Specific interaction of aniline blue with (1 → 3)-β-d-glucan

Interaction between aniline blue and curdlan, a (1 → 3)-β-d-glucan, has been studied using absorption and fluorescence spectroscopy. The evidence suggests that a minor, weakly fluorescent component of commercial dyes forms a strongly fluorescent complex with curdlan, with an excitation maximum of 395 nm and an emission maximum of 495 nm. This component was partially purified by TLC on silica gel. Of many polysaccharides surveyed, a number showed weak interactions with the major component of aniline blue but only (1 → 3)-β-d-glucans such as pachyman, curdlan and laminaran induced fluorescence in the minor component. Fluorescence was less with laminaran than with curdlan and decreased with increasing alkali concentration suggesting conformational control of the dye-binding mechanism. As little as 5 μg/ml of curdlan induced easily detectable fluorescence increases in aniline blue, and this was used to demonstrate the presence of (1 → 3)-β-d-glucan in a fungal cell wall preparation. (1 → 3)-β-d-glucan in cereal grain sections was located as bright yellow-green fluorescent particles. In barley these stained particles, located in association with the sub-aleurone endosperm cell wall, showed a fluorescence excitation maximum at 395 nm and emission maximum of 495 nm.     

Water-soluble (1→3), (1→4)-β-D-glucans from barley (Hordeum vulgare) endosperm. II. Fine structure

Water-soluble (1→3),(1→4)-β-d-glucans isolated from barleys grown in Australia and the UK were depolymerised using a purified (1→3),(1→4)-β-d-glucan 4-glucanohydrolase (EC 3.2.1.73). Oligomeric products were quantitatively separated by high resolution gel filtration chromatography and their structures defined by methylation analysis. Approximately 90% (w/w) of each polysaccharide consists of cellotriosyl and cellotetraosyl residues separated by single (1→3)-linkages but blocks of 5–11 (1→4)-linked glucosyl residues are also present in significant proportions. Periodate oxidation followed by Smith degradation suggested that contiguous (1→3)-linked β-glucosyl residues are either absent, or present in very low frequency. The potential for misinterpretation of data due to incomplete Smith degradation was noted.The irregularly-spaced (1→3)-linkages interrupt the relatively rigid, ribbon-like (1→4)-β-glucan conformation and confer a flexibility and ‘irregular’ shape on the barley (1→3),(1→4)-β-d-glucan, consistent with its solubility in water. Molecular models incorporating the major structural features confirm that the polysaccharide is likely to assume a worm-like conformation in solution. Non-covalent interactions between long blocks of (1→4)-linkages in (1→3),(1→4)-β-d-glucans, or between these blocks and other polysaccharides, offer a possible explanation for the organisation of polysaccharides in the framework of the cell wall.     

Substrate specificities and kinetic properties of two (1→3), (1→4)-β-d-glucan endo-hydrolases from germinating barley (Hordeum vulgare)

Two β-d-glucan endo-hydrolases purified from germinating barley (Hordeum vulgare) hydrolyse (1→4)-β linkages in (1→3),(1→4)-β-d-glucans where the d-glucosyl residue is substituted at O-3, but will not hydrolyse (1→3)-β-d-glucans or (1→4)-β-d-glucans. Methylation analysis of hydrolytic products released from barley (1→3),(1→4)-β-d-glucan indicates that 3-O-β-cellobiosyl-d-glucose and 3-O-β-cellotriosyl-d-glucose are the major oligomers formed. The enzymes exhibit characteristic endo-hydrolase action-patterns on this substrate. Both enzyme can therefore be classified as (1→3),(1→4)-β-d-glucan 4-glucanohydrolases (EC 3.2.1.73). The reduced, pneumococcal polysaccharide RS III, which consists of alternating (1→3)- and (1→4)-linked β-d-glucosyl residues, is hydrolysed by the enzymes to release laminaribiose as a major oligomeric product. Although the kinetic parameters of the two enzymes are similar, one hydrolyses barley (1→3),(1→4)-β-d-glucan at a significantly higher rate than the other and is more stable at elevated temperatures.     

