This study Aimed to evaluate the effects of Lactiplantibacillus plantarum (LP), cellulase (CE) or xylanase (XE) on the fermentation quality and microbial community of the broom sorghum stover silage. It was found that additives can improve the fermentation quality and digestibility of broom sorghum stover. Compared with the CK treatment, the addition of cellulase significantly increased the CP and WSC contents of broom sorghum stover, and also significantly improved thein vitrodry matter digestibility andin vitroneutral detergent fiber digestibility. The microbial community in broom sorghum stover silage also changed with different additive treatments. The microbiota shifted fromPantoeaandNissabactertoLactiplantibacillusandLentilactobacillus. Changes in microorganisms indicate a decrease in the number of harmful microorganisms and more retention of nutrients. All treatments reduced pungent VOCs (e.g., Benzyl isothiocyanate) and enriched aromatic VOCs (e.g., Phenol 4-ethyl-, β-damascenone), with the CE treatment performing best. Ensiling degraded anti-nutritional factors: the CK and LP treatments were superior for tannin reduction, the CE and XE treatments for phytic acid. These additives effectively enhance broom sorghum stover's feed value, with CE showing the most comprehensive effect, providing a basis for its high-value utilization and sustainable livestock production.

PurposeDespite the importance of gerbera in the international market, stem bending is still the biggest problem for gerbera cut flowers during the post-harvest stage, as it reduces the flowers’ vase life and their market value. MethodsTo address this problem, the effects of different vase solutions containing calcium chloride (CaCl2) and sodium selenite (Na2SeO3) on the longevity and stem curvature of gerbera ‘Stanza’ flowers were investigated using repeated-measures ANOVA. The treatments were: distilled water (control, T1), 1 g L–1 (T2) and 2 g L–1 (T3) of CaCl2, 1 mg L–1 (T4) and 2 mg L–1 (T5) of Na2SeO3, and 1.5 g L–1 CaCl2 plus 1.5 mg L–1 Na2SeO3 (T6). Results Gerbera cut flowers subjected to T6 of vase solution exhibited a significantly longer vase life (20 days) and a substantially lower degree of stem bending (25.7°). Moreover, these flowers achieved the lowest significant ion leakage (IL) in petals, and an inhibition in the cellulase (CEL), polygalacturonase (PG), xylanase (XYL), and pectinase (PT) activities, along with noticeably greater levels of catalase (CAT) activity and lignin content until the last day of sampling. Automatic linear modeling analysis revealed CEL, PT, PG, lignin content, and XYL were selected as the most contributing attributes in determining the degree of stem bending. Conclusions It can be recommended that using calcium and/or selenium can be regarded as an effective substitute vase solution to maintain gerbera flowers and reduce bending stems over the post-harvest period.

The highly polymeric structure and complex branching of arabinoxylan (AX) limit its immunomodulatory potential. This study investigated whether enzymatic hydrolysis using xylanase (XYN) and α-l-arabinofuranosidase (ARF) could enhance anti-inflammatory efficacy in DSS-induced colitis. Synergistic ARF-XYN treatment, yielding low-polymerization and debranched oligosaccharides, exhibited superior efficacy in ameliorating colitis symptoms, suppressing pro-inflammatory cytokines (IL-6, TNF-α, IL-1β), and restoring intestinal barrier integrity compared to native AX. Multiomics analyses revealed that ARF-XYN reshaped the gut ecosystem by enriching beneficial bacteria (Akkermansia, Faecalibaculum, Dubosiella) while suppressing pathogenic taxa (Bacteroides, Escherichia-Shigella, and Helicobacter). This microbial restructuring drove a metabolic shift characterized by increased bile acids and short-chain fatty acids, while suppressing inflammatory mediators (prostaglandin B2, histamine, quinolinic acid) and pro-inflammatory lipid metabolites (arachidonic acid, linoleic acid, and their derivatives). These findings demonstrate that precise enzymatic tailoring transforms AX into a potent functional prebiotic ingredient, offering a structure-guided prebiotic strategy for inflammatory bowel disease management through targeted microbiota-metabolite modulation.

Simultaneous production of xylooligosaccharides and acetic acid from xylan-rich biomass by an acetylxylan esterase with two synergistic catalytic domains.

