Improved method of analysis of biomass sugars using high-performance liquid chromatography

The precise quantitative analysis of biomass derived sugars is a very important step in the conversion of biomass feedstocks to fuels and chemicals. However, the most accurate method of biomass sugar analysis is based on the gas chromatography analysis of derivatized sugars either as alditol acetates or trimethylsilanes. The derivatization method is time-consuming but the alternative HPLC method cannot resolve most sugars found in biomass hydrolysates. We have demonstrated for the first time that by careful manipulation of the HPLC mobile phase, biomass monomeric sugars (arabinose, xylose, fructose, glucose, mannose, and galactose) can be analyzed quantitatively and there is excellent baseline resolution of all the sugars. This was demonstrated for both standard sugars and corn stover hydrolysates. Our method can also be used to analyze dimmeric sugars (cellobiose and sucrose).     

The synthesis of exopolysaccharide by Klebsiella aerogenes membrane preparations and the involvement of lipid intermediates

1. Membrane preparations from Klebsiella aerogenes type 8 were shown to transfer glucose and galactose from their uridine diphosphate derivatives to a lipid and to polymer. The ratio of glucose to galactose transfer in both cases was 1:2. This is the same ratio in which these sugars occur in native polysaccharide. Galactose transfer was dependent on prior glucosylation of the lipid. Mutants were obtained lacking (a) glucosyltransferase and (b) galactosyltransferase. The transferase activities in a number of non-mucoid mutants was examined. 2. Glucose transfer was partially inhibited by uridine monophosphate, and incorporation of either glucose or galactose into lipid was decreased in the presence of uridine diphosphate. The sugars are thought to be linked to a lipid through a pyrophosphate bond, and treatment of the lipid intermediates with phenol yielded water-soluble compounds. These could be dephosphorylated with alkaline phosphatase. Transfer of glucuronic acid to lipid or polymer from uridine diphosphate glucuronic acid was much lower than that of the other two sugars. 3. The fate of sugars incorporated into polymer was also followed. Some conversion of glucose into galactose and glucuronic acid occurred. Mutants unable to transfer glucose or galactose to lipid were unable to form polymer. Other mutants capable of lipid glycosylation were in some cases unable to form polymer. A model for capsular polysaccharide synthesis is proposed and its similarity to the formation of other polymers outside the cell membrane is discussed.     

Catabolite inhibition and sequential metabolism of sugars by Streptococcus lactis.

Growth of galactose-adapted cells of Streptococcus lactis ML(3) in a medium containing a mixture of glucose, galactose, and lactose was characterized initially by the simultaneous metabolism of glucose and lactose. Galactose was not significantly utilized until the latter sugars had been exhausted from the medium. The addition of glucose or lactose to a culture of S. lactis ML(3) growing exponentially on galactose caused immediate inhibition of galactose utilization and an increase in growth rate, concomitant with the preferential metabolism of the added sugar. Under nongrowing conditions, cells of S. lactis ML(3) grown previously on galactose metabolized the three separate sugars equally rapidly. However, cells suspended in buffer containing a mixture of glucose plus galactose or lactose plus galactose again consumed glucose or lactose preferentially. The rate of galactose metabolism was reduced by approximately 95% in the presence of the inhibitory sugar, but the maximum rate of metabolism was resumed upon exhaustion of glucose or lactose from the system. When presented with a mixture of glucose and lactose, the resting cells metabolized both sugars simultaneously. Lactose, glucose, and a non-metabolizable glucose analog (2-deoxy-d-glucose) prevented the phosphoenolpyruvate-dependent uptake of thiomethyl-beta-d-galactopyranoside (TMG), but the accumulation of TMG, like galactose metabolism, commenced immediately upon exhaustion of the metabolizable sugars from the medium. Growth of galactose-adapted cells of the lactose-defective variant S. lactis 7962 in the triple-sugar medium was characterized by the sequential metabolism of glucose, galactose, and lactose. Growth of S. lactis ML(3) and 7962 in the triple-sugar medium occurred without apparent diauxie, and for each strain the patterns of sequential sugar metabolism under growing and nongrowing conditions were identical. Fine control of the activities of preexisting enzyme systems by catabolite inhibition may afford a satisfactory explanation for the observed sequential utilization of sugars by these two organisms.     

A simple procedure for the synthesis of alpha-32P-nucleoside triphosphates.

