Characterization of a water-soluble glucan from angelica acutiloba

A water-soluble glucan, AR-Glucan, from the roots of Angelica acutiloba was obtained homogeneous as determined by ultracentrifugal analysis, electrophoresis, and gel filtration. AR-Glucan was composed Of d-glucose, and its MW was estimated to be 13 500. Methylation analysis indicated that AR-Glucan contained 4-O- and 4,6-di-O-substituted glucosyl residues. ¹H and ¹³C NMR data accorded with the results of methylation analysis, and the glycosidic linkages in AR-Glucan were shown to have the α-configuration. The results of β-amylase, α-amylase, and pullulanase treatments of AR-Glucan showed that it contained (1 → 4) linked α-d-glucosyl side chains of long chain length such as amylopectin. Thus, AR-Glucan is a (1 → 4) linked α-d-glucan to which are attached glucosyl side chains at O-6 of the glucosyl residues of the main chain.     

Synthesis of 7-(tetrahydropyran-2-yl)- and 7-(4-acetoxytetrahydropyran-2-yl)-ε-rhodomycinone and 7-(tetrahydropyran-2-yl)-ε-pyrromycinone

The ¹³C.n.m.r spectra of water-soluble and -insoluble glucans synthesized by enzymes isolated from six strains of Streptococcus mutans are interpreted. The glucans are shown to be composed primarily of α(1→3)- and α-(1→6)-linked glucosyl residues, and the relative abundance of each linkage is estimated from peak areas. Treatment of water-insoluble glucans with dextranase is found to result in water-soluble and -insoluble products, the former enriched in α-(1→6)-linkages and the latter in α-(1→3)-linkages. The structural conclusions arrived at by ¹³C-n.m.r. spectroscopy are consistent with data from methylation analysis and ¹H-n.m.r. spectroscopy.     

Purification and structural investigation of a water-soluble polysaccharide from Flammulina velutipes

A novel water-soluble heteropolysaccharide FVP60-B was extracted from the fruiting bodies of Flammulina velutipes with boiling water and purified by Sephacryl S-300 and S-400, which molecular weight was estimated to be 1.3 × 10⁴ Da by HPLC. It is composed of l-fucose, d-mannose, d-glucose and d-galactose in a ratio of 1.16:0.82:1.00:3.08. Sugar analysis, methylation analysis together with ¹H, ¹³C and 2D NMR spectroscopy disclosed that FVP60-B is consisted of a α-(1 → 6)-d-galactopyranan backbone with a terminal fucosyl, terminal glucosyl and α-(1 → 6)-d-mannopyranan units on O-2 of 2,6-O-substituted-d-galactosyl units.     

Enzymatic synthesis of p-nitrophenyl 35-O-β-N-acetylglucosaminyl|-α-maltopentaoside by lysozyme; a novel substrate for human amylase assay

Transglycosylation from di-N-acetylchitobiose to the 3-position at the nonreducing end glucosyl group of p-nitrophenyl α-maltopentaoside was regioselectively induced through the use of hen egg-white lysozome. The enzyme formed p-nitrophenyl 3⁵-O-β-N-acetylglucosaminyl-α-maltopentaoside (5% of the enzyme-catalyzed net decreased of p-nitrophenyl α-maltopentaoside) from di-N-acetylchitobiose as a donor and p-nitrophenyl α-maltopentaoside as an acceptor. The rate of the transglycosylation depended on the concentration of substrate, the temperature and the pH. The hydrolytic actions of human pancreatic and salivary α-amylase on this derivative were examined. The maltopentaoside derivative was shown to be useful as a substrate for α-amylase assay through a coupled reaction involving α-D-glucosidase and glucoamylase.     

Oligomannoside biosynthesis in Mycobacterium smegmatis

A cell-free particulate enzyme system of Mycobacterium smegmatis ATCC 607 was shown to catalyze the incorporation of labeled mannose from GDP-[¹⁴C]mannose into endogenous acceptors to form a series of labeled neutral oligomannosides. These oligomannosides were devoid of amino sugar. The major oligomannoside product was characterized to be a trimannoside, O-α-d-mannopyranosyl-(1 → 2)-O-α-d-mannopyranosyl(1 → 2)-d-mannose and represented 46% of the total labeled oligomannoside product. The higher oligomannosides were shown to have either/or both α(1 → 2) and α(1 → 6) glycosidic linkages. A series of unlabeled endogenous oligosaccharides was isolated from the 105,000g supernatant fractions of the cell-free extracts of M. smegmatis and found to be chromatographically similar to the labeled oligomannosides synthesized by the cell-free system. The nature of the endogenous acceptor was not determined.     