A novel broad-specificity GH51 α-L-arabinofuranosidase from Paenibacillus antarcticus that acts synergistically with other xylanolytic enzymes to completely saccharify arabinoxylans

ABSTRACT Two isoenergetic (12.05 MJ/kg ME) and isonitrogenic (approximately 200 g/kg CP) basal diets were prepared using 670 g/kg of wheat with low fibre and/or high fibre contents. Each basal diet was divided into four portions: one remained as is and fed as control (C); the second was the C supplemented with 100 FXU/kg of a commercial xylanase (XYL); the third was the C plus 20 g/kg of inulin (IN) powder; and the fourth was the C supplemented with both XYL and IN at the same inclusion rates, resulting in a total of eight experimental diets. A study was conducted from 10 to 21 days of age involving 320 female Ross 308 broiler chickens. Each diet, in meal form, was fed ad libitum to eight pens, five birds each, following randomisation. Supplementary XYL increased dietary nitrogen corrected apparent metabolisable energy (AMEn) (p < 0.001), dry matter retention (DMR), daily feed intake (FI), weight gain (WG) and reduced feed conversion ratio (FCR) (p < 0.05). Dietary IN tended (p < 0.01) to increase WG and reduce FCR and dietary wheat did not have an impact on growth performance variables (p > 0.05). Birds fed XYL had reduced relative weight of the pancreas (p < 0.050) and there was XYL by IN interaction (p < 0.05) on relative weight of the caeca as it was greater for those fed XYL and IN. No other changes in relative weight of the gastrointestinal tract organs were observed (p > 0.05). Bird fed XYL or IN had greater (p < 0.05) butyric acid (BA) concentration in caecal content. Feeding XYL led to reduced acetic (AA) and propionic acid (PA) concentrations in caecal excreta (p < 0.05) and to a greater BA:AA ratio (p < 0.001). Dietary IN increased blood serum glutathione peroxidase (GSH-Px) (p < 0.05) and XYL increased concentration of hepatic coenzyme Q10 (p < 0.05). The type of dietary wheat did not have an impact on any of the studied variables, suggesting that birds were able to tolerate the fibre contents in this study. It seems that both IN and XYL, can serve as feed enhancers that potentially may promote antioxidant status of birds and help poultry to cope with various stress factors during production. The study further confirms that supplementing wheat-based diets with XYL may be a strategy to mitigate the reduction in available energy and to increase nutrient availability in broiler diets.

An insect-associated bacterium, Streptomyces sp. SirexAA-E (SirexAA-E) secretes plant biomass-degrading enzymes depending on the available cell wall materials. SirexAA-E readily utilizes xylan, one of the major hemicelluloses. However, the enzyme composition and pathways supporting xylan metabolism in SirexAA-E and other Streptomycetes have not been reported. We aimed to understand changes in both extracellular and intracellular protein productions by cultivating SirexAA-E in defined media containing either xylose, xylobiose, or xylan. Proteomics showed each carbon source gave specific extracellular protein composition when compared to glucose. Furthermore, intracellular proteomics using a pair-wise Tandem Mass Tag (TMT)-labeling LC-MS/MS identified 1,037 proteins with changes in the xylose-, xylobiose-, or xylan-dependent proteome relative to the glucose-derived proteome. We found that numerous proteins related to oxidative phosphorylation were enriched in the pentose-grown proteomes relative to the glucose proteome. This observation is consistent with a high demand for ATP to support protein secretion and intake of xylose and xylooligosaccharides. Additionally, several transporters for carbon uptake were identified in the genome of SirexAA-E by protein homology comparison with Streptomyces coelicolor, and several key transcriptional regulators used by SirexAA-E for catabolizing xylan were found by pull-down proteomics. The current study provides new insights into the extensive extracellular and intracellular responses of a cellulolytic Streptomyces to the major plant hemicellulose.IMPORTANCEStreptomyces sp. SirexAA-E can efficiently degrade cellulose, xylan, and mannan, the major polysaccharide components of woody biomass. Our previous work showed the relative simplicity of the secreted proteome used to degrade cellulose. In this study, we report on the extracellular and intracellular proteomic responses of SirexAA-E during growth on xylan. The substrate-specific proteomic profiles have given a new understanding of the regulation of xylanolytic enzymes and additional metabolic pathways supporting growth on a pentose sugar. These groupings of regulatory and structural proteins provide a blueprint for construction of more robust strains for biomass valorization.

