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The genus Cohnella belongs to a group of Gram-positive endospore-forming bacteria within the Paenibacillaceae family. Although most species were described as xylanolytic bacteria, the literature still lacks some key information regarding their repertoire of xylan-degrading enzymes. The whole genome sequence of an isolated xylan-degrading bacterium Cohnella sp. strain AR92 was found to contain five genes encoding putative endo-1,4-β-xylanases, of which four were cloned, expressed, and characterized to better understand the contribution of the individual endo-xylanases to the overall xylanolytic properties of strain AR92. Three of the enzymes, CoXyn10A, CoXyn10C, and CoXyn11A, were shown to be effective at hydrolyzing xylans-derived from agro-industrial, producing oligosaccharides with substrate conversion values of 32.5%, 24.7%, and 10.6%, respectively, using sugarcane bagasse glucuronoarabinoxylan and of 29.9%, 19.1%, and 8.0%, respectively, using wheat bran-derived arabinoxylan. The main reaction products from GH10 enzymes were xylobiose and xylotriose, whereas CoXyn11A produced mostly xylooligosaccharides (XOS) with 2 to 5 units of xylose, often substituted, resulting in potentially prebiotic arabinoxylooligosaccharides (AXOS). The endo-xylanases assay displayed operational features (temperature optima from 49.9 to 50.4 °C and pH optima from 6.01 to 6.31) fitting simultaneous xylan utilization. Homology modeling confirmed the typical folds of the GH10 and GH11 enzymes, substrate docking studies allowed the prediction of subsites (− 2 to + 1 in GH10 and − 3 to + 1 in GH11) and identification of residues involved in ligand interactions, supporting the experimental data. Overall, the Cohnella sp. AR92 endo-xylanases presented significant potential for enzymatic conversion of agro-industrial by-products into high-value products. Key points • Cohnella sp. AR92 genome encoded five potential endo-xylanases. • Cohnella sp. AR92 enzymes produced xylooligosaccharides from xylan, with high yields. • GH10 enzymes from Cohnella sp. AR92 are responsible for the production of X2 and X3 oligosaccharides. • GH11 from Cohnella sp. AR92 contributes to the overall xylan degradation by producing substituted oligosaccharides.

The hemicellulose content present in corn cobs can help in producing a high amount of xylooligosaccharides (XOS) in an eco-friendly manner. In this work, the XOS was produced from alkali pre-treated corn-cobs having a true yield of 38 ± 1.4% via enzymatic hydrolysis with the help of xylanase from T. lanuginosus VAPS-24. The production process was optimized to achieve a high concentration of XOS using innovative multi-objective optimization through machine learning modeling and finding out the most suitable parameters where xylobiose production is higher than xylose. The Multi-objective connected neural networks (MOCNN) model with tangent sigmoid activation function yielded a correlation coefficient of 96.51%; there were six optimal sets where xylobiose concentration was higher than xylose. The best-optimized conditions yielded 3.03 mg/ml of xylobiose and 1.31 mg/ml of xylose. Therefore, this novel approach of machine learning can target the increasing demand for xylooligosaccharides in the growing industrial market of prebiotics.

A novel xylanase gene of 870bp was isolated and cloned from the thermophilic bacteria Cohnella sp.A01 that encodes an enzyme comprising 290 amino acid residues and belonging to the GH10 family of xylanases. The enzyme was efficiently expressed in Escherichia coli BL21, then purified using one-step chromatography. The recombinant XynB-A01 not only exhibited significant thermostability but also showed to be stable in a broad range of pH (3.0–10.0), the presence of some detergents, and organic solvents. Although XynB-A01 demonstrated significant activity toward both beechwood and oat spelt xylans, it showed a stronger affinity and higher activity toward the former. HPLC analysis of beechwood xylan hydrolysis revealed that xylotetraose, xylobiose, and xylose are the major hydrolysis products. The investigation of XynB-A01 in pulp biobleaching showed effectiveness in the release of reducing sugars and chromophores. Simple purification, stability over a broad range of pH and temperature, cellulase-free character, and the capacity to produce xylooligosaccharides are some of the advantages of the xylanase of Cohnella sp.A01, suggesting that it may be a promising candidate for biobleaching of paper pulps, producing xylooligosaccharides, and applying in other industries.

In this study, crude and partially purified xylanase from Aureobasidium pullulans CCT 1261 were characterized and applied in beechwood xylan hydrolysis to obtain xylooligosaccharides (XOS). Fractional precipitation (0–30 %/30–60 %) with ammonium sulfate allowed partial purification of xylanases in 6.8-fold and recovery of 69.4 %. Both extracts exhibited optimum activity at 50 °C and pH 4.5, and greater stability at 40 °C within the studied range. The thermostability parameters (kd, t1/2, ΔH*, ΔG*, ΔS*) of the extracts indicated the enzyme denaturation by the increment in temperature from 40 °C to 60 °C; its process was endothermic and led to protein unfolding in a more random structure. Crude xylanase showed a lower value of the Michaelis-Menten constant (25 mg/mL) than the partially purified enzyme (50 mg/mL). In hydrolysis reactions, total XOS contents (7.7 mg/mL and 7.9 mg/mL) and XOS yields (25.7 % and 26.5 %) were equal statistically using the crude or partially purified extracts, differing only in the amount of xylose released. The hydrolysates were mainly composed of potential prebiotics, i.e., xylobiose (50.9 % and 53.8 %) and xylotriose (30.9 % and 32.8 %). Based on these results, the use of crude and partially purified xylanase from A. pullulans showed a promising perspective for xylan hydrolysis to produce XOS.

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

The genus Pestalotiopsis are endophytic fungi that have recently been identified as cellulolytic system producers. We herein cloned a gene coding for a xylanase belonging to glycoside hydrolase (GH) family 10 (PesXyn10A) from Pestalotiopsis sp. AN-7, which was isolated from the soil of a mangrove forest. This protein was heterologously expressed by Pichia pastoris as a host, and its enzymatic properties were characterized. PesXyn10A was produced as a glycosylated protein and coincident to theoretical molecular weight (35.3 kDa) after deglycosylation by peptide-NfF-glycosidase F. Purified recombinant PesXyn10A exhibited maximal activity at pH 6.0 and 50 °C, and activity was maintained at 90 % at pH 5.0 and temperatures lower than 30 °C for 24 h. The substrate specificity of PesXyn10A was limited and it hydrolyzed glucuronoxylan and arabinoxylan, but not β-glucan. The final hydrolysis products from birchwood xylan were xylose, xylobiose, and 1,23-α-D-(4-O-methyl-glucuronyl)-1,4-β-D-xylotriose. The addition of metallic salts (NaCl, KCl, MgCl2, and CaCl2) activated PesXyn10A for xylan degradation, and maximal activation by these divalent cations was approximately 160 % at a concentration of 5 mM. The thermostability of PesXyn10A significantly increased in the presence of 50 mM NaCl or 5 mM MgCl2. The present results suggest that the presence of metallic salts at a low concentration, similar to brackish water, exerts positive effects on the enzyme activity and thermal stability of PesXyn10A.

