Mannobiose

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.

Mycotoxins are the secondary fungal metabolites generally produced by wide range of fungi including aflatoxins (AF), ochratoxin A (OTA), fumonisins (FB), zearalenone (ZEN), and deoxynivalenol (DON). Nowadays, they are main concern to food and agricultural commodities due to undesirable health and socio-economic effect. This investigation was designed to synthesized microcapsules loaded the bioactive compounds of date seed and evaluated its inhibitory activities in mice received mold-contaminated diet. The finding revealed that the developed microcapsule is homogenous and mostly spherical with size of 2.58 µm with acceptable PDI of 0.21. The main phytochemical has been confirmed by HPLC analysis were xylose, fructose, mannose, glucose and galactose with the respective values of 41.95, 2.24, 5.27 and 0.169 percent. The invivo analyses manifested that the mice received date seed microcapsules significantly (p < 0.05) improved the average daily weight gain, feed intake, liver enzymes (ALT, ALP and AST) and lipid peroxidation values compare to mice group received mycotoxin-contaminated diet. Furthermore, encapsulation date seed bioactive compounds notably up-regulated the expression of GPx, SOD, IFN-γ and IL-2 genes while down-regulated the iNOS gene. Consequently, the novel microcapsules loaded date seed is suggested to considered as a promising mycotoxin inhibitor.

Low-cost plant substrates, such as soybean hulls, are used for various industrial applications. Filamentous fungi are important producers of Carbohydrate Active enZymes (CAZymes) required for the degradation of these plant biomass substrates. CAZyme production is tightly regulated by several transcriptional activators and repressors. One such transcriptional activator is CLR-2/ClrB/ManR, which has been identified as a regulator of cellulase and mannanase production in several fungi. However, the regulatory network governing the expression of cellulase and mannanase encoding genes has been reported to differ between fungal species. Previous studies showed that Aspergillus niger ClrB is involved in the regulation of (hemi-)cellulose degradation, although its regulon has not yet been identified. To reveal its regulon, we cultivated an A. niger ΔclrB mutant and control strain on guar gum (a galactomannan-rich substrate) and soybean hulls (containing galactomannan, xylan, xyloglucan, pectin and cellulose) to identify the genes that are regulated by ClrB. Gene expression data and growth profiling showed that ClrB is indispensable for growth on cellulose and galactomannan and highly contributes to growth on xyloglucan in this fungus. Therefore, we show that A. niger ClrB is crucial for the utilization of guar gum and the agricultural substrate, soybean hulls. Moreover, we show that mannobiose is most likely the physiological inducer of ClrB in A. niger and not cellobiose, which is considered to be the inducer of N. crassa CLR-2 and A. nidulans ClrB.

β-Mannan is abundant in the human diet and in hemicellulose derived from softwood. Linear or galactose-substituted β-mannan-oligosaccharides (MOS/GMOSs) derived from β-mannan are considered emerging prebiotics that could stimulate health-associated gut microbiota. However, the underlying mechanisms are not yet resolved. Therefore, this study investigated the cross-feeding and metabolic interactions between Bifidobacterium adolescentis ATCC 15703, an acetate producer, and Roseburia hominis A2-183 DSMZ 16839, a butyrate producer, during utilization of MOS/GMOSs. Cocultivation studies suggest that both strains coexist due to differential MOS/GMOS utilization, along with the cross-feeding of acetate from B. adolescentis E194a to R. hominis A2-183. The data suggest that R. hominis A2-183 efficiently utilizes MOS/GMOS in mono- and cocultivation. Notably, we observed the transcriptional upregulation of certain genes within a dedicated MOS/GMOS utilization locus (RhMosUL), and an exo-oligomannosidase (RhMan113A) gene located distally in the R. hominis A2-183 genome. Significantly, biochemical analysis of β-1,4 mannan-oligosaccharide phosphorylase (RhMOP130A), α-galactosidase (RhGal36A), and exo-oligomannosidase (RhMan113A) suggested their potential synergistic role in the initial utilization of MOS/GMOSs. Thus, our results enhance the understanding of MOS/GMOS utilization by potential health-promoting human gut microbiota and highlight the role of cross-feeding and metabolic interactions between two secondary mannan degraders inhabiting the same ecological niche in the gut.

