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Cellobiose and xylobiose are disaccharides composed of two glucose or xylose units with β-1,4 linkages. This study aimed to isolate a Trichoderma reesei mutant that lacks β-glucosidase and β-xylosidase activities for the simultaneous production of these disaccharides. Mutagenesis using Fe-ion beam resulted in a mutant strain, T. reesei T1640; the cellulase production in this strain was as high as that in the parent strain. Genomic analysis revealed that T1640 lost both the β-glucosidase and β-xylosidase activities owing to the translocation of the responsible genes. Hydrolysis of alkali-treated bagasse using the enzymes from T1640 leads to high yields (365 mg/g-biomass) and ratios (72.7% of the total sugars) of cellobiose and xylobiose. [Display omitted] • Trichoderma reesei T1640 was developed using Fe ion irradiation mutagenesis. • T1640 lost both β-glucosidase and β-xylosidase activities through translocation. • T1640 showed a high cellulase production similar to that of the wild parent strain. • One-step co-production of cellobiose and xylobiose from bagasse was achieved. [ABSTRACT FROM AUTHOR]

GH30 xylobiohydrolases, an expanding enzyme category, need deeper insights for optimal use. The primary aim of this study was to characterize a new xylobiohydrolase, Ac GH30A of GH30 family from Acetivibrio clariflavus. The gene encoding Ac GH30A was cloned using pET28a(+) vector and expressed in E. coli BL21(DE3) cells. Ac GH30A was purified by immobilized metal-ion affinity chromatography. SDS-PAGE analysis of Ac GH30A showed molecular mass of ~58 kDa. Ac GH30A showed optimum temperature 80 °C and optimum pH 7.0. Ac GH30A was stable (maintaining >80 % of control activity) in pH range, 4–7 and temperature range, 30 °C -70 °C when incubated for 90 min. Ac GH30A displayed melting temperature, 72 °C and half-life, 21 days at 4 °C. The enzyme activity of Ac GH30A was enhanced by 10 mM Ca2+ and Mg2+ ions by 25 % and 21 %, respectively, whereas 10 mM Co2+, Zn2+, Fe2+, and Cu2+ ions significantly reduced it. Ac GH30A showed activity against various xylan polysaccharides displaying highest V max , 139 U.mg−1 and K M , 0.71 mg.ml−1 against 4- O -methyl glucuronoxylan under optimum conditions. TLC, HPLC and LC-MS analyses of Ac GH30A hydrolyzed products from xylan substrates revealed the release of sole product, xylobiose, confirming it as an obligate xylobiohydrolase. Ac GH30A being a highly thermostable enzyme can be potentially utlilized in various biotechnological applications. [Display omitted] • Recombinant Ac GH30A cloned from Acetivibrio clariflavus showed molecular size 58 kDa. • Thermophilic Ac GH30A showed optimum temperature 80 °C and optimum pH 7.0. • Ac GH30A displayed melting temperature, 72 °C and half-life, 21 days at 4 °C. • Ac GH30A gave V max , 139 U.mg−1 and K M , 0.71 mg.ml−1 with 4- O -methyl glucuronoxylan. • Xylobiohydrolase, Ac GH30A released only xylobiose from xylan polysaccharides. [ABSTRACT FROM AUTHOR]

The fast pyrolysis mechanism of xylose, xylobiose and xylan was investigated with combined fast pyrolysis experiments and quantum chemistry calculations. The results indicate that acyclic d -xylose is an important intermediate for the formation of the major pyrolytic products in xylose pyrolysis, i.e., 1,4-anhydro- d -xylopyranose (ADX), furfural (FF) and hydroxyacetaldehyde (HAA). ADX derives from the successive cyclization of d -xylose which is the only source for ADX formation. 3-Deoxy-xylose is the most important intermediate for FF formation. HAA results from the C C bond scission by retro-aldol and cyclic Grob fragment. In addition, the five-membered intermediate derived from the ring-contraction reaction can transform into FF with high selectivity. The dehydrated xylose is also another source for HAA formation. The fast pyrolysis mechanism of xylobiose is similar to that of xylose, and thus similar pyrolytic product distribution can be obtained from them. However, the pyrolytic products of xylan differ significantly from those of xylose and xylobiose, which is ascribed to its high degree of polymerization and the branches in it. [ABSTRACT FROM AUTHOR]

