Characterization of the extracellular,water-insoluble α-D-glucans of oral streptococci by methylation analysis,and by enzymic synthesis and degradation

Methylation analysis of water-insoluble α-D-glucans synthesized from sucrose by culture filtrates from several strains of Streptococcus spp. has proved that all of the glucans were highly branched and that the chains contained (1→6)- and (1→3)-linked D-glucose residues not involved in branch points. Hydrolysis of the glucans with a specific endo-(1→3)-α-D-glucanase demonstrated that the majority of the (1→3)-linked glucose residues were arranged in sequences. D-Glucose was the major product of the hydrolysis, and a small proportion of nigerose was also released. The use of a specific endo-(1→6)-α-D-glucanase similarly indicated that the glucans also contained sequences of (1→6)-linked α-D-glucose residues, and that those chains were branched. Two D-glucosyltransferases (GTF-S and GTF-I), which reacted with sucrose to synthesize a soluble glucan and a water-insoluble glucan, respectively, were separated from culture filtrates of S. mutans OMZ176. The soluble glucan was characterized as a branched (1→6)-α-D-glucan, whereas the insoluble one was a relatively linear (1→3)-α-D-glucan. The hypothesis is advanced that the glucosyltransferases can transfer glucan sequences by means of acceptor reactions similar to those proposed by Robyt for dextransucrase, leading to the synthesis of a highly branched glucan containing both types of chain. The resulting structure is consistent with the evidence obtained from methylation analysis and enzymic degradations, and explains the synergy displayed when the two D-glucosyltransferases interact with sucrose. Variations in one basic structure can account for the characteristics of water-insoluble glucans from S. sanguis and S. salivarius, and for the strain-dependent diversity of S. mutans glucans.     

A hemicellulosic β-D-glucan from the endosperm of sorghum grain

A hemicellulosic β-D-glucan of $\text{d.p.}$ ≈26 has been isolated from the endosperm of sorghum grain. Methylation analysis, partial hydrolysis with acid, and periodateoxidation studies showed that the glucan is linear and has both (1 → 3)- and (1 → 4)-linked D-glucopyranose residues in the ratio of 3:2. The low, positive, specific rotation and chromium trioxide oxidation studies indicated that the D-glucose residues are β-linked.     

Comparison of Structure and Antitumor Activity of Polysaccharides Isolated from Fukurotake,the Fruiting Body of Volvariella volvacea

An α-linked d-manno-d-galactan, glycogen, and three kinds of branched (1→3)-γ-d-glucans were isolated from Fukurotake, the fruiting body of Volvariella volvacea by successive extractions with cold and hot water, cold and hot alkali, and dimethyl sulfoxide. The mannogalactan, purified from the cold water extract, had a MW of 4x 10⁵, and consisted of an α-(1→6) linked d-galactose backbone, one out of every three d-galactose residues being substituted with a single α-d-mannosyl group. The glycogen, isolated from the hot water extract, had a MW of 12 × 10⁵, and 14 ~ 15 d-glucose residues as an average chain length, as revealed by methylation analysis. The α-(1→4)-linked unit chains of this glycogen were distributed from DP 6 to 13, approximately in equal numbers.

There were three kinds of branched (1→3)-γ-d-glucans, isolated from alkali and dimethyl sulfoxide extractions. They contain a backbone of (1→3)-linked d-glucose residues with side chains of single d-glucosyl groups, but having different degrees of branching. In addition, alkali-extracted glucans contain small but significant proportions of (1→6)-linked sugar units.

Among these polysaccharides, the cold alkali-extracted glucan (degree of branching, 1:5) showed a potent antitumor activity against Sarcoma 180 solid tumors implanted in mice, and chemical modifications changed its original activity, confirming our previous results. Other polysaccharides, such as the mannogalactan and other (1→3)-γ-d-glucans, showed no or lower antitumor activity.     

