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Plant cell walls constitute physical barriers that restrict access of microbial pathogens to the contents of plant cells. The primary cell wall of multicellular plants predominantly consists of cellulose, hemicellulose, and pectin, and its composition can change upon stress. BETA-XYLOSIDASE4 (BXL4) belongs to a seven-member gene family in Arabidopsis (Arabidopsis thaliana), one of which encodes a protein (BXL1) involved in cell wall remodeling. We assayed the influence of BXL4 on plant immunity and investigated the subcellular localization and enzymatic activity of BXL4, making use of mutant and overexpression lines. BXL4 localized to the apoplast and was induced upon infection with the necrotrophic fungal pathogen Botrytis cinerea in a jasmonoyl isoleucine-dependent manner. The bxl4 mutants showed a reduced resistance to B. cinerea, while resistance was increased in conditional overexpression lines. Ectopic expression of BXL4 in Arabidopsis seed coat epidermal cells rescued a bxl1 mutant phenotype, suggesting that, like BXL1, BXL4 has both xylosidase and arabinosidase activity. We conclude that BXL4 is a xylosidase/arabinosidase that is secreted to the apoplast and its expression is upregulated under pathogen attack, contributing to immunity against B. cinerea, possibly by removal of arabinose and xylose side-chains of polysaccharides in the primary cell wall.

The utilization of sugarcane bagasse (SB) in fermentation requires pretreatment processes to render fermentable components available to microorganisms. Pretreatment by using ionic liquids (ILs) is considered promising but the high cost is an impediment in its adoption, therefore, a mixture of IL pretreated and untreated SB was utilized to obtain bacterial multienzyme under solid-state fermentation (SSF). Bacillus aestuarii UE25, a thermophilic strain was utilized for that purpose. Fermentation conditions were optimized by adopting a central composite design. The model showed a good correlation between the predicted and the experimental values for amylase, xylanase, endoglucanase, and β-glucosidase. Volumetric and specific productivity of xylanase (4580 IU ml-1 h-1 , 244.25 IU mg-1 substrate, and 50 IU mg-1 protein) were higher than the other enzymes. Changes in lignin content and reduced cellulose crystallinity due to IL pretreatment, followed by fermentation, were visualized by scanning electron microscopy, Fourier transform infrared spectroscopy, and Nuclear magnetic resonance. The strategy adopted by utilizing a mixture of IL pretreated and untreated SB under SSF proved promising to obtain high titers of different enzymes simultaneously. Since the bacterial strain used is thermophilic, therefore, the multienzyme can find its application in commercial processes which are carried out at high temperatures.

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.

As a promising feedstock, alkali-extracted xylan from lignocellulosic biomass is desired for producing xylose, which can be used for renewable biofuels production. In this study, an efficient pathway has been established for low-cost and high-yield production of xylose by hydrolysis of alkali-extracted xylan from agricultural wastes using an endo-1,4-xylanase (XYLA) from Bacillus safensis TCCC 111022 and a β-xylosidase (XYLO) from B. pumilus TCCC 11573. The optimum activities of recombinant XYLA (rXYLA) and XYLO (rXYLO) were 60 ℃ and pH 8.0, and 30 ℃ and pH 7.0, respectively. They were stable over a broad pH range (pH 6.0-11.0 and 7.0-10.0). rXYLO showed a relatively high xylose tolerance up to 100 mM. Furthermore, the yield of xylose from wheat straw, rice straw, corn stover, corncob and sugarcane bagasse by rXYLA and rXYLO was 63.77%, 71.76%, 68.55%, 53.81%, and 58.58%, respectively. This study demonstrated a strategy to produce xylose from agricultural wastes by integrating alkali-extracted xylan and enzymatic hydrolysis.

International audience; $Dictyoglomus\ thermophilum$ 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.

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 (Ara2Xyl3) and O-β-D-xylopyranosyl-(1 → 4)-[O-4-O-methyl-α-D-glucuronopyranosyl-(l → 2)]-β-D-xylopyranosyl-(1 → 4)-β-D-xylopyranose (MeGlcA2Xyl3), 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 (Ara3Xyl4) 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 (MeGlcA3Xyl4). 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 MeGlcA3Xyl4, while the arabinose branch does not significantly affect the enzyme activity because it degrades Ara3Xyl4 as rapidly as unmodified xylotetraose. The model structure suggested that BhXyl39 enhanced the activity for MeGlcA3Xyl4 by forming a hydrogen bond between glucuronic acid and Lys265. BhXyl52 did not hydrolyze Ara3Xyl4 and MeGlcA3Xyl4 because it has a narrow substrate binding pocket and 2- and 3-hydroxyl groups of xylose at subsite +1 hydrogen bond to the enzyme.