[Display omitted] • Two novel xylanase genes from T. asperellum were expressed for the first time. • XynTR10 and XynTR11 displayed high halophilic and ethanol tolerant. • XynTR11 hydrolyzed xylan/XOS into xylobiose without xylose via transglycosylation. • Glu153/Glu258 and Glu161/Glu252 were the key active sites for XynTR10 and XynTR11. • XynTR11 holds great potential for XOS production from agricultural waste. Effective production of xylooligosaccharides (XOS) with lower proportion of xylose entails unique and robust xylanases. In this study, two novel xylanases from Trichoderma asperellum ND-1 belonging to glycoside hydrolase families 10 (XynTR10) and 11 (XynTR11) were over-expressed in Komagataella phaffii X-33 and characterized to be robust enzymes with high halotolerance and ethanol tolerant. Both enzymes displayed strict substrate specificity towards beechwood xylan and wheat arabinoxylan. (Glu153/Glu258) and (Glu161/Glu252) were key catalytic sites for XynTR10 and XynTR11. Notably, XynTR11 could rapidly degrade xylan/XOS into xylobiose without xylose via transglycosylation. Direct degradation of corncob using XynTR10 and XynTR111 displayed that while XynTR10 yielded 77% xylobiose and 25% xylose, XynTR11 yielded much less xylose (11%) and comparable amounts of xylobiose (63%). XynTR10 or XynTR111 has great potential as a catalyst for bioconversion of xylan-containing agricultural waste into high-value products (biofuel or XOS), which is of significant benefit for the economy and environment. [ABSTRACT FROM AUTHOR]

Biotechnological process for co-production of glucose and xylooligosaccharides from sugarcane bagasse generated a xylobiose-rich sugarcane bagasse xylan hydrolysate. However, sugarcane bagasse xylan hydrolysate is a complex mixture and how to isolate and purify xylobiose from sugarcane bagasse xylan hydrolysate is challenging. In the present study, an efficient combined process consisting of resin adsorption and gel filtration chromatography was developed to isolate and purify xylobiose from sugarcane bagasse xylan hydrolysate using a central composite design-based response surface methodology. The process purified xylobiose to 95.74 ± 1.43%, with a recovery of 93.61 ± 3.50%, and achieved decolourization and desalination rates of 77.59 ± 3.79% and 96.85 ± 1.43%, respectively. Mass balance analysis revealed maximum yields of 64.38 ± 3.47 mg (xylobiose), 6.71 ± 2.20 mg (xylotriose) and 6.71 ± 1.70 mg (xylose) from 100 mg of total sugars in sugarcane bagasse xylan hydrolysate. This process may improve the high-value bio-refinery of sugarcane bagasse. [ABSTRACT FROM AUTHOR]

The feasibility of a simulated moving bed (SMB) technology for the continuous separation of high-purity xylobiose (X2) from the output of a β-xylosidase X1→X2 reaction has recently been confirmed. To ensure high economical efficiency of the X2 production method based on the use of xylose (X1) as a starting material, it is essential to accomplish the comprehensive optimization of the X2-separation SMB process in such a way that its X2 productivity can be maximized while maintaining the X2 product concentration from the SMB as high as possible in consideration of a subsequent lyophilization step. To address this issue, a suitable SMB optimization tool for the aforementioned task was prepared based on standing wave design theory. The prepared tool was then used to optimize the SMB operation parameters, column configuration, total column number, adsorbent particle size, and X2 yield while meeting the constraints on X2 purity, X2 product concentration, and pressure drop. The results showed that the use of a larger particle size caused the productivity to be limited by the constraint on X2 product concentration, and a maximum productivity was attained by choosing the particle size such that the effect of the X2-concentration limiting factor could be balanced with that of pressure-drop limiting factor. If the target level of X2 product concentration was elevated, higher productivity could be achieved by decreasing particle size, raising the level of X2 yield, and increasing the column number in the zones containing the front and rear of X2 solute band. [ABSTRACT FROM AUTHOR]

