C-(polyacetoxy)alkyloxadiazolines and related compounds

The (phenylacetyl)hydrazones of d-galactose, d-glucose, d-mannose, d-arabinose, l-arabinose, d-xylose, and l-sorbose were prepared. The d-galactose and d-arabinose derivatives were converted into their per-O-acetylated derivatives (8 and 9, respectively). The acyclic structure of 8 was proved from its direct preparation by the condensation of(phenylacetyl)hydrazine with penta-O-acetyl-aldehydo-d-galactose. Cyclization of 2,3,4,5,6-penta-O-acetyl-aldehydo-d-galactose (phenylacetyl)hydrazone with boiling acetic anhydride yielded a mixture of two products that could be separated by fractional recrystallization, to give 3-acetyl-5-benzyl-2-(polyacetoxy)alkyl-1,3,4-oxadiazolines; a mechanism for the reaction was proposed. The n.m.r. and mass spectra of some of these derivatives were discussed.     

Engineering nonphosphorylative metabolism to synthesize mesaconate from lignocellulosic sugars in Escherichia coli

Dicarboxylic acids are attractive biosynthetic targets due to their broad applications and their challenging manufacturing process from fossil fuel feedstock. Mesaconate is a branched, unsaturated dicarboxylic acid that can be used as a co-monomer to produce hydrogels and fire-retardant materials. In this study, we engineered nonphosphorylative metabolism to produce mesaconate from d-xylose and l-arabinose. This nonphosphorylative metabolism is orthogonal to the intrinsic pentose metabolism in Escherichia coli and has fewer enzymatic steps and a higher theoretical yield to TCA cycle intermediates than the pentose phosphate pathway. Here mesaconate production was enabled from the d-xylose pathway and the l-arabinose pathway. To enhance the transportation of d-xylose and l-arabinose, pentose transporters were examined. We identified the pentose/proton symporter, AraE, as the most effective transporter for both d-xylose and l-arabinose in mesaconate production process. Further production optimization was achieved by operon screening and metabolic engineering. These efforts led to the engineered strains that produced 12.5 g/l and 13.2 g/l mesaconate after 48 h from 20 g/l of d-xylose and l-arabinose, respectively. Finally, the engineered strain overexpressing both l-arabinose and d-xylose operons produced 14.7 g/l mesaconate from a 1:1 d-xylose and l-arabinose mixture with a yield of 85% of the theoretical maximum. (0.87 g/g). This work demonstrates an effective system that converts pentoses into a value-added chemical, mesaconate, with promising titer, rate, and yield.     

Selective reduction of xylose to xylitol from a mixture of hemicellulosic sugars

The biocatalytic reduction of d-xylose to xylitol requires separation of the substrate from l-arabinose, another major component of hemicellulosic hydrolysate. This step is necessitated by the innate promiscuity of xylose reductases, which can efficiently reduce l-arabinose to l-arabinitol, an unwanted byproduct. Unfortunately, due to the epimeric nature of d-xylose and l-arabinose, separation can be difficult, leading to high production costs. To overcome this issue, we engineered an E. coli strain to efficiently produce xylitol from d-xylose with minimal production of l-arabinitol byproduct. By combining this strain with a previously engineered xylose reductase mutant, we were able to eliminate l-arabinitol formation and produce xylitol to near 100% purity from an equiweight mixture of d-xylose, l-arabinose, and d-glucose.     

A new method for the preparation of crystalline L-arabinose from arabinoxylan by enzymatic hydrolysis and selective fermentation with yeast

