Xylan (Beechwood)

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.

The most commonly used method for the measurement of the level of endo-xylanase in commercial enzyme preparations is the 3,5-dinitrosalicylic acid (DNS) reducing sugar method with birchwood xylan as substrate. It is well known that with the DNS method, much higher enzyme activity values are obtained than with the Nelson-Somogyi (NS) reducing sugar method. In this paper, we have compared the DNS and NS reducing sugar assays using a range of xylan-type substrates and accurately compared the molar response factors for xylose and a range of xylo-oligosaccharides. Purified beechwood xylan or wheat arabinoxylan is shown to be a suitable replacement for birchwood xylan which is no longer commercially available, and it is clearly demonstrated that the DNS method grossly overestimates endo-xylanase activity. Unlike the DNS assay, the NS assay gave the equivalent colour response with equimolar amounts of xylose, xylobiose, xylotriose and xylotetraose demonstrating that it accurately measures the quantity of glycosidic bonds cleaved by the endo-xylanase. The authors strongly recommend cessation of the use of the DNS assay for measurement of endo-xylanase due to the fact that the values obtained are grossly overestimated due to secondary reactions in colour development.

Background: A putative glycosyl hydrolase gene biof1_09 was identified from a metagenomic fosmid library of local biofertilizers in previous report [1]. The gene is renamed as gh43kk in this study. Methods: The gene gh43kk, encoding a putative β-D-xylosidase was amplified by polymerase chain reaction (PCR) and successfully cloned and expressed in Escherichia coli. The expressed recombinant protein was purified by metal affinity chromatography. Its properties were initially verified by enzyme assay and thin layer chromatography (TLC). Results: The purified recombinant protein showed the highest catalytic activities at acidic pH 4 and 50°C toward beechwood xylan, followed by carboxymethylcellulose (CMC). TLC analysis indicated a release of xylose and glucose when xylan and CMC were treated with Gh43kk protein, respectively, whereas glucose and cellobiose were detected when avicel, cellulose and filter paper were used as substrates, suggesting its dual function as xylanase with cellulase activity. The enzyme indicated great stability in a temperature between 10 to 50°C and a wide range of pH from 4 to 8. Enzyme activity of Gh43kk was enhanced in the presence of magnesium and manganese ions, while calcium ions, Ethylenediaminetetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS) inhibited the enzyme activity. Conclusion: These results suggest that Gh43kk could be a potential candidate for application in various bioconversion processes.

In the bio-based era, cellulolytic and hemicellulolytic enzymes are biocatalysts used in many industrial processes, playing a key role in the conversion of recalcitrant lignocellulosic waste biomasses. In this context, many thermophilic microorganisms are considered as convenient sources of carbohydrate-active enzymes (CAZymes). In this work, a functional genomic annotation of Alicyclobacillus mali FL18, a recently discovered thermo-acidophilic microorganism, showed a wide reservoir of putative CAZymes. Among them, a novel enzyme belonging to the family 9 of glycosyl hydrolases (GHs), named AmCel9, was identified; in-depth in silico analyses highlighted that AmCel9 shares general features with other GH9 members. The synthetic gene was expressed in Escherichia coli and the recombinant protein was purified and characterized. The monomeric enzyme has an optimal catalytic activity at pH 6.0 and has comparable activity at temperatures ranging from 40°C to 70°C. It also has a broad substrate specificity, a typical behavior of multifunctional cellulases; the best activity is displayed on β-1,4 linked glucans. Very interestingly, AmCel9 also hydrolyses filter paper and microcrystalline cellulose. This work gives new insights into the properties of a new thermophilic multifunctional GH9 enzyme, that looks a promising biocatalyst for the deconstruction of lignocellulose.

Corncobs of four different corn varieties were physically segregated into two different anatomical portions, namely the corncob outer (CO) and corncob pith (CP). The biomass composition analysis of both the CO and CP was performed by four different methods. The CP showed a higher carbohydrate and lower lignin content (83.32% and 13.58%, respectively) compared with the CO (79.93% and 17.12%, respectively) in all of the methods. The syringyl/guaiacyl (S/G) ratio was observed to be higher in the CP (1.34) than in the CO (1.28). The comprehensive physical characterization of both samples substantiated the lower crystallinity and lower thermal stability that was observed in the CP compared to the CO. These properties make the CP more susceptible to glycanases, as evident from the enzymatic saccharification of CP carried out with a commercial cellulase and xylanase in this work. The yields obtained were 70.57% and 88.70% of the respective theoretical yields and were found to be equal to that of pure cellulose and xylan substrates. These results support the feasibility of the tailored valorization of corncob anatomical portions, such as enzymatic production of xylooligosaccharides from CP without pretreatment combined with the bioethanol production from pretreated CO to achieve an economical biorefinery output from corncob feedstock.

