Anti-inflammatory and immunologically active polysaccharides of Periandra mediterranea

Three polysaccharides, glucans with mean M(r)'s of 1.5 x 10(5), 3.6 x 10(4) and 2.1 x 10(4), were isolated from dried roots of Periandra mediterranea by fractionation on Sephacryl S-300 HR and Sephadex G-25. Chemical and spectroscopic studies indicated that they have a highly branched glucan type structure composed of alpha-(1-->4) linked D-glucopyranose residues with both (3-->4) and (4-->6) branching points. The polysaccharides enhance phagocytosis in vivo, and exhibit anti-inflammatory activity.     

The influence of mutanase and dextranase on the production and structure of glucans synthesized by streptococcal glucosyltransferases

Glucanohydrolases, especially mutanase [alpha-(1-->3) glucanase; EC 3.2.1.59] and dextranase [alpha-(1-->6) glucanase; EC 3.2.1.11], which are present in the biofilm known as dental plaque, may affect the synthesis and structure of glucans formed by glucosyltransferases (GTFs) from sucrose within dental plaque. We examined the production and the structure of glucans synthesized by GTFs B (synthesis of alpha-(1-->3)-linked glucans) or C [synthesis of alpha-(1-->6)- and alpha-(1-->3)-linked glucans] in the presence of mutanase and dextranase, alone or in combination, in solution phase and on saliva-coated hydroxyapatite beads (surface phase). The ability of Streptococcus sobrinus 6715 to adhere to the glucan, which was formed in the presence of the glucanohydrolases was also explored. The presence of mutanase and/or dextranase during the synthesis of glucans by GTF B and C altered the proportions of soluble to insoluble glucan. The presence of either dextranase or mutanase alone had a modest effect on total amount of glucan formed, especially in the surface phase; the glucanohydrolases in combination reduced the total amount of glucan. The amount of (1-->6)-linked glucan was reduced in presence of dextranase. In contrast, mutanase enhanced the formation of soluble glucan, and reduced the percentage of 3-linked glucose of GTF B and C glucans whereas dextranase was mostly without effect. Glucan formed in the presence of dextranase provided fewer binding sites for S. sobrinus; mutanase was devoid of any effect. We also noted that the GTFs bind to dextranase and mutanase. Glucanohydrolases, even in the presence of GTFs, influence glucan synthesis, linkage remodeling, and branching, which may have an impact on the formation, maturation, physical properties, and bacterial binding sites of the polysaccharide matrix in dental plaque. Our data have relevance for the formation of polysaccharide matrix of other biofilms.     

Molecular modeling study of highly branching (1-->3)-alpha-D-glucan, a model polysaccharide for cariogenic glucan, using the N-H mapping method

A systematic search for possible regular helical structures of a highly branching (1-->3)-alpha-D-glucan was done using the n-h mapping technique, combined with MM3-generated relaxed-residue energy map calculations with respect to the conformations of the backbone glycosidic linkages. The alpha-D-glucan, consisting of a (1-->3)-alpha-linked backbone with alpha-D-glucose side residues attaching to an O6 atom of every second backbone residue, was considered as a model polysaccharide of a branching part of the glucan produced by oral bacteria, which was known to be related to dental plaque formation and to contribute to dental caries. The potential energy surfaces of the trisaccharide repeating unit of the branching alpha-D-glucan indicated that (1-->6)-alpha-linked side residues did not appear to interfere significantly with the backbone stereochemistry, probably due to a further separation of the three-bond-linked side residue compared with an ordinary two-bond-linked residue. Based on the n-h maps of the branching alpha-D-glucan, the side residues, when involved in a complete helix, mostly contributed additional stabilizations to particular helical structures. It was found by checking the typical helix models that formation of hydrogen bonds involving side residues was probably a major cause of the stabilization. This hydrogen bonding was expected to increase insolubility for the glucan chain--a typical, physical property observed for the bacterial alpha-D-glucan--by introducing its backbone stereochemistry as an additional stiff feature.     

