Biomass Polysaccharides Research Articles

From Mechanism-Based Retaining Glycosidase Inhibitors to Activity-Based Glycosidase Profiling.

Activity-based protein profiling (ABPP) is an effective technology for the identification and functional annotation of enzymes in complex biological samples. ABP designs are normally directed to an enzyme active site nucleophile, and within the field of Carbohydrate-Active Enzymes (CAZymes), ABPP has been most successful for those enzymes that feature such a residue: retaining glycosidases (GHs). Several mechanism-based covalent and irreversible retaining GH inhibitors have emerged over the past sixty years. ABP designs based on these inhibitor chemistries appeared since the turn of the millennium, and we contributed to the field by designing a suite of retaining GH ABPs modeled on the structure and mode of action of the natural product, cyclophellitol. These ABPs enable the study of both exo- and endo-acting retaining GHs in human health and disease, for instance in genetic metabolic disorders in which retaining GHs are deficient. They are also finding increasing use in the study of GHs in gut microbiota and environmental microorganisms, both in the context of drug (de)toxification in the gut and that of biomass polysaccharide processing for future sustainable energy and chemistries. This account comprises the authors' view on the history of mechanism-based retaining GH inhibitor design and discovery, on how these inhibitors served as blueprints for retaining GH ABP design, and on some current and future developments on how cyclophellitol-based ABPs may drive the discovery of retaining GHs and their inhibitors.

Chitosan-derived N-doped porous carbon with fiber network structure for advanced lithium‑sulfur batteries

The flexible design and construction of advanced carbon-based sulfur hosts contribute to the rapid redox and reaction kinetics of lithium polysulfides (LiPSs) in lithium‑sulfur batteries (LSBs), laying the foundation for the next generation of high-energy density energy storage devices. Herein, chitosan (CS)-derived porous N-doped carbon with fiber network structure was prepared by a simple freeze-drying method combined with sintering process. The effect of inorganic salt additives on the morphology of CS-derived carbon was investigated by SEM. The types and contents of N-doping were investigated through XPS analysis. The influence of morphology and N content on the performance of LSBs has been explored in detail through cycling and CV testing. In addition, the true active sites in the CS-derived carbon were determined by XPS and first principles calculations. As an active species, pyrrolic N exhibits superior adsorption energy and the most suitable degradation energy barrier for LiPSs. As the optimal electrode, the performance of LSBs under different electrolyte to sulfur (E/S) conditions was further investigated to explore the potential application of CS-KOH electrodes in high-energy density batteries. The results unveil that the influence of E/S on Li2S deposition is quite significant, with a limit of 5 μL mg−1. This work provides a new strategy for the preparation of high-performance advanced carbon-based materials with fiber network structures from polysaccharide biomass, and also promotes the application of CS in energy storage.

Combined production of biofuel precursors, platform chemicals, and catalyst material from the integral processing of rice bran

This research reports first-time results regarding the complete processing of rice bran through the combination of extraction, processing hydrothermal, sulfonation, and trans-esterification procedures, generating biofuel precursors – fermentable sugars, bio-oil, hydro-char, and methyl esters – platform chemicals, and a catalyst material. Analyzes of XRD, FTIR, BET, SEM, EDS, calorific power, GC, and HPLC were employed for the product characterizations. Concerning the hydrothermal processing, the “temperature” and “biomass/water mass ratio” demonstrated an interesting influence on the results, where the thermal parameter had a greater impact on directing the process to obtain products resulting from the biomass polysaccharides thermal decomposition. At 260 °C, 11.98 and 11.40 g (100 g biomass)−1 of fermentable sugars and platform chemicals were obtained, respectively. At 300 °C, the production of bio-oil and hydro-char were benefited, yielding 10.48 and 30.19 g (100 g biomass)−1 of each component, respectively. The hydro-char-sulfonated catalyst demonstrated an attractive catalytic capacity in esterifying the free fatty acids of the rice bran oil, while simultaneously presenting some difficulty in transesterifying its triglycerides, indicating limitations in the mass transfer of the heterogeneous reaction. Using 4 wt% of catalyst, 90.1 % of the free fatty acids and 60.3 % of the triglycerides contained in the oil were trans-esterified into methyl esters after 6 h, respectively, with no decrease in its catalytic activity being observed after four reuses. This process combination presents an alternative arrangement for the full processing of lignocellulosic and oily biomasses, in addition to representing an approach matchless for the rice bran.