This study was undertaken to explore alternative applications of the widely known entomopathogenic/endophytic fungus, Beauveria bassiana, besides its sole use as a biocontrol agent. B. bassiana SAN01, was investigated for the production of two glycoside hydrolases, xylanase and endoglucanase under submerged conditions. Among the different biomass tested, wheat bran provided the best results for both xylanase and endoglucanase, and their production levels were further enhanced using response surface methodology. Under optimised conditions, heightened yields of 1061 U/ml and 23.03 U/ml were observed for xylanase and endoglucanase, respectively, which were 3.44 and 1.35 folds higher than their initial yields. These are the highest ever production levels reported for xylanase and endoglucanase from any B. bassiana strain or any known entomopathogenic fungi. Furthermore, the efficacy of xylanase/endoglucanase cocktail in the saccharification of sugarcane bagasse was evaluated. The highest amount of reducing sugar released from the pretreated biomass by the action of the crude Beauveria enzyme cocktail was recorded at 30°C after 8 h incubation. The significant activities of the hydrolytic enzymes recorded with B. bassiana in this study thus present promising avenues for the use of the entomopathogen as a new source of industrial enzymes and by extension, other biotechnological applications.

MeXyl31, a member of glycoside hydrolase family 31 (GH31), is the α-xylosidase isolated from a soil metagenomic library. The enzyme degrades α-xylosyl substrate such as isoprimeverose, α-d-xylopyranosyl-(1→6)-glucopyranose. The crystal structure of MeXyl31 was determined at 1.80 Å resolution. MeXyl31 forms the tetrameric state. The complexed structure with a xylose in the − 1 subsite (α-xylose binding site) shows that the enzyme strictly recognizes α-xylose. Structural comparison between MeXyl31 and its homologue, Aspergillus niger α-xylosidase in GH31, gave insights into the positive subsite of MeXyl31. Firstly, in the tetrameric enzyme, two monomers (a catalytic monomer and the adjacent monomer), are involved in substrate recognition. Secondly, the adjacent monomer composes a part of positive subsites in MeXyl31. Docking simulation and site-directed mutagenesis suggested that the Arg100 from the adjacent monomer is partially involved in the recognizing of a glucopyranose of isoprimeverose.

2,3-Butanediol (2,3-BD) is a valuable platform chemical with extensive industrial applications. The demand for its safe and economic microbial synthesis resulted in increased interest in the isolation of non-pathogenic producers capable of converting cheap and renewable materials. This study reports the isolation of 62 new non-pathogenic Bacillus strains producing 2,3-BD. Three strains were found, including Bacillus velezensis 5RB, B. toyonensis 11RA and B. safensis 14A, which belonged to species not previously reported as 2,3-BD producers. Seventeen strains displayed cellulolytic activity, degrading carboxymethyl cellulose, HE-cellulose and β-glucan, whereas 5 strains were also able to hydrolyze arabinoxylan, arabinan, galactomannan, xyloglucan, xylan and galactan (included in hemicellulose), as well as starch and fructans. The strain capacity to ferment lignocellulosic sugars to 2,3-BD corresponded to the activities of CAZymes engaged in the hydrolysis of the relevant polysaccharides. Regardless of species, Bacillus strains converted glucose, cellobiose, and mannose to 2,3-BD with higher concentration, productivity and yield than arabinose, xylose, and galactose. B. velezensis 5RB was the only strain, which was capable to produce 2,3-BD from all lignocellulosic sugars which corresponded to the presence of the highest extracellular endo-α-(1→5)-l-arabinanase, endo-1,4-β-xylanase, 1,4-β-xylosidase, and endo-β-1,4-galactanase activity. The annotation of its genome showed the presence of 26 genes encoding glycoside-hydrolases allowing biomass degradation. In conclusion, the non-risk new isolated Bacillus strains are promising for 2,3-BD production from agro-industrial residues. When lignocellulose is used, the process would be more efficient if the substrate content is rich in cellulose and low in hemicelluloses.

13 pags., 6 figs., 2 tabs., 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) 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)., This work was supported by grants from the Spanish Ministry of Economy and Competitiveness through throughgrants BIO2016-76601-C3-3-R, BIOFABCEL CTQ2017-84966-C2-2-R. We are grateful to the staff of the Syn-chrotron Radiation Sources at Alba (Barcelona, Spain)and ESRF (Grenoble, France) for providing accessand for technical assistance at BL13-XALOC and Massif beamlines, and to the ‘Xarxa de Referencia en Biotecnologia’ (XRB).

A metagenomic library of mangrove soil samples consisting of approximately 11,000 clones was constructed, and a rare bifunctional cellobiohydrolase/β-xylosidase Cbh2124 was identified by functional screening. Cbh2124 displayed the highest homology (56.43%) with a protein of the glycoside hydrolase 10 (GH10) family from Proteobacteria. Phylogenetic analysis confirmed that Cbh2124 belongs to the GH10 family. The recombinant enzyme showed a strong cellobiohydrolase activity and a relatively high β-xylosidase activity, and its catalytic efficiency to the cellobiose substrate was as high as 1.27 × 10

