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Paenibacillus xylaniclasticus strain TW1, a gram-positive facultative anaerobic bacterium, was isolated as a xylanolytic microorganism from the wastes of a pineapple processing factory. A gene encoding one of its xylanolytic enzymes, a ß-xylosidase, was cloned and sequenced. Sequence analysis revealed that this ß-xylosidase, named PxXyl43A, was composed of a glycoside hydrolase (GH) family 43 subfamily 12 catalytic module and an unknown function module (UM). The full-length PxXyl43A (PxXyl43A) was heterologously expressed in Escherichia coli and purified. Recombinant PxXyl43A exhibited hydrolysis activity against both p-nitrophenyl-ß-D-xylopyranoside (pNPX) and p-nitrophenyl-a-L-arabinofuranoside at specific activities of 250 and 310 mU/mg, respectively. The optimal reaction pH and temperature for pNPX hydrolysis were 7.1 and 54 °C, respectively. At pH 7.0 and 54 °C, the Km and kcat for pNPX were 1.2 mM and 2.8 ± 0.15 s-1, respectively. It was also discovered that the recombinant unknown function module of PxXyl43A (PxXyl43A-UM) could bind to insoluble xylans like birchwood xylan and oat spelt xylan, whereas it did not bind to cellulosic substrates such as ball-milled cellulose, carboxymethyl cellulose or lichenan. The PxXyl43A-UM's binding constant value Ka for oat spelt xylan was 2.0 × 10-5 M-1. These results suggest that PxXyl43A possesses a novel carbohydrate-binding module, named as CBM91, specific for xylan-containing polysaccharides. [ABSTRACT FROM AUTHOR]

Xylan is one of the major structural components of the plant cell wall. Xylan present in the human diet reaches the large intestine undigested and becomes a substrate to species of the gut microbiota. Here, we characterised the capacity of Limosilactobacillus reuteri and Blautia producta strains to utilise xylan derivatives. We showed that L. reuteri ATCC 53608 and B. producta ATCC 27340 produced β-D-xylosidases, enabling growth on xylooligosaccharide (XOS). The recombinant enzymes were highly active on artificial (p-nitrophenyl β-D-xylopyranoside) and natural (xylobiose, xylotriose, and xylotetraose) substrates, and showed transxylosylation activity and tolerance to xylose inhibition. The enzymes belong to glycoside hydrolase family 120 with Asp as nucleophile and Glu as proton donor, as shown by homology modelling and confirmed by site-directed mutagenesis. In silico analysis revealed that these enzymes were part of a gene cluster in L. reuteri but not in Blautia strains, and quantitative proteomics identified other enzymes and transporters involved in B. producta XOS utilisation. Based on these findings, we proposed a model for an XOS metabolism pathway in L. reuteri and B. producta strains. Together with phylogenetic analyses, the data also revealed the extended xylanolytic potential of the gut microbiota. [ABSTRACT FROM AUTHOR]

Two novel xylanolytic enzymes, a xylanase and a β-xylosidase, were simultaneously isolated and characterized from the extracellular medium of Byssochlamys spectabilis ATHUM 8891 (anamorph Paecilomyces variotii ATHUM 8891), grown on Brewer’s Spent Grain as a sole carbon source. They represent the first pair of characterized xylanolytic enzymes of the genus Byssochlamys and the first extensively characterized xylanolytic enzymes of the family Thermoascaceae. In contrast to other xylanolytic enzymes isolated from the same family, both enzymes are characterized by exceptional thermostability and stability at low pH values, in addition to activity optima at temperatures around 65 ◦C and acidic pH values. Applying nano-LC-ESI-MS/MS analysis of the purified SDS-PAGE bands, we sequenced fragments of both proteins. Based on sequence-comparison methods, both proteins appeared conserved within the genus Byssochlamys. Xylanase was classified within Glycoside Hydrolase family 11 (GH 11), while β-xylosidase in Glycoside Hydrolase family 3 (GH 3). The two enzymes showed a synergistic action against xylan by rapidly transforming almost 40% of birchwood xylan to xylose. The biochemical profile of both enzymes renders them an efficient set of biocatalysts for the hydrolysis of xylan in demanding biorefinery applications. [ABSTRACT FROM AUTHOR]

Xylanases hydrolyse efficiently the xylan component of hemicelluloses into single xylose monomers. Hence, these enzymes are suitable to be applied in the bioconversion of lignocellulosic wastes such as sugarcane bagasse to improve the bioethanol process. Misiones rainforest in the northeast of Argentina has a huge biodiversity which becomes the most likely site to screen new fungal isolates for powerful xylanolytic enzymes. The aim of this work was to find efficient xylanolytic Aspergillus with low levels of proteolytic enzymes through the isolation and exploration of native fungi from Misiones. Fungi were isolated from different locations of this subtropical region and 25 Aspergillus isolates were screened based on their ability to produce xylanolytic and proteolytic enzymes. From the principal component and conglomerate analysis, six groups were discriminated. The most promising fungi presented the highest endoxylanase and β-xylosidase activities and the lowest proteolytic activities. These isolates were A. niger LBM 055 and A. niger LBM 134 which reached 29 and 27 UmL−1 of endoxylanases, respectively; 0.6 UmL−1 of β-xylosidases, no casein and very little gelatin proteases. This study proves that the search of new fungi from nature is an important strategy to find novel isolates with the ability to secrete high-performance xylanases and exploit these enzymes in the bioconversion of lignocellulosic wastes.

