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In this study, β-1,3-xylanase (Xyl3088) was designed and prepared by constructing the expression vector plasmid and expressing and purifying the fusion protein. β-1,3-xylo-oligosaccharides were obtained through the specific enzymatic degradation of β-1, 3-xylan from Caulerpa lentillifera. The enzymolysis conditions were established and optimized as follows: Tris-HCl solution 0.05 mol/L, temperature of 37 °C, enzyme amount of 250 μL, and enzymolysis time of 24 h. The oligosaccharides' compositions and structural characterization were identified by thin-layer chromatography (TLC), ion chromatography (IC) and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS). The IC 50 values for scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azino-bis-3-ethyl-benzothiazoline-p-sulfonic acid (ABTS + ), and superoxide anion radical (•O 2- ) were 13.108, 1.258, and 65.926 mg/mL for β-1,3-xylo-oligosaccharides, respectively, and 27.588, 373.048, and 269.12 mg/mL for β-1,4-xylo-oligosaccharides, respectively. Compared with β-1,4-xylo-oligosaccharides, β-1,3-xylo-oligosaccharides had substantial antioxidant activity and their antioxidant effects were concentration dependent. β-1,3-xylo-oligosaccharides also possessed a stronger anti-inflammatory effect on RAW 264.7 cells stimulated by lipopolysaccharide (LPS) than β-1,4-xylo-oligosaccharides. At a working concentration of 100 μg/mL, β-1,3-xylo-oligosaccharides inhibited the release of NO and affected the expression of IL-1β, TNF-α, and other proteins secreted by cells, effectively promoting the release of pro-inflammatory mediators by immune cells in response to external stimuli and achieving anti-inflammatory effects. Therefore, β-1,3-xylo-oligosaccharides are valuable products in food and pharmaceutical industries., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy. This includes valorization of hemicellulosic fraction of plant biomass, the second most abundant biopolymer from plant cell walls, aiming to produce prebiotic oligosaccharides, widely explored in food and feed industries. In this work, we conducted biochemical and biophysical characterization of a prokaryotic two-domain R. champanellensis xylanase from glycoside hydrolase (GH) family 30 (RcXyn30A), and evaluated its applicability for XOS production from glucuronoxylan in combination with two endo-xylanases from GH10 and GH11 families and a GH11 xylobiohydrolase. RcXyn30A liberates mainly long monoglucuronylated xylooligosaccharides and is inefficient in cleaving unbranched oligosaccharides. Crystallographic structure of RcXyn30A catalytic domain was solved and refined to 1.37 Å resolution. Structural analysis of the catalytic domain releveled that its high affinity for glucuronic acid substituted xylan is due to the coordination of the substrate decoration by several hydrogen bonds and ionic interactions in the subsite -2. Furthermore, the protein has a larger β5-α5 loop as compared to other GH30 xylanases, which might be crucial for creating an additional aglycone subsite (+3) of the catalytic site. Finally, RcXyn30A activity is synergic to that of GH11 xylobiohydrolase., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Objective: New characterized carbohydrate-active enzymes are needed for use as tools to discriminate complex carbohydrate structural features. Fungal glycoside hydrolase family 3 (GH3) β-xylosidases have been shown to be useful for the structural elucidation of glucuronic acid (GlcA) and arabinofuranose (Araf) substituted oligoxylosides. A homolog of these GH3 fungal enzymes from the bacterium Segatella baroniae (basonym Prevotella bryantii), Xyl3C, has been previously characterized, but those studies did not address important functional specificity features. In an interest to utilize this enzyme for laboratory methods intended to discriminate the structure of the non-reducing terminus of substituted xylooligosaccharides, we have further characterized this GH3 xylosidase., Results: In addition to verification of basic functional characteristics of this xylosidase we have determined its mode of action as it relates to non-reducing end xylose release from GlcA and Araf substituted oligoxylosides. Xyl3C cleaves xylose from the non-reducing terminus of β-1,4-xylan until occurrence of a penultimate substituted xylose. If this substitution is O2 linked, then Xyl3C removes the non-reducing xylose to leave the substituted xylose as the new non-reducing terminus. However, if the substitution is O3 linked, Xyl3C does not hydrolyze, thus leaving the substitution one-xylose (penultimate) from the non-reducing terminus. Hence, Xyl3C enables discrimination between O2 and O3 linked substitutions on the xylose penultimate to the non-reducing end. These findings are contrasted using a homologous enzyme also from S. baroniae, Xyl3B, which is found to yield a penultimate substituted nonreducing terminus regardless of which GlcA or Araf substitution exists., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Several fungal species produce diverse carbohydrate-active enzymes useful for the xylooligosaccharide biorefinery. These enzymes can be isolated by different purification methods, but fungi usually produce other several compounds which interfere in the purification process. So, the present work has three interconnected aims: (i) compare β-xylosidase production by Fusarium pernambucanum MUM 18.62 with other crop pathogens; (ii) optimise F. pernambucanum xylanolytic enzymes expression focusing on the pre-inoculum media composition; and (iii) design a downstream strategy to eliminate interfering substances and sequentially isolate β-xylosidases, arabinofuranosidases and endo-xylanases from the extracellular media. F. pernambucanum showed the highest β-xylosidase activity among all the evaluated species. It also produced endo-xylanase and arabinofuranosidase. The growth and β-xylosidase expression were not influenced by the pre-inoculum source, contrary to endo-xylanase activity, which was higher with xylan-enriched agar. Using a sequential strategy involving ammonium sulfate precipitation of the extracellular interferences, and several chromatographic steps of the supernatant (hydrophobic chromatography, size exclusion chromatography, and anion exchange chromatography), we were able to isolate different enzyme pools: four partially purified β-xylosidase/arabinofuranoside; FpXylEAB trifunctional GH10 endo-xylanase/β-xylosidase/arabinofuranoside enzyme (39.8 kDa) and FpXynE GH11 endo-xylanase with molecular mass (18.0 kDa). FpXylEAB and FpXynE enzymes were highly active at pH 5-6 and 60-50 °C., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Xylan is the main component of hemicellulose. Complete hydrolysis of xylan requires synergistically acting xylanases, such as β-d-xylosidases. Salt-tolerant β-d-xylosidases have significant application benefits, but few reports have explored the critical amino acids affecting the salt tolerance of xylosidases. Herein, the site-directed mutation was used to demonstrate that negative electrostatic potentials generated by 19 acidic residues in the loop regions of the structural surface positively correlated with the improved salt tolerance of GH39 β-d-xylosidase JB13GH39P28. These mutants showed reduced negative potentials on structural surfaces as well as a 13-43% decrease in stability in 3.0-30.0% (w/v) NaCl. Six key residue sites, D201, D259, D297, D377, D395, and D474, were confirmed to influence both the stability and activity of GH39 β-d-xylosidase. The activity of the GH39 β-d-xylosidase was found promoting by SO 4 2- and inhibiting by NO 3 - . Values of K m and K cat / K m decreased aggravatedly in 30.0% (w/v) NaCl when mutation operated on residues E179 and D182 in the loop regions of the catalytic domain. Taken together, mutation on acidic residues in loop regions from catalytic and noncatalytic domains may cause the deformation of catalytic pocket and aggregation of protein particles then decrease the stability, binding affinity, and catalytic efficiency of the β-d-xylosidase.

Hemicellulose is a highly abundant, ubiquitous, and renewable natural polysaccharide, widely present in agricultural and forestry residues. The enzymatic hydrolysis of hemicellulose has generally been accomplished using β-xylosidases, but concomitantly increasing the stability and activity of these enzymes remains challenging. Here, we rationally engineered a β-xylosidase from Bacillus clausii to enhance its stability by computation-aided design combining ancestral sequence reconstruction and structural analysis. The resulting combinatorial mutant rXYLO M25I/S51L/S79E exhibited highly improved robustness, with a 6.9-fold increase of the half-life at 60 °C, while also exhibiting improved pH stability, catalytic efficiency, and hydrolytic activity. Structural analysis demonstrated that additional interactions among the propeller blades in the catalytic module resulted in a much more compact protein structure and induced the rearrangement of the opposing catalytic pocket to mediate the observed improvement of activity. Our work provides a robust biocatalyst for the hydrolysis of agricultural waste to produce various high-value-added chemicals and biofuels.