This study evaluated the effects of a combination of xylanase, amylase, and protease (XAP), with probiotics (3 Bacillus spp.) supplementation on apparent ileal digestibility (AID) and apparent total tract digestibility (ATTD) of nutrients in Cobb 500 broilers from 0 to 21 d. A completely randomized 2 × 4 factorial design (2 levels of fiber; 4 types of supplements) with 8 replicate cages (6 birds/cage) was used. Each low and high-fiber diet contained 500 FTU/kg Buttiauxella sp. phytase and was supplemented with: (a) none (control), (b) XAP (2,000 U xylanase + 200 U amylase + 4,000 U protease/kg diet), (c) probiotics (75,000 CFU/g of Bacillus spp.), or (d) XAP + probiotics. High fiber decreased (P < 0.05) nitrogen-corrected apparent metabolizable energy (AMEn), AID of all amino acids (AA), AID and ATTD of dry matter (DM), crude protein (CP), starch, and gross energy (GE). High fiber increased (P < 0.01) the flow of total non-starch polysaccharides (NSP) in both ileum and total tract. The XAP + probiotics increased (P < 0.01) AMEn as well as AID and ATTD of DM, CP, GE, starch, while alone, XAP yielded similar improvement except for DM compared with control. The supplemental XAP alone improved (P < 0.01) the digestibility of most of the AAs compared with control. Moreover, XAP + probiotics increased (P < 0.05) AID of all AA except arginine and serine compared with control. A fiber × supplements interaction (P < 0.05) was found for AID of histidine and threonine, and their digestibility in high-fiber diet was improved to a level comparable to low-fiber diet by XAP + probiotics. The flow of NSP in XAP group was 5 to 6% lower than in control while NSP flow in XAP + probiotic group was further 4% lower than that of XAP group (P < 0.01). The results infer that the combination of XAP and probiotics can effectively optimize the nutrient digestibility in broilers fed both low and high-fiber diets.

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 KM, Vmax, kcat and kcat/KM 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.

Xylanases are the enzymes that catalyze the breakdown of the main hemicellulose present in plant cell walls. They have attracted attention due to their biotechnological potential for the preparation of industrially interesting products from lignocellulose. While many xylanases have been characterized from bacteria and filamentous fungi, information on yeast xylanases is scarce and no yeast xylanase belonging to glycoside hydrolase (GH) family 30 has been described so far. Here, we cloned, expressed and characterized GH30 xylanase

Two α-L-arabinofuranosidases (BfdABF1 and BfdABF3) and a β-D-xylosidase (BfdXYL2) genes were cloned from

PcAxy43B is a modular protein comprising a catalytic domain of glycoside hydrolase family 43 (GH43), a family 6 carbohydrate-binding module (CBM6), and a family 36 carbohydrate-binding module (CBM36) and found to be a novel multifunctional xylanolytic enzyme from Paenibacillus curdlanolyticus B-6. This enzyme exhibited α-l-arabinofuranosidase, endoxylanase, and β-d-xylosidase activities. The α-l-arabinofuranosidase activity of PcAxy43B revealed a new property of GH43, via the release of both long-chain cereal arabinoxylan and short-chain arabinoxylooligosaccharide (AXOS), as well as release from both the C(O)(2) and C(O)(3) positions of AXOS, which is different from what has been seen for other arabinofuranosidases. PcAxy43B liberated a series of xylooligosaccharides (XOSs) from birchwood xylan and xylohexaose, indicating that PcAxy43B exhibited endoxylanase activity. PcAxy43B produced xylose from xylobiose and reacted with p-nitrophenyl-β-d-xylopyranoside as a result of β-xylosidase activity. PcAxy43B effectively released arabinose together with XOSs and xylose from the highly arabinosyl-substituted rye arabinoxylan. Moreover, PcAxy43B showed significant synergistic action with the trifunctional endoxylanase/β-xylosidase/α-l-arabinofuranosidase PcAxy43A and the endoxylanase Xyn10C from strain B-6, in which almost all products produced from rye arabinoxylan by these combined enzymes were arabinose and xylose. In addition, the presence of CBM36 was found to be necessary for the endoxylanase property of PcAxy43B. PcAxy43B is capable of hydrolyzing untreated cereal biomass, corn hull, and rice straw into XOSs and xylose. Hence, PcAxy43B, a significant accessory multifunctional xylanolytic enzyme, is a potential candidate for application in the saccharification of cereal biomass. IMPORTANCE Enzymatic saccharification of cereal biomass is a strategy for the production of fermented sugars from low-price raw materials. In the present study, PcAxy43B from P. curdlanolyticus B-6 was found to be a novel multifunctional α-l-arabinofuranosidase/endoxylanase/β-d-xylosidase enzyme of glycoside hydrolase family 43. It is effective in releasing arabinose, xylose, and XOSs from the highly arabinosyl-substituted rye arabinoxylan, which is usually resistant to hydrolysis by xylanolytic enzymes. Moreover, almost all products produced from rye arabinoxylan by the combination of PcAxy43B with the trifunctional xylanolytic enzyme PcAxy43A and the endoxylanase Xyn10C from strain B-6 were arabinose and xylose, which can be used to produce several value-added products. In addition, PcAxy43B is capable of hydrolyzing untreated cereal biomass into XOSs and xylose. Thus, PcAxy43B is an important multifunctional xylanolytic enzyme with high potential in biotechnology.