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     

Étude complémentaire de la structure de trois galactoxyloglucanes (amyloïdes) de graines

D-Galacto-D-xylo-D-glucans (amyloids) from Balsamina, Tropaeolum, and Tamarindus seeds behave in a similar manner in the presence of various glycosidase preparations: slow depolymerization by enzymes from several germinated or non-germinated seeds, and hydrolysis into monosaccharides and oligosaccharides by commercial cellulase and hemicellulase preparations from fungi. A purified cellulase from Penicillium notatum gave a dialyzable fraction almost exclusively composed of α-D-xylopyranosyl-(1→6)-D-glucose residues and a nondialyzable fraction composed of chains of β-D-(1→4)[withsome (1→3)]-glucopyranosyl residues; β-D-galacto-pyranosyl-(1→2)-α-D-xylosyl groups are linked to some of the β-D-glucosyl residues at 0-6. The presence of (1→3)-linkages in the D-glucan chain of the Balsamina was verified by methylation and sequential periodate oxidation-borohydride reduction; the distribution of the substituents on the D-glucan chain is not regular. The main D-glucan backbone, where the β-D-glucosyl residues are partly linked at 0-6 to β-D-galactosyl-(1→2)-D-xylosyl groups, is linked to D-glucan chains where almost all the D-glucose units are linked at 0-6 by one α-D-xylosyl group. The presence of 3,6-di-O-methyl-D-glucose after permethylation and hydrolysis suggests that the xyloglucan chains are linked to 0-2 of the D-glucosyl units of the galactoxyloglucan backbone.     

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.     

New insights into the structure of (1→3,1→6)-β-D-glucan side chains in the Candida glabrata cell wall

β-Glucan is a (1→3)-β-linked glucose polymer with (1→6)-β-linked side chains and a major component of fungal cell walls. β-Glucans provide structural integrity to the fungal cell wall. The nature of the (1-6)-β-linked side chain structure of fungal (1→3,1→6)-β-D-glucans has been very difficult to elucidate. Herein, we report the first detailed structural characterization of the (1→6)-β-linked side chains of Candida glabrata using high-field NMR. The (1→6)-β-linked side chains have an average length of 4 to 5 repeat units spaced every 21 repeat units along the (1→3)-linked polymer backbone. Computer modeling suggests that the side chains have a bent curve structure that allows for a flexible interconnection with parallel (1→3)-β-D-glucan polymers, and/or as a point of attachment for proteins. Based on these observations we propose new approaches to how (1→6)-β-linked side chains interconnect with neighboring glucan polymers in a manner that maximizes fungal cell wall strength, while also allowing for flexibility, or plasticity.     

Separation and characterization of the soluble and insoluble components of insoluble laminaran

Insoluble laminaran, a (1→3)-β-D-glucan from Laminaria hyperborea (L. cloustoni), has been fractionated by differential solubility into soluble and insoluble fractions. These fractions were degraded with a purified exo-(1→3)-β-D-glucanase from Basidiomycete sp. QM806 giving, as primary hydrolysis products, D-glucose, gentiobiose, laminarabiose, and 1-O-β-laminarabiosylmannitol. Gentiobiose was obtained in only trace amounts from the insoluble fraction of laminaran, suggesting the absence of branching. Successive application of periodate oxidation, reduction, mild acid hydrolysis, and enzymic degradation indicated that the branch in the soluble fraction consists of a single β-(1→6)-linked D-glucosyl residue. The results indicate that “insoluble” laminaran is apparently an aggregate of three closely related polysaccharide species: a soluble, branched, reducing component (soluble laminarose); an insoluble, unbranched, reducing component (insoluble laminarose); and an unbranched, nonreducing component (laminaritol) that has a monosubstituted mannitol residue at the reducing terminal. Laminaritol was found to be about equally distributed between the soluble and insoluble fractions. The average d.p. of the laminaran components is 20–25 residues, as determined from the relative amounts of enzymic hydrolysis products and from periodate-oxidation data.     

Structural investigations of the extracellular polysaccharide elaborated by s19, a Xanthomonas-type bacterium

The extracellular, acidic heteropolysaccharide from Xanthomonas S19 consists of D-glucuronic acid, D-glucose, D-galactose, and D-mannose residues in the approximate molar ratios of 1.6:3:1:1, plus acetyl groups liked to C-2 and/or C-3 of a large proportion of the glucose residues. Methylation studies showed that the glucose is present as non-reducing end-group also as 1,2- and 1,4-linked units, the galactose residues are solely 1,3-linked, a major proportion of the mannose residues are 1,2,4-linked and the rest 1,2-linked. A high proportion of the glucuronic acid units are 1,4-linked. Periodate oxidation confirmed the presence of these linkages. The disaccharides D-Glc-(1→4)-D-Glc,D-Glc-(1→2)-D-Man, D-Glc-(1→3)-D-Gal, D-Gal-(1→2)-D-Glc, D-GlcA-(1→4)-D-GlcA, and β-D-GlcA-(1→4)-D-Man were isolated from a partial hydrolysate of the polysaccharide, and characterised. The similarities and differences between this polysaccharide and those from other Xanthomonas species are discussed.     

