Galactopyranosyl Residues Research Articles

Isolation, physical, structural characterization and in vitro prebiotic activity of a galactomannan extracted from endosperm splits of Chinese Sesbania cannabina seeds

The purpose of this study was to identify and determine the physical and structural characterization of the water-soluble galactomannan extracted from endosperm splits of Chinese S. cannabina seeds. The Sesbania galactomannan (SP) was extracted and purified using a novel method with a high yield (40.3 ± 7.2%). The molecular structure of SP was determined by monosaccharide composition, FTIR and NMR spectroscopy. The structural data showed that SP was galactomannan which composed by a β-(1/4)-linked mannose backbone with galactopyranosyl residues attached through α-(1/6) linkages. The constant mannose/galactose (M/G) ratio and average molecular weight (Mw) of SP was 1.6:1 and 2.16 × 105 g/mol, respectively. The physical results revealed that SP had many branches on the backbone and existed as a random coil state in aqueous solution. SP was a good biopolymer which had smooth and clearer surface with homogeneous composition, and had some degree of crystallinity and prebiotic activity. As a consequence, SP could be a potential prebiotic and was expected to be suitable for applications in food, pharmaceutical, biomedical or cosmetic industries as a promising new biomaterial.

High-level expression of a thermophilic and acidophilic β-mannanase from Aspergillus kawachii IFO 4308 with significant potential in mannooligosaccharide preparation

An engineered thermophilic and acidophilic β-mannanase (ManAK) from Aspergillus kawachii IFO 4308 was highly expressed in Pichia pastoris. Through high cell density fermentation, the maximum yield reached 11,600 U/mL and 15.5 g/L, which is higher than most extreme β-mannanases. The recombinant ManAK was thermostable with a temperature optimum of 80 °C, and acid tolerant with a pH optimum of 2.0. ManAK could efficiently degrade locust bean gum, konjac gum, and guar gum into small molecular mannooligosaccharide (<2000 Da), even at high initial substrate concentration (10%), and displayed different Mw distributions in their end products. Docking analysis demonstrated that the catalytic pocket of ManAK could only accommodate a galactopyranosyl residue in subsite −1, which might be responsible for the distinct hydrolysis product compositions from locust bean gum and guar gum. These superior properties of ManAK strongly facilitate mannooligosaccharide preparation and application in food and feed area.

Composition and biosynthetic machinery of the Blumeria graminis f. sp. hordei conidia cell wall

Infection of barley with the powdery mildew causal agent, Blumeria graminis f. sp. hordei (Bgh), can lead to devastating damage to barley crops. The recent emergence of fungicide resistance imposes a need to develop new antifungal strategies. The enzymes involved in cell wall biosynthesis are ideal targets for the development of fungicides. However, in order to narrow down any target proteins involved in cell wall formation, a greater understanding of the cell wall structure and composition is required. Here, we present a detailed carbohydrate analysis of the Bgh conidial cell wall, a full annotation of Carbohydrate Active enZymes (CAZy) in the Bgh genome, and a comprehensive expression profile of the genes involved in cell wall metabolism. Glycosidic linkage analysis has revealed that the cell wall polysaccharide fraction of Bgh conidia predominantly consists of glucosyl residues (63.1%) and has a greater proportion of galactopyranosyl residues compared to other species (8.5%). Trace amounts of xylosyl residues were also detected, which is unusual in ascomycetes. Transcripts of the genes involved in cell wall metabolism show high expression of chitin deacetylases, which assist fungi in evading the host defence system by deacetylating chitin to chitosan. The data presented suggest that the cell wall components of the conidia and the corresponding obligate biotrophic CAZy gene profile play a key role in the infection process.

Cooperation between β-galactosidase and an isoprimeverose-producing oligoxyloglucan hydrolase is key for xyloglucan degradation in Aspergillusoryzae.