Fine structure of (1→3),(1→4)-β-d-glucan from Zea shoot cell-walls

The insoluble material that remains after extraction of Zea shoots with cold buffer was treated successively with 3m LiCl and hot water. The polysaccharides solubilized by these treatments were mostly (1→3),(1→4)-β-d-glucans. The β-d-glucan from the hot-water-soluble fraction was hydrolyzed by Bacillus subtilis (1→3),(1→4)-β-d-glucan 4-glucanohydrolase. The oligosaccharides were characterized by methylation analysis of the enzymic fragments and by methylation analysis of secondary fragments generated by treatment of the isolated oligosaccharides with Streptomyces QM B814 cellulase. The results demonstrate that the native polysaccharide consists mainly of cellotriosyl and cellotetraosyl residues joined by single (1→3) linkages. Evidence is presented to show that certain other glucosyl sequences are also present in the native polysaccharide including (a) two, three, or four contiguous (1→3)-linkages; (b) blocks of more than four (1→4)-linked glucose residues; (c) regions having alternating (1→3)- and (1→4)-linkages.     

The molecular structures of some glucans from the cell walls of Schizosaccharomyces pombe

Extraction of the cell walls of Schizosaccharomyces pombe with dilute alkali at 4° yields a mixture of polysaccharides including galactomannan, (1→3)-α-d-glucan, and a branched (1→3)-β-d-glucan. The alkali-insoluble residue contains a lightly branched (1→3)-β-d-glucan, together with smaller amounts of an extremely highly branched (1→6)-β-d-glucan. The properties of the three distinct β-d-glucans are compared with those isolated from other yeasts.     

A 13C-nuclear magnetic resonance study of polysaccharide gels. Molecular architecture in the gels consisting of fungal,branched (1 → 3)-β-d-glucans (lentinan and schizophyllan) as manifested by conformational changes induced by sodium hydroxide

To gain further insight into the architecture of the gel network of some branched (1 → 3)-β-d-glucans, a ¹³C-n.m.r. study of sodium hydroxide-induced, conformational change was performed. The branched d-glucans examined were lentinan from Lentinusedodes, a lower-molecular-weight fraction thereof, and schizophyllan from Scilizophyllum commune; these (1 → 3)-β-d-glucans have two branches for every five d-glucopyranosyl residues (lentinan), or one for every three or four (schizophyllan) at 0-6. In contrast to the gel oflinear (1 → 3)-β-d-glucan (curdlan), all of the ¹³C signals due to the β-d-(1 → 3)-linked d-glucosyl residues were completely suppressed in the gel state. As the peak intensity and line width of the ¹³C-resonance peaks for the gel state are strongly influenced by the degree of cross-linking, such a complete loss of the peak areas can be explained in terms of a higher degree of cross-linking than that of the linear d-glucans. As demonstrated previously, the cross-links involve physical association of the helical segments, such as the double- or triple-stranded helices. These helix forms were found to be converted, at 0.2M sodium hydroxide, into the random-coil form (gel-to-sol transition), which gives rise to full peak-areas, because of complete breaking of the physical cross-links. Also, in contrast to the linear d-glucan, such helix-coil transition of the branched d-glucans proceeded in a noncooperative way: the peak intensity and line width gradually changed with the concentration of sodium hydroxide. This behavior is best interpreted in terms of distribution of the various degrees of cross-linking. Some loose cross-links are readily broken in the lower range of concentration of alkali (0.09M), and others are resistant until complete conversion into the random coil occurs (0.2M). This result is consistent with the view that the primary structure of the branched (1 → 3)-β-d-glucans is hi-highly branched, as in a tree-like structure.     

Discovery and characterization of a fructosylated capsule polysaccharide and sialylated lipopolysaccharide in a virulent strain of Actinobacillus suis

We are developing a serotyping system for Actinobacillus suis based on its capsule (K) and lipopolysaccharide O-chain (O) structures. Previously, we have shown that less virulent strains of this swine pathogen express a (1→6)-β-D-glucan as both K- and O-chain polysaccharides and were serologically classified as K:1/O:1. Here, we show that representative A. suis strains with a high (H91-0380; serotype K:2/O:2) and intermediate (C84; serotype K:2/O:1) degree of virulence possess a capsule polysaccharide (K:2) composed of an O-acetylated diglycosyl phosphate repeat decorated with fructose: [→4)-3-O-Ac-β-D-GlcpNAc-(1→3)-[β-D-Fruf-(2→2)]-α-D-Galp-(1→PO(4)(-)→]. In addition, the serotype O:2 lipopolysaccharide was shown to express a sialylated O-chain [→3)-β-D-Galp-(1→4)-[Neu5Ac-(2→3)-α-D-Galp-(1→6)]-β-D-Glcp-(1→6)-β-D-GlcpNAc-(1→]. As (1→6)-β-D-glucan is ubiquitous in the environment, low levels of antibodies in the animals are predicted to prevent disease by K:1/O:1 strains. The greater potential associated with K:2/O:2 and K:2/O:1 strains is most likely due to the absence of (1→6)-β-D-glucan as the K antigen and, in the case of K:2/O:2, the presence of sialic acid in the lipopolysaccharide, a nonulosonic acid known to promote evasion of host recognition.     