A novel xylanase gene (RuXyn854) was identified from the rumen metagenome and was heterologously expressed in Escherichia coli to produce xylo-oligosaccharides (XOSs) as a prebiotic in this study. RuXyn854, a member of glycosyl hydrolase family 10, demonstrated peak enzymatic activity at pH 7.0 and 50 °C. RuXyn854 retains more than 50% of its activity after treatment at 100 °C for 10 min, highlighting the enzyme's excellent heat resistance. RuXyn854 showed a preferential hydrolyzation of xylan, especially rice straw xylan. RuXyn854 activity was significantly increased in the presence of 15 mM Mn2+, 0.25% Tween-20, and 0.25% Triton X-100 (125%, 20%, and 26%, respectively). The reaction temperature (30, 40, and 50 °C), dosage (0.20, 0.27, and 0.34 U), and time (90, 120, and 150 min) of RuXyn854 affected the XOS yield and composition, with a higher yield at 0.27 U, 50 °C, and 120-150 min. Xylobiose, xylotriose, and xylotetraose were characterized as the predominant XOS products resulting from the enzymatic hydrolysis of wheat straw xylan by RuXyn854, with xylose present at a mere 0.49% of the total yield. The prebiotic potential of XOSs was assessed through in vitro fermentation with established probiotic strains of Bifidobacterium bifidum and Lactobacillus brevis. The results showed that, regardless of incubation time, XOSs stimulated the growth and xylanolytic enzyme secretion of the two probiotics compared to the controls. These results demonstrate that the feature of RuXyn854 to withstand temperatures up to 100 °C is impressive, and its ability to hydrolyze wheat xylan into XOSs promotes the growth of probiotics.

This study aimed to develop an effective technology for the conversion of wheat bran to xylooligosaccharides (XOSs) and monosaccharides. A glycoside hydrolysis (GH) 30_7 endoxylanase (PpXyn30B) and a GH43_36 α-l-arabinofuranosidase (PpAbf43A) from Penicillium parvum 4-14 were subjected to recombinant expression and biochemical analyses. Wheat bran was fractionated into soluble arabinoxylans (WBAX 1 and 2) and insoluble residue (WBR) using alkaline hydrogen peroxide pretreatment and graded ethanol (50% and 75%) precipitation. The combination of PpXyn30B, PpAbf43A, and EpABF62A (GH62) released approximately 40% of the XOSs (primarily xylobiose and xylotriose) and arabinose from WBAX1 and WBAX2. Integration of low-intensity acid treatment with the enzymatic hydrolysis significantly elevated the conversion ratios of XOSs (>75%) and arabinose (>95%). Moreover, the acid treatment facilitated the saccharification of insoluble arabinoxylan in WBR using an enzyme cocktail. These findings provide new insights into the xylanolytic enzymes and pathways involved in the valorization of cereal biomass.

A 28-day study involving 448 male Ross 308 broilers aimed to determine the effect of dietary rice bran (RB) and xylanase (XYL) in maize- and wheat-based diets on chicken growth, N-corrected apparent metabolizable energy (AMEn), and nutrient availability. Two isonitrogenic and isocaloric maize- or wheat-based basal diets (BDs) were formulated matching breeding recommendations. Each diet was then split in four parts: two parts BD was substituted with 75 g/kg RB and then one of the RB substituted and one of the original parts was supplemented with 16,000 XYL units/kg, resulting in a total of eight experimental dietary treatments. Each diet was fed to seven pens of eight birds per pen following randomization. The data were analyzed by ANOVA using a 2 × 2 × 2 factorial design (cereal type × RB × XYL). Enzyme supplemented RB-free wheat-based diet had greater AMEn (p = 0.002) and fiber digestibility (p = 0.007) compared to the rest. Feeding RB reduced daily feed intake (p = 0.015) and weight gain (p &lt; 0.001) of chicks. Birds fed wheat-based diets had greater feed efficiency, coupled with an increase in starch digestibility (SD) and energy conversion ratio (ECR). The observed differences in feed efficiency were explained only by SD and ECR.

