Functional characterization and oligomerization of a recombinant xyloglucan-specific endo-β-1,4-glucanase (GH12) from Aspergillus niveus

Xyloglucan is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists of a cellulosic backbone (beta-1,4-linked glucosyl residues) that is frequently substituted with side chains. This report describes Aspergillus nidulans strain A773 recombinant secretion of a dimeric xyloglucan-specific endo-β-1,4-glucanohydrolase (XegA) cloned from Aspergillus niveus. The ORF of the A. niveus xegA gene is comprised of 714 nucleotides, and encodes a 238 amino acid protein with a calculated molecular weight of 23.5 kDa and isoelectric point of 4.38. The optimal pH and temperature were 6.0 and 60 °C, respectively. XegA generated a xyloglucan-oligosaccharides (XGOs) pattern similar to that observed for cellulases from family GH12, i.e., demonstrating that its mode of action includes hydrolysis of the glycosidic linkages between glucosyl residues that are not branched with xylose. In contrast to commercial lichenase, mixed linkage beta-glucan (lichenan) was not digested by XegA, indicating that the enzyme did not cleave glucan β-1,3 or β-1,6 bonds. The far-UV CD spectrum of the purified enzyme indicated a protein rich in β-sheet structures as expected for GH12 xyloglucanases. Thermal unfolding studies displayed two transitions with mid-point temperatures of 51.3 °C and 81.3 °C respectively, and dynamic light scattering studies indicated that the first transition involves a change in oligomeric state from a dimeric to a monomeric form. Since the enzyme is a predominantly a monomer at 60 °C, the enzymatic assays demonstrated that XegA is more active in its monomeric state.     

Degradative GH5 β-1,3-1,4-glucanase PpBglu5A for glucan in Paenibacillus polymyxa KF-1

A novel β-1,3-1,4-glucanase in the glycoside hydrolase family 5 (GH5) has been identified in the secretome of Paenibacillus polymyxa KF-1. The recombinant GH5 enzyme PpBglu5A shows broad substrate specificity, with strong lichenase activity, medium β-1,3-glucanase activity, and minimal cellulase activity. Barley β-glucan, lichenan, curdlan, and carboxymethyl cellulose are hydrolyzed to varying degrees by PpBglu5A, with the highest catalytic activity being observed with barley β-glucan. Hydrolysates from barley β-glucan or lichenan are primarily glucan oligosaccharides with degrees of polymerization from 2 to 4. PpBglu5A also hydrolyzes oat bran into oligosaccharides mainly consisted of di-, tri-, and tetra- oligosaccharides that are useful in the preparation of gluco-oligosaccharides. In addition to hydrolytic activity, transglycosylation was also observed with PpBglu5A and cellotriose as substrate. An in vitro assay indicated that the recombinant PpBglu5A has antifungal activity and can inhibit the growth of Canidia albicans. These results suggest that PpBglu5A exhibits unique properties and may be useful as an antifungal agent.     

An endo-(1→3)-β-d-glucanase from the scallop Chlamys albidus: catalytic properties, cDNA cloning and secondary-structure characterization

An endo-(1→3)-β-d-glucanase (L₀) with molecular mass of 37 kDa was purified to homogeneity from the crystalline style of the scallop Chlamys albidus. The endo-(1→3)-β-d-glucanase was extremely thermolabile with a half-life of 10 min at 37 °C. L₀ hydrolyzed laminaran with K_m ∼ 0.75 mg/mL, and catalyzed effectively transglycosylation reactions with laminaran as donor and p-nitrophenyl β d-glucoside as acceptor (K_m ∼ 2 mg/mL for laminaran) and laminaran as donor and as acceptor (K_m ∼ 5 mg/mL) yielding p-nitrophenyl β d-glucooligosaccharides (n = 2-6) and high-molecular branching (1→3),(1→6)-β-d-glucans, respectively. Efficiency of hydrolysis and transglycosylation processes depended on the substrate structure and decreased appreciably with the increase of the percentage of β-(1→6)-glycosidic bonds, and laminaran with 10% of β-(1→6)-glycosidic bonds was the optimal substrate for both reactions. The CD spectrum of L₀ was characteristic for a protein with prevailing β secondary-structural elements. Binding L₀ with d-glucose as the best acceptor for transglycosylation was investigated by the methods of intrinsic tryptophan fluorescence and CD. Glucose in concentration sufficient to saturate the enzyme binding sites resulted in a red shift in the maximum of fluorescence emission of 1-1.5 nm and quenching the Trp fluorescence up to 50%. An apparent association constant of L₀ with glucose (K_a = 7.4 × 10⁵ ± 1.1 × 10⁵ M⁻¹) and stoichiometry (n = 13.3 ± 0.7) was calculated. The cDNA encoding L₀ was sequenced, and the enzyme was classified in glycoside hydrolases family 16 on the basis of the amino acid sequence similarity.     

