Surface-enhanced Raman spectroscopy for the characterization of xylanases enzyme.

Xylanases are essential hydrolytic enzymes which break down the plant cell wall polysaccharide, xylan composed of D-xylose monomers. Surface-enhanced Raman Spectroscopy (SERS) was utilized for the characterization of interaction of xylanases with xylan at varying concentrations. The study focuses on the application of SERS for the characterization of enzymatic activity of xylanases causing hydrolysis of Xylan substrate with increase in its concentration which is substrate for this enzyme in the range of 0.2% to 1.0%. SERS differentiating features are identified which can be associated with xylanases treated with different concentrations of xylan. SERS measurements were performed using silver nanoparticles as SERS substrate to amplify Raman signal intensity for the characterization of xylan treated with xylanases. Principal Component Analysis (PCA) and Partial Least Square Discriminant Analysis (PLS-DA) were applied to analyze the spectral data to analyze differentiation between the SERS spectra of different samples. Mean SERS spectra revealed significant differences in spectral features particularly related to carbohydrate skeletal mode and O-C-O and C-C-C ring deformations. PCA scatter plot effectively differentiates data sets, demonstrating SERS ability to distinguish treated xylanases samples and the PC-loadings plot highlights the variables responsible for differentiation. PLS-DA was employed as a quantitative classification model for treated xylanase enzymes with increasing concentrations of xylan. The values of sensitivity, specificity, and accuracy were found to be 0.98%, 0.99%, and 100% respectively. Moreover, the AUC value was found to be 0.9947 which signifies the excellent performance of PLS-DA model. SERS combined with multivariate techniques, effectively characterized and differentiated xylanase samples as a result of interaction with different concentrations of the Xylan substrate. The identified SERS features can help to characterize xylanases treated with various concentrations of xylan with promising applications in the bio-processing and biotechnology industries.

High-efficiency conversion of corn bran to ethanol at 150 L scale.

Fractionated corn bran was processed to maximize ethanol production from starch, cellulose, and xylan. After various bench-scale experiments, an optimized process with dilute acid pretreatment (1.5 % w/w H2SO4) at 90 °C for 60 min was utilized followed by enzymatic hydrolysis using cellulase and hemicellulase for 48 hr. After simultaneous saccharification (regarding starch) and fermentation at 150 L using an engineered yeast, which consumes both glucose and xylose to make ethanol, the 86 % total sugar conversion yield was achieved, including conversions of 95 % for starch, 77 % for cellulose and 77 % for xylan. Also, an accurate mass balance was formulated for ethanol-producing carbohydrates including starch, cellulose, and xylan from feedstock to final ethanol. A highly efficient process of converting corn fiber to ethanol was successfully scaled up to 150 L.

Genome-wide analysis and identification of the TBL gene family in Eucalyptus grandis.

The TRICHOME BIREFRINGENCE-LIKE (TBL) gene encodes a class of proteins related to xylan acetylation, which has been shown to play an important role in plant response to environmental stresses. This gene family has been meticulously investigated in Arabidopsis thaliana, whereas there have been no related reports in Eucalyptus grandis. In this study, we identified 49 TBL genes in E. grandis. A conserved amino acid motif was identified, which plays an important role in the execution of the function of TBL gene family members. The expression of TBL genes was generally upregulated in jasmonic acid-treated experiments, whereas it has been found that jasmonic acid activates the expression of genes involved in the defense functions of the plant body, suggesting that TBL genes play an important function in the response of the plant to stress. The principle of the action of TBL genes is supported by the finding that the xylan acetylation process increases the rigidity of the cell wall of the plant body and thus improves the plant's resistance to stress. The results of this study provide new information about the TBL gene family in E. grandis and will help in the study of the evolution, inheritance, and function of TBL genes in E. grandis, while confirming their functions.

Enrichment of Aquatic Xylan-Degrading Microbial Communities.

