Human umbilical cord hyaluronate neutral sugar content and carbohydrate-protein linkage studies

Paper chromatography of neural sugars and gas chromatography of their aldononitrile acetates indicated the presence of fucose, arabinose and a small amount of glucose in purified human umbilical cord hyaluronate. The molar ratios of serine, threonine and aspartic acid to neural sugars were not unity, suggesting the non-involvement of the neutral sugars and the amino acids in a carbohydrate-protein linkage. The same was indicated by an increase in the percentage of the aforementioned amino acids and by the absence of sugar alditols in umbilical cord hyaluronate reduced with NaBH₄-PdCl₂, after alkali treatment. This reduction caused a decrease in the intrinsic viscosity and molecular weight to about one-half and an appreciable decrease in the specific rotation of hyaluronate, suggesting a separation of the two antiparallel chains of the double helical hyaluronate. The umbilical cord hyaluronate contained bound silicon and it is possible that this bound silicon may cross-link the two chains at interspersed intervals through the uronic acid moiety and/or through neutral sugars.     

Human umbilical cord hyaluronate. Neutral sugar content and carbohydrate-protein linkage studies.

Paper chromatography of neutral sugars and gas chromatography of their aldononitrile acetates indicated the presence of fucose, arabinose and a small amount of glucose in purified human umbilical cord hyaluronate. The molar ratios of serine, threonine and aspartic acid to neutral sugars were not unity, suggesting the non-involvement of the neutral sugars and the amino acids in a carbohydrate-protein linkage. The same was indicated by an increase in the percentage of the aforementioned amino acids and by the absence of sugar alditols in umbilical cord hyaluronate reduced eith NaBH4 -PdCl2, after alkali treatment. This reduction caused a decrease in the intrinsic viscosity and molecular wieght to about one-half and an appreciable decrease in the specific rota tion of hyaluronate, suggesting a separation of the two antiparallel chains o the double helical hyaluronate. The umbilical cord hyluronate containe contained bound silicon and it is possible that this bound silicon may cross-link the two chains at interspersed intervals through the uronic acid moiety and/or through neutral sugars.     

Engineering of Saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of L-arabinose

For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as l-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of l-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of l-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the l-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h(-1) g [dry weight](-1)) and ethanol production (0.29 g h(-1) g [dry weight](-1)) and a high ethanol yield (0.43 g g(-1)) during anaerobic growth on l-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.     

The rapid,quantitative determination of neutral sugars (as aldononitrile acetates) and amino sugars (as O-methyloxime acetates) in glycoproteins by gas-liquid chromatography

O-Methyloximes have been prepared from 2-amino-2-deoxy-d-glucose, -d-mannose, and -d-galactose. The acetates of these derivatives yield stable compounds which are readily separated quantitatively by gas-liquid chromatography on a number of polar phases. The above compounds along with the aldononitrile acetates of neutral sugars can be easily separated from one another on a single column in one chromatographic run. The procedures developed were tested on a number of glycoproteins of known composition as reported by other workers utilizing more classical methodologies, resulting in excellent agreement in terms of sugar composition. An improved method is also described for converting neutral sugars to oximes which can be either converted to the trimethylsilyl derivatives or, upon acetylation, derivatized to aldononitrile acetates.     

Hierarchy in Pentose Sugar Metabolism in Clostridium acetobutylicum

Bacterial metabolism of polysaccharides from plant detritus into acids and solvents is an essential component of the terrestrial carbon cycle. Understanding the underlying metabolic pathways can also contribute to improved production of biofuels. Using a metabolomics approach involving liquid chromatography-mass spectrometry, we investigated the metabolism of mixtures of the cellulosic hexose sugar (glucose) and hemicellulosic pentose sugars (xylose and arabinose) in the anaerobic soil bacterium Clostridium acetobutylicum. Simultaneous feeding of stable isotope-labeled glucose and unlabeled xylose or arabinose revealed that, as expected, glucose was preferentially used as the carbon source. Assimilated pentose sugars accumulated in pentose phosphate pathway (PPP) intermediates with minimal flux into glycolysis. Simultaneous feeding of xylose and arabinose revealed an unexpected hierarchy among the pentose sugars, with arabinose utilized preferentially over xylose. The phosphoketolase pathway (PKP) provides an alternative route of pentose catabolism in C. acetobutylicum that directly converts xylulose-5-phosphate into acetyl-phosphate and glyceraldehyde-3-phosphate, bypassing most of the PPP. When feeding the mixture of pentose sugars, the labeling patterns of lower glycolytic intermediates indicated more flux through the PKP than through the PPP and upper glycolysis, and this was confirmed by quantitative flux modeling. Consistent with direct acetyl-phosphate production from the PKP, growth on the pentose mixture resulted in enhanced acetate excretion. Taken collectively, these findings reveal two hierarchies in clostridial pentose metabolism: xylose is subordinate to arabinose, and the PPP is used less than the PKP.     

