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.     

Molluscicidal saponins from Gundelia tournefortii

From the roots of Gundelia tournefortii seven saponins have been isolated mainly by DCCC. The main saponins (A and B) were characterized, mainly by ¹³C and ¹H NMR spectroscopy, as oleanolic acid 3-O-(2-[α-l-arabinopyranosyl(1 → 3) -β-d-gentiotriosyl(1 → 6) -β-d-glucopyranosyl]gb-d-xylopyranoside) (saponin A) and oleanolic acid 3-O-(2-[α-l-arabinopyranosyl] (1 → 3)-β-d-gentiobiosyl (1 → 6)-β-d-glucopyranosyl β-d-xylopyranoside) (saponin B). The other saponins are also derived from oleanolic acid and contain more sugar units. The saponin mixture and the saponins A and B possess strong molluscicidal activity against the schistosomiasis transmitting snail Biomphalaria glabrata.     

A galactoglucomannan from the leaf and stem tissues of red clover (Trifolium pratense)

A galactoglucomannan has been isolated by fractionation of the alkali-soluble hemicelluloses of the leaf and stem tissues of red clover (Trifolium pratense L.). The hemicellulose contains galactose, glucose, and mannose residues in the molar ratios 0.25:1.0:1.1 and accounts for ca. 25% of the mannose residues present in the clover tissues. Structural studies showed that the hemicellulose has a main chain of β-(1→4)-linked D-glucopyranosyl and D-mannopyranosyl residues, to which are attached α-(1→6)-linked D-galactopyranosyl residues.     

Endo-(1→4)-β-d-glucanases from sclerotium rolfsii. Purification,substrate specificity,and mode of action

Three purified endo-(1→4)-β-d-glucanases (EC 3.2.1.4), A, B, and C, from Sclerotium rolfsii culture filtrates showed homogeneity in disc-gel electrophoresis and in analytical isoelectric-focusing in polyacrylamide gel. The three endo-d-glucanases are glycoproteins, endo B and endo C being composed of a single polypeptide chain, and endo A of two dissimilar polypeptide chains that are covalently bound by a disulfide bridge. Endo B and endo C do not contain half-cystine residues. With carboxymethylcellulose as substrate, the liquifying activity of the three enzymes was inhibited by cellobiose but not by d-glucose. The specificity of the enzymes is restricted to β-(1→4) linkages, but they showed some differences in the mode of attack on cellodextrins, phosphoric acid-swollen cellulose, and lichenan to give cellobiose, cellotriose, and small proportions of d-glucose. Endo B in addition showed endo-d-xylanase activity.     

Action of alpha-1,6-glucan glucohydrolase on oligosaccharides derived from dextran

The action of α-1,6-glucan glucohydrolase on α-(1→6)-D-glucosidic linkages in oligosaccharides that also contain an α-(1→2)-, α-(1→3)-, or α-(1→4)-D-glucosidic linkage has been investigated. The enzyme could hydrolyse α-(1→6)-D-glucosidic linkages from the non-reducing end, including those adjacent to an anomalous linkage. α-(1→6)-D-Glucosidic linkages at branch points were not hydrolysed, and the enzyme could neither hydrolyse nor by-pass the anomalous linkages. These properties of α-1,6-glucan glucohydrolase explain the limited hydrolysis of dextrans by the exo-enzyme. Hydrolysis of the main chain of α-(1→6)-D-glucans will always stop one D-glucose residue away from a branch point. The extent of hydrolysis by α-1,6-glucan glucohydrolase of some oligosaccharide products of the action on dextran of Penicillium funiculosum and P. lilacinum dextranase, respectively, has been compared. Differences in the specificity of the two endo-dextranases were revealed. The Penicillium enzymes may hydrolyse dextran B-512 to produce branched oligosaccharides that retain the same 1-unit and 2-unit side-chains that occur in dextran.     