Arabinoxylan (`Cellace') corn fiber, containing 28.1% (w/w) as l-arabinose and 32.8% (w/w) as d-xylose, was hydrolyzed by a crude enzyme containing -xylanase, -xylosidase and -l-arabinofuranosidase originating from the extracellular culture broth of Penicillium funiculosum. The resultant hydrolysate contained l-arabinose, d-xylose and small amounts of other mono- and oligosaccharides. The l-arabinose and d-xylose were 21.3% (w/w) and 18.7% (w/w), respectively, based on the initial arabinoxylan. Williopsis saturnus var. saturnus, which metabolizes d-xylose without using l-arabinose, was aerobically cultured in the hydrolysate at 30 °C for 96 h. The sugar solution after removal of yeast cells contained l-arabinose and d-xylose which were 20.3% (w/w) and 0.002% (w/w), respectively, of the initial arabinoxylan. The solution was decolorized with activated carbon, and deionized with cation- and anion-exchange resins. The clear sugar solution thus obtained was composed of l-arabinose and d-xylose which were 19.3% (w/w) and 0.001% (w/w), respectively, of the initial arabinoxylan. After concentration of the sugar solution under reduced pressure, followed by crystallization of l-arabinose, 16% (w/w) l-arabinose (based on the initial arabinoxylan) was obtained as crude crystals. No d-xylose was detected in the final preparation.     

Studies of two low-molecular-weight endo-1(1→4)-β-d-xylanases constitutively synthesised by the cellulolytic fungus Trichoderma koningii

Two endo-(1→4)-β-d-xylanases (xylanases 1 and 2), which were constitutively synthesised by the fungus Trichoderma koningii, were purified to homogeneity on gel-filtration media and by isoelectric focusing. They had molecular weights of 29,000 (xylanase 1) and 18,000 (xylanase 2), and isoelectric pHs of 7.24 (xylanase 1) and 7.3 (xylanase 2); neither enzyme was associated with carbohydrate. Xylanase 1 had an optimum at the remarkably high temperature of 60–65°. Each enzyme liberated a different range of oligosaccharides from oat-straw arabinoxylan, but only xylanase 1 released l-arabinose and d-xylose. Both xylanases were free from cellulase activity.     

Induction of aldose reductase and xylitol dehydrogenase activities in Candida tenuis CBS 4435

In this study the ability of various sugars and sugar alcohols to induce aldose reductase (xylose reductase) and xylitol dehydrogenase (xylulose reductase) activities in the yeast Candida tenuis was investigated. Both enzyme activities were induced when the organism was grown on d-xylose or l-arabinose as well as on the structurally related sugars d-arabinose or d-lyxose. Mixtures of d-xylose with the more rapidly metabolizable sugar d-glucose resulted in a decrease in the levels of both enzymes formed. These results show that the utilization of d-xylose by C. tenuis is regulated by induction and catabolite repression. Furthermore, the different patterns of induction on distinct sugars suggest that the synthesis of both enzymes is not under coordinate control.     

Ethanol production from pentoses and sugar-cane bagasse hemicellulose hydrolysate by Mucor and Fusarium species

The fermentation of carbohydrates and hemicellulose hydrolysate by Mucor and Fusarium species has been investigated, with the following results. Both Mucor and Fusarium species are able to ferment various sugars and alditols, including d-glucose, pentoses and xylitol, to ethanol. Mucor is able to ferment sugar-cane bagasse hemicellulose hydrolysate to ethanol. Fusarium F5 is not able to ferment sugar-cane bagasse hemicellulose hydrolysate to ethanol. During fermentation of hemicellulose hydrolysates, d-glucose was utilized first, followed by d-xylose and l-arabinose. Small amounts of xylitol were produced by Mucor from d-xylose through oxidoreduction reactions, presumably mediated by the enzyme aldose reductase¹ (alditol: NADP⁺ 1-oxidoreductase, EC 1.1.1.21). For pentose fermentation, d-xylose was the preferred substrate. Only small amounts of ethanol were produced from l-arabinose and d-arabitol. No ethanol was produced from l-xylose, d-arabinose or l-arabitol.     

Formation of glucose from hexoses, pentoses, polyols and related substances in kidney cortex

1. Slices of rat kidney cortex, on incubation in a saline medium, formed d-glucose from the following substances: d-fructose, d-galactose, d-mannose, l-sorbose, l-arabinose, d-xylose, glycerol, myo-inositol, l-iditol, sorbitol, xylitol, ribitol, methylglyoxal, dihydroxyacetone, l-glyceraldehyde, d-glyceraldehyde, dl-glyceraldehyde, dl-glycerate. Values for the rates of glucose formation from these precursors are given. 2. No glucose was formed from l-rhamnose, d-arabitol, d-arabinose, d-ribose, l-fucose, d-lyxose, mannitol, dulcitol, d-glucuronate, propane-1,2-diol and propan-2-ol. 3. The pathways of glucose formation from the various precursors are discussed (Scheme 1). 4. l-Glyceraldehyde inhibited the formation of glucose from d-glyceraldehyde.     