There is increasing attention on the exploration of waste feedstocks as economically viable substrates for the production of prebiotic oligosaccharides, especially xylooligosaccharides, as excellent candidates for the maintenance and promotion of gut microbiota. XOS, an emerging prebiotic that has several functional attributes and beneficial health effects, is mainly produced by different processes, especially enzymatic hydrolysis through the valorisation of xylan enriched lignocellulosic materials. The present study deals with the enzymatic production of xylooligosaccharide (XOS) from xylan rich cauliflower stalk, a novel source. Delignification with alkali (NaOH) was found to be more efficient than acid and autohydrolysis, resulting in a higher extraction yield of xylan (18.42%). Alkaline extraction for 120 minutes at 1.25 M alkali concentration produced maximum xylan yield. FTIR analysis of xylan extracted from cauliflower stalk by an alkaline (NaOH) pretreatment method showed typical absorption bands at 1729 cm-1 that correspond to acetyl groups exhibiting the typical xylan specific band. Enzymatic hydrolysis was carried out with indigenously produced crude endoxylanase obtained from Aspergillus niger MTCC 9687 and the effects of substrate concentration, enzyme concentration, pH, time and temperature were investigated. High resolution MS analysis showed the presence of xylobiose as the major XOS. The major 1H spectral signals of XOS liberated from enzymatically hydrolysed alkali extracted cauliflower stalk xylan showed the presence of β-anomeric protons in the spectral region of 4.0-4.7 ppm. Prebiotic efficacy of cauliflower stalk derived XOS alone and synbiotic combinations with known probiotic strains (Lactiplantibacillus plantarum, Bifidobacterium bifidum, Lactobacillus delbrueckii ssp. Helveticus) were evaluated. Butyrate was found to be the major short chain fatty acid produced by XOS supplemented fermentation media. All the synbiotic combinations showed significantly higher antioxidant and antimicrobial activities and reduced the viability of human bone cancer MG-63 cells. The individual profiles of antimicrobial components of XOS were identified as dihydroxy benzoic acid and aspartic acid by HPLC coupled to a photodiode array detector.

Xylooligosaccharides (XOS) are considered as prebiotics, as they help in enhancing the growth of beneficial microbes found in the gut. In the present study, partially purified xylanase from Thermomyces lanuginosus VAPS-24 was used for the production of XOS from xylan extracted from corn cobs. The mild alkali treatment with 20% NaOH yielded 32 ± 1.8% (w/w) xylan and it was structurally characterized by FTIR. The corn cob xylan (2% w/v), when treated with partially purified 20 U VAPS-24 xylanase for 8 h at 45°C, yielded 6.9 ± 0.03 mg/ml of XOS. The XOS contained mainly xylobiose (X2) and xylotriose (X3) which were purified and analyzed using 13C-NMR spectroscopy which showed predominant presence of (1–4) linked β-d-xylose residues. HPLC analysis showed characteristic peaks of xylotriose, xylobiose, and xylose in the XOS mixture. The produced XOS showed concentration-dependent antioxidant activities and probiotic growth promotion. Further, the genes responsible for the metabolism of XOS in probiotics were identified and their probable interactions and coexpression of genes were studied in silico.

Xylanase is a versatile tool in the food, fiber biobleaching and biofuel industries. Here, to discover new enzyme with special properties, we cloned three xylanases (Xyn11A, Xyn11B, and Xyn11C) by mining the genome of the xylanase producing fungus strain Fusarium sp. 21, biochemically characterized these enzyme and explored their potential application in juice processing. Both Xyn11A and Xyn11B had an optimal pH of 6.0 and optimal temperature of 45 °C, and retained >90% of the residual activity at pH range of 5–10.5 for 24 h. Xyn11C displayed the maximum activity at pH 5.0 and 45 °C and outstanding pH stability with a minimal loss of activity in the pH range of 2.0–10.5. These three xylanases displayed a strong specificity towards beechwood and corncob xylan, with no activity for other substrates. Xyn11A showed much a higher activity against corncob xylan, while Xyn11B and Xyn11C presented higher activities against beechwood xylan. Xyn11A catalyzed the hydrolysis of beechwood xylan with a Km of 4.25 ± 0.29 mg·mL−1 and kcat/Km of 30.34 ± 0.65 mL·s−1·mg−1, while the hydrolysis of corncob xylan had Km and kcat/Km values of 14.73 ± 1.43 mg·mL−1and 26.48 ± 0.11 mL·s−1·mg−1, respectively. Xyn11B and Xyn11C hydrolyzed beechwood xylan with Km of 9.8 ± 0.69 mg·mL−1, and 4.89 ± 0.38 mg·mL−1and kcat/Km values of 45.07 ± 1.66 mL−1·mg−1, and 26.95 ± 0.67 mL·s−1·mg−1, respectively. Beechwood xylan hydrolysates catalyzed by these three xylanases contained xylobiose, xylotriose and xylooligosaccharides (XOS). The clarification of orange juice was improved when treated with these three xylanases. Conclusively, the desirable pH stability and substrate specificity make Xyn11A, Xyn11B and Xyn11C have high potential for application in fiber biobleaching, wine and fruit juice clarification, as well as probiotic XOS production.

Alkaliphilic xylanase from Neosartorya spinosa UZ-2-11 was purified using a three-step of purification scheme of ammonium sulphate precipitation followed by Sephadex G-100 gel filtration and DEAE-cellulose ion-exchange chromatography, and compared its properties with N. tatenoi KKU-CLB-3-2-4-1 of our previous report. The purified xylanase from N. spinosa UZ-2-11 exhibited maximum activity at pH 9.0 and 45 °C which was similar to endo-xylanase from N. tatenoi KKU-CLB-3-2-4-1. However, this enzyme was stable in a range of pH 6.0–11.0. It was also more stable at a high temperature of 50 °C where the activity was still up to 50% after heating for 120 min. The xylanase was purified 7.89-fold with 3.0% of yield to obtain a specific activity of 11.88 U/mg. The molecular weight of xylanase from this fungus was 27.68 kDa. The Km and Vmax values of the purified xylanase were 0.24 mg/mL and 15.85 μmol/min/mg, respectively. The xylanase activity was moderately inhibited by Hg2+ at a concentration of 10 mM, which was different to the case of N. tatenoi KKU-CLB-3-2-4-1 where Hg2+ was a strong inhibitor. In addition, the hydrolysed birchwood xylan was obtained mailnly xylobiose, xylotriose, xylotetraose and xylopentaose as end products, suggesting that it was an endo-xylanase.

This study aimed to develop a method for the purification of a xylanase called SMXL1 produced by Stenocarpella maydis and its biochemical characterization. The enzyme was purified using a Rotofor preparative chamber and one chromatographic step in an ion exchange column coupled to equipment FPLC. Posteriorly the protein was characterized, and its effect on the birchwood xylan degradation was determine by HPLC. The purified enzyme showed a molecular weight of 55 kDa calculated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purification process obtained a yield of 6.5  0.3 %. The activity was stable at a pH range of 4 to 10 and temperatures of 45 to 60 °C. The optimum values of temperature and pH were 55 °C and 4, respectively. The Michaelis constant (Km) value was 2.61 mg/mL and the Vmax was 3.02 µmol/mL/min using birchwood xylan as substrate and the Michaelis-Menten equation. The enzyme is inhibited by the cations Mn2+ and by Fe3+ and degrades the birchwood xylan being the principal products the xylobiose and the xylose. This work is the first report of the purification and biochemical characterization of a xylanase called SMXL1 produced by S. maydis.

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.

