Structural features of the hemicellulose a from the stem of Mimosa bracatinga

The hemicellulose A from the stem of Mimosa bracatinga contains residues of d-xylose and 4-O-methyl-d-glucuronic acid. Smith-degradation and methylation data indicate it to consist of a backbone of (1 → 4)-linked β-d-xylopyranosyl residues with side chains consisting of single units of 4-O-methyl--d-glucosyluronic acid attached to position 3. The branched structure was also indicated by Smith-degradation and methylation analysis of the oligosaccharides obtained by enzymic hydrolysis of the hemicellulose.     

Structure of hardwood glucuronoxylans: modifications and impact on pulp retention during wood kraft pulping

The behaviour of different hardwood glucuronoxylans during the kraft pulping process was investigated. Woods and pulps xylans were isolated and characterized by size exclusion chromatography, methylation (linkage) analysis and ¹H NMR. Eucalyptus globulus and Eucalyptus urograndis showed xylan retention significantly higher than that of Betula pendula. The higher retention of Eucalyptus xylans was assigned to (i) their higher average molecular weight (31 against 24 KDa in B. pendula) and to (ii) the presence of O-2 substituted 4-O-methyl--d-glucuronic acid groups ([→2)-GlcpA-(1→]) with galactopyranosyl/glucopyranosyl residues belonging to fragments of galactan/glucan chains that were absent in B. pendula xylans. A significant part of uronic acids, particularly [→2)-GlcpA-(1→] units, remain in fibres until the end of pulping. The acetylation degree and distribution of acetyl groups between Xylp units, in general terms, was similar in the three types of xylans. Unexpectedly, about 20% of the acetyl groups persisted in pulps xylans till the end of pulping.     

Pustulan and branched β-galactofuranan from the phytopathogenic fungus Guignardia citricarpa, excreted from media containing glucose and sucrose

Guignardia citricarpa is a phytopathogenic fungus and the causal agent of citrus black spot. Incubation in a semi-defined media resulted in formation of exopolysaccharides [EPS(s)]. A medium containing glucose gave rise to a (1→6)-linked β-glucan (200 kD), pustulan, which was characterized by NMR and methylation analysis. A sucrose-containing medium provided a homogalactan (376 kD) and methylation analysis showed nonreducing end- (20%), 6-O- (53%) and 5,6-di-O-substituted Galf units (27%). An HMQC spectrum of the homogalactan showed C-1/H-1 signals at δ 108.2/4.820, 108.3/4.820 and 107.1/5.079, corresponding to three types of β- -Galf units. A DEPT analysis showed inverted signals (CH₂) at δ 67.8 and 67.2, corresponding to 6-O-substituted β- -Galf units, whereas a C-5 signal at δ 77.0 suggests 5-O-substitution, confirming a novel structure for a β-galactofuranan.     

Structural characterization and antioxidant activity of a polysaccharide from the fruiting bodies of cultured Cordyceps militaris

The water-soluble crude polysaccharides were obtained from the fruiting bodies of cultured Cordyceps militaris by hot water extraction followed by ethanol precipitation. The polysaccharides were successively purified by chromatography on DEAE–cellulose-52 and Sephacryl S-100 HR columns, giving main three polysaccharide fractions termed P50-1, P70-1, and P70-2. Structural features of P70-1 were investigated by a combination of chemical and instrumental analysis, such as partial acid hydrolysis, methylation analysis, periodate oxidation – Smith degradation, GC–MS, ¹³C NMR, HPAEC-PAD, and FT-IR. The results indicated that P70-1 has a backbone of (1 → 6)-linked β-d-mannopyranosyl residues, which occasionally branches at O-3. The branches were mainly composed of (1 → 4)-linked -d-glucopyranosyl and (1 → 6)-linked β-d-galactopyranosyl residues, and terminated with β-d-galactopyranosyl residues and -d-glucopyranosyl residues. In the in vitro antioxidant assay, P70-1 was found to possess hydroxyl radical-scavenging activity with an IC₅₀ value of 0.548 mg/ml.     

