Interactive Effects of Arabinoxylan Oligosaccharides and Green Tea Polyphenols on Obesity Management and Gut Microbiota Modulation in High-Fat Diet-Fed Mice.

Dietary fiber and polyphenols have been shown to possess antiobesity properties. However, their combined effects need further investigation. This study investigated the individual and combined effects of arabinoxylan oligosaccharides (AXOS) from rice bran and green tea polyphenols (GTP) in high-fat diet-induced obese mice. We found that the combination of AXOS and GTP (A + G) significantly reduced overall fat mass and improved lipid profiles, although the effects were not synergistic. AXOS and GTP regulated lipid metabolism in different tissues and exhibited counteractive effects on gut microbiota. AXOS decreased α diversity and promoted Bifidobacterium, with GTP counteracting these effects. In vitro fermentation confirmed that GTP counteracted AXOS-induced microbiota changes in a dose-dependent manner. This study highlights the potential of tailored combinations of dietary fiber and polyphenols to treat obesity while considering their complex microbial interplay.

BnXTH1 regulates cadmium tolerance by modulating vacuolar compartmentalization and the cadmium binding capacity of cell walls in ramie (Boehmeria nivea).

Xyloglucan endotransglucosylase/hydrolases (XTH) are cell wall-modifying enzymes important in plant response to abiotic stress. However, the role of XTH in cadmium (Cd) tolerance in ramie remains largely unknown. Here, we identified and cloned BnXTH1, a member of the XTH family, in response to Cd stress in ramie. The BnXTH1 promoter (BnXTH1p) demonstrated that MeJA induces the response of BnXTH1p to Cd stress. Moreover, overexpressing BnXTH1 in Boehmeria nivea increased Cd tolerance by significantly increasing the Cd content in the cell wall and decreasing Cd inside ramie cells. Cadmium stress induced BnXTH1-expression and consequently increased xyloglucan endotransglucosylase (XET) activity, leading to high xyloglucan contents and increased hemicellulose contents in ramie. The elevated hemicellulose content increased Cd chelation onto the cell walls and reduced the level of intracellular Cd. Interestingly, overexpressing BnXTH1 significantly increased the content of Cd in vacuoles of ramie and vacuolar compartmentalization genes. Altogether, these results evidence that Cd stress induced MeJA accumulation in ramie, thus, activating BnXTH1 expression and increasing the content of xyloglucan to enhance the hemicellulose binding capacity and increase Cd chelation onto cell walls. BnXTH1 also enhances the vacuolar Cd compartmentalization and reduces the level of Cd entering the organelles and soluble solution.

Mapping secondary substrate-binding sites on the GH11 xylanase from Bacillus subtilis.

Xylanases are of significant interest for biomass conversion technologies. Here, we investigated the allosteric regulation of xylan hydrolysis by the Bacillus subtilis GH11 endoxylanase. Molecular dynamics simulations (MDS) in the presence of xylobiose identified binding to the active site and two potential secondary binding sites (SBS) around surface residues Asn54 and Asn151. Arabinoxylan titration experiments with single cysteine mutants N54C and N151C labeled with the thiol-reactive fluorophore acrylodan or the ESR spin-label MTSSL validated the MDS results. Ligand binding at the SBS around Asn54 confirms previous reports, and analysis of the second SBS around N151C discovered in the present study includes residues Val98/Ala192/Ser155/His156. Understanding the regulation of xylanases contributes to efforts for industrial decarbonization and to establishing a sustainable energy matrix.

Structure and function characterization of the α-L-arabinofuranosidase from the white-rot fungus Trametes hirsuta.

