Beechwood Xylan Research Articles

Properties of Water‐Soluble Xylan from Different Agricultural By‐Products and their Production of Xylooligosaccharides

AbstractXylooligosaccharides (XOS) are non‐digestible oligosaccharides with a significant role as functional food ingredients in the food industry. The present research is undertaken to evaluate water soluble xylan (WSX) extraction from eight different sources using water at ambient temperature (25 ± 2 °C) and structural characterization (FTIR, NMR, XRD, and TGA). Enzymatic production of XOS from extracted substrates and its characterization using HPLC‐RID and FTIR is determined. WSX from orange fleshed sweet potato peel shows highest yield (9%), while the least (2.48%) is from Teff seed coat. FTIR structural identification of xylan confirms the presence of β‐glycosidic linkages and acetyl groups; whereas 1H NMR validates the occurrence of β‐D‐1,4‐xylopyranose backbone with α‐L‐arabinofuranose and 4‐O‐methylglucuronic acid substituents. XRD and TGA characteristics of the extracts indicate similarities in the eight sources in comparison with standard beech wood xylan. WSX is hydrolyzed with endoxylanase to obtain XOS (highest yield of 57.16 ± 1.01% in Cassava cake; lowest yield of 21.72 ± 0.72% in Bambara groundnut). HPLC‐RID reveals xylobiose as the main monomeric sugar component (DP 2) and β‐linkages are identified by FTIR in all the sources. The study suggests the potential commercial production of XOS from the analyzed agricultural by‐products.

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.

TtCel7A: A Native Thermophilic Bifunctional Cellulose/Xylanase Exogluclanase from the Thermophilic Biomass-Degrading Fungus Thielavia terrestris Co3Bag1, and Its Application in Enzymatic Hydrolysis of Agroindustrial Derivatives.

The biomass-degrading thermophilic ascomycete fungus Thielavia terrestris Co3Bag1 produces TtCel7A, a native bifunctional cellulase/xylanase GH7 family. The purified TtCel7A, with an estimated molecular weight of 71 kDa, was biochemically characterized. TtCel7A displayed an optimal pH of 5.5 for both activities and an optimal temperature of 60 and 50 °C for cellulolytic and xylanolytic activities, respectively. The half-lives determined for cellulase activity were 140, 106, and 41 min at 50, 60, and 70 °C, respectively, whereas the half-lives observed for xylanase activity were 24, 10, and 1.4 h at 50, 60, and 70 °C, respectively. The KM and Vmax values were 3.12 mg/mL and 50 U/mg for cellulase activity and 0.17 mg/mL and 42.75 U/mg for xylanase activity. Circular dichroism analysis suggests changes in the secondary structure of TtCel7A in the presence of CMC as the substrate, whereas no modifications were observed with beechwood xylan. TtCel7A displayed the excellent capability to hydrolyze CMC, beechwood xylan, and complex substrates such as oat bran, wheat bran, and sugarcane bagasse, with glucose and cellobiose being the main products released; also, slightly less endo cellulase and xylanase activities were observed. Thus, suggesting TtCel7A has an exo- and endomode of action. Based on the characteristics of the enzyme, it might be considered a good candidate for industrial applications.

BsLPMO10A from Bacillus subtilis boosts the depolymerization of diverse polysaccharides linked via β-1,4-glycosidic bonds

