Polyol conversion specificity of Bacillus pallidus

The conversion specificity of Bacillus pallidus Y25 for polyols, including elusive rare sugar alcohols, was investigated. B. pallidus cells showed transformation potential for several rare polyols, including allitol, L-mannitol, D/L-talitol, and D-iditol, and converted them to their corresponding ketoses. This indicates that the bacterium had two polyol dehydrogenases specific for polyols that have D-erythro and D-threo configurations. By combination with intrinsic isomerases, polyols were converted directly to various aldoses, including L-xylose, L-talose, D-altrose, and L-glucose.     

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of oligosaccharides and oligosaccharide alditols obtained by hydrolysis of agaroses and carrageenans, two important types of red seaweed polysaccharides

MALDI-TOF mass spectrometry analyses of several oligosaccharides (aldoses) and oligosaccharide alditols derived from agaroses, kappa- and iota-carrageenans using different matrices (2,5-dihydroxybenzoic acid, nor-harmane, ferulic acid, and the ionic liquid matrices 2,5-dihydroxybenzoic acid-n-butylamine and ferulic acid-n-butylamine) were conducted. These carbohydrates were selected as model compounds to study the MALDI prompt and post-source decay (PSD) fragmentation processes of both families of oligosaccharides. Sulfated alditols showed in the negative-ion mode the molecular ion as [M−Na]⁻ together with the species yielded by their prompt fragmentation (mainly desulfation) while the sulfated oligosaccharides (aldoses) showed mainly glycosidic prompt fragmentation (glycosidic C-cleavages and desulfation). Non-sulfated aldoses and alditols, which could only be analyzed in positive-ion mode ([M+Na]⁺), did not suffer any prompt fragmentation. The former yielded cross-ring fragmentation in the PSD mode. Best results were obtained by using 2,5-dihydroxybenzoic acid and/or nor-harmane as matrices for all the compounds studied.     

O-methyl oximes of sugars. Analysis as O-trimethylsilyl derivatives by gas-liquid chromatography and mass spectrometry

The mass spectra of aldoses, partially methylated aldoses, deoxyaldoses, and ketoses containing 3–7 carbons, were recorded on the ethers of the trimethylsilyl O-methyl oxime derivatives. Each compound gave a distinctive spectrum indicating the carbon-chain length and the location of substituents. The gas-liquid chromatographic properties of most compounds in this study were also examined.     

Production of D-arabitol by Metschnikowia reukaufii AJ14787

A potent producer of D-arabitol was isolated by screening of natural sources and identified as Metschnikowia reukaufii AJ14787. Resting cells of this strain can efficiently produce D-arabitol from D-glucose with a weight yield of more than 60%, and can also produce D-arabitol from several other types of sugars such as polyols, ketoses, and aldoses. To improve productivity, various culture conditions such as temperature and the concentrations of D-glucose and nitrogen sources were examined. Under optimal conditions, 206 g/l of D-arabitol was produced from D-glucose with a weight yield of 52% in 100 hours.     

Carbon-13-enriched carbohydrates. Preparation of erythrose,threose, glyceraldehyde,and glycolaldehyde with 13C-enrichment in various carbon atoms

Two-, three-, and four-carbon aldononitriles were prepared, and catalytically reduced with palladium-barium sulfate (5%) to the coresponding aldoses in high yields at pH 1.7 ± 0.1 and atmospheric pressure. Carbon-13-enriched glycolaldehyde, glyceraldehyde, erythrose, and threose were prepared with enrichment in various carbon atoms, permitting unequivocal assignment of chemical shifts for all carbons and determination of the proportions of cyclic hemiacetals and linear gem-diol forms in solution. Carbon-carbon and carbon-hydrogen coupling-constants for the furanose ring and linear hydrates of these short-chain aldoses are reported and discussed.     

Resolution of the enantiomers of aldoses by liquid chromatography of diastereoisomeric 1-(N-acetyl-α-methylbenzylamino)-1-deoxyalditol acetates

Acyclic diastereoisomers, namely, 1-(N-acetyl-α-methylbenzylamino)-1-deoxyalditol acetates are readily obtained by reductive amination of aldoses with chiral α-methylbenzylamine (MBA) in the presence of sodium cyanoborohydride, and may be separated by reversed-phase 1.c. and, more effectively, by adsorption 1.c. According to this procedure, enantiomers of the common, neutral aldoses are resolved. In adsorption 1.c., l-l_* [defined as an adduct of an l-aldose and l-(-)-MBA] is eluted before d-l_* for erythrose, xylose, ribose, glucose, 4-O-methylglucose, galactose, and fucose, and the elution order is the reverse for arabinose, lyxose, mannose, rhamnose, and glyceraldehyde. This behavior is probably related to the configuration of C-2 of the aldoses.     

