Methanolysis of cholesteryl esters: conditions for quantitative preparation of methyl esters.

We have investigated the conditions required to obtain a quantitative yield of methyl esters from cholesteryl esters by alkaline methanolysis. Methanolysis of the cholesteryl ester for 60 min at room temperature with 1 m methoxide reagent ensured complete reaction. Some ester hydrolysis always accompanied methanolysis and necessitated acid-catalyzed methylation of the resultant fatty acids after completion of the alcoholysis. Analysis of the composition of methyl ester product and remaining cholesteryl ester substrate before methanolysis had gone to completion showed selective hydrolysis of some fatty acid cholesteryl esters and illustrates the importance of obtaining a quantitative yield of methyl esters following methanolysis.     

Degradation of polysaccharides containing uronic acid residues

A method for the selective degradation of polysaccharides containing uronic acid residues is described. It involves methylation of hydroxyl and carboxyl groups, base-catalysed elimination, and mild hydrolysis with acid. The degraded product is etherified with trideuteriomethyl or ethyl groups and hydrolysed, and the resulting mixture of etherified sugars is analysed, as the alditol acetates, by g.l.c.—m.s. Comparison of this analysis with the methylation analysis of the original polysaccharide gives information on the nature of the sugar residues on either side of the uronic acid residue.     

Structural analysis of colanic acid from Escherichia coli by using methylation and base-catalysed fragmentation. Comparison with polysaccharides from other bacterial sources

Essentially the same methanolysis products were obtained after methylation of the slime and capsular polysaccharides from Escherichia coli K12 (S53 and S53C sub-strains) and the slime polysaccharides from E. coli K12 (S61), Aerobacter cloacae N.C.T.C. 5290 and Salmonella typhimurium SL1543. These were the methyl glycosides of 2-O-methyl-l-fucose, 2,3-di-O-methyl-l-fucose, 2,3-di-O-methyl-d-glucuronic acid methyl ester, 2,4,6-tri-O-methyl-d-glucose, 2,4,6-tri-O-methyl-d-galactose and the pyruvic acid ketal, 4,6-O-(1'-methoxycarbonylethylidene)-2,3-O-methyl-d-galactose. All were identified as crystalline derivatives from an E. coli polysaccharide. The structure of the ketal was proved by proton-magnetic-resonance and mass spectrometry, and by cleavage to pyruvic acid and 2,3-di-O-methyl-d-galactose. All these polysaccharides are therefore regarded as variants on the same fundamental structure for which the name colanic acid is adopted. Although containing the same sugar residues, quite different methanolysis products were obtained after methylation of the extracellular polysaccharide from Klebsiella aerogenes (1.2 strain). The hydroxypropyl ester of E. coli polysaccharide, when treated with base under anhydrous conditions, underwent beta-elimination at the uronate residues with release of a 4,6-O-(1'-alkoxycarbonylethylidene)-d-galactose. Together with the identification of 3-O-(d-glucopyranosyluronic acid)-d-galactose as a partial hydrolysis product, this establishes the nature of most, if not all, of the side chains as O-[4,6-O-(1'-carboxyethylidene)-d-galactopyranosyl]-(1-->4)-O-(d-glucopyranosyluronic acid)-(1-->3)-d-galactopyranosyl...     

Carbohydrate composition analysis of bacterial polysaccharides: optimized acid hydrolysis conditions for HPAEC-PAD analysis.

The capsular polysaccharide from Haemophilus influenzae type b (polyribosyl ribitol-phosphate; PRP) and the capsular polysaccharides from Streptococcus pneumoniae types 6B, 14, 18C, and 23F (Pn6B, Pn14, Pn18C, and Pn23F) were subjected to acid hydrolysis using hydrofluoric (HF) and/or trifluoroacetic acid (TFA) and high-pH anion-exchange chromatography with pulsed amperometric detection in an effort to identify optimum hydrolysis conditions for composition analysis of their carbohydrate components. With the exception of PRP, composition analyses of polysaccharides containing a phosphate moiety in the repeating unit structure (Pn6B, Pn18C, and Pn23F) are significantly improved by subjecting the sample to HF hydrolysis (65 degrees C, 1 h) followed by TFA hydrolysis (98 degrees C, 16 h). This results in essentially quantitative hydrolysis of the phosphodiester bond to the carbohydrate components, which otherwise remained predominantly phosphorylated and poorly accounted for in the analysis. Optimum analysis of PRP was achieved following a 2-h hydrolysis with TFA at 80 degrees C, whereas Pn14 showed optimum results after a 16-h hydrolysis with TFA at 98 degrees C. These analyses also provide information about the relative susceptibility to acid hydrolysis of the various glycosidic and phosphodiester bonds in these polysaccharides, with evidence to suggest that the acid lability of a given bond can be dramatically different from one polysaccharide to another.     