Xylanases have been applied in many industrial fields. To improve the activity and thermostability of the xylanase CDBFV from Neocallimastix patriciarum (GenBank accession no. KP691331), submodule C2 from hyperthermophilic CBM9_1-2 was inserted into the N- and/or C-terminal regions of the CDBFV protein (producing C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2) by genetic engineering. CDBFV and the hybrid proteins were successfully expressed in Escherichia coli BL21 (DE3). Enzymatic property analysis indicates that the C2 submodule had a significant effect on enhancing the thermostability of the CDBFV. At the optimal temperature (60.0 °C), the half-lives of the three chimeras C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2 are 1.5 times (37.5 min), 4.9 times (122.2 min), and 3.8 times (93.1 min) longer than that of wild-type CDBFV (24.8 min), respectively. More importantly, structural analysis and molecular dynamics (MD) simulation revealed that the improved thermal stability of the chimera CDBFV-C2 was on account of the formation of four relatively stable additional hydrogen bonds (S42-S462, T59-E277, S41-K463, and S44-G371), which increased the protein structure’s stability. The thermostability characteristics of CDBFV-C2 make it a viable enzyme for industrial applications.

Notoginsenoside R1 and ginsenoside Rg1 are the main active ingredients of

α-Xylosidases release the α-D-xylopyranosyl side chain from di- and oligosaccharides derived from xyloglucans and are involved in xyloglucan degradation. In this study, an extracellular α-xylosidase, named AxyB, is identified and characterized in Aspergillus oryzae. AxyB belongs to the glycoside hydrolase family 31 and releases D-xylose from isoprimeverose (α-D-xylopyranosyl-(1 → 6)-D-glucopyranose) and xyloglucan oligosaccharides. In the hydrolysis of xyloglucan oligosaccharides (XLLG, Glc

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.

Xylanase enzyme has been classified as an enzyme belonging to the glycoside hydrolase family. The catalytic action of xylanase is focused on the degradation of xylan, a substrate for this enzyme comprising of a complex arrangement of monosaccharides interlinked with the help of ester and glycosidic bonds. Xylan represents the second most profuse renewable polysaccharide present on earth. Breakage of the β- 1, 4-glycoside linkage in the xylan polymer is what makes xylanase enzyme an important biocatalyst favoring various applications including treatment of pulp for improving paper quality, improvement of bread quality, treatment of lignocelluloses waste, production of xylose sugar and production of biological fuels. Most recently, xylanase has been exploited in the food industry for the purpose of fruit juice clarification. Turbidity caused by the colloidal polysaccharides present in the freshly squeezed fruit juice poses a setback to the fruit juice industry since the commercial product must be clear and free of excess polysaccharides to improve juice quality and storage life. This review gives an overview of the recent advancements made in regards to xylanase enzyme being used commercially with main focus on its role in fruit juice clarification.

The hyperthermophilic bacterium Caldicellulosiruptor kristjansonii encodes an unusual enzyme, CkXyn10C-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 CkXyn10C-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.

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.

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

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.

A GH43 bifunctional β-xylosidase encoding gene (XylRBM26) was cloned from Massilia sp. RBM26 and successfully expressed in Escherichia coli. Recombinant XylRBM26 exhibited β-xylosidase and α- l -arabinofuranosidase activities. When 4-nitrophenyl-β- d -xylopyranoside was used as a substrate, the enzyme reached optimal activity at pH 6.5 and 50°C and remained stable at pH 5.0–10.0. Purified XylRBM26 presented good salt tolerance and retained 96.6% activity in 3.5 M NaCl and 77.9% initial activity even in 4.0 M NaCl. In addition, it exhibited high tolerance to xylose with Ki value of 500 mM. This study was the first to identify and characterize NaCl-tolerant β-xylosidase/α- l -arabinofuranosidase from the gut microbiota. The enzyme's salt, xylose, and alkali stability and resistance to various chemicals make it a potential biocatalyst for the saccharification of lignocellulose, the food industry, and industrial processes conducted in sea water.

