GH11 Xylanase Research Articles

Preparation and characterization of ferulic oligosaccharides from different sources by cell-free GH10 and GH11 xylanases

The feruloyl oligosaccharides (FOs) produced by the decomposition of plant hemicellulose have broad potential applications in the food and biomedical areas. FOs were prepared through the specific enzymatic degradation of insoluble dietary fiber from different sources by cell-free GH10 and GH11 xylanases. The cell-free GH10 and GH11 xylanases were obtained by the heterologous expression in Escherichia coli. The enzymatic hydrolysis conditions were optimized as follows: temperature 50 °C, pH 5.5, hydrolysis time 12 h, GH10 xylanase addition 101.74 U, and GH11 xylanase addition 121.60 U. The compositions and structural characterization of wheat bran FOs (WB-FOs), corncob FOs (CC-FOs), and buckwheat straw FOs (BS-FOs) were identified by fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR), high-performance liquid chromatography (HPLC), and electrospray ionization tandem mass spectrometry (ESI-MS). Degrees of polymerization (DP) of WB-FOs, CC-FOs, and BS-FOs were 3–11, 3–7, and 3–6, respectively. Ultraviolet (UV) radiation was investigated in vitro. The results demonstrated that BS-FOs possessed excellent UV resistance and photostability, followed by effectiveness in WB-FOs and CC-FOs. These results have improved our understanding of the relationship between FOs with different structural types and their UV radiation.

Deciphering genome sequence of Paenibacillus illinoisensis strain YWY-3.1: A chitinase, cellulase, and amylase producer

The chitin-degrading Paenibacillus illinoisensis YWY-3.1 was isolated from the lake water at Yok Don National Park, Vietnam. Previous evaluations reported that strain YWY-3.1 had high activities of chitinases, cellulases, and amylases; it also produced remarkable levels of numerous promoting traits for plant growth. This study aimed to sequence and analyze the genome sequence of strain YWY-3.1 for further explorations and applications in agriculture and related fields. The whole genome sequencing revealed the P. illinoisensis YWY-3.1 genome contained 6,451,623 bp, 47.3 % GC, 5786 coding sequences, 88 tRNA, and 1 rRNA. It shared 95.49 % ANI and 87.1 % dDDH to those of P. illinoisensis NBRC 15959 (GCA 004000925). Among the coding sequences, COG deduced 4,991, and KEGG predicted 2819. The genome harbored 338 carbohydrate-active enzymes, including 195 GHs, 44 GTs, 13 PLs, 37 CEs, 3 AAs, and 46 CBMs; among them, 6 GH18 chitinases, 5 GH5 cellulases, 2 GH10 xylanases, 1 GH11 xylanase, and 2 GH13 α-amylases were identified. It contained 49 genes involved in environmental adaptation and plant growth. Moreover, 7 BGCs, with 3 being novel clusters, were identified from the genome. This work provided insight into the genomic information of P. illinoisensis YWY-3.1 and potential gene resources, especially chitinases, cellulases, xylanases, α-amylases, and novel BGCs for further explorations and applications.

Molecular Identification and Engineering a Salt-Tolerant GH11 Xylanase for Efficient Xylooligosaccharides Production.

This study identified a salt-tolerant GH11 xylanase, Xynst, which was isolated from a soil bacterium Bacillus sp. SC1 and can resist as high as 4 M NaCl. After rational design and high-throughput screening of site-directed mutant libraries, a double mutant W6F/Q7H with a 244% increase in catalytic activity and a 10 °C increment in optimal temperature was obtained. Both Xynst and W6F/Q7H xylanases were stimulated by high concentrations of salts. In particular, the activity of W6F/Q7H was more than eight times that of Xynst in the presence of 2 M NaCl at 65 °C. Kinetic parameters indicated they have the highest affinity for beechwood xylan (Km = 0.30 mg mL-1 for Xynst and 0.18 mg mL-1 for W6F/Q7H), and W6F/Q7H has very high catalytic efficiency (Kcat/Km = 15483.33 mL mg-1 s-1). Molecular dynamic simulation suggested that W6F/Q7H has a more compact overall structure, improved rigidity of the active pocket edge, and a flexible upper-end alpha helix. Hydrolysis of different xylans by W6F/Q7H released more xylooligosaccharides and yielded higher proportions of xylobiose and xylotriose than Xynst did. The conversion efficiencies of Xynst and W6F/Q7H on all tested xylans exceeded 20%, suggesting potential applications in the agricultural and food industries.

