Polysaccharides of Soybean Seeds

Methylation of hot-water-extract fraction of soybean seed polysaccharides by the Haworth’s method and then by the Purdie’s method followed by fractional extraction afforded a fully methylated arabinogalactan which gave on complete hydrolysis 2,3,5-tri-, and 2,3-di-O-methyl l-arabinose and 2,3,6-tri-, and 2,6-di-O-methyl d-galactose in the approximate proportion of 1 : 1 : 3 ~ 4: 1, and on controlled acid methanolysis 2,3,5-tri-, and 2,3-di-O-methyl l-arabinose in about equal proportion. Residual undegraded polysaccharide gave mainly 2,3,6-tri-O-methyl d-galactose on remethylation and hydrolysis.     

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.     

Studies on Mannan in Wood Pulp

The glucomannan isolated from holocellulose pulp of Akamatsu (Pinus densiflora Sieb. et Zucc.) as its triacetate was methylated and the following methylated sugars were obtained by hydrolysis: the 2,3,4,6-tetra-O-methyl ethers of d-glucose and d-mannose (I part) and the 2,3,6-tri-O-methyl ethers of d-mannose and d-glucose (34–37 parts). Periodate oxidation of the glueomannan showed that 1.00 mole of periodate was consumed per mole of hexose unit and 3 moles of formic acid liberated for every 33 hexose units.     

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.     

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.     

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.     

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.     

High levels of expression of multiple enzymes in the Smirnoff-Wheeler pathway are important for high accumulation of ascorbic acid in acerola fruits

ABSTRACT

Acerola fruits contain abundant ascorbic acid (AsA). The gene expression levels of three upstream enzymes in the primary AsA biosynthesis pathway were correlated with AsA contents in the fruits of two acerola cultivars. Multiple overexpression of the enzymes increased AsA contents, suggesting their high expression is important for high AsA accumulation in acerola fruits and the breeding of AsA-rich plants.

Abbreviations: AsA: ascorbic acid; PMI: phosphomannose isomerase; PMM: phosphomannomutase; GMP: GDP-d-mannose pyrophosphorylase; GME: GDP-d-mannose 3?,5?-epimerase; GGP: GDP-l-galactose phosphorylase; GPP: l-galactose-1-phosphate phosphatase; GDH: l-galactose dehydrogenase; GLDH: l-galactono-1,4-lactone dehydrogenase     

Studies on Utilization of Agar

Some chemical and physical properties of agarose (AG) and agaropectin (AP) isolated from agar of various red seaweeds were studied. The two components were isolated by acrinol. Methanol containing sodium iodide was superior to the mixed solvent of ethanol and acetone (1:1) as solvent for removing acrinol. Greater value of the ratio of intrinsic viscosity of both AG and AP in the solution of 0.1 m sodium chloride against that in the mixed solution of 4 m urea and 0.001 m sodium thiocyanate made water holding capacity greater except sample whose molecular weight is very small. Water holding capacity of AG was decreased with increasing ratio of d-galactose plus 6-O-methyl-d-galactose against 3,6-anhydro-l-galactose, and with lower liquefying temperature of gel. In the case of AP, however, these relations were not always distinct.     

Mucopolysaccharides Isolated from Human and Cow Colostrums

Mucopolysaccharides were isolated from both human and cow colostrums. Each of the fractionated mucopolysaccharides was considered to be homogeneous from behaviors in chromatography, electrophoresis and sedimentation pattern. The fractions isolated from human colostrum were found to contain 51.0~78.3% carbohydrates consisting of d-galactose, 2-amino-2-deoxy-d-glucose, N-acetylneuraminic acid, l-fucose and d-glucose, and 31.6~11.0% peptides consisting of 16 kinds of amino acids. The sedimentation constants, s_{20, w}, of these fractions were in the range of 0.75 to 1.73 S. The fraction isolated from cow colostrum was found to contain 19.3% carbohydrates consisting of d-galactose, 2-amino-2-deoxy-d-glucose and N-acetylneuraminic acid, and 65.2% peptides or proteins consisting of 18 kinds of amino acids. The sedimentation constant, s_{20, w}, of the fraction was 3.68 S.     