Chemical analysis of grapefruit (Citrus paradisi) pectic polysaccharides demonstrated that galacturonic acid constitutes 78% by weight of the total carbohydrates found. The remaining 22% was accounted for by a number of sugars which include galactose, glucose, arabinose, xylose, and mannose and, by weight, galactose accounted for almost 50% of the total neutral sugar components found in these pectic polysaccharides. Treatment of pectic polysaccharides with galactose oxidase followed by reduction of oxidized galactose residues with tritiated potassium borohydride resulted in the labeling of pectic polysaccharides. Analysis of the labeled polysaccharides demonstrated that of the total radioactivity incorporated more than 90% was recovered in the galactose residues. These results clearly demonstrate the successful utilization of the galactose oxidase/tritiated potassium borohydride method in labeling plant pectic polysaccharide.     

De novo prediction of three-dimensional structures for major protein families

The family 10 xylanase from Streptomyces olivaceoviridis E-86 contains a (beta/alpha)(8)-barrel as a catalytic domain, a family 13 carbohydrate binding module (CBM) as a xylan binding domain (XBD) and a Gly/Pro-rich linker between them. The crystal structure of this enzyme showed that XBD has three similar subdomains, as indicated by the presence of a triple-repeated sequence, forming a galactose binding lectin fold similar to that found in the ricin toxin B-chain. Comparison with the structure of ricin/lactose complex suggests three potential sugar binding sites in XBD. In order to understand how XBD binds to the xylan chain, we analyzed the sugar-complex structure by the soaking experiment method using the xylooligosaccharides and other sugars. In the catalytic cleft, bound sugars were observed in the xylobiose and xylotriose complex structures. In the XBD, bound sugars were identified in subdomains alpha and gamma in all of the complexes with xylose, xylobiose, xylotriose, glucose, galactose and lactose. XBD binds xylose or xylooligosaccharides at the same sugar binding sites as in the case of the ricin/lactose complex but its binding manner for xylose and xylooligosaccharides is different from the galactose binding mode in ricin, even though XBD binds galactose in the same manner as in the ricin/galactose complex. These different binding modes are utilized efficiently and differently to bind the long substrate to xylanase and ricin-type lectin. XBD can bind any xylose in the xylan backbone, whereas ricin-type lectin recognizes the terminal galactose to sandwich the large sugar chain, even though the two domains have the same family 13 CBM structure. Family 13 CBM has rather loose and broad sugar specificities and is used by some kinds of proteins to bind their target sugars. In such enzyme, XBD binds xylan, and the catalytic domain may assume a flexible position with respect to the XBD/xylan complex, inasmuch as the linker region is unstructured.     

Determination of fermentable carbohydrate from the upper gastrointestinal tract by using colectomized rats.

The primary aim of this study was to characterize the carbohydrate that would be supplied to the colon for fermentation under physiological conditions. Colectomized rats were fed fiber-free diets or diets containing 5% (wt/wt) gum arabic. Four (fucose, galactose, glucosamine, and galactosamine) of 11 analyzed sugars accounted for 77% of the total sugar in ileal excreta from colectomized rats fed fiber-free diets. The three sugars in gum arabic, rhamnose, arabinose, and galactose, accounted for 84% of the total sugars in gum arabic ileal excreta. Comparisons of the sugar compositions of the ileal excreta, the water-soluble fractions of the excreta, and three gel filtration fractions of the water-soluble material with those of the water-soluble fraction of rat mucosa, the acetone-soluble fraction of pancreas, and pancreatin suggested that the major source of endogenous carbohydrate is mucin. Gum arabic increased the daily excretion of the four mucin-derived sugars (fucose, galactose, glucosamine, and galactosamine) by the colectomized rats from 473 mumol per day to 634 mumol per day. We conclude that mucin is the major endogenous carbohydrate excreted from the upper gut and that gum arabic increases the amount of this endogenous carbohydrate.     

Determination of fermentable carbohydrate from the upper gastrointestinal tract by using colectomized rats.

The primary aim of this study was to characterize the carbohydrate that would be supplied to the colon for fermentation under physiological conditions. Colectomized rats were fed fiber-free diets or diets containing 5% (wt/wt) gum arabic. Four (fucose, galactose, glucosamine, and galactosamine) of 11 analyzed sugars accounted for 77% of the total sugar in ileal excreta from colectomized rats fed fiber-free diets. The three sugars in gum arabic, rhamnose, arabinose, and galactose, accounted for 84% of the total sugars in gum arabic ileal excreta. Comparisons of the sugar compositions of the ileal excreta, the water-soluble fractions of the excreta, and three gel filtration fractions of the water-soluble material with those of the water-soluble fraction of rat mucosa, the acetone-soluble fraction of pancreas, and pancreatin suggested that the major source of endogenous carbohydrate is mucin. Gum arabic increased the daily excretion of the four mucin-derived sugars (fucose, galactose, glucosamine, and galactosamine) by the colectomized rats from 473 mumol per day to 634 mumol per day. We conclude that mucin is the major endogenous carbohydrate excreted from the upper gut and that gum arabic increases the amount of this endogenous carbohydrate.     