Steroidal saponins from the leaves of Beaucarnea recurvata

Thirteen steroidal saponins were isolated from the leaves of Beaucarnea recurvata Lem. Their structures were established using one- and two-dimensional NMR spectroscopy and mass spectrometry. Six of them were identified as: 26-O-β-d-glucopyranosyl (25S)-furosta-5,20(22)-diene 1β,3β,26-triol 1-O-α-l-rhamnopyranosyl-(1 → 2) β-d-fucopyranoside, 26-O-β-d-glucopyranosyl (25S)-furosta-5,20(22)-diene 1β,3β,26-triol 1-O-α-l-rhamnopyranosyl-(1 → 2)-4-O-acetyl-β-d-fucopyranoside, 26-O-β-d-glucopyranosyl (25R)-furosta-5,20(22)-diene-23-one-1β,3β,26-triol 1-O-α-l-rhamnopyranosyl-(1 → 2) β-d-fucopyranoside, 26-O-β-d-glucopyranosyl (25S)-furosta-5-ene-1β,3β,22α,26-tetrol 1-O-α-l-rhamnopyranosyl-(1 → 4)-6-O-acetyl-β-d-glucopyranoside, 26-O-β-d-glucopyranosyl (25S)-furosta-5-ene-1β,3β,22α,26-tetrol 1-O-α-l-rhamnopyranosyl-(1 → 2) β-d-fucopyranoside, and 24-O-β-d-glucopyranosyl (25R)-spirost-5-ene-1β,3β,24-triol 1-O-α-l-rhamnopyranosyl-(1 → 2)-4-O-acetyl-β-d-fucopyranoside. The chemotaxonomic classification of B. recurvata in the family Ruscaceae was discussed.     

Isolation and structural characterization of a polysaccharide FCAP1 from the fruit of Cornus officinalis

A water-soluble polysaccharide, FCAP1, was isolated from an alkaline extract from the fruits of Cornus officinalis. Its molecular weight was 34.5 kDa. Monosaccharide composition analysis revealed that it was composed of fucose, arabinose, xylose, mannose, glucose, and galactose in a molar ratio of 0.29:0.19:1.74:1:3.30:1.10. On the basis of partial acid hydrolysis and methylation analysis, FCAP1 was shown to be a highly branched polysaccharide with a backbone of β-(1→4)-linked-glucose partially substituted at the O-6 position with xylopyranose residues. The branches were composed of (1→3)-linked-Ara, (1→4)-linked-Man, (1→4,6)-linked-Man, (1→4)-linked-Glc, and (1→2)-linked-Gal. Arabinose, fucose, and galactose were located at the terminal of the branches. The structure was further elucidated by a specific enzymatic degradation with an endo-β-(1→4)-glucanase and MALDI-TOF-MS analysis. Oligosaccharides generated from FCAP1 indicated that FCAP1 contained XXXG-type and XXG-type xyloglucan fragments.     

Lactosylamidine-based affinity purification for cellulolytic enzymes EG I and CBH I from Hypocrea jecorina and their properties

Selective adsorption and separation of β-glucosidase, endo-acting endo-β-(1→4)-glucanase I (EG I), and exo-acting cellobiohydrolase I (CBH I) were achieved by affinity chromatography with β-lactosylamidine as ligand. A crude cellulase preparation from Hypocrea jecorina served as the source of enzyme. When crude cellulase was applied to the lactosylamidine-based affinity column, β-glucosidase appeared in the unbound fraction. By contrast, EG I and CBH I were retained on the column and then separated from each other by appropriately adjusting the elution conditions. The relative affinities of the enzymes, based on their column elution conditions, were strongly dependent on the ligand. The highly purified EG I and CBH I, obtained by affinity chromatography, were further purified by Mono P and DEAE chromatography, respectively. EG I and CBH I cleave only at the phenolic bond in p-nitrophenyl glycosides with lactose and N-acetyllactosamine (LacNAc). By contrast, both scissile bonds in p-nitrophenyl glycosides with cellobiose were subject to hydrolysis although with important differences in their kinetic parameters.     