Hydrolysis of fourteen native dextrans by arthrobacter isomaltodextranase and correlation with dextran structure

The isomaltodextranase (EC 3.2.1.94) from Arthrobacter globiformis T6 hydrolysed thirteen dextrans to various extents (11?64% after 13 days) at initially large but gradually decreasing rates. Dextran B-1355 fraction S was, unlike the other dextrans, hydrolysed by the dextranase initially at the lowest rate among the dextrans used, but the rate was maintained for a long period with little decrease, so that the hydrolysis reached as high as 85% after 13 days. Paper chromatography of these dextran digests revealed that this dextranase produces in addition to isomaltose, one or two trisaccharides [isomaltose residues substituted by (1 →2)-, (1→3)-, or (1→4)-α-D-glucopyranosyl groups at the non-reducing D-glucopyranosyl residues] from every dextran used. It is evident that the non-(1→6)-linkages of these trisaccharide products constitute the “anomalous” linkages of the corresponding dextrans. The relative amounts of these trisaccharide products appear to indicate the approxima te relative amounts of a particular linkage among the dextrans, or the relative amounts of two kinds of linkages of each dextran. The kinds and the relative amounts of “anomalous” linkages of some dextrans were established on the basis of the trisaccharides produced by isomaltodextranase.     

Purification,characterization, and action-pattern studies on the endo-(1 → 3)-β-d-glucanase from Rhizopus arrhizus QM 1032

The extracellular (1 → 3)-β-d-glucanase [1 → 3)-β-d-glucan glucanohydrolase, EC 3.2.1.6] produced by Rhizopus arrhizus QM 1032 was purified 305-fold in 70% overall yield. This preparation was found to be homogeneous by ultracentrifugation (sedimentation velocity and studies), electrophoresis on acrylamide gel with normal, sodium dodecyl sulfate, and urea-acetic acid gels, and upon isoelectric focusing. The amino acid composition of the enzyme has been determined and it possesses a carbohydrate moiety composed of mannose and galactose (in the ratio ≈5:1) that is linked to the protein through a 2-acetamido-2-deoxyglucose residue. The molecular number was confirmed by electrophoresis on gels of sodium dodecyl sulfate. The enzyme does not posses subunit structure. It hydrolyzes it substrates with retention of configuration and possesses transglycosylating ability. The rates of hydrolysis of a wide variety of substrates were determined, and its action pattern on a series of oligosaccharides containing mized (1 → 3-, (1 → 4)-, and (1 → 6)-β-d-glucopyranosyl residues was investigated. The enzyme favors stretches of β-d-(1 → 3) linkages, but it can hydrolyze β-d-(1 → 4) linkages that are flanked on the non-reducing side with stretches of β-d-(1 → 3) links. The enzyme will not act on (1 → 6)-β-d-glucosyl linkages located in stretches of β-d-(1 → 3) and will not act on (1 → 3) β-d-glycosidic linkages involving sugars other than d-glucose.     

Acetolysis of disaccharides: Comparative kinetics and mechanism

The initial acetolysis rates of several disaccharides were compared using an assay procedure which involves adding portions of the reaction mixture to an alkaline sodium borohydride solution. After reduction, glycosidically-linked hexose was determined by the phenol-sulfuric acid method. For D-glucose disaccharides, β linkages were cleaved faster than α linkages, suggesting anchimeric assistance from the trans C-2 acetoxyl group. The acetolysis reaction rates for the various β-linked D-glucose disaccharides decreased in the order (1→6) ? (1→3) > (1→2) > (1»4). For the various α-linked disaccharides the order was (1→6) ? (1→4) > (1»3)> (1→2). The acetolysis rates for D-mannose disaccharides were in the order α-(1»6) ? α-(1→3) > β-(1»4) > α-(1»2). Turanose (3-O-α-D-glucopyranosyl-D-fructose) was cleaved at a much faster rate than either D-mannobiose or D-glucobiose with α-(1»2) or α-(1»3) linkages. A reaction mechanism is supported which features an acyclic intermediate, and, for certain -disaccharides, C-2 acetoxyl anchimeric assistance.     