In order to better understand the pyrolysis behavior of hemicellulose and unveil pyrolytic products distribution at molecular level, xylobiose was selected as a model compound of hemicellulose and relevant degradation processes were theoretically investigated by employing density functional theory (DFT) method. Several important reaction routes including directly bond breaking, Retro-Aldol reaction and Retro-Diels-Alder mechanism were analyzed, and the influence from adjacent xylose on the pyrolytic mechanism of xylobiose was interpreted systematically. The results indicate that the ring-opening of xylose unit dominates in the initial stage, followed by the cleavage of β -1,4-glycosides bond. Retro-Aldol reaction is kinetically favored and therefore leads to the formation of glycolaldehyde. Furfural, on the other hand, is generated via the cleavage of C1 O bond and subsequent ring forming between C2 and C5, which has a lower reaction energy barrier than that of 1-hydroxy-2-propanone. Aldehyde prefers to generate via Retro-Aldol reaction and decarbonylation. Although dehydration reaction is hard to occur in the initial pyrolysis process, it is still the major reaction channel to obtain formaldehyde and 1-hydroxy-2-propanone. [ABSTRACT FROM AUTHOR]

Xylobiose (X2), which is currently available from xylooligosaccharides (XOS), has been reported to have outstanding prebiotic function and to be highly suitable for application in food industries. This has sparked an interest in the economical production of X2 of high purity (> 99%) in food and prebiotic industries. To address such issue, we developed a highly-efficient chromatographic method for the recovery of X2 from XOS with high purity and high recovery. As a first step for this work, an eligible adsorbent for a large-scale separation between X2 and other XOS components was selected. For the selected adsorbent, a single-column experiment was carried out to determine the intrinsic parameters of all the XOS components, which were then used in the optimal design of the continuous X2-recovery process based on a simulated moving bed (SMB) chromatographic method. Finally, the performance of the designed X2-recovery SMB process was verified by the relevant SMB experiments, which confirmed that the developed process in this study could recover X2 from XOS with the purity of 99.5% and the recovery of 92.3% on a continuous-separation mode. The results of this study will be useful in enabling the economical production of high-purity X2 on a large scale. [ABSTRACT FROM AUTHOR]

Xylobiose is a good prebiotic and is used as an additive in the food and feed industries. The fungal strain Aspergillus fumigatus FC2-2, isolated from soil, secretes xylanases that can convert birchwood xylan and extracted xylan from sugarcane bagasse to xylobiose as the main component. A xylanase AfxynA produced by the strain FC2-2 was purified and characterized. The AfxynA hydrolyzed the xylooligosaccharides, birchwood xylan, and sugarcane bagasse xylan, mainly into xylobiose. The half-life of the purified AfxynA at 50 °C and 55 °C was 4207.9 min and 1001.6 min, respectively. The N-terminal sequence of the purified AfxynA was determined to be AGTPSSTG, and the AfxynA was found to belong to the glycosyl hydrolase family 11. The cDNA gene encoding AfxynA was cloned and overexpressed in Escherichia coli , and the gene was site-directed mutated to replace the codons encoding Asn70, Arg121, or Gln135 by Ala. The mutant enzymes N70A, R121A, and Q135A showed reduced enzyme activity in comparison with the native enzyme. More interestingly, the hydrolysis product of birchwood xylan by these mutant xylanases showed less proportion of xylobiose than that by the native enzyme. The above results indicated that N70, R121, and Q135 of AfxynA are required for xylanase activity and formation of xylobiose as a major product. [ABSTRACT FROM AUTHOR]