Acylated flavonol tetraglycosides from Delphinium gracile

An ethanol extract of the aerial parts of Delphinium gracile DC. yielded five flavonol glycosides quercetin-3-O-{[β-d-xylopyranosyl (1 → 3)-4-O-(E-p-caffeoyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranosyl (1 → 2)]}-β-d-glucopyranoside (1), quercetin-3-O-{[β-d-xylopyranosyl (1 → 3)-4-O-(E-p-coumaroyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranosyl (1 → 2)]}-β-d-glucopyranoside (2), quercetin-3-O-{[β-d-xylopyranosyl (1 → 3)-4-O-(Z-p-coumaroyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranosyl (1 → 2)]}-β-d-glucopyranoside (3), kaempferol-3-O-{[β-d-glucopyranosyl (1 → 3)-4-O-(E-p-coumaroyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranoside-7-O-(4-O-acetyl)-α-l-rhamnopyranoside (4) kaempferol-3-O-{[β-d-glucopyranosyl (1 → 3)-4-O-(E-p-coumaroyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranoside-7-O-(4-O-acetyl)-α-l-rhamnopyranoside (5) in addition to 4-(β-d-glucopyranosyloxy)-6-methyl-2H-pyran-2-one (6) and rutin. Structures were elucidated by spectroscopic methods.     

Enzymatic liberation of lycotetraose from the Solanum glycoalkaloid alpha-tomatine

The branched tetrasaccharide, O-β-d-glucopyranosyl-(1 → 2)-O-[β-d-xylopyranosyl-(1 → 3)]-O-β-d-glucopyranosyl-(1 → 4)-d-galactose (lycotetraose) is a key constituent of many biologically interesting natural products. Described herein is a convenient enzymatic preparation of lycotetraose from the readily available Solanum glycoalkaloid α-tomatine. The preparation makes use of the recombinant endo-glycosidase, tomatinase, from the plant pathogen Fusarium oxysporum f. sp. lycopersici.     

Studies on the chemical structure and antitumor activity of an exopolysaccharide from Rhizobium sp. N613

The molecular structure of the rhizobium exopolysaccharide (REPS) was analyzed by enzymolysis, periodate oxidation, and Smith degradation, and by IR and NMR spectroscopy. The results indicated that REPS was a β-glucan with a backbone of β-d-(1→4)-linked glucose residues and branches of β-d-(1→6)-linked glucose residues. The branch was attached to the main chain at the 6-O-position. The molar ratio of 1→4 and 1→6 was 2:1. The terminal C3 of the (1→6)-Glc branch had an O-acetyl group. The molecular weight was estimated to be 35 kDa by Sephadex G-100 column chromatography. The antitumor activity of REPS was evaluated in mice bearing sarcoma 180, hepatoma 22, and Ehrlich ascites carcinoma tumor, respectively. At doses of 10-60 mg/kg, it was observed that tumor formation decreased significantly (P <0.01), but the relative spleen and thymus weight, the phagocytic function of monocytes, lymphocyte proliferation, and serum hemolysis antibody increased significantly (P <0.05). Results of these studies demonstrated that the REPS polysaccharide possessed antitumor activity.     

Degradation of carbohydrate moieties of arabinogalactan-proteins by glycoside hydrolases from Neurospora crassa

Arabinogalactan-proteins (AGPs) are a family of plant proteoglycans having large carbohydrate moieties attached to core-proteins. The carbohydrate moieties of AGPs commonly have β-(1→3)(1→6)-galactan as the backbone, to which other auxiliary sugars such as l-Ara and GlcA are attached. For the present study, an α-l-arabinofuranosidase belonging to glycoside hydrolase family (GHF) 54, NcAraf1, and an endo-β-(1→6)-galactanase of GHF 5, Nc6GAL, were identified in Neurospora crassa. Recombinant NcAraf1 (rNcAraf1) expressed in Pichia pastoris hydrolyzed radish AGPs as well as arabinan and arabinoxylan, showing relatively broad substrate specificity toward polysaccharides containing α-l-arabinofuranosyl residues. Recombinant Nc6GAL (rNc6GAL) expressed in P. pastoris specifically acted on β-(1→6)-galactosyl residues. Whereas AGP from radish roots was hardly hydrolyzed by rNc6GAL alone, β-(1→6)-galactan side chains were reduced to one or two galactan residues by a combination of rNcAraf1 and rNc6GAL. These results suggest that the carbohydrate moieties of AGPs are degraded by the concerted action of NcAraf1 and Nc6GAL secreted from N. crassa.     

