Effects of salts on production and on O-acetylation of glucuronan excreted by the Rhizobium meliloti M5N1 CS strain

Acetylation determined by ¹H-nuclear magnetic resonance spectroscopy and production of the glucuronan excreted by the Rhizobium meliloti M5N1 CS strain during cultivation in RCS medium with and without added magnesium salts have been studied. These salts induce an increase in the degree of substitution and the molar ratio of 2,3-di-O-acetyl residues. A decrease in production is observed after 75 h of fermentation as the magnesium salt concentration increases. The presence of manganese and sodium salts in the culture induces inhibition of exopolysaccharide (EPS) production. However, the structure of the EPS is similar to that of the EPS produced by standard fermentation, without modification in the degree of substitution.     

Isolation and characterization of a (1 → 3)-β-glucan endohydrolase from germinating barley (Hordeum vulgare): amino acid sequence similarity with barley (1 → 3, 1 → 4)-β-glucanases

A (1 → 3)-β-glucan 3-glucanohydrolase (EC 3.2.1.39) has been purified approx. 190-fold from extracts of germinating barley. The enzyme has an apparent M_r 32 000, a pI of 8.6, and a pH optimum of 5.6. Analysis of hydrolysis products released from the (1 → 3)-β-glucan, laminarin, shows that the enzyme is an endohydrolase. Sequence analysis of the 46 NH₂-terminal amino acids of the (1 → 3)-β-glucanase reveals 54% positional identity with barley (1 → 3,1 → 4)-β-glucanases (EC 3.2.1.73) and suggests a common evolutionary origin for these two classes of β-glucan endohydrolases. The barley (1 → 3)-β-glucanase also exhibits significant similarity with a (1 → 3)-β-glucanase from tobacco.     

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.     

Triterpenoidal saponins from Dumasia Truncata

Extraction of the aerial parts of Dumasia truncata Sieb et Zucc. afforded two new triterpenoidal saponins, together with four known ones. The structures of the new compounds were elucidated by spectral analysis as 3-O--l-rhamnopyranosyl-(1 → 3)-β-d-glucuronopyranosy-28-O-β-d-glucopyransoyl hederagenin and 3-O-β-d-xylopyranosyl-(1 → 2)-[-l-rhamnopyranosyl(1 → 3)]-β-d-glucuronopyranosyl oleanic acid.     

Structural investigation of Ceratozamia spinosa mucilage

The polysaccharide fraction from Ceratozamia spinosa appears to be made up mainly by a chemically homogeneous polysaccharide but with a wide range of molecular weight. By NMR and chemical degradative methods, it is shown to consist essentially of a backbone of alternate → 4)-β- -GlcpA-(1 → and → 2)-- -Manp-(1 → units. On the 4 position of the latter, β- -GlcpA residues are linked. End units of - -Ara f, β- -Xylp, - -Rhap, and - -3-OMe-Rhap are linked to C-3 and/or C-4 positions of β- -Glc pA residues.     

Simultaneous and comparative assessment of parent and mutant strain of Rhizobium meliloti for nutrient limitation and enhanced polyhydroxyalkanoate (PHA) production using optimization studies

Nutrient limitation conditions, optimization and comparison of polyhydroxyalkanoate (PHA) yields and biomass production by parent and mutant strains of Rhizobium meliloti were investigated. Complex interactions among concentrations of sucrose (5–55 g/l), urea (0.05–0.65 g/l) inoculum (10–250 ml/l) and K₂HPO₄ (0.5–2 g/l), were studied using central composite rotatable design (CCRD) experiments. Phosphate-limiting medium (0.33 g K₂HPO₄/l) in the presence of excess carbon (sucrose 42.5 g/l) results in more production of PHA (2.2 g/l) in the parent strain. In comparison, the mutant strain required moderate levels of sucrose (30 g/l), along with excess of phosphate (1 g/l) for high PHA content of cell biomass (80%) and PHA yield (3.3 g/l). Optimised PHA production (biomass 4.8 g/l and PHA 3.09 g/l) by the parent strain occurred at: sucrose 51.58 g/l, urea 0.65 g/l, K₂HPO₄ 0.48 g/l and inoculum 10 ml/l. In the mutant strain, higher yields of biomass (9.05 g/l) and PHA (5.66 g/l) were obtained in Optimised medium containing: sucrose 55 g/l, urea 0.65 g/l, K₂HPO₄ 1.0 g/l and inoculum 150.58 ml/l.     