α-L-Arabinofuranosidases (Abfs) play a crucial role in the degradation of hemicelluloses, especially arabinoxylans (AX). Most of the available characterized Abfs are from bacteria, while fungi, as natural decomposers, contain Abfs with little attention given. An arabinofuranosidase (ThAbf1), belonging to the glycoside hydrolase 51 (GH51) family, from the genome of the white-rot fungus Trametes hirsuta, was recombinantly expressed, characterized, and functionally determined. The general biochemical properties showed that the optimal conditions for ThAbf1 were pH 6.0 and 50°C. In substrate kinetics assays, ThAbf1 preferred small fragment arabinoxylo-oligosaccharides (AXOS) and could surprisingly hydrolyze di-substituted 23,33-di-L-arabinofuranosyl-xylotriose (A2,3XX). It also synergized with commercial xylanase (XYL) and increased the saccharification efficiency of arabinoxylan. The crystal structure of ThAbf1 indicated the presence of an adjacent cavity next to the catalytic pocket which led to the ability of ThAbf1 to degrade di-substituted AXOS. The narrow binding pocket prevents ThAbf1 from binding larger substrates. These findings have strengthened our understanding of the catalytic mechanism of GH51 family Abfs and provided a theoretical foundation for the development of more efficient and versatile Abfs to accelerate the degradation and biotransformation of hemicellulose in biomass. KEY POINTS: • ThAbf1 from Trametes hirsuta degraded di-substituted arabinoxylo-oligosaccharide. • ThAbf1 performed detailed biochemical characterization and kinetics. • ThAbf1 structure has been obtained to illustrate the substrate specificity.

Fusion of Oligopeptide to the C Terminus of α-Glucuronidase from Thermotoga maritima Improves the Catalytic Efficiency for Hemicellulose Biotransformation.

Fusion protein combined the oligopeptide (HQAFFHA) with the C terminus of α-glucuronidase from Thermotoga maritima was produced in E. coli and purified for characterization and applications of glucuronic and glucaric acid production. The fusion protein with oligopeptide exhibited a 2.97-fold higher specific activity than individual protein. Their catalytic efficiency kcat/Km and kcat increased from 469.3 ± 2.6 s-1 (g mL-1)-1 and 62.4 ± 0.9 s-1 to 2209.5 ± 26.3 s-1 (g mL-1)-1 and 293.9 ± 4.9 s-1, respectively. Fusion protein had similar temperature and pH profiles to those without oligopeptide, but the thermal stability decreases and the pH stability shifts to alkaline. Using beech xylan hydrolysate as a substrate, the glucuronic acid yield of fusion enzyme increased by 9.94% compared with its parent at 65 °C pH 8.5 for 10 h, and can hydrolyze corn cob xylan with xylanase to obtain glucuronic acid, and can be combined with uronate dehydrogenase to obtain high-added value glucaric acid. Homologous modeling analysis revealed the factors contributing to the high catalytic efficiency of fusion enzyme. These results show that the peptide fusion strategy described here may be useful for improving the catalytic efficiency and stability of other industrial enzymes, and has great potential for producing high value-added products from agricultural waste.

Expression of an extremophilic xylanase in Nicotiana benthamiana and its use for the production of prebiotic xylooligosaccharides.

A gene construct encoding a xylanase, which is active in extreme conditions of temperature and alkaline pH (90 °C, pH 10.5), has been transitorily expressed with high efficiency in Nicotiana benthamiana using a viral vector. The enzyme, targeted to the apoplast, accumulates in large amounts in plant tissues in as little as 7 days after inoculation, without detrimental effects on plant growth. The properties of the protein produced by the plant, in terms of resistance to temperature, pH, and enzymatic activity, are equivalent to those observed when Escherichia coli is used as a host. Purification of the plant-produced recombinant xylanase is facilitated by exporting the protein to the apoplastic space. The production of this xylanase by N. benthamiana, which avoids the hindrances derived from the use of E. coli, namely, intracellular production requiring subsequent purification, represents an important step for potential applications in the food industry in which more sustainable and green products are continuously demanded. As an example, the use of the enzyme producing prebiotic xylooligosdaccharides from xylan is here reported.