Ferulic acid possesses certain antioxidant and antibacterial properties. Additionally, ferulic acid esterase (FAE) and cellulolytic enzymes have been associated with synergistic degradation of ferulic acid ester bonds, thereby facilitating greater release of ferulic acid from lignocellulose, which could have important effects on silage quality and aerobic stability. This study examined the effects of ensiling Broussonetia papyrifera with FAE-producing Lactiplantibacillus plantarum (LP), cellulase (CE) and xylanase (XY) under aerobic exposure conditions. The following treatments were used: distilled water (CK), LP, LP + CE, LP + XY and LP + XY + CE. After 60 days of silage treatment, the samples were unsealed for aerobic exposure for 1, 3, 5, or 7 days. Compared with the CK treatment, the addition of FAE-producing L. plantarum significantly (P < 0.05) led to lower pH, reduced dry matter loss of the silage and increased lactic acid (LA) concentration after 60 d of ensiling (especially for the LP + CE and LP + CE + XY groups). During the aerobic exposure stage, the combined treatment with LP and enzymes effectively inhibited the increase in pH, significantly reduced the rate of dry matter loss and increased the LA concentration and aerobic stability of the silage (P < 0.05). Moreover, the LP + CE and LP + CE + XY treatment groups exhibited higher ferulic acid levels than the other groups did, corresponding with greater aerobic stability, especially for the LP + CE group, which remained stable. In this group, the pH values showed minimal change, increasing by only 0.31 (4.24-4.55) after 7 days of aerobic exposure. In addition, the LP and enzyme co-treatment was linked to shifts in the microbial community of the silage during aerobic exposure, with increased relative abundance of Lactiplantibacillus plantarum, and its abundance positively correlated with lactic acid and ferulic acid concentrations, while negatively correlated with ammonia nitrogen; and inhibited proliferation of spoilage-related bacteria (Enterobacter, Gluconobacter and Cladosporium). The combination of FAE-producing L. plantarum and cellulase can be used as an effective method to increase the preservation efficiency and aerobic stability of B. papyrifera silage.

The present study explores the potential of wild elephant grass (EG), for co-production of ethanol and xylitol. Alkaline H 2 O 2 -pretreated-EG was hydrolyzed by a tailor-made cocktail of recombinant bacterial crude cellulolytic and xylanolytic enzymes, used for co-fermentation. Candida tropicalis (MTCC 230) was adapted in medium having both C5 and C6 sugars. Three significant parameters, inoculum size, S:N in medium and orbital shaking speed (rpm), were optimized using response surface methodology (RSM) and artificial neural network linked genetic algorithm (ANN-GA) for bioethanol and xylitol production. The predictive capabilities of both models were compared. ANN-GA predicted optimum conditions were 10% (v/v) initial inoculum size, the S:N ratio 37.4 and rpm 250 gave 27.4 g/L (0.42 g/g glucose ) ethanol and 5.1 g/L (0.44 g/g xylose ) xylitol titres with K L a of 194 h −1 . The ANN-GA optimized parameters gave 22.3% and 13.3% higher ethanol and xylitol yields, respectively, than those predicted by the RSM-based model. The current innovative method of co-producing ethanol and xylitol from EG offers a promising alternative to traditional bioethanol production.

Annotated draft genome data of Bacillus safensis GMA1 isolated from hot springs in Mexico reveals its xylanolytic potential