The glycoside hydrolase family 39 (GH39) proteins are renowned for their extremophilic and multifunctional enzymatic properties, yet the molecular mechanisms underpinning these unique characteristics continue to be an active subject of research. In this study, we introduce WsuXyn, a GH39 protein with a molecular weight of 58 kDa, originating from the thermophilic Geobacillus sp. WSUCF1. Previously reported for its exceptional thermostable β-xylosidase activity, WsuXyn has recently demonstrated a significant endoxylanase activity (3752 U·mg -1 ) against beechwood xylan, indicating towards its bifunctional nature. Physicochemical characterization revealed that WsuXyn exhibits optimal endoxylanase activity at 70 °C and pH 7.0. Thermal stability assessments revealed that the enzyme is resilient to elevated temperatures, with a half-life of 168 h. Key kinetic parameters highlight the exceptional catalytic efficiency and strong affinity of the protein for xylan substrate. Moreover, WsuXyn-mediated hydrolysis of beechwood xylan has achieved 77 % xylan conversion, with xylose as the primary product. Structural analysis, amalgamated with docking simulations, has revealed strong binding forces between xylotetraose and the protein, with key amino acid residues, including Glu278, Tyr230, Glu160, Gly202, Cys201, Glu324, and Tyr283, playing pivotal roles in these interactions. Therefore, WsuXyn holds a strong promise for biodegradation and value-added product generation through lignocellulosic biomass conversion., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rohit Rai reports financial support was provided by Science and Engineering Research Board. Rajesh K Sani reports financial support was provided by National Science Foundation. Rohit Rai reports financial support was provided by Lovely Professional University. Kian Mau Goh reports financial support was provided by University of Technology Malaysia., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The Xylo-1 xylosidase, which belongs to the GH43 family, exhibits a high salt tolerance. The present study demonstrated that the catalytic activity of Xylo-1 increased by 195% in the presence of 5 M NaCl. Additionally, the half-life of Xylo-1 increased 25.9-fold in the presence of 1 M NaCl. Through comprehensive analysis including circular dichroism, fluorescence spectroscopy, and molecular dynamics simulations, we elucidated that the presence of Na + ions increased the contact frequency between the surface acidic amino acids and the surrounding water molecules. This resulted in the stabilization of the surrounding hydration layer of Xylo-1. Additionally, Na + ions also stabilized the substrate-binding conformation and the fluctuation of water molecules within the active site, which enhanced the catalytic activity of Xylo-1 by increasing the nucleophilic attack by the water molecules. Ultimately, the optimal reaction conditions for the production of xylose by synergistic catalysis with Xylo-1 and xylanase were determined. The results demonstrated that the conversion yield of the method was high for various sources of xylan, indicating the method could have potential industrial applications. This study explored the structure-activity relationship of catalysis in Xylo-1 under high-salt conditions, provides novel insights into the mechanism of halophilic enzymes, and serves as a reference for the industrial application of Xylo-1.

Improving the activity of β-xylosidase at high temperature and organic solvents is important for the conversion of xylan, phytochemicals and some hydroxyl-containing substances to produce xylose and bioactive substances. In this study, a β-xylosidase R333H and an endoxylanase were simultaneously co-immobilized on the metal-organic framework UiO-66-NH 2 . Compared with the single R333H immobilization system, the co-immobilization enhanced the activity of R333H at high temperature and high concentration of acetone, and the relative activities at 95 °C and 50% acetone solution were >95%. The K m value of co-immobilized R333H towards p-Nitrophenyl-β-D-xylopyranoside (pNPX) shifted from 2.04 to 0.94 mM, which indicated the enhanced affinity towards pNPX. After 5 cycles, the relative activities of the co-immobilized enzymes towards pNPX and corncob xylan were 52% and 70% respectively, and the accumulated amount of reducing sugars obtained by co-immobilized enzymes degrading corncob xylan in 30% (v/v) acetone solution was 1.7 times than that with no acetone., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The complete degradation of abundant xylan derived from plants requires the participation of β-xylosidases to produce the xylose which can be converted to xylitol, ethanol, and other valuable chemicals. Some phytochemicals can also be hydrolyzed by β-xylosidases into bioactive substances, such as ginsenosides, 10-deacetyltaxol, cycloastragenol, and anthocyanidins. On the contrary, some hydroxyl-containing substances such as alcohols, sugars, and phenols can be xylosylated by β-xylosidases into new chemicals such as alkyl xylosides, oligosaccharides, and xylosylated phenols. Thus, β-xylosidases shows great application prospects in food, brewing, and pharmaceutical industries. This review focuses on the molecular structures, biochemical properties, and bioactive substance transformation function of β-xylosidases derived from bacteria, fungi, actinomycetes, and metagenomes. The molecular mechanisms of β-xylosidases related to the properties and functions are also discussed. This review will serve as a reference for the engineering and application of β-xylosidases in food, brewing, and pharmaceutical industries.

