Pectin Isolated from the Midrib of Leaves of Nicotiana tabacum

A pectin isolated from tobacco midrib contained residues of d-galacturonic acid (83.7%), L-rhamnose (2.2%), l-arabinose (2.4%) and d-galactose (11.2%) and small amounts of d-xylose and d-glucose. Methylation analysis of the pectin gave 2, 3, 5-tri- and 2, 3-di-O-methyl-l-arabinose, 3, 4-di- and 3-O-methyl-l-rhamnose and 2, 3, 6-tri-O-methyl-d-galactose. Reduction with lithium aluminum hydride of the permethylated pectin gave mainly 2, 3-di-O-methyl-d-galactose and the above methylated sugars. Partial acid hydrolysis gave homologous series of β-(1 → 4)-linked oligosaccharides up to pentaose of d-galactopyranosyl residues, and 2-O-(α-d-galactopyranosyluronic acid)-l-rhamnose, and di- and tri-saccharides of α-(1 → 4)-linked d-galactopyranosyluronic acid residues.

These results suggest that the tobacco pectin has a backbone consisting of α-(1 → 4)-linked d-galactopyranosyluronic acid residues which is interspersed with 2-linked l-rhamnopyranosyl residues. Some of the l-rhamnopyranosyl residues carry substituents on C-4. The pectin has long chain moieties of β-(1 → 4)-linked d-galactopyranosy] residues.     

An Acidic Polysaccharide Isolated from the Midrib of Leaves of Nicotiana tabacum

An acidic polysaccharide (APS-H) purified from the hemicellulosic fraction of the midrib of Nicotiana tabacum was composed of d-galacturonic acid, l-rhamnose, l-arabinose and d-galactose in a molar ratio of 31.8: 15.4: 9.9: 42.9. Its molecular weight was estimated to be 90,000 by gel filtration chromatography. APS-H had a pectin-like structure in which the rhamnogalacturonan backbone was composed of (1 → 2)-linked l-rhamnopyranosyl and (1 → 4)-linked d-galacturonosyl residues in a ratio of approximately 1: 2.1. It also contained (1 → 4)-linked d-galactan and (1 → 5)-linked l-arabinofuranosyl moieties as the side chains. Branch points occurred mainly at C-4 of (1 → 2)-linked l-rhamnosyl residues in the backbone and at C-6 of (1 → 4)-linked d-galactosyl residues in the side chains.     

An Arabinogalactan-protein from Rape Leaves

A l-fucose-containing arabinogalactan-protein that strongly inhibited hemagglutination by eel anti-H agglutinin of human O erythrocytes was purified from hot phosphate-buffered saline extracts of mature leaves of rape, Brassica campestris. The purified glycoconjugate consisted of 90% of the polysaccharide moiety comprising l-fucose, l-arabinose, d-galactose, 4-O-methyl-d-glucuronic acid, and d-glucuronic acid, and 4% of the hydroxyproline-rich protein portion. Upon methylation, periodate oxidation, and enzymatic degradation, we found that consecutive β-(→3)-linked d-galactopyranosyl residues constituted a backbone chain of the polysaccharide moiety, to which the side chains of β-(→6)-linked d-galactopyranosyl residues were attached through O-6. Most of l-arabinofuranosyl residues were linked as single units through 0-3 to the side chains while a small quantity of the sugar was present as (1→2)-, (1→3)-, or (1→5)-linked inter-chain residues. Single residues of α-l-fucopyranose, apparently attached to (1→2)-linked l-arabinofuranosyl residues, reacted with eel anti-H precipitin and Aleuria aurantia l-fucose-specific lectin, and were assumed to be crucial in the expression of the H-like activity. The uronosyl residues were also located at the non-reducing terminal ends. Reductive alkaline degradation of the arabinogalactan-protein provided indications that the polysaccharide chains were mainly conjugated through serine-O-glycosidic linkages to the polypeptide core. In an immunoprecipitation test, the rape leaf arabinogalactan-protein cross-reacted with antisera raised against radish leaf arabinogalactan-protein, indicating that these cruciferous arabinogalactan-proteins share common immunodeterminant(s) in their molecules.     

