Search Results (36)

Modern agriculture generates large amounts of straw, thus posing significant residue management challenges. In this context, traditional disposal methods such as residue incineration can cause severe environmental harm. From a circular economy perspective, rice and wheat straw are valuable lignocellulosic resources from which high-value bioproducts can be derived, including xylooligosaccharides (XOS). Efficient conversion of this biomass depends on the enzymatic degradation of xylan, the main hemicellulose in cereal straw. In this study, four GH10 endoxylanases were evaluated, of which X11 and its hybrid variant X11C2 showed the best performance, particularly at pH 9.0. X11 showed robustness under harsh conditions and a tendency to release short sugars such as xylose and xylobiose. Both rice and wheat straw exhibited partial saccharification, but wheat straw released higher amounts of soluble sugars, indicating a higher susceptibility to enzymatic hydrolysis. Given the growing interest in XOS as prebiotics with multiple health benefits, the enzymatic hydrolysis of low-cost agricultural residues—supported by appropriate pretreatment—represents a promising and sustainable strategy for XOS production. Full article

Wet coffee pulp residues (WCPRs) are typically underutilized, and their accumulation increases alongside coffee production, generating significant environmental impacts. This study proposes a sustainable valorization approach through hydrothermal treatment followed by membrane filtration for the production of xylooligosaccharides (XOSs). Extractive-free WCPR contained 35.4% structural carbohydrates (20.4% cellulose and 15.0% hemicellulose) and 27.0% lignin. Hydrothermal treatments (180 °C, 3 °C min⁻¹, 15–60 min) were performed with and without citric acid as an organic catalyst. The acid-assisted treatment (T4) enhanced hemicellulose depolymerization and xylose release (16 g·kg⁻¹ dry biomass), whereas milder, non-acidic conditions (T3) promoted the selective formation and recovery of short-chain XOS, reaching cumulative biomass-normalized yields of up to 14 g·kg⁻¹ of xylobiose (X2) and 9 g·kg⁻¹ of xylotriose (X3). Subsequent membrane processing (UF–DF–NF) enabled progressive purification and enrichment of XOS fractions. Diafiltration was identified as the main step governing XOS enrichment, whereas nanofiltration primarily refined separation by directing monomeric sugars to the permeate rather than substantially increasing XOS yields. Additionally, Multiple Factor Analysis (MFA) integrated process and compositional variables, explaining 79.6% of the total variance. Dimension 1 represented process intensity and xylose transport, while Dimension 2 reflected molecular-weight-driven XOS fractionation. The acid-assisted process (T4) exhibited a distinct multivariate signature, characterized by enhanced carbohydrate mobilization and improved XOS recovery with reduced dependence on dilution. Overall, coupling hydrothermal pretreatment with membrane fractionation proved to be an efficient, and environmentally friendly strategy for coffee by-product valorization, consistent with hemicellulose-first biorefinery models and the principles of the circular bioeconomy. Full article

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. Full article

Xylooligosaccharides (XOS) are functional oligosaccharides with recognized prebiotic properties and growing industrial relevance, typically obtained through enzymatic depolymerization of xylan-rich lignocellulosic substrates. In this study, a recombinant endo-β-1,4-xylanase (XynA) from Clostridium acetobutylicum was employed for XOS production. The xynA gene was cloned into the expression vector pET-21a(+) and heterologously expressed in Escherichia coli BL21(DE3) under induction with isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant protein, with an estimated molecular mass of 37.5 kDa, was verified by SDS-PAGE and Western blot analysis. Functional characterization via thin-layer chromatography revealed that XynA efficiently hydrolyzed beechwood xylan and rye arabinoxylan, predominantly yielding xylobiose. Additionally, the enzyme catalyzed the conversion of xylotriose into xylobiose and trace amounts of xylose. Notably, XynA demonstrated hydrolytic activity against autohydrolysed and alkali-pretreated coconut husk biomass, facilitating the release of XOS. These results underscore the potential of C. acetobutylicum XynA as a biocatalyst for the valorization of lignocellulosic residues into high-value oligosaccharides. Full article