Xylanolytic enzyme systems in ascomycetous yeasts remain underexplored, despite the presence of yeasts in various xylan-rich ecological niches. In this study, we investigated the secreted xylanolytic machineries of three Blastobotrys species-B. mokoenaii, B. illinoisensis, and B. malaysiensis-by integrating genome annotation, bioinformatics, and secretome analyses of cultures grown on beechwood glucuronoxylan. Our findings demonstrate that these yeasts effectively hydrolyze xylan through the secretion of xylanases from the glycoside hydrolase (GH) family 11, which play a central role in cleaving the xylan backbone. Additionally, the yeasts produce a diverse array of other CAZymes, including members of GH families 3, 5, and 67, with putative roles in xylan degradation. We also report on the heterologous expression and functional characterization of the GH30_7 xylanase BmXyn30A from B. mokoenaii, which exhibits both glucuronoxylanase and xylobiohydrolase activities. We demonstrate additive effects betweenGHfamily 30 BmXyn30A andGHfamily 11 BmXyn11A during the hydrolysis of beechwood glucuronoxylan, where the enzymes exhibit complementary roles that enhance the deconstruction of this complex hemicellulose substrate. These findings broaden our understanding of the xylanolytic systems in yeasts and underscore the potential of Blastobotrys species as cell factories and natural xylanase producers. The enzymes they produce hold promise for biorefining applications, enabling efficient utilization of renewable xylan-rich plant biomass resources. KEY POINTS: • Extracellular GH11 xylanases dominate glucuronoxylan degradation in Blastobotrys yeasts. • Yeast GH30_7 enzyme shows multifaceted activity, supporting complex xylan breakdown. • Blastobotrys yeasts show promise as cell factories for industrial biotechnologyapplications.

Trichoderma sp. SG2, isolated from the Black Belt soils of Alabama, USA, is a potent natural producer of β-glucosidase and a broad spectrum of cellulolytic and xylanolytic enzymes. This study explored the saccharification of lignocellulosic biomass using crude enzymes from Trichoderma sp. SG2, various pretreatment strategies, mixed feedstock approaches to enhance sugar yield, and enzyme supplementation to reduce costs. Among the pretreatment methods tested for switchgrass, the most effective was sequential H₃PO₄–ethanol, followed by NaOH–H₂SO₄, H₃PO₄–acetone, H₂SO₄–NaOH, and single-agent treatments (H₂SO₄ alone or NaOH alone). Sugar yields were significantly improved by combining pretreated switchgrass with paper powder as a mixed feedstock. The highest glucose (15.8 g/L) and xylose (3.8 g/L) yields were achieved at 10% pretreated switchgrass after 72 h. A key finding was the significant cost reduction and enhanced saccharification efficiency achieved by supplementing SG2 crude enzyme with 50% of the recommended commercial enzyme dosage. Acid-pretreated switchgrass hydrolysis with SG2 enzyme and commercial enzyme supplementation emerged as the most effective strategy. These results highlight Trichoderma sp. SG2 as a promising candidate for developing cost-effective enzyme cocktails for lignocellulosic biomass hydrolysis where 30 to 40% cost of ethanol production process is accounted for enzyme cost.

ABSTRACT This study was performed to evaluate the impact of fibrolytic enzymes on the chemical composition, fermentation profile, microbial population, and in vitro dry matter (DM) and neutral detergent fibre (NDF) degradability of silage from two Urochloa species. The experiment followed a completely randomised 4 (fibrolytic enzymes) × 2 (species) factorial design with three replicates. The fibrolytic enzymes were: control (CT), cellulase (CE) (0.1% fresh matter), xylanase (XL) (400 mg/kg of DM), and a blend (CEXL) (cellulase + xylanase). The species tested were U. decumbens cv. Basilisk and U. brizantha cv. BRS Piatã. The CE treatment resulted in a lower pH and a greater lactic acid bacteria population. The CEXL treatment increased crude protein content, while CE and CEXL reduced ammonia, NDF, acid detergent fibre, and lignin levels compared with CT. The XL treatment increased the potentially soluble fraction and the DM degradation rate, while CE and the blend increased the soluble fraction of DM. Silage treated with CEXL had lower indigestible NDF, while CE increased the degradation rate. The U. brizantha exhibited a greater NDF degradability and degradation rate than U. decumbens. Therefore, CE is recommended to improve the chemical composition, fermentation profile, and degradability of Urochloa silages.