Analytical and structural features of the gum exudate from Combretum hartmannianum schweinf.

The gum exudate from Combretum hartmannianum is water-soluble, forms very viscous solutions, and contains galactose (22%), arabinose (43%), mannose (10%), xylose (6%), rhamnose (4%), glucuronic acid (6%), 4-O-methylglucuronic acid (2%), and galacturonic acid (7%). The acidic components produced on hydrolysis of the gum were 6-O-(β-D-glucopyranosyluronic acid)-D-galactose, and two saccharides that had the same chromatographic mobility, and contained mannose and galacturonic acid, and galactose and 4-O-methylglucuronic acid, respectively. Methylation and methanolysis of the gum indicated the presence of terminal uronic acid, rhamnose, xylose, galactose, arabinofuranose, and arabinopyranose. Controlled, acid hydrolysis indicated the presence of (1→3)-linked arabinopyranose side-chains and (1→6)-linked galactose residues. C. hartmannianum gum, when subjected to two Smith-degradations, yielded Polysaccharides I and II, both of which contained galactose, arabinose, and mannose. Insufficient crude gum was available for a complete structural study, but the molecule was shown to contain long, sparsely branched chains of (1→6)-linked galactose residues, to which are attached (1→3)-linked arabinose and (1→3)-linked mannose side-chains.     

An improved methodology for the quantification of uronic acid units in xylans and other polysaccharides

Uronic acids can be quantified either by a colorimetric determination after treatment with concentrated sulfuric acid and carbazole or by gas chromatography after methanolysis and subsequent acetylation. Both methods suffer from incomplete hydrolysis, an unavoidable degradation of the products to be analysed, and an inability to separate and quantify different types of uronic acids. In the present work, the fundamental chemistry involved in the two methods has been evaluated, and some modifications to increase their accuracy are suggested. By combining the two methods, a complete quantification of all individual types of uronic acids present in a sample can be achieved.     

Determination of saccharide content in pneumococcal polysaccharides and conjugate vaccines by GC-MSD

A simple and sensitive gas chromatographic method was designed for quantitative analysis of Streptococcus pneumoniae capsular polysaccharides, activated polysaccharides, and polysaccharide conjugates. Pneumococcal serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F polysaccharide or conjugate were subjected to methanolysis in 3N hydrochloric acid in methanol followed by re-N-acetylation and trimethylsilylation. Derivatized samples were chromatographed and detected using gas chromatography with mass selective detector. Gas chromatographic results were compared with colorimetric values with agreement of 92 to 123% over the range of all samples tested. Monosaccharides released during methanolysis included hexoses, uronic acids, 6-deoxy-hexoses, amino sugars, and alditols. Quantitative recovery of monosaccharides was achieved for all serotypes by the use of a single methanolysis, derivatization, and chromatography procedure. Response factors generated from authentic monosaccharide standards were used for quantitation of pneumococcal polysaccharides and conjugates with confirmation of peak assignments by retention time and mass spectral analysis. This method allows saccharide quantitation in multivalent pneumococcal vaccine intermediates and final drug products with low-level detection (10 pg) and peak purity.     