Occurrence of Superoxide Dismutase in Bovine Milk

Acidic heteropolysaccharides, d-glucurono-d-xylo-d-mannans were isolated from the water- and alkaline extracts of the fruit body of Tremella fuciformis Berk. Similar polysaccharides were isolated from the growing culture of the haploid cells of two strains (T–19 and T–7) of T. fuciformis, when they were cultured in sucrose or glucose-yeast extract medium. The extracellular polysaccharides contain, d-glucuronic acid, d-xylose and d-mannose [molar ratios, 1.3: 1.0: 3.5 (T–7) and 0.8: 1.0: 2.1 (T–19)], and, in addition, small proportions of l-fucose and O-acetyl groups. Methylation and Smith degradation studies indicated that both fruit body and extracellular polysaccharides are built up of α-(1 → 3)-linked d-mannan backbone chain to which β-linked d-glucuronic acid and single or short chains of β-(1 → 2)-linked d-xylose residues are attached at the C–2 position. l-fucose residues in the extracellular polysaccharides may form the single branches. The structural features of these polysaccharides are discussed in comparison with the similar polysaccharides from other fungi.     

An Arabinogalactan from Extracellular Polysaccharides of Suspension-cultured Tobacco Cells

The structure of an arabinogalactan, separated from extracellular polysaccharides of cultured tobacco cells, has been investigated by methylation analysis of the original polysaccharide and of the products obtained after mild acid hydrolysis and after controlled Smith degradation.

The arabinogalactan consists of l-arabinose, d-galactose and l-rhamnose in the molar ratio of 47: 45: 8. The arabinogalactan has a main chain of (1→3)-linked d-galactopyranosyl residues, half of which are substituted at the 6-position. Most of the side chains consist of three (1→6)-linked D-galactopyranosyl residues, to which l-arabinose residues are attached at C-3. The l-arabinofuranosyl and pyranosyl residues are present as end groups, and l-arabinopyranosyl residues are attached to C-5 of l-arabinofuranosyl residues. Non-reducing terminal l-rhamnopyranosyl residues are also present.     

Structures of the Oligosaccharides from the Enzymic Hydrolyzate of Rice-straw Arabinoxylan by a Xylanase of Aspergillus niger

A trisaccharide consisting of two d-xylose units and one l-arabinose unit, and a tetrasaccharide consisting of three d-xylose units and one l-arabinose unit were isolated from the hydrolyzate of rice-straw arabinoxylan by the xylanase I produced by Asp. niger.

The structures of the trisaccharide and the tetrasaccharide were determined to be 3¹-α-l-arabinofuranosylxylobiose ([α]_d^? 80°) and 3¹-α-l-arabinofuranosylxylotriose ([α]_d^? 84°), respectively, by chemical and enzymic methods.

According to the structures of two arabinose-xylose mixed oligosaccharides, it was shown that the rice-straw arabinoxylan is composed of chain of 1,4-linked βd-xylopyranose residues and some of xylose residues have side-chain of 1,3-linked α-l-arabinofuranose.     

Structure of Cell Wall Polysaccharide Isolated from Cotyledon of Tora-bean (Phaseolus vulgaris)

The cell wall polysaccharide of cotyledon of Tora-bean (Phaseolus vulgaris), which surrounds starch granules, was isolated from saline-extraction residues of homogenized cotyledon, as alkali-insoluble fibrous substance. Alkali-insoluble residue, which had been treated with α-amylase (Termamyl), had a cellulose-like matrix under the electron microscope. It was composed of l-arabinose, d-xylose, d-galactose and d-glucose (molar ratio, 1.0: 0.2: 0.1: 1.2) together with a trace amount of l-fucose. Methylation followed by hydrolysis of the polysaccharide yielded 2, 3, 5-tri-O-methyl-l-arabinose (3.3 mol), 2, 3, 4-tri-O-methyl-d-xylose (1.0 mol), 2, 3-di-O-methyl-l-arabinose (3.7 mol), 3, 4-di-O-methyl-d-xylose (1.0 mol), 2-O-methyl-l-arabinose and 2, 3, 6-tri-O-methyl-d-glucose (12.7 mol), 2, 6-di-O-methyl-d-glucose (1.2 mol) and 2, 3-di-O-methyl-d-glucose (1.0 mol).