The galactosylation of xyloglucan blocks many of the enzymatic processes targeting this oligosaccharide. We found that the expression of a gene encoding Aspergillusoryzae β-galactosidase (LacA) is induced in the presence of xyloglucan oligosaccharides. With detailed analyses of the substrate specificity of purified recombinant LacA, we show that LacA cleaves galactopyranosyl residues from xyloglucan oligosaccharides, but not from xyloglucan polysaccharide, and plays a vital role in xyloglucan degradation. LacA acts cooperatively with the isoprimeverose-producing oligoxyloglucan hydrolase IpeA to hydrolyze xyloglucan oligosaccharides. Galactosylation of the xylopyranosyl side chain at the nonreducing end of oligoxyloglucan saccharides completely abolishes IpeA activity while LacA efficiently removes the galactopyranosyl residue. Conversely, an isoprimeverose unit at the nonreducing end of the main chain of xyloglucan oligosaccharides blocks LacA activity, while IpeA can still remove the isoprimeverose moiety. This is the first study reporting the cooperative action of β-galactosidase and isoprimeverose-producing oligoxyloglucan hydrolase on xyloglucan oligosaccharide degradation. Our findings shed light on the true role of LacA and the enzymatic coordination between β-galactosidase and other hydrolases on xyloglucan degradation.

Isolation and characterization of galactomannans from Prosopis affinis as potential gum substitutes

Galactomannans from the endosperm of mature seeds of Prosopis affinis were isolated following different extraction procedures in yields of up to 64%. The polysaccharides were characterized by HPLC, GC-MS, and NMR spectroscopy, and their physicochemical properties were studied by SEC/MALLS-QUELS/RI and rheological methods. The monosaccharide composition of the extracted galactomannans showed only mannose and galactose in a ∼1.5 ratio, similar to galactomannans found in guar gum and obtained from other Prosopis species. The structural characterization showed that these polysaccharides are composed by a β-(1→4)-linked mannose backbone with galactopyranosyl residues attached through α-(1→6) linkages. The weight-average molecular weight (Mw) of the P. affinis galactomannans went from (1.6 ± 0.2) x 106 to (1.8 ± 0.1) x 106 g/mol, and the intrinsic viscosity, in the 9.9 to 10.7 dL/g range, was comparable to that reported for other extracts from the Prosopis genre. Although the molecular weight distribution and intrinsic viscosity were dependent on the extraction procedure, the mannose-to-galactose ratio was almost invariant. Conformation plots showed that the polymer extracted at higher temperatures is more cohesive, a characteristic that could be attributed to a greater number of polymer-polymer interactions. Our findings show that the P. affinis galactomannan has nearly monomodal molecular weight distribution, functional properties similar to those of guar gum, and can be isolated from an abundant and readily available non-traditional source. It therefore represents a promising new biomaterial for the food and pharmaceutical industries that can be produced from the regional native flora in a sustainable way.

An Aspergillus nidulans GH26 endo-β-mannanase with a novel degradation pattern on highly substituted galactomannans

The activity and substrate degradation pattern of a novel Aspergillus nidulans GH26 endo-β-mannanase (AnMan26A) was investigated using two galactomannan substrates with varying amounts of galactopyranosyl residues. The AnMan26A was characterized in parallel with the GH26 endomannanase from Podospora anserina (PaMan26A) and three GH5 endomannanases from A. nidulans and Trichoderma reesei (AnMan5A, AnMan5C and TrMan5A). The initial rates and the maximal degree of enzymatically catalyzed conversion of locust bean gum and guar gum galactomannans were determined. The hydrolysis product profile at maximal degree of conversion was determined using DNA sequencer-Assisted Saccharide analysis in High throughput (DASH). This is the first reported use of this method for analyzing galactomannooligosaccharides. AnMan26A and PaMan26A were found to have a novel substrate degradation pattern on the two galactomannan substrates. On the highly substituted guar gum AnMan26A and PaMan26A reached 35–40% as their maximal degree of conversion whereas the three tested GH5 endomannanases only reached 8–10% as their maximal degree of conversion. α-Galactosyl-mannose was identified as the dominant degradation product resulting from AnMan26A and PaMan26A action on guar gum, strongly indicating that these two enzymes can accommodate galactopyranosyl residues in the −1 and in the +1 subsite. The degradation of α-64-63-di-galactosyl-mannopentaose by AnMan26A revealed accommodation of galactopyranosyl residues in the −2, −1 and +1 subsite of the enzyme. Accommodation of galactopyranosyl residues in subsites −2 and +1 has not been observed for other characterized endomannanases to date. Docking analysis of galactomannooligosaccharides in available crystal structures and homology models supported the conclusions drawn from the experimental results. This newly discovered diversity of substrate degradation patterns demonstrates an expanded functionality of fungal endomannanases, than hitherto reported.