Cycloartane triterpene glycosides from Astragalus trigonus

Three new cycloartane glycosides, trigonoside I, II and III, and the known astragalosides I and II were isolated from the roots of Astragalus trigonus. The structures of the new glycosides were totally elucidated by high field (600 MHz) NMR analyses as cycloastragenol-6-O-β-xylopyranoside, cycloastragenol-3-O-[-l-arabinopyranosyl(1 → 2)-β-d-xylopyranosyl]-6-O-β- d-xylopyranoside and cycloastragenol-3-O-[-l-arabinopyranosyl(1 → 2)-β-d-(3-O-acetyl)-xylopyranosyl]-6-O-β-d-xylopyranoside.     

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.     

A spirostane hexaglycoside from Agave cantala fruits.

A new steroidal glycoside, agaveside D, isolated from the fruits of Agave cantata was characterized as 3β-{- -rhamnopyranosyl-(1→2), β- -glycopyranosyl-(1→3)-β- -glucopyranosyl[β- -xylopyransoyl-(1→4)-- -rhamnopyranosyl-(1→2)]-β- -glucopyranosyl}-25R-5- spirostane on the basis of chemical degradation and spectrometry.     

Investigation of the acetolysis products of the sulphated polysaccharide of Aeodes ulvoidea

Investigation of the acetolysis products of the sulphated polysaccharide of the seaweed Aeodes ulvoidea led to the isolation and characterization of the following oligosaccharides: 3-O-α- -galactopyranosyl- -galactose (1), 3-O-(2-O-methyl-α- -galactopyranosyl)- -galactose (2), 4-O-β- -galactopyranosyl-2-O-methyl- -galactose (3), 4-O-β- -galactopyranosyl-2-O-methyl- -galactose (4), O-β- -galactopyranosyl-(1→4)-O-α- -galactopyranosyl-(1→3)- -galactose (5), O-α- -galactopyranosyl-(1→3)-O-β- -galactopyranosyl-(1→4)- -galactose (6), O-α- -galactopyranosyl-(1→3)-O-β- -galactopyranosyl-(1→4)-2-O-methyl- -galactose (7), O-(2-O-methyl-α- -galactopyranosyl)-(1→3)-O-β- -galactopyranosyl-(1→4)-2-O-methyl- -galactose (10), and O-α- -galactopyranosyl-(1→3)-O-β- -galactopyranosyl-(1→4)-O-α- -galactopyranosyl-(1→3)- -galactose. In addition, the isolation of a tetrasaccharide possessing alternating - and -galactose residues demonstrates the hitherto unexpected presence of -galactose in the polysaccharide. The structure of the polysaccharide is discussed.     

Enzymatic transglycosylation of xylose using a glycosynthase

The application of the hyperactive glycosynthase derived from Agrobacterium sp. β-glucosidase (AbgE358G-2F6) to the synthesis of xylo-oligosaccharides by using -d-xylopyranosyl fluoride as donor represents the first successful use of glycosynthase technology for xylosyl transfer. Transfer to p-nitrophenyl β-d-glucopyranoside yields di- and trisaccharide products with β-(1→4) linkages in 63% and 35% yields, respectively. By contrast, transfer to p-nitrophenyl β-d-xylopyranoside yielded the β-(1→3) linked disaccharide and β-d-Xyl-(1→4)-β-d-Xyl-(1→3)-β-d-Xyl-pNP as major products in 42% and 30% yields, respectively. Transfer of xylose to β-d-Xyl-(1→4)-β-d-Xyl-pNP yielded the β-(1→4) linked trisaccharide in 98% yield, thereby indicating that transfers to xylo-disaccharides occur with formation of β-(1→4) bonds. Xylosylation of carbamate-protected deoxyxylonojirimycin produced a mixture of di- and tri-‘saccharide’ products in modest yields.     

Capsular and extracellular polysaccharides from Rhizobium microsymbionts of Acacia decurrens

The capsular polysaccharide produced by a Rhizobium isolated from a root nodule of Acacia decurrens is composed of 3-O-methyl- -rhamnose: -rhamnose: - mannose: -glucose: -galacturonic acid in the molar ratios of 1:2:2:4:1. The extracellular polysaccharide is similarly constituted. Structural analyses indicate a decasaccharide repeating-unit in which the -rhamnosyl groups occur as single-unit side-chains. The 3-O-methyl- -rhamnosyl and one of the α- -rhamnosyl groups are (1→6)-linked to two of the -glucosyl residues. The other α- -rhamnosyl group is (1→4)-linked to the -galacturonic acid residue. The main-chain residues are all (1→3)-linked, and are partially identified as -(1→3)-α- -GalpA-(1→3)-α- -Manp- (1→3)-α- -Glcp-(1→3)-.     