Differential selectivity of the Escherichia coli cell membrane shifts the equilibrium for the enzyme-catalyzed isomerization of galactose to tagatose

An Escherichia coli galactose kinase gene knockout (DeltagalK) strain, which contains the l-arabinose isomerase gene (araA) to isomerize d-galactose to d-tagatose, showed a high conversion yield of tagatose compared with the original galK strain because galactose was not metabolized by endogenous galactose kinase. In whole cells of the DeltagalK strain, the isomerase-catalyzed reaction exhibited an equilibrium shift toward tagatose, producing a tagatose fraction of 68% at 37 degrees C, whereas the purified l-arabinose isomerase gave a tagatose equilibrium fraction of 36%. These equilibrium fractions are close to those predicted from the measured equilibrium constants of the isomerization reaction catalyzed in whole cells and by the purified enzyme. The equilibrium shift in these cells resulted from the higher uptake and lower release rates for galactose, which is a common sugar substrate, than for tagatose, which is a rare sugar product. A DeltamglB mutant had decreased uptake rates for galactose and tagatose, indicating that a methylgalactoside transport system, MglABC, is the primary contributing transporter for the sugars. In the present study, whole-cell conversion using differential selectivity of the cell membrane was proposed as a method for shifting the equilibrium in sugar isomerization reactions.     

Glucose and sucrose act as agonist and mannose as antagonist ligands of the G protein-coupled receptor Gpr1 in the yeast Saccharomyces cerevisiae

Several examples of G protein-coupled receptors have recently been suggested to respond to common sugars in millimolar concentrations. This low affinity has made it difficult to demonstrate direct receptor-ligand interaction. In the yeast Saccharomyces cerevisiae, rapid activation of the cAMP pathway by glucose and sucrose requires the GPCR Gpr1. Our results obtained by cysteine scanning mutagenesis and SCAM (substituted cysteine accessibility method) of residues in TMD VI provide strong evidence that glucose and sucrose directly interact as ligands with Gpr1. The affinity for sucrose is much higher. Structurally similar sugars such as galactose, mannose, and fructose do not act as agonists, but mannose acts as an antagonist for both sucrose and glucose. These results support the idea that Gpr1 directly senses sugars and that sugars can effectively bind GPCRs with a low affinity in a binding pocket formed by the transmembrane domains. The ligand repertoire of GPCRs can thus be extended to common sugars in millimolar concentrations.     

Influence of essential oil of Hyssopus officinalis on the chemical composition of the walls of Aspergillus fumigatus (Fresenius)

The cell walls of the growing hyphae of Aspergillus fumigatus (Fresenius) cultured in the presence or absence of the essential oil of Hyssopus officinalis were isolated and their chemical composition analysed. The presence of the essential oil led to a reduction in levels of neutral sugars, uronic acid and proteins, whereas amino sugars, lipids and phosphorus levels were increased. HPLC analysis of the neutral sugars showed that they consisted mainly of glucose, mannose and galactose, while the amino sugars consisted of glucosamine and galactosamine. The presence of the essential oil in the culture medium induced marked changes in the content of galactose and galactosamine. Cell walls were fractionated by treatment with alkali and acid. The essential oil induced similar alterations in the various fractions with a more marked effect on the major constituents. The alterations were related to changes in the structure of the cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

The use of lectins for determination of absolute configurations of small amounts of sugars eluted from chromatograms

A simple procedure for the determination of the absolute configuration (i.e., assignment to the D- or L-enantiomeric series) of glucose, mannose, galactose, fucose, arabinose, and rhamnose is described, based on inhibition by these sugars of 125I-labeled lectin binding to the glycoconjugates immobilized on the wells of plastic microculture plates. The method works well with 10 to 100-micrograms amounts of the sugars isolated after paper chromatography of the glycoprotein or polysaccharide hydrolysates.     

Determination of the carbohydrate content of porcine pancreatic amylase

Porcine pancreatic α-amylase (α-1,4 glucan 4-glucanohydrolase, EC 3.2.1.1) is shown to be a glycoprotein. Both molecular forms I and II of amylase contain 0.75 mole of fucose, 0.45 mole of galactose and 0.52 mole of mannose per mole of enzyme. Analyses of amino sugars indicate 1.2 and 0.8 residues of glucosamine bound per mole of amylase I and amylase II, respectively. A method for identification and quantitation of neutral sugars by gas chromatography of the trimethylsilyl derivatives of their corresponding alditols is proposed.     