β-(1→4)-d-glucan synthesis from UDOP-[14C]-d- glucose by a solubilized enzyme from Lupinus albus

The particulate enzyme responsible for the synthesis of β-(1→4)-d-glucans from UDP-[¹⁴C-d-glucose has been solubilized and some of its properties have been characterized. Mg²⁺ markedly enhanced synthesis of β-(1→4)-d-glucans and inhibited synthesis of β-(1→3)-d-glucans. The optimal pH for synthesis of β-(1→4)-d-glucans is near pH 8 and the synthesis was enhanced in these preparations by d-glucose, methyl-β-d-glucopyranoside and cellobiose.     

Structural analysis and ensymic solubilization of barley endosperm cell-walls

Barley endosperm cell-walls were prepared and analysed. The-carbohydrate portion, which constituted most of the wall material, consisted of 10 % of l-arabinose, 13% Of d-xylose, 74% of d-glucose and 2.5% of d-mannose. Mixed-linkage β-d-glucan represented 70-72% of this material; the remaining 2-4% Of d-glucose maybe present as cellulose and glucomannan. Water and alkali-extracted β-d-glucans contained similar ratios of (1 → 3)- to (1 → 4)-Linkages, namely 3 to 7. The walls, which had a protein content of approximately 5 %, contained unidentfied, alkali-labile linkages. An endo-(1 → 3)β-d-glucanase from malted barley, and a fungal endo-(1 → 4)-β-d-glucanase, caused extensive solubilization of the wall polysaccharides.     

Synthesis of analogs of a metabolite of sodium 2-pyridinethiolate N-oxide (pyrithione)

Antitumor activities of two (1 → 6)-branched (1 → 3)-β-d-glucans, isolated from the fruiting body of Auricularia auricula-judae (“kikurage”, an edible mushroom), and other branched polysaccharides containing a backbone chain of (1 → 3)-α-d-glucosidic or (1 → 3)-α-d-mannosidic linkages [and their corresponding (1 → 3)-d-glycans, derived by mild, Smith degradation] were compared. Among these polysaccharides, a water-soluble, branched (1 → 3)-β-d-glucan (glucan I) of A. auriculajudae exhibited potent, inhibitory activity against implanted Sarcoma 180 solid tumor in mice. The alkali-insoluble, branched (1 → 3)-β-d-glucan (glucan II), a major constituent of the fruiting body, showed essentially no inhibitory activity. When the latter glucan, having numerous branches attached, was modified by controlled, periodate oxidation, borohydride reduction, and mild, acid hydrolysis, the resulting, water-soluble, degraded glucan, having covalently linked polyhydroxy groups attached at O-6 of the (1 → 3)-linked d-glucosyl residues, exhibited potent antitumor activity. Further investigations using the glucan-polyalcohol indicated that the attachment of the polyhydroxy groups to the (1 → 3)-β-d-glucan backbone may enhance the antitumor potency of the glucan. On the other hand, partial introduction     

Broad-spectrum biofilm inhibition by Kingella kingae exopolysaccharide

Cell-free extracts prepared from Kingella kingae colony biofilms were found to inhibit biofilm formation by Aggregatibacter actinomycetemcomitans, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Candida albicans, and K. kingae. The extracts evidently inhibited biofilm formation by modifying the physicochemical properties of the cell surface, the biofilm matrix, and the substrate. Chemical and biochemical analyses indicated that the biofilm inhibition activity in the K. kingae extract was due to polysaccharide. Structural analyses showed that the extract contained two major polysaccharides. One was a linear polysaccharide with the structure →6)-α-d-GlcNAcp-(1→5)-β-d-OclAp-(2→, which was identical to a capsular polysaccharide produced by Actinobacillus pleuropneumoniae serotype 5. The second was a novel linear polysaccharide, designated PAM galactan, with the structure →3)-β-d-Galf-(1→6)-β-d-Galf-(1→. Purified PAM galactan exhibited broad-spectrum biofilm inhibition activity. A cluster of three K. kingae genes encoding UDP-galactopyranose mutase (ugm) and two putative galactofuranosyl transferases was sufficient for the synthesis of PAM galactan in Escherichia coli. PAM galactan is one of a growing number of bacterial polysaccharides that exhibit antibiofilm activity. The biological roles and potential technological applications of these molecules remain unknown.     