Structural and conformational insights of a putative β-1,4-xylosidase (PsGH43F) of glycoside hydrolase family 43 from Pseudopedobacter saltans were investigated by computational and Circular Dichroism (CD) analyses. PsGH43F was cloned and expressed in E. coli BL21 (DE3) cells and the purified enzyme gave the size ~50 kDa on SDS-PAGE analysis. Multiple Sequence Alignment of PsGH43F sequence followed by superposition of modeled structure with homologous structures displayed the presence of three conserved catalytic amino acid residues, Asp33, Asp149 and Glu212. The secondary structure analysis by CD showed 2.72 % α-helix and 36.06 % β-strands. The homology modeled structure of PsGH43F displayed a 5-bladed β-propeller fold for catalytic module at N-terminal and a β-sandwich structure for CBM6 at the C-terminal. Ramachandran plot displayed 99.5 % of residues in the allowed regions. MD simulation of PsGH43F revealed the compactness and stability of the structure. Molecular docking studies of PsGH43F with xylo-oligosaccharides revealed its maximum binding affinity for xylobiose. MD simulation of PsGH43F-xylobiose complex confirmed the increased structural and conformational stability in presence of substrate. The Hydrodynamic diameter analysis of PsGH43F by DLS was in the range, 0.25-0.28 μm., Competing Interests: Declaration of competing interest The authors declare that there were no financial or personal relationships with other people or organizations that could inappropriately influence this work., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Nowadays, alkali-tolerant β-xylosidases and their molecular mechanism of pH adaptability have been poorly studied. Here, a novel GH43 β-xylosidase (XYLO) was isolated from Bacillus clausii TCCC 11004, and the recombinant β-xylosidase (rXYLO) was most active at pH 8.0 and stable in a broad pH range (7.0-11.0), exhibiting superior alkali tolerance. Molecular dynamics simulation indicated that XYLO showed a notable overall structural stability and an enlargement of substrate binding pocket under alkaline condition, resulting in the formation of a new hydrogen bond between substrate and Arg286 of XYLO, and the tight binding played a key role in improving the XYLO activity with the increasing pH. Moreover, rXYLO with an endo-xylanase resulted in high xylose yields by hydrolyzing alkali-extracted xylan from agricultural wastes. This work would provide an alkali-tolerant β-xylosidase, enhance the understanding for the relationship of structure and activity adapted to the high-alkaline environment, and promote its application in xylose production., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Xylan is the most common hemicellulose in plant cell walls, though the structure of xylan polymers differs between plant species. Here, to gain a better understanding of fungal xylan degradation systems, which can enhance enzymatic saccharification of plant cell walls in industrial processes, we conducted a comparative study of two glycoside hydrolase family 3 (GH3) β-xylosidases (Bxls), one from the basidiomycete Phanerochaete chrysosporium (PcBxl3), and the other from the ascomycete Trichoderma reesei (TrXyl3A). A comparison of the crystal structures of the two enzymes, both with saccharide bound at the catalytic center, provided insight into the basis of substrate binding at each subsite. PcBxl3 has a substrate-binding pocket at subsite -1, while TrXyl3A has an extra loop that contains additional binding subsites. Furthermore, kinetic experiments revealed that PcBxl3 degraded xylooligosaccharides faster than TrXyl3A, while the K M values of TrXyl3A were lower than those of PcBxl3. The relationship between substrate specificity and degree of polymerization of substrates suggested that PcBxl3 preferentially degrades xylobiose (X 2 ), while TrXyl3A degrades longer xylooligosaccharides. Moreover, docking simulation supported the existence of extended positive subsites of TrXyl3A in the extra loop located at the N-terminus of the protein. Finally, phylogenetic analysis suggests that wood-decaying basidiomycetes use Bxls such as PcBxl3 that act efficiently on xylan structures from woody plants, whereas molds use instead Bxls that efficiently degrade xylan from grass. Our results provide added insights into fungal efficient xylan degradation systems., Competing Interests: Conflict of interest The authors declare that there is no conflict of interests with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The pulp and paper industry discharges massive amount of wastewater containing hazardous organochlorine compounds released during different processing stages. Therefore, some cost-effective and nonpolluting practices such as enzymatic treatments are required for the potential mitigation of effluents released in the environment. Various xylanolytic enzymes such as xylanases, laccases, cellulases and hemicellulases are used to hydrolyse raw materials in the paper manufacturing industry. These enzymes are used either individually or in combination, which has the efficient potential to be considered for bio-deinking and bio-bleaching components. They are highly dynamic, renewable, and high in specificity for enhancing paper quality. The xylanase act on the xylan and cellulases act on the cellulose fibers, and thus increase the bleaching efficacy of paper. Similarly, hemicellulase enzyme like endo-xylanases, arabinofuranosidase and β-D-xylosidases have been described as functional properties towards the biodegradation of biomass. In contrast, laccase enzymes act as multi-copper oxidoreductases, bleaching the paper by the oxidation and reduction process. Laccases possess low redox potential compared to other enzymes, which need some redox mediators to catalyze. The enzymatic process can be affected by various factors such as pH, temperature, metal ions, incubation periods, etc. These factors can either increase or decrease the efficiency of the enzymes. This review draws attention to the xylanolytic enzyme-based advanced technologies for pulp bleaching in the paper industry., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Xylanases (EC 3.2.1.8) have been considered as a potential green solution for the sustainable development of a wide range of industries including pulp and paper, food and beverages, animal feed, pharmaceuticals, and biofuels because they are the key enzymes that degrade the xylosidic linkages of xylan, the major component of the second most abundant raw material worldwide. Therefore, there is a critical need for the industrialized xylanases which must have high specific activity, be tolerant to organic solvent or detergent and be active during a wide range of conditions, such as high temperature and pH. In this study, an extracellular xylanase was purified from the culture broth of Aspergillus niger VTCC 017 for primary structure determination and properties characterization. The successive steps of purification comprised centrifugation, Sephadex G-100 filtration, and DEAE-Sephadex chromatography. The purified xylanase (specific activity reached 6596.79 UI/mg protein) was a monomer with a molecular weight of 37 kDa estimating from SDS electrophoresis. The results of LC/MS suggested that the purified protein is indeed an endo-1,4-β-D-xylanase. The purified xylanase showed the optimal temperature of 55 °C, and pH 6.5 with a stable xylanolytic activity within the temperature range of 45-50 °C, and within the pH range of 5.0-8.0. Most divalent metal cations including Zn 2+ , Fe 2+ , Mg 2+ , Cu 2+ , Mn 2+ showed some inhibition of xylanase activity while the monovalent metal cations such as K + and Ag + exhibited slight stimulating effects on the enzyme activity. The introduction of 10-30% different organic solvents (n-butanol, acetone, isopropanol) and several detergents (Triton X-100, Tween 20, and SDS) slightly reduced the enzyme activity. Moreover, the purified xylanase seemed to be tolerant to methanol and ethanol and was even stimulated by Tween 80. Overall, with these distinctive properties, the putative xylanase could be a successful candidate for numerous industrial uses., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Xylooligosaccharides having various degrees of polymerization such as xylobiose, xylotriose, and xylotetraose positively affect human health by interacting with gut proteins. The present study aimed to identify proteins present in gut microflora, such as xylosidase, xylulokinase, etc., with the help of retrieved whole-genome annotations and find out the mechanistic interactions of those with the above substrates. The 3D structures of proteins, namely Endo-1,4-beta-xylanase B (XynB) from Lactobacillus brevis and beta-D-xylosidase (Xyl3) from Bifidobacterium adolescentis, were computationally predicted and validated with the help of various bioinformatics tools. Molecular docking studies identified the effectual binding of these proteins to the xylooligosaccharides, and the stabilities of the best-docked complexes were analyzed by molecular dynamic simulation. The present study demonstrated that XynB and Xyl3 showed better effectual binding toward Xylobiose with the binding energies of - 5.96 kcal/mol and - 4.2 kcal/mol, respectively. The interactions were stabilized by several hydrogen bonding having desolvation energy (- 6.59 and - 7.91). The conformational stabilities of the docked complexes were observed in the four selected complexes of XynB-xylotriose, XynB-xylotetraose, Xyl3-xylobiose, and Xyn3-xylotriose by MD simulations. This study showed that the interactions of these four complexes are stable, which means they have complex metabolic activities among each other. Extending these studies of understanding, the interaction between specific probiotics enzymes and their ligands can explore the detailed design of synbiotics in the future., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Fungal xylanases belonging to family GH30_7, initially categorized as endo-glucuronoxylanases, are now known to differ both in terms of substrate specificity, as well as mode of action. Recently, TtXyn30A, a GH30_7 xylanase from Thermothelomyces thermophila, was shown to possess dual activity, acting on the xylan backbone in both an endo- and an exo- manner. Here, in an effort to identify the structural characteristics that append these functional properties to the enzyme, we present the biochemical characterization of various TtXyn30A mutants as well as its crystal structure, alone, and in complex with the reaction product. An auxiliary catalytic amino acid has been identified, while it is also shown that glucuronic acid recognition is not mediated by a conserved arginine residue, as shown by previously determined GH30 structures., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

The hyperthermophilic bacterium Caldicellulosiruptor kristjansonii encodes an unusual enzyme, Ck Xyn10C-GE15A, which incorporates two catalytic domains, a xylanase and a glucuronoyl esterase, and five carbohydrate-binding modules (CBMs) from families 9 and 22. The xylanase and glucuronoyl esterase catalytic domains were recently biochemically characterized, as was the ability of the individual CBMs to bind insoluble polysaccharides. Here, we further probed the abilities of the different CBMs from Ck Xyn10C-GE15A to bind to soluble poly- and oligosaccharides using affinity gel electrophoresis, isothermal titration calorimetry, and differential scanning fluorimetry. The results revealed additional binding properties of the proteins compared to the former studies on insoluble polysaccharides. Collectively, the results show that all five CBMs have their own distinct binding preferences and appear to complement each other and the catalytic domains in targeting complex cell wall polysaccharides. Additionally, through renewed efforts, we have achieved partial structural characterization of this complex multidomain protein. We have determined the structures of the third CBM9 domain (CBM9.3) and the glucuronoyl esterase (GE15A) by X-ray crystallography. CBM9.3 is the second CBM9 structure determined to date and was shown to bind oligosaccharide ligands at the same site but in a different binding mode compared to that of the previously determined CBM9 structure from Thermotoga maritima . GE15A represents a unique intermediate between reported fungal and bacterial glucuronoyl esterase structures as it lacks two inserted loop regions typical of bacterial enzymes and a third loop has an atypical structure. We also report small-angle X-ray scattering measurements of the N-terminal CBM22.1-CBM22.2-Xyn10C construct, indicating a compact arrangement at room temperature.