Synthesis of selenium nanowires morphologically directed by Shinorhizobial oligosaccharides

Shinorhizobial cyclosophoraose (cyclic β-(1→2)-glucan) or succinoglycan monomer (SGM 2), which has one acetyl, pyruvyl, and succinyl group, functions as a morphology-directing agent for the synthesis of pure trigonal selenium nanowires by using ascorbic acid (vitamin C) as the reducing agent. The synthesis was achieved in water at room temperature. Under these experimental conditions, the diameters of the as-prepared Se nanowires were varied in the range of 34-120 nm by cyclosophoraose and of 33-66 nm by SGM 2, in which the nanowires were characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Raman spectroscopy. Through this study, we propose that Shinorhizobial cyclic and linear oligosaccharides have morphologically directing functions for the synthesis of single-crystalline selenium nanowires by green chemical methods.     

Cytotoxic triterpenoid saponins acylated with monoterpenic acids from fruits of Gleditsia caspica Desf.

Four bisdesmosidic triterpenoid saponins named caspicaosides A-D, were isolated from the fruits of Gleditsia caspica Desf. Their structures were determined by NMR spectroscopy including HOHAHA, ¹H-¹H COSY, ROE, HMQC, HMBC experiments and HRFAB-MS as well as acid hydrolysis. The four 3,28-O-bisdesmosidic triterpenoid saponins comprised echinocystic acid as the aglycone and common oligosaccharide moieties at C3 and C28. The saccharide moiety at C-3 was identified as β-d-xylopyranosyl-(1 → 2)-α-l-arabinopyranosyl-(1 → 6)-β-d-glucopyranosyl while that at C-28 was determined as β-d-xylopyranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-[α-l-rhamnopyranosyl-(1 → 6)-]β-d-glucopyranosyl. The pentasaccharide moiety linked to C-28 was acylated with monoterpenic acid and or monoterpene-arabinoside moieties at C-2 or C-2 and C-3 of the terminal rhamnose unit. The isolated saponins were assayed for their in vitro cytotoxicities against the three human tumor cell lines HepG2, A549 and HT29 using MTT method. The results showed that caspicaosides B and C bearing two and three monoterpene units, respectively, exhibited significant cytotoxic activities against the used cell lines with IC₅₀ values 1.5-6.5 μM. Caspicaosides A and D with one monoterpene unit exhibited significant cytotoxic activities on HepG2 cell line with IC₅₀ values equal to 4.5 and 5.4 μM, respectively, and IC₅₀ values >10 μM against the other two cell lines. The number of monoterpene units seems to play a main role in determining the activity.     

Flavonol tetraglycosides from fruits of Styphnolobium japonicum (Leguminosae) and the authentication of Fructus Sophorae and Flos Sophorae

The dried fruits and seeds of Styphnolobium japonicum (L.) Schott (syn. Sophora japonica L.) are used in traditional Chinese medicine and known as Fructus Sophorae or Huai Jiao. The major flavonoids in these fruits and seeds were studied by LC-MS and other spectroscopic techniques to aid the chemical authentication of Fructus Sophorae. Among the flavonoids were two previously unreported kaempferol glycosides: kaempferol 3-O-β-glucopyranosyl(1 → 2)-β-galactopyranoside-7-O-α-rhamnopyranoside and kaempferol 3-O-β-xylopyranosyl(1 → 3)-α-rhamnopyranosyl(1 → 6)[β-glucopyranosyl(1 → 2)]-β-glucopyranoside, the structures of which were determined by NMR. Two further tetraglycosides were identified for the first time in S. japonicum as kaempferol 3-O-β-glucopyranosyl(1 → 2)[α-rhamnopyranosyl(1 → 6)]-β-glucopyranoside-7-O-α-rhamnopyranoside and kaempferol 3-O-β-glucopyranosyl(1 → 2)[α-rhamnopyranosyl(1 → 6)]-β-galactopyranoside-7-O-α-rhamnopyranoside; the latter was the main flavonoid in mature seeds. The chromatographic profiles of 27 recorded flavonoids were relatively consistent among fruits of similar ages collected from five trees of S. japonicum, and those of maturing unripe and ripe fruits were similar to a market sample of Fructus Sophorae, and thus provide useful markers for authentication of this herbal ingredient. The flower buds (Huai Mi) and flowers (Huai Hua) of S. japonicum (collectively Flos Sophorae) contained rutin as the main flavonoid and lacked the flavone glycosides that were present in flower buds and flowers of Sophora flavescens Ait., reported to be occasional substitutes for Flos Sophorae. The single major flavonoid in fruits of S. flavescens was determined as 3′-hydroxydaidzein.     