The transition towards a sustainable society involves the utilization of lignocellulosic biomass as a renewable feedstock for materials, fuel, and base chemicals. Lignocellulose consists of cellulose, hemicellulose, and lignin, forming a complex, recalcitrant matrix where efficient enzymatic saccharification is pivotal for accessing its valuable components. This study investigated microbial communities from brackish Lauwersmeer Lake, in The Netherlands, as a potential source of xylan-degrading enzymes. Environmental sediment samples were enriched with wheat arabinoxylan (WAX) and beechwood glucuronoxylan (BEX), with enrichment on WAX showing higher bacterial growth and complete xylan degradation compared to BEX. Metagenomic sequencing revealed communities consisting almost entirely of bacteria (>99%) and substantial shifts in composition during the enrichment. The first generation of seven-day enrichments on both xylans led to a high accumulation of Gammaproteobacteria (49% WAX, 84% BEX), which were largely replaced by Alphaproteobacteria (42% WAX, 69% BEX) in the fourth generation. Analysis of the protein function within the sequenced genomes showed elevated levels of genes associated with the carbohydrate catabolic process, specifically targeting arabinose, xylose, and xylan, indicating an adaptation to the primary monosaccharides present in the carbon source. The data open up the possibility of discovering novel xylan-degrading proteins from other sources aside from the thoroughly studied Bacteroidota.

Plant Cell Wall Polysaccharide O-Acetyltransferases.

Plant cell walls are largely composed of polysaccharide polymers, including cellulose, hemicelluloses (xyloglucan, xylan, mannan, and mixed-linkage ß-1,3/1,4-glucan), and pectins. Among these cell wall polysaccharides, xyloglucan, xylan, mannan, and pectins are often O-acetylated, and polysaccharide O-acetylation plays important roles in cell wall assembly and disease resistance. Genetic and biochemical analyses have implicated the involvement of three groups of proteins in plant cell wall polysaccharide O-acetylation: trichome birefringence-like (TBL)/domain of unknown function 231 (DUF231), reduced wall acetylation (RWA), and altered xyloglucan 9 (AXY9). Although the exact roles of RWAs and AXY9 are yet to be identified, members of the TBL/DUF231 family have been found to be O-acetyltransferases responsible for the O-acetylation of xyloglucan, xylan, mannan, and pectins. Here, we provide a comprehensive overview of the occurrence of O-acetylated cell wall polysaccharides, the biochemical properties, structural features, and evolution of cell wall polysaccharide O-acetyltransferases, and the potential biotechnological applications of manipulations of cell wall polysaccharide acetylation. Further in-depth studies of the biochemical mechanisms of cell wall polysaccharide O-acetylation will not only enrich our understanding of cell wall biology, but also have important implications in engineering plants with increased disease resistance and reduced recalcitrance for biofuel production.

Polysaccharides Analysis of Cell Wall Using Carbohydrate Gel Electrophoresis.

The plant cell wall is rich in polysaccharides with high heterogeneity. Investigating the composition and structure of cell wall polysaccharides is crucial for understanding the functionalities of plant cell walls. Carbohydrate electrophoresis is a sensitive and rapid method to analyze polysaccharides qualitatively and quantitatively. The process includes digesting the polysaccharides with appropriate cleavage enzymes, labeling the reducing ends of the released oligosaccharides with a highly charged fluorophore, and separating the labeled oligosaccharides in a polyacrylamide gel via high-voltage electrophoresis. The generated fluorescence can be calculated as compared to that of oligosaccharide standards. Therefore, this is a convenient method for polysaccharide characterization that can be performed in most laboratories. Here, we introduce the detailed operational steps and precautions, which are helpful for researchers to quickly obtain the structural information of polysaccharides.

Identification of glycosyltransferases mediating 2-O-arabinopyranosyl and 2-O-galactosyl substitutions of glucuronosyl side chains of xylan.

Xylan is one of the major hemicelluloses in plant cell walls and its xylosyl backbone is often decorated at O-2 with glucuronic acid (GlcA) and/or methylglucuronic acid (MeGlcA) residues. The GlcA/MeGlcA side chains may be further substituted with 2-O-arabinopyranose (Arap) or 2-O-galactopyranose (Gal) residues in some plant species, but the enzymes responsible for these substitutions remain unknown. During our endeavor to investigate the enzymatic activities of Arabidopsis MUR3-clade members of the GT47 glycosyltransferase family, we found that one of them was able to transfer Arap from UDP-Arap onto O-2 of GlcA side chains of xylan, and thus it was named xylan 2-O-arabinopyranosyltransferase 1 (AtXAPT1). The function of AtXAPT1 was verified in planta by its T-DNA knockout mutation showing a loss of the Arap substitution on xylan GlcA side chains. Further biochemical characterization of XAPT close homologs from other plant species demonstrated that while the poplar ones had the same catalytic activity as AtXAPT1, those from Eucalyptus, lemon-scented gum, sea apple, 'Ohi'a lehua, duckweed and purple yam were capable of catalyzing both 2-O-Arap and 2-O-Gal substitutions of xylan GlcA side chains albeit with differential activities. Sequential reactions with XAPTs and glucuronoxylan methyltransferase 3 (GXM3) showed that XAPTs acted poorly on MeGlcA side chains, whereas GXM3 could efficiently methylate arabinosylated or galactosylated GlcA side chains of xylan. Furthermore, molecular docking and site-directed mutagenesis analyses of Eucalyptus XAPT1 revealed critical roles of several amino acid residues at the putative active site in its activity. Together, these findings establish that XAPTs residing in the MUR3 clade of family GT47 are responsible for 2-O-arabinopyranosylation and 2-O-galactosylation of GlcA side chains of xylan.