BACKGROUND Non-starch polysaccharides in wheat are dominated by arabinoxylan (AX). Endo-1,4-β-xylanase (EC 3.2.1.8) is the most important enzyme for degrading AX. This paper investigated the ability of endo-1,4-β-xylanase extracted from wheat malt to degrade non-water-extractable wheat-derived arabinoxylan (WUAX). RESULTS The enzyme was observed to break down wheat-derived WUAX effectively, substantially increasing the concentration of water-extractable arabinoxylan (WEAX) in the system for up to 6 h. A considerable quantity of arabinose xylooligosaccharide (AXOS) was also produced, suggesting that the enzyme could produce oligosaccharides too. The molecular weight of the product WEAX was between 23 and 27 kDa and the content of oligosaccharides changed with degradation time. This suggests that endo-1,4-β-xylanase can not only degrade WUAX into WEAX and xylooligosaccharides but can also degrade the xylooligosaccharides with larger molecular weights into xylobiose and xylotriose. The viscosity of the degradation product increased significantly in the first 2 h, then decreased with longer degradation times. The concentration of WEAX in the reaction system increased throughout the reaction but at gradually lower rates, indicating that the endo-1,4-β-xylanase degraded WEAX better than it degraded WUAX. Rheological tests showed that solutions prepared from the WEAX that was produced had properties of a pseudoplastic fluid. CONCLUSION The results showed that the wheat malt endo-1,4-β-xylanase, which we had previously tested on WEAX, was also effective in degrading wheat-derived WUAX. This study can therefore provide a theoretical basis for the subsequent role of the enzyme in other sources of xylan, and provide guidance for the quality control of beer in the brewing process. © 2021 Society of Chemical Industry.

Anaeromyces robustus is an anaerobic rumen microorganism which can produce plant cell wall degrading enzymes. In this study, a new GH10 xylanase gene xylAr10 from A. robustus was identified, cloned and expressed in Pichia pastoris GS115. The recombinant protein ArXyn10 was characterized after being purified by Ni–NTA. The optimal pH and temperature of ArXyn10 was determined at 5.5 and 40 °C, respectively. ArXyn10 was stable at the pH range of 4.0–8.0, and could maintain high stability from 35 to 45 °C. The hydrolysis products released from beechwood xylan by ArXyn10 showed chromatographic mobility similar to xylobiose and xylotriose according to thin-layer chromatography analysis. It was shown that the addition of 7.5 mg of ArXyn10 in 100 g high-gluten wheat flour during bread making could increase the reducing sugar content by 10.80%, indicating that xylo-oligosaccharides were produced. With the addition of ArXyn10, the hardness and chewiness of the bread decreased and the quality was improved. The new discovered xylanase ArXyn10 have potential application prospect in bread making.

Sugarcane processing roughly generates 54 million tonnes sugarcane bagasse (SCB)/year, making SCB an important material for upgrading to value-added molecules. In this study, an integrated scheme was developed for separating xylan, lignin and cellulose, followed by production of xylo-oligosaccharides (XOS) from SCB. Xylan extraction conditions were screened in: (1) single extractions in NaOH (0.25, 0.5, or 1 M), 121 °C (1 bar), 30 and 60 min; (2) 3 × repeated extraction cycles in NaOH (1 or 2 M), 121 °C (1 bar), 30 and 60 min or (3) pressurized liquid extractions (PLE), 100 bar, at low alkalinity (0–0.1 M NaOH) in the time and temperature range 10–30 min and 50–150 °C. Higher concentration of alkali (2 M NaOH) increased the xylan yield and resulted in higher apparent molecular weight of the xylan polymer (212 kDa using 1 and 2 M NaOH, vs 47 kDa using 0.5 M NaOH), but decreased the substituent sugar content. Repeated extraction at 2 M NaOH, 121 °C, 60 min solubilized both xylan (85.6% of the SCB xylan), and lignin (84.1% of the lignin), and left cellulose of high purity (95.8%) in the residuals. Solubilized xylan was separated from lignin by precipitation, and a polymer with β-1,4-linked xylose backbone substituted by arabinose and glucuronic acids was confirmed by FT-IR and monosaccharide analysis. XOS yield in subsequent hydrolysis by endo-xylanases (from glycoside hydrolase family 10 or 11) was dependent on extraction conditions, and was highest using xylan extracted by 0.5 M NaOH, (42.3%, using Xyn10A from Bacillus halodurans), with xylobiose and xylotriose as main products. The present study shows successful separation of SCB xylan, lignin, and cellulose. High concentration of alkali, resulted in xylan with lower degree of substitution (especially reduced arabinosylation), while high pressure (using PLE), released more lignin than xylan. Enzymatic hydrolysis was more efficient using xylan extracted at lower alkaline strength and less efficient using xylan obtained by PLE and 2 M NaOH, which may be a consequence of polymer aggregation, via remaining lignin interactions. Supplementary Information The online version contains supplementary material available at 10.1186/s13068-021-01993-z.

The heteropolysaccharide xylan is a valuable source of sustainable chemicals and materials from renewable biomass sources. A complete hydrolysis of this major hemicellulose component requires a diverse set of enzymes including endo-β-1,4-xylanases, β-xylosidases, acetylxylan esterases, α-l-arabinofuranosidases, and α-glucuronidases. Notably, the most studied xylanases from glycoside hydrolase family 11 (GH11) have exclusively been endo-β-1,4- and β-1,3-xylanases. However, a recent analysis of a metatranscriptome library from a microbial lignocellulose community revealed GH11 enzymes capable of releasing solely xylobiose from xylan. Although initial biochemical studies clearly indicated their xylobiohydrolase mode of action, the structural features that drive this new activity still remained unclear. It was also not clear whether the enzymes acted on the reducing or nonreducing end of the substrate. Here, we solved the crystal structure of MetXyn11 in the apo and xylobiose-bound forms. The structure of MetXyn11 revealed the molecular features that explain the observed pattern on xylooligosaccharides released by this nonreducing end xylobiohydrolase.

Made available in DSpace on 2021-06-25T10:31:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-11-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Xylooligosaccharides (XOS) acts as a prebiotic, a food component that is not digested but stimulates the growth of beneficial microorganisms present in the intestine, such as bifidobacteria and lactobacilli, improving the health of the host. This study obtained XOS by comparing optimized conditions for acid hydrolysis and autohydrolysis of sugarcane bagasse, showing that is possible to produce XOS with minimum degradation of sugras. The acid hydrolysis process varied the parameters applying a 23 factorial design using 1–3% (m/v) sulfuric or acetic acid; a temperature of 100–160 °C and a reaction time of 15–55 min. The autohydrolysis was performed by 22 factorial design using the same range of temperature and time. The acetic acid hydrolysis of the sugarcane bagasse resulted in the conversion of xylan into XOS of 18.41% (m/m) with 1% (m/v) acid at 100 °C for 15 min, with xylopentaose/xylohexaose predominance. The hydrolysis of sugarcane bagasse with sulfuric acid resulted in 90.13% (m/m) of XOS with 2% (m/v) of acid at 79.55 °C for 35 min, with xylobiose prevalence. Autohydrolysis of bagasse resulted in 13.67% (m/m) xylan conversion into XOS at 172.4 °C for 35 min, with xylotetraose prevalence. The degradation sugar products were low for the conditions studied, with 0.31%, 0.16%, and 0.01% (m/m) of furfural using sulfuric, acetic, and autohydrolysis, successively. The results demonstrated that XOS with different degrees of polymerization can be produced applying specific pretreatment conditions of autohydrolysis, sulfuric, and acetic acid, with the minimum content of sugar degradation. Institute for Research in Bioenergy (IPBEN) São Paulo State University (UNESP) School of Pharmaceutical Sciences (FCF) Department of Bioprocesses and Biotechnology São Paulo State University (UNESP) Institute for Research in Bioenergy (IPBEN) São Paulo State University (UNESP) School of Pharmaceutical Sciences (FCF) Department of Bioprocesses and Biotechnology São Paulo State University (UNESP) FAPESP: 2017/22401–8 FAPESP: 2019/12997–6