The recombinant xylanase B of Thermotoga maritima is highly xylan specific and produces exclusively xylobiose from xylans, a unique character for industrial applications

The xynB of a hyperthermophilic Eubacterium, Thermotoga maritima MSB8, coding xylanase B (XynB) was previously expressed in E. coli and the recombinant protein was characterized using the synthetic substrates [J. Biosci. Bioeng. 92 (2001) 423]. In this study, the same xylanase B was purified to homogeneity with a recovery yield of about 43% using heat treatment followed by the Ni-NTA affinity chromatography. The specificity of XynB towards different natural substrates was evaluated. XynB was highly specific towards xylans tested but exhibited low activities towards lichenan (19%), gellan gum (7.3%), laminarin (3.4%) and carboxymethylcellulose (CMC, 1.4%). The apparent K_m values of birchwood xylan and soluble oat-spelt xylan was 0.11 and 0.079 mg/ml, respectively. The XynB hydrolyzed xylooligosaccharides to yield predominantly xylobiose (X₂) and a small amount of xylose (X₁), suggesting that XynB was possibly an endo-acting xylanase. Analysis of the products from birchwood xylan degradation confirmed that the enzyme was an endo-xylanase with xylobiose and xylose as the main degradation products. HPLC results showed that hydrolyzed products of birchwood xylan by XynB yielded up to 66% of the total reaction product as xylobiose. These results clearly indicated that xylobiose could be mass-produced efficiently by the recombinant hyperthermostable XynB of T. maritima. Additionally, conversion of xylobiose (50 mM) to xylose was observed, while xylotriose (X₃) and xylotetraose (X₄) were detected in small amounts, indicating that the enzyme converted xylobiose to xylose based on the transglycosylation reaction. The increased binding ability of XynB to Avicel and/or insoluble xylan was also observed indicating the possibilities of roles of surface-aromatic amino acid residues for such action. However, further investigations are required to prove this speculation.     

Six new C21 steroidal glycosides from Asclepias curassavica L

Six new C₂₁ steroidal glycosides, named curassavosides A–F (3–8), were obtained from the aerial parts of Asclepias curassavica (Asclepiadaceae), along with two known oxypregnanes, 12-O-benzoyldeacylmetaplexigenin (1) and 12-O-benzoylsarcostin (2). By spectroscopic methods, the structures of the six new compounds were determined as 12-O-benzoyldeacylmetaplexigenin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (3), 12-O-benzoylsarcostin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (4), sarcostin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (5), sarcostin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-digitoxopyranoside (6), 12-O-benzoyldeacylmetaplexigenin 3-O-β-d-glucopyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (7), and 12-O-benzoylsarcostin 3-O-β-d-glucopyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (8), respectively. All compounds (1–8) were tested for in vitro cytotoxicity; only compound 3 showed weak inhibitory activity against Raji and AGZY cell lines.     

Fractional and structural characterization of wheat straw hemicelluloses

Six hemicellulosic fractions were extracted successively from dewaxed wheat straw with sodium hydroxide at increasing strength from 0.25 to 2.00M, and the chemical composition are reported. The structure of the hemicellulosic fraction 2 was investigated using acid hydrolysis, methylation analysis and ¹³C-NMR experiments. The hemicelluloses were confirmed to be a (1→4)-linked β-D-xylan with D-glucopyranosyluronic acid (or 4-O-methyl--D-glucopyranosyluronic acid) group attached at position 2, and L-arabinofuranosyl and D-xylopyranosyl groups attached at position 3. For every 26 D-xylopyranosyl residues in the main chain, there was one uronic acid unit. For 13 such D-xylopyranosyl residues, there was one L-arabinofuranosyl group, and for 18 such D-xylopyranosyl residues, there was one D-xylopyranosyl group.     

