Search

Background There is a continued need for improved enzymes for industry. β-xylosidases are enzymes employed in a variety of industries and although many wild-type and engineered variants have been described, enzymes that are highly tolerant of the products produced by catalysis are not readily available and the fundamental mechanisms of tolerance are not well understood. Results Screening of a metagenomic library constructed of mDNA isolated from horse manure compost for β-xylosidase activity identified 26 positive hits. The fosmid clones were sequenced and bioinformatic analysis performed to identity putative β-xylosidases. Based on the novelty of its amino acid sequence and potential thermostability one enzyme (XylP81) was selected for expression and further characterization. XylP81 belongs to the family 39 β-xylosidases, a comparatively rarely found and characterized GH family. The enzyme displayed biochemical characteristics (KM—5.3 mM; Vmax—122 U/mg; kcat—107; Topt—50 °C; pHopt—6) comparable to previously characterized glycoside hydrolase family 39 (GH39) β-xylosidases and despite nucleotide identity to thermophilic species, the enzyme displayed only moderate thermostability with a half-life of 32 min at 60 °C. Apart from acting on substrates predicted for β-xylosidase (xylobiose and 4-nitrophenyl-β-D-xylopyranoside) the enzyme also displayed measurable α-L-arabainofuranosidase, β-galactosidase and β-glucosidase activity. A remarkable feature of this enzyme is its ability to tolerate high concentrations of xylose with a Ki of 1.33 M, a feature that is highly desirable for commercial applications. Conclusions Here we describe a novel β-xylosidase from a poorly studied glycosyl hydrolase family (GH39) which despite having overall kinetic properties similar to other bacterial GH39 β-xylosidases, displays unusually high product tolerance. This trait is shared with only one other member of the GH39 family, the recently described β-xylosidases from Dictyoglomus thermophilum. This feature should allow its use as starting material for engineering of an enzyme that may prove useful to industry and should assist in the fundamental understanding of the mechanism by which glycosyl hydrolases evolve product tolerance.

The genus Cohnella belongs to a group of Gram-positive endospore-forming bacteria within the Paenibacillaceae family. Although most species were described as xylanolytic bacteria, the literature still lacks some key information regarding their repertoire of xylan-degrading enzymes. The whole genome sequence of an isolated xylan-degrading bacterium Cohnella sp. strain AR92 was found to contain five genes encoding putative endo-1,4-β-xylanases, of which four were cloned, expressed, and characterized to better understand the contribution of the individual endo-xylanases to the overall xylanolytic properties of strain AR92. Three of the enzymes, CoXyn10A, CoXyn10C, and CoXyn11A, were shown to be effective at hydrolyzing xylans-derived from agro-industrial, producing oligosaccharides with substrate conversion values of 32.5%, 24.7%, and 10.6%, respectively, using sugarcane bagasse glucuronoarabinoxylan and of 29.9%, 19.1%, and 8.0%, respectively, using wheat bran-derived arabinoxylan. The main reaction products from GH10 enzymes were xylobiose and xylotriose, whereas CoXyn11A produced mostly xylooligosaccharides (XOS) with 2 to 5 units of xylose, often substituted, resulting in potentially prebiotic arabinoxylooligosaccharides (AXOS). The endo-xylanases assay displayed operational features (temperature optima from 49.9 to 50.4 °C and pH optima from 6.01 to 6.31) fitting simultaneous xylan utilization. Homology modeling confirmed the typical folds of the GH10 and GH11 enzymes, substrate docking studies allowed the prediction of subsites (− 2 to + 1 in GH10 and − 3 to + 1 in GH11) and identification of residues involved in ligand interactions, supporting the experimental data. Overall, the Cohnella sp. AR92 endo-xylanases presented significant potential for enzymatic conversion of agro-industrial by-products into high-value products. Key points • Cohnella sp. AR92 genome encoded five potential endo-xylanases. • Cohnella sp. AR92 enzymes produced xylooligosaccharides from xylan, with high yields. • GH10 enzymes from Cohnella sp. AR92 are responsible for the production of X2 and X3 oligosaccharides. • GH11 from Cohnella sp. AR92 contributes to the overall xylan degradation by producing substituted oligosaccharides.

A novel xylanase gene of 870bp was isolated and cloned from the thermophilic bacteria Cohnella sp.A01 that encodes an enzyme comprising 290 amino acid residues and belonging to the GH10 family of xylanases. The enzyme was efficiently expressed in Escherichia coli BL21, then purified using one-step chromatography. The recombinant XynB-A01 not only exhibited significant thermostability but also showed to be stable in a broad range of pH (3.0–10.0), the presence of some detergents, and organic solvents. Although XynB-A01 demonstrated significant activity toward both beechwood and oat spelt xylans, it showed a stronger affinity and higher activity toward the former. HPLC analysis of beechwood xylan hydrolysis revealed that xylotetraose, xylobiose, and xylose are the major hydrolysis products. The investigation of XynB-A01 in pulp biobleaching showed effectiveness in the release of reducing sugars and chromophores. Simple purification, stability over a broad range of pH and temperature, cellulase-free character, and the capacity to produce xylooligosaccharides are some of the advantages of the xylanase of Cohnella sp.A01, suggesting that it may be a promising candidate for biobleaching of paper pulps, producing xylooligosaccharides, and applying in other industries.

In this study, crude and partially purified xylanase from Aureobasidium pullulans CCT 1261 were characterized and applied in beechwood xylan hydrolysis to obtain xylooligosaccharides (XOS). Fractional precipitation (0–30 %/30–60 %) with ammonium sulfate allowed partial purification of xylanases in 6.8-fold and recovery of 69.4 %. Both extracts exhibited optimum activity at 50 °C and pH 4.5, and greater stability at 40 °C within the studied range. The thermostability parameters (kd, t1/2, ΔH*, ΔG*, ΔS*) of the extracts indicated the enzyme denaturation by the increment in temperature from 40 °C to 60 °C; its process was endothermic and led to protein unfolding in a more random structure. Crude xylanase showed a lower value of the Michaelis-Menten constant (25 mg/mL) than the partially purified enzyme (50 mg/mL). In hydrolysis reactions, total XOS contents (7.7 mg/mL and 7.9 mg/mL) and XOS yields (25.7 % and 26.5 %) were equal statistically using the crude or partially purified extracts, differing only in the amount of xylose released. The hydrolysates were mainly composed of potential prebiotics, i.e., xylobiose (50.9 % and 53.8 %) and xylotriose (30.9 % and 32.8 %). Based on these results, the use of crude and partially purified xylanase from A. pullulans showed a promising perspective for xylan hydrolysis to produce XOS.