Action of transglucosidase from Aspergillus niger on maltoheptaose and [U-C]maltose

Oligosaccharides synthesized from a mixture of maltoheptaose and [U-¹³C]maltose with transglucosidase [EC 2.4.1.24] from Aspergillus niger were investigated. When the reaction mixture was incubated at 15 °C for 1 h, several types of oligosaccharides with DP (degree of polymerization) 2 to DP8 containing α-d-Glcp-(1→6)-maltoheptaose were detected by liquid chromatography-mass spectrometry (LC-MS) and methylation analysis. Most of these compounds consisted of α-(1→4) linkages in the main chain and α-(1→6) linkages at the non-reducing ends. However, when the reaction mixture was incubated for 96 h, most of these products were converted into oligosaccharides with DP2 to DP5 consisting of only α-(1→6) linkages. These results suggested that A. niger transglucosidase rapidly transferred glucosyl residues to maltooligosaccharides, and gradually hydrolyzed both α-(1→4) linkages and α-(1→6) linkages at the non-reducing end, and transformed these into smaller molecules of mainly α-(1→6) linkages.     

Structure of l-arabino-d-galactan-containing glycoproteins from radish leaves

Two l-arabino-d-galactan-containing glycoproteins having a potent inhibitory activity against eel anti-H agglutinin were isolated from the hot saline extracts of mature radish leaves and characterized to have a similar monosaccharide composition that consists of l-arabinose, d-galactose, l-fucose, 4-O-methyl-d-glucuronic acid, and d-glucuronic acid residues. The chemical structure features of the carbohydrate components were investigated by carboxyl group reduction, methylation, periodate oxidation, partial acid hydrolysis, and digestion with exo- and endo-glycosidases, which indicated a backbone chain of (1→3)-linked β-d-galactosyl residues, to which side chains consisting of α-(1→6)-linked d-galactosyl residues were attached. The α-l-arabinofuranosyl residues were attached as single nonreducing groups and as O-2- or O-3-linked residues to O-3 of the β-d-galactosyl residues of the side chains. Single α-l-fucopyranosyl end groups were linked to O-2 of the l-arabinofuranosyl residues, and the 4-O-methyl-β-d-glucopyranosyluronic acid end groups were linked to d-galactosyl residues. The O-α-l-fucopyranosyl-(1→2)-α-l-arabinofuranosyl end-groups were shown to be responsible for the serological, H-like activity of the l-arabino-d-galactan glycoproteins. Reductive alkaline degradation of the glycoconjugates showed that a large proportion of the polysaccharide chains is conjugated with the polypeptide backbone through a 3-O-d-galactosylserine linkage.     

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.     

Structural characterization of a highly branched polysaccharide from the seeds of Plantago asiatica L.

In this paper, polysaccharides were extracted from the seeds of Plantago asiatica L. with hot water and separated into three fractions PLP-1 (18.9%), PLP-2 (52.6%) and PLP-3 (28.5%) by Sephacryl™ S-400 HR column chomatography. The main fraction PLP-2's structure was elucidated using oxalic acid hydrolysis, partial acid hydrolysis, methylation, GC, GC-MS, 1D and 2D NMR. PLP-2 was composed of Rha, Ara, Xyl, Man, Glc and Gal, in a molar ratio of 0.05:1.00:1.90:0.05:0.06:0.10. Its uronic acid was GlcA. PLP-2 was highly branched heteroxylan which consisted of a β-1,4-linked Xylp backbone with side chains attached to O-2 or O-3. The side chains consisted of β-T-linked Xylp, α-T-linked Araf, α-T-linked GlcAp, β-Xylp-(1 → 3)-α-Araf and α-Araf-(1 → 3)-β-Xylp, etc. Based on these results, the structure of PLP-2 was proposed.     