Cauloside D a new triterpenoid glycoside from Caulophyllum robustum maxim: Identification of cauloside A

The structure of cauloside D, one of the main saponins isolated from Caulophyllum robustum roots, was shown to be 3-O-α-l-arabinopyranosyl hederagenin-28-O-α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranosyl(1→6)-β-d-glucopyranoside with the aid of methylation and enzymatic hydrolysis by the digestive juice of the Eulota maackii. Cauloside A was shown to be identical with saponin A, isolated from C. robustum Maxim. previously. The composition of the digestive juice of E. maakii was shown to include enzymes that catalyse the cleavage of α-arabinosidic, β-1,6-glucosidic and acyl-O-β-glucosidic linkages.     

Flavonol triglycosides from Magnolia utilis

Three undescribed flavonol triglycosides, rhamnetin-3-O-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-β-d-glucopyranoside (champaluangoside A), rhamnetin-3-O-α-l-rhamnopyranosyl-(1→2)-[α-l-rhamnopyranosyl-(1→6)]-β-d-galactopyranoside (champaluangoside B) and rhamnocitrin-3-O-α-l-rhamnopyranosyl-(1→2)-[α-l-rhamnopyranosyl-(1→6)]-β-d-glucopyranoside (champaluangoside C), were isolated from Magnolia utilis in addition to eleven known compounds; quercetrin-3-O-α-l-rhamnopyranosyl-(1→2)-[α-l-rhamnopyranosyl-(1→6)]-β-d-glucopyranoside, oxytroflavoside G, magnoloside A, magnoloside M, magnoloside D, manglieside A, manglieside B, 1,2-di-O-β-d-glucopyranosyl-4-allylbebzene, syringrin, benzyl β-d-allopyranoside and (+)-syringaresinol-O-β-d-glucopyranoside. The structure elucidation of these compounds was based on analyses of physical and spectroscopic data.     

The structure of the neutral polysaccharide gum secreted by Rhizobium strain cb744

A previous investigation of the structure of the extracellular polysaccharide gum from the nitrogen-fixing Rhizobium strain cb744 (a member of the slow-growing Cowpea group) indicated that there were two β-(1→4)-linked d-glucopyranosyl residues for each α-(1→4)-linked d-mannopyranosyl residue, and that each mannose was substituted at O-6 by a β-d-galactopyranosyl residue having 71% of the galactose present as 4-O-methylgalactose. The present study shows that, although the gum appeared to have a simple tetrasaccharide repeating unit, it is composed of two closely associated components. One is a (1→4)-linked α-d-mannan substituted at each O-6 by a β-d-galactopyranosyl residue (71% 4-O-methylated). The second component is a (1→4)-linked β-d-glucan. The existence of the two polysaccharides was established by separation of the β-d-galactosidase-treated gum on a column of concanavalin A-Sepharose 4B. The d configurations were determined and the anomeric attribution of the linkages confirmed by the use of enzymes. The interaction between the two gum components is discussed.     

STD-NMR studies of two acceptor substrates of GlfT2, a galactofuranosyltransferase from Mycobacterium tuberculosis: Epitope mapping studies

The major structural component of the mycobacterial cell wall, the mycolyl–arabinogalactan–peptidoglycan complex, possesses a galactan core composed of approximately 30 galactofuranosyl (Galf) resides attached via alternating β-(1→6) and β-(1→5) linkages. Recent studies have shown that the entire galactan is synthesized by two bifunctional galactofuranosyltransferases, GlfT1 and GlfT2. We report here saturation transfer difference (STD) NMR studies GlfT2 using two trisaccharide acceptor substrates, β-d-Galf-(1→6)-β-d-Galf-(1→5)-β-d-Galf-O(CH₂)₇CH₃ (2) and β-d-Galf-(1→5)-β-d-Galf-(1→6)-β-d-Galf-O(CH₂)₇CH₃ (3), as well as the donor substrate for the enzyme, UDP-Galf. Epitope mapping demonstrated a greater enhancement toward the ‘reducing’ ends of both trisaccharides, and that UDP-galactofuranose (UDP-Galf) made more intimate contacts through its nucleotide moiety. This observation is consistent with the greater flexibility required within the active site of the reaction between the growing polymer acceptor and the UDP-Galf donor. The addition of UDP-Galf to either 2 or 3 in the presence of GlfT2 generated a tetrasaccharide product, indicating that the enzyme was catalytically active.     

