requires cost-effective degradation strategies. The main objective of this study was to investigate the effect of the growth conditions of Cellulomonas phragmiteti in the production of (hemi)cellulosic supernatants. To meet this aim, different lignocellulosic residues were used as carbon sources for growth using defined mineral or nutritive culture media. Cell-free culture supernatants with xylanolytic activity were produced in all the conditions evaluated, but the highest xylanase activity (15.3 U/mL) was achieved in Luria-Bertani (LB) medium containing 1% waste paper. Under these conditions, almost negligible β-glucosidase, cellobiohydrolase, β-xylosidase, and α-arabinofuranosidase activity was detected. The xylanolytic supernatant showed tolerance to salt and displayed maximal catalytic efficiency at pH 6 and 45°C, along with good activity in the ranges of 45-55°C and pH 5-8. As it showed good stability at 45°C, the supernatant was employed for the hydrolysis of birchwood xylan (50 g/L) under optimal conditions, releasing 10.7 g/L xylose in 72 h. Thus, C. phragmiteti was found to produce a xylanolytic enzymatic supernatant efficiently by utilizing the cheap and abundant lignocellulosic residue of waste paper, and the produced supernatant has promising attributes for industrial applications.

Lignocellulose biomasses (LCB), including spent mushroom substrate (SMS), pose environmental challenges if not properly managed. At the same time, these renewable resources hold immense potential for biofuel and chemicals production. With the mushroom market growth expected to amplify SMS quantities, repurposing or disposal strategies are critical. This study explores the use of SMS for cultivating microbial communities to produce carbohydrate-active enzymes (CAZymes). Addressing a research gap in using anaerobic digesters for enriching microbiomes feeding on SMS, this study investigates microbial diversity and secreted CAZymes under varied temperatures (37°C, 50°C, and 70°C) and substrates (SMS as well as pure carboxymethylcellulose, and xylan). Enriched microbiomes demonstrated temperature-dependent preferences for cellulose, hemicellulose, and lignin degradation, supported by thermal and elemental analyses. Enzyme assays confirmed lignocellulolytic enzyme secretion correlating with substrate degradation trends. Notably, thermogravimetric analysis (TGA), coupled with differential scanning calorimetry (TGA-DSC), emerged as a rapid approach for saccharification potential determination of LCB. Microbiomes isolated at mesophilic temperature secreted thermophilic hemicellulases exhibiting robust stability and superior enzymatic activity compared to commercial enzymes, aligning with biorefinery conditions. PCR-DGGE and metagenomic analyses showcased dynamic shifts in microbiome composition and functional potential based on environmental conditions, impacting CAZyme abundance and diversity. The meta-functional analysis emphasised the role of CAZymes in biomass transformation, indicating microbial strategies for lignocellulose degradation. Temperature and substrate specificity influenced the degradative potential, highlighting the complexity of environmental–microbial interactions. This study demonstrates a temperature-driven microbial selection for lignocellulose degradation, unveiling thermophilic xylanases with industrial promise. Insights gained contribute to optimizing enzyme production and formulating efficient biomass conversion strategies. Understanding microbial consortia responses to temperature and substrate variations elucidates bioconversion dynamics, emphasizing tailored strategies for harnessing their biotechnological potential.

Despite substantial lignocellulose conversion during mycelial growth, previous transcriptome and proteome studies have not yet revealed how secretomes from the edible mushroom Agaricus bisporus develop and whether they modify lignin models in vitro. To clarify these aspects, A. bisporus secretomes collected throughout a 15-day industrial substrate production and from axenic lab-cultures were subjected to proteomics, and tested on polysaccharides and lignin models. Secretomes (day 6-15) comprised A. bisporus endo-acting and substituent-removing glycoside hydrolases, whereas β-xylosidase and glucosidase activities gradually decreased. Laccases appeared from day 6 onwards. From day 10 onwards, many oxidoreductases were found, with numerous multicopper oxidases (MCO), aryl alcohol oxidases (AAO), glyoxal oxidases (GLOX), a manganese peroxidase (MnP), and unspecific peroxygenases (UPO). Secretomes modified dimeric lignin models, thereby catalyzing syringylglycerol-β-guaiacyl ether (SBG) cleavage, guaiacylglycerol-β-guaiacyl ether (GBG) polymerization, and non-phenolic veratrylglycerol-β-guaiacyl ether (VBG) oxidation. We explored A. bisporus secretomes and insights obtained can help to better understand biomass valorization.

A series of polymers, including chitosan (CS), carboxymethylcellulose (CMC) and a chitosan–gelatin (CS–GEL) hybrid polymer, were functionalized with ferulic acid (FA) derived from the enzymatic treatment of arabinoxylan through the synergistic action of two enzymes, namely, xylanase and feruloyl esterase. Subsequently, the ferulic acid served as the substrate for laccase from Agaricus bisporus (AbL) in order to enzymatically functionalize the above-mentioned polymers. The successful grafting of the oxidized ferulic acid products onto the different polymers was confirmed through ultraviolet–visible (UV–Vis) spectroscopy, attenuated total reflectance (ATR) spectroscopy, scanning electron microscopy (SEM) and nuclear magnetic resonance (NMR) spectroscopy. Additionally, an enhancement of the antioxidant properties of the functionalized polymers was observed according to the DDPH and ABTS protocols. Finally, the modified polymers exhibited strong antimicrobial activity against bacterial populations of Escherichia coli BL21DE3 strain, suggesting their potential application in pharmaceutical, cosmeceutical and food industries.

Endo-type chitinase is the principal enzyme involved in the breakdown of N-acetyl-d-glucosamine-based oligomeric and polymeric materials through hydrolysis. The gene (966-bp) encoding a novel endo-type chitinase (ChiJ), which is comprised of an N-terminal chitin-binding domain type 3 and a C-terminal catalytic glycoside hydrolase family 19 domain, was identified from a fibrolytic intestinal symbiont of the earthworm Eisenia fetida, Cellulosimicrobium funkei HY-13. The highest endochitinase activity of the recombinant enzyme (rChiJ: 30.0 kDa) toward colloidal shrimp shell chitin was found at pH 5.5 and 55 °C and was considerably stable in a wide pH range (3.5–11.0). The enzyme exhibited the highest biocatalytic activity (338.8 U/mg) toward ethylene glycol chitin, preferentially degrading chitin polymers in the following order: ethylene glycol chitin > colloidal shrimp shell chitin > colloidal crab shell chitin. The enzymatic hydrolysis of N-acetyl-β-d-chitooligosaccharides with a degree of polymerization from two to six and colloidal shrimp shell chitin yielded primarily N,N′-diacetyl-β-d-chitobiose together with a small amount of N-acetyl-d-glucosamine. The high chitin-degrading ability of inverting rChiJ with broad pH stability suggests that it can be exploited as a suitable biocatalyst for the preparation of N,N′-diacetyl-β-d-chitobiose, which has been shown to alleviate metabolic dysfunction associated with type 2 diabetes.