The genus Pestalotiopsis are endophytic fungi that have recently been identified as cellulolytic system producers. We herein cloned a gene coding for a xylanase belonging to glycoside hydrolase (GH) family 10 (PesXyn10A) from Pestalotiopsis sp. AN-7, which was isolated from the soil of a mangrove forest. This protein was heterologously expressed by Pichia pastoris as a host, and its enzymatic properties were characterized. PesXyn10A was produced as a glycosylated protein and coincident to theoretical molecular weight (35.3 kDa) after deglycosylation by peptide-NfF-glycosidase F. Purified recombinant PesXyn10A exhibited maximal activity at pH 6.0 and 50 °C, and activity was maintained at 90 % at pH 5.0 and temperatures lower than 30 °C for 24 h. The substrate specificity of PesXyn10A was limited and it hydrolyzed glucuronoxylan and arabinoxylan, but not β-glucan. The final hydrolysis products from birchwood xylan were xylose, xylobiose, and 1,23-α-D-(4-O-methyl-glucuronyl)-1,4-β-D-xylotriose. The addition of metallic salts (NaCl, KCl, MgCl2, and CaCl2) activated PesXyn10A for xylan degradation, and maximal activation by these divalent cations was approximately 160 % at a concentration of 5 mM. The thermostability of PesXyn10A significantly increased in the presence of 50 mM NaCl or 5 mM MgCl2. The present results suggest that the presence of metallic salts at a low concentration, similar to brackish water, exerts positive effects on the enzyme activity and thermal stability of PesXyn10A.

Substrate specificities of glycoside hydrolase families 8 (Rex), 39 (BhXyl39), and 52 (BhXyl52) β-xylosidases from Bacillus halodurans C-125 were investigated. BhXyl39 hydrolyzed xylotriose most efficiently among the linear xylooligosaccharides. The activity decreased in the order of xylohexaose > xylopentaose > xylotetraose and it had little effect on xylobiose. In contrast, BhXyl52 hydrolyzed xylobiose and xylotriose most efficiently, and its activity decreased when the main chain became longer as follows: xylotetraose > xylopentaose > xylohexaose. Rex produced O-β-D-xylopyranosyl-(1 → 4)-[O-α-L-arabinofuranosyl-(1 → 3)]-O-β-D-xylopyranosyl-(1 → 4)-β-D-xylopyranose (Ara2Xyl3) and O-β-D-xylopyranosyl-(1 → 4)-[O-4-O-methyl-α-D-glucuronopyranosyl-(l → 2)]-β-D-xylopyranosyl-(1 → 4)-β-D-xylopyranose (MeGlcA2Xyl3), which lost a xylose residue from the reducing end of O-β-D-xylopyranosyl-(1 → 4)-[O-α-L-arabinofuranosyl-(1 → 3)]-O-β-D-xylopyranosyl-(1 → 4)-β-D-xylopyranosyl-(1 → 4)-β-D-xylopyranose (Ara3Xyl4) and O-β-D-xylopyranosyl-(1 → 4)-[O-4-O-methyl-α-D-glucuronopyranosyl-(1 → 2)]-β-D-xylopyranosyl-(1 → 4)-β-D-xylopyranosyl-(1 → 4)-β-D-xylopyranose (MeGlcA3Xyl4). It was considered that there is no space to accommodate side chains at subsite −1. BhXyl39 rapidly hydrolyzes the non-reducing-end xylose linkages of MeGlcA3Xyl4, while the arabinose branch does not significantly affect the enzyme activity because it degrades Ara3Xyl4 as rapidly as unmodified xylotetraose. The model structure suggested that BhXyl39 enhanced the activity for MeGlcA3Xyl4 by forming a hydrogen bond between glucuronic acid and Lys265. BhXyl52 did not hydrolyze Ara3Xyl4 and MeGlcA3Xyl4 because it has a narrow substrate binding pocket and 2- and 3-hydroxyl groups of xylose at subsite +1 hydrogen bond to the enzyme.

As one of the major components of lignocellulosic biomass, the current understanding on pyrolysis mechanism of hemicellulose is relatively limited compared to that of cellulose due to its random and complicated structure. Here, we reported an in-depth study on hemicellulose pyrolysis by combining advanced experimental techniques and quantum chemical calculation, using xylose, xylobiose and xylan as the model compounds. The structural evolution during pyrolysis was firstly characterized via in-situ diffuse reflectance infrared Fourier transform spectroscopy. The corresponding two-dimensional perturbation correlation infrared spectroscopy analysis shows that the dissociation of side chains dominates in the initial stage of xylan pyrolysis, followed by the cleavage of glycosidic bond and the opening of xylopyranose ring. The release behaviors of condensable vapors from hemicellulose pyrolysis obtained by thermogravimetric analyzer coupled with photoionization mass spectrometry indicate that the pyrolysis of xylan polymer with more side chains is prone to produce small compounds via ring rupture reactions compared to that of xylose and xylobiose. The possible reaction pathways of xylan pyrolysis were proposed based on the experimental results and were further confirmed via quantum chemical calculation. This work expands the size of model compounds from common monomers and dimers without side chains to larger tetramers with side chains. It was found that the dissociation of side chains and the cleavage of glycosidic bonds are competitive reactions. The backbone of xylan is decomposed following the dissociation of side chains to yield furfural, dianhydro xylose and other small compounds.

Xylooligosaccharides (XOS) are considered as prebiotics, as they help in enhancing the growth of beneficial microbes found in the gut. In the present study, partially purified xylanase from Thermomyces lanuginosus VAPS-24 was used for the production of XOS from xylan extracted from corn cobs. The mild alkali treatment with 20% NaOH yielded 32 ± 1.8% (w/w) xylan and it was structurally characterized by FTIR. The corn cob xylan (2% w/v), when treated with partially purified 20 U VAPS-24 xylanase for 8 h at 45°C, yielded 6.9 ± 0.03 mg/ml of XOS. The XOS contained mainly xylobiose (X2) and xylotriose (X3) which were purified and analyzed using 13C-NMR spectroscopy which showed predominant presence of (1–4) linked β-d-xylose residues. HPLC analysis showed characteristic peaks of xylotriose, xylobiose, and xylose in the XOS mixture. The produced XOS showed concentration-dependent antioxidant activities and probiotic growth promotion. Further, the genes responsible for the metabolism of XOS in probiotics were identified and their probable interactions and coexpression of genes were studied in silico.

Cellobiose dehydrogenase (CDH) is a flavocytochrome catalyzing oxidation of the reducing end of cellobiose and cellooligosaccharides, and has a key role in the degradation of cellulosic biomass by filamentous fungi. Here, we use a lineup of glucose/xylose-mixed β-1,4-linked disaccharides and trisaccharides, enzymatically synthesized by means of the reverse reaction of cellobiose phosphorylase and cellodextrin phosphorylase, to investigate the substrate recognition of CDH. We found that CDH utilizes β-D-xylopyranosyl-(1→4)-D-glucopyranose (Xyl-Glc) as an electron donor with similar K m and k cat values to cellobiose. β-D-Glucopyranosyl-(1→4)-D-xylopyranose (Glc-Xyl) shows a higher K m value, while xylobiose does not serve as a substrate. Trisaccharides show similar behavior; i.e., trisaccharides with cellobiose and Xyl-Glc units at the reducing end show similar kinetics, while the enzyme was less active towards those with Glc-Xyl, and inactive towards those with xylobiose. We also use docking simulation to evaluate substrate recognition of the disaccharides, and we discuss possible molecular mechanisms of substrate recognition by CDH.