Some structural features of the d-glucan from the seed of Mirabilis jalapa

The cotyledon of the seed of Mirabilis jalapa was found to contain a d-glucan. Methylation, periodate oxidation, and graded and enzymic hydrolysis studies were conducted to elucidate its structure. For every 38 d-glucosyl residues therein, 34 are (1→4)- and 3 are (1→3)-linked; the d-glucosyl unit at the branch point is linked through O-1, O-2, and O-4. In some places in the chain, there are at least three (1→3)-linked d-glucosyl residues in a sequence. Both α- and β-d-glucosidic linkages are present in the polysaccharide, the former preponderating. The d-glucan gave with iodine a faint blue color that had λ_max 420 nm.     

The crystal structure of methyl 3,4-O-isopropylidene-2,6-di-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-α-d-galactopyranoside at 97 K

The crystal structure of methyl 3,4-O-isopropylidene-2,6-di-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-α-d-galactopyranoside (1), C₃₈H₅₄O₂₄ · (C₄H₈O₂)_0.32 was determined by X-ray diffraction;1 crystallises in space group P2₁ with a = 12.480(3), b = 8.821(3), c = 21.182(4)Å, β = 98.46(3)°, and Z = 2. The structure was solved by Patterson-search and Fourier-recycling procedures and refined to R_w(R) = 0.048(0.063), using 4348 [3112 with I> 2σ(I)] independent reflections. The β-d-galactosyl rings are slightly distorted and, due to the isopropylidene group, the α-d-galactoside ring is severely distorted. The conformation near the β-(1→6) and β-(1→2) linkages between the pyranoid rings is not significantly affected by the acetyl groups, but the anomeric C-O-C bridge angles have unusual values. The C-6O-6 bond in the β-d-galactosyl group (1→2)-linked to the α-d-galactoside residue has an unusual gauche—trans conformation with respect to C-4 and O-5. The CH₃-(C = O)-O-C moieties are planar within 0.01Å, and 32.6% of all unit cells contain a molecule of ethyl acetate.     

Structural studies of plant gum from sap of the lac tree, Rhus vernicifera

The plant gum isolated from sap of the lac tree, Rhus vernicifera (China), was separated into two fractions having mol. wt. 84,000 and 27,700 by aqueous-phase gel-permeation chromatography. The fractions contain d-galactose (65 mol%), 4-O-methyl-d-glucuronic acid (24 mol%), d-glucuronic acid (3 mol%), l-arabinose (4 mol%), and l-rhamnose (3 mol%). Smith degradation of the carboxyl-reduced polysaccharides gives products of halved molecular weight, and these consist of a β-(1→3)-linked galactopyranan main chain and side chains made up of galactopyranose residues. Peripheral groups, such as α-d-Galp-, α-d-Galp-(1→6)-β-d-Galp-, 4-O-methyl-β-d-GlcpA-, and 4-O-methyl-β-d-GlcpA-(1→6)-β-d-Galp-, are attached to this interior core through β-(1→3)- or β-(1→6)-linkages.     

Polysaccharides from Hemileia vastatrix uredospores

The polysaccharidic fractions isolated from Hemileia vastatrix uredospores by alkali treatment, expressed as a percentage of the initial uredospore weight, gave the following yields: 1 M NaOH soluble at 22°C (7.1); 1 M NaOH soluble at 60°C (5.0); and insoluble residue (7.6). Both alkali-soluble fractions contained mannose and glucose as the major constituents, with glycosidic linkages of the β-1 → 4 and β-1 → 3 types. The alkaliinsoluble residue contained predominantly glucosamine, and had infrared and X-ray spectra indistingushable from those of crustacean chitin. Electron microscope observations revealed that the insoluble residue consisted of the cell wall spines connected by a thin layer of microfibrils.     