Plant cell wall-derived oligosaccharides, such as xylo-oligosaccharides (XOS), serve as key signaling molecules regulating plant growth and immunity. The bioactivity of XOS is closely tied to their degree of polymerization (DP), yet the molecular mechanisms underlying DP-specific effects remain poorly understood. Here, we investigated the transcriptional and phenotypic responses of lettuce (Lactuca sativa) to foliar application of four high-purity XOS variants: xylobiose (XOSY, DP2), xylotriose (XOSB, DP3), xylotetraose (XOSD, DP4), and xylopentose (XOSW, DP5). Phenotypic analyses revealed that high-DP XOS (XOSD and XOSW) significantly enhanced aboveground biomass and root system development, with XOSD showing the most pronounced effects, including a 31.74% increase in leaf area and a 20.71% increase in aboveground biomass. Transcriptomic profiling identified extensive transcriptional reprogramming across treatments, with XOSD eliciting the highest number of differentially expressed genes (DEGs). Functional enrichment analyses indicated that XOSD and XOSW upregulated genes involved in plant hormone signaling, starch and sucrose metabolism, and cell wall biosynthesis, while downregulating photosynthesis-related genes. Notably, MapMan and KEGG pathway analyses revealed that XOSD significantly activated biotic stress-related pathways, including MAPK signaling, β-1,3-glucanase activity, and PR protein pathways. In contrast, XOSY treatment primarily upregulated genes linked to basal immunity, highlighting distinct mechanisms employed by low- and high-DP XOS. These findings demonstrate that XOS with varying DP differentially modulate growth- and immunity-related processes in lettuce. High-DP XOS, particularly XOSD, not only promote plant biomass accumulation but also enhance immune responses, highlighting their potential as biostimulants for sustainable agriculture. This study provides a molecular framework for understanding the DP-specific bioactivity of XOS and their dual role in optimizing plant growth and defense. Full article

The filamentous fungus Penicillium verruculosum (anamorph Talaromyces verruculosus) has been shown to be an efficient producer of secreted cellulases, used in biorefinery processes. Understanding the mechanisms of regulation of cellulase gene expression in the fungus P. verruculosum is a current task in industrial biotechnology, since it allows for targeted changes in the composition of the complex secreted by the fungus. Expression of cellulase genes in fungi is regulated mainly at the level of transcription via pathway-specific transcription factors (TF), the majority of which belong to the Zn(II)2Cys6 family of zinc binuclear cluster proteins. Transcriptional regulation of cellulase genes may have a species-specific pattern and involves several transcription factors. In this study, we used a qPCR method and transcriptome analysis to investigate the effect of knockouts and constitutive expression of genes encoding homologues of the regulatory factors XlnR and ClrB from P. verruculosum on the transcription of cbh1, egl2, and bgl1 genes, encoding three key cellulases, cellobiohydrolase, endoglucanase, and β-glucosidase, in the presence of various inducers. We have shown that the transcription factor XlnR of the filamentous fungus P. verruculosum is strictly responsible for the transcription of the main cellulolytic genes (cbh1, egl2, and bgl1) in the presence of xylose and xylobiose, but not in the presence of cellobiose. ClrB/Clr-2, a homologue from P. verruculosum, does not represent the main transcription factor regulating transcription of cellulolytic genes in the presence of selected inducers, unlike in the cases of Aspergillus nidulans, Aspergillus niger, and Penicillium oxalicum; apparently, it has a different function in fungi from the genus Talaromyces. We have also shown that constitutive expression of the transcription factor XlnR resulted in 3.5- and 2-fold increases in the activity of xylanase and β-glucosidase in a B1-XlnR enzyme preparation, respectively. In a practical sense, the obtained result can be used for the production of enzyme preparations based on the P. verruculosum B1-XlnR strain used for the bioconversion of renewable cellulose-containing raw materials into technical sugars. Full article