Caldicellulosiruptor species are hyperthermophilic, Gram-positive anaerobes and the most thermophilic cellulolytic bacteria so far described. They have been engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. Xylooligomers, such as xylobiose and xylotriose, that result from the breakdown of plant biomass more strongly inhibit cellulase activity than do glucose or cellobiose. High concentrations of xylobiose and xylotriose are present in C. bescii fermentations after 90 h of incubation, and removal or breakdown of these types of xylooligomers is crucial to achieving high conversion of plant biomass to product. In previous studies, the addition of exogenous β-d-xylosidase substantially improved the performance of glucanases and xylanases in vitro. β-d-Xylosidases are, in fact, essential enzymes in commercial preparations for efficient deconstruction of plant biomass. In addition, the combination of xylanase and β-d-xylosidase is known to exhibit synergistic action on xylan degradation. In spite of its ability to grow efficiently on xylan substrates, no extracellular β-d-xylosidase was identified in the C. bescii genome. Here, we report that the coexpression of a thermal stable β-d-xylosidase from Thermotoga maritima and a xylanase from Acidothermus cellulolyticus in a C. bescii strain containing the A. cellulolyticus E1 endoglucanase significantly increased the activity of the exoproteome as well as growth on xylan substrates. The combination of these enzymes also resulted in increased growth on crystalline cellulose in the presence of exogenous xylan. IMPORTANCE Caldicellulosiruptor species are bacteria that grow at extremely high temperature, more than 75°C, and are the most thermophilic bacteria so far described that are capable of growth on plant biomass. This native ability allows the use of unpretreated biomass as a growth substrate, eliminating the prohibitive cost of preprocessing/pretreatment of the biomass. They only grow under strictly anaerobic conditions, and the combination of high temperature and the lack of oxygen reduces the cost of fermentation and contamination by other microbes. They have been genetically engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. In this study, we introduced genes from other cellulolytic bacteria and identified a combination of enzymes that improves growth on plant biomass. An important feature of this study is that it measures growth, validating predictions made from adding enzyme mixtures to biomass.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to analyze the polymerization distribution of xylo-oligosaccharides from birchwood and beechwood xylans following hydrolysis by the xylanase (XynA) from Bacillus subtilis, which was obtained by recombinant expression and purified by Ni2+ affinity column chromatography. The hydrolysis products of birchwood and beechwood xylans were analyzed by thin layer chromatography (TLC) and MALDI-TOF-MS. In the birchwood hydrolysate, the main neutral xylo-oligosaccharides were xylobiose (X2 ) and xylotriose (X3 ), and the acidic xylo-oligosaccharides (polymerization degree 4-12) were attached via a single methyl-glucuronic acid sidechain (MeG). Both X2 and X3 were identified in the beechwood xylan hydrolysate and acidic xylo-oligosaccharides (polymerization degree 4-16), which were structurally similar to those in the birchwood xylan hydrolysate. Therefore, the recombinant xylanase, XynA, has the potential to produce xylobiose (X2 ) and xylotriose (X3 ) as well as acidic xylo-oligosaccharides (MeGXn ) that would be applied in food industry. PRACTICAL APPLICATIONS: Xylo-oligosaccharides are a novel functional food additive, which has great potential in improving the quality of food. Xylo-oligosaccharides with methyl-glucuronic acid sidechains (MeG) are acidic xylo-oligosaccharides (MeGXn), which can be applied in the preparation of drugs for the treatment of cystitis and mucopolysaccharidosis. In this study, xylanase XynA was first obtained by gene cloning and expression and then used to hydrolyze the birchwood and beechwood xylans. The polymerization distribution of xylo-oligosaccharides generated during the enzymatic digestion was then determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Through enzyme hydrolysis, we are able to produce xylobiose and xylotriose for food additives.

To develop an endo-β-1,4-xylanase with high specificity for production of prebiotic xylooligosaccharides that optimally works at moderate temperature desirable to reduce the energy cost in the production process. The xylB gene, encoding for a glycosyl hydrolase family 11 xylanase from a thermoresistant fungus, Aspergillus niger BCC14405 was expressed in a methylotrophic yeast P. pastoris KM71 in a secreted form. The recombinant XylB showed a high specific activity of 3852 and 169 U mg−1 protein on beechwood xylan and arabinoxylan, respectively with no detectable side activities against different forms of cellulose (Avicel O PH101 microcrystalline cellulose, phosphoric acid swollen cellulose and carboxymethylcellulose). The enzyme worked optimally at 45 °C, pH 6.0. It showed a specific cleavage pattern by releasing xylobiose (X2) as the major product from xylooligosaccharides (X3 to X6) substrates. The highest XOS yield of 708 mg g−1 substrate comprising X2, X3 and X6 was obtained from beechwood xylan hydrolysis. The enzyme is potent for XOS production and for saccharification of lignocellulosic biomass.

Xylooligosaccharides (XOS), a novel functional food and feed ingredient, can be produced from lignocellulosic biomass. In this study, spent mushroom substrate (SMS) gathered after Pleurotus ostreatus cultivation was investigated for its potential as a new raw material for XOS production using crude xylanases produced in-house from Aspergillus flavus KUB2. Xylan was extracted from SMS using the alkaline extraction method. The highest true recovery of xylan (20.76%) and the relative recovery of xylan (83.73%) were obtained from SMS extracted with 4 M NaOH. Enzymatic hydrolysis of SMS-extracted xylan using crude fungal xylanases from A. flavus KUB2 produced a maximum total XOS in the range 1.37–1.48 mg/ml, which was mainly composed of XOS with a low degree of polymerization (xylobiose and xylotriose). XOS derived from SMS-extracted xylan positively influenced the growth of probiotic bacteria, suggesting the prebiotic nature of XOS. The results indicated that XOS with prebiotic properties can be produced from SMS xylan using crude xylanases without any purification, which offers economic potential for food and feed applications.

Penelitian ini berhasil mengisolasi dan menskrining isolat jamur potensial pendegradasi xilan tongkol jagung yang diidentifikasi sebagai Fusarium oxysporum (HM210091.1). Produksi xilanase oleh F. oxysporum melalui fermentasi substrat padat dengan menggunakan tongkol jagung sebagai substratnya mampu menghasilkan aktivitas xilanase maksimum pada kondisi ukuran partikel substrat 60 mesh, rasio kadar air 2 mL/g substrat, suhu inkubasi 30oC, pH awal 6, jumlah inokulum 5x107/3 g substrat, dan waktu inkubasi 2 hari. Aktivitas xilanase meningkat sekitar 4 kali hingga mencapai 7.92 U/mL setelah optimasi. Xilanase ekstrak kasar yang dimurnikan secara parsial melalui presipitasi amonium sulfat (30-70%) yang dilanjutkan dengan dialisis menghasilkan faktor pemurnian sebesar 1.64 kali lipat dan pemulihan sebesar 62.91%. Aktivitas relatif xilanase pada xilan tongkol jagung terhadap aktivitas xilanase pada xilan beechwood komersial adalah sebesar 37.58%. Xilanase memiliki aktivitas optimum pada pH 5.5 dan suhu 50oC, dan memiliki kestabilan paling tingi pada pH 6 dan suhu 30oC setelah 1 jam inkubasi pada pH dan suhu optimum. Proses hidrolisis xilan tongkol jagung dengan konsentrasi 6% oleh xilanase sebanyak 100 U/g substrat pada kondisi pH dan suhu optimum xylanase (pH 5.5 dan suhu 50oC) mampu menghasilkan xilobiosa sebesar 28.71 g/100 g xilan murni setelah 12 jam inkubasi. Proses pemurnian hidrolisat dapat mempertahankan 91.08% jumlah xilobiosa. Xilobiosa murni berhasil diperoleh melalui tahap separasi lebih lanjut dengan menggunakan kromatografi kolom arang aktif, namun pemulihannya hanya sebesar 59.29%. In this present study, a potential corncob xylan-degrading fungal strain as xylanase producer was isolated and screened from soil, and identified as Fusarium oxysporum (HM210091.1). The production of xylanase by F. oxysporum under solid state fermentation using corncob powder as the solid substrate reached the maximum xylanase activity when using particle size of substrate of 60 mesh, water content ratio of 2 mL/g substrate, incubation temperature of 30oC, initial pH of 6, amount of inoculum of 5x107 spore/3 g substrate, and incubation time of 2 days. The xylanase activity increased about 4 times up to 7.92 U/mL after optimization. The crude xylanase was partially purified using ammonium sulphate precipitation (30-70%) followed by dialysis and resulting purification factor of 1.64 fold as well as yield of 62.91%. The relative activity of partial purified xylanase towards alkali-extracted corncob xylan was found 37.58% with respect to commercial beechwood xylan. The xylanase activity was optimum at pH 5.5 and temperature 50oC, and the most stable at pH 6 and temperature 30oC after 1 h incubation at optimum pH and temperature. The potential application of partial purified xylanase of F. oxysporum in hydrolyzing alkali-extracted corncob xylan to produce xylobiose was also demonstrated. Hydrolysis of 6% of corncob xylan using 100 U/g substrate of enzyme loading under optimum pH and temperature conditions (pH 5.5 and 50oC, respectively) achieved the yield of xylobiose up to 28.71 g/100 g pure xylan after 12 h incubation. The purification of hydrolysate could retain 91.08% of xylobiose. Further separation step using activated charcoal column chromatography was able to get a pure xylobiose, but could only recover 59.29% of xylobiose.