Lytic polysaccharide monooxygenase (LPMO) is known as an oxidatively cleaving enzyme in recalcitrant polysaccharide deconstruction. Herein, we report a novel AA10 LPMO derived from Bacillus subtilis (BsLPMO10A). A substrate specificity study revealed that the enzyme exhibited an extensive active-substrate spectrum, particularly for polysaccharides linked via β-1,4 glycosidic bonds, such as β-(Man1 → 4Man), β-(Glc1 → 4Glc) and β-(Xyl1 → 4Xyl). HPAEC-PAD and MALDI-TOF-MS analyses indicated that BsLPMO10A dominantly liberated native oligosaccharides with a degree of polymerization (DP) of 3–6 and C1-oxidized oligosaccharides ranging from DP3ox to DP6ox from mixed linkage glucans and beechwood xylan. Due to its synergistic action with a variety of glycoside hydrolases, including glucanase IDSGLUC5-38, xylanase TfXYN11-1, cellulase IDSGLUC5-11 and chitinase BtCHI18-1, BsLPMO10A dramatically accelerated glucan, xylan, cellulose and chitin saccharification. After co-reaction for 72 h, the reducing sugars in Icelandic moss lichenan, beechwood xylan, phosphoric acid swollen cellulose and chitin yielded 3176 ± 97, 7436 ± 165, 649 ± 44, and 2604 ± 130 μmol/L, which were 1.47-, 1.56-, 1.44- and 1.25-fold higher than those in the GHs alone groups, respectively (P < 0.001). In addition, the synergy of BsLPMO10A and GHs was further validated by the degradation of natural feedstuffs, the co-operation of BsLPMO10A and GHs released 3266 ± 182 and 1725 ± 107 μmol/L of reducing sugars from Oryza sativa L. and Arachis hypogaea L. straws, respectively, which were significantly higher than those produced by GHs alone (P < 0.001). Furthermore, BsLPMO10A also accelerated the liberation of reducing sugars from Celluclast® 1.5 L, a commercial cellulase cocktail, on filter paper, A. hypogaea L. and O. sativa L. straws by 49.58 % (P < 0.05), 72.19 % (P < 0.001) and 54.36 % (P < 0.05), respectively. This work has characterized BsLPMO10A with a broad active-substrate scope, providing a promising candidate for lignocellulosic biomass biorefinery.

Expression and Characterization of a Small, Xylan/Cellulose-degrading GH43 Protein Derived from Biofertilizer Metagenome

Background: A putative glycosyl hydrolase gene biof1_09 was identified from a metagenomic fosmid library of local biofertilizers in previous report [1]. The gene is renamed as gh43kk in this study. Methods: The gene gh43kk, encoding a putative β-D-xylosidase was amplified by polymerase chain reaction (PCR) and successfully cloned and expressed in Escherichia coli. The expressed recombinant protein was purified by metal affinity chromatography. Its properties were initially verified by enzyme assay and thin layer chromatography (TLC). Results: The purified recombinant protein showed the highest catalytic activities at acidic pH 4 and 50°C toward beechwood xylan, followed by carboxymethylcellulose (CMC). TLC analysis indicated a release of xylose and glucose when xylan and CMC were treated with Gh43kk protein, respectively, whereas glucose and cellobiose were detected when avicel, cellulose and filter paper were used as substrates, suggesting its dual function as xylanase with cellulase activity. The enzyme indicated great stability in a temperature between 10 to 50 °C and a wide range of pH from 4 to 8. Enzyme activity of Gh43kk was enhanced in the presence of magnesium and manganese ions, while calcium ions, Ethylenediaminetetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS) inhibited the enzyme activity. Conclusion: These results suggest that Gh43kk could be a potential candidate for application in various bioconversion processes.

Improving Barrier Properties of Xylan-Coated Food Packaging Papers with Alkyl Ketene Dimer

In order to improve the hydrophobicity of xylan hemicellulose, a simple procedure of its chemical modification with alkyl ketene dimer (AKD), a non-toxic, cost-effective, and eco-friendly chemical, was performed. For this purpose, the reaction products of beech wood xylan and different amounts of hydrophobic AKD were used for paper surface treatment. Thus, the coatings of about 4.5 g/m2 were applied on both sides of base paper in single and three successive layers. To obtain a complete reaction between AKD and xylan hemicellulose, the coated papers were thermal cured (about 110 °C) and the effects of AKD content on the barrier (water, oil, and water vapours) and mechanical properties were analysed. The structural analyses by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FT-IR) of coated samples emphasized the presence of β-keto-ester compounds as a result of the reaction between xylan hemicelluloses and AKD. This is confirmed by the improving of barrier properties as the AKD content in coating dispersion is higher. The good barrier performance and improved strength properties were obtained for the coated papers with xylan hemicellulose and 1% AKD applied on paper surface in three successive layers (about 4.5 g/m2). In this case, the water vapours transmission rate (WVTR) was 35% lower than those untreated and the resistance to air passing through coated papers was over 3 times higher compared with base paper. There are no results reported on the chemical reaction of xylan hemicelluloses with AKD as well as its application in coatings for paper packaging. In this context, the obtained results in this study can contribute to expand the applications area of hemicelluloses offering a sustainable strategy for the developing of food packaging papers with appropriate barrier properties using biopolymer coating materials.