Novel approach on the evaluation of enzyme-aided alkaline extraction of polysaccharide from Hordeum vulgare husk and molecular insight on the multifunctional scaffold.

Hordeum vulgare husk, a cereal grain, is rich in dietary fiber and prebiotics beneficial for the gut microbiota and host organism. This study investigates the effects of barley husk-derived water-soluble xylan (BH-WSX) on gut homeostasis and the microbiome. We enzymatically extracted BH-WSX and evaluated its prebiotic and antioxidant properties. A 40.0 % (w/v) xylan yield was achieved, with the extracted xylan having a molecular mass of 212.0885 and a xylose to glucuronic acid molar ratio of 6:1. Specialized optical rotation research indicated that the isolated xylan is composed of monomeric sugars such as D-xylose, glucose, and arabinose. Fourier Transform Infrared (FTIR) spectroscopy revealed that the xylan comprises ß (1 â†’ 4) linked xylose units, randomly substituted with glucose residues, α-arabinofuranose, and acetyl groups. Nuclear Magnetic Resonance (NMR) analysis showed that the barley husk extract's backbone is substituted with 4-O-methyl glucuronic acid at the O2 position. Thermogravimetric analysis indicated that WSX exhibits a single sharp peak at 266 °C on the Differential Thermal Gravimetry (DTG) curve. Furthermore, a combination of in vitro, in vivo models, and molecular docking analysis elaborated on the anti-adhesion properties of BH-WSX. This study presents a novel approach to utilizing barley husk as an efficient source of functional polysaccharides for food-related industrial applications.

Aspergillus labruscus ITAL 22.223 xylanase - immobilization and application for the obtainment of corncob xylan targeting xylitol production.

Corncob is an agro-residue rich in lignocellulosic material that can be used for the xylitol production, through its enzymatic conversion obtaining fermentable sugars and their subsequent fermentation. In light of the above, this study targeted the immobilization of Aspergillus labruscus xylanase and the use of the derivative to hydrolyze the corncob xylan for the obtainment of xylose, and its subsequent use for the production of xylitol. The extracellular xylanase was immobilized using different supports (sodium alginate, DEAE-Cellulose, DEAE-Sephadex and CM-Sephadex). Among all supports used, the best results were obtained with the DEAE-Cellulose derivative showing an efficiency of immobilization of 97-99%, yield of 93-95% and recovered activity of 81-100%. The sodium alginate derivative showed 3 cycles of reuse, with drop in activity of about 65% in the 3rd cycle using both CaCl2 and MnCl2 as crosslinkers. The best enzymatic activity for the DEAE-Cellulose derivative was observed at 55ºC and pH 5.0. This derivative presented reuse of 10 cycles using commercial xylan as substrate, and 4 cycles using corncob xylan. This derivative was used in an enzymatic reactor to hydrolyze corncob xylan, obtaining 2.7 mg/mL of xylose after 48 h of operation under optimal condition of temperature and pH. The xylose obtained from the corncob was fermented by Candida tropicalis for 96 h with consumption of 60%. The HPLC analyses indicated a production of 1.02 mg/mL of xylitol with 48 h of fermentation. In conclusion, this is the first report on the immobilization of the A. labrucus xylanase as an alternative for the obtainment of xylose from corncob xylan, and the subsequent production of xylitol.

Quantitative analysis of intermolecular forces in cellulose microfibrils and hemicellulose with AFM nano-colloidal probes.