Exploring competitive inhibition of a family 10 xylanase derived from Hu sheep rumen microbiota by Oryza sativa xylanase inhibitor protein: In vitro and in silico perspectives.

The catalytic domain of family GH10 xylanase, XYN-LXY_CD derived from Hu sheep rumen microbiota was expressed in Pichia pastoris X33. The special activity of reXYN-LXY_CD in the culture supernatant was 232.56 U/mg. The optima of reXYN-LXY_CD were 53 °C and pH 7.0. Recombinant Oryza sativa xylanase inhibitor protein (rePOsXIP) competitively inhibited reXYN-LXY_CD with an inhibition constant (Ki) value of 237.37 nM. The concentration of hydrolysates released from beechwood xylan by reXYN-LXY_CD reduced when rePOsXIP was added into the hydrolytic system. Fluorescence of reXYN-LXY_CD was statically quenched by rePOsXIP in a dose-dependent manner. The details in intermolecular interaction between XYN-LXY_CD and OsXIP were investigated by using molecular dynamics (MD) simulations, binding free energy computation and non-covalent interactions (NCI) analysis. Hydrogen bonding and van der Waals played indispensable roles in the XYN-LXY_CD/OsXIP interaction. The α-7 helix of OsXIP tightly occupied the catalytic pocket of XYN-LXY_CD with hydrogen bonding such as K239OsXIP-N261/Q292/E197XYN-LXY_CD (E197, the acid-base catalytic residue), D236OsXIP-K327XYN-LXY_CD and Q242OsXIP-E211/Q212XYN-LXY_CD. Based on the quantum theory of atoms in molecules (QTAIM), the Laplacian of electron density and core-valence bifurcation index of HZ3K239-OE2E197 were 0.1025 a.u. and 0.002218, respectively. Elucidating the mechanism underlying xylanase-inhibitor interactions might help construct XYN-LXY_CD mutants that gain resistance to XIPs and high catalytic activity, which would be more efficient in feed additives in livestock.

Experimental and in silico studies of competitive inhibition of family GH10 Aspergillus fumigatus xylanase A by Oryza sativa xylanase inhibitor protein.

The family GH10 Aspergillus fumigatus xylanase A (AfXylA10) gene, afxyla10 was cloned and recombinantly expressed in Pichia pastoris X33. The optimum temperature and pH of reAfXylA10 was 53 °C and 7.0, and Mn2+ remarkably activated the catalytic activity. The recombinant Oryza sativa xylanase inhibitor protein, rePOsXIP significantly inhibited reAfXylA10 with inhibition constant (Ki) of 177.94 nM via competitive inhibition and decreased the concentration of hydrolysate from beechwood xylan. Optimal inhibition of rePOsXIP on reAfXylA10 occurred at 45 °C for 40 min. The fluorescence of reAfXylA10 was statically quenched by rePOsXIP, indicating the formation of reAfXylA10-rePOsXIP complex during their interaction. Furthermore, molecular dynamics (MD) simulations were performed to obtain the detailed information on enzyme-inhibitor interaction. The binding free energy (ΔG) of AfXylA10-OsXIP complex was -30 ± 9 kcal/mol by MM-PBSA calculation, and the α-7 helix of OsXIP anchored in the catalytic cleft of AfXylA10 by competition with the xylan substrate. K239OsXIP stably interacted with the catalytic site E140AfXylA10 through hydrogen bond and vdW interaction. Intermolecular hydrogen bonds T104AfXylA10/V99AfXylA10-Q5OsXIP, R256AfXylA10-E235OsXIP, D155AfXylA10-Y243OsXIP and D145AfXylA10-R194OsXIP on the upper of the TIM barrel were essential for strengthening the stability of complex. Therefore, these non-covalent interactions (NCI) played key role in the interaction between AfXylA10 and OsXIP.