Acylated triterpene saponins from the roots of Securidaca longepedunculata

Four triterpene saponins, 3-O-β-d-glucopyranosylpresenegenin 28-O-β-d-apiofuranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 4)-[β-d-apiofuranosyl-(1 → 3)]-α-l-rhamnopyranosyl-(1 → 2)-{4-O-[(E)-3,4,5-trimethoxycinnamoyl]}-β-d-fucopyranosyl ester, 3-O-β-d-glucopyranosylpresenegenin 28-O-β-d-apiofuranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 4)-[β-d-apiofuranosyl-(1 → 3)]-α-l-rhamnopyranosyl-(1 → 2)-[(6-O-acetyl)-β-d-glucopyranosyl-(1 → 3)]-{4-O-[(E)-3,4,5-trimethoxycinnamoyl]}-β-d-fucopyranosyl ester, 3-O-β-d-glucopyranosylpresenegenin 28-O-β-d-apiofuranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 4)-[β-d-apiofuranosyl-(1 → 3)]-α-l-rhamnopyranosyl-(1 → 2)-[β-d-galactopyranosyl-(1 → 3)]-{4-O-[(E)-3,4,5-trimethoxycinnamoyl]}-β-d-fucopyranosyl ester, and 3-O-β-d-glucopyranosylpresenegenin 28-O-β-d-apiofuranosyl-(1 → 3)-[α-l-arabinopyranosyl-(1 → 4)]-β-d-xylopyranosyl-(1 → 4)-[β-d-apiofuranosyl-(1 → 3)]-α-l-rhamnopyranosyl-(1 → 2)-{4-O-[(E)-3,4,5-trimethoxycinnamoyl]}-β-d-fucopyranosyl ester, were isolated from the roots of Securidaca longepedunculata, together with three known compounds. Their structures were established mainly by 2D NMR techniques and mass spectrometry.     

Triterpenoid saponins from a cytotoxic root extract of Sideroxylonfoetidissimum subsp. gaumeri

Evaluation of the cytotoxicity of an ethanolic root extract of Sideroxylonfoetidissimum subsp. gaumeri (Sapotaceae) revealed activity against the murine macrophage-like cell line RAW 264.7. Systematic bioassay-guided fractionation of this extract gave an active saponin-containing fraction from which four saponins were isolated. Use of 1D (¹H, ¹³C, DEPT135) and 2D (COSY, TOCSY, HSQC, and HMBC) NMR, mass spectrometry and sugar analysis gave their structures as 3-O-(β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl)-28-O-(α-l-rhamnopyranosyl-(1 → 3)[β-d-xylopyranosyl-(1 → 4)]-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-16α-hydroxyprotobassic acid, 3-O-β-d-glucopyranosyl-28-O-(α-l-rhamnopyranosyl-(1 → 3)[β-d-xylopyranosyl-(1 → 4)]-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-16α-hydroxyprotobassic acid, 3-O-(β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl)-28-O-(α-l-rhamnopyranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 4)[β-d-apiofuranosyl-(1 → 3)]-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-16α-hydroxyprotobassic acid, and the known compound, 3-O-β-d-glucopyranosyl-28-O-(α-l-rhamnopyranosyl-(1 → 3)[β-d-xylopyranosyl-(1 → 4)]-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-protobassic acid. Two further saponins were obtained from the same fraction, but as a 5:4 mixture comprising 3-O-(β-d-glucopyranosyl)-28-O-(α-l-rhamnopyranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 4)[β-d-apiofuranosyl-(1 → 3)]-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-16α-hydroxyprotobassic acid and 3-O-(β-d-apiofuranosyl-(1 → 3)-β-d-glucopyranosyl)-28-O-(α-l-rhamnopyranosyl-(1 → 3)[β-d-xylopyranosyl-(1 → 4)]-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-16α-hydroxyprotobassic acid, respectively. This showed greater cytotoxicity (IC₅₀ = 11.9 ± 1.5 μg/ml) towards RAW 264.7 cells than the original extract (IC₅₀ = 39.5 ± 4.1 μg/ml), and the saponin-containing fraction derived from it (IC₅₀ = 33.7 ± 6.2 μg/ml).     