Dihydroxamic acid complexes of iron (III): ligand pKa and coordinated water hydrolysis constants

A series of dihydroxamic acid ligands of the formula [RN(OH)C(O)]₂(CH₂)_n, (n = 2, 4, 6, 7, 8; R = CH₃, H) has been studied in 2.0 M aqueous sodium perchlorate at 25.0 °C. These ligands may be considered as synthetic analogs to the siderophore rhodotorulic acid. Acid dissociation constants (pK_a) have been determined for the ligands and for N-methylacetohydroxamic acid (NMHA). The pK_a1 and pK_a2 values are: n = 2, R = CH₃ (8.72, 9.37); N = 4, R = CH₃ (8.79, 9.37); N = 6, R = CH₃; N = 7, R = CH₃ (8.95, 9.47); N = 8, R = CH₃ (8.93, 9.45); N = 8, R = H (9.05, 9.58). Equilibrium constants for the hydrolysis of coordinated water (log K) have been estimated for the 1:1 feeric complexes of the ligands n = 2, 4, 8; R = CH₃. The N = 8 ligand forms a monomeric complex with Fe(III) while the n = 2 and 4 ligands form dimeric complexes. For hydrolysis of the n = 8 monomeric complex, log K₁ = −6.36 and log K₂ = −9.84. Analysis of the spectrophotometric data for the dimeric complexes indicates deprotonation of all four coordinated waters. The successive hydrolysis constants, log K_1–4, for the dimeric complexes are as follows: n = 2 (−6.37, −5.77, −10.73, −11.8); n = 4 (−5.54, −5.07, −11.57, −10.17). The log K₂ values for the dimers are unexpectedly high, higher in fact than log K₁, inconsistent with the formation of simple ternary hydroxo complexes. A scheme is proposed for the hydrolysis of the ferric dihydroxamate dimers, which includes the possible formation of μ-hydroxo and μ-oxo bridges.     

Carbon–carbon-linked (pyrazolylphenyl)oxazolidinones with antibacterial activity against multiple drug resistant gram-positive and fastidious gram-negative bacteria

In an effort to expand the spectrum of activity of the oxazolidinone class of antibacterial agents to include Gram-negative bacteria, a series of new carbon–carbon linked pyrazolylphenyl analogues has been prepared. The -N-substituted methyl pyrazole (10) in the C3-linked series exhibited very good Gram-positive activity with MICs ≤0.5–1 μg/mL and moderate Gram-negative activity with MICs=2–8 μg/mL against Haemophilus influenzae and Moraxella catarrhalis. This analogue was also found to have potent in vivo activity with an ED₅₀=1.9 mg/kg. β-Substitution at the C3-linked pyrazole generally results in a loss of activity. The C4-linked pyrazoles are slightly more potent than their counterparts in the C3-linked series. Most of the analogues in the C4-linked series exhibited similar levels of activity in vitro, but lower levels of activity in vivo than 10. In addition, incorporation of a thioamide moiety in selected C4-linked pyrazole analogues results in an enhancement of in vitro activity leading to compounds several times more potent than eperezolid, linezolid and vancomycin. The thioamide of the N-cyanomethyl pyrazole analogue (34) exhibited an exceptional in vitro activity with MICs of ≤ 0.06–0.25 μg/mL against Gram-positive pathogens and with MICs of 1 μg/mL against fastidious Gram-negative pathogens.     

Characterization of a novel, ultra-large xylanolytic complex (xylanosome) from Streptomyces olivaceoviridis E-86

A novel, ultra-large xylanolytic complex (xylanosome) from Streptomyces olivaceoviridis E-86 was purified to homogeneity by ammonium sulfate precipitation and Sephacryl S-300 gel filtration chromatography. The purified xylanosome appeared as a single protein band on the non-denaturing (native) polyacrylamide gel electrophoresis (PAGE) gel with a molecular mass of approximately 1200 kDa. The optimal temperature and pH for xylanase activity was 60 °C and pH 6.0, respectively. The xylanase activity was stable within pH 4.1–10.3. It was stable up to 60 °C at pH 6.0. The xylanosome was highly specific towards oat-spelt xylan, and showed low activity towards corncob powder, but exhibited very low activity towards lichenan, CMC and p-nitrophenyl derivatives. Apparent K_m values of the xylansosome for birchwood, beechwood, soluble oat-spelt and insoluble oat-spelt xylans were 2.5, 3.6, 1.7 and 4.9 mg ml⁻¹, respectively. The main hydrolysis products of birchwood xylan were xylotriose, xylobiose and xylose. Analysis of the products from wheat arabinoxylan degradation by xylanosome confirmed that the enzyme had endoxylanase and debranching activities, with xylotriose, xylobiose, xylose and arabinose as the main degradation products. These unique properties of the purified xylanosome from Streptomyces olivaceoviridis E-86 make this enzymatic complex attractive for biotechnological applications.     