Metabolite labelling reveals hierarchies in Clostridium acetobutylicum that selectively channel carbons from sugar mixtures towards biofuel precursors

Clostridial fermentation of cellulose and hemicellulose relies on the cellular physiology controlling the metabolism of the cellulosic hexose sugar (glucose) with respect to the hemicellulosic pentose sugars (xylose and arabinose) and the hemicellulosic hexose sugars (galactose and mannose). Here, liquid chromatography–mass spectrometry and stable isotope tracers in Clostridium acetobutylicum were applied to investigate the metabolic hierarchy of glucose relative to the different hemicellulosic sugars towards two important biofuel precursors, acetyl‐coenzyme A and butyryl‐coenzyme A. The findings revealed constitutive metabolic hierarchies in C. acetobutylicum that facilitate (i) selective investment of hemicellulosic pentoses towards ribonucleotide biosynthesis without substantial investment into biofuel production and (ii) selective contribution of hemicellulosic hexoses through the glycolytic pathway towards biofuel precursors. Long‐term isotopic enrichment demonstrated incorporation of both pentose sugars into pentose‐phosphates and ribonucleotides in the presence of glucose. Kinetic labelling data, however, showed that xylose was not routed towards the biofuel precursors but there was minor contribution from arabinose. Glucose hierarchy over the hemicellulosic hexoses was substrate‐dependent. Kinetic labelling of hexose‐phosphates and triose‐phosphates indicated that mannose was assimilated but not galactose. Labelling of both biofuel precursors confirmed this metabolic preference. These results highlight important metabolic considerations in the accounting of clostridial mixed‐sugar utilization.     

Studies on lectins. XXXVII. Isolation and characterization of the lectin from Jimson-weed seeds (Datura stramonium L.)

The lectin of Jimson-weed seeds (Datura stramonium L.) was isolated by affinity chromatography on a polysaccharide mixture from mycelium of Aspergillus niger. The lectin yields two bands on disc electrophoresis, it has sedimentation coefficient s20,w = 3.8 S and its apparent molecular weight estimated by thin layer gel chromatography is 120,000. The lectin reduced with mercaptoethanol yields on polyacrylamide gel electrophoresis in the presence of dodecyl sulfate three zones corresponding to subunits of molecular weight 72,000, 45,000 and 25,000. The lectin contains large amounts of cystine, glycine, 6.3% of hydroxyproline residues, 4.5% glucosamine and 28% of neutral sugar, predominantly arabinose. The lectin is nonspecific in human erythrocyte ABO system, it is not inhibited by simple sugars but is inhibited by a partial hydrolysate of chitin-containing mixture of polysaccharides from Aspergillus niger.     

Composition of Fatty Acids and Carbohydrates in Leptospira

The fatty acid and monosaccharide composition of four pathogenic and two saprophytic strains of Leptospira was analyzed by gas chromatography (GC) and GC-mass spectrometry. Among the fatty acids, palmitic acid was most abundant and constituted 30 to 50% of the total fatty acids. Even-numbered unsaturated acids including octadecenoic, hexadecenoic, octadecadienoic, and tetradecadienoic acids comprised 40 to 60% of the total fatty acids. Tetradecanoic acid was about 5% in saprophytic strains, but 1% or less in pathogenic strains. The amount of chloroform-methanol extract of L. biflexa strain Ancona was 14 to 20% of the dry weight of the cell. Tetradecadienoic acid was found in the chloroform-methanol insoluble fraction, suggesting the presence of the acid in a bound form. GC analysis of monosaccharides revealed the existence of arabinose, xylose, rhamnose, mannose, galactose, glucose, glucosamine, and muramic acid in the cells. Among the neutral sugars, glucose was a minor component and was especially low in pathogenic strains. Total pentose content was about two to three times greater than total hexose.     

Pentose transport by the ruminal bacterium Butyrivibrio fibrisolvens

Abstract Butyrivibrio fibrisolvens is a fibrolytic ruminal bacterium that degrades hemicellulose and ferments the resulting pentose sugars. Washed cells of strain D1 accumulated radiolabelled xylose ( K _m= 1.5 μ M) and arabinose ( K _m= 0.2 μ M) when the organism was grown on xylose, arabinose, or glucose, but cultures grown on sucrose or cellobiose had little capacity to transport pentose. Glucose and xylose inhibited transport of each other non-competitively. Both sugars were utilized preferentially over arabinose, but since they did not inhibit transport of arabinose, it appeared that the preference was related to an internal metabolic step. Although the protonmotive force was completely abolished by ionophores, cells retained some ability to transport pentose. In contrast, the metabolic inhibitors iodoacetate, arsenate, and fluoride had little effect on protonmotive force but caused a large decrease in intracellular ATP and xylose and arabinose uptake. These results suggested that high-affinity, ATP-dependent mechanisms were responsible for pentose transport and hexose sugars affected the utilization of xylose and arabinose.     

