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.     

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.     

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.     

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.     

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.     

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.     

Isolation of Radiosensitive Mutants of Micrococcus radiodurans

Sulfated polysaccharides (SP) isolated from freshwater green algae, Spirogyra neglecta (Hassall) Kützing, and fractionated SPs were examined to investigate their molecular characteristics and immunomodulatory activity. The crude and fractionated SPs (F₁, F₂, and F₃) consisted mostly of carbohydrates (68.5–85.3%), uronic acids (3.2–4.9%), and sulfates (2.2–12.2%) with various amounts of proteins (2.6–17.1%). d-galactose (23.5–27.3%), d-glucose (11.5–24.8%), l-fucose (19.0–26.7%), and l-rhamnose (16.4–18.3%) were the major monosaccharide units of these SPs with different levels of l-arabinose (3.0–9.4%), d-xylose (4.6–9.8%), and d-mannose (0.4–2.3%). The SPs contained two sub-fractions with molecular weights (M_w) ranging from 164 × 10³ to 1460 × 10³ g/mol. The crude and fractionated SPs strongly stimulated murine macrophages, producing considerable amounts of nitric oxide and various cytokines via up-regulation of their mRNA expression by activation of nuclear factor-kappa B and mitogen-activated protein kinases pathways. The main backbone of the most immunoenhancing SP was (1→3)-l-Fucopyranoside, (1→4,6)-d-Glucopyranoside, and (1→4)-d-Galactopyranoside.     

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

Delipidated cell walls from Aureobasidium pullulans were fractionated systematically.

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

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

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

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

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.     

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 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.     

Detailed Structure of an Acidic Polysaccharide in Soy Sauce,Confirmed by Use of Two Kinds of Purified Pectinases

The partially degraded polysaccharide obtained by means of mild acid hydrolysis of APS–I, an acidic polysaccharide in soy sauce, was incubated with an endo-polygalacturonase, and some acidic sugars liberated were fractionated and purified from the enzymatic hydrolyzate. They were identified as d-galacturonic acid (Gal A), its α-l,4-linked dimer and trimer, d-xylose (Xyl) β1→3 Gal A and Xyl β1→3 Gal A α1→4 Gal A, and an acidic polymer composed of the above-mentioned sugars through methylation analysis and other methods.

Methyl-esterized APS–I was subjected to β-elimination with pectin lyase or by heat treatment. The high molecular fraction which was highly resistant to β-elimination, was concluded to be β-1,4-linked d-galactan of which reducing-end group was attached to d-galacturonic acid. On the basis of these findings and previous knowledges, the construction of APS–I was also discussed.     

Cloning of the d-Xylose Uptake Gene Linked to the xyl A Gene in Escherichia coli

An Escherichia coli mutant (MX-5) deficient in d-xylose utilization was isolated. The d-xylose uptake and d-xylose isomerase activities of the mutant were much lower than those of the parental strain (C600). The genes responsible for the d-xylose uptake by E. coli were cloned onto vector plasmid pBR322, and the resultant hybrid plasmid was designated as pXP5. Hybrid plasmid pXP5 improved the growth rate of the mutant (MX-5) on d-xylose, and also both the d-xylose uptake and d-xylose isomerase activities of the mutant were recovered when pXP5 was introduced into the mutant cells. Based on these results, it was suggested that one (xyl T) of the d-xylose transport genes could be closely linked to the d-xylose isomerase gene (xylA) known to be present at 80 min on E. coli chromosomal DNA.     

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.     

Isolation and Constitution of a Xylan from Bamboo

A xylan from bamboo culm was isolated by extraction with aikali of chlorite holocellulose and fractional precipitation as a copper complex. The structure was investigated by means of examination of acid components by controlled hydrolysis, methylation analysis, and periodate oxidation. As a result, 4-O-methyl-α-D-glucuronic acid and 2-O-(4-O-methyl-α-D-glucopyranosyluronic acid) D-xylose were isolated and identified as acid components of the bamboo xylan. Hydrolysis of the fully methylated products afforded 2,3,5-tri-O- methyl-L-arabinose (1.6 moles), 2,3,4-tri-O-methyl-D-xylose (1.2 moles), 2,3,4,6-tetra-O-methyl-D-glucose(0.4 moles), 2,3-di-O-methyl-D-xylose (35.8 moles) and mono-O-methyl-D-xylose (2.6 moles). In addition to the above methylated sugars, 2,3,4-tri-O-methyl-D-glucuronic acid and partially methylated aldobiouronic acid were separated by cellulose column chromatography and identified. These results suggest that the bamboo xylan consists mainly of a linear backbone of 1,4-linked β-D-xylopyranose unit, to which L-arabinofuranose and 4-O-methyl-D-glucuronic acid were attached as a single side chain unit at C₂ or C₃.