Highlights: [•] Bacterial strain MW8 was isolated from soil with remarkable xylanase activity. [•] The strain was classified as a novel subspecies of Streptomyces thermocarboxydus. [•] The fermentation medium for xylanase production was optimized. [•] Purified xylanase was an endo-β-1,4-xylanase with distinct NH2-termial sequence. [•] The xylanase exhibited remarkable heat stability and was active over broad pH ranges. [ABSTRACT FROM AUTHOR]

Abstract: The effects of xylo-oligosaccharides (XOS) and xylose on the hydrolytic activities of cellulases, endoglucanase II (EGII, originating from Thermoascus aurantiacus), cellobiohydrolase I (CBHI, from T. aurantiacus), and cellobiohydrolase II (CBHII, from Trichoderma reesei) on Avicel and nanocellulose were investigated. After the addition of XOS, the amounts of cellobiose, the main product released from Avicel and nanocellulose by CBHI, decreased from 0.78 and 1.37mg/ml to 0.59 and 1.23mg/ml, respectively. During hydrolysis by CBHII, the amounts of cellobiose released from the substrates were almost cut in half after the addition of XOS. Kinetic experiments showed that xylobiose and xylotriose were competitive inhibitors of CBHI. The results revealed that the strong inhibition of cellulase by XOS can be attributed to the inhibitory effect of XOS especially on cellobiohydrolase I. The results indicate the necessity to totally hydrolyze xylo-oligosaccharides into the less inhibitory product, xylose, to increasing hydrolytic efficiency. [Copyright &y& Elsevier]

Abstract: An extreme halophilic xylanase was purified from cultures of Chromohalobacter sp. TPSV 101 by ultrafiltration, hydroxylapatite and gel filtration chromatography. SDS-PAGE of the xylanase showed an apparent homogeneity and molecular weight of 15kDa. The xylanase had maximum activity at pH 9.0 and 65°C in the presence of 15–25% NaCl and was stable in the range of pH 7.0–9.0, temperature between 50 and 70°C. The enzyme was stable at 50–65°C for 1h retaining 100% activity and by retaining 60% activity at 80°C. The xylanase was completely inhibited by Hg2+ ions and was partially inhibited by Ca2+, Cu2+ and Pb2+ ions, whereas Zn2+, Mn2+ and Co2+ ions enhanced its activity. Both EGTA and EDTA enhanced its activity. It was active in solutions containing water-insoluble organic solvents and osmolytes. Kinetic experiments indicated that the enzyme had K m and V max values of 0.2mg/ml and 1. 17μmol/ml/min for oat spelt xylan. The major products of the oat spelt xylan hydrolysis were xylose and xylobiose; after prolonged incubation xylose was the major end product. [Copyright &y& Elsevier]

Abstract: As a first step toward understanding the energetics of removal of cello-oligomers from the cellulose surface, we have performed umbrella sampling calculations to determine the free energy required for separation of repeating units of cellulose and hemicellulose from each other. Molecular dynamics (MD) simulations were performed for both the stacked and non-stacked arrangements of the cellobiose pair system and the xylobiose pair system. These stacked and non-stacked arrangements were taken as representative systems for the crystalline and amorphous domains in cellulose and hemicellulose. In addition, similar calculations were also carried out to determine the energetics involved in the separation of the cellobiose–xylobiose molecule pair in the non-stacked arrangement. The potential of mean force profiles exhibit a single minimum in all cases and are qualitatively similar. Our results show that the location of the minimum as well as the depth of the well can be correlated with the size of the disaccharide molecules. [Copyright &y& Elsevier]