Development of a more environmentally sustainable society is based on the maximum use of renewable carbon sources and their valorization of environmentally-friendly green technologies. This includes a thorough use of plant biomass and agricultural residues for the production of value-added bioproducts. Xylan is the second most abundant biopolymer in nature which can be sustainable converted into pentoses and xylooligosaccharides, that have wide applications in the food, feed, pharmaceutical, and cosmetic industry. Within the scope of present study, we biochemically characterized two-domain GH10 xylanase from Jonesia denitrificans (JdXyn10A) and evaluated its applicability for production of xylooligosaccharides (XOS). JdXyn10A has a specific activity of 84 ± 2 U/mg and 65 ± 5 U/mg when acting on beechwood glucuronoxylan and rye arabinoxylan, respectively. The enzyme is stable in a wide pH range and is tolerant to high concentrations of NaCl and ethanol. Interestingly, the profile of products released by the enzyme is predominant in xylobiose and xylotriose, with a very low fraction of xylose which is desirable for XOS production. The efficiencies of enzymatic conversion of beechwood glucuronoxylan and rye arabinoxylan are 47.67 % and 26.01 %, respectively, after 6 h of enzymatic hydrolysis only. Structural comparison between the JdXyn10A homology model and the structure from its homologous that while the glycone region of its active site is well preserved, the aglycone region presents structural differences in the +2 subsite that may explain why JdXyn10A does not release xylose.

Eucalyptus pellita is known as attractive biomass, and it has been utilized for eucalyptus oil, furniture, and pulp and paper production that causes a significant amount of byproducts. Liquid hot water treatment depending on combined severity factor (CSF) was subjected to isolate hemicellulose fraction from E. pellita and to produce xylooligosaccharides (XOS). The xylan extraction ratio based on the initial xylan content of the feedstock was maximized up to 77.6% at 170 °C for 50 min condition (CSF: 1.0), which had accounted for XOS purity of 76.5% based on the total sugar content of the liquid hydrolysate. In this condition, the sum of xylobiose, xylotriose, and xylotetraose which has a low degree of polymerization (DP) of 2 to 4 was determined as 80.6% of the total XOS. The highest XOS production score established using parameters including the xylan extraction ratio, XOS purity, and low DP XOS ratio was 5.7 at CSF 1.0 condition. XOS production score evaluated using the CSF is expected to be used as a productivity indicator of XOS in the industry (R-squared value: 0.92).

A novel and eco-friendly approach was developed for the scouring and bleaching of flax roves with xylanase, cellulase and mixed enzymes (xylanase and cellulase) in supercritical carbon dioxide fluid (SC-CO2). The results showed that significant improvements in the whiteness and residual gum content of flax roves were achieved in SC-CO2 since xylanase hydrolysed xylan into low molecular oligosaccharides, xylobiose and xylose by hydrolyzing and cleaving β-1,4-glucosidic bonds of xylan. Simultaneously, the hydrolysis reaction of lignin through the hydrophobic and electrostatic interactions at β-O-4 linkages could be effectively facilitated by the catalyst of cellulase in SC-CO2. Furthermore, the whiteness and residual gum content of flax roves were notably influenced by the dosage of enzyme, enzyme ratio, scouring and bleaching temperature, pressure, time and CO2 flow. An optimized enzymatic process for the scouring and bleaching of flax roves was recommended with a dosage of enzyme 3%, an enzyme ratio of 2: 1 (xylanase: cellulase), a system temperature of 50 °C, a pressure of 20 MPa, a CO2 flow of 30 g/min for 90 min in SC-CO2. The whiteness value of flax roves reached to 42.3%, and the value of residual gum content was decreased to 16.95% in the optimum condition.

Enzymatic transformation of xylans into renewable fuels and value-added products is mediated by xylanases. Here we describe the biochemical and X-ray structural characterization of Thermobacillus composti GH10 xylanase (TcXyn10A) at 2.1 A resolution aiming to unravel details of its recognition of glucurono- and arabinoxylan at a molecular level. TcXyn10A improves the efficiency of pretreated lignocellulosic biomass hydrolysis by a commercial enzyme cocktail causing a 15.35 % increase in xylose release and 4.38 % glucose release after 24 h of reaction. The enzyme releases predominantly xylobiose and xylotriose, as well as MeGlcA3 × 3 (from beechwood glucuronoxylan) and a range of decorated xylooligosaccharides (XOS) from rye arabinoxylan, with Ara2 × 2 being the major product. The enzyme liberates XOS with the yields of 29.09 % for beechwood glucuronoxylan and 16.98 % for rye arabinoxylan. Finally, TcXyn10A has a high thermal stability, halotolerance, and resistance to ethanol, biochemical properties that can be desirable for a number of industrial applications.

Cave sets the example of extreme ecological niche and habitat for diversified microorganisms. Present study involved in the isolation of endoxylanase producing novel strainBacillus velezensisAG20 from the Krem Phyllut cave, Meghalaya, India. Culture dependent studies, molecular phylogentics, RNA secondary folding pattern based on 16S rDNA substantiated the identity of this novel strain.Bacillus velezensisAG20 revealed the superbug quality having resistance against various class of broad-spectrum antibiotics.Bacillus velezensisAG20 revealed biofilm formation over the cell surface in FESEM. Highest cell biomass and xylanase production supported in TB medium, further purified partially to 5.3 fold with 21% yield. Molecular weight of the purified xylanase found to be 45 kDa. Enzyme kinetics and pattern of hydrolysis revealed the evidence for the selection of linear birchwood xylan with Vmax= 21.0 ± 3.0 U/ml, Km = 1.25 mg/ml, Kcat= 1.75/s at optimum pH 7 and temperature 50°C also found significant statistically in Taguchi’s orthogonal design. Conversely, ruled out any exoacting activity against synthetic pNP-xylopyranoside substrate. Endo-xylanase isolated fromBacillus velezensisAG20 was moderately thermostable over temperatures 50 and 60°C. Time dependent hydrolysis of agro-waste sugar cane bagasse depicted the production of xylooligosaccharides (XOS) predominantly xylobiose, xylotriose and xylotetrose. Purified mixed XOS hold their prebiotic potential by promoting the growth of probioticsBifidobacteriumandLactobacillusas well as high stability (~90%) against systemic fluids. Mixed XOS (300 μg/ml) displayed anti-proliferation activities by reducing the growth of HT-29 and Caco-2 cells significantly 90% and 75%, respectively, after 48 h.IMPORTANCEExtremophiles dwelling inside the caves have laden with the extraordinary capabilities of bioconversion by nature. The pristine ecological niche inside the cave, absence of proper light and air, supports the livelihood of novel microorganisms. In India, Meghalaya is hoisting longest caves in the East Khasi Hills, providing conducive environment for novel bacterial strains. With the prime objective of isolating novel bacterial strains that produce extracellular xylanase our studies have been carried out. Considering the present industrial demand for nutraceutical, prebiotics, anti-proliferating agents and biofuels by the conversion of lignocellulosic biomass (LCB), novel enzymes are required. Xylanases from bacterial origin play a significant role in conversion of LCB into oligosaccharides. Therefore, exploration and characterization of xylanase producing novel isolate from cave may pave the new arena for the production of prebiotic and anti-inflammatory oligosaccharides from agro-waste.