A gas chromatographic method for the determination of aldose and uronic Acid constituents of plant cell wall polysaccharides

A major problem in determining the composition of plant cell wall polysaccharides has been the lack of a suitable method for accurately determining the amounts of galacturonic and glucuronic acids in such polymers. A gas chromatographic method for aldose analysis has been extended to include uronic acids. Cell wall polysaccharides are depolymerized by acid hydrolysis followed by treatment with a mixture of fungal polysaccharide-degrading enzymes. The aldoses and uronic acids released by this treatment are then reduced with NaBH₄ to alditols and aldonic acids, respectively. The aldonic acids are separated from the alditols with Dowex-1 (acetate form) ion exchange resin, which binds the aldonic acids. The alditols, which do not bind, are washed from the resin and then acetylated with acetic anhydride to form the alditol acetate derivatives. The aldonic acids are eluted from the resin with HCl. After the resin has been removed, the HCl solution of the aldonic acids is evaporated to dryness, converting the aldonic acids to aldonolactones. The aldonolactones are reduced with NaBH₄ to the corresponding alditols, dried and acetylated. The resulting alditol acetate mixtures produced from the aldoses and those from the uronic acids are analyzed separately by gas chromatography. This technique has been used to determine the changes in composition of Red Kidney bean (Phaseolus vulgaris) hypocotyl cell walls during growth, and to compare the cell wall polysaccharide compositions of several parts of bean plants. Galacturonic acid is found to be a major component of all the cell wall polysaccharides examined.     

Influence of Polyols on the Stability and Kinetic Parameters of Invertase from Candida utilis: Correlation with the Conformational Stability and Activity

Invertase (β-d-fructofuranoside fructohydrolase-E.C. 3.2.1.26) is a sucrose hydrolyzing enzyme found in microbial, plant and animal sources. Invertase from Candida utilis is a dimeric glycoprotein composed of two identical monomer subunits with an apparent molecular mass of 150 kDa. We investigated the mechanism of stabilization of invertase with polyols (glycerol, xylitol, and sorbitol). Activity, thermodynamic and kinetic measurements of invertase were performed as a function of polyol concentration and showed that polyols act as very effective stabilizing agents. The result from the solvent-invertase interaction shows preferential exclusion of the polyols from the protein domain leading to preferential hydration of protein. Apparent thermal denaturation temperature of the protein (T _m ) rose from 75 °C to a maximum of 85 °C in 30% glycerol. The stabilization has been attributed to the preferential hydration of the enzyme.     

Identification of ketoses by use of their peracetylated oxime derivatives: A G.L.C.-M.S. approach

A method based on peracetylated oxime (PAKO) derivatives has been developed for rapid g.l.c.-m.s. survey of ketoses. This derivatization procedure (and the chromatographic analysis of these derivatives) is identical to one previously employed to identify aldoses by means of peracetylated aldononitrile (PAAN) derivatives. The production of chemically different derivatives from the aldoses and ketoses by the same derivatization procedure greatly simplifies the chromatographic separation of the derivatives of the ketoses from those of the aldoses, and also results in distinctively different, mass-spectral fragmentation-pathways for the two sets of derivatives. Both the electron-impact (e.i.) and ammonia chemical-ionization (c.i.) mass spectra of PAKO derivatives have been examined. Extensive differences between the fragmentation-pathways of the PAAN and the PAKO derivatives have been observed both by e.i.m.s. and ammonia c.i.m.s. The g.l.c.-m.s. of these PAKO derivatives, in conjunction with various, isotopic variants of the derivatization process, can yield extensive structural information with regard to the starting saccharides associated with the known, or unknown, g.l.c. peaks. The g.l.c. and mass-spectral properties of highly O-methylated PAKO derivatives of d-fructose are compared, and contrasted, to those of the PAKO derivatives of non-O-methylated saccharides. The chromatographic properties of derivatives of oligosaccharides that result from the PAAN-PAKO derivatization procedure have also been studied.     

Glass capillary or fused-silica gas chromatography—mass spectrometry of several monosaccharides and related sugars: Improved resolution

The gas chromatographic separation of several monosaccharides and related sugars derivatized by methoxylation and trimethylsilylation reactions was optimized with glass capillary (SP-2250) and fused silica (SP-2100) columns. Individual sugars included aldoses, ketoses, polyols, acidic forms and N-acetylated amino sugars. Peaks were detected by selected ion monitoring (SIM). The fused silica column gave complete resolution of all peaks (two per hexose and one per hexitol) arising from glucose, galactose, mannose, fructose, sorbitol, mannitol and dulcitol. The resolution of these sugars with the glass capillary column was not as good, but full differentiation was possible on the basis of SIM. Because the fused silica column gave a better resolution of 33 sugars tested and was more easily installed than the glass capillary column, it was utilized for quantitative analysis. A deuterated algal sugar mixture used for quantitation by isotope dilution was found to contain glucose, galactose, mannose, xylose, arabinose, ribose and rhamnose. Full recoveries were obtained of various amounts of glucose, galactose, mannose, fructose and xylose added to human serum.     