Hevea brasiliensis coniferaldehyde-5-hydroxylase (HbCAld5H) regulates xylogenesis, structure and lignin chemistry of xylem cell wall in Nicotiana tabacum.

KEY MESSAGE: The HbCAld5H1 gene cloned from Hevea brasiliensis regulates the cambial activity, xylem differentiation, syringyl-guaiacyl ratio, secondary wall structure, lignification pattern and xylan distribution in xylem fibres of transgenic tobacco plants. Molecular characterization of lignin biosynthesis gene coniferaldehyde-5-hydroxylase (CAld5H) from Hevea brasiliensis and its functional validation was performed. Both sense and antisense constructs of HbCAld5H1 gene were introduced into tobacco through Agrobacterium-mediated genetic transformation for over expression and down-regulation of this key enzyme to understand its role affecting structural and cell wall chemistry. The anatomical studies of transgenic tobacco plants revealed the increase of cambial activity leading to xylogenesis in sense lines and considerable reduction in antisense lines. The ultra-structural studies showed that the thickness of secondary wall (S2 layer) of fibre had been decreased with non-homogenous lignin distribution in antisense lines, while sense lines showed an increase in S2 layer thickness. Maule color reaction revealed that syringyl lignin distribution in the xylem elements was increased in sense and decreased in antisense lines. The immunoelectron microscopy revealed a reduction in LM 10 and LM 11 labelling in the secondary wall of antisense tobacco lines. Biochemical studies showed a radical increase in syringyl lignin in sense lines without any significant change in total lignin content, while S/G ratio decreased considerably in antisense lines. Our results suggest that CAld5H gene plays an important role in xylogenesis stages such as cambial cell division, secondary wall thickness, xylan and syringyl lignin distribution in tobacco. Therefore, CAld5H gene could be considered as a promising target for lignin modification essential for timber quality improvement in rubber.

Isolation of Clostridium from Yunnan-Tibet hot springs and description of Clostridium thermarum sp. nov. with lignocellulosic ethanol production.

Lignocellulose is considered a major source of renewable energy that serve as an alternative to the fossil fuels. Members of the genus Clostridium are some of the many microorganisms that have the ability to degrade lignocellulose efficiently to sugar, which can be further converted to biofuel. In this study, we isolated twelve Clostridium strains from hot spring samples of Yunnan and Tibet, of which isolates SYSU GA15002T and SYSU GA17076 showed low 16S rRNA gene sequence identity profiles to any of the validly named Clostridium strains (<94.0%). Studies using a polyphasic taxonomy approach concluded that the two isolates represent one novel species of the genus Clostridium, for which we propose the name Clostridium thermarum sp. nov., with SYSU GA15002T as the type strain of the species. Isolate SYSU GA15002T has an optimum growth temperature at 45°C. Fermentation of the substrates cellobiose, cellulose, xylan and untreated straw powder by this strain results in the production of ethanol, along with acetate and formate. The complete pathways for the conversion of cellulose and xylan to ethanol is also predicted from the genome of isolate SYSU GA15002T, which revealed a single step conversion of lignocellulosic biomass through consolidated bioprocessing. This paper is a comprehensive study encompassing isolation, polyphasic taxonomy, lignocellulose biodegradation and the genomic information of Clostridium in Yunnan-Tibet hot springs.

Conservation of Xylose Fermentability in Phlebia Species and Direct Fermentation of Xylan by Selected Fungi.