Presence of 1→3-linked 2-O-β-d-xylopyranosyl-α-l-arabinofuranosyl side chains in cereal arabinoxylans

The presence of a fairly uncommon side chain 2-O-β-d-xylopyranosyl-α-l-arabinofuranosyl in arabinoxylans (AX) from eight different cereal by-products was investigated, using ¹H NMR spectroscopy and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) after Shearzyme^® (GH10 endo-1,4-β-d-xylanase) hydrolysis. This disaccharide side group was present in significant amounts in AX extracted from corn cobs and barley husks. For the first time, it was also detected in AX from oat spelts and rice husks, and in lesser amounts in wheat straw AX. Arabinoxylo-oligosaccharide (AXOS) containing the 2-O-β-d-Xylp-α-l-Araf side chain was purified from the oat spelt AX hydrolysate and the structure was fully analyzed using 1D and 2D NMR spectroscopy. The AXOS was identified as β-d-Xylp-(1→2)-α-l-Araf-(1→3)-β-d-Xylp-(1→4)-d-Xyl. To our knowledge, such a structure with 2-O-β-d-Xylp-α-l-Araf attached to the O-3 of the nonreducing end of xylobiose has not been described previously. New information on substitution of AX from various cereal by-products was obtained by combining NMR and enzyme-assisted HPAEC-PAD analysis.     

Bioactive saponins from Microsechium helleri and Sicyos bulbosus

Eleven oleanane-type saponins (1-11) have been isolated from Microsechium helleri and Sicyos bulbosus roots and were evaluated for their antifeedant, nematicidal and phytotoxic activities. Saponins {3-O-β-d-glucopyranosyl (1 → 3)-β-d-glucopyranosyl-2β,3β,16α,23-tetrahydroxyolean-12-en-28-oic acid 28-O-α-l-rhamnopyranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 4)-[β-d-xylopyranosyl-(1 → 3)]-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranoside} (1), and {3-O-β-d-glucopyranosyl-2β,3β,16α,23-tetrahydroxyolean-12-en-28-oic acid 28-O-α-l-rhamnopyranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 4)-[β-d-xylopyranosyl-(1 → 3)]-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranoside} (2) were also isolated from M. helleri roots together with the two known compounds 3 and 4. Seven known structurally related saponins (5-11) were isolated from S. bulbosus roots. The structures of these compounds were established as bayogenin and polygalacic glycosides using one- and two-dimensional NMR spectroscopy and mass spectrometry. Compounds 7, 10, bayogenin (12) and polygalacic acid (13) showed significant (p < 0.05) postingestive effects on Spodoptera littoralis larvae, compounds 5-11 and 12 showed variable nematicidal effects on Meloydogyne javanica and all tested saponins had variable phytotoxic effects on several plant species (Lycopersicum esculentum, Lolium perenne and Lactuca sativa). These are promising results in the search for natural pesticides from the Cucurbitaceae family.     

Step-wise enzymatic preparation and structural characterization of singly and doubly substituted arabinoxylo-oligosaccharides with non-reducing end terminal branches