Structural elucidation and biological studies of a novel exopolysaccaride from Klebsiella pneumoniae PB12

An exopolysaccharide (KNPS) of an average molecular weight ∼1.8×105Da was isolated from the culture medium of Klebsiella pneumoniae PB12. Structural characterization of KNPS was carried out using sugar and methylation analysis, Smith degradation and 1D/2D NMR experiments. Sugar analysis showed that the KNPS composed of arabinose, galactose, 3-O-methyl-galctose and glucose in a molar ratio of nearly 4:3:1:1. The proposed repeating unit of the KNPS has a backbone chain consisting of two (1→6)-galactopyranosyl residues, two (1→5)-arabinofuranosyl residues, one (1→6)-glucopyranosyl residue and one (1→3)-arabinopyranosyl residue, out of which one (1→6)-galactopyranosyl residue was branched at O-2 position with a (1→2)-linked-galactopyranosyl residue terminated with non reducing arabinofuranosyl residue and one (1→5)-arabinofuranosyl residue branched at O-3 position with non reducing end 3-O-Me-galactopyranosyl residue. KNPS was found non-toxic toward human lymphocyte up to the dosage of 100μg/ml. KNPS enhanced malondialdehyde (MDA), reactive oxygen species (ROS), and have the potential to alter the ratio of oxidized glutathione (GSSG) and reduced glutathione (GSH) levels in the cellular system.

Structure characterization of an arabinogalactan from green tea and its anti-diabetic effect

A water-soluble polysaccharide, 7WA, with an average molecular mass of 7.1×104Da, was isolated from the leaves of green tea. Monosaccharide composition analysis indicated that 7WA mainly contained Arabinose and Galactose in the molar ratio of 1.0:0.96. By using the methods of methylation analysis, partial hydrolysis, and NMR, 7WA was characterized to possess a backbone consisting of 1,3- and 1,6-linked galactopyranosyl residues, with branches attached to O-3 of 1,6-linked galactose residues, and O-4 and O-6 of 1,3-linked galactose residues. The results of glucose-stimulated insulin secretion (GSIS) showed that 7WA significantly augmented insulin secretion at high glucose level (25mM), however, such effect was not seen at low glucose level (5mM). The mechanism study results indicated 7WA, a type II arabinogalactan from Green Tea, enhances GSIS through cAMP-PKA pathway.

A heteropolysaccharide, l-fuco-d-manno-1,6-α-d-galactan extracted from Grifola frondosa and antiangiogenic activity of its sulfated derivative

The tumor growth and metastasis are angiogenesis dependent, thus blockade of angiogenesis is a promising approach for treatment of cancer. Herein we reported the structural and biological features of a novel water-soluble polysaccharide named GFPW from the fruit body of Grifola frondosa. Chemical and spectral analysis revealed that GFPW with an average molecular weight of 15.7kDa, possessed a backbone consisting of α-1,6-linked galactopyranosyl residues, with branches attached to O-2 of α-1,3-linked fucose residues and α-terminal mannose. By the chlorosulfonic acid–pyridine method, we prepared a sulfated derivative of GFPW, Sul-GFPW, with a substitution degree of 0.33. According to the 13C NMR spectrum, the substitution position was deduced at C-2 and C-3. The angiogenesis assays in vitro showed that Sul-GFPW significantly inhibited endothelial cell proliferation in a dose- and time-dependent manner, and reduced endothelial cell migration and tube formation as well.

The core carbohydrate structure of Acacia seyal var. seyal (Gum arabic)

The structure of gum arabic (Acacia seyal) has been studied using methylation analysis and 2D (COSY, TOCSY, HMQC and HMBC) NMR spectroscopy. Galacturonic acid (13.66%) is a major component not previously identified. The backbone is made up of 1,3-linked galactopyranosyl (Galp) residues substituted at O-2, O-6 or O-4 positions, which are terminated with mainly arabinofuranosyl (Araf), galacturonopyranosyl (GalpA), rhamnopyranosyl (Rhap), but occasionally with arabinopyranosyl (Arap), and glucuronopyranosyl (GlcpA) residues. There are long side chains of →3)-α-l-Araf-(1→ and →2)-α-l-Araf-(1→ linked to the backbone.