Automated hypoiodite oxidation of carbohydrates

An automated system is described for the hypoiodite oxidation of aldoses and substituted aldoses to the corresponding aldonic acids. Automated determination of the glyoxylic acid and formaldehyde obtained on oxidation with periodate enables the 3-O-, 4-O-, and 6-O-substituted aldonic acids to be distinguished. The method is applied to the analysis of oligosaccharides in column eluates.     

In silico studies on the substrate specificity of an l-arabinose isomerase from Bacillus licheniformis

l-Arabinose isomerase (BLAI) from Bacillus licheniformis was found to be active only with l-arabinose, unlike other l-arabinose isomerases (l-AIs) active with a variety of aldoses. Therefore, the differences in molecular interactions and substrate orientation in the active site of l-AIs have been examined and the residue at position 346 is proposed to be responsible for the unique substrate specificity of BLAI.     

High-resolution preparative gel electrophoresis: separation and recovery of functional messenger RNA species

Certain open-chain polyols were shown to interfere with the determination of phosphorus of the Lowry-Lopez method by forming a complex with Mo₇O₂₄^6?. The ability to interfere with the assay increased with increasing chain length of the polyols: Ethylene glycol and glycerol did not react at all; i-erythritol reacted to a small extent, but hexitols and perseitol formed stronger complexes. Depending on the polyol, interference occurred even at 0.2 mm (hexitols) or 2 mm (xylitol) concentrations. At these concentrations the polyols interfered only to a small extent with the phosphorus assays based on the use of Triton X-100 and molybdate. The complex formation was exploited in the development of a colorimetric polyol assay.     

Chromatography of sugars in body fluids : III. Stepwise detection of sugars with aniline citrate on paper and thin-layer chromatograms

The paper chromatographic detection method of White and Hess for urinary aldoses using aniline citrate has been studied and modified. As the reactivity of individual sugars is determined mainly by their molecular weight and structure, our modification is based on a gradual increase in the reaction temperature (room temperature, 37 or 55 and 105°) and variations in heating time. This technique enables reaction conditions to be obtained that lead to a full display of both the transient and final characteristics of the fluorescence and colour of the individual spots in the sugar spectrum of any analyzed material.Compared with other reagents based on aniline or other aromatic amines, aniline citrate possesses unusually wide operational flexibility. The procedure is suitable for paper and thin-layer chromatography and is especially valuable in the analysis of urinary and tissue sugar extracts for the identification of largely overlapping spots with different reaction times, the differentiation of oligosaccharides, particularly those with different linkages, and obtaining additional data for the identification of unknown metabolites.     

Simultaneous measurement of urinary polyols using gas chromatography/mass spectrometry

In the present study, we simultaneously measured several polyols, such as adonitol, arabitol, dulcitol, glucose, myo-inositol, mannitol, sorbitol, and xylitol, in urine by gas chromatography/mass spectrometry-positive chemical ionization. We also examined possible relationship between the levels of these metabolites and age in normal individuals. In order to proceed to its quantification by GC/MS, 200 microL of a urine sample were diluted with 3 ml of distilled water, lyophilized, acetylated, and then analyzed them. Using this method, we were able to quantify as little as 0.5-1.0 ng/microL, and we made the calibration curves to be linear from 0.25 to 250 ng/microL (r(2)>0.991). Analytical recoveries were over 89.4%, and the inter-day and intra-day variability for accuracy and reproducibility was less than 20%. In the normal urine sample, the levels of polyols were gender-differentiated and age-related. This simple GC/MS method is sensitive and allows the measurement of wide ranges of polyols using small amounts of urine. We conclude that the quantitation of urinary polyols using GC/MS appears to be a clinically useful method for assessing polyol-pathway activity.     