Xylan is one of the major structural components of the plant cell wall. Xylan present in the human diet reaches the large intestine undigested and becomes a substrate to species of the gut microbiota. Here, we characterised the capacity of Limosilactobacillus reuteri and Blautia producta strains to utilise xylan derivatives. We showed that L. reuteri ATCC 53608 and B. producta ATCC 27340 produced β-D-xylosidases, enabling growth on xylooligosaccharide (XOS). The recombinant enzymes were highly active on artificial (p-nitrophenyl β-D-xylopyranoside) and natural (xylobiose, xylotriose, and xylotetraose) substrates, and showed transxylosylation activity and tolerance to xylose inhibition. The enzymes belong to glycoside hydrolase family 120 with Asp as nucleophile and Glu as proton donor, as shown by homology modelling and confirmed by site-directed mutagenesis. In silico analysis revealed that these enzymes were part of a gene cluster in L. reuteri but not in Blautia strains, and quantitative proteomics identified other enzymes and transporters involved in B. producta XOS utilisation. Based on these findings, we proposed a model for an XOS metabolism pathway in L. reuteri and B. producta strains. Together with phylogenetic analyses, the data also revealed the extended xylanolytic potential of the gut microbiota.

A novel endo-1,4-β-xylanase-encoding gene was identified in Alicyclobacillus mali FL18 and the recombinant protein, named AmXyn, was purified and biochemically characterized. The monomeric enzyme worked optimally at pH 6.6 and 80 °C on beechwood xylan with a specific activity of 440.00 ± 0.02 U/mg and a good catalytic efficiency (k cat /K M  = 91.89 s -1 mLmg -1 ). In addition, the enzyme did not display any activity on cellulose, suggesting a possible application in paper biobleaching processes. To develop an enzymatic mixture for xylan degradation, the association between AmXyn and the previously characterized β-xylosidase AmβXyl, deriving from the same microorganism, was assessed. The two enzymes had similar temperature and pH optima and showed the highest degree of synergy when AmXyn and AmβXyl were added sequentially to beechwood xylan, making this mixture cost-competitive and suitable for industrial use. Therefore, this enzymatic cocktail was also employed for the hydrolysis of wheat bran residue. TLC and HPAEC-PAD analyses revealed a high conversion rate to xylose (91.56 %), placing AmXyn and AmβXyl among the most promising biocatalysts for the saccharification of agricultural waste., 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 Author(s). Published by Elsevier B.V. All rights reserved.)

The effect of the response surface methodology was applied to the production of Caulobacter crescentus (strain NA1000) β-xylosidases using corn cob. The components of the medium that presented the greatest influence on the enzyme production were chosen for optimization including the concentration of the corn cob residue and temperature variation. Optimal concentrations were determined by a central composite rotational design and a combination of 3.5% (w/v) corn cob concentration and 27 °C temperature was found to be optimal. When C. crescentus was cultivated using the optimized conditions, a maximum activity of 393.36 U/mL of β-xylosidases was achieved in 24-h cultures with a yield of 95% in real test conditions compared to the predicted one. In parallel, there was an increase of 3.6 times in the production of intracellular xylanases when compared to cultures without statistical application. In the C. crescentus genome, 5 genes that encode β-xylosidases are present. In order to evaluate which of them would be induced in the optimized conditions, the quantitative expression (qPCR) of the xynB1–xynB5 genes was evaluated in the presence of 1 or 3.5% corn cob (w/v) and surprisingly, all showed a constitutive expression in relation to the control. Assays of Western Blot performed with a polyclonal antiserum against C. crescentus β-xylosidase II in the optimized condition also did not show mass variation of the referred protein. These data strongly suggest that post-transcriptional controls are operating in the induced condition to increase the activity of C. crescentus β-xylosidases I-V, but β-xylosidase II. To our knowledge, this is the first time that these data are reported in literature for a bacterial system. [ABSTRACT FROM AUTHOR]

β-Xylosidase is a major enzyme of the xylanolytic complex, cleaving xylose from the non-reducing end of xylobiose and short chain xylo-oligomers. As a result of its action, the inhibitory effect of the resulting xylo-olimers on xylanase is removed, which is important for the achievement of complete enzymatic hydrolysis of xylan. The research was carried out with β-xylosidase, produced in a submerged cultivation of Aspergillus niger B 03, provided by Biovet AD. The enzyme was purified by subsequent ultrafiltration and gel chromatography on Sephadex G 75 and Sephadex G100. Ultrafiltration was performed in an Amicon ultrafiltration unit through a 10 kDa molecular weight cut-off polysulfone membrane, and the chromatographic isolation was performed on fast protein liquid chromatography (FPLC), Pharmacia Biotech. β-xylosidase purification was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in 12.5 % resolving gel. An extracellular β-xylosidase produced by Aspergillus niger B 03 was isolated by column gel chromatography with Sephadex G 75 and Sephadex G 100 to a homogeneous form, according to SDS-PAGE. The enzyme had a molecular weight of 90 500 Da. The optimal pH and temperature for the enzyme action were determined to be pH 3.50 and 70 0C, respectively. The enzyme showed significant thermal stability at 50 0C. The kinetic parameters of β-xylosidase were determined with p-nitophenyl-β-D-xylopyranoside as a substrate, the Km value was 0.35 mM and Vmax was 3.03 µmol/(min. mL). The presence of 10 mM Mn2+ significantly activated the enzyme, β-xylosidase activity was increased by about 1.5 fold. It was found that xylose was a competitive inhibitor of the isolated β-xylosidase and Ki value was determined to be 1.86 mM. The purification and the determination of the main biochemical characteristics of β-xylosidase allowed the proper application of the enzyme for enzymatic hydrolysis of xylan along with xylanase. As a result the degree of xylan hydrolysis was significantly increased, and reached 70%. [ABSTRACT FROM AUTHOR]