Water- and alkali-soluble glucans from oak lichen

A water-soluble glucan, [α]²_D +217° (water), and an alkali-soluble glucan,

+152° (sodium hydroxide), have been isolated from the oak lichen Evernia prunastri (L.) Ach. On the basis of methylation analysis, periodate oxidation, and partial acid hydrolysis, the water-soluble polysaccharide has been shown to be a neutral, slightly branched glucan with a main chain composed of (1→3)- and (1→4)- linked glucopyranose residues in the ratio 1?:1. Branching occurs most probably at position 2 of (1→4)-linked glucopyranose residues. On the basis of optical rotation and i.r. spectral data, and enzymic hydrolysis, the α-D configuration has been assigned to the glycosidic linkages. Likewise, the alkali-soluble polysaccharide was shown to be a neutral, branched glucan with a main chain composed of (1→3)- and (1→4)-linked α-D-glucopyranose residues in the ratio 6:1. Each of the (1→4)-linked units was a branch point involving position 6. The presence of some β-D linkages is not excluded since hydrolysis with β-D-glucosidase occurred to a small extent.     

Isolation and analysis of neutral glucans from Ecklonia radiata and Cystophora scalaris

Neutral glucans were isolated from the stipes and fronds of Eklonia radiata and Cystophora scalaris. Partial acid hydrolysis revealed the presence of gentiobiose and laminara-oligosaccharides. Methylation analysis, periodate oxidation, and enzyme studies indicated that the glucans contain β-(1→3) and β-(1→6) linkages. Methylation studies showed that branching in these glucans occurs via a 1,3,6-tri-O- substituted residue with a frequency of one branch point per seven glycosyl residues. In contrast to laminaran from Laminaria digitata, the intrachain (1→3)- and (1→6)- glucopyranoside occur in a molar ratio of 1:1. Enzymic hydrolysis confirmed the absence of long segments of (1→3)-linked residues in the glucans.     

Fractionation of the β-Linked Glucans of Bradyrhizobium japonicum and Their Response to Osmotic Potential

Bradyrhizobium japonicum USDA 110 synthesized both extracellular and periplasmic polysaccharides when grown on mannitol minimal medium. The extracellular polysaccharides were separated into a high-molecular-weight acidic capsular extracellular polysaccharide fraction (90% of total hexose) and three lower-molecular-weight glucan fractions by liquid chromatography. Periplasmic glucans, extracted from washed cells with 1% trichloroacetic acid, gave a similar pattern on liquid chromatography. Linkage analysis of the major periplasmic glucan fractions demonstrated mainly 6-linked glucose (63 to 68%), along with some 3,6- (8 to 18%), 3- (9 to 11%), and terminal (7 to 8%) linkages. The glucose residues were β-linked as shown by ¹H-nuclear magnetic resonance analysis. Glucan synthesis by B. japonicum cells grown on mannitol medium with 0 to 350 mM fructose as osmolyte was measured. Fructose at 150 mM or higher inhibited synthesis of periplasmic and extracellular 3- and 6-linked glucans but had no effect on the synthesis of capsular acidic extracellular polysaccharides.     

Construction of Chimeric Glucansucrases for Analyzing Substrate-binding Regions That Affect the Structure of Glucan Products

A gene that encodes dextransucrase S (dsrS) from Leuconostoc mesenteroides NRRL B-512F encodes a glucansucrase dextransucrase S (DSRS) which mainly produces water-soluble glucan (dextran), while the dsrT5 gene derived from dsrT of the B-512F strain encodes an enzyme dextransucrase T5 (DSRT5), which mainly produces water-insoluble glucan. Tyr340-Asn510 of DSRS and Tyr307-Asn477 of DSRT5 (Site 1), Lys696-Gly768 of DSRS and Lys668-Gly740 of DSRT5 (Site 2), and Asn917-Lys1131 of DSRS and Asn904-Lys1118 of DSRT5 (Site 3) were exchanged and six different chimeric enzymes were constructed. Water-soluble glucan produced by recombinant DSRS was composed of 64% 6-linked glucopyranoside (Glcp), 9% 3,6-linked Glcp, and 13% 4-linked Glcp. Water-insoluble glucan produced by recombinant DSRT5 was composed of 47% 6-linked Glcp and 43% 3-linked Glcp. All of the chimeric enzymes produced glucans different from the ones produced by their parental enzymes. Some of the glucans produced by chimeric enzymes were extremely changed. The Site 1 chimeric enzyme of DSRS (STS1) produced water-soluble glucan composed mostly of 6-linked Glcp. That of DSRT5 (TST1) produced water-insoluble glucan composed mostly of 4-linked Glcp. The Site 3 chimeric enzyme of DSRS (STS3) produced mainly water-insoluble glucan, DSRT5 (TST3) produced mainly water-soluble glucans, and all of the glucan fractions consisted of 3-Glcp, 4-Glcp, and 6-Glcp. The amounts of the three linkages in the water-soluble glucan produced by TST3 were about 1:1:1. Site 1 was assumed to be important for making or avoiding making α-1,4 linkages, while Site 3 was assumed to be important for determining the kinds of glucosyl linkages made.     