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.     

Bis(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)amine obtained by a fusion reaction

A unique, alkali-soluble polysaccharide has been isolated from the cell walls of the basidiomycete Coprinus macrorhizus microsporus. The polysaccharide, which is primarily a glucan, contains a large proportion of α-(1→4)-linked d-glucose residues and a smaller amount of β-(1→3) and (1→6) linkages, as suggested by methylation, partial acid hydrolysis, periodate oxidation, and enzymic studies. Hydrolysis of the methylated polysaccharide gave equimolar amounts of 2,4-di- and 2,3-di-O-methyl-d-glucose; no 2,6-di-O-methyl-d-glucose was identified, indicating the absence of branch points joined through O-1, O-3, and O-4. The isolation and identification of 2-O-α- glucopyranosylerythritol from the periodate-oxidized polysaccharide suggests that segments of the a-(1→4)-linked d-glucose residues are joined by single (1→3)-linkages. An extracellular enzyme-preparation from Sporotrichum dimorphosporum (QM 806) containing both β-(1→3)- and α-(1→4)-d-glucanohydrolase activity released 76% of the reducing groups from the polysaccharide. The polysaccharide also contains minor proportions of xylose, mannose, 2-amino-2-deoxyglucose, and amino acids.     

Blood Group Substance-degrading Enzymes Obtained from Streptomyces sp.

The blood group B substance-degrading activity of Streptomyces 9917S₂ is induced by galactosides as α-galactosidase activity is. Purification of the α-galactosidase was attempted by chromatography on DEAE-Sephadex and Sephadex. The purified preparation was shown to be free from α- and β-glucosidases, β-galactosidase, α- and β-glucosaminidases, and α- and β-galactosaminidases activities. The blood group B substance-degrading activity was present only in this fraction. This enzyme preparation cleaves α-(1→3)- and α-(1→6)-galactosidic linkages. The activity is inhibited by d-galactose, melibiose, and raffinose and also by l-arabinose and d-xylose.     

Structural investigations of the galactan of the snail Lymnaea stagnalis

A galactan, isolated from the spawn of the snail Lymnaea stagnalis, contained d-galactose and 0.9% of nitrogen, but neither l-galactose nor phosphate groups. The [α]_D²⁰ values of the galactan and its first Smith-degradation product were +19.5° and +20°, respectively. During each of two consecutive Smith-degradations of the galactan, 1 mol of periodate was consumed and 0.45 mol of formic acid was liberated per mol of “anhydrogalactose” unit. Methylation analyses of the galactan and its first Smith-degradation product yielded equal proportions of 2,3,4,6-tetra-O-methyl- and 2,4-di-O-methyl-galactose. Only small quantities of 2,4,6- (4.9 mol%) and 2,3,4-tri-O-methylgalactose (0.7 mol%) were formed from the galactan, whereas the first Smith-degraded product gave 15.6 and 20.4 mol%, respectively. The product of the second Smith-degradation disintegrated and the following oligosaccharides were identified: β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→3)-β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→6)-β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→6)-d-Gal-β-d-Gal-(1→3)-β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→3)-[β-d-Gal-(1→6)]-β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→3)-β-d-Gal-(1→6)-β-d-Gal-(1→1)-l-Gro, and β-d-Gal-(1→3)-β-d-Gal-(1→3)-β-d-Gal-(1→1)-l-Gro. Thus, the galactan is highly branched with the backbone containing sequences of either exclusively (1→6)-linked or of more or less regularly alternating (1→3)- and (1→6)-linked units. The side chains vary in length and in the degree of branching. In immunoprecipitin studies, a high degree of species-specificity was seen when various snail galactans were tested with the antiserum to the Lymnaea stagnalis galactan.