Structural analysis of anti-tumor heteropolysaccharide GFPS1b from the cultured mycelia of Grifola frondosa GF9801

A 21-kDa heteropolysaccharide, coded as GFPS1b, was obtained from the cultured mycelia of Grifola frondosa GF9801 by hot-water extraction, ethanol precipitation, and fractioned by DEAE Sepharose Fast-flow, followed by the purification with Sephadex G-100 column chromatography using an AKTA purifier. It exhibited more potent anti-proliferative activity on MCF-7 cells than other polysaccharide fractions. GFPS1b was an acidic polysaccharide with approximately 16.60% protein and 4.3% uronic acid. Gas chromatography of absolute acid hydrolysate of GFPS1b suggested that it was composed of D-glucose, D-galactose, and L-arabinose with a molar ratio of 4:2:1. Periodate oxidation, Smith degradation, partial acid hydrolyzation, methylation analysis, FT-IR, and (1)H, (13)C NMR spectroscopy analysis revealed that GFPS1b had a backbone consisting of alpha-(1-->4)-linked D-galacopyranosyl and alpha-(1-->3)-linked D-glucopyranosyl residues substituted at O-6 with glycosyl residues composed of alpha-L-arabinose-(1-->4)-alpha-D-glucose (1--> linked residues.     

Structural characterization of the O-antigenic polysaccharide of the lipopolysaccharide from Rhizobium etli strain CE3. A unique O-acetylated glycan of discrete size, containing 3-O-methyl-6-deoxy-L-talose and 2,3,4-tri-O-,methyl-l fucose

The O-antigenic polysaccharide of the Rhizobium etli CE3 lipopolysaccharide (LPS) was structurally characterized using chemical degradations (Smith degradation and beta-elimination of uronosyl residues) in combination with alkylation analysis, electrospray, and matrix-assisted laser desorption ionization-time of flight mass spectrometry, tandem mass spectrometry, and (1)H COSY and TOCSY nuclear magnetic resonance spectroscopy analyses of the native polysaccharide and the derived oligosaccharides. The polysaccharide was found to be a unique, relatively low molecular weight glycan having a fairly discrete size, with surprisingly little variation in the number of repeating units (degree of polymerization = 5). The polysaccharide is O-acetylated and contains a variety of O-methylated glycosyl residues, rendering the native glycan somewhat hydrophobic. The molecular mass of the major de-O-acetylated species, including the reducing end 3-deoxy-d-manno-2-octulosonic acid (Kdo) residue, is 3330 Da. The polysaccharide is comprised of a trisaccharide repeating unit having the structure -->4)-alpha-d-GlcpA-(1-->4)-[alpha-3-O-Me-6-deoxy-Talp-(1--> 3)]-alpha -l-Fucp-(1-->. The nonreducing end of the glycan is terminated with the capping sequence alpha-2,3, 4-tri-O-Me-Fucp-(1-->4)-alpha-d-GlcpA-(1-->, and the reducing end of the molecule consists of the non-repeating sequence -->3)-alpha-l-Fucp-(1-->3)-beta-d-Manp-(1-->3)-beta-QuiNA cp-(1-->4)-a lpha-Kdop-(2-->, where QuiNAc is N-acetylquinovosamine (2-N-acetamido-2,6-dideoxyglucose). The reducing end Kdo residue links the O-chain polysaccharide to the core region oligosaccharide, resulting in a unique location for a Kdo residue in LPS, removed four residues distally from the lipid A moiety. Structural heterogeneity in the O-chain arises mainly from the O-acetyl and O-methyl substitution. Methylation analysis using trideuteriomethyl iodide indicates that a portion of the 2,3,4-tri-O-methylfucosyl capping residues, typically 15%, are replaced with 2-O-methyl- and/or 2,3-di-O-methylfucosyl residues. In addition, approximately 25% of the 3,4-linked branching fucosyl residues and 10% of the 3-linked fucosyl residues are 2-O-methylated. A majority of the glucuronosyl residues are methyl-esterified at C-6. These unique structural features may be significant in the infection process.     