Improving the catalytic activity and thermostability of Aspergillus niger xylanase through computational design

Xylanase plays the most important role in catalyzing xylan to xylose moieties. GH11 xylanases have been widely used in many fields, but most GH11 xylanases are mesophilic enzymes. To improve the catalytic activity and thermostability of Aspergillus niger xylanase (Xyn-WT), we predicted potential key mutation sites of Xyn-WT through multiple computer-aided enzyme engineering strategies. We introduce a simple and economical Ni affinity chromatography purification method to obtain high-purity xylanase and its mutants. Ten mutants (Xyn-A, Xyn-B, Xyn-C, E45T, Q93R, E45T/Q93R, A161P, Xyn-D, Xyn-E, Xyn-F) were identified. Among the ten mutants, four (Xyn-A, Xyn-C, A161P, Xyn-F) presented improved thermal stability and activity, with Xyn-F(A161P/E45T/Q93R) being the most thermally stable and active. Compared with Xyn-WT, after heat treatment at 55 °C and 60 °C for 10 min, the remaining enzyme activity of Xyn-F was 12 and 6 times greater than that of Xyn-WT, respectively, and Xyn-F was approximately 1.5 times greater than Xyn-WT when not heat treated. The pH adaptation of Xyn-F was also significantly enhanced. In summary, an improved catalytic activity and thermostability of the design variant Xyn-F has been reported.

Integrity of xylan backbone affects plant responses to drought.

Drought is a major factor affecting crops, thus efforts are needed to increase plant resilience to this abiotic stress. The overlapping signaling pathways between drought and cell wall integrity maintenance responses create a possibility of increasing drought resistance by modifying cell walls. Here, using herbaceous and woody plant model species, Arabidopsis and hybrid aspen, respectively, we investigated how the integrity of xylan in secondary walls affects the responses of plants to drought stress. Plants, in which secondary wall xylan integrity was reduced by expressing fungal GH10 and GH11 xylanases or by affecting genes involved in xylan backbone biosynthesis, were subjected to controlled drought while their physiological responses were continuously monitored by RGB, fluorescence, and/or hyperspectral cameras. For Arabidopsis, this was supplemented with survival test after complete water withdrawal and analyses of stomatal function and stem conductivity. All Arabidopsis xylan-impaired lines showed better survival upon complete watering withdrawal, increased stomatal density and delayed growth inhibition by moderate drought, indicating increased resilience to moderate drought associated with modified xylan integrity. Subtle differences were recorded between xylan biosynthesis mutants (irx9, irx10 and irx14) and xylanase-expressing lines. irx14 was the most drought resistant genotype, and the only genotype with increased lignin content and unaltered xylem conductivity despite its irx phenotype. Rosette growth was more affected by drought in GH11- than in GH10-expressing plants. In aspen, mild downregulation of GT43B and C genes did not affect drought responses and the transgenic plants grew better than the wild-type in drought and well-watered conditions. Both GH10 and GH11 xylanases strongly inhibited stem elongation and root growth in well-watered conditions but growth was less inhibited by drought in GH11-expressing plants than in wild-type. Overall, plants with xylan integrity impairment in secondary walls were less affected than wild-type by moderately reduced water availability but their responses also varied among genotypes and species. Thus, modifying the secondary cell wall integrity can be considered as a potential strategy for developing crops better suited to withstand water scarcity, but more research is needed to address the underlying molecular causes of this variability.

Heteroxylan hydrolysis by a recombinant cellulase-free GH10 xylanase from the alkaliphilic bacterium Halalkalibacterium halodurans C-125.