Shearzyme (GH10 endo-1,4-β-d-xylanase) and two different α-l-arabinofuranosidases (AXH-m and AXH-d3) were used stepwise to manufacture arabinoxylo-oligosaccharides (AXOS) with α-l-Araf (1→2)-monosubstituted β-d-Xylp residues or α-l-Araf (1→2)- and (1→3) doubly substituted β-d-Xylp residues from wheat arabinoxylan (AX) in a rather straightforward way. Four major AXOS (d-I, d-II, m-I and m-II) were formed in two separate hydrolyses. The AXOS were purified and the structures were confirmed using TLC, HPAEC-PAD, MALDI-TOF-MS and 1D and 2D NMR spectroscopy. The samples were identified as d-I: α-l-Araf-(1→2)-[α-l-Araf-(1→3)]-β-d-Xylp-(1→4)-β-d-Xylp-(1→4)-d-Xylp, d-II: α-l-Araf-(1→2)-[α-l-Araf-(1→3)]-β-d-Xylp-(1→4)-d-Xylp, m-I: α-l-Araf-(1→2)-β-d-Xylp-(1→4)-β-d-Xylp-(1→4)-d-Xylp and m-II: α-l-Araf-(1→2)-β-d-Xylp-(1→4)-d-Xylp. To our knowledge, this is the first report on structural ¹H and ¹³C NMR analysis of xylobiose-derived AXOS d-II and m-II. The latter compound has not been reported previously. The doubly substituted AXOS were produced for the first time in good yields, as d-I and d-II corresponded to 11.8 and 5.6 wt% of AX, respectively. Singly α-l-Araf (1→2)-substituted AXOS could also be prepared in similar yields by treating the doubly substituted AXOS further with AXH-d3.     

Galactans from Cryptonemia species. Part II: Studies on the system of galactans of Cryptonemia seminervis (Halymeniales) and on the structure of major fractions

Cryptonemia seminervis biosynthesizes a family of d,l-hybrid galactans based on the classical 3-linked β-d-galactopyranosyl→4-linked α-d- and α-l-galactopyranosyl alternating sequence (A-units→B-units) with major amounts of α-d- and α-l-galactose and 3,6-anhydro-d- and l-galactose and lesser percentages of 3,6-anhydro-2-O-methyl-l-galactose, 2-O-methyl-, 4-O-methyl- and 6-O-methylgalactoses. The dispersion of structures in this family is based on five structural factors, namely: (a) the amount and position of substituent groups as sulfate (major), pyruvic acid ketals, methoxyl and glycosyl side-chain (4-O-methyl galactopyranosyl and/or xylosyl); (b) the ratio galactose/3,6-anhydrogalactose in the B-units; (c) the ratio d,l-galactoses and d,l-3,6-anhydrogalactoses also in the B-units, (d) the formation of diads and (e) the sequence of the diads in the linear backbone. Considering these variables it is not unexpected to find in the fractions studied at least 18 structural units producing highly complex structures. Structural studies carried out in two major fractions (S2S-3 and S2S-4) showed that these galactans were formed mainly by β-d-galactopyranosyl 2-sulfate (20 and 11.9 mol %), β-d-galactopyranosyl 2-sulfate 4,6-O-(1′-carboxyethylidene) (8.9 and 6.0 mol %) and β-d-galactopyranosyl 2,6-sulfate (5.4 and 18.6 mol %), together with 3,6-anhydro-α-l-galactopyranosyl (11.4 and 7.3 mol %) and 3,6-anhydro-α-l-galactopyranosyl 2-sulfate (4.9 and 15.4 mol %) and minor quantities of 12-15 other structural units.Preparative alkaline treatment carried out on fraction (S2S-3) produced a quantitative formation of 3,6-anhydro α-l-galactopyranosyl units from precursor units (α-l-galactose 6-sulfate and α-l-galactose 2,6-sulfate). Kinetic studies on this 3,6-anhydro cyclization show a rate constant of 5.2 × 10⁴ s⁻¹ indicating diads of the type G→L6S/2,6S. Data from chemical, spectroscopic and kinetic studies suggest that, in S2S-3, the agaran block in the d,l-hybrid galactan is composed of the following diads: G(6R)→L6S/2,6S and G2S(P)(2,6S)→LA(2S)(2R)(2M) and the carrageenan block of G2S(P)→D(2S)(2,3S)(3S)(3,6S) in a molar ratio of agaran to carrageenan structures of ∼2:1.     

O-Methylated mannogalactan from the microalga Coccomyxa mucigena, symbiotic partner of the lichenized fungus Peltigera aphthosa

A structural study of the carbohydrates from Coccomyxa mucigena, the symbiotic algal partner of the lichenized fungus Peltigera aphthosa, was carried out. It produced an O-methylated mannogalactan, with a (1 → 6)-linked β-galactopyranose main-chain partially substituted at O-3 by β-Galp, 3-OMe-α-Manp or α-Manp units. There were no similarities with polysaccharides previously found in the lichen thallus of P. aphthosa. Moreover, the influence of lichenization in polysaccharide production by symbiotic microalgae and the nature of the photobiont in carbohydrate production in lichen symbiosis are also discussed.