It is an interesting research topic to study the interfacial interactions between hemicellulose and cellulose, specifically how hemicellulose's structure affects its binding to cellulose nanofibers. Our research proposes that dispersion interaction play an important role in this interfacial interaction, more so than electrostatic forces when considering the adherence of cellulose to xylan. To quantify these interactions, the Atomic Force Microscope (AFM) colloidal probe technique is applied to measure the intermolecular forces between cellulose nanofibers, which are attached to the probe and xylan. These measured forces are then analyzed in relation to the length, diameter and functional groups of the nanocellulose, as well as the molecular weight and side chains of the xylan. Moreover, the predominance of dispersion forces by contrasting the adhesive forces before and after the grafting of a large nonpolar group onto xylan. This modification significantly reduces contact between the cellulose and xylan backbone, thereby markedly diminishing the dispersion interactions. Parallel to the AFM experiments, molecular dynamics (MD) simulations corroborate the experimental results and support our hypotheses. Collectively, these findings contribute to a deeper understanding of polysaccharide interactions within lignocellulose.

Fibrobacter sp. HC4, a newly isolated strain, demonstrates a high cellulolytic activity as revealed by enzymatic measurements and in vitro assay.

Despite their low quantity and abundance, the cellulolytic bacteria that inhabit the equine large intestine are vital to their host, as they enable the crucial use of forage-based diets. Fibrobacter succinogenes is one of the most important intestinal cellulolytic bacteria. In this study, Fibrobacter sp. HC4, one cellulolytic strain newly isolated from the horse cecum, was characterized for its ability to utilize plant cell wall fibers. Fibrobacter sp. HC4 consumed only cellulose, cellobiose, and glucose and produced succinate and acetate in equal amounts. Among genes coding for CAZymes, 26% of the detected glycoside hydrolases (GHs) were involved in cellulolysis. These cellulases belong to the GH5, GH8, GH9, GH44, GH45, and GH51 families. Both carboxymethyl cellulase and xylanase activities of Fibrobacter sp. HC4 were detected using the Congo red method and were higher than those of F. succinogenes S85, the type strain. The in vitro addition of Fibrobacter sp. HC4 to a fecal microbial ecosystem of horses with large intestinal acidosis significantly enhanced fibrolytic activity as measured by the increase in gas and volatile fatty acids production during the first 48 h. According to this, the pH decreased and the disappearance of dry matter increased at a faster rate with Fibrobacter sp. HC4. Our data suggest a high specialization of the new strain in cellulose degradation. Such a strain could be of interest for future exploitation of its probiotic potential, which needs to be further determined by in vivo studies.IMPORTANCECellulose is the most abundant of plant cell wall fiber and can only be degraded by the large intestine microbiota, resulting in the production of volatile fatty acids that are essential for the host nutrition and health. Consequently, cellulolytic bacteria are of major importance to herbivores. However, these bacteria are challenged by various factors, such as high starch diets, which acidify the ecosystem and reduce their numbers and activity. This can lead to an imbalance in the gut microbiota and digestive problems such as colic, a major cause of mortality in horses. In this work, we characterized a newly isolated cellulolytic strain, Fibrobacter sp. HC4, from the equine intestinal microbiota. Due to its high cellulolytic capacity, reintroduction of this strain into an equine fecal ecosystem stimulates hay fermentation in vitro. Isolating and describing cellulolytic bacteria is a prerequisite for using them as probiotics to restore intestinal balance.

Cryptococcus laurentii: a wild yeast for xylanase production from agricultural by-products.

The development of technologies that allow the production of enzymes at a competitive cost is of great importance for several biotechnological applications, and the use of agro-industrial by-products is an excellent alternative to minimize costs and reduce environmental impacts. This study aimed to produce endo-xylanases using agro-industrial substrates rich in hemicellulose as sources of xylan in culture media. For this purpose, the yeast Cryptococcus laurentti and five lignocellulosic materials (defatted rice bran, rice husk, corn cob, oat husks, and soybean tegument), with and without pretreatment, were used as a source of xylan for enzyme production. To insert the by-products in the culture medium, they were dried and treated (if applicable) with 4% (w.v-1) NaOH and then added in a concentration of 2% (w.v-1). The cultures were agitated for 96 h, and the aliquots were removed to determine the enzymatic activities. Among the by-products studied, the maximum activity (8.7 U. mL-1 at pH 7.3) was obtained where rice bran was used. In contrast, corn cob was the by-product that resulted in lower enzyme production (1.6 U.mL-1). Thus, the defatted rice bran deserves special attention in front of the other by-products used since it provides the necessary substrate for producing endo-xylanases by yeast.

Carbohydrate-active enzymes involved in rice cell wall metabolism.