In efforts to lower the cost of total conversion of lignocellulosic materials, utilization of hemicellulose must be considered. White-rot fungus Phlebia sp. MG-60 can produce ethanol directly from cellulose and has fermentation ability for glucose, cellulose, and xylose. Therefore, white-rot fungi can be considered a good candidate for consolidated bioprocessing to give bioethanol from lignocellulosic biomass, although little information is available on the direct fermentation of xylan. In the present study, some Phlebia species were selected as candidates because of their ability to ferment xylose to ethanol more efficiently than Phlebia sp. MG-60. This process indicated that the basidiomycetes that can produce ethanol from xylose are closely related genetically within the Phlebia genus. The selected Phlebia species showed higher ethanol productivity from corn core and beechwood xylans than Phlebia sp. MG-60. The ethanol yields from corn core xylan in culture with Phlebia acerina HHB11146, Phlebia ludoviciana HHB9640, and Phlebia subochracea HHB8494 were 46.2%, 46.7%, and 39.7% of theoretical maximum, and those from beechwood xylan were 19.09%, 17.7%, and 21.4% of the theoretical maximum, respectively.

Significance of a family-6 carbohydrate-binding module in a modular feruloyl esterase for removing ferulic acid from insoluble wheat arabinoxylan.

Ruminiclostridium josui Fae1A is a modular enzyme consisting of an N-terminal signal peptide, family-1 carbohydrate esterase module (CE1), family-6 carbohydrate-binding module (CBM6), and dockerin module in that order. Recombinant CE1 and CBM6 polypeptides were collectively and separately produced as RjFae1A, RjCE1, and RjCBM6. RjFae1A showed higher feruloyl esterase activity than RjCE1 towards insoluble wheat arabinoxylan, but the latter was more active towards small synthetic substrates than the former. This suggests that CBM6 in RjFae1A plays an important role in releasing ferulic acid from the native substrate. RjCBM6 showed a higher affinity for soluble wheat arabinoxylan than for rye arabinoxylan and beechwood xylan in native affinity polyacrylamide gel electrophoresis. Isothermal titration calorimetry analysis demonstrated that RjCBM6 recognized a xylopyranosyl residue at the nonreducing ends of xylooligosaccharides. Moreover, it showed exceptional affinity for 23-α-l-arabinofuranosyl-xylotriose (A2XX) among the tested branched arabinoxylooligosaccharides. Fluorometric titration analysis demonstrated that xylobiose and A2XX competitively bound to RjCBM6, and both bound to the same site in RjCBM6. RjCBM6's preference for the xylopyranosyl residue at the nonreducing end of xylan chains explains why the positive effect of CBM6 on RjFae1A activity was observed only during short incubation but not after extended incubation.

Refolding and purification of cGMP-grade recombinant human neurturin from Escherichia coli inclusion bodies.

Neurturin is a potent neurotrophic factor that has been investigated as a potential therapeutic agent for the treatment of neurodegenerative diseases, including Parkinson's disease, and, more recently, for the treatment of type II diabetes. However, purification of neurturin for clinical applications has been hampered by its low solubility in aqueous solutions. Here we describe the development of a scalable manufacturing process for recombinant neurturin from E. coli. inclusion bodies. Neurturin was refolded from solubilized inclusion bodies by fed-batch dilution refolding with a titer of 90 mg per liter refold and a refold yield of 89%. A two-step purification process using cation exchange and hydrophobic interaction chromatography, followed by formulation using tangential flow filtration resulted in an overall process yield of about 56 mg purified neurturin per liter refold. Solubility of neurturin during the purification process was maintained by the addition of 15% (w/v) glycerol to all buffers. For clinical applications and parenteral administration glycerol was replaced by 15% (w/v) sulfobutyl ether-beta-cyclodextrin (i.e. Captisol) in the drug substance formulation buffer. The final purified product had low or undetectable levels of product-related impurities and concentrations of process-related contaminants such as host cell proteins, host cell DNA, endotoxins and Triton X-100 were reduced more than 10,000-fold or below the limit of detection. Bioactivity of purified recombinant neurturin was demonstrated in a cell-based assay by activation of the MAPK signaling pathway.

A polyphasic approach to characterize Weissella cibaria and Weissella confusa strains.