Methylation analysis, Smith degradation and enzymatic fragmentation with cellulase and α-l-arabinofuranosidase showed that the l-arabinose-rich alkali-insoluble polysaccharide possesses a unique structural feature, consisting of β-(1 → 4)-linked glucan backbone, which was attached with side chains of d-xylose residue and β-d-galactoxylose residue at O-6 positions and α-(1 → 5)-linked l-arabinosyl side cains (DP=8) at O-3 positions of β-(1 → 4)-linked d-glucose residues, respectively.     

A Modified Procedure for the Synthesis of Sucrose

A modified procedure for synthesis of sucrose was carried out by using purified crystalline 1,3,4,6-tetra-O-benzoyl-d-fructofuranose in place of 1,3,4,6-tetra-O-acetyl-d-fructofuranose; namely, reaction of 1,2-anhydro-3,4,6-tri-O-acetyl-α-d-glucopyranose with tetrabenzoyl-d-fructose at 110~118°C for 3 hr followed by deacylation, and by chromatographic separation of the deacylated product afforded crude sucrose. Acetylation of the synthetic product gave octaacetate sucrose in the yield of 5.0%.     

The Structures of Water-soluble Polysaccharides Isolated from Bamboo Shoot

The structures of two polysaccharides reported in the previous paper were studied by means of methylation analysis and the Smith degradation. As a result, it was concluded that the water-soluble xylan consisted essentially of a (l→4)-linked β-d-xylopyranosyl chain and contained l-arabinofuranosyl residues linked through the C–l as terminal side units. Unambiguous information concerning the residues of d-glactose and d-glucuronic acid as the constituents of the xylan has not been obtained. For the arabinogalactan, evidence was obtained for an interesting structure having a backbone chain of (l→3)-linked β-d-galactopyranosyl residues to which the terminal arabinose residues were attached at the C–6 position as the most prevalent side chains.     

Methylation Analysis of Fractions from the Leuconostoc mesenteroides NRRL B-1299 Dextran

Methylation analysis of five fractions of the dextran elaborated by Leuconostoc mesenteroides NRRL B-1299 has shown that each fraction was a highly branched dextran with the branches being joined mainly through C-2. Detection of a small amount of 4-O-mono-methyl-d-glucose has suggested that parts of the d-glucose residues were doubly branched at both C-2 and C-3. Detection of a larger amount of 2,4,6-tri-O-methyl-d-glucose in the hydrolyzates of the methylated products of the borate insoluble fractions has shown a greater percentage of linear α-1,3-linked d-glucose residues in these fractions. It is suggested that the solubility of the dextran is closely related to the content of linear α-1,3-linked d-glucose residues.     

Polyol Dehydrogenases in the Soluble Fraction of Acetobacter melanogenum

Polyol dehydrogenases of Acetobacter melanogenum were investigated. Three polyol dehydrogenases, i. e. NAD⁺-linked d-mannitol dehydrogenase, NAD⁺-linked sorbitol dehydrogenase and NADP⁺-linked d-mannitol dehydrogenase, in the soluble fraction of the organism were purified 12-fold, 8-fold and 88-fold, respectively, by fractionation with ammonium sulfate and DEAE-cellulose column chromatography. NAD⁺-linked sorbitol dehydrogenase reduced 5-keto-d-fructose (5KF) to l-sorbose in the presence of NADH, whereas NADP⁺-linked d-mannitol dehydrogenase reduced the same substrate to d-fructose in the presence of NADPH. It was also shown that NAD⁺-linked d-mannitol dehydrogenase was specific for the interconversion between d-mannitol and d-fructose and that this enzyme was very unstable in alkaline conditions.     

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.     