Chemical Characterization and Structure of Exopolysaccharides from Submerged Culture of New Medicinal Mushroom from China, Phellinus mori (Higher Basidiomycetes)

Two different exopolysaccharides (EPSs) were first time-fractionated from submerged mycelia culture of Phellinus mori (PM) by gel-filtration chromatography: PM-EPS1 and PM-EPS3. Results of compositional analysis using high-performance anion exchange chromatography indicated that both PM-EPS1 and PM-EPS3 were heteropolysaccharides consisting of mannose, glucose, galactose, and rhamnose. Gel permeation chromatography analysis showed that the weight-average molecular mass of PM-EPS1 and PM-EPS3 were 4,990 g/mol and 27,890 g/mol, respectively. Fourier transform infrared spectral analysis of PM-EPS1 and PM-ESP3 revealed prominent characteristic groups corresponding to carbohydrates. 1H and 13C nuclear magnetic resonance spectroscopy analysis indicated that PM-EPS3 had a (1→4)-linked mannopyranosyl backbone, with branches of (1→4)-linked glucosyl residues and (1→3, 4)-linked galactopyranosyl residues. Preliminary activity tests in vitro revealed that the hydroxyl radical scavenging activity and total superoxide dismutase activities of PM-EPS3 obviously increased with an increase in concentration at the range 0.1-2.5 mg/mL.

Novel isolation of water-soluble polysaccharides from the fruiting bodies of Pleurotus ostreatus mushrooms

Novel water-soluble polysaccharides have been isolated from the fruiting bodies of the edible mushroom Pleurotus ostreatus. Three polysaccharide fractions were obtained by ethanol precipitation from cold water, hot water and hot aqueous NaOH extracts. The fractions were purified by size exclusion chromatography showing a unique carbohydrate occurring in each fraction: PC from the cold fraction, PH from the hot fraction and PB from the hot aqueous NaOH fraction. The analysis of the methylated alditol acetates and the NMR studies revealed that all the polysaccharides displayed a linear backbone. PC was formed by α-(1→3),(1→6)-linked galactopyranosyl residues whereas PH and PB consisted of glucose-linked units. PH was exclusively composed of glucopyranosyl units bound by α-(1→4) linkages whereas PB was a β-linked glucan showing (1→3) and (1→6) glycosidic bonds. The analysis of molecular arrangement by complexation with Congo red showed that only the β-linked polysaccharide (PB) displayed a triple helix conformation.

Structural characterization of an immunoregulatory polysaccharide from the fruiting bodies of Lepista sordida

Abstract A water-soluble polysaccharide, named as LSP, was extracted and fractioned from the fruiting bodies of Lepista sordida by DEAE-cellulose anion exchange and Sepharose CL-6B column chromatography. Its molecular weight (Mw) was estimated to be about 4 × 10 4 Da by using high-performance gel permeation chromatography (HPGPC). According to Fourier transform infrared (FT-IR) spectroscopy, partial acid hydrolysis, periodate oxidation and Smith degradation, methylation and GC–MS analysis, the results indicated LSP had a backbone consisting of (1 → 6)-linked-α- d -glucopyranosyl and (1 → 2,6)-linked-α- d -glucopyranosyl residues, which was terminated with terminal (1 → )-α- d -galactopyranosyl residue at the O-3 position of (1 → 2,6)-linked-α- d -glucopyranosyl residue along the main chain in the ratio of 2:1.1:0.9. Preliminary tests in vitro showed LSP had potent activation effects on murine macrophages, supported by the fact that the elevated NO production and TNF-α secretion, and its branches played a crucial role for macrophages activation.