This study aimed to develop a method for the purification of a xylanase called SMXL1 produced by Stenocarpella maydis and its biochemical characterization. The enzyme was purified using a Rotofor preparative chamber and one chromatographic step in an ion exchange column coupled to equipment FPLC. Posteriorly the protein was characterized, and its effect on the birchwood xylan degradation was determine by HPLC. The purified enzyme showed a molecular weight of 55 kDa calculated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purification process obtained a yield of 6.5  0.3 %. The activity was stable at a pH range of 4 to 10 and temperatures of 45 to 60 °C. The optimum values of temperature and pH were 55 °C and 4, respectively. The Michaelis constant (Km) value was 2.61 mg/mL and the Vmax was 3.02 µmol/mL/min using birchwood xylan as substrate and the Michaelis-Menten equation. The enzyme is inhibited by the cations Mn2+ and by Fe3+ and degrades the birchwood xylan being the principal products the xylobiose and the xylose. This work is the first report of the purification and biochemical characterization of a xylanase called SMXL1 produced by S. maydis.

PcAxy43B is a modular protein comprising a catalytic domain of glycoside hydrolase family 43 (GH43), a family 6 carbohydrate-binding module (CBM6), and a family 36 carbohydrate-binding module (CBM36) and found to be a novel multifunctional xylanolytic enzyme from Paenibacillus curdlanolyticus B-6. This enzyme exhibited α-l-arabinofuranosidase, endoxylanase, and β-d-xylosidase activities. The α-l-arabinofuranosidase activity of PcAxy43B revealed a new property of GH43, via the release of both long-chain cereal arabinoxylan and short-chain arabinoxylooligosaccharide (AXOS), as well as release from both the C(O)(2) and C(O)(3) positions of AXOS, which is different from what has been seen for other arabinofuranosidases. PcAxy43B liberated a series of xylooligosaccharides (XOSs) from birchwood xylan and xylohexaose, indicating that PcAxy43B exhibited endoxylanase activity. PcAxy43B produced xylose from xylobiose and reacted with p-nitrophenyl-β-d-xylopyranoside as a result of β-xylosidase activity. PcAxy43B effectively released arabinose together with XOSs and xylose from the highly arabinosyl-substituted rye arabinoxylan. Moreover, PcAxy43B showed significant synergistic action with the trifunctional endoxylanase/β-xylosidase/α-l-arabinofuranosidase PcAxy43A and the endoxylanase Xyn10C from strain B-6, in which almost all products produced from rye arabinoxylan by these combined enzymes were arabinose and xylose. In addition, the presence of CBM36 was found to be necessary for the endoxylanase property of PcAxy43B. PcAxy43B is capable of hydrolyzing untreated cereal biomass, corn hull, and rice straw into XOSs and xylose. Hence, PcAxy43B, a significant accessory multifunctional xylanolytic enzyme, is a potential candidate for application in the saccharification of cereal biomass. IMPORTANCE Enzymatic saccharification of cereal biomass is a strategy for the production of fermented sugars from low-price raw materials. In the present study, PcAxy43B from P. curdlanolyticus B-6 was found to be a novel multifunctional α-l-arabinofuranosidase/endoxylanase/β-d-xylosidase enzyme of glycoside hydrolase family 43. It is effective in releasing arabinose, xylose, and XOSs from the highly arabinosyl-substituted rye arabinoxylan, which is usually resistant to hydrolysis by xylanolytic enzymes. Moreover, almost all products produced from rye arabinoxylan by the combination of PcAxy43B with the trifunctional xylanolytic enzyme PcAxy43A and the endoxylanase Xyn10C from strain B-6 were arabinose and xylose, which can be used to produce several value-added products. In addition, PcAxy43B is capable of hydrolyzing untreated cereal biomass into XOSs and xylose. Thus, PcAxy43B is an important multifunctional xylanolytic enzyme with high potential in biotechnology.

The Acetivibrioclariflavus (basonym: Clostridium clariflavum) glycoside hydrolase family 30 cellulosomal protein encoded by the Clocl_1795 gene was highly represented during growth on cellulosic substrates. In this report, the recombinantly expressed protein has been characterized and shown to be a non-reducing terminal (NRT)-specific xylobiohydrolase (AcXbh30A). Biochemical function, optimal biophysical parameters, and phylogeny were investigated. The findings indicate that AcXbh30A strictly cleaves xylobiose from the NRT up until an α-1,2-linked glucuronic acid (GA)-decorated xylose if the number of xyloses is even or otherwise a single xylose will remain resulting in a penultimate GA-substituted xylose. Unlike recently reported xylobiohydrolases, AcXbh30A has no other detectable hydrolysis products under our optimized reaction conditions. Sequence analysis indicates that AcXbh30A represents a new GH30 subfamily. This new xylobiohydrolase may be useful for commercial production of industrial quantities of xylobiose.

Las xilanasas son enzimas que hidrolizan el xilano, produciendo azúcares fermentables tales como xilosa y xilobiosa con aplicaciones industriales muy diversas entre las que destaca el biobleaching y la producción de bioetanol de segunda generación. El objetivo principal del presente estudio se enfoca en la optimización de la producción de xilanasa bacteriana por parte de la bacteria Bacillus sp. K1. La misma ha sido previamente aislada de muestras de madera en putrefacción recolectadas en las premisas de la Universidad de Lakehead para su Departamento de Biología. Las variables investigadas para la optimización fueron físicas tales como pH, temperatura y volumen de inóculo; y de composición del medio de fermentación tales como fuente de carbono, fuente de nitrógeno orgánico e inorgánico y relación porcentual entre dichos componentes. Adicionalmente, se determinó el efecto de iones metálicos (sodio, potasio, magnesio, calcio, ferroso, niqueloso, cúprico, cobaltoso y manganoso) y surfactantes (Polisorbato-20, dodecil sulfato de sodio y Triton X-100) en la actividad enzimática de la xilanasa producida. La mayor producción de xilanasa fue tras 36h de fermentación y las condiciones físicas óptimas asociadas a la producción de xilanasa fueron pH 6, temperatura 35 ºC y volumen de inóculo 1%. La composición óptima del medio de fermentación determinada está constituida por: Afrecho de trigo 4%, Glucosa 0,5%, NH4NO3 0,5%, K2HPO4 0,1%, KCl 0,1%, MgSO4 0,05%, peptona 0,5%, SDS 0,1%. La actividad enzimática generada es de 264,96±7,89 IU/L, con un 248,79% de incremento con respecto al inicial. Los inhibidores más importantes fueron los iones cúprico y manganoso con 32,00% y 49,16% de inhibición comparado con el experimento de control.