AIM: To study Weissella cibaria and Weissella confusa strains, lactic acid bacteria (LAB) members naturally present in food products, but not yet included in Qualified Presumption of Safety (QPS) list of European Food Safety Authority (EFSA). METHODS AND RESULTS: We carried out a comparative genome analysis of 23 sequenced W. cibaria and 7 W. confusa genomes, in parallel with a physiological and functional characterization of several strains previously isolated from sourdough-like maize bran fermentation. The genome analysis revealed the absence of dedicated pathogenicity factors. Some putative virulence genes found in Weissella genomes were also present in other LAB strains, considered safe by EFSA and commonly used as probiotics. The physiological tests carried out on our strains corroborated the genomic results. Moreover, the following functional traits of interest to application in the food sector were identified: the majority of tested strains displayed high acidification rate, high reducing ability, production of exopolysaccharides (EPS), arabinoxylan degradation ability, growth in the presence of fructo-oligosaccharides (FOS), bile and gastric juice tolerance, and antifungal activity. CONCLUSIONS: These results provide evidence for the possible use of selected strains of W. cibaria and W. confusa in the food sector. SIGNIFICANCE AND IMPACT OF THE STUDY: This polyphasic study adds to the body of knowledge on the functional and applicable characteristics of these controversial species of LAB. This knowledge contributes to design new selected cultures included in the QPS list required for food applications.

Higher expression of the strawberry xyloglucan endotransglucosylase/hydrolase genes FvXTH9 and FvXTH6 accelerates fruit ripening.

Fruit softening in Fragaria (strawberry) is proposed to be associated with the modification of cell wall components such as xyloglucan by the action of cell wall-modifying enzymes. This study focuses on the in vitro and in vivo characterization of two recombinant xyloglucan endotransglucosylase/hydrolases (XTHs) from Fragaria vesca, FvXTH9 and FvXTH6. Mining of the publicly available F. vesca genome sequence yielded 28 putative XTH genes. FvXTH9 showed the highest expression level of all FvXTHs in a fruit transcriptome data set and was selected with the closely related FvXTH6 for further analysis. To investigate their role in fruit ripening in more detail, the coding sequences of FvXTH9 and FvXTH6 were cloned into the vector pYES2 and expressed in Saccharomyces cerevisiae. FvXTH9 and FvXTH6 displayed xyloglucan endotransglucosylase (XET) activity towards various acceptor substrates using xyloglucan as the donor substrate. Interestingly, FvXTH9 showed activity of mixed-linkage glucan:xyloglucan endotransglucosylase (MXE) and cellulose:xyloglucan endotransglucosylase (CXE). The optimum pH of both FvXTH9 and FvXTH6 was 6.5. The prediction of subcellular localization suggested localization to the secretory pathway, which was confirmed by localization studies in Nicotiana tabacum. Overexpression showed that Fragaria × ananassa fruits infiltrated with FvXTH9 and FvXTH6 ripened faster and showed decreased firmness compared with the empty vector control pBI121. Thus FvXTH9 and also FvXTH6 might promote strawberry fruit ripening by the modification of cell wall components.

A systems genetics analysis in Eucalyptus reveals coordination of metabolic pathways associated with xylan modification in wood-forming tissues.

Acetyl- and methylglucuronic acid decorations of xylan, the dominant hemicellulose in secondary cell walls (SCWs) of woody dicots, affect its interaction with cellulose and lignin to determine SCW structure and extractability. Genes and pathways involved in these modifications may be targets for genetic engineering; however, little is known about the regulation of xylan modifications in woody plants. To address this, we assessed genetic and gene expression variation associated with xylan modification in developing xylem of Eucalyptus grandis × Eucalyptus urophylla interspecific hybrids. Expression quantitative trait locus (eQTL) mapping identified potential regulatory polymorphisms affecting gene expression modules associated with xylan modification. We identified 14 putative xylan modification genes that are members of five expression modules sharing seven trans-eQTL hotspots. The xylan modification genes are prevalent in two expression modules. The first comprises nucleotide sugar interconversion pathways supplying the essential precursors for cellulose and xylan biosynthesis. The second contains genes responsible for phenylalanine biosynthesis and S-adenosylmethionine biosynthesis required for glucuronic acid and monolignol methylation. Co-expression and co-regulation analyses also identified four metabolic sources of acetyl coenxyme A that appear to be transcriptionally coordinated with xylan modification. Our systems genetics analysis may provide new avenues for metabolic engineering to alter wood SCW biology for enhanced biomass processability.