Xylooligosaccharides (XOS) are emerging prebiotic ingredients used for enriching food products due to their health-promoting properties. This study investigated XOS chemical stability after high-pressure processing (HPP) combined with heat treatment. First, orange juice was enriched with XOS (xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose), and then HPP-treated at 100, 300, and 600 MPa applying non-thermal (30 °C) and thermal (100 °C) conditions. Ascorbic acid (vitamin C) degradation was monitored to evaluate the extend of the severity of the treatments due to its low stability under high-energy process conditions. Also, quality attributes associated with fruit juices such as physicochemical properties (pH, soluble solid content, and ζ-potential), organic acid content (malic and citric acids), total phenolic content, antioxidant activity, and sugar content (glucose, fructose, and sucrose) were determined. The use of HPP combined with heat treatment degraded ascorbic acid up to 37% and promoted sucrose hydrolysis into glucose and fructose. However, our findings demonstrated that the XOS were chemically stable to HPP in the range of 100–600 MPa even applying thermal treatment. Therefore, XOS are promising ingredients for the formulation of functional plant-based beverages since their integrity was preserved under high mechanical and thermal stress conditions of 600 MPa and 100 °C. [Display omitted] • Xylooligosaccharides (XOS) were chemically stable to severe mechanical and thermal stress conditions. • High-pressure processing (HPP) combined with heat treatment degraded ascorbic acid up to 37%. • HPP treatments (100–600 MPa) at 100 °C promoted sucrose hydrolysis into glucose and fructose. • Non-thermal HPP treatment is a promising stabilization technique for beverages enriched with XOS. [ABSTRACT FROM AUTHOR]

Abstract: In this study, a thermostable recombinant xylanase B (XynB) from Thermotoga maritima MSB8 was immobilized on nickel-chelated Eupergit C 250L. This immobilized XynB was then used to hydrolyze the autohydrolysis explosion liquor of corncob (AELC) in a packed-bed enzyme reactor for continuous production of xylooligosaccharides, especially xylobiose. When tested in batch hydrolysis of AELC, the immobilized XynB still retained its relative activity of 92.5% after 10 cycles of hydrolysis at 90°C. The immobilized XynB retained 83.6% of its initial hydrolysis activity even after 168h of hydrolysis reaction at 90°C and demonstrated a half-life time of 577.6h (24days) for continuous hydrolysis. HPLC showed that xylobiose (49.8%) and xylose (22.6%) were the main hydrolysis products yielded during continuous hydrolysis. Xylobiose was adsorbed on an activated charcoal column and eluted with a linear gradient of 15% (v/v) ethanol to yield xylobiose with 84.7% of recovery. Also, the purity of xylobiose was up to 97.2% as determined by HPLC. Therefore, the immobilized XynB was suitable for the efficient production of xylobiose from AELC. This is the first report on the immobilization of xylanase for xylobiose production. [Copyright &y& Elsevier]

Abstract: Immobilization of XynB from the hyperthermophilic Thermotoga maritima on nickel-chelate IDA (iminodiacetic acid disodium salt monohydrate)-Eupergit C 250L (Ni-E, nickel-chelate Eupergit C 250L) resulted in high immobilization yield (76.3%), catalytic efficiency (77.9%) and bond protein (96.6%). Thermostability of Ni-E XynB was significantly improved by 1moll−1 phosphate buffer (pH 7.0) treatment. Chemical surface modifications were followed by FT-NIR (Fourier transform near infrared) spectroscopy, strongly indicated that a chemical reaction between the amines or some other groups and epoxy groups took place in Eupergit C 250L during immobilization. Characterization of the immobilized XynB was further evaluated. The optimum pH was not affected by immobilization, but the optimum pH range of the immobilized XynB was pH 4.6–6.6 while that of the free XynB was pH 5.6–7.5. The free XynB had an optimum temperature of 90°C, whereas that of the immobilized XynB was shifted to 100°C. Immobilization increased both pH stability and thermostability when compared with the free enzyme. Autohydrolysis liquor of corncob (ALC) was used as the substrate for enzymatic hydrolysis by the immobilized XynB in a packed-bed enzyme reactor. The major hydrolysis product is xylobiose. The immobilization procedure developed provides a promising solution for application of xylanases in continuous hydrolysis of ALC. [Copyright &y& Elsevier]