LXYL-P1-2 is one of the few xylosidases that efficiently catalyze the reaction from 7-β-xylosyl-10-deacetyltaxol (XDT) to 10-deacetyltaxol (DT), and is a potential enzyme used in Taxol industrial production. Here we report the crystal structure of LXYL-P1-2 and its XDT binding complex. These structures reveal an enzyme/product complex with the sugar conformation different from the enzyme/substrate complex reported previously in GH3 enzymes, even in the whole glycohydrolases family. In addition, the DT binding pocket is identified as the structural basis for the substrate specificity. Further structure analysis reveals common features in LXYL-P1-2 and Taxol binding protein tubulin, which might provide useful information for designing new Taxol carrier proteins for drug delivery. Yang, Chen et al. report crystallographic structures of GH3 beta-glucosidase LXYL-P1-2 that converts XDT into DT, a precursor for an anticancer drug taxol, and the complex of LXYL-P1-2 with XDT. This study provides insight into the catalytic mechanism for GH3 enzymes. [ABSTRACT FROM AUTHOR]

Aims: This study focuses on the development of a new strategy xylooligosaccharide (XOS) production from aqueous ammonia‐pretreated rice straw (A‐PRS), followed by β‐xylosidase hydrolysis produced by the newly identified strain Weissella cibaria FB069. Methods and Results: We report a higher efficiency of A‐PRS, including the removal of lignin and increase in cellulose and xylan content, compared to that of the alkali and stream explosion methods. Using the ammonia pretreatment method, rice straw was used to obtain 32·4% xylan. The crude xylanase from W. cibaria was used to hydrolyse A‐PRS over different hydrolysis times. The highest XOS yield (131 mg XOS per gram rice straw) was observed after 10 h. XOS produced from the PRS was tested on stimulation effect on Bifidobacterium and Lactobacillus. Conclusion: The possibility of XOS production from PRS using β‐xylosidase with strong prebiotic properties. Significance and Impact of the Study: We investigated the new strain for signification production of XOS. The two‐stage process here described could help to further explore the optimization conditions for prebiotic production. Additionally, the stimulation effect of XOS from alternative source has a promising prospect in functional food. [ABSTRACT FROM AUTHOR]

Highlights • Bee-originated proteins are predominant proteinaceous components in loquat honey. • Loquat nectar proteome mainly consisted of xylosidase, thaumatin, and chitinase which were not detected in loquat honey. • The zymography of nectar-originated chitinases is a potential marker for honey botanical origin authentication. Abstract Honey, as a commercial product, is a target of adulteration through inappropriate production practices and deliberate mislabelling of botanical origin. Floral nectar protein could be a good marker for determining the source flowers of honey, especially monofloral honeys. Here, nectar and monofloral honey from Eriobotrya japonica Lindl. (loquat) were systematically compared, especially regarding proteomic and enzymatic activity. Using two-dimensional electrophoresis and mass spectrometry, only bee-originated proteins were detected in loquat honey. Xylosidase, thaumatin, and two kinds of chitinases were detected in loquat floral nectar, and their activity in loquat nectar and honey were quantified. Following gel electrophoresis, loquat honey had similar chitinase activity profiles to loquat nectar, but both were clearly distinguishable from Camellia sinensis nectar and Brassica napus honey. To our knowledge, this is the first examination of nectar-origin enzyme activity in honey. Zymography of chitinases is a potential marker for determining or authenticating the botanical origin of honeys. [ABSTRACT FROM AUTHOR]

Abstract An intracellular β-xylosidase (AbXyl), from the thermoalkaline Anoxybacillus sp. 3M, was purified and characterized. The homodimeric enzyme (140 kDa) was optimally active at 65 °C and pH 5.5, exhibited half life of 10 h at 60 °C, 78 and 88% residual activity after 24 h, at pH 4.5 and 8.0, respectively. Fe2+, Cu2+, Al3+, Ag+ and Hg2+ inhibited the enzyme; the activity was moderately stimulated by SDS and not influenced by β-mercaptoethanol. In the presence of p -nitrophenyl-β- d -xylopyranoside, AbXyl exhibited Km of 0.19 mM, Kcat of 453.29 s−1, Kcat Km−1 of 2322 s−1 mM and was moderately influenced by xylose (Ki 21.25 mM). The enzyme hydrolyzed xylo-oligomers into xylose and catalyzed transxylosilation reactions also in presence of alcohols as acceptors, producing xylo-oligosaccharides and alkyl-xylosides. Finally AbXyl was applied towards a statistically optimized process of brewery's spent grain bioconversion, highlighting the important role of this biocatalyst in reaching high yields of fermentable sugars. [ABSTRACT FROM AUTHOR]

The early-lineage, aerobic, zoosporic fungi from the Chytridiomycota constitute less than 1% of the described fungi and can use diverse sources of nutrition from plant or animal products. One of the ancestral sources of fungal nutrition could be products following enzymatic degradation of plant material. However, carbohydrate-active enzymes from these ancient fungi have been less studied. A GH11 xylanase (RrXyn11A) (EC 3.2.1.8) and a GH43 xylosidase (RrXyl43A) (EC 3.2.1.37) were identified from an early-lineage aerobic zoosporic fungus, Rhizophlyctis rosea NBRC 105426. Both genes were heterologously expressed in Pichia pastoris and the recombinant enzymes were purified and characterized. The optimal pH for recombinant RrXyn11A and RrXyl43A was pH 7. RrXyn11A had high stability over a wide range of pH (4-8) and temperature (25-70 °C). RrXyn11A also showed high substrate specificity on both azurine-cross-linked (AZCL) arabinoxylan and AZCL xylan. RrXyl43A had β-xylosidase and minor α-L-arabinofuranosidase activity. This enzyme showed low product inhibition and retained 51% activity in the presence of 100 mM xylose. A combination of RrXyn11A and RrXyl43A exhibited significantly higher hydrolytic and polymer degradation capability and xylose release on wheat bran and beechwood xylan compared to treatment with commercial enzymes. This study was the first to heterologously express and characterize the GH11 xylanase (RrXyn11A) and GH43 xylosidase (RrXyl43A) from the ancient fungus, R. rosea. Meanwhile, this study also demonstrated that the enzymes from the ancient fungus R. rosea can be easily handled and heterologously expressed in Pichia, which presents a promising path to a new source of enzymes for biomass degradation. [ABSTRACT FROM AUTHOR]