The Structure of Arabinoxylan and Arabinoglucuronoxylan Isolated from Rice Endosperm Cell Wall

A neutral and an acidic arabinoxylan fraction (H-l and H-2) were obtained from rice endosperm cell wall. The results of methylation analysis and partial hydrolysis of these fractions showed that both of them have highly branched structures in which approximately 6 out of 7 (H-l) and 5 out of 6 (H-2) of the (1→4)-linked d-xylose residues are branched. Most of the side chains in H-l consists of single α-l-arabinofuranose residues, whereas some of them in H-2 were substituted with α-d-glucuronic acid or 4-O-methyl-α-d-glucuronic acid residues, both attached to the O-2 position of d-xylose residues. These highly branched arabinoxylans are not readily hydrolyzed by an endoxylanase of Streptomyces sp.     

Studies on the Non-Starchy Polysaccharides of the Endosperm of Naked Barley

The structure of F-4 β-glucan, a minor component of water soluble non-starchy polysaccharides from the endosperm of naked barley, was elucidated. Hydrolysate of the methylated F-4 β-glucan gave 2,3,6-tri-O-methyl-d-glucose and 2,4,6-tri-O-methyl-d-glucose as main components with small amounts of 2,3,4,6-tetra-O-methyl-d-glucose and unidentified di-O-methyl-d-glucose. This result indicated that the main chain of F-4 β-glucan consisted of 1,4- and 1,3-linked β-d-glucopyranose residues with proportions of approximately 2:1 (β-1,4 linkage to be predominant) with some branching.     

Biochemical characteristics of maltose phosphorylase MalE from Bacillus sp. AHU2001 and chemoenzymatic synthesis of oligosaccharides by the enzyme

ABSTRACT

Maltose phosphorylase (MP), a glycoside hydrolase family 65 enzyme, reversibly phosphorolyzes maltose. In this study, we characterized Bacillus sp. AHU2001 MP (MalE) that was produced in Escherichia coli. The enzyme exhibited phosphorolytic activity to maltose, but not to other α-linked glucobioses and maltotriose. The optimum pH and temperature of MalE for maltose-phosphorolysis were 8.1 and 45°C, respectively. MalE was stable at a pH range of 4.5–10.4 and at ≤40°C. The phosphorolysis of maltose by MalE obeyed the sequential Bi–Bi mechanism. In reverse phosphorolysis, MalE utilized d-glucose, 1,5-anhydro-d-glucitol, methyl α-d-glucoside, 2-deoxy-d-glucose, d-mannose, d-glucosamine, N-acetyl-d-glucosamine, kojibiose, 3-deoxy-d-glucose, d-allose, 6-deoxy-d-glucose, d-xylose, d-lyxose, l-fucose, and l-sorbose as acceptors. The k_cat(app)/K_m(app) value for d-glucosamine and 6-deoxy-d-glucose was comparable to that for d-glucose, and that for other acceptors was 0.23–12% of that for d-glucose. MalE synthesized α-(1→3)-glucosides through reverse phosphorolysis with 2-deoxy-d-glucose and l-sorbose, and synthesized α-(1→4)-glucosides in the reaction with other tested acceptors.     

Functions,structures, and applications of cellobiose 2-epimerase and glycoside hydrolase family 130 mannoside phosphorylases

Carbohydrate isomerases/epimerases are essential in carbohydrate metabolism, and have great potential in industrial carbohydrate conversion. Cellobiose 2-epimerase (CE) reversibly epimerizes the reducing end d-glucose residue of β-(1→4)-linked disaccharides to d-mannose residue. CE shares catalytic machinery with monosaccharide isomerases and epimerases having an (α/α)₆-barrel catalytic domain. Two histidine residues act as general acid and base catalysts in the proton abstraction and addition mechanism. β-Mannoside hydrolase and 4-O-β-d-mannosyl-d-glucose phosphorylase (MGP) were found as neighboring genes of CE, meaning that CE is involved in β-mannan metabolism, where it epimerizes β-d-mannopyranosyl-(1→4)-d-mannose to β-d-mannopyranosyl-(1→4)-d-glucose for further phosphorolysis. MGPs form glycoside hydrolase family 130 (GH130) together with other β-mannoside phosphorylases and hydrolases. Structural analysis of GH130 enzymes revealed an unusual catalytic mechanism involving a proton relay and the molecular basis for substrate and reaction specificities. Epilactose, efficiently produced from lactose using CE, has superior physiological functions as a prebiotic oligosaccharide.     