Method for isolation of high molecular weight genomic DNA from Botryococcus biomass.

The development of high molecular weight (HMW) genomic DNA (gDNA) extraction protocols for non-model species is essential to fully exploit long-read sequencing technologies in order to generate genome assemblies that can help answer complex questions about these organisms. Obtaining enough high-quality HMW gDNA can be challenging for these species, especially for tissues rich in polysaccharides such as biomass from species within the Botryococcus genus. The existing protocols based on column-based DNA extraction and biochemical lysis kits can be inefficient and may not be useful due to variations in biomass polysaccharide content. We developed an optimized protocol for the efficient extraction of HMW gDNA from Botryococcus biomass for use in long-read sequencing technologies. The protocol utilized an initial wash step with sorbitol to remove polysaccharides and yielded HMW gDNA concentrations up to 220 ng/μL with high purity. We then demonstrated the suitability of the HMW gDNA isolated from this protocol for long-read sequencing on the Oxford Nanopore PromethION platform for three Botryococcus species. Our protocol can be used as a standard for efficient HMW gDNA extraction in microalgae rich in polysaccharides and may be adapted for other challenging species.

Characterization of Degraded Konjac Glucomannan from an Isolated Bacillus licheniformis Strain with Multi-Enzyme Synergetic Action.

The large molecular weight and high viscosity of natural konjac glucomannan (KGM) limit its industrial application. Microbial degradation of low-molecular-weight KGM has health benefits and various biological functions; however, the available KGM strains used in the industry have microbial contamination and low degradation efficiencies. Therefore, exploring novelly adaptable strains is critical for industrial processes. Here, the Bacillus licheniformis Z7-1 strain isolated from decaying konjac showed high efficiency for KGM degradation. The monosaccharide composition of the degradation products had a reduced molar ratio of mannose to glucose, indicating that Z7-1 preferentially degraded glucose in KGM. The degraded component was further characterized by ESI-MS, Fourier-transform infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM), and it also exhibited good antibacterial activity against various food-spoilage bacteria. Genome sequencing and zymolytic analysis revealed that abundant carbohydrate-active enzymes exist in the Z7-1 genome, with at least five types of extracellular enzymes responsible for KGM degradation, manifesting multi-enzyme synergetic action. The extracellular enzymes had significant thermal stability, indicating their potential application in industry. This study provides an alternative method for obtaining low-molecular-weight KGM with antibacterial functions and supports foundational knowledge for its development as a biocatalyst for the direct conversion of biomass polysaccharides into functional components.

High-strength alginate fibers wet-spun from pre-crosslinked sodium alginate solutions

Facing the severe problem of microplastic pollution, there is an urgent need to develop biodegradable fibers to replace the petrochemical fibers. Sodium alginate, a biomass polysaccharide, has gained widespread attentions recently for the fiber manufacture. However, the limited mechanical strength of alginate fibers restricts their usages as load-bearing fabrics and reinforcement fibers. Here, we develop a novel strategy to prepare alginate multifilaments using pre-crosslinked sodium alginate solutions. The increase in the pre-crosslinking ratio effectively hinders the disentanglement of sodium alginate chains at high stretches, causing an increase in the shear viscosity of the solution ascertained from the capillarity-driven thinning process from 4.5 Pa·s to 9.9 Pa·s and facilitating the high alignment and orientation of sodium alginate chains. The resultant fibers possess a breaking strength of 474 MPa, elongation at break of 16 %, Young's modulus of 14.4 GPa, and toughness of 51.8 MJ/m3, exceeding most biomass fibers without reinforcement additives. The high orientation degree of 0.865 and high spinnability of alginate multifilaments enable their applications in multi-channel encryption fabrics that exhibit distinct information under various optical conditions. This rheological regulation of spinning solutions provides a facile yet effective strategy to enhance the mechanical performance and broaden application scenarios of alginate fibers.