This study investigated the characterization of a novel multifunctional enzyme, RuXyn394, derived from the metagenome of beef cattle rumen, and its impact on the in vitro microbial fermentation of wheat straw. RuXyn394, a member of the glycosyl hydrolase 11 family, displayed optimal activity under diverse pH and temperature conditions: xylanase at pH 5.5 and 50 °C, acetyl esterase at pH 6.5 and 60 °C, exoglucanase at pH 7.0 and 50 °C, and endoglucanase at pH 6.0 and 50 °C. The enzyme’s xylanase, endoglucanase, and exoglucanase activities exhibited remarkable pH stability across the range of pH 3–8 and maintained a relatively stable performance at temperatures from 20 to 50 °C, 20 to 60 °C, and 20 to 70 °C, respectively. The xylanase function, with the highest k_cat/K_m ratio, was identified as the predominant activity of RuXyn394. The enzyme’s various functions responded uniquely to metal ions; notably, the addition of 5 mM K⁺ significantly boosted the activities of xylanase, exoglucanase, and endoglucanase by 55.5%, 53.5%, and 16.4%, respectively, without affecting its acetyl esterase activity. Over the course of three time points (30 min, 60 min, 120 min), the degradation products of wheat straw xylan, including xylopentaose, xylotetraose, xylotriose, xylobiose, xylose, and total xylooligosaccharides, constituted an average of 18.4%, 33.7%, 20.6%, 22.9%, 4.3%, and 95.7% of the total products, respectively. RuXyn394 effectively hydrolyzed wheat straw, resulting in augmented volatile fatty acid production and ammonia-N levels during in vitro microbial fermentation. These findings indicate the potential of RuXyn394 as a novel and highly efficient enzyme preparation, offering promising prospects for the valorization of wheat straw, an agricultural by-product, in ruminant diets. Full article

This study identified a salt-tolerant GH11 xylanase, Xyn^st, 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 Xyn^st 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 Xyn^st in the presence of 2 M NaCl at 65 °C. Kinetic parameters indicated they have the highest affinity for beechwood xylan (K_m = 0.30 mg mL⁻¹ for Xyn^st and 0.18 mg mL⁻¹ for W6F/Q7H), and W6F/Q7H has very high catalytic efficiency (K_cat/K_m = 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 Xyn^st did. The conversion efficiencies of Xyn^st and W6F/Q7H on all tested xylans exceeded 20%, suggesting potential applications in the agricultural and food industries. Full article

A gene encoding a polysaccharide-degrading enzyme was cloned from the genome of the bacterium Nocardiopsis halotolerans. Analysis of the amino acid sequence of the protein showed the presence of the catalytic domain of the endo-1,4-β-xylanases of the GH11 family. The gene was amplified by PCR and ligated into the pPic9m vector. A recombinant producer based on Pichia pastoria was obtained. The production of the enzyme, which we called NhX1, was carried out in a 10 L fermenter. Enzyme production was 10.4 g/L with an activity of 927 U/mL. Purification of NhX1 was carried out using Ni-NTA affinity chromatography. The purified enzyme catalyzed the hydrolysis of xylan but not other polysaccharides. Endo-1,4-β-xylanase NhX1 showed maximum activity and stability at pH 6.0–7.0. The enzyme showed high thermal stability, remaining active at 90 °C for 20 min. With beechwood xylan, the enzyme showed K_m 2.16 mg/mL and V_max 96.3 U/mg. The products of xylan hydrolysis under the action of NhX1 were xylobiose, xylotriose, xylopentaose, and xylohexaose. Endo-1,4-β-xylanase NhX1 effectively saccharified xylan-containing products used for the production of animal feed. The xylanase described herein is a thermostable enzyme with biotechnological potential produced in large quantities by P. pastoria. Full article