Engineering mesophilic GH11 xylanase from Cellulomonas flavigena by rational design of N-terminus substitution.

Xylanase, a glycoside hydrolase, is widely used in the food, papermaking, and textile industries; however, most xylanases are inactive at high temperatures. In this study, a xylanase gene, CFXyl3, was cloned from Cellulomonas flavigena and expressed in Escherichia coli BL21 (DE3). To improve the thermostability of xylanase, four hybrid xylanases with enhanced thermostability (designated EcsXyl1-4) were engineered from CFXyl3, guided by primary and 3D structure analyses. The optimal temperature of CFXyl3 was improved by replacing its N-terminus with the corresponding area of SyXyn11P, a xylanase that belongs to the hyperthermostable GH11 family. The optimal temperatures of the hybrid xylanases EcsXyl1-4 were 60, 60, 65, and 85°C, respectively. The optimal temperature of EcsXyl4 was 30C higher than that of CFXyl3 (55°C) and its melting temperature was 34.5°C higher than that of CFXyl3. After the hydrolysis of beechwood xylan, the main hydrolysates were xylotetraose, xylotriose, and xylobiose; thus, these hybrid xylanases could be applied to prebiotic xylooligosaccharide manufacturing.

Recombinant acetylxylan esterase of Halalkalibacterium halodurans NAH-Egypt: molecular and biochemical study

Acetylxylan esterase plays a crucial role in xylan hydrolysis as the acetyl side-groups restrict endoxylanase action by stearic hindrance. In this study, an acetylxylan esterase (AXE-HAS10: 960 bp & 319 a.a) putative ORF from Halalkalibacterium halodurans NAH-Egypt was extensively studied through heterologous overexpression in Escherichia coli, biochemical characterization, and structural modeling. The AXE-HAS10 tertiary structure was predicted by the Local Meta Threading Server. AXE-HAS10 belongs to the carbohydrate esterase Family 7. Purified to homogeneity AXE-HAS10 showed specific activity (36.99 U/mg), fold purification (11.42), and molecular mass (41.39 kDa). AXE-HAS10 showed optimal pH (8.5) and temperature (40 oC). After 15 h of incubation at pH 7.0–9.0, AXE-HAS10 maintained 100% activity. After 120 min at 35 and 40 oC, the retained activity was 80 and 50%, respectively. At 10 mM Mn2+, Fe3+, K+, and Ca2+ after 30 min, retained activity was 329 ± 15, 212 ± 5.2, 123 ± 1.4, and 120 ± 3.0%, respectively. After 30 min of preincubation with triton x-100, SDS, and CTAB at 0.1% (v/v), the retained activity was 150 ± 19, 88 ± 4, and 82 ± 7%, respectively. At 6.0 M NaCl after 30 min, retained activity was 58%. A 1.44-fold enhancement of beechwood xylan hydrolysis was achieved by AXE-HAS10 and Penicillium chrysogenum DSM105774 β-xylanase concurrently. Present data underpins AXE-HAS10 as a promising AXE for industrial exploitation.

Optimization, purification, and characterization of xylanase production by a newly isolated Trichoderma harzianum strain by a two-step statistical experimental design strategy