Studies on Sophora alopecuroides Gum

The seeds of Sophora alopecuroides L, contained 12.8 per cent of endosperm, from which the water-soluble polysaccharide gum was isolated and purified by fractionation with alcohol three times. Hydrolysis of the polysacchatides with sulphuric acid produced galactose and mannose in the molar ratio of 1:2.37, as determined by the phenolsulphuric acid method and by paper chromatographic method. The total sugar content reached 67-70%. The polysaccharides, after complete hydrolysis, reduction with NaBH4 and acetylation, ridded acetates. An analysis on them by gas-chromatography showed that they contained galactose and mannose in the molar ratio of 1:1.83, and the total sugar content reached 56%. In order to identify the linkages of the polysaccharides isolated from the seeds of Sophora alopecuroides, they were methylated by using the methods of Hokomori, Conval and Albersheim. The fully methylated polysaccharides were hydrolyzed and then reduced with NaBH4, followed by acetylation. Finally they yielded acetates. An analysis on IR data, the results of acid hydrolysis, periodate oxidation and Smith type degradation, as well as the acetates by GC, GC-MS and 1HNMR showed that the linkages of Sophora alopecuroides. gum may be, The purified polysaccharides seem to be homogenous after ultracentrifugation analysis. The sedimentation coefficient of the gum obtained from repeated purification with alcohol was 5.31, its intrinsic viscosity was 6.25 dl/g and its molecular weight was estimated to be 635000. By the light-scattering method, however, its molecular weight was estimated to be 715000. The crude polysacchafides contained 44 per cent of the water-soluble polysaccharides.     

Comparison of Cell Wall Polysaccharide Hydrolysis by a Dilute Acid/Enzymatic Saccharification Process and Rumen Microorganisms

Evaluation of biomass crops for breeding or pricing purposes requires an assay that predicts performance in the bioenergy conversion process. Cell wall polysaccharide hydrolysis was compared for a dilute sulfuric acid pretreatment at 121°C followed with cellulase hydrolysis for 72?h conversion assay (CONV) with in vitro rumen microflora incubation for 72?h (RUMEN) for a set of maize (Zea mays L.) stover samples with a wide range in cell wall composition. Residual polysaccharides from the assays were analyzed for sugar components and extent of hydrolysis calculated. Cell wall polysaccharide hydrolysis was different for all sugar components between the CONV and RUMEN assays. The CONV assay hydrolyzed xylose-, arabinose-, galactose-, and uronic acid-containing polysaccharides to a greater degree than did the RUMEN assay, whereas the RUMEN assay was more effective at hydrolyzing glucose- and mannose-containing polysaccharides. Greater hydrolysis of hemicelluloses and pectins by CONV can be attributed to the acid hydrolysis mechanism of the CONV assay for noncellulosic polysaccharides, whereas the RUMEN assay was dependent on enzymatic hydrolysis. While CONV and RUMEN hydrolysis were correlated for most polysaccharide components, the greatest correlation was only r?=?0.70 for glucose-containing polysaccharides. Linear correlations and multiple regressions indicated that polysaccharide hydrolysis by the RUMEN assay was negatively associated with lignin concentration and ferulate ether cross linking as expected. Corresponding correlations and regressions for CONV were less consistent and occasionally positive. Use of rumen microbial hydrolysis to characterize biomass performance in a conversion process may have some limited usefulness for genetic evaluations, but such assays would be unreliable for biomass pricing.     

Modification of polysaccharides containing uronic acid residues

Klebsiella Type 47 capsular polysaccharide has side chains attached to the main chain viaD-glucuronic acid residues. The side chains have been removed to yield an essentially linear polysaccharide by the following sequence of reactions: (1) substitution of hydroxyl and car?yl groups with methyl vinyl ether; (2) β-elimination by treatment with base; (3) removal of modified uronic acid residues and protecting groups by mild acid hydrolysis. The possibility of modifying other uronic acid-containing polysaccharides by this method is discussed.     

A microapparatus for liquid hydrogen fluoride solvolysis: Sugar and amino sugar composition of Erysiphe graminis and Triticum aestivum cell walls

The assembly and use of a simple and safe apparatus for HF solvolysis of microgram amounts of cell walls, polysaccharides, or glycoproteins are described. Using this apparatus the cell wall composition of Erysiphe graminis was compared with that of its wheat host. The HF solvolysis combined with TFA posthydrolysis considerably increased sugar yields compared with TFA hydrolysis alone, due mainly to increased yields of glucose from wheat, and glucosamine from Erysiphe, corresponding to cellulose and chitin, respectively. A potentially useful method for determining amounts of fungal hyphae in plant tissue is also provided.     

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.     