Highlights • First report investigating the production of xylanolytic enzymes and XOS by A. pullulans. • A. pullulans strains produced endo-β-1,4-xylanase with low β-xylosidase activity. • XOS produced by A. pullulans were mainly composed of xylobiose and xylotriose. • Endo-β-1,4-xylanase was produced using low-cost substrates as carbon source. • Untreated rice bran presents as a potential inducer to endo-β-1,4-xylanase production. Abstract Endo-β-1,4-xylanases are microbial-produced enzymes capable of converting lignocellulosic biomass in value-added products of biotechnological interest, such as xylooligosaccharides (XOS). Although endo-β-1,4-xylanase production using lignocellulosic substrates is commonly studied, there are few reports investigating the production of both xylanolytic enzymes and XOS by microbial species. In this paper, the production of xylanolytic enzymes and XOS by three Aureobasidium pullulans strains was evaluated during submerged cultivation, as well as the use of untreated and alkali pretreated agricultural substrates as sources of xylan for endo-β-1,4-xylanase production. The CCT 1261 strain showed the highest endo-β-1,4-xylanase activity (74.94 U/mL), specific activity (158.65 U/mg protein), productivity (P , 1.25 U/mL.h), maximum specific growth rate (μ max , 0.18 1/h), and maximum biomass concentration (X max , 4.10 g/L); and a low β-xylosidase production (0.19 U/mL). XOS production by the CCT 1261 strain was verified in the first 12 h of cultivation, which reached the total concentration of 1.68 mg/mL, and the XOS produced were comprised mainly of xylobiose and xylotriose. Among the agricultural substrates evaluated, untreated rice bran showed the highest amount of hemicellulose (29.6%) and a low lignin content (12.2%), which implied in the highest endo-β-1,4-xylanase activity (11.69 U/mL) when used as substrate in submerged cultivation. Endo-β-1,4-xylanase could be produced by A. pullulans in an economical way using a by-product of rice grain milling, which can be further improved by optimization of the submerged cultivation conditions. [ABSTRACT FROM AUTHOR]

Abstract 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. Graphical abstract Image 1 Highlights • Novel endo-1,4-xylanase and β-xylosidase were heterologously expressed and characterized. • Synergistic hydrolysis of xylan and SHPC by rXyn162 and rXylCD2 was observed. • XOS with small degrees of polymerization were majorly present in SHPC hydrolysate. • rXyn162 and rXylCD2 enhanced SHPC hydrolysis by commercial cellulase. [ABSTRACT FROM AUTHOR]

Plant architecture, lodging resistance, and yield are closely associated with height. In this paper, we report the identification and characterization of two allelic EMS-induced mutants of Zea mays, xyl-1, and xyl-2 that display dwarf phenotypes. The mutated gene, ZmXYL, encodes an α-xylosidase which functions in releasing xylosyl residue from a β-1,4-linked glucan chain. Total α-xylosidase activity in the two alleles is significantly decreased compared to wild-type plants. Loss-of-function mutants of ZmXYL resulted in a decreased xylose content, an increased XXXG content in xyloglucan (XyG), and a reduced auxin content. We show that auxin has an antagonistic effect with XXXG in promoting cell divisions within mesocotyl tissue. xyl-1 and xyl-2 were less sensitive to IAA compared to B73. Based on our study, a model is proposed that places XXXG, an oligosaccharide derived from XyG and the substrate of ZmXYL, as having a negative impact on auxin homeostasis resulting in the dwarf phenotypes of the xyl mutants. Our results provide a insight into the roles of oligosaccharides released from plant cell walls as signals in mediating plant growth and development., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)

Biomass hydrolysis constitutes a bottleneck for the biotransformation of lignocellulosic residues into bioethanol and high-value products. The efficient deconstruction of polysaccharides to fermentable sugars requires multiple enzymes acting concertedly. GH43 β-xylosidases are among the most interesting enzymes involved in hemicellulose deconstruction into xylose. In this work, the structural and functional properties of β-xylosidase EcXyl43 from Enterobacter sp. were thoroughly characterized. Molecular modeling suggested a 3D structure formed by a conserved N-terminal catalytic domain linked to an ancillary C-terminal domain. Both domains resulted essential for enzymatic activity, and the role of critical residues, from the catalytic and the ancillary modules, was confirmed by mutagenesis. EcXyl43 presented β-xylosidase activity towards natural and artificial substrates while arabinofuranosidase activity was only detected on nitrophenyl α-L-arabinofuranoside (pNPA). It hydrolyzed xylobiose and purified xylooligosaccharides (XOS), up to degree of polymerization 6, with higher activity towards longer XOS. Low levels of activity on commercial xylan were also observed, mainly on the soluble fraction. The addition of EcXyl43 to GH10 and GH11 endoxylanases increased the release of xylose from xylan and pre-treated wheat straw. Additionally, EcXyl43 exhibited high efficiency and thermal stability under its optimal conditions (40 °C, pH 6.5), with a half-life of 58 h. Therefore, this enzyme could be a suitable additive for hemicellulases in long-term hydrolysis reactions. Because of its moderate inhibition by monomeric sugars but its high inhibition by ethanol, EcXyl43 could be particularly more useful in separate hydrolysis and fermentation (SHF) than in simultaneous saccharification and co-fermentation (SSCF) or consolidated bioprocessing (CBP). [ABSTRACT FROM AUTHOR]