The depolymerization of xylo-oligosaccharides to xylose by β-xylosidase and the subsequent ethanol production from xylose are important for reducing inhibition towards cellulase and hemicellulase and for taking full advantage of all sugars in lignocellulose hydrolysate. The activity of β-xylosidase in most cellulases produced by filamentous fungi is usually deficient, and thus recombinant xylose-fermenting Saccharomyces cerevisiae capable of secreting highly active β-xylosidase is increasingly recognized as an excellent candidate for addressing this issue. To this end, a prominent chassis cell, BSPX042, constructed in our previous work with a well-modified downstream metabolic pathway of xylose through multiple chromosome manipulations and beneficial mutations after evolution engineering, was selected as the host. The xylose isomerase coding gene Ru-xylA (where Ru represents the rumen), which was screened from the metagenomics library of bovine rumen contents, was shown to exhibit a high activity level in S. cerevisiae, and the glycoside hydrolase family 3 β-xylosidase gene xyl3A from Penicillium oxalicum was coexpressed in BSPX042. The recombinant strain BSGIBX with the highest extracellular activity of β-xylosidase was determined by comparing different signal peptides. The hydrolysis of xylobiose and xylotriose by BSGIBX culture further confirmed its β-xylosidase activity. BSGIBX can grow and produce ethanol with xylo-oligosaccharides as the sole carbon source. When xylo-oligosaccharides were pretreated by effective xylanase, the majority of the xylo-oligosaccharides were consumed rapidly and produced ~ 19.4 g L−1 ethanol at 48 h with a yield of 0.473 g g−1 consumed sugars in mixture with glucose. These results indicate the successful coexpression of β-xylosidase and xylose isomerase in ethanol-producing S. cerevisiae, and provide theoretical support for our future work in robust industrial yeast strains.

A one-step method to immobilize xylanase onto cellulosic material by fusion of expansin from Bacillus subtilis to xylanase LC9 without the requirement of prior purification of enzyme has been developed. Fusion enzyme EXLX-R2-XYN was specifically adsorbed onto corncob residue with high loading capacity due to bio-affinity adsorption of expansin onto cellulose. The immobilization yield was close to 100%, with a recovered activity of 82.4%. The immobilized EXLX-R2-XYN retained 45.3% of its activity after incubation at 70 °C for 3 h, whereas only 16.3% of the activity was left in free form under the same conditions. The conversion yield of XOS by using immobilized EXLX-R2-XYN reached up to 515 mg/g xylan from 2% corncob extracted xylan, which was higher than that of the free enzyme. The hydrolysis products were mainly xylobiose (57.5%) and xylotriose (38.4%), without undesirable xylose production. After five cycles of hydrolysis, more than 70% of conversion was obtained.

In the current study, we attempted to enhance the xylanase activity of Trichoderma reesei ATCC66589 by using disparity mutagenesis, wherein a plasmid harboring proofreading-impaired DNA polymerase δ was inserted. Following selection on xylan-rich media and successive plasmid curing, a mutant showing conidiospores strikingly different from those of the parent strain, with many small humped-surface spheres, was generated. Xylanase and β-xylosidase activities of the mutant XM1, cultivated in xylan medium, were 15.8- and 11.0-fold higher than those of the parent strain, respectively. Furthermore, xylanase activity was generated approximately 24 h in advance compared to that in the parent. In contrast, when cultivated in Avicel medium, its xylanase and β-xylosidase activities were 0.14- and 0.33-fold, respectively, compared to those in the parent. Among the xylan component sugars and related polyols, D-xylose and xylobiose exerted a distinct inductive effect on the xylanase activity in Avicel media, while xylitol and L-arabinose did not. Mutagenesis involved in xylose catabolism is suggestive of changes at the gene transcription level. Although the induction mechanism remains unclear in details, disparity mutagenesis may be useful for obtaining T. reesei mutants with high xylanase activity.

BSH-1 is an O‑acetyl-arabinoxylan obtained from bamboo shavings. This study investigated its fermentation behavior by human colonic microbiota in vitro. Results showed that BSH-1 remarkably modulated the composition of human colonic microbiota, mainly by increasing the growth of potential beneficial genera (i.e. Bifidobacterium, Lactobacillus, Bacteroides, Prevotella_7, Parabacteroides) and by decreasing the growth of potential harmful genera (i.e. Fusobacterium, Lachnospiraceae_UCG-008, Bilophila and Desulfovibrio). BSH-1 significantly promoted the production of short-chain fatty acids, especially acetic, propionic and n-butyric acids. After 48 h fermentation, the concentration of n-butyric acid in BSH-1 fermentation culture was increased by 2.41 times compared to the blank. During fermentation, the activity of acetyl xylan esterase, arabinofuranosidase, xylanase and xylosidase was enhanced. Moreover, free arabinose, xylose, xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose were detected. These results suggest that BSH-1 could potentially be a functional ingredient to improve gut health.

Bioconversion of lignocellulosic biomass into value-added products relies on polysaccharides depolymerization by carbohydrate active enzymes. This work reports biochemical characterization of Paludibacter propionicigenes xylanase from GH10 (PpXyn10A) and its application for enzymatic xylooligosaccharides (XOS) production from commercial heteroxylans and liquor of hydrothermally pretreated corn cobs (PCC). PpXyn10A is tolerant to ethanol and NaCl, and releases xylobiose (X2) and xylotriose (X3) as the main hydrolytic products. The conversion rate of complex substrates into short XOS was approximately 30% for glucuronoxylan and 8.8% for rye arabinoxylan, after only 4 h; while for PCC, PpXyn10A greatly increased unbranched XOS yields. B. adolescentis fermentation with XOS from beechwood glucuronoxylan produced mainly acetic and lactic acids. Structural analysis shows that while the glycone region of PpXyn10A active site is well preserved, the aglycone region has aromatic interactions in the +2 subsite that may explain why PpXyn10A does not release xylose.