A gene construct encoding a xylanase, which is active in extreme conditions of temperature and alkaline pH (90°C, pH 10.5), has been transitorily expressed with high efficiency in Nicotiana benthamiana using a viral vector. The enzyme, targeted to the apoplast, accumulates in large amounts in plant tissues in as little as 7 days after inoculation, without detrimental effects on plant growth. The properties of the protein produced by the plant, in terms of resistance to temperature, pH, and enzymatic activity, are equivalent to those observed when Escherichia coli is used as a host. Purification of the plant-produced recombinant xylanase is facilitated by exporting the protein to the apoplastic space. The production of this xylanase by N. benthamiana, which avoids the hindrances derived from the use of E. coli, namely, intracellular production requiring subsequent purification, represents an important step for potential applications in the food industry in which more sustainable and green products are continuously demanded. As an example, the use of the enzyme producing prebiotic xylooligosdaccharides from xylan is here reported.

The immune system produces a diverse collection of antiglycan antibodies that are critical for host defense. At present, however, we know very little about the binding properties, origins, and sequences of these antibodies because of a lack of access to a variety of defined individual antibodies. To address this challenge, we used a glycan microarray with over 800 different components to screen a panel of 516 human monoclonal antibodies that had been randomly cloned from different B-cell subsets originating from healthy human subjects. We obtained 26 antiglycan antibodies, most of which bound microbial carbohydrates. The majority of the antiglycan antibodies identified in the screen displayed selective binding for specific glycan motifs on our array and lacked polyreactivity. We found that antiglycan antibodies were about twice as likely than expected to originate from IgG⁺ memory B cells, whereas none were isolated from naïve, early emigrant, or immature B cells. Therefore, our results indicate that certain B-cell subsets in our panel are enriched in antiglycan antibodies, and IgG⁺ memory B cells may be a promising source of such antibodies. Furthermore, some of the newly identified antibodies bound glycans for which there are no reported monoclonal antibodies available, and these may be useful as research tools, diagnostics, or therapeutic agents. Overall, the results provide insight into the types and properties of antiglycan antibodies produced by the human immune system and a framework for the identification of novel antiglycan antibodies in the future.

Acidic xylooligosaccharides (XOS), also called aldouronics, are hetero-oligomers of xylose randomly branched with 4-O-methyl-D-glucuronic acid residues linked by α(1 → 2) bonds, which display bioactive properties. We have developed a simple and integrated method for the production of acidic XOS by enzymatic hydrolysis of a glucurono-xylan from beechwood. Among the enzymes screened, Depol 670L (a cellulolytic preparation from Trichoderma reesei) displayed the highest activity (70.3 U/mL, expressed in reducing xylose equivalents). High-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) analysis revealed the formation of a neutral fraction (corresponding to linear XOS, mainly xylose and xylobiose) and a group of more retained products (acidic XOS), which were separated using strong anion-exchange cartridges. The acidic fraction contained a major product, characterized by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and mono- and two-dimensional nuclear magnetic resonance spectroscopy (NMR) as 2′-O-α-(4-O-methyl-α-D-glucuronosyl)-xylobiose (X2_MeGlcA). Starting from 2 g of beechwood xylan, 1.5 g of total XOS were obtained, from which 225 mg (11% yield) corresponded to the aldouronic X2_MeGlcA. The acidic XOS exhibited higher antioxidant activity (measured by the ABTS^·+ discoloration assay) than xylan, whilst neutral XOS displayed no antioxidant activity. This work demonstrates that it is possible to obtain a safe and natural antioxidant by enzymatic biotransformation of hardwood hemicellulose.