Xylanases are hydrolytic enzymes with a wide range of applications in several industries such as biofuels, paper and pulp, food, and feed. The objective of this study was to optimize the culture conditions and medium components for maximal xylanase production from a newly isolated Trichoderma harzianum strain using the Plackett–Burman Design (PBD) and Box Behnken Design (BBD) experimental strategies. Xylanase production was enhanced 4.16-fold to 153.80 U/ml by BBD compared to a preliminary one-factor-at-a-time (OFAT) activity of 37.01 U/ml and 2.24-fold compared to the PBD (68.70 U/ml). The optimal conditions for xylanase production were: 6 days of fermentation, incubation temperature of 70 °C, pH 5.0, agitation of 160 rpm, and 1.2% wheat bran and ammonium sulphate. The experimental design effectively provided conditions for the production of an acidic-thermostable enzyme with exciting potential for application in animal feed improvement. The acidic-thermostable xylanase was purified from the submerged culture and SDS-PAGE analysis revealed a molecular weight of 72 kDa. This protein had maximum xylanolytic activity at pH 6.0 and 65 °C and was stable for 4 h retaining > 70% activity and exhibited substrate specificity for beechwood xylan with a Km of 5.56 mg/ml and Vmax of 1052.63 µmol/min/mg. Enzyme activity was enhanced by Fe2+, Mg2+, and Zn2+. There was an absence of strong inhibitors of xylanase activity. Overall, these characteristics indicate the potential for at least two industrial applications.

Heterologous expression of family GH11 Aspergillus niger xylanase B (AnXylB11) in Pichia pastoris and competitive inhibition by riceXIP: An experimental and simulation study

The family GH11 Aspergillus niger JL15 xylanase B (AnXylB11) was heterologously expressed in Pichia pastoris X33. The recombinant AnXylB11 (reAnXylB11) was secreted into the culture medium with a molecular weight of approximately 33.0 kDa. The optimal temperature and pH of reAnXylB11 were 40 ℃ and 5.0, respectively. reAnXylB11 released xylobiose (X2)-xylohexaose (X6) from beechwood xylan, with xylotriose (X3) as the primary product. The hydrolysates showed significant antioxidant activity. reAnXylB11 was also competitively inhibited by recombinant rice xylanase inhibitory protein (rePriceXIP), with an inhibition constant (Ki) of 106.9 nM. Molecular dynamics (MD) simulations, non-covalent interactions (NCI), and binding free energy calculation and decomposition were conducted to decipher the interactional features between riceXIP and AnXyB11. Representative conformation of riceXIP-AnXylB11 complex showed that a U-shaped long loop between α4 and β5 (K143-L152) of riceXIP was protruded into the catalytic groove and formed tight interaction with many key residues of AnXylB11. The binding free energy of riceXIP-AnXylB11 was calculated to be − 46.1 ± 10.5 kcal/mol, with Coulomb and van der Waals forces contributing the most. NCI analysis showed that the hydrogen bonding networks such as R148riceXIP-E98AnXyl11B, K143riceXIP-D138AnXyl11B and R148riceXIP-E189AnXyl11B provided terrific contributions to the interface interaction. The Laplacian of electron density values of atom pairs R148riceXIP@ 2HH1-E89AnXylB11@OE2 and N142riceXIP@ 1HD2-D138AnXylB11@OD1 were 0.12190 and 0.16009 a.u., respectively. Exploring the interactional features between xylanase and inhibitor protein may aid in constructing mutant xylanase that is insensitive to xylanase inhibitory proteins (XIs).

Biochemical characterization of a novel acidophilic β-xylanase from Trichoderma asperellum ND-1 and its synergistic hydrolysis of beechwood xylan.

Acidophilic β-xylanases have attracted considerable attention due to their excellent activity under extreme acidic environments and potential industrial utilizations. In this study, a novel β-xylanase gene (Xyl11) of glycoside hydrolase family 11, was cloned from Trichoderma asperellum ND-1 and efficiently expressed in Pichia pastoris (a 2.0-fold increase). Xyl11 displayed a maximum activity of 121.99 U/ml at pH 3.0 and 50°C, and exhibited strict substrate specificity toward beechwood xylan (Km = 9.06 mg/ml, Vmax = 608.65 μmol/min/mg). The Xyl11 retained over 80% activity at pH 2.0–5.0 after pretreatment at 4°C for 1 h. Analysis of the hydrolytic pattern revealed that Xyl11 could rapidly convert xylan to xylobiose via hydrolysis activity as well as transglycosylation. Moreover, the results of site-directed mutagenesis suggested that the Xyl11 residues, Glu127, Glu164, and Glu216, are essential catalytic sites, with Asp138 having an auxiliary function. Additionally, a high degree of synergy (15.02) was observed when Xyl11 was used in association with commercial β-xylosidase. This study provided a novel acidophilic β-xylanase that exhibits excellent characteristics and can, therefore, be considered a suitable candidate for extensive applications, especially in food and animal feed industries.

