Studies on Protopectinase-C Mode of Action: Analysis of the Chemical Structure of the Specific Substrate in Sugar Beet Protopectin and Characterization of the Enzyme Activity

Chemical structures of pectic substances degraded by protopectinase-C (PPase-C) were characterized to identify the releasing mechanism of pectin from sugar beet protopectin by the action of that enzyme. The substrate of PPase-C was a polysaccharide isolated from sugar beet pulp by extraction with NaOH and sequential digestions with rhamnogalacturonase (PPase-T), β-1,4-D-galactanase, and α-L-arabinofuranosidase. The structure of this polysaccharide was analyzed by gas-liquid chromatography (GLC), NMR analysis, and gas chromatography-mass spectrometry (GC-MS), and it was identified as α-1,5-L-arabinan. According to our results, arabinan chains seemed to be connected to rhamnogalacturonan through a chain of β-l,4-D-galactan. PPase-C hydrolyzed both linear α-1,5-L-arabinan and ramified L-arabinan in a random manner, producing L-arabinose. From these results, PPase-C could be classified as arabinan endo-1,5-α-L-arabinase [EC 3.2.1.99]. Moreover, PPase-C seemed to split the L-arabinan of the polysaccharides connecting the rhamnogalacturonan to the other constituents of the plant cell wall in sugar beet pulp, releasing water-soluble pectin.     

Acid and Enzymatic Hydrolysis of an Acidic Polysaccharide from Soy Sauce

Mild acid hydrolysis of an acidic polysaccharide (APS-I) from soy sauce resulted in a degraded polysaccharide (DPS), the mixture of neutral sugar, D-galacturonic acid, its α-1,4-linked homologous di- and trisaccharides, and acidic oligosaccharides containing residues of D-galacturonic acid and L-rhamnose. Besides the above-mentioned sugars, an aldobiouronic acid containing D-xylose moiety was also yielded in the enzymatic hydrolysates with a crude polysaccharidase preparation. However, only a β-l, 4-galactobiose was isolated from the lower molecular fraction of enzymatic digest of APS-I with a typical hemicellulase preparation. DPS containing 83% of D-galacturonic acid was able to be degraded by endo-polygalacturonase, but APS-I was not because of its highly was discussed on the basis of these results, periodate oxidation study.     

Isolation of Oligosaccharides Corresponding to the Branching-point of Konjac Mannan

Ultracentrifugically homogeneous glucomannan acetate derived from konjac mannan was subjected to acetolysis. Besides β-1,4-linked oligosaccharides composed of D-mannose and/or D-glucose, three oligosaccharides corresponding to the branching point of the polysaccharide were isolated and identified as (1) 3-O-β-D-mannopyranosyl-D-mannose, (2) O-β-D-mannopyranosyl-(1→4)-O-β-D-mannopyranosyl-(1→3)-D-mannose, and (3) O-β-D- mannopyranosyl-(1→3)-O-β-D-mannopyranosyl-(1→4)-D-glucose. The average chain length (CL) was, moreover, determined to be about 46 by methylation analysis. The structural pattern of the glucomannan, including the branching point, is discussed.     

Structural Analysis of an Extracellular Polysaccharide Bioflocculant of Klebsiella pneumoniae

The glycoside composition and sequence of an extracellular polysaccharide flocculant of Klebsiella pneumoniae H12 was analyzed. GC and HPLC analysis of the acid-hydrolysate identified its constituent monosaccharides as D-Glc, D-Man, D-Gal, and D-GlcA in an approximate molar ratio of 3.9:1.0:2.3:3.6. To analyze the glycoside sequence, the polysaccharide was partially hydrolyzed by acid and enzyme treatment. GC, HPLC, TLC, MALDI-TOF/MS, and 1H- and 13C- NMR spectroscopy characterized the obtained oligosaccharides.

The results clarified the partial structure of H12 polysaccharide as a linear polymer of a unit of pentasaccharide with a side chain of one D-GlcA to D-Glc moiety (see below). Although the existence of other sequences or other constituent glycosides could not be fully excluded, H12 polysaccharide must be a novel types as such a complicated unit for a polymer has not so far been reported. The partial structure of a H12 polysaccharide flocculant is also discussed in this report.

