A New Preparation of 2,3,6-Tri-O-Methyl d-Galactose

d-galactose was incompletely methylated with methyl sulphate and sodium hydroxide, and two trimethylgalactoses were chromatographically separated from the products. Gas-liquid chromatographic examination, periodate oxidation and melting points of them or their suitable derivatives showed that one of them was 2,3,6-tri-O-methyl d-galactose, and the other was presumed to be 2,4,6-tri-O-methyl d-galactose, For confirmation of 2,3,6- tri-O-methyl d-galactose, 2,3-di-O-methyl l-threose and its aldonophenylhydrazide were prepared from 2,3-di-O-methyl l-arabinose as authentic sample.     

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.     

Sugar Composition of Cell Wall Polysaccharides of Suspension-cultured Tobacco Cells

Neutral sugar composition of cell walls of suspension-cultured tobacco cells was examined with the advance of culture age by an anion-exchange chromatography. Isolated cell walls gave on hydrolysis the following sugars: 2% of l-rhamnose, 6% of d-mannose, 26% of l-arabinose, 13% of d-galactose, 8% of d-xylose and 47% of d-glucose as neutral sugars. Little changes in composition of cell wall polysaccharides were recognized with the advance of culture age. Sugar composition of the extra-cellular polysaccharides was similar to that of hemicellulose fraction from cell walls. Pectinic acid gave on hydrolysis 2-O-(α-d-galactopyranosyluronic acid)-l-rhamnose, d-galacturonic acid and its oligosaccharides.     

Sugar Specificity in the Induction and on the Activity of Streptomyces α-d-Galactosidases

The α-d-galactosidases of six Streptomyces strains were examined on their inducer susceptibility, substate specificity, and inhibitor susceptibility. In all strains examined, α-d-galactosidase was induced by d-galactose, but neither by d-fucose nor by l-arabinose. α-d-Fucosidase activity was always induced accompanying with α-d-galactosedase activity. β-l-Arabinosidase activity, however, was never observed. These α-d-galactosidases were purified to electrophoretically pure degree by successive ammonium sulfate and ethanol precipitation, and ion exchange and gel filtration chromatography. The purified preparations from six strains were different from each other in their chromatographic behaviors and in some physical properties, but they all showed strong α-d-fucosidase activity as well. The α-d-galactosidase activities were strongly inhibited by d-galactose and l-arabinose, but scarcely by d-fucose. On the other hand, their α-d-fucosidase activities were inhibited by d-fucose as well as by d-galactose and l-arabinose.     

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.     

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.     

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.     

Identification and characterization of d-arabinose reductase and d-arabinose transporters from Pichia stipitis

d-xylose and l-arabinose are the major constituents of plant lignocelluloses, and the related fungal metabolic pathways have been extensively examined. Although Pichia stipitis CBS 6054 grows using d-arabinose as the sole carbon source, the hypothetical pathway has not yet been clarified at the molecular level. We herein purified NAD(P)H-dependent d-arabinose reductase from cells grown on d-arabinose, and found that the enzyme was identical to the known d-xylose reductase (XR). The enzyme activity of XR with d-arabinose was previously reported to be only 1% that with d-xylose. The k_cat/K_m value with d-arabinose (1.27 min^?1 mM^?1), which was determined using the recombinant enzyme, was 13.6- and 10.5-fold lower than those with l-arabinose and d-xylose, respectively. Among the 34 putative sugar transporters from P. stipitis, only seven genes exhibited uptake ability not only for d-arabinose, but also for d-glucose and other pentose sugars including d-xylose and l-arabinose in Saccharomyces cerevisiae.     

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.     