Strucrural investigation of Klebsiella serotype K7 polysaccharide

Methylation analysis of and partial hydrolysis studies on the Klebsiella K7 capsular polysaccharide and its carboxyl-reduced derivative indicated the recurrence of D-glucopyranuronic acid, D-mannopyranose, and D-glucopyranose residues, linearly linked in a specific manner, in the molecular structure. D-Galactopyranose and pyruvic acid residues are linked to the main chain on the D-mannose residues (at O-3) and the D-glucose residues (at O-4 and O-6), respectively; the simplest interpretation of this evidence is that nine sugar residues and pyruvic acid constitute a repeating unit. The sequence →3)-β-D-GlcAp-(1→2)-α-D-Manp-(1→2)-α-D-Manp-(1→3)-D-Glcp→ was demonstrated by the isolation from the polysaccharide of an aldotetraouronic acid of this structure.     

A new synthetic hypoglycaemic polysaccharide.

The synthetic (1-->6)-alpha-D-glucopyranan with branching and without branching were tested as a new hypoglycaemic drug. (1-->6)-alpha-D-glucopyranan having an alpha-D-glucopyranosyl branch at the C-3 position (1) showed a remarkable hypoglycaemic activity on i.p. injection to mice. The polysaccharide having both alpha- and beta-glucopyranosyl branches (2) also lowered the blood sugar (glucose) level in mice. On the other hand, the synthetic linear (1-->6)-alpha-D-glucopyranan (3) and alpha-D-glucopyranosyl branched polysaccharide (4) did not have a hypoglycaemic function, indicating that the branching glucose units are essential for the biological activity.     

Highly hydrolytic reuteransucrase from probiotic Lactobacillus reuteri strain ATCC 55730

Lactobacillus reuteri strain ATCC 55730 (LB BIO) was isolated as a pure culture from a Reuteri tablet purchased from the BioGaia company. This probiotic strain produces a soluble glucan (reuteran), in which the majority of the linkages are of the alpha-(1-->4) glucosidic type ( approximately 70%). This reuteran also contains alpha-(1-->6)- linked glucosyl units and 4,6-disubstituted alpha-glucosyl units at the branching points. The LB BIO glucansucrase gene (gtfO) was cloned and expressed in Escherichia coli, and the GTFO enzyme was purified. The recombinant GTFO enzyme and the LB BIO culture supernatants synthesized identical glucan polymers with respect to linkage type and size distribution. GTFO thus is a reuteransucrase, responsible for synthesis of this reuteran polymer in LB BIO. The preference of GTFO for synthesizing alpha-(1-->4) linkages is also evident from the oligosaccharides produced from sucrose with different acceptor substrates, e.g., isopanose from isomaltose. GTFO has a relatively high hydrolysis/transferase activity ratio. Complete conversion of 100 mM sucrose by GTFO nevertheless yielded large amounts of reuteran, although more than 50% of sucrose was converted into glucose. This is only the second example of the isolation and characterization of a reuteransucrase and its reuteran product, both found in different L. reuteri strains. GTFO synthesizes a reuteran with the highest amount of alpha-(1-->4) linkages reported to date.     

Synthesis and conformational analysis of a pentasaccharide corresponding to the cell-wall polysaccharide of the Group A Streptococcus

The synthesis and conformational analysis of a pentasaccharide corresponding to a fragment of the cell-wall polysaccharide (CWPS) of the bacteria Streptococcus Group A are described. The polysaccharide consists of alternating alpha-(1 --> 2)- and alpha-(1 --> 3)-linked L-rhamnopyranose (Rhap) residues with branching 2-acetamido-2-deoxy-D-glucopyranose (GlcpNAc) residues linked beta-(1 --> 3) to alternate rhamnose rings. The pentasaccharide is of interest as a possible terminal unit on the CWPS, for use in a vaccine. The syntheses employed a trichloroacetimidate glycosyl donor. Molecular dynamics (MD) calculations of the pentasaccharide with the force fields CVFF and PARM22, both in gas phase and with explicit water present, gave different predictions for the flexibility and preferred conformational space. Metropolis Monte Carlo (MMC) calculations with the HSEA force field were also performed. Experimental data were obtained from 1D transient NOE measurements. Complete build-up curves were compared to those obtained by full relaxation matrix calculations in order to derive a model of the conformation. Overall, the best fit between experimental and calculated data was obtained with MMC simulations using the HSEA force field. Molecular dynamics and MMC simulations of a tetrasaccharide corresponding to the Group A-variant polysaccharide, which differs in structure from Group A in lacking the GlcpNAc residues, were also performed for purposes of comparison.     