Improved strains of recombinant Escherichia coli for ethanol production from sugar mixtures

Hemicellulose hydrolysates of agricultural residues often contain mixtures of hexose and pentose sugars. Ethanologenic Escherichia coli that have been previously investigated preferentially ferment hexose sugars. In some cases, xylose fermentation was slow or incomplete. The purpose of this study was to develop improved ethanologenic E. coli strains for the fermentation of pentoses in sugar mixtures. Using fosfomycin as a selective agent, glucose-negative mutants of E. coli KO11 (containing chromosomally integrated genes encoding the ethanol pathway from Zymomonas mobilis) were isolated that were unable to ferment sugars transported by the phosphoenolpyruvate-dependent phosphotransferase system. These strains (SL31 and SL142) retained the ability to ferment sugars with independent transport systems such as arabinose and xylose and were used to ferment pentose sugars to ethanol selectively in the presence of high concentrations of glucose. Additional fosfomycin-resistant mutants were isolated that were superior to strain KO11 for ethanol production from hexose and pentose sugars. These hyperproductive strains (SL28 and SL40) retained the ability to metabolize all sugars tested, completed fermentations more rapidly, and achieved higher ethanol yields than the parent. Both SL28 and SL40 produced 60 gl^–1 ethanol from 120 gl^–1 xylose in 60 h, 20% more ethanol than KO11 under identical conditions. Further studies illustrated the feasibility of sequential fermentation. A mixture of hexose and pentose sugars was fermented with near theoretical yield by SL40 in the first step followed by a second fermentation in which yeast and glucose were added. Such a two-step approach can combine the attributes of ethanologenic E. coli for pentoses with the high ethanol tolerance of conventional yeasts in a single vessel.     

Xylose and arabinose utilization by the rumen bacterium Butyrivibrio fibrisolvens

Abstract The rumen bacterium Butyrivibrio fibrisolvens strain D1 co-utilized xylose and glucose in batch culture, but there was a marked preference for glucose over arabinose. When both pentoses were provided, xylose was preferred over arabinose. Strain D1 co-utilized a combination of either pentose and cellobiose, but preferred pentoses over maltose. Pentose sugars were depleted less rapidly in the presence of sucrose than controls containing only pentose. In contrast, B. fibrisolvens strain A38 exhibited a strong preference for disaccharides, including maltose, over either xylose or arabinose. Theoretical maximum growth yields for strain D1v in single-substrate continuous culture were highest for sucrose and cellobiose and the maintenance energy coefficient for arabinose was at least 3.8-fold greater than for other substrates. We suggest that B. fibrisolvens may have evolved a mechanism to utilize certain sugars before arabinose in order to avoid this high maintenance energy expenditure.     

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.     

Crystallization and partial characterization of Staphylococcus aureus hyaluronate lyase

Staphylococcus aureus, strain 1801, hyaluronate lyase was purified and crystallized to homogeneity as ascertained by chromatography and disc-gel electrophoresis. Purification procedures included sequential ammonium sulfate fractionation, acetone precipitation, Sephadex gel filtration, and ion exchange chromatography. During its passage through the cation exchange column, the hyaluronate lyase was resolved into two minor and one major fraction. The major peak, which was found to be cationic, was further characterized and designated as Fraction III. Carbohydrate analysis showed the presence of neutral sugars galactose, glucose, and mannose in the ratio of 1:3:6. Amino sugars galactosamine and glucosamine (or mannosamine) were present in a ratio of 1:1. Quantitative amino acid analysis of the Fraction III showed a relative abundance of the basic amino acids lysine and histidine.     

Feruloylated pectic substances from sugar-beet pulp

Pectins have been isolated from an ethanol-insoluble residue of sugar-beet pulp by sequential extraction with water, oxalate, hot dilute acid, andcold dilute alkali in yields of 2.2, 0.53, 20, and 11%, respectively. They were purified by chromatography on DEAE-cellulose at pH 4.8,or by precipitation with copper sulphate (alkali-soluble pectin). The pectins had fairly low molecular weights, a high degree of acetylation, and relatively high contents of neutral sugars, but there were clear differences beteen the four fractions. The main neutral sugars in each pectin were arabinose and galactose, and rhamnose, fucose, xylose, mannose, and glucose were also present. The fractions were homogeneous in ion-exchange and gel-filtration chromatography. Polyphenols (1–2%) and possibly proteins (3–6%) were associated with the purified pectins. In addition, feruloyl groups (up to 0.6%) were linked mainly to the acid-soluble and alkali-soluble pectins.     

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.     