Enzymatic bioconversion of beechwood xylan into the antioxidant 2′-O-α-(4-O-methyl-D-glucuronosyl)-xylobiose

Acidic xylooligosaccharides (XOS), also called aldouronics, are hetero-oligomers of xylose randomly branched with 4-O-methyl-D-glucuronic acid residues linked by α(1 → 2) bonds, which display bioactive properties. We have developed a simple and integrated method for the production of acidic XOS by enzymatic hydrolysis of a glucurono-xylan from beechwood. Among the enzymes screened, Depol 670L (a cellulolytic preparation from Trichoderma reesei) displayed the highest activity (70.3 U/mL, expressed in reducing xylose equivalents). High-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) analysis revealed the formation of a neutral fraction (corresponding to linear XOS, mainly xylose and xylobiose) and a group of more retained products (acidic XOS), which were separated using strong anion-exchange cartridges. The acidic fraction contained a major product, characterized by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and mono- and two-dimensional nuclear magnetic resonance spectroscopy (NMR) as 2′-O-α-(4-O-methyl-α-D-glucuronosyl)-xylobiose (X2_MeGlcA). Starting from 2 g of beechwood xylan, 1.5 g of total XOS were obtained, from which 225 mg (11% yield) corresponded to the aldouronic X2_MeGlcA. The acidic XOS exhibited higher antioxidant activity (measured by the ABTS·+ discoloration assay) than xylan, whilst neutral XOS displayed no antioxidant activity. This work demonstrates that it is possible to obtain a safe and natural antioxidant by enzymatic biotransformation of hardwood hemicellulose.

Purification of xylanase from Kretzschmaria zonata with potential interest in the production of xylooligosaccharides

Xylanases are widely applied for different biotechnological purposes, especially to produce xylooligosaccharides (XOS), prebiotics for the food industry. The fungus Kretzschmaria zonata produced xylanase after growth on corncob. The partial purification of the enzyme was performed through a Q-Sepharose anion exchange column. Enzyme purity was performed with electrophoresis gel and xylanolytic activity was confirmed through a zymogram. Xylanase was biochemically characterized and applied for XOS production using 2 % of beechwood xylan. The partially purified xylanase showed higher activity at pH 5.0 and 50 °C, being stable at pH values from 4 to 8. It was inhibited in the presence of CuSO4 and sodium dodecyl sulphate, and it showed higher affinity for beechwood xylan as a substrat. The xylanase was able to hydrolyze beechwood xylan, resulting mainly in xylobiose and xylotriose, which have potential for applications in the food industry as prebiotics.

Use of corn cob through xylan extraction and its potential use as a substrate for the quantification of xylanase enzymatic activity

In Mexico, it is estimated that the amount of residues produced by the planting of the ten main crops is approximately 45 million tons of dry matter, of which just over 25 million tons are corn cob. The corn cob contains large amounts of hemicellulose, mainly composed of xylan, which can have various industrial uses. For this reason, in the present study, fresh and dry corn cob was used with two particle sizes: 0.105 mm and 0.42 mm, to carry out an exhaustive alkaline extraction of xylan and where eight fractions of hemicelluloses extracts were obtained. Each of the fractions were subjected to enzymatic hydrolysis for eight hours with a commercial xylanase (Livanol Devisco 1500) and as internal control, beechwood xylan (Sigma Aldrich, X4252) was used, of which only 6.9 ± 2.25 g/L were released. reducing sugars. It was found that the fractions from the steam treatment and the fraction from the alkaline extraction precipitated with alcohol, both for fresh corn cob, were released a quantity of reducing sugars very similar to commercial xylan with 7.10 ± 2.02 and 7.24 ± 0.37 g/L, respectively. Finally, it was possible to determine that although the 8 fractions obtained in the present study can be used as substrates to determine the xylanase enzymatic activity, the best was the fraction of the alkaline extraction since 0.236 ± 0.03 IU/mL were quantified, while for the commercial substrate (beechwood xylan) an activity of 0.287 ± 0.01 IU/mL was obtained.