A β-xylosidase from Colletotrichum graminicola (Bxcg) was purified. The enzyme showed high halotolerance, retaining about 63% of the control activity in the presence of 2.5 mol L −1 NaCl. The presence of NaCl has not affected the optimum reaction temperature (65 °C), but the optimum pH was slightly altered (from 4.5 to 5.0) at high salt concentrations. Bxcg was fully stable at 50 °C for 24 h and over a wide pH range even in the presence of NaCl. In the absence of salt Bxcg hydrolyzed p -nitrophenyl-β- d -xylopyranoside with maximum velocity of 348.8 ± 11.5 U mg −1 and high catalytic efficiency (1432.7 ± 47.3 L mmol −1 s −1 ). Bxcg revealed to be a bifunctional enzyme with both β-xylosidase and α- l -arabinofuranosidase activities, and hydrolyzed xylooligosaccharides containing up to six pentose residues. The enzyme showed high synergistic effect (3.1-fold) with an endo-xylanase for the hydrolysis of beechwood xylan, either in the absence or presence of 0.5 mol L −1 NaCl, and was tolerant to different organic solvents and surfactants. This is the first report of a halotolerant bifunctional β-xylosidase/α- l -arabinofuranosidase from C. graminicola , and the enzyme showed attractive properties for application in lignocellulose hydrolysis, particularly under high salinity and/or in the presence of residues of pretreatment steps. [ABSTRACT FROM AUTHOR]

Two GH43 β-xylosidases, RS223-BX from a rice straw metagenomic library, and BoXA from Bacteroides ovatus , that share similar amino acid sequences (81% identical) and 19 of 20 active-site residues, were compared by using site-directed mutagenesis of Asp and His residues implicated in metal binding. Thus, RS223-BX is strongly activated by divalent-metal cations and the previously published X-ray structure of this enzyme shows that a Ca 2+ cation is chelated by an active-site Asp carboxyl group and an active-site His. Mutation to Ala causes 90% loss of activity for the Asp mutant and 98% loss of activity for the His mutant, indicating their importance to catalysis. For the other enzyme (BoXA), mutation to Ala causes 20% loss of activity for the His mutant and 40% gain of activity for the Asp mutant, indicating the lack of importance for activity of the native residues and the lack of metal-dependency, given that the Asp residue occupies the active site to secure the metal cation in known metal ion dependent GH43 xylosidases. The high activity of the BoXA mutants compared to that of the analogous RS223-BX mutants further undermines the possibility that BoXA maintains a tightly bound metal cofactor resistant to EDTA extraction. The results strengthen our conclusion that the very similar proteins differ in one being metal ion dependent and one not. [ABSTRACT FROM AUTHOR]

Xylobiose consists of two molecules of xylose and has been highly recognized as a food supplement because it possesses high prebiotic functions. β-xylosidase exhibits enzymatic activity to hydrolyze xylobiose, and the enzyme can also catalyze the reverse reaction in the presence of high concentrations of xylose. Previously, β-xylosidase from Bacillus pumilus IPO (BpXynB), belonging to GH family 43, was employed to produce xylobiose from xylose. To improve the enzymatic efficiency, this study determined the high-resolution structure of BpXynB in a complex with xylobiose and engineered BpXynB based on the structures. The structure of BpXynB deciphered the residues involved in the recognition of the xylobiose. A site-directed mutation at the residue for xylobiose recognition increased the yield of xylobiose by 20% compared to a similar activity of the wild type enzyme. The complex structure of the mutant enzyme and xylobiose provided the structural basis for a higher yield of the engineered protein. This engineered enzyme would enable a higher economic production of xylobiose, and a similar engineering strategy could be applied within the same family of enzymes. [ABSTRACT FROM AUTHOR]

A β-xylosidase gene (Bxyl BOH3 ) was identified in a solventogenic bacterium Clostridium sp. strain BOH3, which is a member of the glycoside hydrolase family 43 (GH43). The recombinant enzyme of Bxyl BOH3 was expressed in Escherichia coli with a high specific activity of 48.7 U/mg and a low K m value of 2.71 mM at an optimal temperature of 40 °C and pH of 5.0-6.0, indicating its high affinity for the substrate and enzymatic potency. Moreover, activity of β-xylosidase can be enhanced (>1.6 times) by addition of Zn 2+ . The concentration of reducing sugar (including xylose and xylooligosaccharides) with the synergism of xylanase and recombinant β-xylosidase was enhanced up to 129.8% (11.93 g/L) after hydrolysis of 30 g/L of xylan for 24 h when comparing with only xylanase (9.19 g/L). Application of this recombinant β-xylosidase together with xylanase improved xylan hydrolysis efficiency, thus leading to increased biofuels productivity from xylan fermented by Clostridium species. [ABSTRACT FROM AUTHOR]

β-Xylosidase plays an important role in xylan degradation by relieving the end product inhibition of endo -xylanase caused by xylo -oligosaccharides. β-Xylosidase has a wide range of applications in food, feed, paper and pulp, pharmaceutical industries and in bioconversion of lignocellulosic biomass. Hence, in the present study focused on purification, biochemical characterization and partial sequencing of purified β-xylosidase from xylanolytic strain Aspergillus niger ADH-11. Acetone precipitation followed by GPC using Sephacryl S-200 yielded 20.59-fold purified β-xylosidase with 58.30% recovery. SDS-PAGE analysis of purified β-xylosidase relieved a monomeric subunit with a molecular weight 120.48 kDa. Kinetic parameters of purified β-xylosidase viz Km, Vmax, Kcat and catalytic efficiency were assessed. Purified β-xylosidase was additionally active on p -nitrophenyl-β- d -glucopyranoside substrate also. Moreover, peptide mass fingerprinting analysis support our biochemical studies and showed that the purified protein is a novel β-xylosidase with β-glucosidase activity and belongs to the bi-functional GH3 superfamily. Besides, tolerance of purified β-xylosidase towards glucose and xylose was also assessed. [ABSTRACT FROM AUTHOR]