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.     

A study of fucoidan from the brown seaweed Chorda filum.

Fucoidan fractions from the brown seaweed Chorda filum were studied using solvolytic desulfation. Methylation analysis and NMR spectroscopy were applied for native and desulfated polysaccharides. Homofucan sulfate from C. filum was shown to contain poly-alpha-(1-->3)-fucopyranoside backbone with a high degree of branching, mainly of alpha-(1-->2)-linked single units. Some fucopyranose residues are sulfated at O-4 (mainly) and O-2 positions. Some alpha-(1-->3)-linked fucose residues were shown by NMR to be 2-O-acetylated. The 1H and 13C NMR spectra of desulfated, deacetylated fucan were completely assigned. The spectral data obtained correspond to a quasiregular polysaccharide structure with a branched hexasaccharide repeating unit. Other fucoidan fractions from C. filum have more complex carbohydrate composition and give rather complex methylation patterns. [formula: see text]     

Characterization of two cell-wall polysaccharides from Fusicoccum amygdali

1. The nature of two polysaccharides (s(0) (20) values 6S and 2S respectively in 1m-sodium hydroxide), comprising a fragment (fraction BB, [alpha](D) +236 degrees in 1m-sodium hydroxide), previously isolated from cell walls of Fusicoccum amygdali, has been investigated. 2. Both the major (2S) and minor (6S) components were affected by incubation with alpha-amylase. The 6S polysaccharide was also attacked by exo-beta-(1-->3)-glucanase, which is evidence that it contained both alpha-(1-->4)- and beta-(1-->3)-glucopyranose linkages. By fractionation of the products of alpha-amylase-treated fraction BB it was possible to obtain a water-insoluble polysaccharide, fraction P ([alpha](D) +290 degrees in 1m-sodium hydroxide, 67% of fraction BB) and a water-soluble polysaccharide, fraction Q ([alpha](D) +16 degrees in 1m-sodium hydroxide, 11% of fraction BB), both of which sedimented as single boundaries with s(0) (20) values (in 1m-sodium hydroxide) of 1.7S and 4.6S respectively. 3. Evidence from periodate oxidation, methylation analysis, i.r. spectroscopy and partial acid hydrolysis showed that fraction P consisted of linear chains of alpha-(1-->3)-glucopyranose units with blocks of one or two alpha-(1-->4)-glucopyranose units interspersed at intervals along the main chain. The 2S polysaccharide, from which fraction P is derived, evidently also contains longer blocks of alpha-(1-->4)-glucopyranose units, that are susceptible to alpha-amylase action. 4. Fraction Q consisted of glucose (88%) with small amounts of galactose, mannose and rhamnose. Evidence from digestion with exo- and endo-beta-(1-->3)-glucanases, periodate oxidation and methylation analysis suggests that fraction Q consists of a branched galactomannorhamnan core, to which is attached a beta-(1-->3)-, beta-(1-->6)-glucan. In the cell wall, chains of alpha-(1-->4)-linked glucopyranose units are linked to fraction Q to form the 6S component of fraction BB.     

