Oat Spelt Xylan Research Articles

Synergistic xylan decomposition by a reducing-end xylose-releasing exo-oligoxylanase with other xylanolytic enzymes derived from Paenibacillus xylaniclasticus strain TW1.

Paenibacillus xylaniclasticus strain TW1 is a promising tool for decomposing xylan-containing lignocellulosic biomass, since this strain possesses various genes encoding cellulolytic/hemicellulolytic enzymes. In this study, PxRex8A from the TW1 strain was found to be a reducing-end xylose-releasing exo-oligoxylanase of glycoside hydrolase family 8, which cleaves xylose from xylooligosaccharides of corn core xylan. In a synergistic assay, the efficient decomposition of oat spelt xylan (OSX) and beech wood xylan was exemplified in the combination of endo-β-1,4-xylanase (PxXyn11A) and PxRex8A from the TW1 strain in a molar ratio of 4:1. Furthermore, it was found that the addition of β-d-xylosidase/α-l-arabinofuranosidase (PxXyl43A) from this strain with PxXyn11A and PxRex8A achieved twice the amount of reducing sugars (1.1mg/mL) against OSX after 24 h compared to PxXyn11A alone (0.5mg/mL). These results demonstrate that synergy effect of PxRex8A and PxXyl43A with PxXyn11A promotes xylan degradation into xylose.

The CBM91 module enhances the activity of β-xylosidase/α-L-arabinofuranosidase PphXyl43B from Paenibacillus physcomitrellae XB by adopting a unique loop conformation at the top of the active pocket

Carbohydrate-binding module (CBM) family 91 is a novel module primarily associated with glycoside hydrolase (GH) family 43 enzymes. However, our current understanding of its function remains limited. PphXyl43B is a β-xylosidase/α-L-arabinofuranosidase bifunctional enzyme from physcomitrellae patens XB belonging to the GH43_11 subfamily and containing CBM91 at its C terminus. To fully elucidate the contributions of the CBM91 module, the truncated proteins consisting only the GH43_11 catalytic module (rPphXyl43B-dCBM91) and only the CBM91 module (rCBM91) of PphXyl43B were constructed, respectively. The result showed that rPphXyl43B-dCBM91 completely lost hydrolysis activity against both p-nitrophenyl-β-D-xylopyranoside and p-nitrophenyl-α-L-arabinofuranoside; it also exhibited significantly reduced activity towards xylobiose, xylotriose, oat spelt xylan and corncob xylan compared to the control. Thus, the CBM91 module is crucial for the β-xylosidase/α-L-arabinofuranosidase activities in PphXyl43B. However, rCBM91 did not exhibit any binding capability towards corncob xylan. Structural analysis indicated that CBM91 of PphXyl43B might adopt a loop conformation (residues 496–511: ILSDDYVVQSYGGFFT) to actively contribute to the catalytic pocket formation rather than substrate binding capability. This study provides important insights into understanding the function of CBM91 and can be used as a reference for analyzing the action mechanism of GH43_11 enzymes and their application in biomass energy conversion.

Isolation and potency of Actinomycetes from rhizosphere of nutmeg (Myristica fragrans Houtt)

Nutmeg (Myristica fragrans Houtt) is commonly cultivated by people in the forests of Moluccas Islands. This plant grows well on relatively infertile soil types. This is presumably due to the presence of symbiotic microbes in the root of nutmeg. The research aimed to isolate, characterize and test the potential of Actinomycetes from rhizosphere of nutmeg. Soil sample were taken from the nutmeg forest in Ambon Island. The Actinomycetes isolation using humic acid vitamin, continued with yeast malt agar (YMA) media. The testing of antibacterial and antifungal activities using YMA media, while cellulolytic activity, phosphate solubilizing, and xylanolytic activity using carboxyl methyl cellulose, Picovskaya agar, and birchwood agar or oat spelt xylan agar. A total of 12 isolates of Actinomycetes were isolated and dominated by Streptomyces with various types of aerial mycelia. The substrate mycelium looks brown and cream, while the aerial mycelium looks white and gray. These isolates had the highest inhibitory power against Escherichia coli and Fusarium oxysporum with indexes of 16.5 mm and 16.0 mm, respectively. The other isolates have the ability of cellulolytic, phosphate solubilizing, and xylanolytic with indexes 3.26, 3.87, and 1.2, respectively. The Actinomycetes isolates that were found can be used as starter to improve the biofertilizer formula for nutmeg.