Purpose This study investigated the influence of inter-domain interactions on the substrate affinity and hydrolysis product specificity of xylanase. Methods Genes encoding a GH10 endo-xylanase from Streptomyces chartreusis L1105 xynA and its truncated derivative were cloned and expressed in Escherichia coli. The catalytic activities of the enzyme (xynA) and the derivative xynADCBM, lacking the carbohydrate binding module (CBM), were assessed to evaluate the role of CBM in xynA. Results Recombinant xynA (44 kDa) was found to be optimally active on beechwood xylan at 65 °C with pH 7.7, while xynADCBM (34 kDa) exhibited optimal activity at 65 °C with pH 7.2. Additionally, xynA and xynADCBM were found to be highly thermostable at 40–60 °C, each retaining 80% of their original activity after 30 min. The xynADCBM without the CBM domain was highly efficient at hydrolyzing xylan to produce xylobiose (over 67%), which may be because the CBM domain facilitates substrate binding with xylanase. Meanwhile, the xylan hydrolysis efficiency of xynADCBM was higher than that of xynA. Conclusion These findings showed that the CBM domain with non-catalytic activity has no significant effect on the characteristics of the enzyme at optimum pH and pH tolerance. It has also been suggested that the derivative xynADCBM without CBM components can promote hydrolysis of xylan to yield xylooligosaccharides, which has great potential economic benefits.

A xylanase from Talaromyces thermophiles F1208 (T-Xyn) was used specifically to explore the effects of disulfide bond on hydrolytic activity. The T-Xyn-C122S-C166S mutant does not have the C122-C166 disulfide bond present in wild-type T-Xyn, whereas T-Xyn-T38C-S50C and T-Xyn-T38C-S50C-C122S-C166S mutants have an introduced disulfide bond, C38-C50, to T-Xyn and T-Xyn-C122S-C166S, respectively. The optimum pH of T-Xyn-T38C-S50C and T-Xyn-T38C-S50C-C122S-C166S was lower than that of T-Xyn and T-Xyn-C122S-C166S. The introduction of a disulfide bond caused a decrease in the optimum temperature and thermal stability of T-Xyn. The existence of a disulfide bond has a strong influence on the hydrolysis characteristics of T-Xyn, which caused changes in the composition and proportion of the hydrolysate products. T-Xyn-T38C-S50C produces the highest level of xylose when using beechwood xylan as the substrate, whereas T-Xyn produces the highest level of xylobiose and T-Xyn-T38C-S50C-C122S-C166S produces the largest amount of xylotriose. When birchwood xylan was used as the substrate, the introduction of a disulfide bond increased the content of xylose, decreased the content of xylotriose and a high degree of polymerization (DP ≥ 5) was observed. The hydrolysis of oat-spelt xylan is more complex with the introduction of the disulfide bond causing an increase in the degradation rate of xylotriose.

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 (μmax, 0.18 1/h), and maximum biomass concentration (Xmax, 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.

Highly efficient and stable enzymes are required for application in biotechnology, to meet the technical, environmental, and economic industrial demands. Xylanases are hemicellulolytic enzymes that degrade the heteroxylan constituent of the lignocellulosic plant cell wall. In this study, an acidic xylanase designated Pjxyn (pH 4.0) from Penicillium janthinellum was engineered by the introduction of a disulfide bridge. This strategy exploited the influence of the bridge on hydrolysis characteristics and enhanced hydrolysis was achieved. Three mutants [PjxynS(27)S(39), PjxynS(27)S(186), and PjxynS(39)S(186)] produced more xylose and xylobiose as hydrolysis products compared with the wild-type Pjxyn, when commercial xylans and lab-prepared water-insoluble corncob-xylan were used as the substrates, especial for the PjxynS(27)S(39) mutant, the content of xylose and xylobiose was 87.62% (using beechwood xylan) and 69.91% (using oat-spelt xylan) higher than that in the hydrolysis products of Pjxyn. Moreover, each mutant combined with the xylanase mutant T-XynFM effectively decreased the production of xylose with an optimum xylobiose yield. The findings demonstrate the potential industrial value of engineering xylanase to improve its hydrolytic properties and thermostability.

As an industrially important biological macromolecule, xylanase hydrolyzes xylan to produce xylooligosaccarides (XOS). XOS, with a degree of polymerization (DP) 2 to 4, are important prebiotics used as food ingredients. In this study, xylanase (5536 U/g substrate) was produced by Pichia stipitis using corncob and wheat bran mixture under solid state fermentation. Crude xylanase were purified and biochemically characterized. XOS hydrolyzed by crude and purified xylanases were quantified. Molecular weight of the purified enzyme was around 31.6 kDa on SDS-PAGE. Enzyme kinetics showed Km and Vmax values of 4.52 mg/mL and 9.17 μmol/min/mL, respectively. The optimal conditions were pH 6.0 and 50 °C. Xylanase was stable at pH 5–8 for 60 min by retaining 57% activity and at 50 °C for 80 min by retaining 65% activity. Cooper and potassium had no inhibitory effect on xylanase activity. Xylan hydrolysates produced by purified xylanase contained 92% XOS consisting of 14% xylotetroase (DP 4), 49% xylotriose (DP 3) and 29% xylobiose (DP 2). These findings indicate the potential of applying purified xylanase for industrial XOS production.

A GH30-8 endoxylanase was identified from an environmental Bacillus subtilis isolate following growth selection on aspen wood glucuronoxylan. The putative endoxylanase was cloned for protein expression and characterization in the Gram-positive protease deficient protein expression host B. subtilis WB800. The extracellular activity obtained was 55 U/mL, which was 14.5-fold higher than that obtained with the native species. The apparent molecular mass of BsXyn30 was estimated as 43 kDa by SDS-PAGE. BsXyn30 showed an optimal activity at pH 7.0 and 60 °C. Recombinant BsXyn30 displayed maximum activity against aspen wood xylan, followed by beechwood xylan but showed no catalytic activity on arabinose-substituted xylans. Analysis of hydrolyzed products of beechwood xylan by thin-layer chromatography and mass spectroscopy revealed the presence of xylooligosaccharides with a single methyl-glucuronic acid residue. BsXyn30 exhibited very low activity for hydrolysis xylotetraose and xylopentaose, but had no detectable activity against xylobiose and xylotriose. Using BsXyn30 as an additive in breadmaking, a decrease in water-holding capacity, an increase in dough expansion as well as improvements in volume and specific volume of the bread were recorded. Thus, the present study provided the basis for the application of GH30 xylanase in breadmaking.