Xylan is a biopolymer readily available from forest resources. Various modification methods, including oxidation with sodium periodate, have been shown to facilitate the engineering applications of xylan. However, modification procedures are often optimized for semicrystalline high molecular weight polysaccharide cellulose rather than for lower molecular weight and amorphous polysaccharide xylan. This paper elucidates the procedure for the periodate oxidation of xylan into dialdehyde xylan and its further reduction into a dialcohol form and is focused on the modification work up. The oxidation-reduction reaction decreased the molecular weight of xylan while increased the dispersity more than 50%. Unlike the unmodified xylan, all the modified grades could be solubilized in water, which we see essential for facilitating the future engineering applications of xylan. The selection of quenching and purification procedures and pH-adjustment of the reduction step had no significant effect on the degree of oxidation, molecular weight and only a minor effect on the hydrodynamic radius in water. Hence, it is possible to choose the simplest oxidation-reduction route without time consuming purification steps within the sequence.

The lignocellulosic enzymes of Trichoderma asperellum have been intensely investigated toward efficient conversion of biomass into high-value chemicals/industrial products. However, lack of genome data is a remarkable hurdle for hydrolase systems studies. The secretory enzymes of newly isolated T. asperellum ND-1 during lignocellulose degradation are currently poorly known. Herein, a high-quality genomic sequence of ND-1, obtained by both Illumina HiSeq 2000 sequencing platforms and PacBio single-molecule real-time, has an assembly size of 35.75 Mb comprising 10,541 predicted genes. Secretome analysis showed that 895 proteins were detected, with 211 proteins associated with carbohydrate-active enzymes (CAZymes) responsible for biomass hydrolysis. Additionally, T. asperellum ND-1, T. atroviride IMI 206040, and T. virens Gv-298 shared 801 orthologues that were not identified in T. reesei QM6a, indicating that ND-1 may play critical roles in biological-control. In-depth analysis suggested that, compared with QM6a, the genome of ND-1 encoded a unique enzymatic system, especially hemicellulases and chitinases. Moreover, after comparative analysis of lignocellulase activities of ND-1 and other fungi, we found that ND-1 displayed higher hemicellulases (particularly xylanases) and comparable cellulases activities. Our analysis, combined with the whole-genome sequence information, offers a platform for designing advanced T. asperellum ND-1 strains for industrial utilizations, such as bioenergy production.

Second-generation biofuel production strategies require utilization of the largest possible fraction of the available sugars in renewable biomass. Xylose can make up as much as 35% of plant dry cell weight (DCW), primarily in the form of the hemicellulose polymer xylan. Xylo-oligosaccharides (XOS) are breakdown products of xylan released during pre-treatment and hydrolysis that may inhibit the fermentation process. To enable growth on xylan and the removal of inhibitory XOS, CRISPR-Cas9 was used to confer xylanase and GH43 xylosidase activity to xylose-assimilating Saccharomyces cerevisiae strains. Xylosidase activity was engineered to be cell free or tethered to the yeast cell surface.

Microbiome plasticity in chickens provides an attractive target for future potential therapeutic platforms. However, a better understanding of the chicken microbial ecology and how interventions such as xylanase addition can modulate this is needed. Interest in xylanase as an essential additive for livestock in manipulating growth performance has been reported as early as 1980s. In this paper, we describe how a new xylanase leads to an array of benefits in broilers, not only by improving broiler performance and nutrient digestibility, but also by clearly improving gut health. Gut health improvements are demonstrated by better intestinal morphology, different volatile fatty acid profile in the gut and even promotion of beneficial over pathogenic micro-organisms in the intestinal tract. Our study confirms that supplementation of xylanase at the lowest dose of 30,000 U/kg (10 g/t) and above significantly improves broiler performance and digestibility. Furthermore, the observation that xylanase can modulate broilers’ intestinal morphology and microbial content beneficially, by stimulating Lactobacilli growth and thus exerting a prebiotic effect, provides novel and useful insights for future applications.

Fast pyrolysis is an industrially attractive method to produce fuels and chemicals from biomass; however, to gain better control over the process, the reactions and interactions between the components and decomposition products need elucidation. This study investigated primary reactions during fast pyrolysis of biomass. Pyrolysis of the three main biomass components (cellulose, hemicellulose and lignin) and their blends was carried out with a micro-pyrolyser connected to a Gas Chromatograph-Mass Spectrometer/Flame Ionisation Detector (GC–MS/FID). The blends of the individual components were prepared in similar proportions to that of native biomass (birchwood) and were pyrolysed at 600 °C for 2 s. The results showed that the two-component blends decrease the production of saccharides to a large extent. This was especially noticeable for levoglucosan when cellulose was mixed with either hemicellulose or lignin. Similarly, in the presence of cellulose, the formation of phenolic compounds from lignin was inhibited by 62 %. However, no differences were found in yields of the main products for the xylan-lignin blend compared to those from the individual components. The yields of volatile products from the cellulose-xylan blend were promoted for a majority of the product categories and were most pronounced for the aldehydes. Furthermore, while the formation of the phenols and saccharides was slightly inhibited for the three-component blend, the aldehydes, ketones and furans showed an increased production compared to the weighed sum of products expected, based on the pyrolysis of the individual components. The native biomass showed a similar trend as the three-component blend in all product categories except for the saccharides, which were inhibited to a large extent. This study provides a better understanding of the interactions occurring between different components during fast pyrolysis of biomass.