Carbohydrates of the inner bark of Pseudotsuga menziesii

A holocellulose fraction was isolated from the inner bark of Psedotsuga menziesii and analyzed. It was composed of acid-insoluble lignin (3·1 acid-soluble lignin (4·1%), l-arabinose (2·6%), d-xylose (6.3%). d-mannose(9·5%), d-galactose (2·3%), and d-glcose (61·1%). The presence of these sugars and their configurations were positively established by the preparation of crystalline derivatives. The holocellulose was fractionated into its component polysaccharides, a xylan, a galactoglucomannan, a glucomannan, and a glucan-rich residue.     

Specificity of sugar-phospholipid interactions

Previous studies by Lefevre et al. (6) have shown that phospholipids stimulate uptake of glucose and other sugars by lipid solvents and enhance transfer of glucose through solvent layers into water. In this paper the specificity of the process for different sugars is investigated by following uptake from thin films of sugars or from glass fiber strips coated with radioactive sugars. Hexoses were taken up slowly to molar ratios of sugar to lipid phosphorus of about 1:1. Pentoses and deoxy sugars were taken up 5–10 times more rapidly to molar ratios of between 1.5 and 2.5:1. Relative rates of formation of the complexes at 25 °C were (d-glucose = 1.0):l-fucose, 9.1; d-ribose, 6.1; d-arabinose, 5.5; l-rhamnose, 3.8; l-arabinose, 3.7; d-xylose, 3.6; d-lyxose, 3.1; 3-O-methyl-d-glucose, 1.52; d-mannose, 1.36; d-galactose, 1.13; sucrose, 0.03; and lactose, 0.015. Radioactive sugars bound to phospholipids exchanged readily with unlabeled sugar in the anhydrous state and the sugars passed slowly into the aqueous phase when the complexes were shaken with water. The relative rates of dissociation (d-glucose = 1.0): l-arabinose, 2.82; d-arabinose, 2.49; l-rhamnose, 2.26; l-fucose, 1.96; d-xylose, 1.65; 3-O-methyl-d-glucose, 0.37; d-galactose, 0.28 were in the same general order as formation, suggesting that a common intermediate may be involved in both processes. In general, sugars with high mutarotation rates reacted most rapidly indicating a possible relationship between the structural features which favor interaction with phospholipids and those which enhance mutarotation.     

Structural features of an acidic polysaccharide from the mucin of Drosera binata

The polysaccharide from the mucin secreted by the leaves of Drosera binata is composed of l-arabinose, d-xylose, d-galactose, d-man     

Model studies pertaining to the hydrazinolysis of glycopeptides and glycoproteins: hydrazinolysis of the 1-N-acetyl and 1-N-(l-β-aspartyl) derivatives of 2-acetamido-2-deoxy-β-d-glucopyranosylamine

The products of hydrazinolysis of the 1-N-acetyl and 1-N-(l-β-aspartyl) derivatives of 2-acetamido-2-deoxy-β-d-glucopyranosylamine could not be converted quantitatively into 2-amino-2-deoxy-d-glucose under mild conditions. Proton and ¹³C-n.m.r. measurements indicated that the hydrazone of 2-amino-2-deoxy-d-glucose was a major product of the hydrazinolysis of 2-acetamido-1-N-acetyl-2-deoxy-β-d-glucopyranosylamine. Control experiments showed that acetohydrazide is slowly converted into 4-amino-3,5-dimethyl-1,2,4-triazole under-the conditions of hydrazinolysis, and that 2-amino-2-deoxy-d-glucose reacts slowly with acetohydrazide in dilute acetic acid. The implications of these results in relation to the hydrazinolysis of glycopeptides and glycoproteins are discussed.     