The search for affordable enzymes with exceptional characteristics is fundamental to overcoming industrial and environmental constraints. In this study, a recombinant GH10 xylanase (Xyn10-HB) from the extremelyalkaliphilic bacterium Halalkalibacteriumhalodurans C-125 cultivated at pH 10 was cloned and expressed in E. coli BL21(DE3). Removal of the signal peptide improved the expression, and an overall activity of 8 U/mL was obtained in the cell-free supernatant. The molecular weight of purified Xyn10-HB was estimated to be 42.6 kDa by SDS-PAGE. The enzyme was active across a wide pH range (5-10) with optimal activity recorded at pH 8.5 and 60°C. It also presented good stability with a half-life of 3h under these conditions. Substrate specificity studies showed that Xyn10-HB is a cellulase-free enzyme that conventionally hydrolyse birchwood and oat spelts xylans (Apparent Km of 0.46mg/mL and 0.54mg/mL, respectively). HPLC analysis showed that both xylans hydrolysis produced xylooligosaccharides (XOS) with a degree of polymerization (DP) ranging from 2 to 9. The conversion yield was 77% after 24h with xylobiose and xylotriose as the main end-reaction products. When assayed on alkali-extracted wheat straw heteroxylan, the Xyn10-HB produced active XOS with antioxidant activity determined by the DPPH radical scavenging method (IC50 of 0.54mg/mL after 4h). Owing to its various characteristics, Xyn10-HB xylanase is a promising candidate for multiple biotechnological applications.

Antioxidant capacity of xylooligosaccharides generated from beechwood xylan by recombinant family GH10 Aspergillus niger xylanase A and insights into the enzyme's competitive inhibition by riceXIP

In this study, the family GH10 xylanase AnXylA10 derived from Aspergillus niger JL15 strain was expressed in Pichia pastoris X33. The recombinant xylanase, reAnXylA10 exhibited optimal activity at 40 ℃ and pH 5.0. The hydrolysates generated from beechwood xylan using reAnXylA10 primarily consisted of xylobiose (X2) to xylohexaose (X6) and demonstrated remarkable antioxidant capacity. Furthermore, the rice xylanase inhibitory protein (riceXIP) was observed to competitively inhibit reAnXylA10, exhibiting an inhibition constant (Ki) of 140.6 nM. Molecular dynamics (MD) simulations of AnXylA10-riceXIP complex revealed that the α-7 helix (Q225-S238) of riceXIP intruded into the catalytic pocket of AnXylA10, thereby obstructing substrate access to the active site. Specifically, residue K226 of riceXIP formed robust interactions with E136 and E242, the two catalytic sites of AnXylA10, predominantly through high-occupied hydrogen bonds. Based on QTAIM, electron densities for the atom pairs K226riceXIP@HZ1-E136AnXylA10@OE2 and K226riceXIP@HZ3-E242AnXylA10@OE1 were determined to be 0.04628 and 0.02914 a.u., respectively. Binding free energy of AnXylA10-riceXIP complex was −59.0±7.6 kcal/mol, significantly driven by electrostatic and van der Waals forces. Gaining insights into the interaction between xylanase and its inhibitors, and mining the inhibition mechanism in depth, will facilitate the design of innovative GH10 family xylanases that are both highly efficient and resistant to inhibitors.

Sugarcane bagasse derived xylooligosaccharides produced by an arabinofuranosidase/xylobiohydrolase from Bifidobacterium longum in synergism with xylanases

Arabinoxylan is a major hemicellulose in the sugarcane plant cell wall with arabinose decorations that impose steric restrictions on the activity of xylanases against this substrate. Enzymatic removal of the decorations by arabinofuranosidases can allow a more efficient arabinoxylan degradation by xylanases. Here we produced and characterized a recombinant Bifidobacterium longum arabinofuranosidase from glycoside hydrolase family 43 (BlAbf43) and applied it, together with GH10 and GH11 xylanases, to produce xylooligosaccharides (XOS) from wheat arabinoxylan and alkali pretreated sugarcane bagasse. The enzyme synergistically enhanced XOS production by GH10 and GH11 xylanases, being particularly efficient in combination with the latter family of enzymes, with a degree of synergism of 1.7. We also demonstrated that the enzyme is capable of not only removing arabinose decorations from the arabinoxylan and from the non-reducing end of the oligomeric substrates, but also hydrolyzing the xylan backbone yielding mostly xylobiose and xylose in particular cases. Structural studies of BlAbf43 shed light on the molecular basis of the substrate recognition and allowed hypothesizing on the structural reasons of its multifunctionality.