Xyloglucan exoglycosidases in the monocot model Brachypodium distachyon and the conservation of xyloglucan disassembly in angiosperms.

KEY MESSAGE: Brachypodium distachyon has a full set of exoglycosidases active on xyloglucan, including α-xylosidase, ß-galactosidase, soluble and membrane-bound ß-glucosidases and two α-fucosidases. However, unlike in Arabidopsis, both fucosidases are likely cytosolic. Xyloglucan is present in primary walls of all angiosperms. While in most groups it regulates cell wall extension, in Poaceae its role is still unclear. Five exoglycosidases participate in xyloglucan hydrolysis in Arabidopsis: α-xylosidase, ß-galactosidase, α-fucosidase, soluble ß-glucosidase and GPI-anchored ß-glucosidase. Mutants in the corresponding genes show alterations in xyloglucan composition. In this work putative orthologs in the model grass Brachypodium distachyon were tested for their ability to complement Arabidopsis mutants. Xylosidase and galactosidase mutants were complemented, respectively, by BdXYL1 (Bd2g02070) and BdBGAL1 (Bd2g56607). BdBGAL1, unlike other xyloglucan ß-galactosidases, is able to remove both galactoses from XLLG oligosaccharides. In addition, soluble ß-glucosidase BdBGLC1 (Bd1g08550) complemented a glucosidase mutant. Closely related BdBGLC2 (Bd2g51280), which has a putative GPI-anchor sequence, was found associated with the plasma membrane and only a truncated version without GPI-anchor complemented the mutant, proving that Brachypodium also has soluble and membrane-bound xyloglucan glucosidases. Both BdXFUC1 (Bd3g25226) and BdXFUC2 (Bd1g28366) can hydrolyze fucose from xyloglucan oligosaccharides but were unable to complement a fucosidase mutant. Fluorescent protein fusions of BdXFUC1 localized to the cytosol and both proteins lack a signal peptide. Signal peptides appear to have evolved only in some eudicot lineages of this family, like the one leading to Arabidopsis. These results could be explained if cytosolic xyloglucan α-fucosidases are the ancestral state in angiosperms, with fucosylated oligosaccharides transported across the plasma membrane.

Delineating thermophilic xylanase from Bacillus licheniformis DM5 towards its potential application in xylooligosaccharides production.

In this present study novel endoxylanase producing Bacillus licheniformis DM5 isolated, identified based on 16S rDNA from Garampani hotspring, Assam, India and enzyme was purified. RNA secondary structure predicted the similarity of B. licheniformis DM5 with B. licheniformis ATCC14580. Highest production of xylanase from B. licheniformis DM5 was achieved in the TY medium with cell densities 12 g/l and extracellular protein concentration containing xylanase 400 mg/l. Partially purified extracellular xylanase displayed optimum pH 6.5 and temperature 50 °C. Thermostability of the xylanase at the elevated temperature showed stability between 50 and 60 °C retaining its 99% activity. Kinetic parameters of thermophilic xylanase revealed Km 1.5 ± 0.2 mg/ml, Vmax 2.7 ± 0.2 U/ml and and Kcat 1.8 ± 0.2 s-1 against beechwood xylan but ruled out any exo-acting activity against synthetic pNP-xylopyranoside substrate. Time dependent enzymatic hydrolysis of beechwood xylan and preprocessed agrowaste corncob exhibited the release of xylotriose and xylobiose oligosaccharide (XOS) significantly high. Xylobiose and xylotriose exhibited higher binding affinities with BIAXP transporter protein of probiotic bacteria explaining their easy uptake by the cells. Mixed oligosaccharides also exhibited better prebiotic activity by promoting growth of Bifidobacterium infantis and Lactobacillus delbrueckii. Mixed XOS when tested for their cytotoxicity on Hela cell lines in in vitro MTT assay displayed significant lowering of cell viability after 48 h and 24 h at 100 µg/ml to 60% and 50%, respectively. In contrast, cytotoxicity wasn't observed against normal cervical cell line (VK2/E6E7-ATCC-CRL-2616). Therefore, thermophilic endoxylanase from B. licheniformis DM5 could be attributed for the production of prebiotic and anti-inflammatory XOS from agrowaste.