The culture of Chlorella with livestock wastewater and the application of its oligosaccharides in promoting rice seed germination under high salinity conditions

Algal oligosaccharides, derived from polysaccharides in algal biomass, exhibit bioactive properties that enhance plant antioxidant enzyme activity and free radical scavenging. Currently, algal oligosaccharides are mainly produced from large algae, while studies on preparing algal oligosaccharides from microalgae are relatively insufficient. This study focuses on utilizing Chlorella cultivated under controlled conditions using diluted livestock wastewater to produce algal oligosaccharides and investigates their impact on rice seed germination under salt stress. Results show that culturing Chlorella with diluted livestock wastewater not only stimulated algal growth but also efficiently removed nitrogen, phosphorus, and other nutrients. Algal oligosaccharides demonstrated significant improvements in rice seed germination, vigor indices, biomass accumulation, and nitrogen absorption under salt stress. Physiological indicators revealed elevated osmotic adjustment substances, increased chlorophyll content, and alleviated oxidative damage in rice seedlings treated with algal oligosaccharides. The study introduces an innovative method of using livestock wastewater for algal oligosaccharides production, emphasizing its dual benefits including efficient resource utilization and significant cost reduction. The findings also support the theoretical application of Chlorella-derived algal oligosaccharides in enhancing rice cultivation under salt stress.

Preparation of bright yellow color sodium alginate solution

Sodium alginate (SA) is a marine polysaccharide biomass material that is environmentally friendly and exhibits color-changing properties under certain conditions. In this study, we have discovered sodium alginate solution to be chromogenic under four conditions, namely alkali-chromogenic, thermo-chromogenic, force-chromogenic and photo-chromogenic. Under simple strong alkaline conditions, sodium alginate forms clusters of blue light-absorbing chromogenic aggregates, which exhibit a bright yellow color at a certain size. Under different temperature conditions, SA shows varying shades of yellow, and the color tends to stabilize after 48 h of resting. The aggregates can be dispersed by stirring, which changes SA from yellow to colorless. The yellow color can then be recovered after resting. Additionally, exposure to sunlight can cause the yellow SA to fade, but the color can be restored by reheating. Therefore, the force-chromogenic and photo-chromogenic properties are reversible. This makes it a promising material for use in color-developing and indicating materials. It is expected to become a sodium alginate cluster pigment with broad application prospects in the future.

Mechanochemical Hydrolysis of Polysaccharide Biomass: Scope and Mechanistic Insights.

Mechanical forces can affect chemical reactions in a way that thermal reactions cannot do, which may have a variety of applications. In biomass conversion, the selective conversion of cellulose and chitin is a grand challenge because they are the top two most abundant resources and recalcitrant materials that are insoluble in common solvents. However, recent works have clarified that mechanical forces enable the depolymerization of these polysaccharides, leading to the selective production of corresponding monomers and oligomers. This article reviews the mechanochemical hydrolysis of cellulose and chitin, particularly focusing on the scope and mechanisms to show a landscape of this research field and future subjects. We introduce the background of mechanochemistry and biomass conversion, followed by recent progress on the mechanochemical hydrolysis of the polysaccharides. Afterwards, a considerable space is devoted to the mechanistic consideration on the mechanochemical reactions.

Comparison of simultaneous saccharification and fermentation with LPMO-supported hybrid hydrolysis and fermentation.

Enzymatic saccharification is used to convert polysaccharides in lignocellulosic biomass to sugars which are then converted to ethanol or other bio-based fermentation products. The efficacy of commercial cellulase preparations can potentially increase if lytic polysaccharide monooxygenase (LPMO) is included. However, as LPMO requires both a reductant and an oxidant, such as molecular oxygen, a reevaluation of process configurations and conditions is warranted. Saccharification and fermentation of pretreated softwood was investigated in demonstration-scale experiments with 10m3 bioreactors using an LPMO-containing cellulase preparation, a xylose-utilizing yeast, and either simultaneous saccharification and fermentation (SSF) or hybrid hydrolysis and fermentation (HHF) with a 24-hour or 48-hour initial phase and with 0.15 vvm aeration before addition of the yeast. The conditions used for HHF, especially with 48h initial phase, resulted in better glucan conversion, but in poorer ethanol productivity and in poorer initial ethanol yield on consumed sugars than the SSF. In the SSF, hexose sugars such as glucose and mannose were consumed faster than xylose, but, in the end of the fermentation >90% of the xylose had been consumed. Chemical analysis of inhibitory pretreatment by-products indicated that the concentrations of heteroaromatic aldehydes (such as furfural), aromatic aldehydes, and an aromatic ketone decreased as a consequence of the aeration. This was attributed mainly to evaporation caused by the gas flow. The results indicate that further research is needed to fully exploit the advantages of LPMO without compromising fermentation conditions.