Production of l-allose and d-talose from l-psicose and d-tagatose by l-ribose isomerase

l-ribose isomerase (L-RI) from Cellulomonas parahominis MB426 can convert l-psicose and d-tagatose to l-allose and d-talose, respectively. Partially purified recombinant L-RI from Escherichia coli JM109 was immobilized on DIAION HPA25L resin and then utilized to produce l-allose and d-talose. Conversion reaction was performed with the reaction mixture containing 10% l-psicose or d-tagatose and immobilized L-RI at 40 °C. At equilibrium state, the yield of l-allose and d-talose was 35.0% and 13.0%, respectively. Immobilized enzyme could convert l-psicose to l-allose without remarkable decrease in the enzyme activity over 7 times use and d-tagatose to d-talose over 37 times use. After separation and concentration, the mixture solution of l-allose and d-talose was concentrated up to 70% and crystallized by keeping at 4 °C. l-Allose and d-talose crystals were collected from the syrup by filtration. The final yield was 23.0% l-allose and 7.30% d-talose that were obtained from l-psicose and d-tagatose, respectively.     

Fermentative Production of l-Arginine

Most of the bacteria, which were examined for the sensitivity to l-arginine analogs (l-canavanine, l-homoarginine, d-arginine and arginine hydroxamate), were insensitive to the analogs at a concentration of 8 mg/ml. Corynebacterium glutamicum DSS-8 isolated as d-serine-sensitive mutant from an isoleucine auxotroph KY 10150, was found to be sensitive to d-arginine and arginine hydroxamate. Furthermore, DSS-8 produced l-arginine in a cultural medium. l-Arginine analog-resistant mutants were derived from DSS-8 by N-methyl-N′-nitro-N-nitrosoguanidine (NTG) treatment. Most of them were found to produce a large amount of l-arginine. An isoleucine revertant from one of these mutants produced 19.6 mg/ml of l-arginine in the medium containing 15% (as sugar) of molasses.

The mechanism of the sensitivity to l-arginine analogs and that of the production of l-arginine in the d-serine-sensitive mutant, DSS-8, were investigated. DSS-8 seems to be a mutant having increased permeability to d- and l-arginine.     

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.     

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.     

Elimination,Replacement and Isomerization Reactions by Intact Cells Containing Tyrosine Phenol Lyase

Tyrosine phenol lyase catalyzes a series of α,β-elimination, β-replacement and racemization reactions. These reactions were studied with intact cells of Erwinia herbicola ATCC 21434 containing tyrosine phenol lyase.

Various aromatic amino acids were synthesized from l-serine and phenol, pyrocatechol, resorcinol or pyrogallol by the replacement reaction using the intact cells. l(d)-Tyrosine, 3,4-dihydroxyphenyl-l(d)-alanine (l(d)-dopa), l(d)-serine, l-cysteine, l-cystine and S-methyl-l-cysteine were degraded to pyruvate and ammonia by the elimination reaction. These amino acids could be used as substrate, together with phenol or pyrocatechol, to synthesize l-tyrosine or l-dopa via the replacement reaction by intact cells. l-Serine and d-serine were the best amino acid substrates for the synthesis of l-tyrosine or l-dopa. l-Tyrosine and l-dopa synthesized from d-serine and phenol or pyrocatechol were confirmed to be entirely l-form after isolation and identification of these products. The isomerization of d-tyrosine to l-tyrosine was also catalyzed by intact cells.

Thus, l-tyrosine or l-dopa could be synthesized from dl-serine and phenol or pyrocatechol by intact cells of Erwinia herbicola containing tyrosine phenol lyase.     

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.     

Synthesis of N-{2-O-2-Acetamido-1-O-acyl (or 1,6-di-O-acyl)-2,3-dideoxy-α-d-glucopyranose-3-yl]-d-lactoyl}-l-alanyl-d-isoglutamine Methyl Esters,and Their Immunoadjuvant Activities

A variety of 1-O-acyl and 1,6-di-O-acyl derivatives of N-acetylmuramoyl-l-alanyl-G-isoglutamine methyl esters were synthesized from N-[2-O-(2-acetamido-2,3-dideoxy-4,6-O-iso- propylidene-d-glucopyranose-3-yl)-d-lactoyl]-l-alanyl-d-isoglutamine methyl ester, and their biological activities were examined in guinea-pigs and mice.     

Contributions of tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase to the conversion of d-tryptophan to nicotinamide analyzed by using tryptophan 2,3-dioxygenase-knockout mice

We investigated the contribution percentage of tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) to the conversion of d-tryptophan to nicotinamide in TDO-knockout mice. The calculated percentage conversions indicated that TDO and IDO oxidized 70 and 30%, respectively, of the dietary l-tryptophan. These results indicate that both TDO and IDO biosynthesize nicotinamide from d-tryptophan and l-tryptophan in mice.     

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.