In vivo anti-tussive activity and structural features of a polysaccharide fraction from water extracted Withania somnifera

Anti-tussive drugs are amongst the most widely used medications worldwide; however no new class of drugs has been introduced into the market for many years. The present study aims at evaluating the structural features and in vivo anti-tussive activity of a polysaccharide fraction from water extracted Withania somnifera. Herein, we have analyzed water extracted material of Withania somnifera using chemical, chromatographic, spectroscopic and biological methods. A polysaccharide fraction (F3) containing arabinosyl, galactosyl and galacturonosyl residues were obtained by anion exchange chromatography of the water extracted material. This polymer is branched and contained (1,5)-/(1,3,5)-linked arabinofuranosyl, (1,3)-/(1,6)-/(1,3,6)-linked galactopyranosyl residues together with small amount of terminal rhamnopyranosyl and terminal arabinofuranosyl residues. Peroral administration of this pectic arabinogalactan in a dose of 50 mg kg(-1) body weight (b.w.) decreased the number of cough efforts induced by citric acid in guinea pigs like that of codeine. This study provides a scientific basis for the past and present ethnomedical uses of this plant.

Structural Characterization of an Arabinogalactan from Platycodon grandiflorum Roots and Antiangiogenic Activity of Its Sulfated Derivative

A water-soluble polysaccharide, PGAW1, with an average molecular mass of 9.2 kDa determined by high performance gel permeation chromatography (HPGPC), was isolated from radix of Platycodon grandiflorum . Monosaccharide composition analysis indicated that PGAW1 contains Ara and Gal in the molar ratio of 1.42:1.0. Using methylation analysis, partial hydrolysis, endo-1,4-β-d-galactanase digestion, NMR and ESI-MS, PGAW1 was determined to possess a backbone consisting of 1,4- and 1,6-linked galactopyranosyl residues, with branches attached to O-3 of 1,6-linked galactose residues. By the chlorosulfonic acid-pyridine method we produced a sulfated derivative of PGAW1, Sul-w1, with a substitution degree of 1.52. The substitution was at O-6 on 1,4-linked Gal residues according to the (13)C NMR spectra. Bioactivity test showed that Sul-w1 could inhibit tube formation by human microvascular endothelial cells (HMEC) in a dose-dependent manner.

Isolation, purification and structure of a new water-soluble polysaccharide from Zizyphus jujuba cv. Jinsixiaozao

A novel water-soluble polysaccharide (ZSP3c) was isolated from Zizyphus jujuba cv. Jinsixiaozao and its structure was investigated. ZSP3c was obtained by extraction with hot water followed by purification using DEAE-SepharoseCL-6B anion-exchange, SepharoseCL-6B and Sephadex G-200 column chromatography. It was eluted as a single symmetrical narrow peak on high-performance gel-permeation chromatography (HPGPC) and the average molecular weight (Mw) was estimated to be 1.4 × 10 5 Da. The ZSP3c was composed of l-rhamnose, d-arabinose and d-galactose in a molar ratio of 1:2:8. The structural features of ZSP3c were investigated by partial hydrolysis with acid, methylation analysis and NMR spectroscopy analysis. The results revealed that the main backbone chain of ZSP3c was (1 → 4)- d-galacturonopyranosyl residues interspersed with (1 → 2)- l-rhamnopyranosyl residues and (1 → 2,4)- l-rhamnopyranosyl residues. The neutral side chains of arabinofuranosyl residues and galactopyranosyl residues were attached to the backbone at the O-4 position of rhamnopyranosyl residues.

Study of Macrophage Activation and Structural Characteristics of Purified Polysaccharide from the Fruiting Body of Cordyceps militaris

Cordyceps militaris, an entomophathogenic fungus belonging to the class Ascomycetes, has been reported to have beneficial biological activities such as hypoglycemic, anti-inflammatory, anti-tumor, anti-metastatic, hypolipidemic, immunomodulatory, and antioxidant effect. In this study, the crude water-soluble polysaccharides CMP, which was obtained from the fruiting body of C. militaris by hot water extraction and ethanol precipitation, was fractionated by DEAE cellulose and Sepharose CL-6B column chromatography. This process resulted in three polysaccharide fractions, termed CMP Fr I, CMP Fr II, and CMP Fr III. Of these fractions, CMP Fr II, with an average molecular weight of 127 kDa, was able to upregulate effectively the phenotypic functions of macrophages such as NO production and cytokine expression. The chemical property of the stimulatory polysaccharide, CMP Fr II, was determined based on monosaccharide composition, which consisted of glucose (56.4 %), galactose (26.4 %), and mannose (17.2%). Its structural characteristics were investigated by a combination of chemical and instrumental analyses, including methylation, reductive cleavage, acetylation, Fourier transform infrared spectroscopy (FT-IR), and gas chromatography-mass spectrometry (GC-MS). Results indicated that CMP Fr II consisted of the (1-->4) or (1-->2) linked glucopyranosyl or galactopyranosyl residue with a (1-->2) or (1-->6) linked mannopyranosyl, glucopyranosyl or galactopyranosyl residue as a side chain. The configuration of the beta-linkage and random coil conformation of CMP Fr II were confirmed using a Fungi Fluor kit and Congo Red reagent, respectively.