Recognition of a Single β-D-Xylopyranose Molecule by Xylanase GH11 from Thermoanaerobacterium saccharolyticum

The endo-β-1,4-xylanase glycosyl hydrolase (GH11) decomposes the backbone of xylan into xylooligosaccharides or xylose. These enzymes are important for industrial applications in the production of biofuel, feed, food, and value-added materials. β-D-xylopyranose (XYP, also known as β-D-xylose) is the fundamental unit of the substrate xylan, and understanding its recognition is fundamental for the initial steps of GH11’s molecular mechanism. However, little is known about the recognition of a single XYP molecule by GH11. In this study, the crystal structures of GH11 from Thermoanaerobacterium saccharolyticum (TsaGH11) complexed with an XYP molecule were determined at a resolution of 1.7–1.9 Å. The XYP molecule binds to subsite −2 of the substrate-binding cleft. The XYP molecule is mainly stabilized by a π–π interaction with the conserved Trp36 residue. The O2 and O3 atoms of XYP are stabilized by hydrogen bond interactions with the hydroxyl groups of Tyr96 and Tyr192. The conformation of the thumb domain of TsaGH11 does not play a critical role in XYP binding, and XYP binding induces a shift in the thumb domain of TsaGH11 toward the XYP molecule. A structural comparison of TsaGH11 with other GH11 xylanases revealed that the XYP molecule forms π–π stacking with the center between the phenyl and indoline ring of Trp36, whereas the XYP molecule unit from xylobiose or xylotetraose forms π–π stacking with the indoline of Trp36, which indicates that the binding modes of the substrate and XYP differ. These structural results provide a greater understanding of the recognition of XYP by the GH11 family.

Enhancing the acid stability of the recombinant GH11 xylanase xynA through N-terminal substitution to facilitate its application in apple juice clarification

The utilization of xylanase in juice clarification is contingent upon its stability within acidic environments. We generated a mutant xynA-1 by substituting the N-terminal segment of the recombinant xylanase xynA to investigate the correlation between the N-terminal region of xylanase and its acid stability. The enzymatic activity of xynA-1 was found to be superior under acidic conditions (pH 5.0). It exhibited enhanced acid stability, surpassing the residual enzyme activity values of xynA at pH 4.0 (53.07 %), pH 4.5 (69.8 %), and pH 5.0 (82.4 %), with values of 60.16 %, 77.74 %, and 87.3 %, respectively. Additionally, the catalytic efficiency of xynA was concurrently improved. Through molecular dynamics simulation, we observed that N-terminal shortening induced a reduction in motility across most regions of the protein structure while enhancing its stability, particularly Lys131-Phe146 and Leu176-Gly206. Furthermore, the application of treated xynA-1 in the process of apple juice clarification led to a significant increase in clarity within a short duration of 20 min at 35 °C while ensuring the quality of the apple juice. This study not only enhances the understanding of the N-terminal region of xylanase but also establishes a theoretical basis for augmenting xylanase resources employed in fruit juice clarification.

Two-domain GH30 xylanase from human gut microbiota as a tool for enzymatic production of xylooligosaccharides: Crystallographic structure and a synergy with GH11 xylosidase

Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy. This includes valorization of hemicellulosic fraction of plant biomass, the second most abundant biopolymer from plant cell walls, aiming to produce prebiotic oligosaccharides, widely explored in food and feed industries. In this work, we conducted biochemical and biophysical characterization of a prokaryotic two-domain R. champanellensis xylanase from glycoside hydrolase (GH) family 30 (RcXyn30A), and evaluated its applicability for XOS production from glucuronoxylan in combination with two endo-xylanases from GH10 and GH11 families and a GH11 xylobiohydrolase. RcXyn30A liberates mainly long monoglucuronylated xylooligosaccharides and is inefficient in cleaving unbranched oligosaccharides. Crystallographic structure of RcXyn30A catalytic domain was solved and refined to 1.37 Å resolution. Structural analysis of the catalytic domain releveled that its high affinity for glucuronic acid substituted xylan is due to the coordination of the substrate decoration by several hydrogen bonds and ionic interactions in the subsite −2. Furthermore, the protein has a larger β5-α5 loop as compared to other GH30 xylanases, which might be crucial for creating an additional aglycone subsite (+3) of the catalytic site. Finally, RcXyn30A activity is synergic to that of GH11 xylobiohydrolase.