Characterization of glucuronic acid containing glycolipid in Aspergillus fumigatus mycelium

A glucuronic acid containing glycerolipid was isolated from the filamentous fungi Aspergillus fumigatus. This acidic glycolipid was extracted from the membrane of mycelium and purified by two successive chromatographic steps on DEAE-Sephadex and Silica columns. Chemical structural analysis was performed using methylation, gas-chromatography, gas-chromatography-mass spectrometry, nano-electrospray mass spectrometry and ¹H/¹³C NMR spectra. The corresponding structure is a 3-(O-α-glucuronyl)-1,2-diacyl-sn-glycerol, where acyl chains are mainly C_16:0, C_18:0, C_18:1, and C_18:2. This α-GlcA-diacylglycerol is not present in fungal conidia. This acidic glycerolipid is described here for the first time in a fungal species. Two homologs of UDP-glucose dehydrogenase that convert UDP-glucose into UDP-glucuronic acid, are present in A. fumigatus genome, UGD1 and UGD2. Gene deletion showed that only UGD1 is essential for the biosynthesis of GlcA-DG. However, no particular phenotype has been observed in the Ugd1Δ mutant. Biological function of this acidic glycolipid remains unknown in A. fumigatus.     

Structural and immunological studies of a major polysaccharide from spores of Ganoderma lucidum (Fr.) Karst

A polysaccharide isolated from spores of the fungus, Ganoderma lucidum, was found to be a complex glucan. On the basis of compositional and methylation analyses, periodate oxidation, Smith degradation, 1D and 2D NMR, and ESIMS experiments of the native polysaccharide and its degraded products, the polysaccharide was shown to have a backbone of beta-(1-->3)-linked D-glucopyranosyl residues, with branches of mono-, di- and oligosaccharide side chains substituting at the C-6 of the glucosyl residues in the main chain. Conformational analysis in aqueous solution and immunological activities of the native and degraded glucans were also investigated. The results suggested that the degree of substitution on the main chain and the length of side chains may be very important factors in determining the conformation and the biological activities of beta-(1-->3)-linked glucans.     

Structural investigation of the arabinoxyloglucan from Nicotiana tabacum

The structure of tobacco arabinoxyloglucan has been further studied by methylation analysis, by ¹H-, and ¹³C-n.m.r., and by fd. mass spectrometry, after complete digestion by cellulase. The results showed the polysaccharide molecule to be composed of two parts; a hexasaccharide component (AraXyl₂Glc₃, 1) and an unsubstituted (1→4)-β-d-glucan region (4-O-linked glucosyl residues) in the molar ratio of ～ 1:2. Some heterogeneities of this structure in the arabinofuranosyl sub-group were also found.     

Novel succinylated and large-sized osmoregulated periplasmic glucans of Pseudomonas syringae pv. syringae

Osmoregulated periplasmic glucans (OPGs) are intrinsic components of the Gram-negative bacterial envelope and are important for bacterial-host interactions. The OPGs of Pseudomonas syringae pv. syringae have been known to be highly branched linear glucans ranging from 6 to 13 glucose residues devoid of any substituents, while having backbone structure similar to those of Escherichia coli and Erwinia chrysanthemi. Here, we report for the first time succinylated and large-sized OPGs from P. syringae pv. syringae. The glucans were isolated with trichloroacetic acid treatment and various chromatographic techniques. These were further characterized by thin-layer chromatography, matrix-assisted laser desorption/ionization time of flight mass spectrometer, and 1D ¹H nuclear magnetic resonance spectroscopy. The results demonstrate that novel anionic glucans with one succinyl residue at the C-6 position of the glucose unit as well as neutral glucans including large-sized glucans with up to 28 degrees of polymerization are produced in P. syringae pv. syringae. Furthermore, the succinylated and large-sized OPGs of P. syringae pv. syringae are necessary for hypoosmotic adaptation.     

Structural studies of extracellular glucans of Streptococcus mutans by proton magnetic resonance

Proton magnetic resonance spectra at 100 MHz were obtained for water-soluble and water-insoluble glucans from 11 strains of Streptococcus mutans. The percentages of α-D-(1→6) and non-α-D-(1→6)-, namely, α-D-(1→3)-, linkages were calculated from the anomeric-proton resonances in the 4.7-4.8 and 5.0-5.1 p.p.m. range, respectively. The average content of α-D(1→6) linkages in the polymer fractions precipitating from solution during synthesis of the glucans was generally much lower than that of fractions remaining in solution. The frequent appearance of the α-D-(1→3) resonances as doublets in the spectra suggested neighboring-group effects among the possible α-D-(1→3) and α-D-(1→6) linkage-configurations. These effects were confirmed from 100-MHz spectra of products of a dextranase-degraded, water-insoluble glucan, and a 270-MHz spectrum of an undegraded glucan. It was thus possible to assign the doublet resonances to α-D-(1→3), homogeneous, heterogeneous, and branch configurations, although complete differentiation among proportions of each configuration in the glucan chains could not be achieved.     