An endo-xylanase (PsGH10A) of family 10 glycoside hydrolase from Pseudopedobacter saltans was cloned, expressed and purified. Substrate specificity analysis of PsGH10A showed activity against β-1,4-xylans. It showed maximum activity against beechwood xylan (59.7 ± 1.1 U/mg) followed by xylan (Mw. 20,000–30,000) (57.1 ± 0.7 U/mg). PsGH10A displayed maximum activity at pH 6.0 and 40 °C. The enzyme was stable in the pH range, from 6.0 to 7.5 and showed thermostability up to 40 °C. The kinetic parameters of PsGH10A using beechwood xylan determined were Km 6.2 mg/mL and Vmax 72 U/mg. The activity of PsGH10A was 29 ± 2.4% enhanced by 2 mM Mn2+ ions and inhibited by less than 50 ± 1.6% by 2 mM Zn2+, Pb2+ or Cu2+ ions. The time-dependent TLC analysis of hydrolyzed products of beechwood xylan released by PsGH10A showed the release of xylose, xylobiose and xylotetraose as end products confirming the endolytic mode of action. PsGH10A hydrolyzed products of xylan and substituted xylan indicate production of series of short-chain xylooligosaccharides and arabinoxylo-saccharides. PsGH10A also showed saccharification of AFEX pretreated poplar and sugarcane bagasse. Therefore, it can be used for various biotechnological applications such as for prebiotic xylooligosaccharides and bioethanol production from pretreated agrowaste biomass. This is the first report on β-1,4-xylanase cloned from Pseudopedobacter saltans.

Solid-state cultivations of genetically modified strains of Aspergillus nidulans A773, using soybean fibre as substrate, were carried out to produce xylanase and arabinofuranosidase, and these enzymes were subsequently used to obtain xylooligosaccharides using the same agro-residue. First, the best fungi cultivation conditions (moisture content, pH, temperature and addition of maltose) were optimized one-by-one for obtain the crude enzyme extracts. Subsequently, the application of xylanase on soybean fibre to obtain xylooligosaccharides was optimized by central composite design, defining best enzyme concentration and reaction temperature. The best condition obtained (50 °C and 117 U g−1 of soybean fibre) was used to evaluate the co-production of xylooligosaccharides by the addition of different concentrations of arabinofuranosidase. The highest yield of xylooligosaccharides obtained was 28% (mass fraction of xylan), showing final concentrations (in mg g−1 arabino-xylan) of 138.36 xylobiose (X2), 96.96 xylotriose (X3), and 53.04 xylotetraose (X4), in 9 h enzymatic reactions. The conversion of arabino-xylans into different xylooligosaccharides suggests the potential to use recombinant A. nidulans A773 enzymes to obtain prebiotics using a sugar-rich, low-cost soybean residue.

A novel glycosyl hydrolase family 11 xylanase gene, xynMF13A, was cloned from Phoma sp. MF13, a xylanase-producing fungus isolated from mangrove sediment. xynMF13A was heterologously expressed in Pichia pastoris, and the recombinant XynMF13A (rXynMF13A) was purified by Ni-affinity chromatography. The temperature and pH optima of purified rXynMF13A were 45 °C and pH 5.0, respectively. rXynMF13A showed a high level of salt tolerance, with maximal enzyme activity being seen at 0.5 M NaCl and as much as 53% of maximal activity at 4 M NaCl. The major rXynMF13A hydrolysis products from corncob xylan were xylobiose, xylotriose, xylotetraose, and xylopentaose, but no xylose was found. These hydrolysis products suggest an important potential for XynMF13A in the production of xylooligosaccharides (XOs). Furthermore, rXynMF13A had beneficial effects on Chinese steamed bread production, by increasing specific volume and elasticity while decreasing hardness and chewiness. These results demonstrate XynMF13A to be a novel xylanase with potentially significant applications in baking, XOs production, and seafood processing.

The purpose of study was utilization of sugarcane bagasse, waste product of sugarcane industry, for production of xylooligosaccharides (XOS) and their evaluation of their prebiotic potential. The XOS production was carried out in two steps: alkaline extraction of xylan (5, 10 and 15%KOH and NaOH) followed by acid hydrolysis (0.25 and 0.50M H2SO4; 20, 40 and 60 min) of xylan, and quantified using high performance thin layer chromatography (HPTLC). The prebiotic potency of XOS was evaluated for probiotics viz. Lactobacillus brevis, Lactobacillus acidophilus and Lactobacillus viridescens in comparison to standard fructooligosaccharides (FOS). The chemical compositional analysis indicated that bagasse contain 28.42% hemicellulose out of which 21.46% was estimated to be xylan. Maximum yield of xylan (20.5%) was obtained with 15%NaOH treatment. The best treatments for xylan hydrolysis were found to be 0.25M H2SO4 for 20 and 40 min with concentrations of xylose, xylobiose and xylotriose to be 2.014, 2.106 and 1.228 mg ml− 1, respectively, in 20 min hydrolysis and 2.138, 1.502 and 0.824 mg ml− 1, respectively, in 40 min hydrolysis. XOS were found to be better prebiotics than standard FOS. Pure xylobiose was found to have highest positive effect on growth of all three bacteria tested indicating that effects of XOS were due to presence of xylobiose, xylotriose and XOS with higher degree of polymerization in xylan hydrolysates. Sugarcane bagasse xylan can be converted into XOS only by controlled acid hydrolysis leading to increased production of XOS which can be used as good prebiotics in drugs and food ingredients after their purification eliminating all the acidic and alkaline residues and also side products.

Pozol is a traditional Mexican refreshing acid beverage made of hot-alkali treated and fermented maize dough. It has been demonstrated that non-amylolytic lactic acid bacteria as Weissella sp. are important in this process. The aim of this work was to study Weissella spp. strains isolated from pozol from the carbon source perspective. For this purpose, Weissella confusa L9, W. confusa L17, W. confusa Snc40, W. paramesenteroides 3a and W. cibaria 3LA were studied. All the microorganisms were able to metabolize xylose; they also showed β-glucosidase and β-galactosidase activities. The ability to grow in xylan was tested; all the strains showed maximum growth at 24 h but W. confusa L9, W. confusa L17 and W. confusa Snc40 grew from 7.09 to 8.66, 7.08 to 7.84 and 7.26 to 7.94 Log CFU/ml, respectively. W. confusa L9 growth in partially hydrolyzed xylan was evaluated. The evolution of xylooligosaccharides throughout the fermentation was performed. Xylose accumulation was observed (0.28–0.28 mg/ml at 24 h). A preference for xylobiose and xylotriose was observed. β-xylosidase activity was found in W. confusa L9 since consumption of xylooligosaccharides by this microorganism was demonstrated. Hemicellulose is therefore an alternative carbon source sustaining pozol fermentation.