Improvement of thermostability and catalytic efficiency of xylanase from Myceliophthora thermophilar by N-terminal and C-terminal truncation.

Extracting xylanase from thermophilic filamentous fungi is a feasible way to obtain xylanase with good thermal stability. The transcriptomic data of Myceliophthora thermophilic destructive ATCC42464 were differentially expressed and enriched. By comparing the sequences of Mtxylan2 and more than 10 xylanases, the N-terminal and C-terminal of Mtxylan2 were truncated, and three mutants 28N, 28C and 28NC were constructed. GH11 xylan Mtxylan2 was identified by transcriptomic analysis, the specific enzyme activity of Mtxylan2 was 104.67 U/mg, and the optimal temperature was 65°C. Molecular modification of Mtxylan2 showed that the catalytic activity of the mutants was enhanced. Among them, the catalytic activity of 28C was increased by 9.3 times, the optimal temperature was increased by 5°C, and the residual enzyme activity remained above 80% after 30 min at 50-65°C, indicating that redundant C-terminal truncation can improve the thermal stability and catalytic performance of GH11 xylanase.

Activity-stability trade-off observed in variants at position 315 of the GH10 xylanase XynR

XynR is a thermostable alkaline GH10 xylanase, for which we have previously examined the effects of saturation mutagenesis at position 315 on enzyme alkaliphily, and found that at pH 10, the activities of variants could be ordered as follows: T315Q > T315S = T315N > T315H = wild-type XynR (WT) > 15 other variants. In this study, we sought to elucidate the mechanisms underlying the variable activity of these different variants. Crystallographic analysis revealed that the Ca2+ ion near position 315 in WT was absent in the T315Q variant. We accordingly hypothesized that the enhancement of alkaliphily in T315Q, and probably also in the T315H, T315N, and T315S variants, could be ascribed to an activity-stability trade-off associated with a reduction in stability due to the lack of this Ca2+ ion. Consistent with expectations, the alkaline resistance of T315H, T315N, T315Q, and T315S, evaluated through the pH-dependence of stability at 0 mM CaCl2 under alkaline conditions, was found to be lower than that of WT: the residual activity at pH 11 of WT was 78% while those of T315H, T315N, T315Q, and T315S were 0, 9, 0, and 43%, respectively. In addition, the thermostabilities of these four variants, as assessed using the denaturing temperatures (Tm) at 0 mM CaCl2 based on ellipticity at 222 nm in circular dichroism measurements, were lower than that of WT by 2–8 °C. Furthermore, the Tm values of WT and variants at 5 mM CaCl2 were higher than those at 0 mM CaCl2 by 6–11 °C. Collectively, our findings in this study indicate that mutation of the T residue at position 315 of XynR to H, N, Q, and S causes an increase in the alkaliphily of this enzyme, thereby reducing its stability.

Comparative Analysis of Room Temperature Structures Determined by Macromolecular and Serial Crystallography

Temperature directly influences the function and structure of proteins. Crystal structures determined at room temperature offer more biologically relevant structural information regarding flexibility, rigidity, and thermal motion than those determined by conventional cryocrystallography. Crystal structures can be determined at room temperature using conventional macromolecular crystallography (MX) or serial crystallography (SX) techniques. Among these, MX may theoretically be affected by radiation damage or X-ray heating, potentially resulting in differences between the room temperature structures determined by MX and SX, but this has not been fully elucidated. In this study, the room temperature structure of xylanase GH11 from Thermoanaerobacterium saccharolyticum was determined by MX (RT-TsaGH11-MX). The RT-TsaGH11-MX exhibited both the open and closed conformations of the substrate-binding cleft within the β-sandwich fold. The RT-TsaGH11-MX showed distinct structural changes and molecular flexibility when compared with the RT-TsaGH11 determined via serial synchrotron crystallography. The notable molecular conformation and flexibility of the RT-TsaGH11-MX may be induced by radiation damage and X-ray heating. These findings will broaden our understanding of the potential limitations of room temperature structures determined by MX.