Synthesis of Some New α- and β-(l→2) Linked Disaccharides Containing L-Quinovose (6-Deoxy L-Glucose)

Hepta-O-acetyl-2-0-β-l-quinovopyranosyl-α-d-glucose (VI) and hepta-O-acetyl-2-O-α-l-quinovopyranosyl-β-d-gIucose (VIII) were prepared by the coupling of 2,3,4-tri-O-acetyl-α-l-quinovopyranosyl bromide (IV) with l,3,4,6-tetra-O-acetyl-α-D-glucose (V) in the presence of mercuric cyanide and mercuric bromide in absolute acetonitrile.

Similarly, hepta-O-acetyW-O-α-l-quinovopyranosyl-α-d-galactose (X) and hepta-O-acetyl-2-O-β-L-quinovopyranosyl-α-d-galactose (XI) were prepared by the reaction of IV with 1,3,4,6-tetra-O-acetyl-α-d-galactose (IX).

Removal of the protecting groups of VI, VIII, X and XI afforded the corresponding disaccharides. On treatment with hydrogen bromide, VI, VIII, X and XI gave the corresponding acetobromo derivatives.     

The Production of Xylitol,l-Arabinitol and Ribitol by Yeasts

The polyalcohol production from the pentoses such as d-xylose, l-arabinose and d-ribose by various genera and species of yeasts was examined. Candida polymorpha dissimilated aerobically these three pentoses and produced xylitol from d-xylose, l-arabinitol from l-arabinose and ribitol from d-ribose at good yield of 30~40% of sugar consumed. The result suggests that these polyalcohols would be major products from pentoses by yeasts, but some unidentified minor polyalcohols were also produced.     

Stereoselective Synthesis of 2-Amino-2-deoxy-d-arabinose and 2-Deoxy-d-ribose

An efficient method for the stereoselective synthesis of 2-amino-2-deoxy-d-arabinose and 2-deoxy-d-ribose is described.

The key step in this method was accomplished by the nucleophilic addition of methyl isocyanoacetate to 2,3-O-isopropylidene-d-glyceraldehyde with high erythro-selectivity (nearly 100%).

Subsequent intermolecular cyclization predominantly gave the desired oxazoline derivative (trans-form), in which two new chiral centers were formed. The oxazoline derivative was efficiently converted to both 2-amino-2-deoxy-d-arabinose and 2-deoxy-d-ribose.     

Arabinogalactan-Proteins Reacting with Eel Anti-H Agglutinin from Leaves of Cruciferous Plants

Glycoconjugates reacting with eel anti-H agglutinin were purified from extracts of leaves of three species of cruciferous plants (radish, turnip, and rape) by precipitation with ethanol, ion-exchange chromatography, and gel filtration. High voltage paper electrophoresis or ultracentrifugal analysis revealed that the purified specimens were homogeneous. Their apparent molecular weights were estimated to range from 0.5 to 1.5 x 10⁵. They consisted of a novel l-fucose-containing acidic arabinogalactan-protein composed of residues of l-arabinose, d-galactose, l-fucose, 4-O-methyl-d-glucuronic acid, and d-glucuronic acid in similar molar proportions, and containing polypeptide portions with abundant hydroxyproline, serine, threonine, and alanine. All the arabinogalactan-proteins exhibited potent inhibitory activity against the hemagglutination of human O erythrocytes by eel anti-H agglutinin.     

Studies on the Pentose Isomerases of Lactic Acid Bacteria

Production of d-xylose and l-arabinose isomerases by lactic acid bacteria was greatly promoted by the addition of manganese ions in cultural medium. Effective concentration of the ions was 5 × 1O^-3 m. Ferrous ions were also effective for the production of d-xylose isomerase and cobaltous ions were somewhat effective for the production of l-arabinose isomerase. Zinc and cadmium ions inhibited bacterial growth. It was possible to increase the production of isomerase by changing MnSO₄ concentration to 5× 10^-3 m (0.l1 %) in place of 0.001 per cent in the normal medium.