Structural and biochemical analysis reveals how ferulic acid improves catalytic efficiency of Humicolagrisea xylanase

Humicolagrisea var. thermoidea is an aerobic and thermophilic fungus that secretes the GH11 xylanase HXYN2 in the presence of sugarcane bagasse. In this study, HXYN2 was expressed in Pichiapastoris and characterized biochemically and structurally in the presence of beechwood xylan substrate and ferulic acid (FA). HXYN2 is a thermally stable protein, as indicated by circular dichroism, with greater activity in the range of 40–50 °C and pH 5.0–9.0, with optimal temperature and pH of 50 °C and 6.0, respectively. FA resulted in a 75% increase in enzyme activity and a 2.5-fold increase in catalytic velocity, catalytic efficiency, and catalytic rate constant (kcat), with no alteration in enzyme affinity for the substrate. Fluorescence quenching indicated that FA forms a complex with HXYN2 interacting with solvent-exposed tryptophan residues. The binding constants ranged from moderate (pH 7.0 and 9.0) to strong (pH 4.0) affinity. Isothermal titration calorimetry, structural models and molecular docking suggested that hydrogen bonds and hydrophobic interactions occur in the aglycone region inducing conformational changes in the active site driven by initial and final enthalpy- and entropy processes, respectively. These results indicate a potential for biotechnological application for HXYN2, such as in the bioconversion of plant residues rich in ferulic acid.

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.

A novel cold-active GH8 xylanase from cellulolytic myxobacterium and its application in food industry

Although xylanase have a wide range of applications, cold-active xylanases have received less attention. In this study, a novel glycoside hydrolase family 8 (GH8) xylanase from Sorangium cellulosum with high activity at low temperatures was identified. The recombinant xylanase (XynSc8) was most active at 50 °C, demonstrating 20% of its maximum activity and strict substrate specificity towards beechwood and corncob xylan at 4 °C with Vmax values of 968.65 and 1521.13 μmol/mg/min, respectively. Mesophilic XynSc8 was active at a broad range of pH and hydrolyzed beechwood and corncob xylan into xylooligosaccharides (XOS) with degree of polymerization greater than 3. Moreover, incorporation of XynSc8 (0.05–0.2 mg/kg flour) provided remarkable improvement (28–30%) in bread specific volume and textural characteristics of bread compared to commercial xylanase. This is the first report on a novel cold-adapted GH8 xylanase from myxobacteria, suggesting that XynSc8 may be a promising candidate suitable for bread making.

Exploring competitive inhibition of a family 10 xylanase derived from Hu sheep rumen microbiota by Oryza sativa xylanase inhibitor protein: In vitro and in silico perspectives

The catalytic domain of family GH10 xylanase, XYN-LXY_CD derived from Hu sheep rumen microbiota was expressed in Pichia pastoris X33. The special activity of reXYN-LXY_CD in the culture supernatant was 232.56 U/mg. The optima of reXYN-LXY_CD were 53 °C and pH 7.0. Recombinant Oryza sativa xylanase inhibitor protein (rePOsXIP) competitively inhibited reXYN-LXY_CD with an inhibition constant (Ki) value of 237.37 nM. The concentration of hydrolysates released from beechwood xylan by reXYN-LXY_CD reduced when rePOsXIP was added into the hydrolytic system. Fluorescence of reXYN-LXY_CD was statically quenched by rePOsXIP in a dose-dependent manner. The details in intermolecular interaction between XYN-LXY_CD and OsXIP were investigated by using molecular dynamics (MD) simulations, binding free energy computation and non-covalent interactions (NCI) analysis. Hydrogen bonding and van der Waals played indispensable roles in the XYN-LXY_CD/OsXIP interaction. The α-7 helix of OsXIP tightly occupied the catalytic pocket of XYN-LXY_CD with hydrogen bonding such as K239OsXIP-N261/Q292/E197XYN-LXY_CD (E197, the acid-base catalytic residue), D236OsXIP-K327XYN-LXY_CD and Q242OsXIP-E211/Q212XYN-LXY_CD. Based on the quantum theory of atoms in molecules (QTAIM), the Laplacian of electron density and core-valence bifurcation index of HZ3K239-OE2E197 were 0.1025 a.u. and 0.002218, respectively. Elucidating the mechanism underlying xylanase-inhibitor interactions might help construct XYN-LXY_CD mutants that gain resistance to XIPs and high catalytic activity, which would be more efficient in feed additives in livestock.