The disaccharide composition of heparins and heparan sulfates

Heparin and heparan sulfate can be cleaved selectively at their N-sulfated glucosamine residues by direct treatment with nitrous acid at pH 1.5. These polymers can also be cleaved selectively at their N-acetylated glucosamine residues by first N-deacetylating with hydrazine and then treating the products with nitrous acid at pH 4. These procedures have been combined and optimized for the conversion of these glycosaminoglycan chains into their disaccharide units. A modified hydrazinolysis procedure in which the glycosaminoglycans were heated with hydrazine:water (70:30) containing 1% hydrazine sulfate gave rapid rates of N-deacetylation and minimal conversion of the uronic acid residues to their hydrazide derivatives. Under these conditions, N-deacetylation was complete in 4 h and the beta-eliminative cleavage of the polymer chains that occurs during hydrazinolysis (P. N. Shaklee and H. E. Conrad (1984) Biochem. J. 217, 187-197) was eliminated. Treatment of the N-deacetylated polymer with nitrous acid at pH 3 for 15 h at 25 degrees C then gave simultaneous cleavage at the N-unsubstituted glucosamine residues and the N-sulfated glucosamine residues. These deamination conditions minimized, but did not eliminate, the side reaction in which nitrous acid-reactive glucosamine residues undergo ring contraction without glucosaminide bond cleavage. Thus, the disaccharides were obtained in a yield of 90% of those originally present in the glycosaminoglycan chains. Since the ring contraction side reaction occurs randomly at the diazotized glucosamine residues, the disaccharides formed in the pH 3 nitrous acid reaction were recovered in proportions equal to those in the original glycosaminoglycan chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Utilization of degraded peach gum polysaccharide by Aspergillus flavus

d-Galactose, d-mannose, d-xylose, l-arabinose, and d-glucuronic acid and its γ-lactone were examined as carbon sources for the culture of Aspergillus flavus. d-Mannose was taken up the most rapidly and d-glucuronic acid and its γ-lactone the least rapidly. A partially degraded polysaccharide from peach tree gum (Prunus persica [L.] Batsch containing the above sugars together with d-glucuronic acid and its 4-O-methyl ether was used as substrate for another A. flavus culture. It was found that d-galactose was the major sugar passing into the culture medium with lower proportions of d-xylose, l-arabinose, 2-O-β-d-glucopyranuronosyl-d-mannose, and 6-O-β-d-glucopyranuronosyl-d-galactose. This indicates that the fungus produces extracellular exo- and endo-glycanohydrolases which may be useful in structural studies on polysaccharides.     

Regioselective O-deacylation of fully acylated glycosides and 1,2-O-isopropylidenealdofuranose derivatives with hydrazine hydrate

On hydrazinolysis in 1:4 acetic acid-pyridine, and in pyridine, partial O-deacylation of fully acylated methyl glycosides and some other glycosyl compounds (23 compounds) was found to be induced, to give, in good yields, products bearing one free hydroxyl group; the results obtained indicated that, among the primary and secondary O-acyl groups, the 2-O-acyl groups were, in general, the most labile toward the nucleophile (hydrazine). Hydrazinolysis of 1,2-O-isopropylidenealdofuranose acylates (3 compounds), on the other hand, gave, in high yield, the corresponding monoacyl derivatives having the protecting group on their primary hydroxyl group. The factors possibly involved in the regioselectivity of the hydrazinolysis were discussed.     

Hydrazinolysis of heparin and other glycosaminoglycans.

Heparin, carboxy-group-reduced heparin, several sulphated monosaccharides and disaccharides formed from heparin, and a tetrasaccharide prepared from chondroitin sulphate were treated at 100 degrees C with hydrazine containing 1% hydrazine sulphate for periods sufficient to cause complete N-deacetylation of the N-acetylhexosamine residues. Under these hydrazinolysis conditions both the N-sulphate and the O-sulphate substituents on these compounds were completely stable. However, the uronic acid residues were converted into their hydrazide derivatives at rates that depended on the uronic acid structures. Unsubstituted L-iduronic acid residues reacted much more slowly than did unsubstituted D-glucuronic acid or 2-O-sulphated L-iduronic acid residues. The chemical modification of the carboxy groups resulted in a low rate of C-5 epimerization of the uronic acid residues. The hydrazinolysis reaction also caused a partial depolymerization of heparin but not of carboxy-group-reduced heparin. Treatment of the hydrazinolysis products with HNO2 at either pH 4 or pH 1.5 or with HIO3 converted the uronic acid hydrazides back into uronic acid residues. The use of the hydrazinolysis reaction in studies of the structures of uronic acid-containing polymers and the implications of the uronic acid hydrazide formation are discussed.     