Male grower pigs fed cereal soluble dietary fibres display biphasic glucose response and delayed glycaemic response after an oral glucose tolerance test.

Acute and sustained soluble dietary fibre (SDF) consumption are both associated with improved glucose tolerance in humans and animal models (e.g. porcine). However, the effects on glucose tolerance in grower pigs, adapted to diets with a combination of SDF have not been studied previously. In this experiment, cereal SDF wheat arabinoxylan (AX) and oat ß-glucan (BG) were fed individually and in combination to determine the effect on glucose tolerance in jugular vein catheterized grower pigs. Five groups of Large White male grower pigs were fed highly digestible diets containing either 10% AX, 10% BG, 5% AX with 5% BG, a model cereal whole wheat flour (WWF), or a control wheat starch diet (WS) with no SDF. Blood was collected via jugular vein catheters over 240 minutes following a feed challenge and an oral glucose tolerance test (OGTT) on two separate days. Postprandial blood samples were used to determine plasma glucose, insulin, non-esterified fatty acids (NEFA), glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), peptide tyrosine tyrosine (PYY), ghrelin, glucagon and cortisol concentrations. No dietary effects on glycaemic response were observed following the feed challenge or the OGTT as determined by the area under the curve (AUC). A biphasic glucose and insulin response was detected for all pigs following the OGTT. The current study showed male grower pigs have tight glycaemic control and glucose tolerance regardless of diet. In addition, pigs fed the combined SDF had a reduced GIP response and delayed insulin peak following the feed challenge. Incretin (GLP-1 and GIP) secretion appeared asynchronous reflecting their different enteroendocrine cell locations and response to nutrient absorption.

Effects of substrate binding site residue substitutions of xynA from Bacillus amyloliquefaciens on substrate specificity.

BACKGROUND: The aromatic residues of xylanase enzyme, W187, Y124, W144, Y128 and W63 of substrate binding pocket from Bacillus amyloliquefaciens were investigated for their role in substrate binding by homology modelling and sequence analysis. These residues are highly conserved and play an important role in substrate binding through steric hindrance. The substitution of these residues with alanine allows the enzyme to accommodate nonspecific substrates. RESULTS: Wild type and mutated genes were cloned and overexpressed in BL21. Optimum pH and temperature of rBAxn exhibited pH 9.0 and 50 °C respectively and it was stable up to 215 h. Along with the physical properties of rBAxn, kinetic parameters (Km 19.34 ± 0.72 mg/ml; kcat 6449.12 ± 155.37 min- 1 and kcat/Km 333.83 ± 6.78 ml min- 1 mg- 1) were also compared with engineered enzymes. Out of five mutations, W63A, Y128A and W144A lost almost 90% activity and Y124A and W187A retained almost 40-45% xylanase activity. CONCLUSIONS: The site-specific single mutation, led to alteration in substrate specificity from xylan to CMC while in case of double mutant the substrate specificity was altered from xylan to CMC, FP and avicel, indicating the role of aromatic residues on substrate binding, catalytic process and overall catalytic efficiency.