Structure of the serine-containing capsular poly-saccharide K40 antigen from Escherichia coli O8:K40:H9

The structure of the K40 antigenic capsular polysaccharide (K40 antigen) of E. coli O8:K40:H9 was elucidated by determination of the composition, ¹H- and ¹³C-n.m.r. spectroscopy, periodate oxidation and Smith degradation, and methylation analysis. The K40 polysaccharide consists of [(O-β- -glucopyranosyluronic acid)-(1→4)-O-(2-acetamido-2-deoxy-- -glucopyranosyl)-(1→6)-O-(2-acetamido-2-deoxy-- -glucopyranosyl)-(1→4)] repeating units. All of the glucuronic acid residues are substituted amidically with -serine.     

Synthesis of a series of ganglioside GM3 analogs containing a deoxy-N-acetylneuraminic acid residue.

Ganglioside GM₃ analogs containing 4-, 7-, 8-, and 9-deoxy-N-acetylneuraminic acids in the place of N-acetylneuraminic acid (Neu5Ac) have been synthesized. Glycosylation of 2-(trimethylsilyl)ethyl O-(6-O-benzoyl-β- - galactopyranosyl)-(1 → 4)-2,6-di-O-benzoyl-β- -glucopyranoside with the methyl 2-thioglycoside derivatives of the respective deoxy-N-acetylneuraminic acids, using dimethyl(methylthio)sulfonium triflate as a promoter, gave the four required 2-(trimethylsilyl)ethyl -sialosyl-(2 → 3b)-β-lactosides. These were converted via O-acetylation, selective removal of the 2-(trimethylsilyl)ethyl group, and subsequent imidate formation, into the corresponding -sialosyl-(2 → 3b)--lactose trichloroacetimidates 15, 17, 19, and 21. Glycosylation of (2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol with 15, 17, 19, and 21 in the presence of boron trifluoride etherate afforded the expected β glycosides, which were transformed in good yields, via selective reduction of the azido group, coupling with octadecanoic acid, O-deacylation, and de-esterification, into the target compounds.     

Purification and characterization of xylanase from Aspergillus ficuum AF-98

The purification and characterization of xylanase from Aspergillus ficuum AF-98 were investigated in this work. The extracellular xylanase from this fungal was purified 32.6-fold to homogeneity throughout the precipitation with 50–80% (NH₄)₂SO₄, DEAE-Sephadex A-50 ion exchange chromatography and Sephadex G-100 chromatography. The purified xylanase (specific activity at 288.7 U/ mg protein) was a monomeric protein with a molecular mass of 35.0 kDa as determined by SDS-PAGE. The optimal temperature and pH for the action of the enzyme were at 45 °C and 5.0, respectively. The xylanase was activated by Cu²⁺ up to 115.8% of activity, and was strongly inhibited by Hg²⁺, Pb²⁺ up to 52.8% and 89%, respectively. The xylanase exhibited K_m and V_max values of 3.267 mg/mL, 18.38 M/min/mg for beechwood xylan and 3.747 mg/mL, 11.1 M/min/mg for birchwood xylan, respectively.     