Ultrastructure and Chemical Analysis of the Cell Wall of Pythium debaryanum1

The ultrastructure and component polysaccharides of the cell wall of Pythium debaryanum IFO-5919 were investigated. From results obtained by means of acid, alkali, Schweitzer reagent and β-1, 3-glucanase treatments and electron microscopy, it was concluded that 1) the acid-extracted fraction was a 1,3-linked branched glucan, 2) the alkali-extracted fraction was a mixture of 1,3-, 1,6-, and 1,3,6-linked highly branched two glucans, 3) the Schweitzer reagent-extracted fraction was a β-1, 4-linked glucan, 4) the cell wall was constructed from two types of cullulosic microfibrils, as a frame and as a finer network, and amorphous β-1, 3-glucan including β-1, 6-linkage, 5) cellulosic microfibrils were covered by matrix material consisting of a mixture of amorphous β-1, 3-linked and β-1, 6-linked branching glucans.     

The origin of the periodate-resistant D-glucose residues in starches and glycogens

Under ordinary analytical conditions, the proportion of periodate-resistant-D-glucose residues in starches and glycogens was consistently about one-third of the proportion of branching points. The resistant D-glucose residues became freely oxidisable after the limit-oxidised glucans had been reduced with sodium borohydride. The results can be explained by assuming that, when a D-glucose residue carrying a branch at position 6 is oxidised, the resulting two aldehyde groups both form 6-membered hemiacetal rings with the closest hydroxyl groups on neighbouring, unoxidised residues in the same, (1→4)-linked chain, whereas when the other D-glucose residues are oxidised, only one of the aldehyde groups shows a strong tendency to form a hemiacetal of this kind. It is suggested that, in the unbranched units, the other aldehyde group preferentially forms a hemiacetal with the primary hydroxyl group in the same unit.     

The production of glucans via glucansucrases from Lactobacillus satsumensis isolated from a fermented beverage starter culture

Several starter cultures used in the production of fermented beverages were screened for lactic acid bacteria that produced water-insoluble polysaccharides from sucrose. The strain producing the greatest amount was identified as Lactobacillus satsumensis by its 16S RNA sequence and was deposited in the ARS culture collection as NRRL B-59839. This strain produced at least two α-d-glucans from sucrose. One was a water-soluble dextran, consisting of predominantly α-(1?→?6)-linked d-glucose units, and the other was a water-insoluble glucan containing both α-(1?→?6)-linked and α-(1?→?3)-linked d-glucose units. The culture fluid was found to contain glucansucrases responsible for the two glucans, and no significant level of fructansucrase was detected. Glucansucrase activity was not present in the culture fluid when the bacteria were grown on glucose, fructose, or raffinose as the carbon source. Although the water-soluble glucans produced by cell-free enzyme and by cell suspensions were essentially identical, the same was not true for the water-insoluble glucans. The water-insoluble glucan produced by cell-free culture fluid contained a higher proportion of α-(1?→?3)-linked d-glucose units than the water-insoluble glucan produced by cell suspensions.     

Antitumor Activities and Immunochemical Properties of the Cell-wall Polysaccharides from Aureobasidium pullulans

Delipidated cell walls from Aureobasidium pullulans were fractionated systematically.

The cell surface heteropolysaccharide contains D-mannose, D-galactose, D-glucose, and D-glucuronic acid (ratio, 8.5:3.9:1.0:1.0). It consists of a backbone of (1→6)-α-linked D-mannose residues, some of which are substituted at O-3 with single or β-(1→6)-linked D-galactofuranosyl side chains, some terminated with a D-glucuronic acid residue, and also with single residues of D-glucopyranose, D-galactopyranose, and D-mannopyranose.