Arabinoxylans are cell wall polysaccharides whose re-modelling and degradation during plant development are mediated by several classes of xylanolytic enzymes. Here, we present the identification and new annotation of twelve putative (1,4)-β-xylanase and six β-xylosidase genes, and their spatio-temporal expression patterns during vegetative and reproductive growth of barley (Hordeum vulgare cv. Navigator). The encoded xylanase proteins are all predicted to contain a conserved carbohydrate-binding module (CBM) and a catalytic glycoside hydrolase (GH) 10 domain. Additional domains in some xylanases define three discrete phylogenetic clades: one clade contains proteins with an additional N-terminal signal sequence, while another clade contains proteins with multiple CBMs. Homology modelling revealed that all fifteen xylanases likely contain a third domain, a β-sandwich folded from two non-contiguous sequence segments that bracket the catalytic GH domain, which may explain why the full length protein is required for correct folding of the active enzyme. Similarly, predicted xylosidase proteins share a highly conserved domain structure, each with an N-terminal signal peptide, a split GH 3 domain, and a C-terminal fibronectin-like domain. Several genes appear to be ubiquitously expressed during barley growth and development, while four newly annotated xylanase and xylosidase genes are expressed at extremely high levels, which may be of broader interest for industrial applications where cell wall degradation is necessary., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Substrate specificities of glycoside hydrolase families 8 (Rex), 39 (BhXyl39), and 52 (BhXyl52) β-xylosidases from Bacillus halodurans C-125 were investigated. BhXyl39 hydrolyzed xylotriose most efficiently among the linear xylooligosaccharides. The activity decreased in the order of xylohexaose > xylopentaose > xylotetraose and it had little effect on xylobiose. In contrast, BhXyl52 hydrolyzed xylobiose and xylotriose most efficiently, and its activity decreased when the main chain became longer as follows: xylotetraose > xylopentaose > xylohexaose. Rex produced O-β-D-xylopyranosyl-(1 → 4)-[O-α-L-arabinofuranosyl-(1 → 3)]-O-β-D-xylopyranosyl-(1 → 4)-β-D-xylopyranose (Ara 2 Xyl 3 ) and O-β-D-xylopyranosyl-(1 → 4)-[O-4-O-methyl-α-D-glucuronopyranosyl-(l → 2)]-β-D-xylopyranosyl-(1 → 4)-β-D-xylopyranose (MeGlcA 2 Xyl 3 ), which lost a xylose residue from the reducing end of O-β-D-xylopyranosyl-(1 → 4)-[O-α-L-arabinofuranosyl-(1 → 3)]-O-β-D-xylopyranosyl-(1 → 4)-β-D-xylopyranosyl-(1 → 4)-β-D-xylopyranose (Ara 3 Xyl 4 ) and O-β-D-xylopyranosyl-(1 → 4)-[O-4-O-methyl-α-D-glucuronopyranosyl-(1 → 2)]-β-D-xylopyranosyl-(1 → 4)-β-D-xylopyranosyl-(1 → 4)-β-D-xylopyranose (MeGlcA 3 Xyl 4 ). It was considered that there is no space to accommodate side chains at subsite -1. BhXyl39 rapidly hydrolyzes the non-reducing-end xylose linkages of MeGlcA 3 Xyl 4 , while the arabinose branch does not significantly affect the enzyme activity because it degrades Ara 3 Xyl 4 as rapidly as unmodified xylotetraose. The model structure suggested that BhXyl39 enhanced the activity for MeGlcA 3 Xyl 4 by forming a hydrogen bond between glucuronic acid and Lys265. BhXyl52 did not hydrolyze Ara 3 Xyl 4 and MeGlcA 3 Xyl 4 because it has a narrow substrate binding pocket and 2- and 3-hydroxyl groups of xylose at subsite +1 hydrogen bond to the enzyme.

Dictyoglomus thermophilum β-d-xylosidase DtXyl is attractive as a potential thermostable biocatalyst able to produce biologically active ginsenosides intermediates from β-(1,2)-D-xylosylated compounds, including Notoginsenoside-R1. DtXyl was expressed as an active N-terminal His-tagged protein, and its crystal structure was solved in presence or absence of d-xylose product. Modelling of notoginsenoside R1 in DtXyl active site led to the identification of several hydrophobic residues interacting in close contact to the substrate hydrophobic core. Unlike other residues involved in substrate binding, these residues are not conserved among GH39 xylosidase family, and their physico-chemical properties can be correlated to the efficient binding and subsequent hydrolysis of Notoginsenoside R1., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Neurospora crassa possesses six putative xylanases and four putative xylosidases. qRT-PCR results showed that the expression of all these xylanolytic enzymes was induced by xylan. Except for two intracellular β-xylosidases, others were shown to be secreted enzymes based on the localization analysis of EGFP-fusion proteins. Among them, GH10-1, GH10-2, GH11-1, and GH11-2 were successfully expressed and characterized as typical endo-β-1,4-xylanases that hydrolyze the xylooligosaccharides with a polymeric degree not less than three or four. Strains deleted for either gh10-1, gh10-2, gh3-7, or gh3-8 displayed decreased growth in xylan and biomass media. Disruption of gh3-7 or gh43-1 resulted in enhanced-xylanolytic enzyme activity when cultivated in biomass medium. Collectively, these results suggest that xylooligosaccharides released by the actions of xylanases and xylosidases not only serve as the carbon sources to maintain the growth of N. crassa, but they also act as inducers to trigger the expression of hydrolytic enzymes in vivo., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Functional screening of metagenomic libraries is an effective approach for identification of novel enzymes. A Caatinga biome goat rumen metagenomic library was screened using esculin as a substrate, and a gene from an unknown bacterium encoding a novel GH3 enzyme, BGL11, was identified. None of the BGL11 closely related genes have been previously characterized. Recombinant BGL11 was obtained and kinetically characterized. Substrate specificity of the purified protein was assessed using seven synthetic aryl substrates. Activity towards nitrophenyl-β-D-glucopyranoside (pNPG), 4-nitrophenyl-β-D-xylopyranoside (pNPX) and 4-nitrophenyl-β-D-cellobioside (pNPC) suggested that BGL11 is a multifunctional enzyme with β-glucosidase, β-xylosidase, and cellobiohydrolase activities. However, further testing with five natural substrates revealed that, although BGL11 has multiple substrate specificity, it is most active towards xylobiose. Thus, in its native goat rumen environment, BGL11 most likely functions as an extracellular β-xylosidase acting on hemicellulose. Biochemical characterization of BGL11 showed an optimal pH of 5.6, and an optimal temperature of 50°C. Enzyme stability, an important parameter for industrial application, was also investigated. At 40°C purified BGL11 remained active for more than 15 hours without reduction in activity, and at 50°C, after 7 hours of incubation, BGL11 remained 60% active. The enzyme kinetic parameters of Km and Vmax using xylobiose were determined to be 3.88 mM and 38.53 μmol.min-1.mg-1, respectively, and the Kcat was 57.79 s-1. In contrast to BLG11, most β-xylosidases kinetically studied belong to the GH43 family and have been characterized only using synthetic substrates. In industry, β-xylosidases can be used for plant biomass deconstruction, and the released sugars can be fermented into valuable bio-products, ranging from the biofuel ethanol to the sugar substitute xylitol., Competing Interests: The authors have declared that no competing interests exist.