Structure of a Physiologically Active Polysaccharide Produced by Bacillus polymyxa S-4

The structure of an acidic polysaccharide elaborated by Bacillus polymyxa S-4 was investigated in relation to its physiological activity, particularly, its hypocholesterolemic effect on experimental animals. The polysaccharide is composed of d-glucose, d-mannose, d-galactose, d-glucuronic acid, and d-mannuronic acid (molar ratio 3:3:1: 2:1). Methylation and fragmentation analyses, such as Smith degradation and partial acid hydrolysis showed that the polysaccharide has a complicated, highly branched structure, consisting mainly of (1 → 3)- and (1 → 4)-d-glycosidic linkages. The backbone chain containing d-glucuronic acid, d-mannose, and d-galactose residues is attached at the C-3, C-4, and C-4 positions, respectively, with side chains of single or a few carbohydrate units, which are terminated with d-glucose or d-mannose residues.     

Change of the Structure of Cell Wall β-l,3-d-Glucan with the Growth of Neurospora crassa Cells

The chemical structure of cell wall β-d-glucans as well as the activities of lytic enzymes such as β-1,3-d-glucanase and β-1,6-d-glucanase changed during the growth of Neurospora crassa.

A dramatic change in the cell wall β-d-glucan structure was observed between cells of the middle logarithmic phase and ones of the late logarithmic phase. The ratio of 1,3-linked glucose residues to non reducing terminal glucose residues decreased from 85 to 55 and the ratio of gentiobiose as a hydrolysis product with exo-β-1,3-d-glucanase increased significantly between the two phases.

Two prominent peaks of β-1,3-d-glucanase as well as the β-1,6-d-glucanase activities appeared in the culture filtrate at different growth stages, the early logarithmic phase and the stationary phase. In the cell wall, β-d-glucosidase activity instead of the β-l,6-d-glucanase and β-1,3-d-glucanase activities was observed in the late logarithmic phase.     

The Acceptor Specificity of Amylomaltase from Escherichia coli IFO 3806

The acceptor specificity of amylomaltase from Escherichia coli IFO 3806 was investigated using various sugars and sugar alcohols. d-Mannose, d-glucosamine, N-acetyl- d-glucosamine, d-xylose, d- allose, isomaltose, and cellobiose were efficient acceptors in the transglycosylation reaction of this enzyme. It was shown by chemical and enzymic methods that this enzyme could transfer glycosyl residues only to the C4-hydroxyl groups of d-mannose, iY-acetyl- d-glucosamine, d-allose, and d-xylose, producing oligosaccharides terminated by 4–0-α-d-glucopyranosyl-d-mannose, 4–0-α-d-glucopyranosyl-yV-acetyl-d-glucosamine, 4-O-α-d-glucopyranosyl-d-allose, and 4–0-α-d-gluco- pyranosyl-d-xylose at the reducing ends, respectively.     

Studies on the Chemical Structure of Konjac Mannan

The electrophoretically homogeneous glucomannan isolated from konjac flour was composed of d-glucose and d-mannose residues in the approximate ratio of 1: 1.6. Controlled acid hydrolysis gave 4-O-β-d-mannopyranosyl-d-mannose, 4-O-β-d-mannopyranosyl-d-glucose_T 4-O-β-d-glucopyranosyl-d-glucose(cellobiose), 4-O-β-d-glucopyranosyl-d-mannose(epicellobiose), O-β-d-mannopyranosyl-(1→4)-O-β-d-mannopyranosyl-(1→4)-d-mannose, O-β-d-glucopyranosyl- (1→4)-O-β-d-mannopyranosyl-(1→4)-d-mannose, O-β-d-mannopyranosyl-(1→4)-O-β-d-glucopy- ranosyl-(1→4)-d-mannose and O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→4)-d-mannose.