Four experiments were conducted to examine the effects of a recombinant bacterial expansin-like protein (BsEXLX1) from Bacillus subtilis and a commercial exogenous fibrolytic enzyme (EFE) preparation for ruminants on hydrolysis of pure substrates (cellulose and xylan) and in vitro digestibility of bermudagrass haylage (BMH). Recombinant Escherichia coli BL21 strain was used to express BsEXLX1; the protein was purified using an affinity column. In experiment 1, carboxymethylcellulose, Whatman #1 filter paper (General Electric, Boston, MA) and oat-spelt xylan substrates were subjected to 4 treatments (1) sodium citrate buffer (control), (2) BsEXLX1 (162 µg/g of substrate), (3) EFE (2.3 mg/g of substrate), and (4) EFE + BsELX1 in 3 independent runs. Samples were incubated at optimal conditions for both additives (pH 5 and 50°C) or at ruminal (pH 6 and 39°C) or ambient (pH 6 and 25°C) conditions for 24 h and sugar release was measured. In experiment 2, digestibility in vitro of BMH was examined after treatment with the following: (1) control (buffer only), (2) BsEXLX1 (162 µg/g of dry matter), (3) EFE (2.2 mg/g of dry matter), and (4) EFE + BsEXLX1 in 3 independent runs at 39°C for 24 h. Experiment 3 examined effects of EFE and BsEXLX1 on simulated preingestive hydrolysis and profile of released sugars from BMH after samples were suspended in deionized water with sodium azide at 25°C for 24 h in 2 independent runs. In experiment 4, the sequence of the BsEXLX1 purified protein was compared with 447 ruminal bacterial genomes to identify similar proteins from the rumen. In experiment 1, compared with EFE alone, EFE and BsEXLX1 synergistically increased sugar release from carboxymethylcellulose and Whatman #1 filter paper under all simulated conditions; however, hydrolysis of xylan was not improved. In experiment 2, compared with EFE alone, treatment with EFE and BsEXLX1 increased neutral detergent fiber and acid detergent fiber digestibility of bermudagrass haylage (by 5.5 and 15%, respectively) and total volatile fatty acid concentrations, and decreased acetate-propionate ratio. In experiment 3, compared with EFE alone. The EFE and BsEXLX1 synergistically reduced concentrations of neutral detergent fiber and acid detergent fiber and increased release of sugars by 9.3%, particularly cellobiose (72.5%). In experiment 4, a similar sequence to that of BsEXLX1 was identified in Bacillus licheniformis, and similar hypothetical protein sequences were identified in Ruminococcus flavefaciens strains along with different protein structures in E. xylanophilum and Lachnospiraceae. This study showed that an expansin-like protein synergistically increased the hydrolysis of pure cellulose substrates and the hydrolysis and digestibility in vitro of BMH.

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.

Simultaneous production of alkaline xylanase and all seven types of pectinases by a bacterial isolate, under solid-state fermentation was checked in this study. Under optimized conditions, high concurrent production of xylanase (22,800 ± 578 IU/g substrate) and pectinase (4,832 ± 189 IU/g substrate) was achieved. The different types of pectinases produced were exo-polymethylgalacturonase (782 IU/g), endo-polymethylgalacturonase (6.42 U/g), exo-polygalacturonase (2,250 IU/g), endo-polygalacturonase (11.57 U/g), polymethylgalacturonate lyase (53.99 IU/g), polygalacturonate lyase (59.78 IU/g), and pectin esterase (5.78 IU/g). Wheat bran resulted in the highest titer of both enzymes. The maximum xylanase-pectinase yield was detected after 7 days of incubation with 2 mM MgSO4 and 1.5 g/L K2 HPO4 at wheat bran to moisture ratio 1:1.5 (w/v), media to flask volume ratio 1:25, pH 7.0, temperature 37 °C, and inoculum size 15%. Xylanase was most stable at pH 8.0, retained more than 75% activity up to 24 H, whereas pectinase was most stable at pH 9.0, having full activity even after 24 H. At 45 °C, the xylanase showed 82% residual activity after 6 H of incubation. The pectinase was 97% and 61% stable up to 3 H at 50 and 55 °C, respectively. This is the first report showing the production of xylanase-pectinases by bacterium along with high titer of seven types of pectinases, suitable for industries.

Lignocellulosic biomass (LB) is the renewable feedstock for the production of fuel/energy, feed/food, chemicals, and materials. LB could also be the versatile source of the functional oligosaccharides, which are non-digestible food ingredients having numerous applications in food, cosmetics, pharmaceutical industries, and others. The burgeoning functional food demand is expected to be more than US$440 billion in 2022. Because of higher stability at low pH and high temperature, oligosaccharides stimulate the growth of prebiotic bifidobacteria and lactic acid bacteria. Xylooligosaccharides (XOS) are major constituents of oligosaccharides consisting of 2-7 xylose monomeric units linked via β-(1,4)-linkages. XOS can be obtained from various agro-residues by thermochemical pretreatment, enzymatic or chemoenzymatic methods. While thermochemical methods are fast, reproducible, enzymatic methods are substrate specific, costly, and produce minimum side products. Enzymatic methods are preferred for the production of food grade and pharmaceutically important oligosaccharides. XOS are potent prebiotics having antioxidant properties and enhance the bio-adsorption of calcium and improving bowel functions, etc. LB can cater to the increasing demand of oligosaccharides because of their foreseeable amount and the advancements in technology to recover oligosaccharides. This paper summarizes the methods for oligosaccharides production from LB, classification, and benefits of oligosaccharides on human health.

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.