Irrespective of their biological origin, most proteins are composed of several elementary domains connected by linkers. These domains are either functionally independent units, or part of larger multidomain structures whose functions are defined by their spatial proximity. Carbohydrate-degrading enzymes provide examples of a range of multidomain structures, in which catalytic protein domains are frequently appended to one or more non-catalytic carbohydrate-binding modules which specifically bind to carbohydrate motifs. While the carbohydrate-binding specificity of these modules is clear, their function is not fully elucidated. Herein, an original approach to tackle the study of carbohydrate-binding modules using the Jo-In biomolecular welding protein pair is presented. To provide a proof of concept, recombinant xylanases appended to two different carbohydrate-binding modules have been created and produced. The data reveal the biochemical properties of four xylanase variants and provide the basis for correlating enzyme activity to structural properties and to the nature of the substrate and the ligand specificity of the appended carbohydrate-binding module. It reveals that specific spatial arrangements favour activity on soluble polymeric substrates and that activity on such substrates does not predict the behaviour of multimodular enzymes on insoluble plant cell wall samples. The results highlight that the Jo-In protein welding system is extremely useful to design multimodular enzyme systems, especially to create rigid conformations that decrease the risk of intermodular interference. Further work on Jo-In will target the introduction of varying degrees of flexibility, providing the means to study this property and the way it may influence multimodular enzyme functions.

Thermophilic bacteria, especially from the genus Bacillus, constitute a huge potential source of novel enzymes that could be relevant for biotechnological applications. In this work, we described the cellulose and hemicellulose-related enzymatic activities of the hot spring Bacillus aerius CCMM B940 from the Moroccan Coordinated Collections of Microorganisms (CCMM), and revealed its potential for hemicellulosic biomass utilization. Indeed, B940 was able to degrade complex polysaccharides such as xylan and lichenan and exhibited activity towards carboxymethylcellulose. The strain was also able to grow on agriculture waste such as orange and apple peels as the sole carbon source. Whole-genome sequencing allowed the reclassification of CCMM B940 previously known as B. aerius into Bacillus paralicheniformis since the former species name has been rejected. The draft genome reported here is composed of 38 contigs resulting in a genome of 4,315,004 bp and an average G + C content of 45.87%, and is an important resource for illuminating the molecular mechanisms of carbohydrate metabolism. The annotated genomic sequences evidenced more than 52 genes encoding glycoside hydrolases and pectate lyases belonging to 27 different families of CAZymes that are involved in the degradation of plant cell wall carbohydrates. Genomic predictions in addition to in vitro experiments have revealed broad hydrolytic capabilities of the strain, thus reinforcing its relevance for biotechnology applications.

XynR is a thermophilic and alkaline GH10 xylanase, identified in the culture broth of alkaliphilic and thermophilic Bacillus sp. strain TAR-1. We previously selected S92E as a thermostable variant from a site saturation mutagenesis library. Here, we attempted to select the alkaliphilic XynR variant from the library and isolated T315N. In the hydrolysis of beechwood xylan, T315N and S92E/T315N exhibited a broader bell-shaped pH-dependent activity than the wild-type (WT) XynR and S92E. The optimal pH values of T315N and S92E/T315N were 6.5-9.5 while those of WT and S92E were 6.5-8.5. On the other hand, T315N and S92E/T315N exhibited a narrower bell-shaped pH dependence of stability: the pHs at which the activity was stable after the incubation at 37°C for 24 h were 6.0-8.5 for T315N and S92E/T315N, but 6.0-10.0 for WT and S92E. These results indicated that the mutation of Thr315 to Asn increased the alkaliphily but decreased the alkaline resistance.