Sugarcane processing roughly generates 54 million tonnes sugarcane bagasse (SCB)/year, making SCB an important material for upgrading to value-added molecules. In this study, an integrated scheme was developed for separating xylan, lignin and cellulose, followed by production of xylo-oligosaccharides (XOS) from SCB. Xylan extraction conditions were screened in: (1) single extractions in NaOH (0.25, 0.5, or 1 M), 121 °C (1 bar), 30 and 60 min; (2) 3 × repeated extraction cycles in NaOH (1 or 2 M), 121 °C (1 bar), 30 and 60 min or (3) pressurized liquid extractions (PLE), 100 bar, at low alkalinity (0–0.1 M NaOH) in the time and temperature range 10–30 min and 50–150 °C. Higher concentration of alkali (2 M NaOH) increased the xylan yield and resulted in higher apparent molecular weight of the xylan polymer (212 kDa using 1 and 2 M NaOH, vs 47 kDa using 0.5 M NaOH), but decreased the substituent sugar content. Repeated extraction at 2 M NaOH, 121 °C, 60 min solubilized both xylan (85.6% of the SCB xylan), and lignin (84.1% of the lignin), and left cellulose of high purity (95.8%) in the residuals. Solubilized xylan was separated from lignin by precipitation, and a polymer with β-1,4-linked xylose backbone substituted by arabinose and glucuronic acids was confirmed by FT-IR and monosaccharide analysis. XOS yield in subsequent hydrolysis by endo-xylanases (from glycoside hydrolase family 10 or 11) was dependent on extraction conditions, and was highest using xylan extracted by 0.5 M NaOH, (42.3%, using Xyn10A from Bacillus halodurans), with xylobiose and xylotriose as main products. The present study shows successful separation of SCB xylan, lignin, and cellulose. High concentration of alkali, resulted in xylan with lower degree of substitution (especially reduced arabinosylation), while high pressure (using PLE), released more lignin than xylan. Enzymatic hydrolysis was more efficient using xylan extracted at lower alkaline strength and less efficient using xylan obtained by PLE and 2 M NaOH, which may be a consequence of polymer aggregation, via remaining lignin interactions. Supplementary Information The online version contains supplementary material available at 10.1186/s13068-021-01993-z.

Collision-induced dissociation (CID) of α-xylose and β-xylose were studied using mass spectrometry and quantum chemistry calculations. Three dissociation channels, namely loss of metal ions, dehydration, and cross-ring dissociation were found. The major dissociation channel of sodium adducts is the loss of sodium ions, and the minor dissociation channels are dehydration and cross-ring dissociation. By contrast, dehydration and cross-ring dissociation are the major dissociation channels of lithium adducts, and the corresponding dissociation mechanisms can be used to determine the anomericity and linkages of xylose in oligosaccharides. These mechanisms include (1) the dehydration branching ratio can be used to differentiate the anomericity of xylose and xylose in oligosaccharides because α-xylose has a larger branching ratio of dehydration than β-xylose, (2) various cross-ring dissociation reactions can be used to identify linkage positions. The oligosaccharide with xylose at the reducing end is predicted to undergo 0,2X, 0,3X, and 0,2A cross-ring dissociation for the 1 → 2, 1 → 3, and 1 → 4 linkages, respectively. Application of these mechanisms to determine the anomericity and linkage positions of xylobiose was demonstrated.

Development of a more environmentally sustainable society is based on the maximum use of renewable carbon sources and their valorization of environmentally-friendly green technologies. This includes a thorough use of plant biomass and agricultural residues for the production of value-added bioproducts. Xylan is the second most abundant biopolymer in nature which can be sustainable converted into pentoses and xylooligosaccharides, that have wide applications in the food, feed, pharmaceutical, and cosmetic industry. Within the scope of present study, we biochemically characterized two-domain GH10 xylanase from Jonesia denitrificans (JdXyn10A) and evaluated its applicability for production of xylooligosaccharides (XOS). JdXyn10A has a specific activity of 84 ± 2 U/mg and 65 ± 5 U/mg when acting on beechwood glucuronoxylan and rye arabinoxylan, respectively. The enzyme is stable in a wide pH range and is tolerant to high concentrations of NaCl and ethanol. Interestingly, the profile of products released by the enzyme is predominant in xylobiose and xylotriose, with a very low fraction of xylose which is desirable for XOS production. The efficiencies of enzymatic conversion of beechwood glucuronoxylan and rye arabinoxylan are 47.67 % and 26.01 %, respectively, after 6 h of enzymatic hydrolysis only. Structural comparison between the JdXyn10A homology model and the structure from its homologous that while the glycone region of its active site is well preserved, the aglycone region presents structural differences in the +2 subsite that may explain why JdXyn10A does not release xylose.

A novel and eco-friendly approach was developed for the scouring and bleaching of flax roves with xylanase, cellulase and mixed enzymes (xylanase and cellulase) in supercritical carbon dioxide fluid (SC-CO2). The results showed that significant improvements in the whiteness and residual gum content of flax roves were achieved in SC-CO2 since xylanase hydrolysed xylan into low molecular oligosaccharides, xylobiose and xylose by hydrolyzing and cleaving β-1,4-glucosidic bonds of xylan. Simultaneously, the hydrolysis reaction of lignin through the hydrophobic and electrostatic interactions at β-O-4 linkages could be effectively facilitated by the catalyst of cellulase in SC-CO2. Furthermore, the whiteness and residual gum content of flax roves were notably influenced by the dosage of enzyme, enzyme ratio, scouring and bleaching temperature, pressure, time and CO2 flow. An optimized enzymatic process for the scouring and bleaching of flax roves was recommended with a dosage of enzyme 3%, an enzyme ratio of 2: 1 (xylanase: cellulase), a system temperature of 50 °C, a pressure of 20 MPa, a CO2 flow of 30 g/min for 90 min in SC-CO2. The whiteness value of flax roves reached to 42.3%, and the value of residual gum content was decreased to 16.95% in the optimum condition.

Enzymatic transformation of xylans into renewable fuels and value-added products is mediated by xylanases. Here we describe the biochemical and X-ray structural characterization of Thermobacillus composti GH10 xylanase (TcXyn10A) at 2.1 A resolution aiming to unravel details of its recognition of glucurono- and arabinoxylan at a molecular level. TcXyn10A improves the efficiency of pretreated lignocellulosic biomass hydrolysis by a commercial enzyme cocktail causing a 15.35 % increase in xylose release and 4.38 % glucose release after 24 h of reaction. The enzyme releases predominantly xylobiose and xylotriose, as well as MeGlcA3 × 3 (from beechwood glucuronoxylan) and a range of decorated xylooligosaccharides (XOS) from rye arabinoxylan, with Ara2 × 2 being the major product. The enzyme liberates XOS with the yields of 29.09 % for beechwood glucuronoxylan and 16.98 % for rye arabinoxylan. Finally, TcXyn10A has a high thermal stability, halotolerance, and resistance to ethanol, biochemical properties that can be desirable for a number of industrial applications.

A xylanase from Talaromyces thermophiles F1208 (T-Xyn) was used specifically to explore the effects of disulfide bond on hydrolytic activity. The T-Xyn-C122S-C166S mutant does not have the C122-C166 disulfide bond present in wild-type T-Xyn, whereas T-Xyn-T38C-S50C and T-Xyn-T38C-S50C-C122S-C166S mutants have an introduced disulfide bond, C38-C50, to T-Xyn and T-Xyn-C122S-C166S, respectively. The optimum pH of T-Xyn-T38C-S50C and T-Xyn-T38C-S50C-C122S-C166S was lower than that of T-Xyn and T-Xyn-C122S-C166S. The introduction of a disulfide bond caused a decrease in the optimum temperature and thermal stability of T-Xyn. The existence of a disulfide bond has a strong influence on the hydrolysis characteristics of T-Xyn, which caused changes in the composition and proportion of the hydrolysate products. T-Xyn-T38C-S50C produces the highest level of xylose when using beechwood xylan as the substrate, whereas T-Xyn produces the highest level of xylobiose and T-Xyn-T38C-S50C-C122S-C166S produces the largest amount of xylotriose. When birchwood xylan was used as the substrate, the introduction of a disulfide bond increased the content of xylose, decreased the content of xylotriose and a high degree of polymerization (DP ≥ 5) was observed. The hydrolysis of oat-spelt xylan is more complex with the introduction of the disulfide bond causing an increase in the degradation rate of xylotriose.