Column chromatographic procedures for the purification of pentose isomerases were carried out. Cation and anion exchange resins were not suitable because of their low exchange capacities and instability of the enzyme at acidic pH range. But the isomerases were successfully purified by DEAE-cellulose column chromatography with high recovery (85~90%). Using a Tris buffer, KCl concentration was increased in gradient. d-Xylose isomerase was eluted at pH 7.0 at 0~0.2 m KCl, and l-arabinose isomerase at pH 8.0 at 0~0.4 m KCl. The purified isomerases, d-xylose isomerase and l-arabinose isomerase, both required manganese ions specifically for their activities.

D-Xylose isomerase and l-arabinose isomerase are different enzymes which can be separated from each other with acetone fractionation at pH 4.8~5.0, heat treatment or chromatography on a colnmn of DEAE-cellulose. In DEAE-cellulose chromatography with a linear gradient elution method, d-xylose isomerase is recovered in the first peak at pH 7.0 (Tris bnffer) with 0~0.2 m KCl, and l-arabinose isomerase is eluted in the second peak at pH 8.0 (Tris buffer) with a larger ionic strength.     

Substrate Specificity of Alkaline Proteinases from Aspergillus sydowi and Aspergillus sulphureus

l-Fucose (l-galactose) dehydrogenase was isolated to homogeneity from a cell-free extract of Pseudomonas sp. No 1143 and purified about 380-fold with a yield of 23 %. The purification procedures were: treatment with polyethyleneimine, ammonium sulfate fractionation, chromatographies on phenyl-Sepharose and DEAE-Sephadex, preparative polyacrylamide gel electrophoresis, and gel filtration on Sephadex G-100. The enzyme had a molecular weight of about 34,000. The optimum pH was at 9 — 10.5 and the isoelectric point was at pH 5.1. l-Fucose and l-galactose were effective substrates for the enzyme reaction, but d-arabinose was not so much. The anomeric requirement of the enzyme to l-fucose was the β-pyranose form, and the reaction product from l-fucose was l-fucono- lactone. The hydrogen acceptor for the enzyme reaction wasNADP⁺, and NAD ⁺ could be substituted for it to a very small degree. Km values were 1.9mm, 19mm, 0.016mm, and 5.6mm for l-fucose, l- galactose, NADP⁺, and NAD⁺, respectively. The enzyme activity was strongly inhibited by Hg^{2 +}, Cd^{2 +}, and PCMB, but metal-chelating reagents had almost no effect. In a preliminary experiment, it was indicated that the enzyme may be usable for the measurement of l-fucose.     

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.     

Studies on the Pentose Isomerases of Lactic Acid Bacteria

d-Xylose isomerase requires manganese ions for its action, but l-arabinose isomerase has a less specific on metal requirement. l-Arabinose isomerase is activated by addition of Mn⁺⁺ or Co⁺⁺, less effectively by addition of Zn⁺⁺, Ca⁺⁺, Mg⁺⁺, Sr⁺⁺ or Cd⁺⁺. Moreover, manganese and potassium ions for d-xylose isomerase, and manganese and cobaltous ions for l-arabinose isomerase were also shown to have protective effect on respective enzymes against thermal inactivation.     

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.     

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.     

l-Asparaginase from Escherichia coli

Crystalline l-asparaginase from Escherichia coli A-I-3 hydrolyzed d-asparagine, l- and d-glutamine but at much slower rates than the rate at which it hydrolyzed l-asparagine. Inhibitions by these substrates and related compounds were revealed to be competitive.

d-Asparagine showed the same affinity for the enzyme both in its hydrolysis and inhibition of l-asparagine hydrolysis. l-Aspartate, d-aspartate and α-N-ethylasparagine inhibited various hydrolysis reactions with the respective inhibitor constants. The enzyme was found to hydrolyze β-methylaspartate as well as β-aspartohydroxamate. These data strongly suggest that the hydrolysis occurred at the same active site of the enzyme molecule with relatively low specificity for the configuration of the substrate molecule and the kind of bonding which it hydrolyzes.