Xylan is one of the major polymeric hemicellulosic constituents of lignocellulosic biomass, and its effective utilization by microorganisms is crucial for the economical production of biofuels. In this study, Paenibacillus physcomitrellae XB exhibited different xylan degradation ability on different substrates of corncob xylan (CCX), oat spelt xylan (OSX), wheat flour arabinoxylan (AX) and beech wood xylan (BWX). The RT-QPCR result showed that two genes (Pph_0602 and Pph_2344) belonging to the glycoside hydrolase family 43 were up-regulated more than 5-fold on CCX and xylose. Substrate-specific assays with purified proteins Ppxyl43A (Pph_0602) and Ppxyl43B (Pph_2344) revealed that both exhibited β-xylosidase activity toward the chromogenic substrate p-nitrophenyl-β-D-xylopyranoside, and α-L-arabinofuranosidase activity toward p-nitrophenyl-α-L-arabinofuranoside, indicating their bifunctionality. By testing their degradation characteristics on different natural substrates, it was found that both Ppxyl43A and Ppxyl43B showed similar degradation ability on CCX and OSX. Both enzymes could hydrolyze xylohexaose and xylobiose completely to xylose, but could not hydrolyze BWX and AX, suggesting they mainly hydrolyze xylo-oligosaccharides by β-xylosidase activity. Further analysis showed that both of them displayed very high pH stability and thermostability on the β-xylosidase activity, but Ppxy143B exhibited wider pH and temperature ranges, higher pH and temperature stability, was less influenced by metal ions, and had a slower start-up response than Ppxyl43A. Given their predicted structure, it is likely that the enzymatic differences between Ppxyl43A and Ppxyl43B might be related to the extra C-terminus domain (GH43_C2) in Ppxyl43B, which could enhance the enzymatic stability while restricting the substrates’ or metal ions’ access to the active sites of Ppxyl43B. In conclusion, both Ppxyl43A and Ppxyl43B were β-xylosidase/α-L-arabinofuranosidase bifunctional enzymes and might be useful in xylan biomass conversion, especially in the hydrolysis of xylo-oligosaccharides into xylose.

A promising approach for production of value-added xylooligosaccharides (XOS) from poplar was developed by combining hydrothermal pretreatment and endo-xylanase post-hydrolysis. Results showed that the 35.4% XOS (DP 2–6) and 17.6% low DP xylans (DP > 6) were obtained at the identified optimal condition (170 °C, 50 min) for hydrothermal pretreatment. Structural features of low DP xylans generated during the hydrothermal pretreatment were examined, revealing that low DP xylans are mainly comprised of 4-O-methylglucuronic xylan and are involved in lignin carbohydrate complexes. Moreover, higher pretreatment intensity promoted the cleavage of side-chain substituents including arabinose and glucuronic acid groups. The subsequent endo-xylanase hydrolysis of the pretreatment liquor hydrolyzed low DP xylans, contributing to a significant improvement in xylobiose and xylotriose proportions. This combined strategy resulted in a XOS with conversion yield of 44.6% containing 78.7% xylobiose and xylotriose starting from the initial xylan in raw poplar.

A GH10 xylanase Srxyn10 from Streptomyce rochei L10904, and its truncated derivative, Srxyn10M, were investigated. Both displayed great salt-tolerant ability, retaining more than 95% and 91% activity after incubation at 37°C for 1h in 3.0M and 5.0M NaCl, respectively. They exhibited a special hydrolytic property of forming xylobiose as the major product and produced fewer xylose compounds when combined with a reported xylanase while digesting corncob xylans. The mutant, Srxyn10M, was constructed from Srxyn10 by deleting the C-terminal carbohydrate-binding module. It possessed a 3.26-fold higher specific activity on beechwood xylan than Srxyn10. Moreover, Srxyn10M showed greater substrate affinity and catalytic efficiency than Srxyn10 when beechwood xylan, birchwood xylan, and oat-spelt xylan were used as substrates. The thermostability was also greatly improved. Therefore, the application potential was markedly enhanced by the improvement of these properties.

Empty fruit bunch (EFB), the largest amount of biomass from a palm oil mill, is a promising renewable source for many value-added products. This study aimed to utilize EFB for production of xylanase by Streptomyces thermocarboxydus ME742 and applied the enzyme to the production of xylooligosaccharides (XOS) from EFB derived xylan (EFB-xylan). The strain ME742 produced the maximum xylanase (473.21 ± 6.55 U/mL) in a medium containing 50 g/L EFB supplemented with 3.0 g/L peptone at 40 °C for 4 days. Xylanase ME742 was purified 23.68 fold with specificity of 1229 U/mg by acetone precipitation and DEAE-Sepharose chromatography. It was stable in a wide range of pH (4.0–9.5) and in elevated temperatures up to 55 °C. Xylanase ME742 effectively hydrolyzed EFB-xylan. HPLC analysis revealed that the hydrolysate was composed of xylose and XOS with degree of polymerization between 2 and 5 and xylobiose was the predominant product. The XOS yield ranging in 95.01–151.19 mg/g EFB-xylan was generated from enzymatic processing for 12–72 h. Interestingly, arabinosyl-xylooligosaccharides were also predicted to be produced from EFB-xylan. XOS mixture exhibited high antioxidant activity (71.36 ± 0.57%) against DPPH at 100 μg/mL concentration and moderate activity (45.18 ± 1.56%) against ABTS radical. These findings reveal the potential use of Streptomyces thermocarboxydus ME742 and xylanase produced in a sustainable bioprocess to add value of EFB waste.

The cost-efficient degradation of xylan to fermentable sugars is of particular interest in second generation bioethanol production, feed, food, and pulp and paper industries. Multiple potentially secreted enzymes involved in polysaccharide deconstruction are encoded in the genome of Paenibacillus sp. A59, a xylanolytic soil bacterium, such as three endoxylanases, seven GH43 β-xylosidases, and two GH30 glucuronoxylanases. In secretome analysis of xylan cultures, ten glycoside hydrolases were identified, including the three predicted endoxylanases, confirming their active role. The two uni-modular xylanases, a 32-KDa GH10 and a 20-KDa GH11, were recombinantly expressed and their activity on xylan was confirmed (106 and 85 IU/mg, respectively), with differences in their activity pattern. Both endoxylanases released mainly xylobiose (X2) and xylotriose (X3) from xylan and pre-treated biomasses (wheat straw, barley straw, and sweet corn cob), although only rGH10XynA released xylose (X1). rGH10XynA presented optimal conditions at pH 6, with thermal stability at 45?50 °C, while rGH11XynB showed activity in a wider range of pH, from 5 to 9, and was thermostable only at 45 °C. Moreover, GH11XynB presented sigmoidal kinetics on xylan, indicating possible cooperative binding, which was further supported by the structural model. This study provides a detailed analysis of the complete set of carbohydrate-active enzymes encoded in Paenibacillus sp. A59 genome and those effectively implicated in hemicellulose hydrolysis, contributing to understanding the mechanisms necessary for the bioconversion of this polysaccharide. Moreover, the two main free secreted xylanases, rGH10XynA and rGH11XynB, were fully characterized, supporting their potential application in industrial bioprocesses on lignocellulosic biomass. Fil: Ghio, Silvina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Suelos; Argentina Fil: Ontañon, Ornella Mailén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Piccinni, Florencia Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Marrero Díaz de Villegas, Rubén. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Talia, Paola Monica. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Grasso, Daniel Hector. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Suelos; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina Fil: Campos, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina

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.

A β-xylosidase gene, xyl43C, from Clostridium clariflavum was heterogeneously expressed in Escherichia coli BL21. Xyl43C showed strong activity toward xylobiose, with specific activity of 76.6 U/mg and Km of 4.97 mM. The optimal pH and temperature of Xyl43C were pH 6.0 and 60 °C, respectively. Xyl43C retained 94.4% activity after incubation at 55 °C for 1 h, and 75.4% at 60 °C for 1 h. It also showed xylose tolerance with IC50 (half maximal inhibitory concentration) of approximately 100 mM. It nearly completely hydrolyzed 2 g/L of xylobiose at enzyme load of 2.51 mg/g xylobiose within 30 min and converted 40 g/L of corncob xylan into xylose at enzyme load of 1.48 mg/g xylan, with a yield of 60.9%. In conclusion, Xyl43C is an efficient xylose-tolerant β-xylosidase, with promising application potential in saccharification of xylan in biofuels industry.