Red alga dulse possesses a unique xylan, which is composed of a linear &beta, (1&rarr, 3)/&beta, 4)-xylosyl linkage. We previously prepared characteristic xylooligosaccharide (DX3, (&beta, 3)-xylosyl-xylobiose)) from dulse. In this study, we evaluated the prebiotic effect of DX3 on enteric bacterium. Although DX3 was utilized by Bacteroides sp. and Bifidobacterium adolescentis, Bacteroides Ksp. grew slowly as compared with &beta, 4)-xylotriose (X3) but B. adolescentis grew similar to X3. Therefore, we aimed to find the key DX3 hydrolysis enzymes in B. adolescentis. From bioinformatics analysis, two enzymes from the glycoside hydrolase family 43 (BAD0423: subfamily 12 and BAD0428: subfamily 11) were selected and expressed in Escherichia coli. BAD0423 hydrolyzed &beta, 3)-xylosyl linkage in DX3 with the specific activity of 2988 mU/mg producing xylose (X1) and xylobiose (X2), and showed low activity on X2 and X3. BAD0428 showed high activity on X2 and X3 producing X1, and the activity of BAD0428 on DX3 was 1298 mU/mg producing X1. Cooperative hydrolysis of DX3 was found in the combination of BAD0423 and BAD0428 producing X1 as the main product. From enzymatic character, hydrolysis of X3 was completed by one enzyme BAD0428, whereas hydrolysis of DX3 needed more than two enzymes.

Highly efficient and stable enzymes are required for application in biotechnology, to meet the technical, environmental, and economic industrial demands. Xylanases are hemicellulolytic enzymes that degrade the heteroxylan constituent of the lignocellulosic plant cell wall. In this study, an acidic xylanase designated Pjxyn (pH 4.0) from Penicillium janthinellum was engineered by the introduction of a disulfide bridge. This strategy exploited the influence of the bridge on hydrolysis characteristics and enhanced hydrolysis was achieved. Three mutants [PjxynS(27)S(39), PjxynS(27)S(186), and PjxynS(39)S(186)] produced more xylose and xylobiose as hydrolysis products compared with the wild-type Pjxyn, when commercial xylans and lab-prepared water-insoluble corncob-xylan were used as the substrates, especial for the PjxynS(27)S(39) mutant, the content of xylose and xylobiose was 87.62% (using beechwood xylan) and 69.91% (using oat-spelt xylan) higher than that in the hydrolysis products of Pjxyn. Moreover, each mutant combined with the xylanase mutant T-XynFM effectively decreased the production of xylose with an optimum xylobiose yield. The findings demonstrate the potential industrial value of engineering xylanase to improve its hydrolytic properties and thermostability.

Pre-hydrolysis liquor (PHL) from the kraft-based dissolving pulp process contains large amounts of hemicelluloses and their degradation products, which makes it a promising candidate for the production of xylose or xylooligosaccharides (XOS). However, it is usually treated as wastewater of the pulp and paper industries. In this study, both acid hydrolysis and enzymatic hydrolysis were applied to prepare xylose and XOS from the PHL, which contained 47.80 g/L of sugars and 14.10 g/L of lignins. Results showed that an optimal xylose yield (30.10 g/L) was obtained at 120 °C for 0.5 h with 4% aqueous sulfuric acid, while the maximum XOS yield (11.63 g/L) was achieved with 0.3% aqueous sulfuric acid at 120 °C for 2.0 h. For the enzymatic hydrolysis, the highest XOS yield of 15.65 g/L was obtained under the optimal condition (60 IU/L PHL, 50 °C, 6 h), and the XOS were mainly composed of xylobiose and xylotriose.

Penicillium purpurogenum grows on a variety of natural carbon sources and secretes to the medium a large number of enzymes that degrade the polysaccharides present in lignocellulose. In this work, the gene coding for a novel xylanase (XynC) belonging to family 30 of the glycoside hydrolases (GH), has been identified in the genome of the fungus. The enzyme has been expressed in Pichia pastoris and characterized. The mature XynC has 454 amino acid residues and a calculated molecular weight of 49 240. The purified protein shows a molecular weight of 67 000, and it is partially deglycosylated using EndoH. Its pH optimum is in the range of 3–5, and the optimal temperature is 45 °C. It is active on both arabinoxylan and glucuronoxylan, similarly to other fungal GH 30 xylanases. It liberates a set of oligosaccharides, which have been detected by thin-layer chromatography, thus indicating that it is an endo-acting xylanase. It hydrolyzes xylooligosaccharides, releasing mainly xylobiose, in contrast to other fungal GH family 30 enzymes which generate chiefly xylose. Highest sequence identity to a characterized family 30 xylanase is found with the enzyme from the fungus Bispora sp (53%). This is the first GH 30 xylanase described from a Penicillium.

An endo-xylanase (PsGH10A) of family 10 glycoside hydrolase from Pseudopedobacter saltans was cloned, expressed and purified. Substrate specificity analysis of PsGH10A showed activity against β-1,4-xylans. It showed maximum activity against beechwood xylan (59.7 ± 1.1 U/mg) followed by xylan (Mw. 20,000–30,000) (57.1 ± 0.7 U/mg). PsGH10A displayed maximum activity at pH 6.0 and 40 °C. The enzyme was stable in the pH range, from 6.0 to 7.5 and showed thermostability up to 40 °C. The kinetic parameters of PsGH10A using beechwood xylan determined were Km 6.2 mg/mL and Vmax 72 U/mg. The activity of PsGH10A was 29 ± 2.4% enhanced by 2 mM Mn2+ ions and inhibited by less than 50 ± 1.6% by 2 mM Zn2+, Pb2+ or Cu2+ ions. The time-dependent TLC analysis of hydrolyzed products of beechwood xylan released by PsGH10A showed the release of xylose, xylobiose and xylotetraose as end products confirming the endolytic mode of action. PsGH10A hydrolyzed products of xylan and substituted xylan indicate production of series of short-chain xylooligosaccharides and arabinoxylo-saccharides. PsGH10A also showed saccharification of AFEX pretreated poplar and sugarcane bagasse. Therefore, it can be used for various biotechnological applications such as for prebiotic xylooligosaccharides and bioethanol production from pretreated agrowaste biomass. This is the first report on β-1,4-xylanase cloned from Pseudopedobacter saltans.