The xynB of a hyperthermophilic Eubacterium, Thermotoga maritima MSB8, coding xylanase B (XynB) was previously expressed in E. coli and the recombinant protein was characterized using the synthetic substrates [J. Biosci. Bioeng. 92 (2001) 423]. In this study, the same xylanase B was purified to homogeneity with a recovery yield of about 43% using heat treatment followed by the Ni-NTA affinity chromatography. The specificity of XynB towards different natural substrates was evaluated. XynB was highly specific towards xylans tested but exhibited low activities towards lichenan (19%), gellan gum (7.3%), laminarin (3.4%) and carboxymethylcellulose (CMC, 1.4%). The apparent Km values of birchwood xylan and soluble oat-spelt xylan was 0.11 and 0.079 mg/ml, respectively. The XynB hydrolyzed xylooligosaccharides to yield predominantly xylobiose (X2) and a small amount of xylose (X1), suggesting that XynB was possibly an endo-acting xylanase. Analysis of the products from birchwood xylan degradation confirmed that the enzyme was an endo-xylanase with xylobiose and xylose as the main degradation products. HPLC results showed that hydrolyzed products of birchwood xylan by XynB yielded up to 66% of the total reaction product as xylobiose. These results clearly indicated that xylobiose could be mass-produced efficiently by the recombinant hyperthermostable XynB of T. maritima. Additionally, conversion of xylobiose (50 mM) to xylose was observed, while xylotriose (X3) and xylotetraose (X4) were detected in small amounts, indicating that the enzyme converted xylobiose to xylose based on the transglycosylation reaction. The increased binding ability of XynB to Avicel and/or insoluble xylan was also observed indicating the possibilities of roles of surface-aromatic amino acid residues for such action. However, further investigations are required to prove this speculation. [Copyright &y& Elsevier]

An extracellular β-xylosidase from a newly isolated Fusarium proliferatum (NRRL 26517) capable of utilizing corn fiber xylan as growth substrate was purified to homogeneity from the culture supernatant by DEAE-Sepharose CL-6B batch adsorption chromatography, CM Bio-Gel A column chromatography, Bio-Gel A-0.5 m gel filtration and Bio-Gel HTP Hydroxyapatite column chromatography. The purified β-xylosidase (specific activity, 53 U/mg protein) had a molecular weight of 91,200 as estimated by SDS–PAGE. The optimum temperature and pH for the action of the enzyme were 60 °C and 4.5, respectively. The purified enzyme hydrolyzed xylobiose and higher xylooligosaccharides but was inactive against xylan substrates. It had a Km value of 0.77 mM (p-nitrophenol-β-d-xyloside, pH 4.5, 50 °C) and was competitively inhibited by xylose with a Ki value of 5 mM. The enzyme did not require any metal ion for activity and stability. Comparative properties of this enzyme with other fungal β-xylosidases are presented. [Copyright &y& Elsevier]

Fusarium proliferatum (NRRL 26517), was isolated by screening soil samples surrounding decaying corn and wood using corn fibre xylan as carbon source. The extracellular xylanase from this fungal strain was purified 975-fold to homogeneity from the culture supernatant by ammonium sulphate treatment, DEAE Bio-Gel A column chromatography, octyl-Sepharose column chromatography and Bio-gel A-0.5 m gel filtration. The purified xylanase (specific activity 591 U mg−1 protein) was a monomeric glycoprotein with a molecular weight (MW) of 22 400 as determined by SDS-PAGE. The optimum pH and temperature for the action of the enzyme were at 5.0–5.5 and 55 °C, respectively. The purified xylanase was fully stable at pH 5.0–7.5 and temperature up to 55 °C. It hydrolyzed a variety of xylan substrates mainly to xylobiose and higher short-chain xylooligosaccharides. No xylose was formed. The enzyme did not require metal ions for activity and stability. [Copyright &y& Elsevier]