Improved voltammetric discrimination of acetaminophen and uric acid in urine using CoO biochar nanocomposite

Elaeagnus angustifolia gum offers a good polysaccharide biomass source for producing porous biochar. The incorporation of CoO cracked nanoplate and the biochar gave a promising application in analysis of clinical urine.

A Multiplexing Activity-Based Protein-Profiling Platform for Dissection of a Native Bacterial Xyloglucan-Degrading System.

Bacteria and yeasts grow on biomass polysaccharides by expressing and excreting a complex array of glycoside hydrolase (GH) enzymes. Identification and annotation of such GH pools, which are valuable commodities for sustainable energy and chemistries, by conventional means (genomics, proteomics) are complicated, as primary sequence or secondary structure alignment with known active enzymes is not always predictive for new ones. Here we report a "low-tech", easy-to-use, and sensitive multiplexing activity-based protein-profiling platform to characterize the xyloglucan-degrading GH system excreted by the soil saprophyte, Cellvibrio japonicus, when grown on xyloglucan. A suite of activity-based probes bearing orthogonal fluorophores allows for the visualization of accessory exo-acting glycosidases, which are then identified using biotin-bearing probes. Substrate specificity of xyloglucanases is directly revealed by imbuing xyloglucan structural elements into bespoke activity-based probes. Our ABPP platform provides a highly useful tool to dissect xyloglucan-degrading systems from various sources and to rapidly select potentially useful ones. The observed specificity of the probes moreover bodes well for the study of other biomass polysaccharide-degrading systems, by modeling probe structures to those of desired substrates.

A Comparison of Dilute Aqueous Isethionic Acid and Sulfuric Acid in Hydrolysis of Three Different Untreated Lignocellulosic Biomass Varieties.

Efficient catalytic hydrolysis of lignocellulosic biomass to sugars is a major challenge in the production of sustainable biofuels and chemical feedstocks. In this study isethionic acid was compared with H2SO4 for hydrolysis of polysaccharides in corn stover, switch grass, and poplar. The catalytic activities of acids were compared by analysis of total reducing sugar (TRS) and glucose yields in a sequence of experiments in water at 90-190 °C using 0.050 mol of H+/L isethionic acid and H2SO4. In comparison to using H2SO4, the use of isethionic acid catalyst lowered the maximum TRS percent yield temperatures by 25, 24, and 21% for corn stover, switch grass, and poplar. A similar effect was observed for glucose percent yields as well. This temperature reduction is due to lowering of the activation energy in the polysaccharide depolymerization reaction and most likely due to hydrogen-bonding-type dipolar interactions between the isethionic acid -OH group and -OH groups in biomass polysaccharides.

Enhanced biomass processing towards acetone-butanol-ethanol fermentation using a ternary deep eutectic solvent

This work evaluated the effectiveness of ternary deep eutectic solvents (DES) composed of cholinium chloride (ChCl), lactic acid (LA) and a diol (ethylene glycol – EG, or 1,6-hexanediol – HEX) in the pretreatment of rice straw (RS) towards the production of biobutanol. The most promising results were obtained with ChCl:LA:EG 1:5:5 at 120 °C for 4 h, which enabled the highest conversion of biomass polysaccharides into glucose and xylose up to 89.3 % and 53.6 % yields, respectively. Subsequently, the produced sugars were efficiently converted into biobutanol through the acetone-butanol-ethanol (ABE) fermentation process, resulting in the production of 95.7 g butanol/kg RS as one of the highest values reported in literature. Thus, the application of the ternary DES ChCl:LA:EG 1:5:5 represents a competitive and environmentally friendly pathway for RS valorization in comparison to conventional processes.