The gene of Pae1263 (2,196 bp, 732 aa) was found from the full-length sequence analysis of bacterium Paenibacillus terrae HPL-003 isolated from soil on Gara Mountain in Korea (CP003107, our previous study). Among the 20 open reading frames (ORFs) related with the xylose substrate, only the recombinant enzyme of ORF Pae1263 showed a 1,4-beta-D-xylosidase activity when all of the ORFs were transformed into E. coli. This gene is considered to be a new 1,4-beta-D-xylosidase because it has up to 93% similarity with other genes of ZP_10240221.1 from Lactococcus raffinolactis 4877 and ZP_11237858.1 from Paenibacillus peoriae in the GenBank blast search. The enzyme activity was confirmed by HPLC in which xylose was produced from xylobiose as a substrate by this recombinant enzyme. Mass production of the recombinant enzyme was done with the construction of the pET22(+)- Pae1263-6H expression vector system from E. coli. This new 1,4-beta-D-xylosidase was highly active at 50°C in a pH range between 6.0 and 8.0 and had thermo-stability for at least 24 h at 50°C and a Km and Vmax of 6.42 mg/mL and 75.76 U/mg on a xylobiose substrate, respectively. [ABSTRACT FROM AUTHOR]

Objectives: To screen the phylogenetically-nearest members of Cellulosimicrobium cellulans for the production of cellulosome-like multienzyme complexes and extracellular β-xylosidase activity against 7-xylosyltaxanes and to get corresponding molecular insights.Results: Cellulosimicrobium (family Promicromonosporaceae) and all genera of the family Cellulomonadeceaec produced both cellulosome-like multienzyme complexes and extracellular β-xylosidase activity, while the other genera of the family Promicromonosporaceae did not. Multiple sequence alignments further indicated that hypothetic protein M768_06655 might be a possible key subunit.Conclusion: This is the first report that many actinobacteria species can produce cellulosome-like multienzyme complexes. The production of cellulosome-like complexes and the extracellular β-xylosidase activity against 7-xylosyltaxanes might be used to differentiate the genus Cellulosimicrobium from other genera of the family Promicromonosporaceae. [ABSTRACT FROM AUTHOR]

In this study, we investigated the secretion mechanism of the hyper-secretion signal peptide-lacking β-xylosidase PtXyl43, a non-classically secreted protein, from the fungus Paecilomyces thermophila in Escherichia coli BL21(DE3). PtXyl43 secretion is a two-step process, and the second step is accompanied by cell periplasmic leakage, indicating that PtXyl43 secretion is the result of semi-specific secretion. Homology modeling of PtXyl43 suggested that PtXyl43 had a canonical GH43 family β-xylosidase structure containing five blades. Seventeen blade deletions or circular mutants were designed to identify the conformational motif(s) involved in secretion. These mutants were expressed as recombinant, codon-optimized proteins in E. coli. Notably, only mutants containing blades 2-4 were effectively secreted. Blades 2-4 are necessary for secretion, but it appears that blade 1 or 5 must be present to maintain the structure of blades 2-4. Simultaneous deletion of blades 1 and 5 dramatically reduces excretion. The covalent and sequential linking of blades of 2, 3 and 4 are important for the excretion of mutants, as separate blades of 2 and 3 or 3 and 4 abolishes excretion. Fusion with PtXyl43 promotes the excretion of GFP from the periplasm to the extracellular milieu, which suggested that PtXyl43 had the potential to carry proteins. This study provides new insights into secretory mechanism of secretable signal peptide-lacking proteins in E. coli. To our knowledge, this is the first to definitively identify the conformational determinants for secretion of a signal peptide-lacking GH43 family β-xylosidase. This finding also has application potential for the secretion of recombinant proteins. [ABSTRACT FROM AUTHOR]

The phytopathogenic fungus Ceratocystis fimbriata produced an extracellular β-xylosidase when grown in mineral medium with wheat bran as carbon source. The enzyme was purified and characterized. The molecular mass was estimated by SDS-PAGE (161.6 kDa). The enzyme showed maximum activity at pH 3.9 and 65 °C, and it showed to be significantly thermostable at 60 °C, maintaining 70% of its maximum activity after 24 h of incubation at this temperature. The K M and V MAX using ρNρβXyl were 0.326 mM and 0.91 × 10 −3 µmol/min, respectively. The β-xylosidase was partially inhibited by 1 mM of aluminum chloride, copper sulfate II, iron sulfate and SDS. The β-xylosidase combined with a xylanase had an additive effect on beechwood xylan hydrolysis, generating xylose as a final product. The supplementation of the enzymatic cocktail Multifect CL® with the β-xylosidase generated a more efficient hydrolysis of alkali-pretreated sugarcane bagasse when compared to the saccharification performed only by the cocktail Multifect CL®, showing an increase of 97.7% and 45.7% in glucose and xylose release, respectively. [ABSTRACT FROM AUTHOR]

Xylooligosaccharide-assimilating Corynebacterium glutamicum strains were constructed using metabolic engineering and cell surface display techniques. First, C. glutamicum was metabolically engineered to create lysine-producing strains. Beta-xylosidase BSU17580 derived from Bacillus subtilis was then expressed on the C. glutamicum cell surface using PorH anchor protein, and enzymes involved in the xylose assimilation pathway were also expressed. Metabolic engineering had no effect on the activity of beta-xylosidase. The engineered strains efficiently consumed xylooligosaccharides and produced 12.4 mM of lysine from 11.9 g/L of xylooligosaccharides as the carbon source. Finally, co-expression of lysine decarboxylase enabled production of 11.6 mM of 1,5-diaminopentane (cadaverine) from 13 g/L of consumed xylooligosaccharides. [ABSTRACT FROM AUTHOR]