Quantitative gas—liquid chromatography of neutral sugars in human serum glycoproteins : Fucose, mannose, and galactose as predictors in ovarian and small cell lung carcinoma

A precise and accurate gas—liquid chromatographic (GLC) method has been developed for the quantitative analysis of the neutral sugars -fucose (6-deoxygalactose), mannose, galactose, and glucose in ethanol precipitates of human serum proteins. The chromatographic conditions and sample preparation resulted in short analysis times (20 min per run) and made routine analyses practicable (twelve samples per day). The alditol acetate derivatization yielded single derivatives for each sugar. Complete separation was achieved on a 2.0 m × 2 mm I.D. column with 2.0% Silar-7 CP on Chromosorb W AW 80–100 mesh. The results of hydrolysis showed that the release of fucose and galactose preceded the release of mannose. Hydrolysis with AG 50W-X8 (H⁺) ion-exchange resin in 0.5 N HCI at 100° for 7 h optimized glycosidic bond cleavage with only minimal destruction of fucose, mannose and galactose. A combination of strong cation- and anion-exchange resin columns was used to remove chromatographic background of peptides, amino acids, amino sugars, and inorganic ions. An average R.S.D. of less than 4% with recovery of <86% for the three sugars was achieved. The homogeneity of the chromatographic peaks for the neutral sugars of normal human serum glycoproteins was confirmed by GLC—mass spectrometry. Significantly elevated ratios of fucose, galactose, and mannose to serum protein were observed for patients with small cell lung and ovarian carcinomas.     

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.     

Galactose transport in Streptococcus thermophilus.

Although Streptococcus thermophilus accumulated [14C]lactose in the absence of an endogenous energy source, galactose-fermenting (Gal+) cells were unable to accumulate [14C]galactose unless an additional energy source was added to the test system. Both Gal+ and galactose-nonfermenting (Gal-) strains transported galactose when preincubated with sucrose. Accumulation was inhibited 50 or 95% when 10 mM sodium fluoride or 1.0 mM iodoacetic acid, respectively, was added to sucrose-treated cells, indicating that ATP was required for galactose transport activity. Proton-conducting ionophores also inhibited galactose uptake, although N,N'-dicyclohexyl carbodiimide had no effect. The results suggest that galactose transport in S. thermophilus occurs via an ATP-dependent galactose permease and that a proton motive force is involved. The galactose permease in S. thermophilus TS2b (Gal+) had a Km for galactose of 0.25 mM and a Vmax of 195 micromol of galactose accumulated per min per g (dry weight) of cells. Several structurally similar sugars inhibited galactose uptake, indicating that the galactose permease had high affinities for these sugars.     

Galactose transport in Streptococcus thermophilus

Although Streptococcus thermophilus accumulated [14C]lactose in the absence of an endogenous energy source, galactose-fermenting (Gal+) cells were unable to accumulate [14C]galactose unless an additional energy source was added to the test system. Both Gal+ and galactose-nonfermenting (Gal-) strains transported galactose when preincubated with sucrose. Accumulation was inhibited 50 or 95% when 10 mM sodium fluoride or 1.0 mM iodoacetic acid, respectively, was added to sucrose-treated cells, indicating that ATP was required for galactose transport activity. Proton-conducting ionophores also inhibited galactose uptake, although N,N'-dicyclohexyl carbodiimide had no effect. The results suggest that galactose transport in S. thermophilus occurs via an ATP-dependent galactose permease and that a proton motive force is involved. The galactose permease in S. thermophilus TS2b (Gal+) had a Km for galactose of 0.25 mM and a Vmax of 195 micromol of galactose accumulated per min per g (dry weight) of cells. Several structurally similar sugars inhibited galactose uptake, indicating that the galactose permease had high affinities for these sugars.     

Sugar-binding sites of the HA1 subcomponent of Clostridium botulinum type C progenitor toxin

Clostridium botulinum type C 16S progenitor toxin contains a hemagglutinin (HA) subcomponent, designated HA1, which appears to play an important role in the effective internalization of the toxin in gastrointestinal epithelial cells and in creating a broad specificity for the oligosaccharide structure that corresponds to various targets. In this study, using the recombinant protein fused to glutathione S-transferase, we investigated the binding specificity of the HA1 subcomponent to sugars and estimated the binding sites of HA1 based on X-ray crystallography and soaking experiments using various sugars. N-Acetylneuraminic acid, N-acetylgalactosamine, and galactose effectively inhibited the binding that occurs between glutathione S-transferase-HA1 and mucins, whereas N-acetylglucosamine and glucose did not inhibit it. The crystal structures of HA1 complex with N-acetylneuraminic acid, N-acetylgalactosamine, and galactose were also determined. There are two sugar-binding sites, sites I and II. Site I corresponds to the electron densities noted for all sugars and is located at the C-terminal β-trefoil domain, while site II corresponds to the electron densities noted only for galactose. An aromatic amino acid residue, Trp176, at site I has a stacking interaction with the hexose ring of the sugars. On the other hand, there is no aromatic residue at site II; thus, the interaction with galactose seems to be poor. The double mutant W176A at site I and D271F at site II has no avidity for N-acetylneuraminic acid but has avidity for galactose. In this report, the binding specificity of botulinum C16S toxin HA1 to various sugars is demonstrated based on its structural features.     