→4)- α-D-Glcp-(1→2)-α-D-Manp-(1→3)-4,6-Pyr-β-D- 3 Galp-(1→4)-β-D-Galp-(1→ ↓

1 β-D-GlcpA     

New Polar Constituents in the Pupae of the Silkworm Bombyx mori L. (II): Developmental Changes of the Constituents

A correlation between the quantitative changes in L-methionine analogs, the ratio of D-serine/L-serine during the pupal stage, and metamorphosis was observed. The glycoside appearing at low blood sugar values during the pupal stage was isolated and characterized as D-glucosyl-L-tyrosine. ¹H-NMR indicated the appearance and increase of this glycoside, and Mirrorcle Ray CV4 equipment was used to take X-ray pictures of the pupal bodies. The results indicate that γ-cyclic di-L-glutamate and L-methionine sulfone might be concerned with ammonia assimilation in the pupae, and that D-glucosyl-L-tyrosine served as a switch for the fatty acid (pupal oil) dissimilation hybrid system.     

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.     

Isolation and Characterization of Thermotolerant Gluconobacter Strains Catalyzing Oxidative Fermentation at Higher Temperatures

Thermotolerant acetic acid bacteria belonging to the genus Gluconobacter were isolated from various kinds of fruits and flowers from Thailand and Japan. The screening strategy was built up to exclude Acetobacter strains by adding gluconic acid to a culture medium in the presence of 1% D-sorbitol or 1% D-mannitol. Eight strains of thermotolerant Gluconobacter were isolated and screened for D-fructose and L-sorbose production. They grew at wide range of temperatures from 10°C to 37°C and had average optimum growth temperature between 30-33°C. All strains were able to produce L-sorbose and D-fructose at higher temperatures such as 37°C. The 16S rRNA sequences analysis showed that the isolated strains were almost identical to G. frateurii with scores of 99.36-99.79%. Among these eight strains, especially strains CHM16 and CHM54 had high oxidase activity for D-mannitol and D-sorbitol, converting it to D-fructose and L-sorbose at 37°C, respectively. Sugar alcohols oxidation proceeded without a lag time, but Gluconobacter frateurii IFO 3264^T was unable to do such fermentation at 37°C. Fermentation efficiency and fermentation rate of the strains CHM16 and CHM54 were quite high and they rapidly oxidized D-mannitol and D-sorbitol to D-fructose and L-sorbose at almost 100% within 24 h at 30°C. Even oxidative fermentation of D-fructose done at 37°C, the strain CHM16 still accumulated D-fructose at 80% within 24 h. The efficiency of L-sorbose fermentation by the strain CHM54 at 37°C was superior to that observed at 30°C. Thus, the eight strains were finally classified as thermotolerant members of G. frateurii.     

Smith Degradation and Methylation of an Acidic Polysaccharide from Soy Sauce

APS–I, an acidic polysaccharide isolated from soy sauce, was subjected to Smith degradation and methylation, and each product obtained was examined by paper chromatography, gas-liquid chromatography and mass spectrometry. Some compounds, such as ethylene glycol, propylene glycol, glycerol, D-threitol, D-galactose, L-rhamnose, D-galacturonic acid and D-threonic acid, were identified as Smith degradation products, and their molar ratios were estimated. Several kinds of methylated sugars were detected in the hydrolysate of methylated APS–I and identified by gas chromatograph-mass spectrometry. On the basis of those findings and previous discussion, the constitution of APS–I was discussed.     

Structures and Properties of a Diastereoisomeric Molecular Compound of (2S,3S)- and (2R,3S)-N-Acetyl-2-amino-3-methylpentanoic Acids

An X-ray crystal structural analysis revealed that (2S,3S)-N-acetyl-2-amino-3-methylpentanoic acid (N-acetyl-L-isoleucine; Ac-L-Ile) and (2R,3S)-N-acetyl-2-amino-3-methylpentanoic acid (N-acetyl-D-alloisoleucine; Ac-D-aIle) formed a molecular compound containing one Ac-L-Ile molecule and one Ac-D-aIle molecule as an unsymmetrical unit. This molecular compound is packed with strong hydrogen bonds forming homogeneous chains consisting of Ac-L-Ile molecules or Ac-D-aIle molecules and weak hydrogen bonds connecting these homogeneous chains in a fashion similar to that observed for Ac-L-Ile and Ac-D-aIle. Recrystallization of an approximately 1:1 mixture of Ac-L-Ile and Ac-D-aIle from water gave an equimolar molecular compound due to its lower solubility than that of Ac-D-aIle or especially Ac-L-Ile. The results suggest that the equimolar mixture of Ac-L-Ile and Ac-D-aIle could be obtained from an Ac-L-Ile-excess mixture by recystallization from water.     