Microwave-assisted ternary deep eutectic solvent pretreatment for improved rice straw saccharification under mild pretreatment conditions

The development of a green and cost-effective pretreatment method to overcome biomass recalcitrance is a challenging assignment for the efficient valorization of lignocellulosic compounds. In this study, a ternary deep eutectic solvent (DES) consisting of choline chloride (ChCl), p-toluene sulfonic acid (pTSA) and ethylene glycol (EG) was applied in rice straw (RS) pretreatment under microwave (MW) irradiation for the further conversion of the polysaccharide fraction. The effect of the pretreatment temperature, time and pTSA content in the DES on lignin extraction and polysaccharide hydrolysis was assessed by a central composite design (CCD). Under the selected pretreatment conditions (80 ºC, 22.5 min, ChCl:pTSA:EG 1:1:9), 61.6% delignification was achieved, as well as nearly complete conversion of the initial glucan in RS (93.4% glucose yield) and a good xylan conversion rate (48.3% xylose yield), representing a total polysaccharide exploitation of 78.3%. Energetical requirement was remarkably lower compared to conventional conductive heating. Importantly, the ternary DES showed good recyclability pattern after two reuses. Overall, this study demonstrated the potential of MW-assisted ternary DES pretreatment for improved valorization of biomass polysaccharides under mild pretreatment conditions, representing an efficient and energetical-saving alternative pathway within the biorefinery framework.

Functional characterization of fungal lytic polysaccharide monooxygenases for cellulose surface oxidation

BackgroundMicrobial lytic polysaccharide monooxygenases (LPMOs) cleave diverse biomass polysaccharides, including cellulose and hemicelluloses, by initial oxidation at C1 or C4 of glycan chains. Within the Carbohydrate-Active Enzymes (CAZy) classification, Auxiliary Activity Family 9 (AA9) comprises the first and largest group of fungal LPMOs, which are often also found in tandem with non-catalytic carbohydrate-binding modules (CBMs). LPMOs originally attracted attention for their ability to potentiate complete biomass deconstruction to monosaccharides. More recently, LPMOs have been applied for selective surface modification of insoluble cellulose and chitin.ResultsTo further explore the catalytic diversity of AA9 LPMOs, over 17,000 sequences were extracted from public databases, filtered, and used to construct a sequence similarity network (SSN) comprising 33 phylogenetically supported clusters. From these, 32 targets were produced successfully in the industrial filamentous fungus Aspergillus niger, 25 of which produced detectable LPMO activity. Detailed biochemical characterization of the eight most highly produced targets revealed individual C1, C4, and mixed C1/C4 regiospecificities of cellulose surface oxidation, different redox co-substrate preferences, and CBM targeting effects. Specifically, the presence of a CBM correlated with increased formation of soluble oxidized products and a more localized pattern of surface oxidation, as indicated by carbonyl-specific fluorescent labeling. On the other hand, LPMOs without native CBMs were associated with minimal release of soluble products and comparatively dispersed oxidation pattern.ConclusionsThis work provides insight into the structural and functional diversity of LPMOs, and highlights the need for further detailed characterization of individual enzymes to identify those best suited for cellulose saccharification versus surface functionalization toward biomaterials applications.

Recent advance in bioactive hydrogels for repairing spinal cord injury: material design, biofunctional regulation, and applications.

Functional hydrogels show potential application in repairing spinal cord injury (SCI) due to their unique chemical, physical, and biological properties and functions. In this comprehensive review, we present recent advance in the material design, functional regulation, and SCI repair applications of bioactive hydrogels. Different from previously released reviews on hydrogels and three-dimensional scaffolds for the SCI repair, this work focuses on the strategies for material design and biologically functional regulation of hydrogels, specifically aiming to show how these significant efforts can promoting the repairing performance of SCI. We demonstrate various methods and techniques for the fabrication of bioactive hydrogels with the biological components such as DNA, proteins, peptides, biomass polysaccharides, and biopolymers to obtain unique biological properties of hydrogels, including the cell biocompatibility, self-healing, anti-bacterial activity, injectability, bio-adhesion, bio-degradation, and other multi-functions for repairing SCI. The functional regulation of bioactive hydrogels with drugs/growth factors, polymers, nanoparticles, one-dimensional materials, and two-dimensional materials for highly effective treating SCI are introduced and discussed in detail. This work shows new viewpoints and ideas on the design and synthesis of bioactive hydrogels with the state-of-the-art knowledges of materials science and nanotechnology, and will bridge the connection of materials science and biomedicine, and further inspire clinical potential of bioactive hydrogels in biomedical fields.