Analysis of the Components Released from Potato Tuber Tissues during Maceration by Pectolytic Enzymes

Endo-pectin lyase and endo-polygalacturonase of Aspergillus japonicus attack the middle lamella of plant tissue and cause tissue maceration. Galacturonides, neutral sugars, and proteins were released from potato tuber tissues during maceration by both purified enzymes. These three components accounted for 92% of the soluble products. The neutral sugars released were rhamnose, arabinose, and galactose with a molar ratio of 1:3:15. They were covalently linked to galacturonides. Over 85% of the galacturonides released by the enzymes were short chain products, which indicated that a large portion of the main chain of pectic substances is a homogalacturonan. The results of chromatography on columns of Sephadex G-100 and DEAE-cellulose suggested that a protein component may be attached to pectic substances. This protein did not contain hydroxyproline and, therefore, was different from the cell wall structural glycoprotein.     

Induction of Xylose Reductase and Xylitol Dehydrogenase Activities in Pachysolen tannophilus and Pichia stipitis on Mixed Sugars

The induction of xylose reductase and xylitol dehydrogenase activities on mixed sugars was investigated in the yeasts Pachysolen tannophilus and Pichia stipitis. Enzyme activities induced on d-xylose served as the controls. In both yeasts, d-glucose, d-mannose, and 2-deoxyglucose inhibited enzyme induction by d-xylose to various degrees. Cellobiose, l-arabinose, and d-galactose were not inhibitory. In liquid batch culture, P. tannophilus utilized d-glucose and d-mannose rapidly and preferentially over d-xylose, while d-galactose consumption was poor and lagged behind that of the pentose sugar. In P. stipitis, all three hexoses were used preferentially over d-xylose. The results showed that the repressibility of xylose reductase and xylitol dehydrogenase may limit the potential of yeast fermentation of pentose sugars in hydrolysates of lignocellulosic substrates.     

Stereoselective Syntheses of Pentose Sugars Under Realistic Prebiotic Conditions

Glycolaldehyde and dl-glyceraldehyde reacted in a water-buffered solution under mildly acidic conditions and in the presence of chiral dipeptide catalysts produced pentose sugars whose configuration is affected by the chirality of the catalyst. The chiral effect was found to vary between catalysts and to be largest for di-valine. Lyxose, arabinose, ribose and xylose are formed in different amounts, whose relative proportions do not change significantly with the varying of conditions. With LL-peptide catalysts, ribose was the only pentose sugar to have a significant D-enantiomeric excess (ee) (≤44%), lyxose displayed an L-ee of ≤66%, arabinose a smaller L-ee of ≤8%, and xylose was about racemic. These data expand our previous findings for tetrose sugars and further substantiate the suggestion that interactions between simple molecules of prebiotic relevance on the early Earth might have included the transfer of chiral asymmetry and advanced molecular evolution.     

Differences in pectic polysaccharides between carrot embryogenic and non-embryogenic calli

Summary Cell walls and media were obtained from three kinds of carrot cell culture, namely, embryogenic callus (EC), non-embryogenic callus (NC) and somatic embryos (SE), and analyzed for their sugar content and sugar composition by electrophoresis and gas chromatography. EC formed large cell clusters while NC formed small clusters. Observations under the light microscope revealed that the intercellular contacts in NC were much more limited than those in EC. The analysis of pectic polysaccharides revealed that the level of neutral sugars was higher than that of acidic sugars in EC, while the opposite was true in NC. Gaschromatographic analysis of neutral sugars in pectic fractions revealed that EC and SE were rich in arabinose, while NC was rich in galactose. On the basis of these results, we discuss the possible involvement of neutral sugars, and of arabinose and galactose in particular, in pectic polysaccharides in intercellular contacts.Abbreviations EC embryogenic callus - NC non-embryogenic callus - SE somatic embryo - MS Murashige and Skoog - PAS periodic acid-Schiff s reagent     

The extracellular proteoglycan produced by Rhodella grisea

Highly viscous extracellular proteoglycan (EPG) has been isolated from culture medium of the unicellular red alga Rhodella grisea (Rhodophyceae) by ethanol precipitation. EPG was composed of xylose (29.3%), 3-O-methyl-xylose (26.0%), uronic acids (17.1%), rhamnose (14.4%), galactose (7.5%), glucose (3.9%), arabinose (1.4%) and mannose (0.4%), and traces of fucose, 4-O-methyl-xylose and 2,3-di-O-methyl-rhamnose or fucose. In addition, the polymer contained proteins (13.1%), sulphates and 13C-CP MAS spectra indicated the presence of acetyl and succinyl groups. The molecular mass was estimated to be 136,000. Ion-exchange chromatography afforded five fractions differing in composition of neutral sugars, uronic acids, and protein content indicating thus the complex structure of the EPG.