A recombinant β-xylosidase (rBxTW1) from the ascomycete Talaromyces amestolkiae and a mutant derived from it, with mostly synthetic activity, have been immobilized as magnetic cross-linked enzyme aggregates (mCLEAs). The mCLEAs of rBxTW1 kept the excellent hydrolytic and O-transxylosylating activities of the free enzyme and had improved thermal and pH stability. The mCLEAs of the mutant also maintained or improved the catalytic properties of the soluble enzyme, synthetizing the O-xylosides of vanillin and (-)-epigallocatechin gallate, and the N- and S-xyloside of 3,5-dibromo-1,2,4-triazole and thiophenol, respectively. The mCLEAs were recyclable across 4 cycles of synthesis of the O-xylosides through a green and highly selective process. The magnetic properties of the scaffold used for immobilization may allow the easy recovery and reuse of the biocatalyst even from reactions containing insoluble lignocellulosic biomass., Competing Interests: Declaration of competing interest None., (Copyright © 2020. Published by Elsevier B.V.)

A biogenic mesoporous silica nanoparticles (MSNs)-based nanocarrier has been used for improving the stability and recyclability of PersiXyn2 as a recombinant xylanase enzyme. The biogenic MSNs (called RKIT-6 henceforth) were synthesized via a soft templating method using rice husk biomass as a renewable silica source. Then bis-(2-aminoethyl) ether modified RKIT-6 (denoted as bis-AE@RKIT-6) was prepared through the furnishing surface with bis-(2-aminoethyl) ether, as a pendant anchoring agent to immobilize PersiXyn2. The nanomaterials were characterized using nitrogen adsorption-desorption isotherms, atomic force microscopy (AFM), X-ray diffraction (XRD), molecular docking (MD) study, and thermogravimetric analysis (TGA). After immobilizing, PersiXyn2@bis-AE@RKIT-6, the optimal temperature of enzyme performance was improved more than 10 °C in comparison with the free enzyme. Such a way that PersiXyn2@bis-AE@RKIT-6 sample could maintain 90% of its maximum activity at the range of 30-60 °C. PersiXyn2@bis-AE@RKIT-6 also enhanced the degradation of lignocellulosic agro-waste (rice straw) and reducing sugar production up to 35% in comparison to the free enzyme. Moreover, PersiXyn2@bis-AE@RKIT-6 could be recycled for ninth runs with a reasonable decrease in its activity. This study presents an efficient nano-biocatalyst which in a more comprehensive sense can be considered as a promising candidate in the fields of animal feed and lignocellulosic biomasses saccharification., Competing Interests: Declaration of competing interest The authors declare that they have no known competing for financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Reducing-end xylose-releasing exo-oligoxylanases (Rex) are GH8 enzymes that depolymerize xylooligosaccharides complementing xylan degradation by endoxylanases in an exo manner. We have studied Paenibacillus barcinonensis Rex8A and showed the release of xylose from xylooligomers decorated with methylglucuronic acid (UXOS) or with arabinose (AXOS). This gives the enzyme a distinctive trait among known Rex, which show activity only on linear xylooligosaccharides. The structure of the enzyme has been solved by X-ray crystallography showing a (α/α) 6 folding common to GH8 enzymes. Analysis of inactived Rex8A-E70A complexed with xylotetraose revealed the existence of at least four binding subsites in Rex8A, with the oligosaccharide occupying subsites -3 to +1. The enzyme shows an extended Leu320-His321-Pro322 loop, common to other Rex, which blocks the binding of longer substrates to positive subsites further than +1 and seems responsible for the lack or diminished activity of Rex enzymes on xylan. Mutants with smaller residues in this loop failed to increase Rex8A activity on the polymer. Analysis of the complexes with AXOS showed the accommodation of arabinose at subsite -2, which cannot be allocated at subsite -1. Arabinose substitutions at the xylose O2 or O3 are accommodated by hydrophobic interaction and seem tolerated rather than recognized by Rex8A. A strained binding of the branch is facilitated by the lack of direct polar interactions of the xylose occupying this subsite, its water-mediated links allowing some conformational flexibility of the sugar. The plasticity of Rex8A is a notable property of the enzyme for its application in xylan deconstruction and upgrading. DATABASE: Structural data are available in PDB database under the accession numbers 6SRD (native form), 6TPP (E70A mutant in complex with EDO), 6TOW (E70A in complex with Xyl4), 6SUD (L320A mutant in complex with xylose), 6SHY (L320A/H321S double mutant in complex with EDO), 6TO0 (E70A in complex with AX3), and 6TRH (E70A in complex with AX4)., (© 2020 Federation of European Biochemical Societies.)

A xylanase from Talaromyces thermophiles F1208 (T-Xyn) was used specifically to explore the effects of disulfide bond on hydrolytic activity. The T-Xyn-C122S-C166S mutant does not have the C122-C166 disulfide bond present in wild-type T-Xyn, whereas T-Xyn-T38C-S50C and T-Xyn-T38C-S50C-C122S-C166S mutants have an introduced disulfide bond, C38-C50, to T-Xyn and T-Xyn-C122S-C166S, respectively. The optimum pH of T-Xyn-T38C-S50C and T-Xyn-T38C-S50C-C122S-C166S was lower than that of T-Xyn and T-Xyn-C122S-C166S. The introduction of a disulfide bond caused a decrease in the optimum temperature and thermal stability of T-Xyn. The existence of a disulfide bond has a strong influence on the hydrolysis characteristics of T-Xyn, which caused changes in the composition and proportion of the hydrolysate products. T-Xyn-T38C-S50C produces the highest level of xylose when using beechwood xylan as the substrate, whereas T-Xyn produces the highest level of xylobiose and T-Xyn-T38C-S50C-C122S-C166S produces the largest amount of xylotriose. When birchwood xylan was used as the substrate, the introduction of a disulfide bond increased the content of xylose, decreased the content of xylotriose and a high degree of polymerization (DP ≥ 5) was observed. The hydrolysis of oat-spelt xylan is more complex with the introduction of the disulfide bond causing an increase in the degradation rate of xylotriose., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The covalent immobilization of xylanase onto the trichlorotriazine-functionalized polyethylene glycol grafted magnetic nanoparticles was exploited to generate a stabilized xylanase with improved catalytic activity and stability. Several tools were deployed to monitor the synthesis and immobilization processes, the loading capacity of nanocarrier, and the structural/chemical characteristics of the nanobiocatalyst. The optimum immobilization yield of xylanase was 260 mg xylanase/g nanocarrier in 20 mM phosphate buffer, pH 6.5 at 25 °C. A forward shift in optimum pH (6.5 to 7.5) and temperature (60 to 70 °C) of xylanase was observed after immobilization and the performance of immobilized enzyme was improved at high temperatures and pHs as affirmed by enhancement of v max (2.69 to 6.01 U/mL) and decreases of E a (14.61 to 13.41 kJ/mol). An increase in K m from 25.51 to 40.42 mg/mL was recorded after immobilization. The obtained results indicated augmented thermal stability of the immobilized xylanase. Notably, it showed good reusability as validated by retention of 50% of its initial activity after nine recycles in enrichment of the pineapple juice clarification after 120 min incubation at 50 °C, pH 4.5. The structural analysis revealed some partial changes in the α-helix and β-sheet content of the enzyme after several recycles., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The synthesis of customized glycoconjugates constitutes a major goal for biocatalysis. To this end, engineered glycosidases have received great attention and, among them, thioglycoligases have proved useful to connect carbohydrates to non-sugar acceptors. However, hitherto the scope of these biocatalysts was considered limited to strong nucleophilic acceptors. Based on the particularities of the GH3 glycosidase family active site, we hypothesized that converting a suitable member into a thioglycoligase could boost the acceptor range. Herein we show the engineering of an acidophilic fungal β-xylosidase into a thioglycoligase with broad acceptor promiscuity. The mutant enzyme displays the ability to form O-, N-, S- and Se- glycosides together with sugar esters and phosphoesters with conversion yields from moderate to high. Analyses also indicate that the pK a of the target compound was the main factor to determine its suitability as glycosylation acceptor. These results expand on the glycoconjugate portfolio attainable through biocatalysis.