Chiral macrocyclic compounds from lactose derivatives

The reaction of benzyl 2,6,6′-tri-O-benzyl-3′,4′-O-isopropylidene-β-lactoside with 1,11-ditosyloxy-3,6,9-trioxaundecane gave benzyl 2,6,6′-tri-O-benzyl-3′,4′-O-isopropylidene-3,2′-O--(3,6,9-trioxaundecane-1,11-diyl)-β-lactoside (2, 47%). Acid hydrolysis of 2 and condensation of the product with 1,14-ditosyloxy-3,6,9,12-tetra-oxatetradecane afforded benzyl 2,6,6′-tri-O-benzyl-3′,4′-O-(3,6,9,12-tetraoxa-tetradecane-1,14-diyl)-3,2′-O-(3,6,9-trioxaundecane-1,11-diyl)-β-lactoside (29%). Similarly, the reaction of benzyl 2,6,2′,4′,6′-penta-O-benzyl-β-lactoside with Ts[OCH₂CH₂]₄OTs gave benzyl 2,6,2′,4′,6′-penta-O-benzyl-3,3′-O-(3,6,9-trioxaundecane-1,11-diyl)-β-lactoside (78%). ¹H-N.m.r. spectroscopy has been used to study the formation of host-guest complexes with some of these macrocyclic compounds and benzyl ammonium thiocyanate.     

Anti-proliferative lichen compounds with inhibitory activity on 12(S)-HETE production in human platelets

Several lichen compounds, i.e. lobaric acid (1), a β-orcinol depsidone from Stereocaulon alpinum L., (+)-protolichesterinic acid (2), an aliphatic -methylene-γ-lactone from Cetraria islandica Laur. (Parmeliaceae), (+)-usnic acid (3), a dibenzofuran from Cladonia arbuscula (Wallr.) Rabenh. (Cladoniaceae), parietin (4), an anthraquinone from Xanthoria elegans (Link) Th. Fr. (Calaplacaceae) and baeomycesic acid (5), a β-orcinol depside isolated from Thamnolia vermicularis (Sw.) Schaer. var. subuliformis (Ehrh.) Schaer. were tested for inhibitory activity on platelet-type 12(S)-lipoxygenase using a cell-based in vitro system in human platelets. Lobaric acid (1) and (+)-protolichesterinic acid (2) proved to be pronounced inhibitors of platelet-type 12(S)-lipoxygenase, whereas baeomycesic acid (5) showed only weak activity (inhibitory activity at a concentration of 100 μg/ml: 1 93.4±6.62%, 2 98,5±1.19%, 5 14.7±2.76%). Usnic acid (3) and parietin (4) were not active at this concentration. 1 and 2 showed a clear dose–response relationship in the range of 3.33–100 μg/ml. According to the calculated IC₅₀ values the highest inhibitory activity was observed for the depsidone 1 (IC₅₀=28.5 μM) followed by 2 (IC₅₀=77.0 μM). The activity of 1 was comparable to that of the flavone baicalein, which is known as a selective 12(S)-lipoxygenase inhibitor (IC₅₀=24.6 μM).     

Structural studies on hairy region of pectic polysaccharide from campion Silene vulgaris (Oberna behen)

Using enzymic digestion with pectinase, controlled Smith degradation and NMR-spectroscopy, some structural features of the hairy region of pectic polysaccharide termed silenan SV from the aerial part of campion Silene vulgaris (Moench) Garke (Oberna behen (L.) Ikonn) were elucidated.

Silenan was subjected to enzymic digestion with pectinase to furnish the polysaccharide fraction (SVP). The contained residues of -galacturonic acid (43%), arabinose, galactose and rhamnose as main constituents. The backbone of the hairy region of silenan was found to consist of -1,4-galactopyranosyl uronic acid and 2-O-glycosylated rhamnopyranose residues. The side chains contained linear regions of residues of -1,5-linked arabinofuranose and β-1,3-, β-1,4-linked galactopyranose. Silenan SV and its fragment SVP were subjected to Smith degradation to give fractions SVS and SVPS. These contain the residues of terminal and 2-substituted -arabinofuranose as well as residues of terminal, 3-, and 2,3-substituted β-galactopyranose. In addition, NMR-spectral data confirmed that the residues of -rhamnopyranose 2-O-glycosylated with the residues of -1,4-galactopyranosyl uronic acid of the backbone occurred in the core of SVPS and, therefore, in the backbone of silenan SV.