Carbon catabolite repression (CCR) enables preferential utilization of easily metabolizable carbon sources, implying the presence of mechanisms to ensure discriminatory gene repression depending on the ambient carbon sources. However, the mechanisms for such hierarchical repression are not precisely understood. In this report, we examined how deletion of pkaA and ganB, which encode cAMP signaling factors, and creA, which encodes a well-characterized repressor of CCR, affects CCR of hemicellulase genes in the filamentous fungus Aspergillus nidulans. β-Xylanase production increased not only in ΔcreA but also in ΔpkaA and ΔganB, with the highest level observed in their double deletants, irrespective of the presence or absence of D-glucose. Expression of the β-xylanase genes in the presence of D-glucose was de-repressed in all the deletion mutants, with significantly higher tolerance against D-glucose repression in ΔpkaA and ΔganB than in ΔcreA. In the presence of galactomannan and D-glucose, partial de-repression of β-mannanase production was detected in ΔcreA, but not in ΔpkaA and ΔganB. The double deletion of creA/pkaA and creA/ganB led to earlier production. Release from D-glucose repression of the β-mannanase genes was partial in the single deletants, while nearly full de-repression was observed in ΔcreAΔpkaA and ΔcreAΔganB. The contribution of PkaA and GanB to CCR by D-xylose of the β-mannanase genes was very minor compared to that of CreA. Consequently, the present study revealed that cAMP signaling plays a major role in CCR of hemicellulase gene expression in a manner that is clearly independent from CreA.

The study of specific glycan uptake and metabolism is an effective tool in aiding with the continued unravelling of the complexities in the human gut microbiome. To this aim fluorescent labelling of glycans may provide a powerful route towards this target. Here, we successfully used the fluorescent label 2-aminobenzamide (2-AB) to monitor and study microbial degradation of labelled glycans. Both single strain and co-cultured fermentations of microbes from the common human-gut derived Bacteroides genus, are able to grow when supplemented with 2-AB labelled glycans of different monosaccharide composition, degrees of acetylation and polymerization. Utilizing a multifaceted approach that combines chromatography, mass spectrometry, microscopy and flow cytometry techniques, it is possible to better understand the metabolism of labelled glycans in both supernatants and at a single cell level. We envisage this combination of complementary techniques will help further the understanding of substrate specificity and the role it plays within microbial communities.

This study aimed to produce manno-oligosaccharides (MOS) from Gleditsia microphylla galactomannans (GMG) using CO₂ and solid acid (Amberlyst-35) in subcritical water. The optimal condition for MOS preparation was 3 MPa CO₂, 0.1 g/g solid acid (relative to GMG) at 150°C for 40 min. The maximum MOS yield with a degree of polymerization from 2 to 4 (M₂-M₄) was 52.19%, which doubled the yield of MOS compared to either using solely CO₂ or solid acid. Solid acid showed excellent performance in producing MOS under subcritical H₂O-CO₂ condition, due to the enhanced mass transfer efficiency and increased H⁺ concentration in the reaction system. The solid acid can be easily separated and reused. Comparing with traditional methods used to produce MOS, this approach has many merits such as higher galactomannan hydrolysis efficiency (largely reduced time and higher MOS yield), purer M₂-M₄ product, lower costs, and more environmental-friendly.