Biochemical characterization of a xylose-tolerant GH43 β-xylosidase from Geobacillus thermodenitrificans

Hemicelluloses are the second most abundant polysaccharide in plant biomass, in which xylan is the main constituent. Aiming at the total degradation of xylan and the obtention of fermentable sugars, several enzymes acting synergistically are required, especially β-xylosidases. In this study, β-xylosidase from Geobacillus thermodenitrificans (GtXyl) was expressed in E. coli BL21 and characterized. The enzyme GtXyl has been grouped within the family of glycoside hydrolases 43 (GH43). Results showed that GtXyl obtained the highest activity at pH 5.0 and temperature of 60 °C. In the additive's tests, the enzyme remained stable in the presence of metal ions and EDTA, and showed high tolerance to xylose, with a relative activity of 55.4% at 400 mM. The enzyme also presented bifunctional activity of β-xylosidase and α-l-arabinofuranosidase, with the highest activity on the substrate p-nitrophenyl-β-d-xylopyranoside. The specific activity on p-nitrophenyl-β-d-xylopyranoside was 18.33 U mg−1 and catalytic efficiency of 20.21 mM−1 s−1, which is comparable to other β-xylosidases reported in the literature. Putting together, the GtXyl enzyme presented interesting biochemical characteristics that are desirable for the application in the enzymatic hydrolysis of plant biomass, such as activity at higher temperatures, high thermostability and stability to metal ions.

Toward a hydrogen-free reductive catalytic fractionation of wheat straw biomass.

The reductive catalytic fractionation (RCF) of lignocellulosic biomass is an attractive method for the conversion of lignin toward valuable low-molecular weight aromatics. A limitation to the upscaling of such technology is represented by the use of pressurized hydrogen gas. Here, the role of hydrogen gas within the RCF of wheat straw biomass is investigated. The use of H2 is shown to enhance lignin depolymerization, by virtue of an improved hydrogenolysis and hydrogenation of lignin fragments, with a yield of phenolic monomers that increased from ~12 wt% of acid-insoluble lignin in the initial biomass under inert atmosphere, up to ~25 wt% under H2 (in methanol, at 250 °C, with Ru/C). The adoption of methanol, ethanol or isopropanol as hydrogen-donor solvents was also investigated in the absence of H2. Ethanol was found to give the highest yield of monophenolics (up to ~20 wt%) owing to a better balance between solvolysis, hydrogenolysis, and hydrogenation of lignin. Nevertheless, a substantial loss of the carbohydrate fraction was observed. The use of a lower temperature (200 °C) in combination with H3PO4 resulted in an improved recovery of cellulose in the pulp and in the solubilization of hemicellulose and lignin, with the formation of monosaccharides (~14 wt% of polysaccharides in the initial biomass) and phenolic monomers (up to 18 wt%, in the absence of H2). Overall, a tradeoff exists between the removal of H2 from the process and the production of low-MW phenolics during RCF.

Orientation of Cel5A and Xyn10B in a fusion construct is important in facilitating synergistic degradation of plant biomass polysaccharides

One approach to achieve efficient and economical saccharification of plant biomass would be using thermostable and multifunctional enzymes from hyperthermophiles such as Thermotoga maritima. Thus, the bifunctional constructs, Cel5A-Xyn10B and Xyn10B-Cel5A, were produced by fusing cellulase Cel5A at the N- and C-terminals of xylanase Xyn10B, respectively. The Cel5A-Xyn10B fusion construct showed cellulase activity of 1483 Uμmol-1 against carboxymethyl cellulose, which was nearly the same as that of Cel5A in the free form. However, xylanase activity of this construct increased by 2-fold against beechwood xylan as compared to that of Xyn10B in free form. The synergistic effect between Cel5A and Xyn10B in the form of Cel5A-Xyn10B fusion resulted an overall increase in the release of reducing sugars. However, Xyn10B-Cel5A showed about 60% decrease in activities of both the component enzymes as compared to their activities in the free form. Both the fusion constructs were active in a wide range of pH from 4.0 to 9.0 and temperatures from 50 to 90°C. Nearly 80% of cellulase and xylanase activities were retained in Cel5A-Xyn10B fusion after incubation at 60°C for 1h. Secondary structures of the component enzymes were retained in the Cel5A-Xyn10B fusion as observed by circular dichroism spectroscopy. Docking and simulation studies suggested that the enhanced xylanase activity in Cel5A-Xyn10B was due to the high binding energy, favorable orientation of the active sites, as well as relative positioning of the active site residues of Cel5A and Xyn10B in closer vicinity, which facilitated the substrate channeling.