The use of agroindustrial waste for chemicals, biofuels and food ingredients production has been studied considering limitations of natural resources. Therefore, sugarcane bagasse was studied to produce arabinoxylan, xylanases, xylooligosaccharides and lignocellulose by the improvement of some technologies. A comparison of two methods of extraction of arabinoxylan was performed. Arabinoxylan was obtained in a soluble fraction in a better extraction by KOH method with higher yield (72.1%) and efficiency (84%) than H 2 O 2 method, which only produced 64.9 and 43.3%, respectively. Lignocellulose was obtained after filtration, as the solid fraction, representing 66.2% (KOH) and 78.8% (H 2 O 2 method) of the total biomass. The endoxylanase was produced by Aspergillus fumigatus CCT7732 in submerged fermentation with bagasse culture medium reaching 40.2 U/mL in optimal conditions, pH 5.0 and 31.5 °C. In addition, NH 4 H 2 PO 4 was efficient as phosphorus and nitrogen sources for xylanases production in the culture, when the more expensive salts K 2 HPO 4 and KH 2 PO 4 were replaced. An enzymatic hydrolysis of arabinoxylan in Xylooligosaccharides (XOS) by xylanases of A. fumigatus resulted in 3.3% (m/v) XOS, 46.9% yield using 7% arabinoxylan, 350 U/g of xylanases at 50 °C in 48 h. These methods provided a prebiotic XOS since mainly xylobiose and xylotriose were produced with potential applications for food and feed. The set of chemical and biological methods applied in bagasse indicated that these methods should be evaluated in scale up of an integrated biosystem including economic studies for industrial purposes. • Arabinoxylan was better extracted from bagasse by KOH method than peroxide. • Alkaline method also can be used to obtain lignocellulose fraction from bagasse. • High level of xylanases can be obtained by A. fumigatus culture using bagasse. • NH 4 H 2 PO 4 can be less expensive than other nitrogen salts for the culture. • High level of XOS was produced with optimized xylanases hydrolyses of arabinoxylan. [ABSTRACT FROM AUTHOR]

• Yield of the endoxylanase (Taxy11) from T. asperellum ND-1 was improved (68.40%). • Glu119, Glu210, and Asp53 were the essential catalytic sites for Taxy11 activity. • A schematic model for Taxy11 shows novel catalysis mechanism in xylobiose production. • Taxy11 efficiently converts corncob xylan into xylobiose-enriched XOSs (50.44%). Here, we proposed an effective strategy to enhance a novel endoxylanase (Taxy11) activity and elucidated an efficient catalysis mechanism to produce xylooligosaccharides (XOSs). Codon optimization and recruitment of natural propeptide in Pichia pastoris resulted in achievement of Taxy11 activity to 1405.65 ± 51.24 U/mL. Analysis of action mode reveals that Taxy11 requires at least three xylose (xylotriose) residues for hydrolysis to yield xylobiose. Results of site-directed mutagenesis indicate that residues Glu119, Glu210, and Asp53 of Taxy11 are key catalytic sites, while Asp203 plays an auxiliary role. The novel mechanism whereby Taxy11 catalyzes conversion of xylan or XOSs into major product xylobiose involves transglycosylation of xylose to xylotriose or xylotetraose as substrate, to form xylotetraose or xylopentaose intermediate, respectively. Taxy11 displayed highly hydrolytic activity toward corncob xylan, producing 50.44 % of xylobiose within 0.5 h. This work provides a cost-effective and sustainable way to produce value-added biomolecules XOSs (xylobiose-enriched) from agricultural waste. [ABSTRACT FROM AUTHOR]