On the basis of data obtained, the hairy regions of silenan were suggested to contain mainly the linear chains of β-1,3-, β-1,4-galactopyranan and -1,5-arabinofuranan. The chains of -1,5-linked arabinofuranose, β-1,3- and β-1,4-linked galactopyranose were shown to be involved in the side chains of the hairy region having branching points at 2,3-substituted β-galactopyranose residues.     

Structural of a glucomannan from Lupinus varius seed

Polysaccharides extracted from seeds of Lupinus varius with hot ethanol 85% (polysaccharide FI) and 0.1 M sodium phosphate buffer, pH 7.5 (polysaccharide FII) were fractionated and purified by ion-exchange and gel-filtration chromatography. According to methylation and hydrolysis analysis, the main chains of FI and FII consisted of (1 → 4)-linked glucomannan; only traces of branched sugar residues were detected. This is the first report on the isolation of glucomannan from L. varius seeds.     

Polysaccharides from the green seaweeds Codium fragile and C. vermilara with controversial effects on hemostasis

Codium fragile and Codium vermilara biosynthesize water-soluble sulfated arabinans and galactans (and/or sulfated arabinogalactans), (1 → 4)-d-glucans and β(1 → 4)-d-mannans. The former polysaccharides are composed by 3-linked β-d-galactopyranose and β-l-arabinopyranose residues, they are highly sulfated and substituted with pyruvic acid ketals. For both seaweeds, they have the same main structural units, but in different percentages. All the room-temperature water extracts from both seaweeds showed a dual haemostatic effect: they prevented coagulation, but they induced platelet aggregation. Anticoagulant activity and platelet aggregation were higher in the samples with polysaccharides richer in sulfate, mainly in those from C. vermilara, which have a higher degree of sulfation and arabinose content.     

Biotransformation of paeonol by Panax ginseng root and cell cultures

Panax ginseng root and cell cultures were shown to biotransform paeonol (1) into its 2-O-β-d-glucopyranoside (2). P. ginseng root cultures were also able to biotransform paeonol (1) into its 2-O-β-d-xylopyranoside (3), 2-O-β-d-glucopyranosyl(1 → 6)-β-d-glucopyranoside (4) and 2-O-β-d-xylopyranosyl(1 → 6)-β-d-glucopyranoside (5), and its demethylated derivate, 2′,4′-dihydroxyacetophenone (6). Compounds 3 and 4 are new glycosides. It is the first example that the administrated compound was converted into its xylopyranoside by plant biotransformation.     

Purification and partial characterization of Cu, Zn containing superoxide dismutase from entomogenous fungal species Cordyceps militaris

Cordyceps militaris mycelium produced mainly Cu, Zn containing superoxide dismutase (Cu, Zn-SOD). Cu, Zn-SOD activity was detectable in the culture filtrates, and intracellular Cu, Zn-SOD activity as a proportion protein was highest in early log phase culture. The effects of Cu²⁺, Zn²⁺, Mn²⁺ and Fe²⁺ on enzyme biosynthesis were studied. The Cu, Zn-SOD was isolated and purified to homogeneity from C. militaris mycelium and partially characterized. The purification was performed through four steps: (NH₄)₂SO₄ precipitation, DEAE-sepharose™ fast flow anion-exchange chromatography, CM-650 cation-exchange chromatography, and Sephadex G-100 gel filtration chromatography. The purified enzyme had a molecular weight of 35070 ± 400 Da and consisted of two equal-sized subunits each having a Cu and Zn element. Isoelectric point value of 7.0 was obtained for the purified enzyme. The N-terminal amino acid sequence of the purified enzyme was determined for 12 amino acid residues and the sequences was compared with other Cu, Zn-SODs. The optimum pH of the purified enzyme was obtained to be 8.2–8.8. The purified enzyme remained stable at pH 5.8–9.8, 25 °C and up to 50 °C at pH 7.8 for 1.5 h incubation. The purified enzyme was sensitive to H₂O₂, KCN. 2.5 mM NaN₃, PMSF, Triton X-100, β-mercaptoethanol and DTT showed no significant inhibition effect on the purified enzyme within 5 h incubation period.