Ng-Methylated Arginines in Broad Bean Seed

l-N^g-Methylarginine, l-N^g,N^g-dimethylarginine and ethanolamine were isolated from basic amino acids fraction of broad bean (Vicia faba L.) seed. The presence of N^g,N′^g-dimethylarginine was also suggested.     

Synthesis of β-D-Fructopyranosyl-(2→6)-D-glucopyranose from D-Glucose and D-Fructose by a Thermal Treatment

The synthesis is reported of β-D-fructopyranosyl-(2→6)-D-glucopyranose that had previously been isolated from a fermented plant extract as a new saccharide. A disaccharide was predominately formed from an equal amount of D-glucose and D-fructose under melting conditions at 140 °C for 60 to 90 min. This saccharide was isolated from the reaction mixture by carbon-Celite column chromatography and preparative HPLC, and was confirmed to be β-D-fructopyranosyl-(2→6)-D-glucopyranose by TOF-MS and NMR analyses.     

Suppressive Effect of Modified Arabinoxylan from Rice Bran (MGN-3) on D-Galactosamine-Induced IL-18 Expression and Hepatitis in Rats

We investigated in this study the effect of modified arabinoxylan from rice bran (MGN-3) and its fractions on D-galactosamine (D-GalN)-induced IL-18 expression and hepatitis in rats. Male Wistar rats were pretreated with MGN-3 or fractions of the MGN-3 hydrolysate, or with saline 1 h before administering D-GalN (400 mg/kg B.W.). The serum transaminase activities, IL-18 mRNA expression level in the liver and IL-18 concentration in the serum were determined 24 h after injecting D-GalN. Both the oral and intraperitoneal administration of MGN-3 (20 mg/kg B.W.) alleviated D-GalN-induced hepatic injury under these experimental conditions. The low-molecular-weight fraction (LMW) of MGN-3 showed the strongest protective effect on D-GalN-induced liver injury, its main sugar component being glucose. Moreover, the D-GalN-induced IL-18 expression was significantly reduced by treating with MGN-3 and LMW. The results suggest that MGN-3 and LMW could provide significant protection against D-GalN liver injury, and that IL-18 might be involved in their protective influence.     

Characterization of a Cellobiose Phosphorylase from a Hyperthermophilic Eubacterium,Thermotoga maritima MSB8

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80°C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70°C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and α-D-glucose-1-phosphate. The K _m for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the k _cat was 5.4 s^-1. In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-β-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s^-1) k _cat followed by 6-deoxy-D-glucose (17 s^-1) and 2-deoxy-D-glucose (16 s^-1). The natural substrate, D-glucose with the k _cat of 8.0 s^-1 had the highest (1.1×10⁴ M^-1 s^-1) k _cat/K _m compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, α-D-glucose-1-phosphate, at higher concentrations.     

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.     

Biotransformation of Cinnamic Acid,p-Coumaric Acid,Caffeic Acid,and Ferulic Acid by Plant Cell Cultures of Eucalyptus perriniana

Biotransformations of phenylpropanoids such as cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid were investigated with plant-cultured cells of Eucalyptus perriniana. The plant-cultured cells of E. perriniana converted cinnamic acid into cinnamic acid β-D-glucopyranosyl ester, p-coumaric acid, and 4-O-β-D-glucopyranosylcoumaric acid. p-Coumaric acid was converted into 4-O-β-D-glucopyranosylcoumaric acid, p-coumaric acid β-D-glucopyranosyl ester, 4-O-β-D-glucopyranosylcoumaric acid β-D-glucopyranosyl ester, a new compound, caffeic acid, and 3-O-β-D-glucopyranosylcaffeic acid. On the other hand, incubation of caffeic acid with cultured E. perriniana cells gave 3-O-β-D-glucopyranosylcaffeic acid, 3-O-(6-O-β-D-glucopyranosyl)-β-D-glucopyranosylcaffeic acid, a new compound, 3-O-β-D-glucopyranosylcaffeic acid β-D-glucopyranosyl ester, 4-O-β-D-glucopyranosylcaffeic acid, 4-O-β-D-glucopyranosylcaffeic acid β-D-glucopyranosyl ester, ferulic acid, and 4-O-β-D-glucopyranosylferulic acid. 4-O-β-D-Glucopyranosylferulic acid, ferulic acid β-D-glucopyranosyl ester, and 4-O-β-D-glucopyranosylferulic acid β-D-glucopyranosyl ester were isolated from E. perriniana cells treated with ferulic acid.     