This glucurono-gluco-galactomannan interacted with antiserum against Elsinoe leucospila, which also reacted with its galactomannan, indicating that both polysaccharides contain a common epitope, i.e., at least terminal β-galactofuranosyl groups and also possibly internal β-(1→6)-linked galactofuranose residues.

It was further separated by DEAE-Sephacel column chromatography to gluco-galactomannan and glucurono-gluco-galactomannan.

The alkali-extracted β-D-glucan was purified by DEAE-cellulose chromatography to afford two antitumor-active (1→3)-β-D-glucans. One of the glucans (M_r, 1–2 × 10⁵) was a O-6-branched (1→3)-β-D-glucan with a single β-D-glucosyl residue, d.b., 1/7, and the other (M_r, 3.5–4.5 × 10⁵) had similar branched structure, but having d.b., 1/5. Side chains of both glucans contain small proportions of β-(1→6)-and β-(1→4)-D-glucosidic linkages.     

13C-nmr spectroscopic investigation of two yeast cell wall β-D-glucans

By means of alkaline extraction of the cell walsl of the yeasts Saccharomyces cerevisiae and Canadida albicans, water-insoluble glucans were obtained. Methylation analysis and ¹³C-nmr investigation in dimethyl sulfoxide solution revealed the similar chemical structure of these glucans, being composed of β(1 → 3) glycosidically linked D -glucopyranosyl units with a small amount of β(1 → 6) linkages. More detailed study in dilute alkaline solutions and in the gel state at neutral pH, however, showed that an ordered helical conformation of the glucan chain is less stable in the case of the S. cerevisiae glucan in comparison with that of C. albicans. Measurements of the shift of the absorption maximum of the glucan complexes with Congo Red also demonstrated such difference. The S. cerevisiae glucan was also inable to form a gel at neutral pH. The difference in stability of helical conformation of the glucans is explained on the basis of the methylation analysis, so that the S. cerevisiae glucan possesses longer side chains, which hinder its adoption of a stable helical conformation.     

Metabolism of the polysaccharides of human dental plaque. Part II. Purification and properties of Cladosporium resinae (1 leads to 3)-alpha-D-glucanase, and the enzymic hydrolysis of glucans synthesised by extracellular D-glucosyltransferases of oral streptococci.

Cladosporium resinae (1 leads to 3)-alpha-D-glucanase has been characterized as an endoglucanase capable of completely hydrolysing insoluble (1 leads to 3)-alpha-D-glucans isolated from fungal cell-walls. D-Glucose was the major product, but a small amount of nigerose was also produced. The enzyme was specific for the hydrolysis of (1 leads to 3) bonds that occur in sequence, and nigerotetraose was the smallest substrate that was rapidly attacked. Isolated (1 leads to 3)-alpha-D-glucosidic linkages that occur in mycodextran, isolichein, dextrans, and oligosaccharides derived from dextran were not hydrolysed. Insoluble glucan synthesised from sucrose by culture filtrates of Streptococcus spp. were all hydrolysed to various limits; the range was 11-61%. A soluble glucan, synthesised by an extracellular D-glucosyltransferase of S. mutans OMZ176, was not a substrate, whereas insoluble glucans synthesised by a different D-glucosyltransferase, isolated from S. mutans strains OMZ176 and K1-R, were extensively hydrolysed (84 and 92%, respectively). It is suggested that dextranase-CB, a bacterial endo(1 leads to 6)-alpha-D-glucanase that does not release D-glucose from any substrate, could be used together with C. resinae (1 leads to 3)-alpha-D-glucanase to determine the relative proportions of (1 leads to 6)-linked to (1 leads to 3)-linked sequences of D-glucose residues in the insoluble glucans produce by oral streptococci. The simultaneous action of the two D-glucanoses was highly effective in solubilizing the glucans.     

Structural characterization of a water-soluble beta-D-glucan from fruiting bodies of Agaricus blazei Murr

A beta-D-glucan, Ab2-2N, was isolated from the hot-water extract of fruiting bodies of Agaricus blazei Murr by ethanol precipitation, anion-exchange and gel-permeation chromatography. Its structure was investigated by composition analysis, methylation analysis, Smith degradation, mild hydrolysis, and NMR spectroscopy. It contains a (1-->6)-linked beta-D-glucopyranosyl backbone, with one side chain consisting of terminal and 3-substituted beta-D-glucopyranosyl residues attached at O-3 for every three backbone residues.