Discovery of a Novel β-xylosidase with Xylanase Activity and Its Application in the Production of Xylitol from Corncob Xylan

Although β-xylosidases with xylanase activity are preferential for the hydrolysis of xylan and production of xylitol, reports on their use are scarce. In this study, a multifunctional β-xylosidase (XYL4) was identified. In addition to β-xylosidase activity, XYL4 also exhibited xylanase and low α-arabinosidase activity. The enzyme was able to hydrolyze bagasse xylan, oat spelt xylan, birchwood xylan, beechwood xylan, and corncob xylan, and showed the highest hydrolysis activity for corncob xylan. Structural modeling analysis indicated that XYL4 had an additional PA14 domain, which may play a key role in binding xylan substrates. Moreover, XYL4 was used to hydrolyze corncob xylan to produce xylose. When enzymatic hydrolysis and whole-cell catalysis were used to hydrolyze 100 g/L of corncob xylan, the xylose yields were 60.26% and 35.85%, respectively. Then, the Candida tropicalis was inoculated with the above hydrolysates for fermentation to produce xylitol. Using enzymatic hydrolysis and whole-cell catalysis, xylitol yields of 77.56% and 73.67% were obtained by C. tropicalis after the optimization of fermentation, respectively.

Heterologous Expression, Purification and Characterization of an Alkalic Thermophilic β-Mannanase CcMan5C from Coprinopsis cinerea.

A endo-1,4-β-mannanase (CcMan5C) gene was cloned from Coprinopsis cinerea and heterologously expressed in Pichia pastoris, and the recombinant enzyme was purified by Ni-affinity chromatography and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). CcMan5C hydrolyzed only locust bean gum galactomannan (LBG) but not α-mannan from S. cerevisiae or Avicel cellulose, oat spelt xylan, or laminarin from Laminaria digitata. CcMan5C exhibited distinctive catalytic features that were different from previously reported β-mannanases. (1) CcMan5C is the first reported fungal β-mannase with an optimal alkalic pH of 8.0-9.0 for hydrolytic activity under assay conditions. (2) CcMan5C is the first reported alkalic fungal β-mannase with an optimal temperature of 70 °C for hydrolytic activity under assay conditions. (3) The organic solvents methanol, ethanol, isopropanol, and acetone at concentrations of 10% or 20% did not inhibit CcMan5C activity, while 10% or 20% isopropanol and acetone even enhanced CcMan5C activity by 9.20-34.98%. Furthermore, CcMan5C tolerated detergents such as Tween 20 and Triton X-100, and its activity was even enhanced to 26.2-45.6% by 1% or 10% Tween 20 and Triton X-100. (4) CcMan5C solution or lyophilized CcMan5C exhibited unchanged activity and even increasing activity after being stored at -20 °C or -80 °C for 12 months and retained above 50% activity after being stored at 4 °C for 12 months. These features make CcMan5C a suitable candidate for the detergent industry and paper and pulp industry.

Characterization of the recombinant GH10 xylanase from Trichoderma orientalis EU7-22 and its synergistic hydrolysis of bamboo hemicellulose with α-glucuronidase and α-L-arabinofuranosidase