Comparative studies of the effects of two commercial enzyme formulations on fiber refining were conducted. Extensive basic characterisation of the enzymes involved, assessment of their hydrolytic activities on different model substrates as well as on different pulps (softwood sulfate, softwood sulfite, hardwood sulfate) were evaluated. Both enzyme formulations showed endoglucanase as well as some xylanase and β-glucosidase activity. In addition, Enzyme A reached a CMC end viscosity of 19.5 mPa compared to 11.1 mPa for Enzyme B. Reducing sugar release almost doubled from 695 μmol mL -1 for hardwood sulfate pulp to 1300 μmol mL -1 for softwood sulfite pulp with Enzyme B under the same conditions. Enzyme A increased the degree of refining even under non-ideal conditions from 23 °SR to up to 50 °SR. Further characterization of hand sheets, made from enzyme pre-treated and refined cellulose fibers with Enzyme A and B, showed that Enzyme A had the best effects leading to hand sheets with increased tensile strength and low air permeability. In summary, the increase in the degree of refining seen for Enzyme A correlated to higher xylanase and β-glucosidase activity and lower endoglucanase activity., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Synergism between enzymes is of crucial importance in cell metabolism. This synergism occurs often through a spatial organisation favouring proximity and substrate channelling. In this context, we developed a strategy for evaluating the impact of the geometry between two enzymes involved in nature in the recycling of the carbon derived from plant cell wall polymers. By using an innovative covalent association process using two protein fragments, Jo and In, we produced two bi-modular chimeric complexes connecting a xylanase and a xylosidase, involved in the deconstruction of xylose-based plant cell wall polymer. We first show that the intrinsic activity of the individual enzymes was preserved. Small Angle X-rays Scattering (SAXS) analysis of the complexes highlighted two different spatial organisations in solution, affecting both the distance between the enzymes (53 Å and 28 Å) and the distance between the catalytic pockets (94 Å and 75 Å). Reducing sugar and HPAEC-PAD analysis revealed different behaviour regarding the hydrolysis of Beechwood xylan. After 24 h of hydrolysis, one complex was able to release a higher amount of reducing sugar compare to the free enzymes (i.e., 15,640 and 14,549 µM of equivalent xylose, respectively). However, more interestingly, the two complexes were able to release variable percentages of xylooligosaccharides compared to the free enzymes. The structure of the complexes revealed some putative steric hindrance, which impacted both enzymatic efficiency and the product profile. This report shows that controlling the spatial geometry between two enzymes would help to better investigate synergism effect within complex multi-enzymatic machinery and control the final product.

Soybean straw cannot be efficiently degraded and utilized by ruminants due to the complex cross-linked structure among cellulose, hemicellulose, and lignin in its cell wall. Xylanase can degrade the xylan component of hemicellulose, destroy the xylan-lignin matrix and, consequently, would theoretically improve the hydrolysis effectiveness of cellulose. Therefore, this study was performed to investigate the effects of recombinant Lentinula edodes xylanase (rLeXyn11A) on fiber structure, hydrolysis, and in vitro ruminal fermentation of soybean straw. Treatment with rLeXyn11A enhanced the hydrolysis of soybean straw with an evident increase in productions of ribose, rhamnose, and xylose. Soybean straw treated by rLeXyn11A had lower hemicellulose content and greater cellulose and lignin contents. The rLeXyn11A could remove xylan, loosen unordered fibrous networks, enhance substrate porosity, and rearrange lignin, consequently increasing the exposure of cellulose and improving the cellulase hydrolysis of soybean straw. Supplemental rLeXyn11A stimulated the dry matter digestion, volatile fatty acids production, and microbial protein synthesis during in vitro ruminal incubation. This paper demonstrated that rLeXyn11A could strengthen the cellulase hydrolysis and in vitro ruminal fermentation of soybean straw by degrading xylan and changing fiber structure, showing its potential for improving the utilization of soybean straw in ruminants., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Magnetic nanoparticles with silica/gold (Au) shell were synthesized to be used as substrates for enzyme immobilization. Successful functionalization was followed by addition of phosphate linkers containing carboxyl end as free end for linking with enzyme. Effect of varied phospho-carboxyl chain lengths based linkers, 3-phosphonopropionic acid (3-PPA) and 16-phosphonopropionic acid (16-PHDA) on kinetic parameters of enzyme were studied. Immobilization of upto 670μg/mg of nanoparticles were observed. Enzyme bound with longer chain linker were observed to have K m (11.8mg/mL) and V max (1.59μmol/min/mg) which was found to be lower than 3-PPA. Reusability assay gave a minimum of 70% activity at 10th time of usage. Thus, an efficient, robust, cost effective and easily separable substrate for restraining the enzyme was prepared., (© 2020 Elsevier Inc. All rights reserved.)

Xylanases, capable of hydrolyzing xylans which are abundant in nature, have been employed as important biocatalyst in many industrial processes. Xylanases with advantageous properties, especially excellent thermostability, are in high demand in industry. In this study, we aim to improve the thermostability of XynCDBFV, a fungal GH11 xylanase. To achieve this aim, we discovered residues 87-QNSS-90 with pronounced flexibility based on B-factor analysis, identified highly conserved residues 87-RGHT-90 in GH11 xylanases by multiple sequence alignment, and constructed four single mutants by substituting residues from 87 to 90 by site-directed mutagenesis. Temperature stability measurements showed promising enhancement of thermostability for all four single mutants, and the thermal tolerant ability from strong to weak is N88 G, S90 T, S89H, Q87R, XynCDBFV. Four single mutants all retained higher than 50% activities after incubation at the optimal temperature 60℃ for 1 h, while the retained activity for XynCDBFV was only 20.94% at the same condition. N88 G retained greater than 60% residual activity after incubation at 65℃ for 1 h, while the residual activity of XynCDBFV decreased rapidly, losing all activity after 45 min of incubation. Molecular dynamics simulations and structural analysis were applied to explore the heat-resistant mechanisms for mutants: novel hydrogen bonding interaction were discovered and accounted for the improved thermostability. Enzyme activity of the single mutants compromised with their thermostability and combined mutations displayed antagonistic effect due to the closed contact of the mutated residues. This study confirms that combining B-factor analysis and multiple sequence alignment is an effective strategy for obtaining a thermostable enzyme, and the negative findings help to recognize limitations in xylanase engineering for preferable properties., (Copyright © 2019 Elsevier Inc. All rights reserved.)

A glycoside hydrolase family 5 (GH5) subfamily 22 gene, designated T81Xyl5_22A, was identified in the genome of the aerobic thermophilic bacterium, Thermogemmatispora sp. T81 (locus A4R35_07040). The gene was cloned and heterologously expressed in Escherichia coli and the gene product characterized biochemically. The recombinant enzyme had an optimal catalytic activity at pH5.0 and 65 °C, and was active against beechwood xylan and rye arabinoxylan. It yielded only xylose molecules as products of beechwood xylan hydrolysis, indicating that it is a GH5 family β-d-xylosidase. Using 4-nitrophenyl β-d-xylopyranoside (pNPX) as a substrate, the K M , Vmax, k cat and k cat /K M kinetic parameters were determined as 0.25 ± 0.03 mM, 889.47 ± 28.54 U/mg, 39.20 s -1 and 156.8 mM -1  s -1 , respectively. Small-angle X-ray scattering (SAXS) data enabled reconstruction of the enzyme's low-resolution molecular envelope and revealed that it formed dimers in solution. As far as we are aware, this is the first description of a thermostable bacterial GH5 family β-d-xylosidase., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Xylan, a prominent component of cellulosic biomass, has a high potential for degradation into reducing sugars, and subsequent conversion into bioethanol. This process requires a range of xylanolytic enzymes. Among them, β-xylosidases are crucial, because they hydrolyze more glycosidic bonds than any of the other xylanolytic enzymes. They also enhance the efficiency of the process by degrading xylooligosaccharides, which are potent inhibitors of other hemicellulose-/xylan-converting enzymes. On the other hand, the β-xylosidase itself is also inhibited by monosaccharides that may be generated in high concentrations during the saccharification process. Structurally, β-xylosidases are diverse enzymes with different substrate specificities and enzyme mechanisms. Here, we review the structural diversity and catalytic mechanisms of β-xylosidases, and discuss their inhibition by monosaccharides.