• Xylooligosaccharides (XOS)-enriched orange juice was high-intensity ultrasound (HIUS) treated. • Nominal power effects of 300, 600, 900, and 1200 W on the XOS stability were evaluated. • The HIUS process intensification reduced the ascorbic acid content from 0.189 to 0.084 mg/mL. • XOS chromatographic profiles were not modified by the HIUS treatment. • XOS were not hydrolyzed into short-chain molecules like xylose up to 1200 W for 10 min. The effects of the high-intensity ultrasound (HIUS) technology at the nominal powers of 300, 600, 900, and 1200 W were evaluated on the chemical stability of xylooligosaccharides (XOS) used to enrich orange juice. The ultrasound energy performance for each nominal power applied to the XOS-enriched orange juice was determined by calculating acoustic powers (W), HIUS intensity (W/cm2), and energy density (kJ/mL). Physicochemical properties (pH and soluble solid content), organic acid content (ascorbic, malic, and citric acids), total phenolic content (TPC), antioxidant activity by the FRAP (Ferric reducing ability of plasma) method, sugar (glucose, fructose, and sucrose), and XOS (xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose) content were determined. The pH and soluble solid content did not change after all HIUS treatments. The HIUS process severity was monitored by quantifying ascorbic acid content after the treatments. A significant linear decrease in the ascorbic acid content was observed in prebiotic orange juice with the HIUS process intensification by increasing nominal power. The malic acid and citric acid contents had similar behavior according to the HIUS process intensification. The nominal power increase from 300 to 600 W increased the concentration of both organic acids, however, the intensification up to 1200 W reduced their concentration in the functional beverage. The TPC and FRAP data corroborated with the results observed for the ascorbic acid content. However, the HIUS processing did not alter sugar and XOS contents. The XOS chromatographic profiles were not modified by the HIUS treatment and presented the same amount of all prebiotic compounds before and after the HIUS treatment. Overall, HIUS technology has been evaluated as a promising stabilization technique for prebiotic beverages enriched with XOS due to their high chemical stability to this emerging technology under severe process conditions. [ABSTRACT FROM AUTHOR]

• A putative Acremonium alcalophilum GH30_7 xylanase gene was cloned and expressed. • Aa Xyn30A is active on different xylans and linear xylooligosaccharides. • Aa Xyn30A releases xylobiose from the non-reducing end of the substrates. • Xylobiohydrolase activity makes Aa Xyn30A a promising tool for xylobiose production. Xylanases of the GH30 family are grouped to subfamilies GH30-7 and GH30-8. The GH30-8 members are of bacterial origin and well characterized, while the GH30-7 members are from fungal sources and their properties are quite diverse. Here, a heterologous expression and characterization of the GH30-7 xylanase Aa Xyn30A from a cellulolytic fungus Acremonium alcalophilum is reported. From various polymeric and oligomeric substrates Aa Xyn30A generates xylobiose as the main product. It was proven that xylobiose is released from the non-reducing end of all tested substrates, thus the enzyme behaves as a typical non-reducing-end acting xylobiohydrolase. Aa Xyn30A is active on different types of xylan, exhibiting the highest activity on rhodymenan (linear β-1,3-β-1,4-xylan) from which also an isomeric xylotriose Xyl-β-1,3-Xyl-β-1,4-Xyl is formed. Production of xylobiose from glucuronoxylan is at later stage accompanied by a release of aldouronic acids differing from those liberated by the bacterial GH30-8 glucuronoxylanases. [ABSTRACT FROM AUTHOR]

• First time reported an endo -xylanase (SipoEnXyn10A) from Streptomyces ipomoeae. • SipoEnXyn10A was neutral, thermo-philic/stable and was 66% identical with an enzyme. • Ratios of xylobiose in hydrolysates (84.52%–87.76%) were higher than most reports. • The yields of xylobiose from corncob and bamboo were the highest in the literature. • It was the first report of producing XOSs from cassava stem and Chinese fir sawdust. Endoxylanases capable of producing high ratios of xylobiose from agricultural and forestry residues in neutral and high temperature conditions are attractive for the prebiotic and alternative sweetener industries. In this study, a putative glycosyl hydrolase gene from Streptomyces ipomoeae was cloned and expressed in Escherichia coli. The recombinant enzyme, named as SipoEnXyn10A, hydrolyzed beechwood xylan in endo -action mode releasing xylobiose as its main end product. It was most active at pH 6.5 and 75–80 °C and showed remarkable stability at 65 °C. The xylobiose yield from 10 g corncob and moso bamboo reached 1.123 ± 0.021 and 0.229 ± 0.005 g, respectively, at pH 6.5 and 70 °C, which was higher than other reports using the same material. Moreover, high ratios of xylobiose in the xylose-based product of about 85% were obtained from corncob, moso bamboo sawdust, cassava stem and Chinese fir sawdust. These results demonstrated that SipoEnXyn10A has potential for industrial application. [ABSTRACT FROM AUTHOR]