Production of tailored xylo-oligosaccharides from beechwood xylan by different enzyme membrane reactors and evaluation of their prebiotic activity

Xylo-oligosaccharides (XOs) from xylans are gaining interest due to their wide use in food, feed and pharmaceutical industry. In this work, the enzymatic production of XOs from beechwood xylan in batch, continuous and semi-continuous enzyme membrane reactor mode was compared. The potential of an inline viscometer for real-time monitoring of the hydrolysis was assessed as well. In all modes, an initial concentration of 7% (w/w) beechwood xylan was applied at a temperature of 50 °C and a pH of 5.8. Initially, the effect of enzyme dosage was investigated in batch mode (0.5, 2.6, 12, 26 and 720 U of Cellic®CTec2.g−1 xylan). Higher enzyme dosages led to a faster hydrolysis rate, but also to an undesirable higher production of xylose. The introduction of enzyme membrane reactors allowed production of XOs with weight average molecular weights of ca. 5–8.9 kDa combined with negligible xylose quantities (<3% in freeze dried product). The semi-continuous configuration has the edge in comparison to the full continuous configuration in terms of enzyme consumption and ease of operation. In vitro fermentations with three different gut bacteria were performed and confirmed the prebiotic properties of the produced XOs with average degree of polymerization of 48.

Rahnella sp., a Dominant Symbiont of the Core Gut Bacteriome of Dendroctonus Species, Has Metabolic Capacity to Degrade Xylan by Bifunctional Xylanase-Ferulic Acid Esterase.

Rahnella sp. ChDrAdgB13 is a dominant member of the gut bacterial core of species of the genus Dendroctonus, which is one of the most destructive pine forest bark beetles. The objectives of this study were identified in Rahnella sp. ChDrAdgB13 genome the glycosyl hydrolase families involved in carbohydrate metabolism and specifically, the genes that participate in xylan hydrolysis, to determine the functionality of a putative endo-1,4-β-D-xylanase, which results to be bifunctional xylanase–ferulic acid esterase called R13 Fae and characterize it biochemically. The carbohydrate-active enzyme prediction revealed 25 glycoside hydrolases, 20 glycosyl transferases, carbohydrate esterases, two auxiliary activities, one polysaccharide lyase, and one carbohydrate-binding module (CBM). The R13 Fae predicted showed high identity to the putative esterases and glycosyl hydrolases from Rahnella species and some members of the Yersiniaceae family. The r13 fae gene encodes 393 amino acids (43.5 kDa), containing a signal peptide, esterase catalytic domain, and CBM48. The R13 Fae modeling showed a higher binding affinity to ferulic acid, α-naphthyl acetate, and arabinoxylan, and a low affinity to starch. The R13 Fae recombinant protein showed activity on α-naphthyl acetate and xylan, but not on starch. This enzyme showed mesophilic characteristics, displaying its optimal activity at pH 6.0 and 25°C. The enzyme was stable at pH from 4.5 to 9.0, retaining nearly 66–71% of its original activity. The half-life of the enzyme was 23 days at 25°C. The enzyme was stable in the presence of metallic ions, except for Hg2+. The products of R13 Fae mediated hydrolysis of beechwood xylan were xylobiose and xylose, manifesting an exo-activity. The results suggest that Rahnella sp. ChDrAdgB13 hydrolyze xylan and its products could be assimilated by its host and other gut microbes as a nutritional source, demonstrating their functional role in the bacterial-insect interaction contributing to their fitness, development, and survival.