Effects of amino acid substitutions at position 315 of GH10 xylanase XynR on its alkaliphily

Effects of amino acid substitutions at position 315 of GH10 xylanase XynR on its alkaliphily

Clustered surface amino acid residues modulate the acid stability of GH10 xylanase in fungi

Acidic xylanases are widely used in industries such as biofuels, animal feeding, and fruit juice clarification due to their tolerance to acidic environments. However, the factors controlling their acid stability, especially in GH10 xylanases, are only partially understood. In this study, we identified a series of thermostable GH10 xylanases with optimal temperatures ranging from 70 to 90 °C, and among these, five enzymes (Xyn10C, Xyn10RE, Xyn10TC, Xyn10BS, and Xyn10PC) exhibited remarkable stability at pH 2.0. Our statistical analysis highlighted several factors contributing to the acid stability of GH10 xylanases, including electrostatic repulsion, π-π stacking, ionic bonds, hydrogen bonds, and Van der Waals interactions. Furthermore, through mutagenesis studies, we uncovered that acid stability is influenced by a complex interplay of amino acid residues. The key amino acid sites determining the acid stability of GH10 xylanases were thus elucidated, mainly concentrated in two surface regions behind the enzyme active center. Notably, the critical residues associated with acid stability markedly enhanced Xyn10RE’s thermostability by more than sixfold, indicating a potential acid-thermal interplay in GH10 xylanases. This study not only reported a series of valuable genes but also provided a range of modification targets for enhancing the acid stability of GH10 xylanases.Key points• Five acid stable and thermostable GH10 xylanases were reported.• The key amino acid sites, mainly forming two enriched surface regions behind the enzyme active center, were identified responsible for acid stability of GH10 xylanases.• The finding revealed interactive amino acid sites, offering a pathway for synergistic enhancement of both acid stability and thermostability in GH10 xylanase modifications.

Mapping secondary substrate-binding sites on the GH11 xylanase from Bacillus subtilis.

Xylanases are of significant interest for biomass conversion technologies. Here, we investigated the allosteric regulation of xylan hydrolysis by the Bacillus subtilis GH11 endoxylanase. Molecular dynamics simulations (MDS) in the presence of xylobiose identified binding to the active site and two potential secondary binding sites (SBS) around surface residues Asn54 and Asn151. Arabinoxylan titration experiments with single cysteine mutants N54C and N151C labeled with the thiol-reactive fluorophore acrylodan or the ESR spin-label MTSSL validated the MDS results. Ligand binding at the SBS around Asn54 confirms previous reports, and analysis of the second SBS around N151C discovered in the present study includes residues Val98/Ala192/Ser155/His156. Understanding the regulation of xylanases contributes to efforts for industrial decarbonization and to establishing a sustainable energy matrix.

An eco-friendly process for low-degree polymerization xylo-oligosaccharides production from corncob via enzyme adsorption-hydrolysis integrated approach by reconstructing fusion protein

Xylo-oligosaccharides (XOS) have gained significant attention for their extensive health-boosting characteristics. However, the tedious procedures involved in lignocellulose pretreatment and XOS purification, coupled with the low efficiency of enzymatic hydrolysis, constrain the broader commercialization of XOS. In this study, the effects of fusion order and linker composition on enzyme activity and thermostability of fusion proteins consisting of a GH11 xylanase mutant (XYNm) and an expansin (EXLX) were investigated. The results indicated that the reconstructed protein XYNm-R2-EXLX exhibited 2.6-fold activity higher than EXLX-R2-XYNm. Furthermore, the denaturation thermodynamics evaluation demonstrated a significant enhancement in the thermostability of XYNm-R2-EXLX. Additionally, the reconstructed fusion protein showed a notable affinity adsorption efficiency on corncob, reaching a remarkable 95.5%. Subsequently, the fusion enzyme-corncob complex underwent direct hydrolysis, and a yield of 58.7 g XOS/kg corncob was obtained by employing RSM optimization techniques. Finally, a membrane-assisted refining process was implemented, resulting in the recovery of 68.2% (w/w) of the initial XOS (X2-X4) with a purity of 96.2%. Here, a chemical free and eco-friendly bioprocess for production of low-degree polymerization XOS was developed, eliminating the need for xylan separation from corncob and simplifying the process for product purification. These findings exhibited the great potential in XOS (X2-X4) production.