The three dimensional structure (3D structure) of GH-11 xylanase from Thermomyces lanuginosus was obtained through homology modeling. To study the enzyme interaction with an end product of enzyme catalysis, the xylanase two sugar molecules xylose and xylobiose has been docked into the active site of GH-11 xylanase through molecular docking. Based on the free binding energy and Inhibition constant, concluded xylose makes more stable complex than xylobiose. Further, the molecular dynamic simulation studies were carried out at different temperature, i.e. 323, 333, 343 and 353 K (i.e. 50, 60, 70 and 80 °C). It has been observed that there was minor structural modification in 3D-structure of xylanase at 323, 333, and 343 K. But the helix and sheets moved out of the initial structure when simulation carried out at during 353 K (80 °C).

Background β-D-xylosidase is a vital exoglycosidase with the ability to hydrolyze xylooligosaccharides to xylose and to biotransform some saponins by cleaving outer β-xylose. β-D-xylosidase is widely used as one of the xylanolytic enzymes in a diverse range of applications, such as fuel, food and the pharmaceutical industry; therefore, more and more studies have focused on the thermostable and xylose-tolerant β-D-xylosidases. Results A thermostable β-xylosidase gene (xln-DT) of 1509 bp was cloned from Dictyoglomus thermophilum and expressed in E.coli BL21. According to the amino acid and phylogeny analyses, the β-xylosidase Xln-DT is a novel β-xylosidase of the GH family 39. The recombinant β-xylosidase was purified, showing unique bands on SDS-PAGE, and had a protein molecular weight of 58.7 kDa. The β-xylosidase Xln-DT showed an optimal activity at pH 6.0 and 75 °C, with p-nitrophenyl-β-D-xylopyranoside (pNPX) as a substrate. Xln-DT displayed stability over a pH range of 4.0-7.5 for 24 h and displayed thermotolerance below 85 °C. The values of the kinetic parameters Km and Vmax for pNPX were 1.66 mM and 78.46 U/mg, respectively. In particular, Xln-DT displayed high tolerance to xylose, with 60% activity in the presence of 3 M xylose. Xln-DT showed significant effects on the hydrolyzation of xylobiose. After 3 h, all the xylobiose tested was degraded into xylose. Moreover, β-xylosidase Xln-DT had a high selectivity for cleaving the outer xylose moieties of natural saponins, such as notoginsenoside R1 and astragaloside IV, which produced the ginsenoside Rg1 with stronger anti-fatigue activity and produced cycloastragenol with stronger anti-aging activity, respectively. Conclusion This study provides a novel GH 39 β-xylosidase displaying extraordinary properties of highly catalytic activity at temperatures above 75 °C, remarkable hydrolyzing activity of xylooligosaccharides and rare saponins producing ability in the pharmaceutical and commercial industries. Electronic supplementary material The online version of this article (10.1186/s12896-018-0440-3) contains supplementary material, which is available to authorized users.

A novel glycosyl hydrolase family 11 xylanase gene, xynMF13A, was cloned from Phoma sp. MF13, a xylanase-producing fungus isolated from mangrove sediment. xynMF13A was heterologously expressed in Pichia pastoris, and the recombinant XynMF13A (rXynMF13A) was purified by Ni-affinity chromatography. The temperature and pH optima of purified rXynMF13A were 45 °C and pH 5.0, respectively. rXynMF13A showed a high level of salt tolerance, with maximal enzyme activity being seen at 0.5 M NaCl and as much as 53% of maximal activity at 4 M NaCl. The major rXynMF13A hydrolysis products from corncob xylan were xylobiose, xylotriose, xylotetraose, and xylopentaose, but no xylose was found. These hydrolysis products suggest an important potential for XynMF13A in the production of xylooligosaccharides (XOs). Furthermore, rXynMF13A had beneficial effects on Chinese steamed bread production, by increasing specific volume and elasticity while decreasing hardness and chewiness. These results demonstrate XynMF13A to be a novel xylanase with potentially significant applications in baking, XOs production, and seafood processing.

The purpose of study was utilization of sugarcane bagasse, waste product of sugarcane industry, for production of xylooligosaccharides (XOS) and their evaluation of their prebiotic potential. The XOS production was carried out in two steps: alkaline extraction of xylan (5, 10 and 15%KOH and NaOH) followed by acid hydrolysis (0.25 and 0.50M H2SO4; 20, 40 and 60 min) of xylan, and quantified using high performance thin layer chromatography (HPTLC). The prebiotic potency of XOS was evaluated for probiotics viz. Lactobacillus brevis, Lactobacillus acidophilus and Lactobacillus viridescens in comparison to standard fructooligosaccharides (FOS). The chemical compositional analysis indicated that bagasse contain 28.42% hemicellulose out of which 21.46% was estimated to be xylan. Maximum yield of xylan (20.5%) was obtained with 15%NaOH treatment. The best treatments for xylan hydrolysis were found to be 0.25M H2SO4 for 20 and 40 min with concentrations of xylose, xylobiose and xylotriose to be 2.014, 2.106 and 1.228 mg ml− 1, respectively, in 20 min hydrolysis and 2.138, 1.502 and 0.824 mg ml− 1, respectively, in 40 min hydrolysis. XOS were found to be better prebiotics than standard FOS. Pure xylobiose was found to have highest positive effect on growth of all three bacteria tested indicating that effects of XOS were due to presence of xylobiose, xylotriose and XOS with higher degree of polymerization in xylan hydrolysates. Sugarcane bagasse xylan can be converted into XOS only by controlled acid hydrolysis leading to increased production of XOS which can be used as good prebiotics in drugs and food ingredients after their purification eliminating all the acidic and alkaline residues and also side products.

Pozol is a traditional Mexican refreshing acid beverage made of hot-alkali treated and fermented maize dough. It has been demonstrated that non-amylolytic lactic acid bacteria as Weissella sp. are important in this process. The aim of this work was to study Weissella spp. strains isolated from pozol from the carbon source perspective. For this purpose, Weissella confusa L9, W. confusa L17, W. confusa Snc40, W. paramesenteroides 3a and W. cibaria 3LA were studied. All the microorganisms were able to metabolize xylose; they also showed β-glucosidase and β-galactosidase activities. The ability to grow in xylan was tested; all the strains showed maximum growth at 24 h but W. confusa L9, W. confusa L17 and W. confusa Snc40 grew from 7.09 to 8.66, 7.08 to 7.84 and 7.26 to 7.94 Log CFU/ml, respectively. W. confusa L9 growth in partially hydrolyzed xylan was evaluated. The evolution of xylooligosaccharides throughout the fermentation was performed. Xylose accumulation was observed (0.28–0.28 mg/ml at 24 h). A preference for xylobiose and xylotriose was observed. β-xylosidase activity was found in W. confusa L9 since consumption of xylooligosaccharides by this microorganism was demonstrated. Hemicellulose is therefore an alternative carbon source sustaining pozol fermentation.