The modified medium composed of the alkaline-pretreated oil palm empty fruit bunch (APEFB) and tuna condensate powder was used for cellulase and xylanase productions by Streptomyces thermocoprophilus strain TC13W. The APEFB contained 74.46% (w/w) cellulose, 15.72% (w/w) hemicellulose, and 6.40% (w/w) lignin. The tuna condensate powder contained 55.49% (w/w) protein and 11.05% (w/w) salt. In the modified medium with only 6.75 g/l tuna condensate powder, 10 g/l APEFB, and 0.5 g/l Tween 80, S. thermocoprophilus strain TC13W produced cellulase 4.9 U/ml and xylanase 9.0 U/ml. The enzyme productions in the modified medium were lower than cellulase (6.0 U/ml) and xylanase (12.0 U/ml) productions in the complex medium (CaCl2 0.1, MgSO4·7H2O 0.1, KH2PO4 0.5, K2HPO4 1.0, NaCl 0.2, yeast extract 5.0, NH4NO3 1.0, Tween 80 0.5). When tuna condensate powder in the modified medium was reduced to 5.0 g/l and Tween 80 was increased to 1.5 g/l, S. thermocoprophilus strain TC13W produced cellulase and xylanase activities of 9.1 and 12.1 U/ml, respectively. This study shows that the cost of enzyme production could be reduced by using pretreated EFB and tuna condensate as a carbon and a nitrogen source, respectively.

β-1,2-Xylosidase activity has not been recorded as an EC subsubclass. In this study, phylogenetic analysis and multiple sequence alignments revealed that characterized β-xylosidases of glycoside hydrolase family (GH) 39 were classified into the same subgroup with conserved amino acid residue positions participating in substrate recognition. Protein-ligand docking revealed that seven of these positions were probably essential to bind xylose-glucose, which is linked by a β-1,2-glycosidic bond. Amino acid residues in five of the seven positions are invariant, while those in two of the seven positions are variable with low frequency. Both the wild-type β-xylosidase rJB13GH39 and its mutants with mutation at the two positions exhibited β-1,2-xylosidase activity, as they hydrolyzed o-nitrophenyl-β-d-xylopyranoside and transformed notoginsenosides R1 and R2 to ginsenosides Rg1 and Rh1, respectively. The results suggest that all of these characterized GH 39 β-xylosidases probably show β-1,2-xylosidase activity, which should be assigned an EC number with these β-xylosidases as representatives.

The secretomes of the strain Cellulosimicrobium cellulans F16 grown on different carbon sources were analyzed by zymography, and the subcellular surface structures were extensively studied by electron microscope. The exo-cellulase and xylanase systems were sparse when cells were grown on soluble oligosaccharides, but were significantly increased when grown on complex and water-insoluble polysaccharides, such as Avicel, corn cob, and birchwood xylan. The cellulosome-like protuberant structures were clearly observed on the cell surfaces of strain F16 grown on cellulose, with diameters of 15-20 nm. Fibrous structures that connected the adjacent cells can be seen under microscope. Moreover, protuberances that adsorbed the cell to cellulose were also observed. As the adhesion of Cellulosimicrobium cellulans cells onto cellulose surfaces occurs via thick bacterial curdlan-type exopolysaccharides (EPS), such surface layer is potentially important in the digestion of insoluble substrates such as cellulose or hemicellulose, and the previously reported xylanosomes are part of such complex glycocalyx layer on the surface of the bacterial cell.

To explore the diversity of glycoside hydrolase family 10 xylanase genes in the sediment of soda lake Dabusu by using high-throughput amplicon sequencing based on the Illumina HiSeq2500 platform. A total of 227,420 clean reads, representing approximately 49.5 M bp, were obtained. Operational taxonomic unit (OTU) classification, with a 95% sequence identity cut-off, resulted in 467 OTUs with 392 annotated as GH10 xylanase, exhibiting 35–99% protein sequence identity with their closest-related xylanases in GenBank. Above 75% of the total OTUs demonstrated less than 80% identity with known xylanases. In addition, xylanases derived from the sediment were found to be affiliated to 12 different phyla, with Bacteroidetes, Proteobacteria, Actinobacteria, Firmicutes, Verrucomicrobia, and Basidiomycota being the dominant phyla. Moreover, barcode sequence had a major effect on abundance with only a minor effect on diversity. High-throughput amplicon sequencing offers insight into xylanase gene diversity at a substantially higher resolution and lesser cost than library cloning and Sanger sequencing, facilitating a more thorough understanding of xylanase distribution and ecology.

With the passage of time, energy sources are decreasing day by day. In order to meet the world's demand, much attention is being paid to the study of enzymes with xylanolytic activity as a potential means of generating energy. A thermophilic xylanolytic bacterium, Bacillus sp., was isolated from naturally decaying material by enrichment culture and serial dilution methods. The bacterium was grown in MH medium at 50°C and pH 7 for 10 h. The xylanolytic Bacillus sp. produced clear yellow haloes around the colonies in the presence of p-nitrophenyl beta-D-xylopyranoside (pNPX) as a substrate. After condition optimization, it was found that the organism produced the higher level of xylosidase activity after 14 h in the presence of arabinose as a carbon source and ammonium sulfate as a nitrogen source in the pH 7 medium of at 55°C. The maximum β-xylosidase activity after optimizing the culture condition was 5.0 U/mL. Later this thermophilic Bacillus sp. was used as a donor in cloning of the β-xylosidase gene. A genomic library of Bacillus sp. was prepared by digesting the genomic DNA of the Bacillus with the restriction endonuclease BamHI, ligating the fragments in the pUC18 cloning vector and then transforming the competent E. coli DH5α cells with the resultant chimeric plasmid. The β-xylosidase gene was identified by screening the transformants in duplicates on LB agar plates overlaid with pNPX as a substrate. Commercial production of β-xylosidase to be used as a methanol-producing enzyme can help to overcome fuel shortages.