Transglycosylation reactions of Bacillus stearothermophilus maltogenic amylase with acarbose and various acceptors

It was observed that Bacillus stearothermophilus maltogenic amylase cleaved the first glycosidic bond of acarbose to produce glucose and a pseudotrisaccharide (PTS) that was transferred to C-6 of the glucose to give an alpha-(1-->6) glycosidic linkage and the formation of isoacarbose. The addition of a number of different carbohydrates to the digest gave transfer products in which PTS was primarily attached alpha-(1-->6) to D-glucose, D-mannose, D-galactose, and methyl alpha-D-glucopyranoside. With D-fructopyranose and D-xylopyranose, PTS was linked alpha-(1-->5) and alpha-(1-->4), respectively. PTS was primarily transferred to C-6 of the nonreducing residue of maltose, cellobiose, lactose, and gentiobiose. Lesser amounts of alpha-(1-->3) and/or alpha-(1-->4) transfer products were also observed for these carbohydrate acceptors. The major transfer product to sucrose gave PTS linked alpha-(1-->4) to the glucose residue. alpha,alpha-Trehalose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4). Maltitol gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the glucopyranose residue. Raffinose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the D-galactopyranose residue. Maltotriose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the nonreducing end glucopyranose residue. Xylitol gave PTS linked alpha-(1-->5) as the major product and D-glucitol gave PTS linked alpha-(1-->6) as the only product. The structures of the transfer products were determined using thin-layer chromatography, high-performance ion chromatography, enzyme hydrolysis, methylation analysis and 13C NMR spectroscopy. The best acceptor was gentiobiose, followed closely by maltose and cellobiose, and the weakest acceptor was D-glucitol.     

In vitro biosynthesis of galactans by membrane-bound galactosyltransferase from radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis> L.) seedlings

We investigated a galactosyltransferase (GalT) involved in the synthesis of the carbohydrate portion of arabinogalactan-proteins (AGPs), which consist of a beta-(1-->3)-galactan backbone from which consecutive (1-->6)-linked beta-Gal p residues branch off. A membrane preparation from 6-day-old primary roots of radish ( Raphanus sativus L.) transferred [(14)C]Gal from UDP-[(14)C]Gal onto a beta-(1-->3)-galactan exogenous acceptor. The reaction occurred maximally at pH 5.9-6.3 and 30 degrees C in the presence of 15 mM Mn(2+) and 0.75% Triton X-100. The apparent K(m) and V(max) values for UDP-Gal were 0.41 mM and 1,000 pmol min(-1) (mg protein)(-1), respectively. The reaction with beta-(1-->3)-galactan showed a bi-phasic kinetic character with K(m) values of 0.43 and 2.8 mg ml(-1). beta-(1-->3)-Galactooligomers were good acceptors and enzyme activity increased with increasing polymerization of Gal residues. In contrast, the enzyme was less efficient on beta-(1-->6)-oligomers. The transfer reaction for an AGP from radish mature roots was negligible but could be increased by prior enzymatic or chemical removal of alpha- l-arabinofuranose (alpha- l-Ara f) residues or both alpha- l-Ara f residues and (1-->6)-linked beta-Gal side chains. Digestion of radiolabeled products formed from beta-(1-->3)-galactan and the modified AGP with exo-beta-(1-->3)-galactanase released mainly radioactive beta-(1-->6)-galactobiose, indicating that the transfer of [(14)C]Gal occurred preferentially onto consecutive (1-->3)-linked beta-Gal chains through beta-(1-->6)-linkages, resulting in the formation of single branching points. The enzyme produced mainly a branched tetrasaccharide, Galbeta(1-->3)[Galbeta(1-->6)] Galbeta(1-->3)Gal, from beta-(1-->3)-galactotriose by incubation with UDP-Gal, confirming the preferential formation of the branching linkage. Localization of the GalT in the Golgi apparatus was revealed on a sucrose density gradient. The membrane preparation also incorporated [(14)C]Gal into beta-(1-->4)-galactan, indicating that the membranes contained different types of GalT isoform catalyzing the synthesis of different types of galactosidic linkage.     

Branched glucan from the fruiting bodies of Piptoporus betulinus (Bull.:Fr.) Karst

A new glucan, namely, piptoporane I, with a molecular mass of 270 kDa was isolated from fruiting bodies of Piptoporus betulinis (Bull.:Fr.) Karst. (Fomitopsidacaeae). Using a combination ofphysicochemical methods, it was established that piptoporane I was a branched glucan with a backbone consisting of alpha-( 1->3)-glucopyranose residues substituted at the C-6 position by single residues of beta-D-glucopyranose by 17.3%. A polysaccharide with such a structure was isolated for the first time from the fungus genus Piptoporus.     