Hemicelluloses of young internodes of Arundo donax

An arabino 4-O-methyl glucurono xylan has been isolated as the dominant form of the hemicellulosic material of the youngest internodes of Arundo donax. The polysaccharide contained d-glucose, d-xylose and l-arabinose in the molar ratios of 0·1:9·2:0·66, respectively, with a uronic acid content of 4·5%. Methylation and periodate-oxidation showed that the xylan has a β-1 → 4-linked xylose backbone and a degree of polymerization ca. 63. The xylan is very similar to that found in the mature tissues.     

Structural studies of an acidic polysaccharide from the mucin secreted by Drosera capensis

The polysaccharide of the mucin secreted by the leaves of Drosera capensis is composed of l-arabinose, d-xylose, d-galactose, d-mannose, and d-glucuronic acid in the molar ratio of 3.6:1.0:4.9:8.4:8.2. For structural elucidation, methylation analysis using g.l.c. and g.l.c.-m.s. was performed on the native, the carboxyl-reduced, and the degraded polysaccharides. Partial hydrolysis, periodate oxidation, chromium trioxide oxidation, and uronic acid degradation were also performed on the native and carboxyl-reduced polysaccharides. Partial hydrolysis of the native and carboxyl-reduced polysaccharides gave various oligosaccharides that were characterized and suggest a structure containing a d-glucurono-d-mannan backbone having a repeating unit → 4)-β-d-GlcpA-(1 → 2)-α-d-Manp-(1 →. l-Arabinose and d-xylose are present as nonreducing furanosyl and pyranosyl end-groups, respectively, both attached to O-3 of d-glucuronic acid residues of the backbone. d-Galactose is present as non-reducing pyranosyl end-group linked to O-3 of d-mannose residues.     

Pentose degradation in archaea: Halorhabdus species degrade D-xylose,L-arabinose and D-ribose via bacterial-type pathways

Extremophiles - The degradation of the pentoses d-xylose, l-arabinose and d-ribose in the domain of archaea, in Haloferax volcanii and in Haloarcula and Sulfolobus species, has been shown to...     

Characterization of a α-l-rhamnosidase from Bacteroides thetaiotaomicron with high catalytic efficiency of epimedin C

In this study, a α-l-rhamnosidase gene from Bacteroides thetaiotaomicron VPI-5482 was cloned and expressed in Escherichia coli. The specific activity of rhamnosidase was 0.57 U/mg in LB medium with 0.1 mM Isopropyl β-d-Thiogalactoside (IPTG) induction at 28 °C for 8 h. The protein was purified by Ni-NTA affinity, which molecular weight approximately 83.3 kDa. The characterization of BtRha was determined. The optimal activity was at 55 °C and pH 6.5. The enzyme was stable in the pH range 5.0–8.0 for 4 h over 60%, and had a 1-h half-life at 50 °C. The Kcat and Km for p-nitrophenyl-α-l-rhamnopyranoside (pNPR) were 1743.29 s⁻¹ and 2.87 mM, respectively. The α-l-rhamnosidase exhibited high selectivity to cleave the α-1,2 and α-1,6 glycosidic bond between rhamnoside and rhamnoside, rhamnoside and glycoside, respectively, which could hydrolyze rutin, hesperidin, epimedin C and 2″-O-rhamnosyl icariside II. Under the optimal conditions, BtRha transformed epimedin C (1 g/L) to icariin by 90.5% in 4 h. This study provides the first demonstration that the α-l-rhamnosidase could hydrolyze α-1,2 glycosidic bond between rhamnoside and rhamnoside.