Retaining β-mannanases are glycoside hydrolases (GHs) that can potentially be applied for synthesis of glycosides by catalysis of transglycosylation reactions. A novel active-site double mutant (R171K/E205D) of the catalytic module (CM) of the family GH5 Trichoderma reesei β-mannanase (TrMan5A) was expressed in Pichia pastoris and purified. TrMan5A, CM and CM-variants R171K and R171K/E205D had pH optima between pH 4.0-5.3 and showed >80% remaining activity after incubation at 40°C for 48 h. The enzymes were screened for transglycosylation capacity toward oligomeric and polymeric donor substrates and alcohol acceptors using mass-spectrometry. Hydrolysis and transglycosylation products were analysed by a novel HPLC procedure using an NH₂ column. R171K/E205D was superior in reactions with mannotetraose and the acceptor allyl alcohol, it had twice as high propensity for transglycosylation as wild-type TrMan5A. Wild-type TrMan5A produced the highest amounts of allyl β-mannosides (with 1-3 mannosyls) from locust bean galactomannan. Applying enzyme synergy, adding the GH27 guar α-galactosidase to the reaction (to cleave off galactomannan side-groups), gave a 2.1-fold increase of allyl mannosides and simultaneously a significant production of allyl galactopyranoside, increasing overall yield of allyl glycosides 4.4-fold, from 2.2% to 9.8%. The enzymatic synthesis of reactive allyl glycosides opens up for production of novel biomaterials and glycopolymers.

In this study, a GH26 endo-mannanase (Man26A) from an Aspergillus niger ATCC 10864 strain, with a molecular mass of 47.8 kDa, was cloned in a yBBH1 vector and expressed in Saccharomyces cerevisiae Y294 strain cells. Upon fractionation by ultra-filtration, the substrate specificity and substrate degradation pattern of the endo-mannanase (Man26A) were investigated using ivory nut linear mannan and two galactomannan substrates with varying amounts of galactosyl substitutions, guar gum and locust bean gum. Man26A exhibited substrate specificity in the order: locust bean gum ≥ ivory nut mannan > guar gum; however, the enzyme generated more manno-oligosaccharides (MOS) from the galactomannans than from linear mannan during extended periods of mannan hydrolysis. MOS with a DP of 2–4 were the major products from mannan substrate hydrolysis, while guar gum also generated higher DP length MOS. All the Man26A generated MOS significantly improved the growth (approximately 3-fold) of the probiotic bacterial strains Streptococcus thermophilus and Bacillus subtilis in M9 minimal medium. Ivory nut mannan and locust bean gum derived MOS did not influence the auto-aggregation ability of the bacteria, while the guar gum derived MOS led to a 50 % reduction in bacterial auto-aggregation. On the other hand, all the MOS significantly improved bacterial biofilm formation (approximately 3-fold). This study suggests that the prebiotic characteristics exhibited by MOS may be dependent on their primary structure, i.e. galactose substitution and DP. Furthermore, the data suggests that the enzyme-generated MOS may be useful as potent additives to dietary foods.

The present investigation was focused on the production of extracellular β-mannanase from newly isolated Penicillium aculeatum APS1 using palm kernel cake and soyabean meal which are the residual by-products of oil extraction industry, under solid state fermentation. On supplementing palm kernel cake with 20% soyabean meal, yield of β-mannanase production was reached to 2807 U/g. Response surface methodology was used for statistical optimization of β-mannanase production. Two independent variables, namely moisture level and incubation time, were found to be significantly contributing for the production process. Under optimized condition of moisture level (52.25%) and incubation time (130 h), the yield of β-mannanase was improved by 1.6 fold and maximum activity of β-mannanase was 4696 U/g. The optimum temperature and pH for crude β-mannanase were 65°C and 6.0, respectively. Crude and partially purified β-mannanase was found to be effective in release of mannooligosachharides by hydrolysis of mannan rich substrates viz. locust bean gum, guar gum and konjac glucomannan. Qualitative and quantitative analysis of MOS was carried out by TLC and ion chromatography. β-Mannanase from Penicillium aculeatum APS1 was found to have properties suitable for applications in feed/food industry.