Here the recombinant GH10 xylanase (HoXyn10) was investigated for biochemical characteristics and synergistic effect with α-glucuronidase (AnGus67) and α-L-arabinofuranosidase (AnAxh62A) on extracted Maso bamboo hemicellulose (BCH) to produce xylooligosaccharides (XOS). The GH10 endo-β-1, 4-xylanase from Trichoderma orientalis EU7–22 is heterologously expressed in Pichia pastoris GS115. The crude HoXyn10 showed three bands on the SDS-PAGE profile and one of the proteins was N-glycosylated. The optimum pH and temperature of HoXyn10 were pH= 6.0 and 65 °C, respectively. It showed a wide range of pH stability and remained more than 80% of optimal activity at pH4.5–8.0 after holding at 40 °C for 60 min. HoXyn10 was highly stable at 50 °C without substrate and the residue xylanase activities were retained 97% and 83% after incubating for 2 h and 18 h, respectively. HoXyn10 displayed high activities and almost the same catalytic efficiency to oat-spelt xylan (OSX), Beechwood xylan (BWX) and wheat arabinoxylan (WAX). Notably, the HoXyn10 exhibited apparent activity on Avicel, but showed no activity on CMC-Na. The hydrolytic products from oligosaccharides (X3-X6) by HoXyn10 were all xylose (X1) and xylobiose (X2). The results from FT-IR and NMR showed the backbone of extracted BCH was (1→4)-linked β-D-xylopyranose and the main side chains were α-L-arabinofuranose residues and 4-O-methyl-D-glucuronic acid residues. Based on the characterized structure, when BCH was synergistically hydrolyzed with HoXyn10, AnGus67 and AnAxh62A using appropriate strategy, the yield of xylooligosaccharide (XOS) and xylose were 50.3% and 14.7%. The results indicated HoXyn10 might be a promising tool for various hemicellulose hydrolysis and Maso bamboo hemicellulose is a promising source for XOS production.

Biochemical and Regulatory Analyses of Xylanolytic Regulons in Caldicellulosiruptor bescii Reveal Genus-Wide Features of Hemicellulose Utilization.

Caldicellulosiruptor species scavenge carbohydrates from runoff containing plant biomass that enters hot springs and from grasses that grow in more moderate parts of thermal features. While only a few Caldicellulosiruptor species can degrade cellulose, all known species are hemicellulolytic. The most well-characterized species, Caldicellulosiruptor bescii, decentralizes its hemicellulase inventory across five different genomic loci and two isolated genes. Transcriptomic analyses, comparative genomics, and enzymatic characterization were utilized to assign functional roles and determine the relative importance of its six putative endoxylanases (five glycoside hydrolase family 10 [GH10] enzymes and one GH11 enzyme) and two putative exoxylanases (one GH39 and one GH3) in C. bescii. Two genus-wide conserved xylanases, C. bescii XynA (GH10) and C. bescii Xyl3A (GH3), had the highest levels of sugar release on oat spelt xylan, were in the top 10% of all genes transcribed by C. bescii, and were highly induced on xylan compared to cellulose. This indicates that a minimal set of enzymes are used to drive xylan degradation in the genus Caldicellulosiruptor, complemented by hemicellulolytic inventories that are tuned to specific forms of hemicellulose in available plant biomasses. To this point, synergism studies revealed that the pairing of specific GH family proteins (GH3, -11, and -39) with C. bescii GH10 proteins released more sugar in vitro than mixtures containing five different GH10 proteins. Overall, this work demonstrates the essential requirements for Caldicellulosiruptor to degrade various forms of xylan and the differences in species genomic inventories that are tuned for survival in unique biotopes with variable lignocellulosic substrates. IMPORTANCE Microbial deconstruction of lignocellulose for the production of biofuels and chemicals requires the hydrolysis of heterogeneous hemicelluloses to access the microcrystalline cellulose portion. This work extends previous in vivo and in vitro efforts to characterize hemicellulose utilization by integrating genomic reconstruction, transcriptomic data, operon structures, and biochemical characteristics of key enzymes to understand the deployment and functionality of hemicellulases by the extreme thermophile Caldicellulosiruptor bescii. Furthermore, comparative genomics of the genus revealed both conserved and divergent mechanisms for hemicellulose utilization across the 15 sequenced species, thereby paving the way to connecting functional enzyme characterization with metabolic engineering efforts to enhance lignocellulose conversion.

Characterization of a GH Family 43 β-Xylosidase Having a Novel Carbohydrate-binding Module from Paenibacillus xylaniclasticus Strain TW1.