The aim of this study was to enhance the production of xylano-pectinolytic enzymes concurrently and also to reduce the fermentation period. In this study, the effect of agro-residues extract-based inoculum on yield and fermentation time of xylano-pectinolytic enzymes was studied. Microbial inoculum and fermentation media were supplemented with xylan and pectin polysaccharides derived from agro-based residues. Enzymes production parameters were optimized through two-stage statistical design approach. Under optimized conditions (temperature 37°C, pH 7.2, K 2 HPO 4 0.22%, MgSO 4 0.1%, gram flour 5.6%, substrate: moisture ratio 1:2, inoculum size 20%, agro-based crude xylan in production media 0.45%, and agro-based crude xylan-pectin in inoculum 0.13%), nearly 28,255 ± 565 and 9,202 ± 193 IU of xylanase and pectinase, respectively, were obtained per gram of substrate in a time interval of 6 days only. The yield of both xylano-pectinolytic enzymes was enhanced along with a reduction of nearly 24 h in fermentation time in comparison with control, using polysaccharides extracted from agro-residues. The activity of different types of pectinase enzymes such as exo-polymethylgalacturonase (exo-PMG), endo-PMG, exo-polygalacturonase (exo-PG), endo-PG, pectin lyase, pectate lyase, and pectin esterase was obtained as 1,601, 12.13, 5637, 24.86, 118.62, 124.32, and 12.56 IU/g, respectively, and was nearly twofold higher than obtained for all seven types in control samples. This is the first report mentioning the methodology for enhanced production of xylano-pectinolytic enzymes in short solid-state fermentation cycle using agro-residues extract-based inoculum and production media., (© 2019 American Institute of Chemical Engineers.)

Objectives: To develop a novel multi-catalytic domain (CD) xylanase Xyn2083 from Clostridium clariflavum by expression of its truncated forms in Escherichia coli and cooperation of xylanase with cellulase in the hydrolysis of waste lignocellulosic resources., Results: Xyn2083 has two glycoside hydrolase family (GH) domains GH11 and GH10. These two catalytic domains functioned synergistically in xylan hydrolysis. The recombinant protein with GH11 domain, Xyn2083GH11, had the highest xylanase activity among three constructed truncated forms. The deletion of N-terminal extra amino acid residues of Xyn2083GH11 decreased catalytic activity as well as the stability of the enzyme. The hydrolysis rates of cellulose and xylan in the pretreated corn cobs were 90.56% and 72.80% with the addition of Xyn2083GH11 and cellulase, whereas those were 67.95% and 34.45% using sole cellulase respectively. The structural analysis of substrates indicated that the addition of Xyn2083GH11 led to a looser structure and more exposure of crystal cellulose for cellulase to approach., Conclusions: Since the native multi-CDs' xylanases are rare, the thermostable Xyn2083 provides a good source for functional studies of two CDs coexisted in one xylanase and for potential applications after modification.

Although core xylose on glycoproteins has been implicated in allergy, infection and other biological processes, research on core xylose modification is rare. The lack of a β-d-xylosidase that can catalytically remove the core xylose directly from glycoproteins is a reason for this. Through functional genomic analysis, we identified a glycoprotein core xylosidase and named it gpcXase I. gpcXase I is located immediately downstream of glycoprotein core fucosidase cFase I in Elizabethkingia meningoseptica. These two genes form a functional operon for glycoprotein core modifications. Three acidic residues (Asp-200, Asp-304 and Glu-649) were identified as key catalytic sites for gpcXase I activity, suggesting a unique triacdic mechanize for its activity. Asp-200 was identified a novel and essential base catalysts in the catalytic process, Asp-304 and Glu-649 was function as catalytic nucleophiles and acid catalysts, respectively. In addition, IgE-specific reactions were detected in 55% of serum samples collected from 40 allergic patients, and the reactions were significantly attenuated by removal of the core xylose of the allergen by treatment with gpcXase I. gpcXase I is a novel tool for basic and clinical glycomics., (Copyright © 2019 Elsevier Inc. All rights reserved.)

β-xylosidase is an essential enzyme for breakdown of xylan to d-xylose. It has a significant potential application value for medicine, food, paper and pulp, and biofuel industries. Due to the negative consequences caused by buried free cysteine residues, mutational substitution of such residues is often accompanied by a notable increase in thermal stability. To characterize the role of cysteine residues in the structure, function and stability of Selenomonas ruminantium β-d-Xylosidase (SXA), we prepared and evaluated wild-type and four cysteines- deficient SXA proteins. Buried cysteine residues were replaced with. In comparison with the wild-type, the Km values of the mutants remained relatively constant while their kcat values decreased. The C101V and C286V displayed higher thermal stability than the wild-type at 55 and 60 °C. Conformational changes of the secondary and tertiary structure as derived from circular dichroism and fluorescence spectroscopy revealed that changing a buried cysteine to a hydrophobic residue could lead to an increase in thermal stability with minimal perturbation of the wild-type protein structure. In addition to experimental methods, the stability of WT SXA and C101V and C286V mutants at 333 K was also studied by MD simulation. Our theoretical data had a good agreement with the experimental results., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This study focuses on enzymatic saccharification of hemicellulose part of the pretreated Finger millet straw (FMS) for production of xylose. The variation in the carbohydrate composition of FMS was analysed when subjected to different pretreatments. The recombinant endo-1,4-β-xylanase (CtXyn11A) was most active on the FMS pretreated with 1% (w/v) NaOH combined with oven heating at 120 °C for 20 min, resulting in a total reducing sugar yield (TRS) of 32 mg/g pretreated biomass. The pretreatment aided in concentrating the holocellulose content from 69.3% of raw powdered FMS to 76.4%. The post-treatment solid biomass yield was 0.36 g/g raw biomass. The two-step optimization of hemicellulose saccharification from the above pretreated FMS with i) endo-1,4-β-xylanase (CtXyn11A) at 55 °C and ii) exo-1,4-β-xylosidase (BoGH43A) at 37 °C, both at pH 7.5 by Box-Behnken design yielded the TRS of 70 mg/g pretreated biomass. The percentage conversion of xylan to xylose by CtXyn11A and BoGH43A was 24.7%., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Aspergillus quadrilineatus RSNK-1 produced a multi-enzymatic system containing (U/gds) β-mannanase (1021), endo-xylanase (1 9 1), α-galactosidase (3.42), β-xylosidase (0.07) and β-glucosidase (0.28) on low-cost copra meal (CM) in SSF. The enzyme preparation was covalently immobilized on aluminum oxide pellets (3 mm) under statistically optimized conditions leading to 73.17% immobilization yield. The immobilized enzyme (Man-AOP) displayed enhanced thermal and pH stability. Man-AOP was characterized by FTIR, SEM and PXRD revealing a covalent interaction. The bio-conjugate was successfully recycled for mannooligosaccharide (MOS) generation from locust bean gum (LBG) up to 10 cycles, yielding an average of 0.95 mg MOS/cycle. Man-AOP was also effective in clarification of apple, kiwi, orange and peach juices and enhanced their reducing sugar content. The bio-conjugate was useful in generation of MOS from mannan and enrichment of fruit juices., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Aims: This work aims to determine the tolerance of xylanase towards enzyme-generated oxidative conditions, such as those produced by the peroxidase or laccase mediator systems (LMS)., Methods and Results: The activity of Thermomyces lanuginosus xylanase was measured after incubation with lignin peroxidase, manganese peroxidase or laccase plus various mediators. The laccase system, using mediators such as 1-hydroxybenzotriazole and violuric acid, resulted in complete loss of xylanase activity, accompanied by an increase in the solution potential. However, an increase in solution potential alone was not sufficient to inactivate xylanase, nor was loss of xylanase activity always accompanied by a significant increase in solution potential, as observed with N-hydroxyphthalimide as the mediator. Neither lignin peroxidase nor manganese peroxidase impacted xylanase activity; only extended treatment with elevated hydrogen peroxide concentration promoted modest xylanase activity loss. The mechanism of inactivation as determined by the tryptophan-modifying reagent N-bromosuccinimide (NBS) indicated that oxidation of just one of the eight tryptophan residues of T. lanuginosus xylanase would be sufficient to result in complete loss of xylanase activity, since xylanase is completely inactivated at 1 : 1 molar ratio of NBS to xylanase., Conclusions: While showing tolerance to peroxidase-based enzyme systems, T. lanuginosus xylanase is readily inactivated in the presence of the LMS. Based upon treatment with NBS as the oxidant, inactivation can be attributed to modification of a single tryptophan residue., Significance and Impact of the Study: The simultaneous application of mixed hydrolytic and oxidative enzyme systems is of importance to biomass processing industries. Understanding the tolerance of xylanase to oxidative conditions will facilitate the design of reaction conditions or enzyme variants to maximize the impact of mixed enzyme systems., (© 2019 The Society for Applied Microbiology.)