β-Xylosidase activity is deficient in most cellulase enzymes secreted by filamentous fungi, which limits effective enzymatic hydrolysis of hemicellulose in lignocellulose materials and resulted in accumulation of xylo-oligosaccharides that inhibit the cellulase and xylanase activitives. An endogenous β-xylosidase gene, xyl3A , was overexpressed using two types of promoters in cellulolytic P. oxalicum RE-10. The mutants RXyl, RGXyl-1 and RGXyl-2 displayed higher β-xylosidase production than native strain RE-10 besides higher cellulase and xylanase activities, especially RGXyl-1, showing the highest β-xylosidase activity of 15.05 ± 1.79 IU/mL, about 29 folds higher than native strain, more than the highest level reported by literature. Enzymatic hydrolysis results indicated the cellulase RGXyl-1 not only increased glucose and xylose yields and thus resulted in high ethanol yield during the simultaneous saccharification and fermentation, but decreased the total enzyme loading compared to starting RE-10, which indicated a good prospect of industrial application in bioconversion of lignocellulose. [ABSTRACT FROM AUTHOR]

Ionic liquids (ILs) are used in lignocellulosic biomass (LCB) pretreatment because of their ability to disrupt the extensive hydrogen-bonding network in cellulose and hemicellulose, and thereby decrease LCB recalcitrance to subsequent enzymatic degradation. However, this approach necessitates the development of cellulases and hemicellulases that can tolerate ∼20% ( w/ v) IL, an amount that either co-precipitates with the sugar polymers after the initial pretreatment or is typically used in single-pot biomass deconstructions. By investigating the secretomes from 4 marine-derived fungal endophytes, we identified a β-xylosidase derived from Trichoderma harzianum as the most promising in terms of tolerating 1-ethyl-3-methylimidazolium-dimethyl phosphate (EMIM-DMP), an IL. When tested with p-nitrophenyl-β- d-xyloside, this extracellular xylosidase retained ∼50% activity even in 1.2 mol·L-1 (20% w/ v) EMIM-DMP after incubation for 48 h. When tested on the natural substrate xylobiose, there was ∼85% of the initial activity in 1.2 mol·L-1 EMIM-DMP after incubation for 9 h and ∼80% after incubation for 48 h. Despite previous findings associating thermostability and IL tolerance, our findings related to the mesophilic T. harzianum β-xylosidase(s) emphasize the need to include the marine habitat in the bioprospecting dragnet for identification of new IL-tolerant LCB-degrading enzymes. [ABSTRACT FROM AUTHOR]

The crystal structure of metagenomicβ-xylosidase/α-Larabinofuranosidase CoXyl43, activated by calcium ions, was determined in its apo and complexed forms with xylotriose or L-arabinose in the presence and absence of calcium. The presence of calcium ions dramatically increases the kcat of CoXyl43 for p-nitrophenyl β-D-xylopyranoside and reduces the Michaelis constant for p-nitrophenyl α-L-arabinofuranoside. CoXyl43 consists of a single catalytic domain comprised of a five-bladed β-propeller. In the presence of calcium, a single calcium ion was observed at the centre of this catalytic domain, behind the catalytic pocket. In the absence of calcium, the calcium ion was replaced with one sodium ion and one water molecule, and the positions of these cations were shifted by 1.3 Å. The histidine-319 side chain, which coordinates to the 2-hydroxyl oxygen atom of the bound xylose molecule in the catalytic pocket, also coordinates to the calcium ion, but not to the sodium ion. The calcium-dependent increase in activity appears to be caused by the structural change in the catalytic pocket induced by the tightly bound calcium ion and coordinating water molecules, and by the protonation state of glutamic acid-268, the catalytic acid of the enzyme. Our findings further elucidate the complex relationship between metal ions and glycosidases. [ABSTRACT FROM AUTHOR]

Lignocellulosic biomass from various types of wood has become a renewable resource for production of biofuels and biobased chemicals. Because xylan is the major component of wood hemicelluloses, highly efficient enzymes to enhance xylan hydrolysis can improve the use of lignocellulosic biomass. In this study, a xylanolytic gene cluster was identified from the crude oil-degrading thermophilic strain Geobacillus thermodenitrificans NG80-2. The enzymes involved in xylan hydrolysis, which include two xylanases (XynA1, XynA2), three β-xylosidases (XynB1, XynB2, XynB3), and one α- l-arabinofuranosidase (AbfA), have many unique features, such as high pH tolerance, high thermostability, and a broad substrate range. The three β-xylosidases were highly resistant to inhibition by product (xylose) accumulation. Moreover, the combination of xylanase, β-xylosidase, and α- l-arabinofuranosidase exhibited the largest synergistic action on xylan degradation (XynA2, XynB1, and AbfA on oat spelt or beechwood xylan; XynA2, XynB3, and AbfA on birchwood xylan). We have demonstrated that the proposed enzymatic cocktail almost completely converts complex xylan to xylose and arabinofuranose and has great potential for use in the conversion of plant biomass into biofuels and biochemicals. [ABSTRACT FROM AUTHOR]