Lignocellulose, the main component of plant cell walls, comprises polyaromatic lignin and fermentable materials, cellulose and hemicellulose. It is a plentiful and renewable feedstock for chemicals and energy. It can serve as a raw material for the production of various value-added products, including cellulase and xylanase. Cellulase is essentially required in lignocellulose-based biorefineries and is applied in many commercial processes. Likewise, xylanases are industrially important enzymes applied in papermaking and in the manufacture of prebiotics and pharmaceuticals. Owing to the widespread application of these enzymes, many prokaryotes and eukaryotes have been exploited to produce cellulase and xylanases in good yields, yet yeasts have rarely been explored for their plant-cell-wall-degrading activities. This review is focused on summarizing reports about cellulolytic and xylanolytic yeasts, their properties, and their biotechnological applications.

Endo-xylanase and β-xylosidase are the major enzymes for hemicellulose hydrolysis, which play a significant role in biomass conversion. In our previous work, the white-rot fungi Pleurotus ostreatus HAUCC 162 and Irpex lacteus CD2 were demonstrated to have strong ability in lignocellulose degradation, and the related lignin degradation enzymes were characterized. However, little was known about their hemicellulases. In this work, a novel endo-1, 4-xylanase and a β-xylosidase from Pleurotus ostreatus HAUCC 162 and Irpex lacteus CD2 were heterologously expressed and characterized. The optima of pH and temperature were 5.0 and 55 °C for rXyn162, and 6.5 and 30 °C for rXylCD2. rXyn162 showed high tolerance to metal ions such as Ca2+, Cr3+, Zn2+, Na+, and Al3+. The recombinant rXyn162 and rXylCD2 exhibited synergistic hydrolysis of oat spelts xylan and sodium hydroxide pretreated cornstalk (SHPC), where the degree of synergy (DS) was 2.26 for SHPC hydrolysis. MALDI-TOF-MS and HPLC analysis showed that xylooligosaccharides (XOS) with small degrees of polymerization (DP2-DP4) were the major XOS hydrolyzate during SHPC degradation by rXyn162 and rXylCD2. In addition, rXyn162 and rXylCD2 could efficiently improve the hydrolysis of SHPC by commercial cellulase. The present study suggested the potential application of rXyn162 and rXylCD2 in the field of biomass pretreatment and biofuel production.

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.

Nanocellulose isolation fromlignocellulose is a tedious and expensive processwith high energy and harsh chemical requirements, primarily due to the recalcitrance of the substrate, which otherwise would have been costeffective due to its abundance. Replacing the chemical steps with biocatalytic processes offers opportunities to solve this bottleneck to a certain extent due to the enzymes substrate specificity and mild reaction chemistry. In this work, we demonstrate the isolation of sulphate-free nanocellulose from organosolv pretreated birch biomass using different glycosyl-hydrolases, along with accessory oxidative enzymes including a lytic polysaccharide monooxygenase (LPMO). The suggested process produced colloidal nanocellulose suspensions (zeta-potential-19.4 mV) with particles of 7-20 nm diameter, high carboxylate content and improved thermostability (T-o= 301 degrees C, T-max= 337 degrees C). Nanocelluloseswere subjected to post-modification using LPMOs of different regioselectivity. The sample from chemical route was the least favorable for LPMO to enhance the carboxylate content, while that from the C1-specific LPMO treatment showed the highest increase in carboxylate content. (c) 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).

Enzymatic degradation of hemi-cellulosic substrates has gained plenty of industrial attentions recently. Complete enzymatic degradation of complex and recalcitrant hemicellulose requires an enzymatic cocktail consisting primarily of endo-1,4-β-xylanase (xyl), β-xylosidase, arabinofuranosidase etc. This article reports, for the first time, the identification, cloning, expression and partial characterization of a potent endo-1,4- β-xylanase gene (pxyl) from the mushroom Termitomyces clypeatus (TC) in E. coli and S. cerevisiae. The cDNA for pxyl was found to be 678 bp that in turn gives rise to a precursor protein (Pxyl) of 225 amino acids long when cloned in prokaryotic expression vector. To characterize additionally, the cDNA was also expressed in S. cerevisiae. Bioinformatics study predicted that the Pxyl contains a 19 amino acid long leader peptide that enables post translational modifications including glycosylation as well as its efficient secretion in the medium. The recombinant protein has been found to be a member of GH11 family containing two distant glutamic acids as catalytic residues. This report describes yet another new and potent source of xylanase for commercial exploitation by industry in future.