Xylan is one of the major polymeric hemicellulosic constituents of lignocellulosic biomass, and its effective utilization by microorganisms is crucial for the economical production of biofuels. In this study, Paenibacillus physcomitrellae XB exhibited different xylan degradation ability on different substrates of corncob xylan (CCX), oat spelt xylan (OSX), wheat flour arabinoxylan (AX) and beech wood xylan (BWX). The RT-QPCR result showed that two genes (Pph_0602 and Pph_2344) belonging to the glycoside hydrolase family 43 were up-regulated more than 5-fold on CCX and xylose. Substrate-specific assays with purified proteins Ppxyl43A (Pph_0602) and Ppxyl43B (Pph_2344) revealed that both exhibited β-xylosidase activity toward the chromogenic substrate p-nitrophenyl-β-D-xylopyranoside, and α-L-arabinofuranosidase activity toward p-nitrophenyl-α-L-arabinofuranoside, indicating their bifunctionality. By testing their degradation characteristics on different natural substrates, it was found that both Ppxyl43A and Ppxyl43B showed similar degradation ability on CCX and OSX. Both enzymes could hydrolyze xylohexaose and xylobiose completely to xylose, but could not hydrolyze BWX and AX, suggesting they mainly hydrolyze xylo-oligosaccharides by β-xylosidase activity. Further analysis showed that both of them displayed very high pH stability and thermostability on the β-xylosidase activity, but Ppxy143B exhibited wider pH and temperature ranges, higher pH and temperature stability, was less influenced by metal ions, and had a slower start-up response than Ppxyl43A. Given their predicted structure, it is likely that the enzymatic differences between Ppxyl43A and Ppxyl43B might be related to the extra C-terminus domain (GH43_C2) in Ppxyl43B, which could enhance the enzymatic stability while restricting the substrates’ or metal ions’ access to the active sites of Ppxyl43B. In conclusion, both Ppxyl43A and Ppxyl43B were β-xylosidase/α-L-arabinofuranosidase bifunctional enzymes and might be useful in xylan biomass conversion, especially in the hydrolysis of xylo-oligosaccharides into xylose.

Camellia oleifera shell (COS), a by-product of processing woody vegetable oil, is rich in hemicellulose and lignin. In this study, we investigated the effects of acid concentration, pretreatment temperature and reaction time on the concentration and yield of xylo-oligosaccharides (XOS) and the degree of polymerization (DP) distribution of XOS when pretreating COS with malic acid (MA). Under moderate condition (2 M MA, 120 ℃, 30 min), the maximum yield of XOS with DP 2–4 was 48.78% (based on the initial xylan) with low xylose, 5-hydroxymethylfurfural (HMF) and furfural, in which xylobiose (X2), xylotriose (X3) and xylotraose (X4) concentrations were 5.22 g/L, 2.75 g/L and 2.91 g/L, respectively. In addition, acid-insoluble lignin (AIL) in the residue after MA pretreatment and milling wood lignin (MWL) were mainly composed of guaiacyl and syringyl. AIL has higher thermal stability than MWL, which can be the stabilizer for producing flame-resistant materials.

Xylooligosaccharides (XOS) can be obtained from sugarcane straw hydrolyzed liquor (SSHL) after pre-treatment by deacetylation followed by hydrothermal process. The fractionation of low molecular weight XOS (LMW XOS) such as xylobiose (X2) and xylotriose (X3) from high molecular weight XOS (HMW XOS) such as xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) is a challenge to be overcome in SSHL for either biotechnological applications or functional foods applications with prebiotic potential. In this study, we proposed and investigated the nanofiltration (NF) membranes technology configured as a serial process for XOS fractionation from SSHL to isolate LMW XOS. In our findings, the first membrane selected was NP030 (polyethersulfone material with MWCO of 500–600 Da) and the second one was TS40 (polypiperazine material with MWCO of 200–300 Da). Then, the selected membranes were operationally optimized for pressure, temperature, and pH: target in NP030 was for increasing the HMW XOS rejection with its best conditions under 40 bar, 20 °C, and pH 4.6, while targets in TS40 were for increasing the LMW XOS rejection and decreasing the xylose (X1) rejection with its best conditions under 20 bar, 50 °C, and pH 4.6. Finally, the operation of diafiltration (DF) was investigated and it was found that 1-time DF in each membrane could improve the performance indices for LMW XOS in the serial membrane-based process, achieving final values of 3.7 g/L of concentration, 20.9% purity, and 46.0% yield. This study brought great contribution to XOS fractionation using NF membranes technology, mainly due to the protocol of investigating DF influence.

A GH10 xylanase Srxyn10 from Streptomyce rochei L10904, and its truncated derivative, Srxyn10M, were investigated. Both displayed great salt-tolerant ability, retaining more than 95% and 91% activity after incubation at 37°C for 1h in 3.0M and 5.0M NaCl, respectively. They exhibited a special hydrolytic property of forming xylobiose as the major product and produced fewer xylose compounds when combined with a reported xylanase while digesting corncob xylans. The mutant, Srxyn10M, was constructed from Srxyn10 by deleting the C-terminal carbohydrate-binding module. It possessed a 3.26-fold higher specific activity on beechwood xylan than Srxyn10. Moreover, Srxyn10M showed greater substrate affinity and catalytic efficiency than Srxyn10 when beechwood xylan, birchwood xylan, and oat-spelt xylan were used as substrates. The thermostability was also greatly improved. Therefore, the application potential was markedly enhanced by the improvement of these properties.

Xylanases produce xylooligosaccharides from xylan and have thus attracted increasing attention for their usefulness in industrial applications. Previously, we demonstrated that the GH11 xylanase XynLC9 from Bacillus subtilis formed xylobiose and xylotriose as the major products with negligible production of xylose when digesting corncob-extracted xylan. Here, we aimed to improve the catalytic performance of XynLC9 via protein engineering. Based on the sequence and structural comparisons of XynLC9 with the xylanases Xyn2 from Trichoderma reesei and Xyn11A from Thermobifida fusca, we identified the N-terminal residues 5-YWQN-8 in XynLC9 as engineering hotspots and subjected this sequence to site saturation and iterative mutagenesis. The mutants W6F/Q7H and N8Y possessed a 2.6- and 1.8-fold higher catalytic activity than XynLC9, respectively, and both mutants were also more thermostable. Kinetic measurements suggested that W6F/Q7H and N8Y had lower substrate affinity, but a higher turnover rate (kcat), which resulted in increased catalytic efficiency than WT XynLC9. Furthermore, the W6F/Q7H mutant displayed a 160% increase in the yield of xylooligosaccharides from corncob-extracted xylan. Molecular dynamics simulations revealed that the W6F/Q7H and N8Y mutations led to an enlarged volume and surface area of the active site cleft, which provided more space for substrate entry and product release and thus accelerated the catalytic activity of the enzyme. The molecular evolution approach adopted in this study provides the design of a library of sequences that captures functional diversity in a limited number of protein variants.