Osmoregulatory Responses of Fungi Inhabiting Standing Litter of the Freshwater Emergent Macrophyte Juncus effusus

Standing litter of emergent macrophytes often forms a major portion of the detrital mass in wetland habitats. Microbial assemblages inhabiting this detritus must adapt physiologically to daily fluctuations in temperature and water availability. We examined the effects of various environmental conditions on the concentrations of osmoregulatory solutes (polyols and trehalose) and the respiratory activities of fungal assemblages inhabiting standing litter of the freshwater emergent macrophyte Juncus effusus. Under field conditions, the concentrations of osmolytes (polyols plus trehalose) in fungal decomposers were negatively correlated with plant litter water potentials (r = −0.75, P < 0.001) and rates of microbial respiration (r = −0.66, P < 0.001). The highest concentration of osmolytes (polyols plus trehalose) occurred in standing litter exposed to desiccating conditions (range from wet to dry, 0.06 to 0.68 μmol · mg of fungal biomass⁻¹). Similar fluctuations in polyol and trehalose concentrations were observed in standing litter wetted and dried under laboratory conditions and for four predominant fungal decomposers of J. effusus grown individually on sterilized Juncus leaves. These studies suggest that fungal inhabitants associated with standing litter of emergent macrophytes can adjust their intracellular solute concentrations in response to daily fluctuations in water availability.     

Construction and Screening of Metagenomic Libraries Derived from Enrichment Cultures: Generation of a Gene Bank for Genes Conferring Alcohol Oxidoreductase Activity on Escherichia coli

Enrichment of microorganisms with special traits and the construction of metagenomic libraries by direct cloning of environmental DNA have great potential for identifying genes and gene products for biotechnological purposes. We have combined these techniques to isolate novel genes conferring oxidation of short-chain (C₂ to C₄) polyols or reduction of the corresponding carbonyls. In order to favor the growth of microorganisms containing the targeted genes, samples collected from four different environments were incubated in the presence of glycerol and 1,2-propanediol. Subsequently, the DNA was extracted from the four samples and used to construct complex plasmid libraries. Approximately 100,000 Escherichia coli strains of each library per test substrate were screened for the production of carbonyls from polyols on indicator agar. Twenty-four positive E. coli clones were obtained during the initial screen. Sixteen of them contained a plasmid (pAK101 to pAK116) which conferred a stable carbonyl-forming phenotype. Eight of the positive clones exhibited NAD(H)-dependent alcohol oxidoreductase activity with polyols or carbonyls as the substrates in crude extracts. Sequencing revealed that the inserts of pAK101 to pAK116 encoded 36 complete and 17 incomplete presumptive protein-encoding genes. Fifty of these genes showed similarity to sequenced genes from a broad collection of different microorganisms. The genes responsible for the carbonyl formation of E. coli were identified for nine of the plasmids (pAK101, pAK102, pAK105, pAK107 to pAK110, pAK115, and pAK116). Analyses of the amino acid sequences deduced from these genes revealed that three (orf12, orf14, and orf22) encoded novel alcohol dehydrogenases of different types, four (orf5, sucB, fdhD, and yabF) encoded novel putative oxidoreductases belonging to groups distinct from alcohol dehydrogenases, one (glpK) encoded a putative glycerol kinase, and one (orf1) encoded a protein which showed no similarity to any other known gene product.     

The mass spectra of some per-O-acetylaldononitriles

The mass spectra of some per-O-acetylaldono- and per-O-acetyldeoxyaldononitriles have been recorded. The major fragmentation-pathways are discussed in terms of the application of electron-impact mass-spectrometry to structural studies of aldoses. Both acetyl and deuterium-labelled acetyl derivatives are included. The spectra are useful in verifying the position of the deoxy group(s) of deoxyaldoses.     

Carbon-13-enriched carbohydrates. Preparation of aldononitriles and their reduction with a palladium catalyst

Cyanide condenses with aldoses at 25° in aqueous solution between pH 7.0–9.0 to produce aldononitriles in high yield. These nitriles may be reduced catalytically over palladium-barium sulfate (5%) at pH 4.2 ± 0.1 and 25° to yield the corresponding aldoses in 60–90% yield, depending on the structure of the nitrile. 1-Amino-1-deoxyalditols are produced in approximately 10% yield, and their formation is favored when hemiacetal formation is hindered in the parent aldose. Generally, the product epimeric aldoses can be separated from contaminating by-products and from each other by ion-exchange and adsorption chromatography. This procedure has been applied to the preparation of [1-¹³C]-enriched pentoses and hexoses.     

Analysis of the monosaccharide compositions of total non-dialyzable urinary glycoconjugates by the dithioacetal method

The component aldoses, uronic acid, and hexosamines in the total non-dialyzable urinary glycoconjugates were determined by the dithioacetal method, and normal levels of these monosaccharides are presented. The molar ratios of fucose/galactose, glucuronic acid/galactose, and galactosamine/glucosamine for cancer patients were lower, and that of mannose/galactose was higher, than normal values.