Overconsumption of fossil fuel reserves and its adverse effects has sparked interest in the production of second-generation biofuels due to the abundance of lignocellulosic waste and potential energy crops. However, processing costs associated with depolymerization of the cellulose crystalline structure have stalled advancement in cellulosic ethanol production. Current investigations range from identification of novel enzymes for lignocellulose hydrolysis to consolidation of enzyme production into a singular alcohol producing microorganism to potentially reduce cost for commercial processing. In this study, a total of 828 non-Saccharomyces and black yeasts were screened for cellulolytic and xylanolytic enzyme activities, whereby 60 isolates were identified that exhibited activity for at least one of the enzymes tested. In doing so, a novel Zalaria obscura strain (Z. obscura Y1223) was identified and assessed for enzyme activity in multiple growth media. Semi-quantitative assays showed that Z. obscura Y1223 produced cellulases optimally in media containing yeast extract, peptone and oat bran, with a pH range between pH 5 and 6 and at 30°C. Maximum xylanase activity (20.5 U/L/OD600) was attained using synthetic complete media supplemented with xylo-oligosaccharides and maximum cellulase activity (7.51 U/L/OD600 endoglucanase, 1.302 U/L/OD600 β-glucosidase) was attained when grown in media containing yeast extract, peptone and oat bran. To our knowledge, this is the first study to quantify the cellulolytic and xylanolytic enzyme activities of a Zalaria spp., which provides key insight into the availability of unexplored cellulolytic enzymes that could inform the design of organisms engineered for consolidated bioprocessing.

Insight into the eminent biotechnological applications of xylanolytic enzymes for sustainable bioprocessing

A study was conducted using 144 Hy-Line Brown laying hens (22 weeks old) to assess the impact of exogenous enzymes on energy utilisation and ileal nutrient digestibility in diets containing 300 g/kg wheat distillers’ dried grains with solubles (DDGS). A basal diet was prepared and divided into eight treatments: a control (C) and diets supplemented with 2000 units/kg xylanase (XYL), 500 units/kg phytase (PHY), and 4000 units/kg protease (PRO), individually and in combination. The diets were fed for nine days to six coops, each housing three birds. Feed intake, weight gain, and feed conversion ratio were recorded. The feed and excreta were analysed for gross energy, and the apparent metabolisable energy (AME) was calculated. On the final day, the birds were euthanised, and ileal digesta were collected, freeze-dried, and analysed for the digestibility coefficients of dry matter (DM), nitrogen, fat, and neutral detergent fibres (NDFs). XYL supplementation improved (p = 0.035) dietary AME but did not affect (p &gt; 0.05) DM, nitrogen, fat, or NDF digestibility. No significant effects (p &gt; 0.05) were observed for PHY or PRO, and no interactions (p &gt; 0.05) were found between enzyme combinations. Substrates in experimental diets involving various enzyme combinations should be given careful consideration.

Myceliophthora thermophila is a thermophilic fungus, known to produce industrially important enzymes in biorefineries. The mechanism underlying cellulase and xylanase expression in filamentous fungi is a complex regulatory network controlled by numerous transcription factors (TFs). These TFs in M. thermophila remain unclear. Here, we identified and characterised a novel cellulase and xylanase regulator MtFKH1 in M. thermophila through comparative transcriptomic and genetic analyses. Five of the eight potential TFs, which showed differential expression levels when grown on Avicel and glucose, were successfully deleted using the newly designed CRISPR/Cas9 system. This system identified the forkhead TF MtFKH1. The disruption of Mtfkh1 elevated the cellulolytic and xylanolytic enzyme activities, whereas the overexpression of Mtfkh1 led to considerable decrease in cellulase and xylanase production in M. thermophila cultivated on Avicel. The loss of Mtfkh1 also exhibited an impairment in sporulation in M. thermophila. Real-time quantitative reverse transcription PCR (RT-qPCR) and the electrophoretic mobility shift assays (EMSAs) demonstrated that MtFKH1 regulates the gene expression and specifically bind to the promoter regions of genes encoding β-glucosidase (bgl1/MYCTH_66804), cellobiohydrolase (cbh1/MYCTH_109566), and xylanase (xyn1/MYCTH_112050), respectively. Furthermore, DNase I footprinting analysis identified binding motif of MtFKH1 in the upstream region of Mtbgl1, with strongest binding affinity. Finally, transcriptomic profiling and Gene Ontology (GO) enrichment analyses of Mtfkh1 deletion mutant revealed that the regulon of MtFKH1 were significantly prevalent in hydrolase activity (acting on glycosyl bonds), polysaccharide binding, and carbohydrate metabolic process functional categories. These findings expand our knowledge on how forkhead transcription factor regulates lignocellulose degradation and provide a novel target for engineering of fungal cell factories with the hyperproduction of cellulase and xylanase.