Plant cell walls are complex, multifunctional structures, built up of polysaccharides and proteins. The configuration and abundance of cell wall constituents determine cellular elongation and plant growth. The emphasis of this review is on rice, a staple crop with economic importance, serving as model for grasses/cereals. Recent advancements have contributed to a better understanding of the grass/cereal cell wall. This review brings together the current knowledge about the organisation and metabolism of the rice cell wall, and addresses gaps and missing information connected to the cell wall of rice and the enzymes involved. Several cell wall fractions, including cellulose, mixed-linkage glucans and glucuronoarabinoxylans, are well-understood in rice and other grasses/grains. Conversely, there are still open questions and missing links when it comes down to xyloglucans, glucomannans, pectin, lignin and arabinogalactan proteins. There is still a large and untapped potential to identify carbohydrate-active enzymes (CAZymes), to characterise their activity and to elucidate their involvement in the metabolism of the mentioned cell wall fractions. With this review, we demonstrate the current state and demarcate the research areas with potential for further investigations.

Characterization of a novel AA16 lytic polysaccharide monooxygenase from Thermothelomyces thermophilus and comparison of biochemical properties with an LPMO from AA9 family.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes which are categorized in the CAZy database under auxiliary activities families AA9-11, 13, 14-17. Secreted by various microorganisms, they play a crucial role in carbon recycling, particularly in fungal saprotrophs. LPMOs oxidize polysaccharides through monooxygenase/peroxygenase activities and exhibit peroxidase and oxidase activities, with variations among different families. AA16, a newly identified LPMO family, is noteworthy due to limited studies on its members, thus rendering the characterization of AA16 enzymes vital for addressing controversies around their functions. This study focused on heterologous expression and biochemical study of an AA16 LPMO from Thermothelomyces thermophilus (formerly known as Myceliophthora thermophila), namely MtLPMO16A. Substrate specificity evaluation of MtLPMO16A showed oxidative cleavage of hemicellulosic substrates and no activity on cellulose, accompanied by a strong oxidase activity. A comparative analysis with an LPMO from AA9 family explored correlations between these families, while MtLPMO16A was shown to boost the activity of some AA9 family LPMOs. The results offer new insights into the AA16 family's action mode and microbial hemicellulose decomposition mechanisms in nature.

Unveiling metabolic pathways of selected plant-derived glycans by Bifidobacterium pseudocatenulatum.

Bifidobacteria are commonly encountered members of the human gut microbiota that possess the enzymatic machinery necessary for the metabolism of certain plant-derived, complex carbohydrates. In the current study we describe differential growth profiles elicited by a panel of 21 newly isolated Bifidobacterium pseudocatenulatum strains on various plant-derived glycans. Using a combination of gene-trait matching and comparative genome analysis, we identified two distinct xylanases responsible for the degradation of xylan. Furthermore, three distinct extracellular α-amylases were shown to be involved in starch degradation by certain strains of B. pseudocatenulatum. Biochemical characterization showed that all three α-amylases can cleave the related substrates amylose, amylopectin, maltodextrin, glycogen and starch. The genes encoding these enzymes are variably found in the species B. pseudocatenulatum, therefore constituting a strain-specific adaptation to the gut environment as these glycans constitute common plant-derived carbohydrates present in the human diet. Overall, our study provides insights into the metabolism of these common dietary carbohydrates by a human-derived bifidobacterial species.

Evolution of glucuronoxylan side chain variability in vascular plants and the compensatory adaptations of cell wall-degrading hydrolases.

Polysaccharide structural complexity not only influences cell wall strength and extensibility but also hinders pathogenic and biotechnological attempts to saccharify the wall. In certain species and tissues, glucuronic acid side groups on xylan exhibit arabinopyranose or galactose decorations whose genetic and evolutionary basis is completely unknown, impeding efforts to understand their function and engineer wall digestibility. Genetics and polysaccharide profiling were used to identify the responsible loci in Arabidopsis and Eucalyptus from proposed candidates, while phylogenies uncovered a shared evolutionary origin. GH30-family endo-glucuronoxylanase activities were analysed by electrophoresis, and their differing specificities were rationalised by phylogeny and structural analysis. The newly identified xylan arabinopyranosyltransferases comprise an overlooked subfamily in the GT47-A family of Golgi glycosyltransferases, previously assumed to comprise mainly xyloglucan galactosyltransferases, highlighting an unanticipated adaptation of both donor and acceptor specificities. Further neofunctionalisation has produced a Myrtaceae-specific xylan galactosyltransferase. Simultaneously, GH30 endo-glucuronoxylanases have convergently adapted to overcome these decorations, suggesting a role for these structures in defence. The differential expression of glucuronoxylan-modifying genes across Eucalyptus tissues, however, hints at further functions. Our results demonstrate the rapid adaptability of biosynthetic and degradative carbohydrate-active enzyme activities, providing insight into plant-pathogen interactions and facilitating plant cell wall biotechnological utilisation.