Structural studies of gum arabic, the exudate polysaccharide from Acacia senegal

A 25.182-MHz ¹³C-n.m.r. spectrum of gum arabic allows unambiguous characterisation of all the C-1 resonances. These assignments have been confirmed by correlation of the modification of the intensities of these signals after controlled acid hydrolysis and characterisation of the released fragments. The resonances of the other carbons have been assigned through partial relaxed T₁ spectra of the polysaccharides obtained by graded degradation of the gum. These results indicate gum arabic to consist mainly of a (1→3)-β- -galactan core with (1→6)-β- -galacto-pyranosyl branches and with - -arabinofuranosyl-(1→3)-- -arabinofuranosyl and - -rhamnopyranosyl-(1→4)-β- -glucopyranosyluronic acid groups attached to positions 3 and 6, respectively, of the branch units.     

Synthesis of part of the antigenic repeating-unit of streptococcus pneumoniae type II

Condensation of methyl 4-O-acetyl-3-O-(2,3,4-tri-O-acetyl-α- -rhamnopyranosyl)-α- -rhamnopyranoside with 2,3,4,6-tetra-O-benzyl-α- -glucopyranosyl chloride gave a mixture of methyl O-[2,3,4,6-tetra-O-benzyl-α- (4) and -β- -glucopyranosyl]-(1→2)-O-[(2,3,4-tri-O-acetyl-α- -rhamnopyranosyl)-(1→3)]-4-O-acetyl-α- -rhamnopyranoside (9) in 43:7 proportion in 63% yield. After chromatographic separation, removal of the benzyl and acetyl groups gave methyl O-α- -glucopyranosyl-(1→2)-[O-α- -rhamnopyranosyl-(1→3)]-α- -rhamnopyranoside and the β anomer. Removal of benzyl groups of 4 was followed by tritylation, acetylation, and detritylation of the α- -glucopyranosyl group, and finally condensation with benzyl (2,3,4-tri-O-benzyl- -glucopyranosyl chloride)uronate gave a mixture of two tetrasaccharides (15 and 16), containing the α- and β- -glucopyranosyluronic acid groups in the ratio 81:19, and an overall yield of 71%. After chromatographic separation, alkaline hydrolysis and hydrogenation of 15 gave methyl O-α- -glucopyranosyluronic acid-(1→6)-O-α- -glucopyranosyl-(1→2)-[O-α- -rhamnopyranosyl-(1→3)]-α- -rhamnopyranoside. The β- anomer was obtained by similar treatment of 16. 6-O-α- -glucopyranosyluronic acid-α,β- -glucopyranose was synthesized as a model compound.     

Saponins from Albizzia lucida.

Three main saponins were isolated from the seeds of Albizzia lucida. Their structures were established by spectral analyses and chemical and enzymatic transformations as 3-O-[β- -xylopyranosyl(1→2)-- -arabinopyranosyl (1→6)] [β- -glucopyranosyl (1→2)] β- -glucopyranosyl echinocystic acid; 3-O-[- -arabinopyranosyl (1→6)][β- -glucopyranosyl (1→2)]-β- -glucopyranosyl echinocystic acid and 3-O-[β- -xylopyranosyl (1→2)-β- -fucopyranosyl (1→6)-2-acetamido-2-deoxy-β- -glucopyranosyl echinocystic acid, characterized as its methyl ester.     

Application of the hydro(solvo)thermal technique to the synthesis of metal carbonyl chalcogenide clusters Part 3. Synthesis, structural characterization, and spectroscopic studies of the clusters [{M(CO)4}n(MS4)] (M = Mo, W; N=1, 2)