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

A novel reducing-end xylose-releasing exo-oligoxylanase (PphRex8A) from Paenibacillus physcomitrellae XB

In order to better understand the function of a putative GH8 xylanase gene in the xylan-degrading bacterium Paenibacillus physcomitrellae XB, a novel reducing-end xylose-releasing exo-oligoxylanase (Rex) PphRex8A of GH8 family was characterized. Phylogenetic analysis showed that it was clustered tightly with other published GH8 Rexs and exhibited the highest amino acid sequence identity (77.4 %) with the Rex of PbRex8 from P. barengoltzii G22. The three-dimensional (3D) structure of PphRex8A was also built based on the template of PbRex8 (5YXT) and three conserved catalytic active sites (Glu71, Asp263, and Asp129) were predicted and further confirmed by the enzymatic inactivity of their mutants (E71A, D129A, and D263A). The hydrolysis assay of PphRex8A showed that it could hydrolyze xylo-oligosaccharides (XOSs) with a degree of polymerization (DP) ≥ 3, such as xylotriose (X3) through xylohexaose (X6), and some natural XOSs, such as corncob xylan (CCX) and oat spelt xylan (OSX) to release xylose. However, it could not hydrolyze p-nitrophenyl-β-D-xylopyranoside (pNPX), which suggested that it mainly released xylose from the reducing-end of XOSs and belonged to a Rex. In addition, PphRex8A also could deconstruct xylans with high DP, such as wheat arabinoxylan (AX) and beech wood xylan (BWX) to produce XOSs with DP3–6. Moreover, PphRex8A had synergistic effects with other xylanolytic enzymes of P. physcomitrellae XB, such as with PphXyn10 or PphXyn11 at a ratio of 1:3, or with PphXyl43B as a ratio of 3:1, significantly increasing the amounts of reducing sugars toward different xylan substrates. Thus, PphRex8A could be an exo-xylanase toward XOSs and could improve the deconstruction capability of high DP xylans, thereby complementing other xylanolytic enzymes to contribute to xylan degradation and improve the efficiency of converting hemicellulose biomass into energy by P. physcomitrellae XB.

Determination Kinetic Parameters of Endo-β-1,4-D-Xylanase from Abdomenal Termites with Xylan Oat and Birchwood

Parameters kinetic (KM, VMax, and kCat) endo-β-1,4-D-xylanase under optimum conditions with oat spelt xylan and birchwood substrate have been investigated in this study. Hydrolysis of endo-β-1,4-D-xylanase using variation of substrate concentration (b/v) ranging from 0.2 to 1.2%. Variation of incubation time is up to 20 hours with 4 hours interval at the optimum temperature of the enzyme, 40°C. The results obtained from this study were the KM of endo-β-1,4-D-xylanase for oat spelt xylan and birchwood were 4.10 mg/ml and 0.681 mg/ml, respectively. VMax values, and kCat for oat spelt xylan substrate of 0.28 mg/ml.jam and 1.7 x 10-3 s-1. While VMax, and kCat for birchwood substrate that is 0.117 mg/U/jam and 7 x 10-4 s-1. From the results of this study we found that endo-β-1,4-D-xylanase can hydrolaze substrates which have differences solubility.

Biochemical and Thermodynamic Studies on a Novel Thermotolerant GH10 Xylanase from Bacillus safensis.

Xylanases have a broad range of applications in agro-industrial processes. In this study, we report on the discovery and characterization of a new thermotolerant GH10 xylanase from Bacillus safensis, designated as BsXyn10. The xylanase gene (bsxyn10) was cloned from Bacillus safensis and expressed in Escherichia coli. The reduced molecular mass of BsXyn10 was 48 kDa upon SDS-PAGE. Bsxyn10 was optimally active at pH 7.0 and 60 °C, stable over a broad range of pH (5.0–8.0), and also revealed tolerance toward different modulators (metal cations, EDTA). The enzyme was active toward various xylans with no activity on the glucose-based polysaccharides. KM, vmax, and kcat for oat spelt xylan hydrolysis were found to be 1.96 g·L−1, 58.6 μmole·min−1·(mg protein)−1, and 49 s−1, respectively. Thermodynamic parameters for oat spelt xylan hydrolysis at 60 °C were ΔS* = −61.9 J·mol−1·K−1, ΔH* = 37.0 kJ·mol−1 and ΔG* = 57.6 kJ·mol−1. BsXyn10 retained high levels of activity at temperatures up to 60 °C. The thermodynamic parameters (ΔH*D, ΔG*D, ΔS*D) for the thermal deactivation of BsXyn10 at a temperature range of 40–80 °C were: 192.5 ≤ ΔH*D ≤ 192.8 kJ·mol−1, 262.1 ≤ ΔS*D ≤ 265.8 J·mol−1·K−1, and 99.9 ≤ ΔG*D ≤ 109.6 kJ·mol−1. The BsXyn10-treated oat spelt xylan manifested the catalytic release of xylooligosaccharides of 2–6 DP, suggesting that BsXyn10 represents a promising candidate biocatalyst appropriate for several biotechnological applications.