Laccase has the enormous potential to be implemented as the multitasking biocatalyst in whole process of paper making. The enzyme can utilize effectively for pulping, delignification of pulps as alone or in the combination with other bleaching enzymes. Laccase has been evaluated for the biografting of pulp fibers, decolorize and stabilize the effluent of the paper mills, biotransformation of high molecular weight (HMW) lignins to lower molecular weight (LMW) aromatic compounds. Further this enzyme has the huge possibilities to apply for deinking of old newsprint (ONP) and pitch removal from varieties of the different pulps. There is no doubt on versatility mode of action of laccases in paper industry, but still there is limited commercialization of this enzyme has been possible, because of laccases has the less redox potential (E0) and needs mediators for the oxidation of non phenolic substrates, production of enzyme is cost intensive at large scale. This review is providing the proper updated information on the state of the art of different applications of laccase in paper industry. It also confer the interpretation to the readers about the areas of extensively studied and the field where there is still much left to be done., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Myceliophthora heterothallica is a thermophilic fungus potentially relevant for the production of enzymes involved in the degradation of plant biomass. A xylanase encoding gene of this species was identified by means of RT-PCR using primers designed based on a xylanase coding sequence (GH11) of the fungus M. thermophila. The obtained gene was ligated to the vector pET28a(+) and the construct was transformed into Escherichia coli cells. The recombinant xylanase (r-ec-XylMh) was heterologously expressed, and the highest activity was observed at 55 °C and pH 6. The enzyme stability was greater than 70% between pH 4.5 and 9.5 and the inclusion of glycerol (50%) resulted in a significant increase in thermostability. Under these conditions, the enzyme retained more than 50% residual activity when incubated at 65 °C for 1 h, and approximately 30% activity when incubated at 70 °C for the same period. The tested cations did not increase xylanolytic activity, and the enzyme indicated significant tolerance to several phenolic compounds after 24 h, as well as high specificity for xylan, with no activity for other substrates such as CMC (carboxymethylcellulose), Avicel, pNPX (p-nitrophenyl-β-D-xylopyranoside) and pNPA (p-nitrophenyl-α-L-arabinofuranoside), and is thus, of potential relevance in pulp bleaching., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Rational design is an important tool for sculpting functional and stability properties of proteins and its potential can be much magnified when combined with in vitro and natural evolutionary diversity. Herein, we report the structure-guided design of a xylose-releasing exo-β-1,4-xylanase from an inactive member of glycoside hydrolase family 43 (GH43). Structural analysis revealed a nonconserved substitution (Lys 247 ) that results in the disruption of the hydrogen bond network that supports catalysis. The mutation of this residue to a conserved serine restored the catalytic activity and crystal structure elucidation of the mutant confirmed the recovery of the proper orientation of the catalytically relevant histidine. Interestingly, the tailored enzyme can cleave both xylooligosaccharides and xylan, releasing xylose as the main product, being the first xylose-releasing exo-β-1,4-xylanase reported in the GH43 family. This enzyme presents a unique active-site topology when compared with closely related β-xylosidases, which is the absence of a hydrophobic barrier at the positive-subsite region, allowing the accommodation of long substrates. Therefore, the combination of rational design for catalytic activation along with naturally occurring differences in the substrate binding interface led to the discovery of a novel activity within the GH43 family. In addition, these results demonstrate the importance of solvation of the β-propeller hollow for GH43 catalytic function and expand our mechanistic understanding about the diverse modes of action of GH43 members, a key and polyspecific carbohydrate-active enzyme family abundant in most plant cell-wall-degrading microorganisms., (© 2018 Wiley Periodicals, Inc.)

A thermostable β-xylosidase gene Tpexyl3 from Thermotoga petrophila DSM 13,995 was cloned and overexpressed by Escherichia coli. Recombinant Tpexyl3 was purified, and its molecular weight was approximately 86.7 kDa. Its optimal activity was exhibited at pH 6.0 and 90 °C. It had broad specificity to xylopyranosyl, arabinopyranosyl, arabinofuranosyl and glucopyranosyl moieties. The β-xylosidase activity of the recombinant Tpexyl3 was 6.81 U/mL in the LB medium and 151.4 U/mL in a 7.5 L bio-reactor. It was applied to transform ginsenoside extract into the pharmacologically active minor ginsenoside 20(S)-Rg3, which was combined with thermostable β-glucosidase Tpebgl3. After transforming under optimal condition, the 20 g/L of ginsenoside extract was transformed into 6.28 g/L of Rg3 within 90 min, with a corresponding molar conversion of 95.0% and Rg3 productivity of 1793.49 mg/L/h, respectively. This study is the highest report of a GH3 family glycosidase with arabinopyranosidase activity and also the first report on the high substrate concentration bioconversion of ginsenoside extract to ginsenoside 20(S)-Rg3 by using two thermostable glycosidases., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Brunfelsia calycina flowers lose anthocyanins rapidly and are therefore well suited for the study of anthocyanin degradation mechanisms, which are unclear in planta. Here, we isolated an anthocyanin-β-glycosidase from B. calycina petals. The MS/MS (Mass Spectrometry) peptide sequencing showed that the enzyme (72 kDa) was a β-xylosidase (BcXyl). The enzyme showed high activity to p-Nitrophenyl-β-d-galactopyranoside (pNPGa) and p-Nitrophenyl-β-d-xylopyranoside (pNPX), while no activity to p-Nitrophenyl-β-d-glucopyranoside (pNPG) or p-Nitrophenyl-β-D-mannopyranoside (pNPM) was seen. The optimum temperature of BcXyl was 40 °C and the optimum pH was 5.0. The enzyme was strongly inhibited by 1 mM D-gluconate and Ag⁺. HPLC (High Performance Liquid Chromatography) analysis showed that BcXyl catalyzed the degradation of an anthocyanin component of B. calycina , and the release of xylose and galactose due to hydrolysis of glycosidic bonds by BcXyl was detected by GC (Gas Chromatography) /MS. A full-length mRNA sequence (2358 bp) of BcXyl (NCBI No. MK411219) was obtained and the deduced protein sequence shared conserved domains with two anthocyanin-β-glycosidases (Bgln and BadGluc, characterized in fungi). BcXyl, Bgln and BadGluc belong to AB subfamily of Glycoside hydrolase family 3. Similar to BcPrx01 , an anthocyanin-degradation-related Peroxidase ( POD ), BcXyl was dramatically activated at the stage at which the rapid anthocyanin degradation occurred. Taken together, we suggest that BcXyl may be the first anthocyanin-β-glycosidase identified in higher plants.