Xylanases, key enzymes for hydrolyzing xylan, have diverse industrial applications. The bioprocessing of agricultural byproducts to produce xylanase through fermentation approaches is gaining importance due to its significant potential to reduce enzyme production costs. In this work, the productivity of Streptomyces thermocarboxydus TKU045 xylanase was enhanced through liquid fermentation employing wheat bran as the sole carbon source. The maximum xylanase activity (25.314 ± 1.635 U/mL) was obtained using the following optima factors: 2% (w/v) wheat bran, 1.4% (w/v) KNO₃, an initial pH of 9.8, an incubation temperature of 37.3 °C, and an incubation time of 2.2 days. Xylanase (Xyn_TKU045) of 43 kDa molecular weight was isolated from the culture supernatant and was biochemically characterized. Analysis through liquid chromatography with tandem mass spectrometry revealed a maximum amino acid identity of 19% with an endo-1,4-β-xylanase produced by Streptomyces lividans. Xyn_TKU045 exhibited optimal activity at pH 6, with remarkable stability within the pH range of 6.0 to 8.0. The enzyme demonstrated maximum efficiency at 60 °C and considerable stability at ≤70 °C. Mg²⁺, Mn²⁺, Ba²⁺, Ca²⁺, 2-mercaptoethanol, Tween 20, Tween 40, and Triton X-100 positively influenced Xyn_TKU045, while Zn²⁺, Fe²⁺, Fe³⁺, Cu²⁺, and sodium dodecyl sulfate exhibited adverse impact. The kinetic properties of Xyn_TKU045 were a K_m of 0.628 mg/mL, a k_cat of 75.075 s⁻¹ and a k_cat/K_m of 119.617 mL mg⁻¹s⁻¹. Finally, Xyn_TKU045 could effectively catalyze birchwood xylan into xylotriose and xylobiose as the major products. Full article

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. Full article

In this study, the α-glucosidase (maltase-glucoamylase: MGAM) and α-amylase inhibitory properties elicited by xylooligosaccharides (XOSs) prepared from dulse xylan were analysed as a potential mechanism to control postprandial hyperglycaemia for type-2 diabetes prevention and treatment. Xylan was purified from red alga dulse powder and used for enzymatic hydrolysis using Sucrase X to produce XOSs. Fractionation of XOSs produced xylobiose (X2), β-(1→3)-xylosyl xylobiose (DX3), xylotriose (X3), β-(1→3)-xylosyl-xylotriose (DX4), and a dulse XOS mixture with n ≥ 4 xylose units (DXM). The different fractions exhibited moderate MGAM (IC₅₀ = 11.41–23.44 mg/mL) and α-amylase (IC₅₀ = 18.07–53.04 mg/mL) inhibitory activity, which was lower than that of acarbose. Kinetics studies revealed that XOSs bound to the active site of carbohydrate digestive enzymes, limiting access to the substrate by competitive inhibition. A molecular docking analysis of XOSs with MGAM and α-amylase clearly showed moderate strength of interactions, both hydrogen bonds and non-bonded contacts, at the active site of the enzymes. Overall, XOSs from dulse could prevent postprandial hyperglycaemia as functional food by a usual and continuous consumption. Full article

Poultry production faces several challenges, with feed efficiency being the main factor that can be influenced through the use of different nutritional strategies. Xylooligosaccharides (XOS) are functional feed additives that are attracting growing commercial interest due to their excellent ability to modulate the composition of the gut microbiota. The aim of the study was to apply crude and purified fungal xylanases, from Trichoderma harzianum, as well as a recombinant glycoside hydrolase family 10 xylanase, derived from Geobacillus stearothermophilus T6, as additives to locally produced chicken feeds. A Box–Behnken Design (BBD) was used to optimize the reducing sugar yield. Response surface methodology (RSM) revealed that reducing sugars were higher (8.05 mg/mL, 2.81 mg/mL and 2.98 mg/mL) for the starter feed treated with each of the three enzymes compared to the treatment with grower feed (3.11 mg/mL, 2.41 mg/mL and 2.62 mg/mL). The hydrolysis products were analysed by thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC) analysis and showed that the enzymes hydrolysed the chicken feeds, producing a range of monosaccharides (arabinose, mannose, glucose, and galactose) and XOS, with xylobiose being the predominant XOS. These results show promising data for future applications as additives to poultry feeds. Full article