Characterization of a beta-D-(1-->3)-glucan from the marine diatom Chaetoceros mülleri by high-resolution magic-angle spinning NMR spectroscopy on whole algal cells

High-resolution magic-angle spinning (hr-MAS) NMR spectroscopy was used to record NMR spectra of a cell paste from the marine diatom Chaetoceros mülleri. This gave information on a cellular storage polysaccharide identified as a beta-D-(1-->3)-linked glucan, using hr-MAS one-dimensional 1H and 13C, two-dimensional 1H,1H-COSY and 13C,1H-correlation spectroscopy. The same structural information was deduced from the liquid state NMR data on the glucan extracted from C. mülleri. The extracted glucan proved to be a beta-D-(1-->3)-linked glucan with a degree of polymerization of 19 and a degree of beta-D-(1-->6) branching of 0.005. The hr-MAS spectrum of the diatom showed several nonglucan resonances in the carbohydrate region of the NMR spectrum (60-103 ppm) that were shown to be noncarbohydrate resonances by means of two-dimensional 13C,1H- and 1H,1H-correlated NMR data.     

Differential inhibition of branching enzyme in a morphological mutant and in wild type Neurospora. Influence of carbon source in the growth medium.

1. A morphological mutant of Neurospora crassa, smco 9, (R2508) that exhibits colonial morphology when grown on sucrose or on maltose, showed a partial reversal of this morphology toward that of the wild type when it was grown on potato starch or on isomaltose. 2. A common feature of both potato starch and isomaltose is the presence of alpha-1, 6 glucosidic linkages. This suggested that these morphological effects might be due to differences in alpha-1,4 glucan: alpha-1,4 glucan 6 glycosyltransferase, (EC 2.4.1.18) commonly known as "the branching enzyme". 3. The branching enzyme was purified from wild type, Neurospora crassa, and from the semicolonial mutant, R2508, both grown on sucrose or on potato starch. It has a molecular weight of 140,000 as estimated by gel filtration on a Bio Gel A 1.5 m column. This enzyme plus phosphorylase a in an unprimed reaction catalyzes the synthesis of a branched polysaccharide in vitro. 4. No branching enzyme activity was apparent in extracts of the mutant R2508, grown on potato starch until a thermolabile inhibitor was removed by fractionation on a DEAE column. 5. This inhibitor has a molecular weight greater than 100,000 as estimated on a P-100 polyacrylamide gel column. The specificity of the inhibitor is not absolute in that it inhibits glycogen synthetase in addition to the branching enzyme in Neurospora.     

Purification and characterization of branching specificity of a novel extracellular amylolytic enzyme from marine hyperthermophilic Rhodothermus marinus

An extracellular enzyme (RMEBE) possessing alpha- (1-->4)-(1-->6)-transferring activity was purified to homogeneity from Rhodothermus marinus by combination of ammonium sulfate precipitation, Q-Sepharose ion-exchange, and Superdex- 200 gel filtration chromatographies, and preparative native polyacrylamide gel electrophoresis. The purified enzyme had an optimum pH of 6.0 and was highly thermostable with a maximal activity at 80 degrees . Its half-life was determined to be 73.7 and 16.7 min at 80 and 85 degrees , respectively. The enzyme was also halophilic and highly halotolerant up to about 2 M NaCl, with a maximal activity at 0.5M. The substrate specificity of RMEBE suggested that it possesses partial characteristics of both glucan branching enzyme and neopullulanase. RMEBE clearly produced branched glucans from amylose, with partial alpha-(1-->4)-hydrolysis of amylose and starch. At the same time, it hydrolyzed pullulan partly to panose, and exhibited alpha-(1-->4)-(1-->6)-transferase activity for small maltooligosaccharides, producing disproportionated alpha-(1-->6)-branched maltooligosaccharides. The enzyme preferred maltopentaose and maltohexaose to smaller maltooligosaccharides for production of longer branched products. Thus, the results suggest that RMEBE might be applied for production of branched oligosaccharides from small maltodextrins at high temperature or even at high salinity.     