Xylooligosaccharides production using multi-substrate specific xylanases secreted by a psychrotolerant Paenibacillus sp. PCH8

Xylanases are industrial enzymes with multiple applications in the food, pharmaceuticals, bio-bleaching, and textiles industries. The present study explores a putative novel bacterium Paenibacillus sp. PCH8 shows xylanolytic activity from Himalayan glacial soil. Genome sequencing and analysis revealed multiple genes encoding xylanases, cellulases, and other lignocellulolytic enzymes. The bacterium utilized oat spelt xylan substrate and showed xylanolytic activity in wide pH (4.0 – 12.0) and temperature (4 to 90 ºC). Proteomic analysis revealed 1,4-β-xylanase, arabinan endo-1,5-α-l-arabinosidase, and 11 hypothetical proteins in partially purified protein fraction. Multi-substrate enzymatic activity (IU/mg) was observed for beechwood (21.42), oat spelt xylan (19.8), CMC (5.17), avicel (7.7), and starch (1.62) in protein fraction. The hydrolysis of xylan led to the formation of xylose, xylobiose, xylotriose, and xylotetraose upon analysis by LC-MS. The xylooligosaccharides (XOS) containing hydrolysate enhanced the growth of probiotic microbes, suggesting prebiotic potential. Thus, the study provides a new source of xylanases from Paenibacillus sp. PCH8, with potential applications in lignocellulosic biomass hydrolysis and XOS production.

Knockdown of has_circ_0010452 Promotes Proliferation and Osteogenic Differentiation via Up-Regulating miR-543 in Human Bone Marrow Mesenchymal Stem Cells

Objective: The aim of this study was to explore the role of has_circ_0010452 in the progression of osteoporosis (OP) targeting miR-543, as well as their functions in regulating proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Methods: The expression levels of circ_0010452 and miR-543 in hBMSCs at different time points of osteogenic differentiation were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). After transfection of circ_0010452 siRNA or miR-543 inhibitor in hBMSCs, the relative expression levels of osteogenic marker proteins, including oat spelt xylan (OSX), osteocalcin (OCN) and collagen I (Col-1), were determined by western blot. Cell proliferation of hBMSCs was valued by Cell Counting Kit 8 (CCK-8) assay. Dual-Luciferase reporter gene assay was performed to verify the relationship between circ_0010452 and miR-543. Subsequently, the regulatory effects of circ_0010452 and miR-543 on osteogenic differentiation and the capability of mineralization were evaluated by alkaline phosphatase (ALP) determination and alizarin red staining, respectively. Results: The expression of circ_0010452 decreased gradually and miR-543 increased in hBMSCs with the prolongation of osteogenic differentiation. circ_0010452 could bind to miR-543, which was negatively regulated by miR-543 in hBMSCs. Moreover, knockdown of circ_0010452 inhibited proliferation and osteogenic differentiation by upregulating miR-543, as well as upregulating expressions of OSX, OCN and Col-1. Furthermore, knockdown of circ_0010452 markedly promoted the capability of mineralization of hBMSCs, which was further reversed by transfection of miR-543 inhibitor. The knockdown of miR-543 partially reversed the inhibitory effect of circ_0010452 on the osteogenesis of hBMSCs. Conclusions: Silence of circ_0010452 promotes the development of OP via binding to miR-543 regulating proliferation and osteogenic differentiation of hBMSCs, thus promoting the progression of osteoporosis.