32 p.-5 fig.-2 tab.-3 fig. supl., 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 four 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., This work was funded by the Comunidad de Madrid [RETOPROSOST-2-CM, P2018/EMT-4459] and MICIU/AEI/FEDER [GLYSUS, RTI2018-093683-B-I00]. The authors acknowledge BioSFerA (RIA 884208) and IBISBA1.0 (H2020 730976) projects and the SusPlast-CSIC Interdisciplinary Platform for their support.

Background Lignin in sugarcane bagasse (SB) hinders its utilization by microorganism, therefore, pretreatment methods are employed to make fermentable components accessible to the microbes. Multivariate analysis of different chemical pretreatment methods can aid to select the most appropriate strategy to valorize a particular biomass. Results Amongst methods tested, the pretreatment by using sodium hydroxide in combination with methyltrioctylammonium chloride, an ionic liquid, (NaOH+IL) was the most significant for xylanase production by Bacillus aestuarii UE25. Investigation of optimal levels of five significant variables by adopting Box-Behnken design (BBD) predicted 20 IU mL− 1 of xylanase and experimentally, a titer of 17.77 IU mL− 1 was obtained which indicated the validity of the model. The production kinetics showed that volumetric productivity of xylanase was much higher after 24 h (833.33 IU L− 1 h− 1) than after 48 h (567.08 IU L− 1 h− 1). The extracted xylan from SB induced more xylanase in the fermentation medium than pretreated SB or commercially purified xylan. Nuclear Magnetic Resonance, Fourier transform infrared spectroscopy and scanning electron microscopy of SB indicated removal of lignin and changes in the structure of SB after NaOH+IL pretreatment and fermentation. Conclusion Combined pretreatment of SB with alkali and methyltrioctylammonium chloride appeared better than other chemical methods for bacterial xylanase production and for the extraction of xylan form SB.

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.

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.

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 xylohexaosexylopentaosexylotetraose 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: xylotetraosexylopentaosexylohexaose. Rex produced O-β-D-xylopyranosyl-(1 → 4)-[O-α-L-arabinofuranosyl-(1 → 3)]-O-β-D-xylopyranosyl-(1 → 4)-β-D-xylopyranose (Ara

The objective of this research was to determine effects of five enzymes on whole wheat bread properties, particularly loaf volume, bread texture, and staling. Enzymes containing conventional α-amylase (α-amyl), cellulase (cel), glucose oxidase, maltogenic α-amylase (m amyl), and xylanase (xyl) were added at three levels. Vital wheat gluten (VWG) was added as an additional, separate treatment at 2.5% (flour weight basis). Enzymes had minimal effect on water absorption and mixing time. Each enzyme increased specific loaf volume for at least one of the usage levels tested (P < 0.01). Among the enzyme treatments, the greatest loaf volume was seen for xyl at the medium and high levels. No enzyme was as effective as VWG at increasing loaf volume. Overall, enzymes did not significantly change cell structure. The greatest reduction in fresh bread hardness was obtained for the high level of xyl. VWG, m amyl, and xyl reduced the rate of bread firming over 7 days. α-Amyl, cel, and m amyl decreased starch retrogradation at day 7 as measured by differential scanning calorimetry (P < 0.01). M amyl nearly eliminated the endothermic peak for recrystallized amylopectin. This study demonstrated the specific application of enzymes in whole wheat bread to increase loaf volume and decrease initial crumb hardness and bread staling. PRACTICAL APPLICATION: This study will provide guidance for practical uses of enzymes in improving whole wheat dough and bread quality.

Gut microbiota participates in diverse metabolic and homeostatic functions related to health and well-being. Its composition varies between individuals, and depends on factors related to host and microbial communities, which need to adapt to utilize various nutrients present in gut environment. We profiled fecal microbiota in 63 healthy adult individuals using metaproteomics, and focused on microbial CAZy (carbohydrate-active) enzymes involved in glycan foraging. We identified two distinct CAZy profiles, one with many Bacteroides-derived CAZy in more than one-third of subjects (n = 25), and it associated with high abundance of Bacteroides in most subjects. In a smaller subset of donors (n = 8) with dietary parameters similar to others, microbiota showed intense expression of Prevotella-derived CAZy including exo-beta-(1,4)-xylanase, xylan-1,4-beta-xylosidase, alpha-l-arabinofuranosidase and several other CAZy belonging to glycosyl hydrolase families involved in digestion of complex plant-derived polysaccharides. This associated invariably with high abundance of Prevotella in gut microbiota, while in subjects with lower abundance of Prevotella, microbiota showed no Prevotella-derived CAZy. Identification of Bacteroides- and Prevotella-derived CAZy in microbiota proteome and their association with differences in microbiota composition are in evidence of individual variation in metabolic specialization of gut microbes affecting their colonizing competence.

Synergistic saccharification ability of hemicellulases (endo-xylanase and β-xylosidase) was evaluated in this study for the bioethanol production from plant biomass. Endo-xylanase and β-xylosidase genes from