Localization of mucilaginous polysaccharides in mulberry leaves

Mulberry tree leaves were shown to have mucilaginous polysaccharides. The extracted water-soluble mucilage was separated into three fractions via a cetylpyridinum chloride complex and purified by anion-exchange chromatography. Five acidic polysaccharides were separated from these fractions, one of which was a major polysaccharide (Mp-3) that was structurally analyzed and used for antibody preparation. The Mp-3 polysaccharide contained rhamnose, galactose, glucose, galacturonic acid, and glucuronic acid in a molar ratio of 1 : 0.2 : 0.5 : 2.3 : 1.5 as constituent monosaccharides. Methylation and gas chromatography-mass spectrometry analysis indicated that the polysaccharide was a rhamnogalacturonan mainly consisting of 1,2,3-linked rhamnose residues, 1,3,4- and 1,4-linked uronic acid residues, and terminal uronic acid residues. Its molecular weight was estimated to be 5.5 x 10(5). Immunohistological observation revealed that the Mp-3 polysaccharide is specifically localized in inner epidermal cells situated in adaxial leaves, and electron microscopy showed that its subcellular location is between the plasma membrane and the cell wall. In young leaves, numerous secretory vesicles were present in a shrunken cytoplasm that was surrounded by fibers. In mature leaves, more than 20% of total epidermal cells were these inner cells in which polysaccharide deposition was significantly increased. The deposits appeared as a rounded electron-dense mass throughout the inner cells by electron microscopy.     

Structural studies of a heteroxylan from Plantago major L. seeds by partial hydrolysis, HPAEC-PAD, methylation and GC-MS, ESMS and ESMS/MS.

The seed mucilage from Plantago major L. contains acidic heteroxylan polysaccharides. For further structural analysis, oligosaccharides were generated by partial acid hydrolysis and then isolated by high-pH anion-exchange chromatography (HPAEC). Each HPAEC fraction was shown by ESMS to contain one major oligosaccharide and several minor components. Partial structures of the oligosaccharides were determined using GC-MS, ESMS and ES tandem mass spectrometry (ESMS/MS). A (1-->4)-linked xylan trisaccharide and (1-->3)-linked xylan oligosaccharides with DP 6-11 suggested that the backbone of the heteroxylan polysaccharide consisted of blocks of (1-->4)-linked and (1-->3)-linked Xylp residues. A (1-->2)-linked Xylp disaccharide and a branched tetrasaccharide were also found, revealing that single Xylp residues are linked to the O-2 of some of the (1-->4)-linked Xylp residues in the backbone. In addition, our results confirm the presence of side chains consisting of the disaccharide GlcpA-(1-->3)-Araf.     

Hydrolysis of wheat straw hemicellulose with trifluoroacetic acid. Fermentation of xylose with Pachysolen tannophilus

Treatment of wheat straw with 1N trifluoroacetic acid (TFA) for 7 h at reflux temperature yielded 23% xylose based upon initial straw weight. This corresponds to about an 80% xylose yield based on the xylan content of the hemicellulose. The cellulose component of wheat straw was largely unaffected, as evidenced by low glucose yields. Decomposition of xylose by prolonged refluxing (23 h) was minimal in 1N TFA compared to 1N HCl. Treatment of wheat straw with refluxing 1N TFA converts about 10% of the lignin initially present in straw into water-soluble lignin fragments. Fermentation of the xylose-rich wheat straw hydrolyzate to ethanol with Pachysolen tannophilus was comparable to the fermentation of reagent grade xylose, indicating that furfural and toxic lignin by-products were not produced by 1N TFA in sufficient amounts to impair cell growth and ethanol production. Cellulase treatment of the wheat straw residue after TFA hydrolysis resulted in a 70-75% conversion of the cellulose into glucose.     

Isolation of two different polysaccharides from halophilic Zoogloea sp.

Summary An extracellular polysaccharide producing bacterium Zoogloea sp. was isolated from marine environments. This strain could produce two different polysaccharides. One (water-soluble polysaccharide : WSP) was from cell-free liquid medium, the other (cell-bound polysaccharide : CBP) was obtained from cell surface. Both polysaccharides contained glucose, galactose and mannose as sugar components, but their molar ratios were different (WSP : 2:2:3, CBP : 1:2:2) and half of the sugar components existed as uronic acid form. Both polysaccharide productions started at the early stage of the logarithmic growth phase. The amount of WSP and CBP was influenced by culture conditions such as additional carbon and nitrogen sources. Isolated Zoogloea sp. showed a high product yield without the increase of cell mass.     