Penicillium oxalicum has received increasing attention as a potential cellulase-producer. In this study, a copper-controlled flippase recombination enzyme/recognition target (FLP/FRT)-mediated recombination system was constructed in P. oxalicum, to overcome limited availability of antibiotic resistance markers. Using this system, two crucial transcription repressor genes atf1 and cxrC for the production of cellulase and xylanase under solid-state fermentation (SSF) were simultaneously deleted, thereby leading to 2.4- to 29.1-fold higher cellulase and 78.9% to 130.8% higher xylanase production than the parental strain under SSF, respectively. Glucose and xylose released from hydrolysis of pretreated sugarcane bagasse achieved 10.6%-13.5% improvement by using the crude enzymes from the engineered strain Δatf1ΔcxrC::flp under SSF in comparison with that of the parental strain. Consequently, these results provide a feasible strategy for improved cellulase and xylanase production by filamentous fungi.

Biodegradable food packaging provides an environmentally-conscious alternative to plastic food packaging options. This study investigated whey protein edible films containing 10-40 g xylan/100 g whey protein isolate (WPI). Transglutaminase (TG) was used as a cross-linking agent in WPI-only and 40 g xylan/100 g WPI films. The food packaging properties investigated were water vapor permeability (WVP), oxygen permeability (OP), tensile stress, and % elongation at break. Thermal properties were studied using differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Crystallinity and microstructure were assessed using X-ray diffraction (XRD) and scanning electron microscopy (SEM), respectively. Composite films containing 40 g xylan/100 g WPI that were also treated with TG showed the greatest improvement in properties important to food packaging. Compared to the WPI-only control films, WVP decreased from 6.41 to 3.89 g mm/m² day kPa (p < 0.05), OP decreased from 21.85 to 7.32 cc μm/m² day kPa (p < 0.05), and tensile stress increased from 6.73 MPa to 15.96 MPa (p < 0.05). The % elongation at break decreased significantly from 12.5% in WPI-only films to 5.8-1.4% in all xylan and TG treated films (p < 0.05). The temperature of melting increased from 121°C in control films to a maximum of 166°C in the 20 g xylan/100 g WPI films, indicating increased intermolecular strength. Film microstructure showed separate organization of xylan within films. Crystallinity was identified with increasing xylan content through XRD analysis, suggesting increased polymer packing.

The use of low-cost feedstock for enzyme production is an environmental and economic solution. Sugarcane bagasse and soybean meal are employed in this study for optimised xylanase production with the concomitant synthesis of proteases. The enzymatic complex is produced by submerged fermentation by Aspergillus niger. Optimisation steps lead to a 2.16-fold increase in enzymatic activity. The fermentation kinetics are studied in Erlenmeyer flasks, a stirred tank reactor and a bubble column reactor, with the xylanase activities reaching 52.9; 33.7 and 60.5 U.mL⁻¹, respectively. The protease production profile is also better in the bubble column reactor, exceeding 7 U.mL⁻¹. The enzyme complex is then evaluated for the synthesis of xylooligosaccharides from sugarcane extracted xylan with a production of 3.1 g.L⁻¹ where xylotriose is the main product. Excellent perspectives are observed for the developed process with potential applications in the animal feed, prebiotics and paper industries.

Xylanases are glycoside hydrolases (GH) that degrade β-1,4-xylan, a linear polysaccharide found as hemicellulose in cell wall of plants. Endoxylanase (Endo-1,4-β-xylanase, EC 3.2.1.8) randomly catalyses xylan to produce varying short xylooligosaccharides (XOS). This study aimed to determine the characteristics of a partially purified endoxylanase from Leohumicola incrustata. Enzyme production was carried out using beechwood (BW) xylan, after which the cell-free crude filtrate was concentrated using the ammonium sulphate precipitation method. The hydrolysed products were analysed by thin-layer chromatography (TLC) and zymography. The result showed that the enzyme produced varying smaller-sized linear xylooligosaccharides with R_f values corresponding to those of xylobiose, xylotriose, xylotetraose, xylopentaose, xylohexaose and other higher oligomers. The endoxylanase had a molecular mass of 72 kDa. The enzyme is stable in the presence of K⁺, Na⁺, Ca²⁺, Fe²⁺, Mg²⁺, Zn²⁺, Co²⁺, pH of 5.0 and temperature of 37^oC. However, the activity gradually decreased after 60 min at 50^oC and retained over 69% activity after 120 min, while at 60 and 70^oC, the enzyme activity sharply decreased (pre-incubation periods). Endoxylanase from L. incrustata is comparable to those of other microorganisms and should be considered an attractive candidate for future industrial applications.