Additional evidence for a linear chain structure has been given by periodate oxidation. On oxidation by periodate, the bamboo xylan consumed 1.09 moles of periodate and produced 0.05 mole of formic acid per anhydroxylose unit.     

An Endo-(l → 6)-β-d-glucanase of Flavobacterium M64 Hydrolyzing the Octasaccharide Repeating Unit of Succinoglycan to Two Tetrasaccharides

An endo-(l → 6)-β-d-glucanase capable of hydrolyzing octasaccharide to two tetrasaccharides was isolated from cells of Flavobacterium M64. The octasaccharide represents the repeating unit of succinoglycan (SG-D). One tetrasaccharide was composed of d-glucose, succinic acid and pyruvic acid (4:1:1, molar ratio), and the other was composed of d-glucose and d-galactose (3:1, molar ratio). This enzyme hydrolyzed the (l → 6)-β-d-glucosidic linkage adjacent to the (1 → 6)-linked β-d-glucose residue in the octasaccharide repeating unit of succinoglycan and also hydrolyzed the octasaccharide repeating units of similar polysaccharides produced by many strains of Agrobacterium and Rhizobium species.     

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.     

Purification and Crystallization of d-Arabinose (l-Fucose) Isomerase from Aerobacter aerogenes by Polyethylene Glycol

d-Arabinose(l-fucose) isomerase (d-arabinose ketol-isomerase, EC 5.3.1.3) was purified from the extracts of d-arabinose-grown cells of Aerobacter aerogenes, strain M-7 by the procedure of repeated fractional precipitation with polyethylene glycol 6000 and isolating the crystalline state. The crystalline enzyme was homogeneous in ultracentrifugal analysis and polyacrylamide gel electrophoresis. Sedimentation constant obtained was 15.4s and the molecular weight was estimated as being approximately 2.5 × 10⁵ by gel filtration on Sephadex G-200.

Optimum pH for isomerization of d-arabinose and of l-fucose was identical at pH 9.3, and the Michaelis constants were 51 mm for l-fucose and 160 mm for d-arabinose. Both of these activities decreased at the same rate with thermal inactivation at 45 and 50°C. All four pentitols inhibited two pentose isomerase activities competitively with same Ki values: 1.3–1.5 mm for d-arabitol, 2.2–2.7 mm for ribitol, 2.9–3.2 mm for l-arabitol, and 10–10.5 mm for xylitol. It is confirmed that the single enzyme is responsible for the isomerization of d-arabinose and l-fucose.     

Characterization of an Extracellular Polysaccharide Elaborated by TX-1, a New Strain of Beijerinckia indica

An extracellular polysaccharide elaborated by a new species of Beijerinckia indica, named TX-1, was composed of D-glucose, L-fucose, D-glycero-D-manno-heptose, and D-glucuronic acid in a molar ratio of 5.0:1.0:2.0:0.9, in addition to 16.2% of the acetyl group. Among the polysaccharides of the Beijerinckia species, the present polysaccharide might be the first acidic type having an L-fucose residue. A methylation analysis, Smith degradation study and fragmentation analysis show that this polysaccharide consisted of non-reducing terminal D-glucose, O-4 substituted D-glucose, O-2 substituted D-glycero-D-manno-heptose, O-4 substituted D-glucuronic acid, O-3 and O-4 substituted D-glucose, and O-3 substituted L-fucose residues. A D-glucuronic acid residue was linked to the O-3 position of the L-fucose residue by an α-glycosidic linkage. Most of the D-glucose residues in the backbone chain were substituted at the O-3 position, with the side chain having non-reducing terminal D-glucose residues. It is suggested by the reaction with Con A that the anomeric configuration of the terminal D-glucose residues was β.     

A Simple Transductional Method for Construction of Adenylate- cyclase or cAMP Receptor Protein-deficient Mutants of Escherichia coli K-12

The substrate specificity of α-d-xylosidase from Bacillus sp. No. 693–1 was further investigated. The enzyme hydrolyzed α-1,2-, α-1,3-, and α-1,4-xylobioses. It also acted on some heterooligosaccharides such as O-α-d-xylopyranosyl-(1→6)-d-glucopyranose, O-α-d-xylopyranosyl-(1→6)-O-β-d-glucopyranosyl-(1→4)-d-glucopyranose, O-α- d-xylopyranosyl-(1→6)-O-d-glucopyranosyl-(1→4)-O-[α-d-xylopyranosyl-(1→6)]-d-glucopyranose, and O-α-d-xylopyranosyl-(1→3)-l-arabinopyranose. The enzyme was unable to hydrolyze tamarinde polysaccharides although it could hydrolyze low molecular weight substrates with similar linkages.