Molecular dynamics simulations of a cyclic-beta-(1-->2) glucan containing an alpha-(1-->6) linkage as a 'molecular alleviator' for the macrocyclic conformational strain

The conformational preferences of a cyclic osmoregulated periplasmic glucan of Ralstonia solanacearum (OPGR), which is composed of 13 glucose units and linked entirely via beta-(1-->2) linkages excluding one alpha-(1-->6) linkage, were characterized by molecular dynamics simulations. Of the three force fields modified for carbohydrates that were applied to select a suitable one for the cyclic glucan, the carbohydrate solution force field (CSFF) was found to most accurately simulate the cyclic molecule. To determine the conformational characteristics of OPGR, we investigated the glycosidic dihedral angle distribution, fluctuation, and the potential energy of the glucan and constructed hypothetical cyclic (CYS13) and linear (LINEAR) glucans. All beta-(1-->2)-glycosidic linkages of OPGR adopted stable conformations, and the dihedral angles fluctuated in this energy region with some flexibility. However, despite the inherent flexibility of the alpha-(1-->6) linkage, the dihedral angles have no transition and are more rigid than that in a linear glucan. CYS13, which consists of only beta-(1-->2) linkages, is somewhat less flexible than other glycans, and one of its linkages adopts a higher energy conformation. In addition, the root-mean-square fluctuation of this linkage is lower than that of other linkages. Furthermore, the potential energy of glucans increases in the order of LINEAR, OPGR, and CYS13. These results provide evidence of the existence of conformational constraints in the cyclic glucan. The alpha-(1-->6)-glycosidic linkage can relieve this constraint more efficiently than the beta-(1-->2) linkage. The conformation of OPGR can reconcile the tendency for individual glycosidic bonds to adopt energetically favorable conformations with the requirement for closure of the macrocyclic ring by losing the inherent flexibility of the alpha-(1-->6)-glycosidic linkage.     

Chemical characterization of the immunomodulating polysaccharide of Aloe vera L

The polysaccharide isolated by alcohol precipitation of Aloe vera mucilaginous gel was found to have a Man:Glc:Gal:GalA:Fuc:Ara:Xyl ratio of 120:9:6:3:2:2:1 with traces of Rha and GlcA. Linkage analysis of the endo-(1-->4)-beta-d-mannanase-treated sample yielded Manp-(1--> (approximately 26%), 4-Manp (approximately 53%), 2,4-Manp (approximately 3%), 3,4-Manp (approximately 1%), 4,6-Manp (approximately 1%), 4-Glcp (approximately 5%), 4-Xylp (approximately 1%), Xylp-(1--> (approximately 2%), Galp-(1--> (approximately 5%), and traces of 4,6-Galp and 3,6-Galp. Hydrolysis with strong acids produced a mixture of short oligosaccharides and an acid-resistant fraction containing greater relative fractions of Manp-(1-->, Araf-(1-->, Xylp-(1-->, and 4-Xylp than the bulk polysaccharide. NMR analysis of oligosaccharides generated by endo-(1-->4)-beta-D-mannanase and acid hydrolysis showed the presence of di-, tri-, and tetrasaccharides of 4-beta-Manp, beta-Glcp-(1-->4)-Man, beta-Glcp-(1-->4)-beta-Manp-(1-->4)-Man, and beta-Manp-(1-->4)-[alpha-Galp-(1-->6)]-Man, consistent with a backbone containing alternating -->4)-beta-Manp-(1--> and -->4)-beta-Glcp-(1--> residues in a approximately 15:1 ratio. Analysis of the sample treated sequentially with endo-(1-->4)-beta-d-mannanase and alpha-D-galactosidase showed that the majority of alpha-Galp-(1--> residues were linked to O-2, O-3, or O-6 of -->4)-beta-Manp-(1--> residues, with approximately 16 -->4)-beta-Manp-(1--> residues between side chains. Our data provide direct evidence of a previously proposed glucomannan backbone, but draw into question previously proposed side-chain structures.