蒸汽爆破对油菜秸秆降解产物的影响

采用DNS法、离子色谱法和气质联用(GC-MS)技术对油菜秸秆蒸汽爆破降解产物进行了分析。结果表明,经蒸汽爆破处理后的油菜秸秆酶解产糖量有明显的提高,为未处理样品直接酶解含量的3.4倍,各种水溶性糖化合物的含量也大大提高,其中葡萄糖和木糖含量分别为未处理样品直接酶解含量的3.5倍和4.7倍之多。水提液乙酸乙酯萃取物中分别检测到脂肪酸类、芳香类和呋喃类物质40、12和1种。通过扫描电镜观察处理后油菜秸秆的表面形貌结构,发现处理后的秸秆表面结构松散,比表面积增大,酶对纤维素的可及性增加。     

The combined enzymatic hydrolysis and fermentation of hemicellulose to 2,3-butanediol

Summary Hemicellulose-rich fractions from several agricultural residues were converted to 2,3-butanediol by a combined enzymatic hydrolysis and fermentation process. Culture filtrates from Trichoderma harzianum E58 were used to hydrolyze the substrates while Klebsiella pneumoniae fermented the liberated sugars to 2,3-butanediol. Approximately 50–60% of a 5% (w/v) xylan preparation could be hydrolyzed and quantitatively converted to 2,3-butanediol using this procedure. Although enzymatic hydrolysis was optimal at pH 5.0 and 50° C, the combined hydrolysis and fermentation was most efficient at pH 6.5 and 30° C. Combined hydrolysis and fermentation resulted in butanediol levels that were 20–40% higher than could be obtained with a separate hydrolysis and fermentation process. The hemicellulose-rich water-soluble fractions obtained from a variety of steam-exploded agricultural residues could be readily used by the combined hydrolysis and fermentation approach resulting in butanediol yields of 0.4–0.5 g/g of reducing sugar utilized.     

Isolation and chemical characterization of algal polysaccharides from the green seaweed Ulva clathrata (Roth) C. Agardh

In order to obtain information on the content and composition of the water-soluble polysaccharides from Ulva clathrata, an extraction at 60°C, in different media, was performed: water, EDTA and HCl (F-I), each followed by a sequential extraction in NaOH 0.1 M (F-II). The extracts were recovered and analyzed for total carbohydrates, proteins, rhamnose, uronic acids and sulfate content. Differences were obtained in the yield and composition in both fractions of the different media (F-I and F-II). Higher yields resulted in the first fraction on all media. HCl extraction was the best in both fractions (14.83 ± 1.5% and 5.96 ± 1.1%, F-I and F-II, respectively). In all cases, F-I was more sulfated ranging from 27.87% to 35.8% and higher in rhamnose content, whereas F-II had higher protein and slightly higher uronic acid content. FTIR spectra showed that soluble polysaccharides from the green seaweed U. clathrata are sulfated polysaccharides, similar to ulvan obtained from other Ulva species and confirmed by the ¹ H-NMR spectrum, where the characteristic signal for the deoxy sugar (rhamnose) is present.     

Diagnostic methods for the determination of iduronic acid in oligosaccharides

A high-performance liquid chromatography (HPLC) method with pulsed amperometric detection (PAD) was used for the determination of the acid hydrolysis products of L-iduronic acid containing oligosaccharides isolated from biological sources. This HPLC-PAD method was compared with gas chromatographic (GLC) methods. Since acid hydrolysis of oligosaccharides can produce a number of products, several uronic acid derivatives were prepared by chemical synthesis. These well characterized standards in conjunction with mass spectrometry allowed for the identification of most of the products of methanolysis or hydrolysis of glycosamino-glycans, which included chondroitin sulfates A and B (dermatan sulfate), heparin, and hyaluronic acid. (4 M) HCl in methanol 100 degrees C for 24 h was found to be optimum for GLC and 1 M aqueous HCl for 4 h at 100 degrees C for HPLC-PAD. All of the monosaccharides, hexosamines, and uronic acids could be separately identified in a single chromatographic step using either technique. Good resolution, high sensitivity (low microgram samples) and rapid analysis makes these methods particularly useful for the determination of small amounts of glycosaminoglycans and other glycoconjugates found in samples isolated from biological sources. These two techniques are specifically designed to allow the qualitative determination of the carbohydrate content and composition of samples whose carbohydrate composition and content is completely unknown.