Production of cell wall-hydrolyzing enzymes and GC-MS extract analysis of Byssochlamys lagunculariae, a new record isolated from Egypt

In the present work, six strains of Byssochlamys lagunculariae were isolated from alkaline wastewater of textile factory polluted with azo dyes in Alexandria, Egypt. Out of the six strains, only one was identified using the sequencing of the internal transcribed spacer gene (ITS). ITS sequence was uploaded to GenBank (MT939911) and a pure culture was maintained in the culture collection of Assiut University Mycological Centre (AUMC) as B. lagunculariae AUMC 14498. The strain shared some morphological characteristics indicating species such as the existence of smooth chlamydospores, ascospore size, and good growth on Czapek’s yeast autolysate agar (CYA) at 30 °C. However, it was different from the type species by providing larger conidia and positive acid production on creatine sucrose agar (CREA) at 30 °C. Extrolite analysis of this strain was performed using gas chromatography mass spectrometry (GC-MS). The extrolite analysis extract revealed that more than fifty chemical compounds were detected, which have an important medical application. All B. lagunculariae strains tested, when grown on oat spelt xylan could utilize oat spelt xylan to produce endoglucanase, exoglucanase and xylanase as well as maize starch and pectin, respectively for amylase and pectinase production. Three strains of B. lagunculariae achieved maximum specific activity for amylase ranged from 75.65 to 114.67 IU/mg, while one of these three was superior for endoglucanase (59.1 IU/mg), exoglucanase (102.84 IU/mg), pectinase (34.33 IU/mg), and xylanase (90.21 IU/mg) production.

Characterisation of biomass degrading xylanolytic enzymes of Penicillium chrysogenum produced using sugarcane bagasse

•Penicillium chrysogenum CCDCA10746 secretome (SPc) produced using raw sugarcane bagasse was used to hydrolyse the hydrothermally pretreated sugarcane bagasse, in order to solubilise xylan to produce xylose with the conversion rate of 67%. The secretome was active in a wide pH range and exhibits increased thermostability (t1/2 8 h) than purified PcX2, a 23 kDa endo-β-1,4-xylanase of GH11 family, identified by mass spectrometry and zymogram. Xylanolytic activity was sensitive to Cu+2 and Zn+2 ions. In trials where enzymes were exposed to lignin-derived phenolic compounds: vanillin, cinnamic acid, and 4-hydroxy-benzoic acid inhibited the 70% activity of xylanase. On contrary, both enzymes showed resistance for tannic acid, additionally PcX2 was resistant to p-coumaric and SPc to gallic acid. PcX2 showed a binding affinity of KM 0.53 mg/mL and Vmax 0.21 U/mg for oat spelt xylan. Ionization of histidine appeared to be a crucial event in substrate binding and catalysis of PcX2 since decrease in fluorescence intensity and optimum activity was observed at pH 6.0.

Contributions and characteristics of two bifunctional GH43 β-xylosidase /α-L-arabinofuranosidases with different structures on the xylan degradation of Paenibacillus physcomitrellae strain XB

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.

Identification, heterologous expression and biochemical characterization of a novel cellulase-free xylanase B from the thermophilic bacterium Cohnella sp.A01

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.

Identification and Characterization of a Novel, Cold-Adapted d-Xylobiose- and d-Xylose-Releasing Endo-β-1,4-xylanase from an Antarctic Soil Bacterium, Duganella sp. PAMC 27433.