Biochemical characterization of an esterase from Thermobifida fusca YX with acetyl xylan esterase activity.

BACKGROUND: Esterases (EC 3.1.1.X) are enzymes that catalyze the hydrolysis ester bonds. These enzymes have large potential for diverse applications in fine industries, particularly in pharmaceuticals, cosmetics, and bioethanol production. METHODS AND RESULTS: In this study, a gene encoding an esterase from Thermobifida fusca YX (TfEst) was successfully cloned, and its product was overexpressed in Escherichia coli and purified using affinity chromatography. The TfEst kinetic assay revealed catalytic efficiencies of 0.58 s-1 mM-1, 1.09 s-1 mM-1, and 0.062 s-1 mM-1 against p-Nitrophenyl acetate, p-Nitrophenyl butyrate, and 1-naphthyl acetate substrates, respectively. Furthermore, TfEst also exhibited activity in a pH range from 6.0 to 10.0, with maximum activity at pH 8.0. The enzyme demonstrated a half-life of 20 min at 70 °C. Notably, TfEst displayed acetyl xylan esterase activity as evidenced by the acetylated xylan assay. The structural prediction of TfEst using AlphaFold indicated that has an α/ß-hydrolase fold, which is consistent with other esterases. CONCLUSIONS: The enzyme stability over a broad pH range and its activity at elevated temperatures make it an appealing candidate for industrial processes. Overall, TfEst emerges as a promising enzymatic tool with significant implications for the advancement of biotechnology and biofuels industries.

Valorization of Grain and Oil By-Products with Special Focus on Hemicellulose Modification.

Hemicellulose is one of the most important natural polysaccharides in nature. Hemicellulose from different sources varies in chemical composition and structure, which in turn affects the modification effects and industrial applications. Grain and oil by-products (GOBPs) are important raw materials for hemicellulose. This article reviews the modification methods of hemicellulose in GOBPs. The effects of chemical and physical modification methods on the properties of GOBP hemicellulose biomaterials are evaluated. The potential applications of modified GOBP hemicellulose are discussed, including its use in film production, hydrogel formation, three-dimensional (3D) printing materials, and adsorbents for environmental remediation. The limitations and future recommendations are also proposed to provide theoretical foundations and technical support for the efficient utilization of these by-products.

A novel thermophilic recombinant obligate xylobiohydrolase (AcGH30A) from Acetivibrio clariflavus orchestrates the deconstruction of xylan polysaccharides.

GH30 xylobiohydrolases, an expanding enzyme category, need deeper insights for optimal use. The primary aim of this study was to characterize a new xylobiohydrolase, AcGH30A of GH30 family from Acetivibrio clariflavus. The gene encoding AcGH30A was cloned using pET28a(+) vector and expressed in E. coli BL21(DE3) cells. AcGH30A was purified by immobilized metal-ion affinity chromatography. SDS-PAGE analysis of AcGH30A showed molecular mass of ~58 kDa. AcGH30A showed optimum temperature 80 °C and optimum pH 7.0. AcGH30A was stable (maintaining >80 % of control activity) in pH range, 4-7 and temperature range, 30 °C -70 °C when incubated for 90 min. AcGH30A displayed melting temperature, 72 °C and half-life, 21 days at 4 °C. The enzyme activity of AcGH30A was enhanced by 10 mM Ca2+ and Mg2+ ions by 25 % and 21 %, respectively, whereas 10 mM Co2+, Zn2+, Fe2+, and Cu2+ ions significantly reduced it. AcGH30A showed activity against various xylan polysaccharides displaying highest Vmax, 139 U.mg-1 and KM, 0.71 mg.ml-1 against 4-O-methyl glucuronoxylan under optimum conditions. TLC, HPLC and LC-MS analyses of AcGH30A hydrolyzed products from xylan substrates revealed the release of sole product, xylobiose, confirming it as an obligate xylobiohydrolase. AcGH30A being a highly thermostable enzyme can be potentially utlilized in various biotechnological applications.