The global development of the bioeconomy is impossible without technologies for comprehensive processing of plant renewable resources. The use of proven pretreatment technologies raises the possibility of the industrial implementation of the enzymatic conversion of polysaccharides from lignocellulose considering the process’s complexity. For instance, a well-tuned kraft pulping produces a substrate easily degraded by cellulases and hemicelulases. Enzymatic hydrolysis of bleached hardwood kraft pulp was carried out using an enzyme complex of endoglucanases, cellobiohydrolases, β-glucosidases, and xylanases produced by recombinant strains of Penicillium verruculosum at a 10 FPU/g mixture rate and a 10% substrate concentration. As a result of biocatalysis, the following products were obtained: sugar solution, mainly glucose, xylobiose, xylose, as well as other minor reducing sugars; a modified complex based on cellulose and xylan. The composition of the biomodified kraft pulp was determined by HPLC. The method for determining the crystallinity on an X-ray diffractometer was used to characterize the properties. The article shows the possibility of producing biomodified cellulose cryogels by amorphization with concentrated 85% H₃PO₄ followed by precipitation with water and supercritical drying. The analysis of the enzymatic hydrolysate composition revealed the predominance of glucose (55–67%) among the reducing sugars with a maximum content in the solution up to 6% after 72 h. The properties and structure of the modified kraft pulp were shown to change during biocatalysis; in particular, the crystallinity increased by 5% after 3 h of enzymatic hydrolysis. We obtained cryogels based on the initial and biomodified kraft pulp with conversion rates of 35, 50, and 70%. The properties of these cryogels are not inferior to those of cryogels based on industrial microcrystalline cellulose, as confirmed by the specific surface area, degree of swelling, porosity, and SEM images. Thus, kraft pulp enzymatic hydrolysis offers prospects not only for producing sugar-rich hydrolysates for microbiological synthesis, but also cellulose powders and cryogels with specified properties. Full article

Resveratrol is a natural polyphenol with antioxidant activity and numerous health benefits. However, in vivo application of this compound is still a challenge due to its poor aqueous solubility and rapid metabolism, which leads to an extremely low bioavailability in the target tissues. In this work, rXynSOS-E236G glycosynthase, designed from a GH10 endoxylanase of the fungus Talaromyces amestolkiae, was used to glycosylate resveratrol by using xylobiosyl-fluoride as a sugar donor. The major product from this reaction was identified by NMR as 3-O-ꞵ-d-xylobiosyl resveratrol, together with other glycosides produced in a lower amount as 4′-O-ꞵ-d-xylobiosyl resveratrol and 3-O-ꞵ-d-xylotetraosyl resveratrol. The application of response surface methodology made it possible to optimize the reaction, producing 35% of 3-O-ꞵ-d-xylobiosyl resveratrol. Since other minor glycosides are obtained in addition to this compound, the transformation of the phenolic substrate amounted to 70%. Xylobiosylation decreased the antioxidant capacity of resveratrol by 2.21-fold, but, in return, produced a staggering 4,866-fold improvement in solubility, facilitating the delivery of large amounts of the molecule and its transit to the colon. A preliminary study has also shown that the colonic microbiota is capable of releasing resveratrol from 3-O-ꞵ-d-xylobiosyl resveratrol. These results support the potential of mutagenic variants of glycosyl hydrolases to synthesize highly soluble resveratrol glycosides, which could, in turn, improve the bioavailability and bioactive properties of this polyphenol. Full article