Xylitol production from xylose mother liquor: a novel strategy that combines the use of recombinant <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and <Emphasis Type="Italic">Candida maltosa</Emphasis>

Background  

Xylose mother liquor has high concentrations of xylose (35%-40%) as well as other sugars such as L-arabinose (10%-15%), galactose (8%-10%), glucose (8%-10%), and other minor sugars. Due to the complexity of this mother liquor, further isolation of xylose by simple method is not possible. In China, more than 50,000 metric tons of xylose mother liquor was produced in 2009, and the management of sugars like xylose that present in the low-cost liquor is a problem.     

中性糖在土壤中的来源与分布特征

糖类(即碳水化合物)是土壤有机质的重要组成部分, 经生物化学降解形成不同结构的单糖。土壤中的中性单糖也叫中性糖, 主要包括木糖、核糖、阿拉伯糖、葡萄糖、半乳糖、甘露糖、岩藻糖和鼠李糖。其中, 植物来源的糖主要为五碳糖, 如木糖和阿拉伯糖; 微生物来源的糖主要包括半乳糖、甘露糖、岩藻糖、鼠李糖等六碳糖。研究中常利用六碳糖和五碳糖的比例指示微生物和植物对土壤有机碳的相对贡献。中性糖是微生物重要的碳源和能量来源, 在团聚体的形成过程中扮演着重要角色。该文整合了近30年土壤中性糖的研究进展, 对比了提取中性糖的常用方法, 分析了不同土地利用类型和不同土壤组分中中性糖的含量、来源和周转特征, 综述了影响中性糖含量和分布的主要环境因素。结果表明, 中性糖在耕地土壤中的绝对含量和相对含量均显著低于针叶林、阔叶林、草地和灌丛4种土地利用类型。(半乳糖+甘露糖)/(阿拉伯糖+木糖)(GM/AX)在不同土地利用间差异不显著, 而(鼠李糖+岩藻糖)/(阿拉伯糖+木糖)(RF/AX)则表明草地土壤中的微生物来源的中性糖含量高于针叶林和耕地。不同密度的土壤组分中, 轻质组分中中性糖的含量比重质组分高, 重质组分中微生物来源的中性糖较多; 就不同粒径(或团聚体)而言, 黏粒(或微团聚体)中微生物来源的中性糖含量更丰富。有关影响土壤中性糖含量和分布的因素的研究, 目前主要集中在人为活动(如耕种和放牧等), 而有关温度、降水等自然环境因素影响的研究较少。     

A Pro to Gly mutation in the hinge of the arabinose-binding protein enhances binding and alters specificity. Sugar-binding and crystallographic studies

The L-arabinose-binding protein (ABP) of Escherichia coli consists structurally of two distinct globular domains connected by a hinge of three separate peptide segments. Arabinose is bound and completely sequestered within the deep cleft between the two domains. With reduced affinity, ABP also binds D-galactose (approximately 2-fold reduction) and D-fucose (approximately 40-fold reduction). Experiments have been conducted to explore the role in sugar binding of the hinge connecting the two domains of ABP. To increase the flexibility of the hinge region, a glycine was substituted for a proline at position 254 by site-directed mutagenesis. Unexpectedly, this mutation resulted in the dramatic enhancement of galactose binding over that of arabinose. The affinity of the mutant ABP for galactose increased by over 20-fold, while that for arabinose and fucose remained relatively unchanged. We have measured association and dissociation rates of the Gly-254 ABP with L-arabinose, D-galactose, and D-fucose and have determined the crystallographic structure of the protein complexed with each of the three sugars. Both the ligand-binding kinetic measurements and structure analysis indicate that the altered specificity is due to an effective increase in the rigidity of the hinge in the closed conformation which is induced upon galactose binding. Stabilizing contacts are formed between the strands of the hinge in the Gly-254 ABP when galactose is bound which are not found in complexes with the other sugars or the liganded wild-type protein.