Structure-Based Modification of D-Alanine-D-Alanine Ligase from Thermotoga maritima ATCC 43589 for Depsipeptide Synthesis

Depsipeptides are peptide-like polymers consisting of amino acids and hydroxy acids, and are expected to be new functional materials for drug-delivery systems and polymer science. In our previous study, D-alanyl-D-lactate, a type of depsipeptide, was enzymatically synthesized using D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589 (TmDdl) by Y207F substitution. Thereafter, in this study, further mutagenesis was introduced, based on structural comparison between TmDdl and a well-characterized D-alanine-D-alanine ligase from Escherichia coli. The S137A/Y207F mutant showed higher D-alanyl-D-lactate and lower D-alanyl-D-alanine synthesizing activity than the Y207F mutant. This suggests that substitution at the S137 residue contributes to product selectivity. Saturated mutagenesis on S137 revealed that the S137G/Y207F mutant showed the highest D-alanyl-D-lactate synthesizing activity. Moreover, the mutant showed broad substrate specificity toward D-amino acid and recognized D-lactate and D,L-isoserine as substrates. On the basis of these characteristics, various depsipeptides can be produced using S137G/Y207F-replaced TmDdl.     

Acceptor Specificity of Cyclodextrin Glycosyltransferase from Bacillus stearothermophilus and Synthesis of α-D-Glucosyl O-β-D-galactosyl-(1→4)-β-D-glucoside

Bacillus stearothermophilus CGTase had a wider acceptor specificity than Bacillus macerans CGTase did and produced large amounts of transfer products of various acceptors such as D-galactose, D-mannose, D-fructose, D- and L-arabinose, d- and L-fucose, L-rhamnose, D-glucosamine, and lactose, which were inefficient acceptors for B. macerans CGTase. The main component of the smallest transfer products of lactose was assumed to be α-D-glucosyl O-β-D-galactosyl-(l→4)-β-D-glucoside.     

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.     

Stereoselective Incorporation of Isoleucine into Cypridina Luciferin in Cypridina hilgendorfii (Vargula hilgendorfii)

The emission of light in the marine ostracod Cypridina hilgendorfii (presently Vargula hilgendorfii) is produced by the Cypridina luciferin-luciferase reaction in the presence of molecular oxygen. Cypridina luciferin has an asymmetric carbon derived from isoleucine, and the absolute configuration is identical to the C-3 position in L-isoleucine or D-alloisoleucine. To determine the stereoselective incorporation of the isoleucine isomers (L-isoleucine, D-isoleucine, L-alloisoleucine, and D-alloisoleucine), we synthesized four ²H-labeled isoleucine isomers and examined their incorporation into Cypridina luciferin by feeding experiments. Judging by these results, L-isoleucine is predominantly incorporated into Cypridina luciferin. This suggests that the isoleucine unit of Cypridina luciferin is derived from L-isoleucine, but not from D-alloisoleucine.     

Preparation of Optically Active Allothreonine by Separating from a Diastereoisomeric Mixture with Threonine

A simple procedure is described to obtain D- and L-allothreonine (D- and L-aThr). A mixture of N-acetyl-D-allothreonine (Ac-D-aThr) and N-acetyl-L-threonine (Ac-L-Thr) was converted to a mixture of their ammonium salts and then treated with ethanol to precipitate ammonium N-acetyl-L-threoninate (Ac-L-Thr·NH₃) as the less-soluble diastereoisomeric salt. After separating Ac-L-Thr·NH₃ by filtration, Ac-D-aThr obtained from the filtrate was hydrolyzed in hydrochloric acid to give D-aThr of 80% de, recrystallized from water to give D-aThr of >99% de. L-aThr was obtained from a mixture of the ammonium salts of Ac-L-aThr and Ac-D-Thr in a similar manner.