Water-soluble (1→3), (1→4)-β-d-glucans from barley (Hordeum vulgare) endosperm. I. Physicochemical properties

Physicochemical methods have been used to define molecular weight, molecular weight distribution, solution behaviour and shape of (1→3), (1→4)-β-d-glucans purified from the 40°C water-extract of barley endosperm by precipitation with 30% saturated ammonium sulphate. The molecular weight and solution properties of a (1→3), (1→4)-β-d-glucan from Australian grown barley (cv. Clipper) are compared with a commercially available preparation. Weight and number average molecular weights are 290 000 and 210 000 respectively for the Clipper (1→3), (1→4)-β-d-glucan and 160 000 and 150 000 respectively for the commercial preparation. The degree of polydispersity is small, but this probably results from the selection of a specific population of (1→3), (1→4)-β-d-glucan molecules during isolation. The higher molecular weight of the Clipper (1→3), (1→4)-β-d-glucan is reflected in higher sedimentation coefficient and intrinsic viscosity values. Viscosity and sedimentation data indicate that the molecules are highly asymmetric, with axial ratios of approximately 100 and 80 for the Clipper (1→3), (1→4)-β-d-glucan and the commercial preparation, respectively. Both polysaccharides appear to exist in solution as extended, worm-like chains.     

Coupling of polysaccharides activated by means of chloroacetaldehyde dimethyl acetal to amines or proteins by reductive amination

A novel method has been developed for the coupling of modified polysaccharides to proteins or other amines. Chloroacetaldehyde dimethyl acetal has been used for the introduction of O-(2,2-dimethoxyethyl) groups into amylose, dextran, and a linear (1→3)-β-d-glucan. In amylose and the (1→3)-β-d-glucan, these groups were attached preponderantly at O-6 and in dextran at O-2. Mild treatment with acid then gave polysaccharide derivatives substituted with aldehyde groups which were coupled in good yields to proteins and other amines by reductive amination with sodium cyanoborohydride in aqueous solution at pH 7. An aminated (1→3)-β-d-glucan derivative that induced antitumor activity in mouse macrophages in vitro is reported.     

Synthesis of the tri- and tetra-saccharides related to the fine structures of lichenan and cereal β-d-glucans

Syntheses, based on silver trifluoromethanesulfonate-promoted Koenigs-Knorr type condensations, are described of the d-glucotrioses, β-d-Glcp-(1→3)-β-d-Glcp-(1→4)-d-Glcp and β-d-Glcp-(1→4)-β-d-Glcp-(1→3)-d-Glcp, and the d-Glucotetraoses, β-d-Glcp-(1→3)-β-d-Glcp-(1→4)-β-d-Glcp-(1→4)-d-Glcp, β-d-Glcp-(1→4)-β-d-Glcp-(1→3)-β-d-Glcp-(1→4)-d-Glcp, and β-d-Glcp-(1→4)-β-d-Glcp-(1→4)-β-d-Glcp-(1→3)-d-Glcp, corresponding to the tri- and tetra-saccharide units in the linear chains of (1→4)- and (1→3)-linked β-d-glucopyranosyl residues of lichenan, and of oat and barley β-d-glucans.     

Purification and properties of an endoglucanase isolated from the cell walls of Zea mays seedlings

An endoglucanase liberated from Zea mays seedling cell-walls by LiCl was purified by using SP-Sephadex, CM-Sephadex, and gel filtration, resulting in a 98-fold increase in specific activity. It has a pH optimum of 4.5–5.0 and is heat stable up to 40–45°. Compounds that interact with sulfhydryl groups did not inhibit the activity of the enzyme, nor did EDTA, suggesting that the enzyme does not require free sulfhydryl groups or metal ions for activity. The endoglucanase has an apparent molecular weight of 20–25,000 and an isoelectric point of ⩾9. Hydrolytic activity against (1»3),(1»4)-β-d-glucans is restricted to isolated sites, with the release of high-molecular-weight products (10–15,000). Action on isolated, inactivated Zea cell-walls caused the release of approximately the same products as observed when the enzyme was incubated with soluble (1→3), (1→4)-β-d-glucans.     