Polyphenolic compounds in the fruits of Egyptian medicinal plants (Terminalia bellerica, Terminalia chebula and Terminalia horrida): Characterization, quantitation and determination of antioxidant capacities

Thirty-four polyphenolic substances in methanol extracts of the fruits of Terminalia bellerica, Terminalia chebula and Terminalia horrida, three plants used in Egyptian folk medicine, were initially identified by HPLC-ESI-MS and quantitated by analytical HPLC after column chromatography on Sephadex LH-20. After purification by semi-preparative HPLC the compounds were identified by their mass and fragmentation patterns using ESI-MS-MS. For several compounds detailed ¹H/¹³C NMR analysis at 600 MHz was performed. Two polyphenolics, namely 4-O-(4″-O-galloyl-α-l-rhamnopyranosyl)ellagic acid and 4-O-(3″,4″-di-O-galloyl-α-l-rhamnopyranosyl)ellagic acid were identified by NMR. Antioxidant capacities of the raw fruit extracts and the major isolated substances were determined using the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), oxygen radical absorbance capacity (ORAC) and ferric reducing ability of plasma (FRAP) in vitro assays and indicated that chebulic ellagitannins have high activity which may correlate with high potential as cancer chemopreventive agents. Therefore, further studies (metabolism, bioavailability and toxicity) of the polyphenolics in Terminalia species using preclinical models and in vivo human intervention trials are warranted.     

Characterization of substrate and product specificity of the purified recombinant glycogen branching enzyme of Rhodothermus obamensis

Background

Glycogen and starch branching enzymes catalyze the formation of α(1 → 6) linkages in storage polysaccharides by rearrangement of preexisting α-glucans. This reaction occurs through the cleavage of α(1 → 4) linkage and transfer in α(1 → 6) of the fragment in non-reducing position. These enzymes define major elements that control the structure of both glycogen and starch.

Methods

The kinetic parameters of the branching enzyme of Rhodothermus obamensis (RoBE) were established after in vitro incubation with different branched or unbranched α-glucans of controlled structure.

Results

A minimal chain length of ten glucosyl units was required for the donor substrate to be recognized by RoBE that essentially produces branches of DP 3–8. We show that RoBE preferentially creates new branches by intermolecular mechanism. Branched glucans define better substrates for the enzyme leading to the formation of hyper-branched particles of 30–70 nm in diameter (dextrins). Interestingly, RoBE catalyzes an additional α-4-glucanotransferase activity not described so far for a member of the GH13 family.

Conclusions

RoBE is able to transfer α(1 → 4)-linked-glucan in C4 position (instead of C6 position for the branching activity) of a glucan to create new α(1 → 4) linkages yielding to the elongation of linear chains subsequently used for further branching. This result is a novel case for the thin border that exists between enzymes of the GH13 family.

General significance

This work reveals the original catalytic properties of the thermostable branching enzyme of R. obamensis. It defines new approach to produce highly branched α-glucan particles in vitro.     

Structure of the O-polysaccharides of the lipopolysaccharides of Mesorhizobium loti HAMBI 1148 and Mesorhizobium amorphae ATCC 19655 containing two O-methylated monosaccharides

The O-polysaccharide of Mesorhizobium loti HAMBI 1148 was obtained by mild acid degradation of the lipopolysaccharide and studied by sugar and methylation analyses, Smith degradation, and ¹H and ¹³C NMR spectroscopies, including 2D ¹H/¹H COSY, TOCSY, ROESY, and H-detected ¹H/¹³C HSQC experiments. The O-polysaccharide was found to have a branched hexasaccharide-repeating unit of the following structure:where 2-acetamido-2-deoxy-4-O-methyl-d-glucose (d-GlcNAc4Me) and methyl group on 2-substituted d-rhamnose (Me) shown in italics are present in ∼80% and ∼40% repeating units, respectively. Similar studies of the O-polysaccharide from Mesorhizobium amorphae ATCC 19655 by sugar analysis and NMR spectroscopy revealed essentially the same structure but a higher content of 3-O-methyl-d-rhamnose (∼70%).     

α-Acid degradation by suspension culture cells of humulus lupulus

In a suspension culture of Humulus lupulus hop α-acids could not be detected. However, the culture was shown to have an in vivo ability to degrade exogenous α-acids and related compounds. It is shown that this is due to peroxidase with a rate constant for α-acid degradation of 2.7 × 10⁴/M · sec.     

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.