Endo-β-1,4-xylanase is a key enzyme in the degradation of β-1,4-d-xylan polysaccharides through hydrolysis. A glycoside hydrolase family 10 (GH10) endo-β-1,4-xylanase (XylR) from Duganella sp. PAMC 27433, an Antarctic soil bacterium, was identified and functionally characterized. The XylR gene (1122-bp) encoded an acidic protein containing a single catalytic GH10 domain that was 86% identical to that of an uncultured bacterium BLR13 endo-β-1,4-xylanase (ACN58881). The recombinant enzyme (rXylR: 42.0 kDa) showed the highest beechwood xylan-degrading activity at pH 5.5 and 40 °C, and displayed 12% of its maximum activity even at 4 °C. rXylR was not only almost completely inhibited by 5 mM N-bromosuccinimide or metal ions (each 1 mM) including Hg2+, Ca2+, or Cu2+ but also significantly suppressed by 1 mM Ni2+, Zn2+, or Fe2+. However, its enzyme activity was upregulated (>1.4-fold) in the presence of 0.5% Triton X-100 or Tween 80. The specific activities of rXylR toward beechwood xylan, birchwood xylan, oat spelts xylan, and p-nitrophenyl-β-d-cellobioside were 274.7, 103.2, 35.6, and 365.1 U/mg, respectively. Enzymatic hydrolysis of birchwood xylan and d-xylooligosaccharides yielded d-xylose and d-xylobiose as the end products. The results of the present study suggest that rXylR is a novel cold-adapted d-xylobiose- and d-xylose-releasing endo-β-1,4-xylanase.

Feruloyl esterase (FAE-1) sourced from a termite hindgut and GH10 xylanases synergy improves degradation of arabinoxylan

Cereal feedstocks have high arabinoxylan content as their main hemicellulose, which is linked to lignin by hydroxycinnamic acids such as ferulic acid. The ferulic acid is linked to arabinoxylan by ester bonds, and generally, the high substitution of ferulic acid leads to a loss of activity of xylanases targeting the arabinoxylan. In the current study, a feruloyl esterase (FAE-1) from a termite hindgut bacteria was functionally characterised and used in synergy with xylanases during xylan hydrolysis. The FAE-1 displayed temperature and pH optima of 60 ℃ and 7.0, respectively. FAE-1 did not release reducing sugars from beechwood xylan (BWX), wheat arabinoxylan (WAX) and oat spelt xylan (OX), however, displayed high activity of 164.74 U/mg protein on p-nitrophenyl-acetate (pNPA). In contrast, the GH10 xylanases; Xyn10 and XT6, and a GH11 xylanase, Xyn2A, showed more than two-fold increased activity on xylan substrates with low sidechain substitutions; BWX and OX, compared to the highly branched substrate, WAX. Interestingly, the FAE-1 and GH10 xylanases (Xyn10D and XT6) displayed a degree of synergy (DS) that was higher than 1 in all enzyme loading combinations during WAX hydrolysis. The 75%XT6:25%FAE-1 synergistic enzyme combination increased the release of reducing sugars by 1.34-fold from WAX compared to the control, while 25%Xyn10D:75%FAE-1 synergistic combination released about 2.1-fold of reducing sugars from WAX compared to controls. These findings suggest that FAE-1 can be used in concert with xylanases, particularly those from GH10, to efficiently degrade arabinoxylans contained in cereal feedstocks for various industrial settings such as in animal feeds and baking.

High–level expression and enzymatic properties of a novel thermostable xylanase with high arabinoxylan degradation ability from Chaetomium sp. suitable for beer mashing

A novel thermostable xylanase gene from Chaetomium sp. CQ31 was cloned and codon–optimized (CsXynBop). The deduced protein sequence of the gene shared the highest similarity of 75% with the glycoside hydrolase (GH) family 10 xylanase from Achaetomium sp. Xz–8. CsXynBop was over–expressed in Pichia pastoris GS115 by high–cell density fermentation, with the highest xylanase yield of 10,017 U/mL. The recombinant xylanase (CsXynBop) was purified to homogeneity and biochemically characterized. CsXynBop was optimally active at pH 6.5 and 85 °C, respectively, and stable over a broad pH range of 5.0–9.5 and up to 60 °C. The enzyme exhibited strict substrate specificity towards oat–spelt xylan (2, 489 U/mg), beechwood xylan (1522 U/mg), birchwood xylan (1067 U/mg), and showed relatively high activity towards arabinoxylan (1208 U/mg), but exhibited no activity on other tested polysaccharides. CsXynBop hydrolyzed different xylans to yield mainly xylooligosaccharides (XOSs) with degree of polymerization (DP) 2–5. The application of CsXynBop (200 U/g malt) in malt mashing substantially decreased the filtration time and viscosity of malt by 42.3% and 8.6%, respectively. These excellent characteristics of CsXynBop may make it a good candidate in beer industry.