In vitro evaluation of the application of an optimized xylanase cocktail for improved monogastric feed digestibility.

Xylanases from glycoside hydrolase (GH) families 10 and 11 are common feed additives for broiler chicken diets due to their catalytic activity on the nonstarch polysaccharide xylan. This study investigated the potential of an optimized binary GH10 and GH11 xylanase cocktail to mitigate the antinutritional effects of xylan on the digestibility of locally sourced chicken feed. Immunofluorescence visualization of the activity of the xylanase cocktail on xylan in the yellow corn of the feed showed a substantial collapse in the morphology of cell walls. Secondly, the reduction in the viscosity of the digesta of the feed by the cocktail showed an effective degradation of the soluble fraction of xylan. Analysis of the xylan degradation products from broiler feeds by the xylanase cocktail showed that xylotriose and xylopentaose were the major xylooligosaccharides (XOS) produced. In vitro evaluation of the prebiotic potential of these XOS showed that they improved the growth of the beneficial bacteria Streptococcus thermophilus and Lactobacillus bulgaricus. The antibacterial activity of broths from XOS-supplemented probiotic cultures showed a suppressive effect on the growth of the extraintestinal infectious bacterium Klebsiella pneumoniae. Supplementing the xylanase cocktail in cereal animal feeds attenuated xylan's antinutritional effects by reducing digesta viscosity and releasing entrapped nutrients. Furthermore, the production of prebiotic XOS promoted the growth of beneficial bacteria while inhibiting the growth of pathogens. Based on these effects of the xylanase cocktail on the feed, improved growth performance and better feed conversion can potentially be achieved during poultry rearing.

A Glycosyl Hydrolase 30 Family Xylanase from the Rumen Metagenome and Its Effects on In Vitro Ruminal Fermentation of Wheat Straw.

The challenge of wheat straw as a ruminant feed is its low ruminal digestibility. This study investigated the impact of a xylanase called RuXyn, derived from the rumen metagenome of beef cattle, on the in vitro ruminal fermentation of wheat straw. RuXyn encoded 505 amino acids and was categorized within subfamily 8 of the glycosyl hydrolase 30 family. RuXyn was heterologously expressed in Escherichia coli and displayed its highest level of activity at pH 6.0 and 40 °C. RuXyn primarily hydrolyzed xylan, while it did not show any noticeable activity towards other substrates, including carboxymethylcellulose and Avicel. At concentrations of 5 mM, Mn2+ and dithiothreitol significantly enhanced RuXyn's activity by 73% and 20%, respectively. RuXyn's activity was almost or completely inactivated in the presence of Cu2+, even at low concentrations. The main hydrolysis products of corncob xylan by RuXyn were xylopentose, xylotriose, and xylotetraose. RuXyn hydrolyzed wheat straw and rice straw more effectively than it did other agricultural by-products. A remarkable synergistic effect was observed between RuXyn and a cellulase cocktail on wheat straw hydrolysis. Supplementation with RuXyn increased dry matter digestibility; acetate, propionate, valerate, and total volatile fatty acid yields; NH3-N concentration, and total bacterial number during in vitro fermentation of wheat straw relative to the control. RuXyn's inactivity at 60 °C and 70 °C was remedied by mutating proline 151 to phenylalanine and aspartic acid 204 to leucine, boosting activity to 20.3% and 21.8% of the maximum activity at the respective temperatures. As an exogenous enzyme preparation, RuXyn exhibits considerable potential to improve ruminal digestion and the utilization of wheat straw in ruminants. As far as we know, this is the first study on a GH30 xylanase promoting the ruminal fermentation of agricultural straws. The findings demonstrate that the utilization of RuXyn can significantly enhance the ruminal digestibility of wheat straw by approximately 10 percentage points. This outcome signifies the emergence of a novel and highly efficient enzyme preparation that holds promise for the effective utilization of wheat straw, a by-product of crop production, in ruminants.