Environmental and biofilm-dependent changes in a Bacillus cereus secondary cell wall polysaccharide

Bacterial species from the Bacillus genus, including Bacillus cereus and Bacillus anthracis, synthesize secondary cell wall polymers (SCWP) covalently associated to the peptidoglycan through a phospho-diester linkage. Although such components were observed in a wide panel of B. cereus and B. anthracis strains, the effect of culture conditions or of bacterial growth state on their synthesis has never been addressed. Herein we show that B. cereus ATCC 14579 can synthesize not only one, as previously reported, but two structurally unrelated secondary cell wall polymers (SCWP) polysaccharides. The first of these SCWP, →4)[GlcNAc(β1-3)]GlcNAc(β1-6)[Glc(β1-3)][ManNAc(α1-4)]GalNAc(α1-4)ManNAc(β1→, although presenting an original sequence, fits to the already described the canonical sequence motif of SCWP. In contrast, the second polysaccharide was made up by a totally original sequence, →6)Gal(α1-2)(2-R-hydroxyglutar-5-ylamido)Fuc2NAc4N(α1-6)GlcNAc(β1→, which no equivalent has ever been identified in the Bacillus genus. In addition, we established that the syntheses of these two polysaccharides were differently regulated. The first one is constantly expressed at the surface of the bacteria, whereas the expression of the second is tightly regulated by culture conditions and growth states, planktonic, or biofilm.     

Polysaccharides of basidiomycetes. Alkali-soluble polysaccharides from the mycelium of white rot fungus <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> (Curt.: Fr.) P. Karst

Two polysaccharides were isolated from submergedly cultured mycelium of the basidiomycete Ganoderma lucidum by extraction with alkali followed by fractionation with Fehling reagent. The polysaccharides were shown to be a linear (1→3)-α-D-glucan and a highly branched xylomannan containing a backbone built up of (1→3)-linked α-D-mannopyranose residues, the majority of which are substituted at O-4 by single β-D-xylopyranose residues or by disaccharide fragments β-D-Manp-(1→3)-β-D-Xylp-(1→. Polysaccharide structures were elucidated by NMR spectroscopy in combination with methylation analysis and periodate oxidation. An interesting feature of the xylomannan is the simultaneous presence of α-D-mannopyranose and β-D-mannopyranose residues, the first forming the backbone, and the second being the non-reducing terminal units of disaccharide side chains.     

中药巴戟天中水溶性多糖的分离及结构鉴定(英文)

中药巴戟天(Morinda officinalis)的根经过水提、醇沉、脱色和离子交换得到水溶性多糖(MOHP-1),经过FIIR、HPLC、NMR和GC-MS分析,最后确定MOHP-1是由果糖以(2→1)糖苷键连接的菊淀粉性多糖,其结构为α-Glcp-(1→2)-[β-Fruf-(2→1)-β-Frucf]n。     

Synthesis of Staphylococcus aureus type 5 capsular polysaccharide repeating unit using novel l-FucNAc and d-FucNAc synthons and immunochemical evaluation

Staphylococcus aureus is a major cause of nosocomial infections. Glycoconjugates of type 5 and 8 capsular polysaccharides have been investigated for vaccine application. The proposed structure of type 5 polysaccharide is: →4-β-d-ManNAcA-(1→4)-α-l-FucNAc(3OAc)-(1→3)-β-d-FucNAc-(1→. The stereocontrolled insertion of these three glycosydic bonds is a real synthetic challenge. In the present paper we report the preparation of two novel versatile l- and d-fucosamine synthons from commercially available starting materials. In addition we applied the two building blocks to the synthesis of type 5 trisaccharide repeating unit. The immunochemical properties of the synthesized trisaccharide were assessed by competitive ELISA and by immunodot blot analysis using sera of mice immunized with type 5 polysaccharide conjugated to CRM₁₉₇. The results suggest that although the type 5 S. aureus trisaccharide is recognized by specific anti polysaccharide antibodies in dot blot, structures longer than the trisaccharide may be needed in order to significantly compete with the native type 5 polymer in the binding with sera from mice immunized with S. aureus type 5 polysaccharide–CRM₁₉₇ conjugate.