Purification, structural analysis and hydroxyl radical-scavenging capacity of a polysaccharide from the fruiting bodies of Russula virescens

Abstract In this study, one water-soluble polysaccharide (RVP) was isolated from the fruiting bodies of Russula virescens. Its molecular weight (Mw) was ∼3.9 ×104 Da, as determined by high-performance size-exclusion chromatography (HPSEC). The RVP was composed of d -galactose and d -mannose in a ratio of 2:1, as detected by gas chromatography (GC). The structural features of RVP were investigated by partial hydrolysis with acid, periodate oxidation and Smith degradation, acetylation, methylation analysis, and NMR spectroscopy (1H, 13C). The results revealed that RVP had a backbone consisting of (1 → 6)-linked-α- d -galactopyranosyl and (1 → 2, 6)-linked-α- d -galactopyranosyl residues that terminated in a single non-reducing terminal (1 → )-α- d -mannopyranosyl residue at the O-2 position of each (1 → 2, 6)-linked-α- d -galactopyranosyl residues along the main chain in the ratio of 1:1:1. Preliminary, in vitro antioxidant activity tests showed that RVP could dose-dependently enhance the scavenging effect on hydroxyl radicals at concentrations from 20 to 160 μg/ml. These results indicated that RVP was mainly composed of water-soluble galactomannan, and it had a significant effect on scavenging of hydroxyl radicals in vitro.

Localization of arabinogalactan-proteins in roots of Echinacea purpurea by immunofluorescent labelling

From the high molecular weight fraction of an aqueous extract from roots of Echinacea purpurea (L.) MOENCH, arabinogalactan-proteins (AGPs) were purified and characterized with special regard to the structure of the polysaccharide moiety. It is highly branched and shows a linkage composition comparable to AGPs from the aerial parts of E. purpurea [1] with 1,3-, 1,6- and 1,3,6-linked galactopyranosyl residues as basic structural elements and arabinofuranosyl residues predominantly as terminal and 1,5-linked residues. Some terminal units of glucuronic acids could also be detected.

Characterization and Immunolocalization of Arabinogalactan-Proteins in Roots ofEchinacea purpurea

From the high molecular weight fraction of an aqueous extract from roots of Echinacea purpurea L. Moench, arabinogalactan-proteins (AGPs), a class of proteoglycans proposed to be involved in cell differentiation and plant growth, were purified and characterized with regard to amino acid composition and structure of the polysaccharide moiety. The protein content of the AGP was 5.0 % (w/w) with the dominating amino acids Glx, Hyp, Asx, Ser, Thr and Ala. The highly branched polysaccharide moiety shows a linkage composition typical of AGPs with 1,3-, 1,6- and 1,3,6-linked galactopyranosyl residues and arabinofuranosyl residues predominantly as terminal and 1,5-linked residues. Terminal units of glucuronopyranose acid were also detected. Furthermore, a new method for the localization of AGPs in plant tissue has been developed. The synthetic (beta- D-Glc)(3) Yariv phenylgycoside (betaGlcY) is known to specifically bind to AGPs. For immunolocalization, polyclonal betaGlcY-antibodies have been generated and were used to label Yariv-treated thin sections of roots from E. purpurea. After addition of the FITC-conjugated secondary antibody, the sections were analyzed by confocal laser scanning microscopy. AGPs are detected mainly in the central cylinder in the area of the xylem. Cell walls of vessels and tracheids are strongly labelled, especially at the inner area of the wall. Furthermore, there is intense labelling of the pit canals.