Aims The aim of the study was to characterize 10 hemicellulolytic enzymes obtained from a wheat straw-degrading microbial consortium. Methods and Results Based on previous metagenomics analyses, 10 glycosyl hydrolases were selected, codon-optimized, synthetized, cloned and expressed in Escherichia coli. Nine of the overexpressed recombinant proteins accumulated in cellular inclusion bodies, whereas one, a 37·5- kDa protein encoded by gene xylM1989, was found in the soluble fractions. The resulting protein, denoted XylM1989, showed β-xylosidase and α-arabinosidase activities. It fell in the GH43 family and resembled a Sphingobacterium sp. protein. The XylM1989 showed optimum activity at 20°C and pH 8·0. Interestingly, it kept approximately 80% of its β-xylosidase activity in the presence of 0·5% (w/v) furfural and 0·1% (w/v) 5-hydroxymethylfurfural. Additionally, the presence of Ca2+, Mg2+ and Mn2+ ions increased the enzymatic activity and conferred complete tolerance to 500 mmol l−1 of xylose. Protein XylM1989 is also able to release sugars from complex polysaccharides. Conclusion We report the characterization of a novel bifunctional hemicellulolytic enzyme obtained through a targeted synthetic metagenomics approach. Significance and Impact of the Study The properties of XylM1989 turn this protein into a promising enzyme that could be useful for the efficient saccharification of plant biomass. [ABSTRACT FROM AUTHOR]

Commercial scale degradation of hemicelluloses into easily accessible sugar residues is practically crucial in industrial as well as biochemical processes. Xylanolytic enzymes have a great number of possible applications in many biotechnological processes and therefore, these enzymes are continuously attracting the attention of scientists. Due to this fact, different β-Xylosidases have been isolated, purified and characterized from several bacteria and fungi. Microorganisms in this respect have gained much momentum for production of these significant biocatalysts with remarkable features. It is difficult to propagate microorganisms for efficient and cost-competitive production of β-Xylosidase from hemicelluloses due to expensive conditions of fermentation. The screening of new organisms with an enhanced production of β-Xylosidases has been made possible with the help of recombinant DNA technology. β-Xylosidase genes haven been cloned and expressed on large scale in both homologous and heterologous hosts with the advent of genetic engineering. Therefore, we have reviewed the literature regarding cloning of β-Xylosidase genes into various hosts for their heterologous production along with sequence similarities among different β-Xylosidases. The study provides insight into the current status of cloning, expression and sequence analysis of β-Xylosidases for industrial applications. [ABSTRACT FROM AUTHOR]

β-Xylosidases are critical for complete degradation of xylan, the second main constituent of plant cell walls. A minor β-xylosidase (BXYL II) from Penicillium janczewskii was purified by ammonium sulfate precipitation (30% saturation) followed by DEAE-Sephadex chromatography in pH 6.5 and elution with KCl. The enzyme presented molecular weight (MW) of 301 kDa estimated by size exclusion chromatography. Optimal activity was observed in pH 3.0 and 70-75 °C, with higher stability in pH 3.0-4.5 and half-lives of 11, 5, and 2 min at 65, 70, and 75 °C, respectively. Inhibition was moderate with Pb and citrate and total with Cu, Hg, and Co. Partially purified BXYL II and BXYL I (the main β-xylosidase from this fungus) were individually immobilized and stabilized in glyoxyl agarose gels. At 65 °C, immobilized BXYL I and BXYL II presented half-lives of 4.9 and 23.1 h, respectively, therefore being 12.3-fold and 33-fold more stable than their unipuntual CNBr derivatives (reference mimicking soluble enzyme behaviors). During long-term incubation in pH 5.0 at 50 °C, BXYL I and BXYL II glyoxyl derivatives preserved 85 and 35% activity after 25 and 7 days, respectively. Immobilized BXYL I retained 70% activity after 10 reuse cycles of p-nitrophenyl-β-D-xylopyranoside hydrolysis. [ABSTRACT FROM AUTHOR]

Cell wall-associated β-xylosidase was isolated fromAspergillus nigerE-1 and identified as XlsIV, corresponding to the extracellular enzyme XlnD reported previously.xlsIVwas transcribed only in the early cultivation period. Cell wall-associated enzyme activity gradually decreased, but extracellular activity increased as the strain grew. These results indicate that XlsIV (XlnD) was secreted into culture after localizing at cell wall. [ABSTRACT FROM PUBLISHER]

A polymer–salt-based aqueous two-phase system (ATPS) was developed for the effective extraction and purification of extracellular β-xylosidase from the fermentation broth of recombinantBacillus megateriumMS941. The effect of molecular weight (MW) of polyethylene glycol (PEG), tie-line length (TLL), volume ratio (VR), crude loading and pH on the recovery performance was evaluated. Under the optimal extraction conditions, β-xylosidase was successfully purified up to 23-fold with a recovery yield of 99% in the bottom salt-rich phase at PEG 4,000/potassium phosphate ATPS comprising TLL of 41.8, VRof 2.3, crude loading (CL) of 30% (w/w) at pH 6. [ABSTRACT FROM AUTHOR]

Homotetrameric β-xylosidase from Selenomonas ruminantium (SXA) is one of the most efficient enzymes known for the hydrolysis of cell wall hemicellulose. SXA shows a rapid rate of activity loss at temperatures above 50 °C. In this study, we have introduced two inter-subunit disulfide bridges with one point mutation. Lys237 was chosen to be replaced with cysteine since it interacts with the same residue in the opposite subunit. While pH optimum, temperature profile and catalytic efficiency of the mutated variant were similar to the native enzyme, the mutated enzyme showed about 40% increase in thermal stability at 55 °C. Our results showed that introduction of a single residue mutation in structure of SXA results in appearance of two disulfide bonds at dimer-dimer interface of the enzyme. Coarse-grained molecular dynamics (CG-MD) simulations also proved lower amounts of root mean square fluctuation (RMSF) for position 237 and potential energy for mutated SXA. Based these results, we suggest that choosing a correct residue for mutation in multi subunit proteins results in multiple site conversions which equals to several simultaneous mutations. Furthermore, CG-MD simulation in agreement with experimental methods showed higher thermostability of mutated SXA which proved applicability of this simulation for thermostability analysis. [ABSTRACT FROM AUTHOR]