The methanothermal reactions of M(CO)₆ (M = Mo, W) with Na₂S₂ gave a series of homonuclear clusters [{M(CO)₄}_n(MS₄)]²⁻ (M=Mo, W; N=1, 2), i.e. (Ph₄P)₂[(CO)₄Mo(MoS₄)] (I), (Ph₄P)₂[(CO)₄W(WS₄)] (II), (Ph₄P)₂[(CO)₄Mo(MoS₄)Mo(CO)₄] (III) and (Ph₄P)₂[(CO)₄W(WS₄)W(CO)₄] (IV). The two dimers, I and II, as well as the two trimers, III and IV, are isostructural to each other, respectively. All compounds crystallize in the triclinic space group with Z=2. The cell dimensions are: a=12.393(8), b=19.303(9), c=11.909(6) Å, =102.39(5), β=111.54(5), γ=73.61(5)°, V=2522(3) ų at T=23 °C for I; a=12.390(3), b=19.314(4), c=11.866(2) Å, =102.66(2), β=111.49(1), γ=73.40(2)°, V=2511(1) ų at T=23 °C for II; a=11.416(3), b=22.524(4), c=10.815(4) Å, =91.03(2), β=100.57(3), γ=88.96(2)°, V=2733(1) ų at T=−100 °C for III, a=11.498(1), b=22.600(4), c=10.864(3) Å, =90.92(2), β=100.85(1), γ=88.58(1)°, V=2771(2) ų at T=23 °C for IV. The dimers are each formed by the coordination of the tetrathiometalate as a bidentate chelating ligand to an M(CO)₄ fragment while addition of another M(CO)₄ fragment to the dimers results in the trimers. All compounds contain both tetrahedral and octahedral metal centers with the formal 6+ and 0 oxidation states, respectively.     

Catalytic function and affinity purification of site-directed mutant β-cyclodextrin glucanotransferase from alkalophilic Bacillus firmus var. alkalophilus

Subsites −3 and −7 in the active site of β-cyclodextrin glucanotransferase (β-CGTase) from alkalophilic Bacillus firmus var. alkalophilus were modified through site-directed mutagenesis to obtain novel mutant CGTases. Four mutant CGTases, H59Q, Y96M, 90-PPI-92, and Δ(154–160) were constructed and produced using a recombinant E. coli with a secretive expression system extracellularly. The secreted mutant β-CGTases were purified by one-step affinity adsorption chromatography using a β-cyclodextrin (CD) polymer as an adsorbent to nearly homogeneous purity. The catalytic activities were modified significantly compared to the wild-type. In particular, the Y96M and Δ(154–160) mutants increased cyclization activity around 1.5 times without any significant reduction of coupling and hydrolyzing activities. Meanwhile, the Y96M and Δ(154–160) mutants exhibited a much higher conversion yield into CDs from 28.6 to 39% without any recognizable change in the CD ratio. The conversion yield into linear maltooligosaccharides was also significantly reduced. The catalytic functions of subsites −3 and −7 in the active site of β-CGTase would appear to be related to the overall productivity of CDs rather than the product specificity.     

Sesquiterpene polyol esters from Celastrus rosthornianus

Two new sesquiterpene polyol esters with β-dihydroagarofuran skeleton were isolated from the root bark of Celastrus rosthornianus. Their structures were elucidated, mainly on the basis of spectral analyses, as 1 β-acetoxy-8β,9-dibenzoyloxy-6-hydroxy-2β(-methylbutanoyloxy)-β-dihydroagarofuran and 1β-acetoxy-9-benzoyloxy-8β-(β-furanocarbonyloxy)-6-hydroxy-2β(-methylbutanoyloxy)-β-dihydroagarofuran. The complete assignments of ¹³C NMR chemical shifts for both compounds on the basis of ¹H-¹³C chemical-shift correlation spectrum were also carried out.     

Botryosphaeran, a new substrate for the production of β-1,3-glucanases by Botryosphaeria rhodina and Trichoderma harzianum Rifai

Botryosphaeria rhodina and Trichoderma harzianum Rifai were grown on botryosphaeran (an exopolysaccharide (EPS) of the β-1,3;1,6-d-glucan type produced by B. rhodina) as sole carbon source with the objective of producing β-glucanases of the 1,3-type. Conditions for β-1,3-glucanase production by T. harzianum were examined by a statistical response surface method, and showed maximal enzyme production at 5 days growth in media containing 1.5 g/l of EPS. Good agreement was obtained between the experimental values of β-1,3-glucanase activity and the corresponding values predicted by the mathematical model. The crude β-1,3-glucanase preparations were active towards a number of different β-1,3-glucans and β-glucosides. The mycelium of B. rhodina also proved to be a good substrate for β-1,3-glucanase production by both fungal species.     

The structures of six urinary oligosaccharides that are characteristic for a patient with morquio syndrome type b

Morquio syndrome type B is an inherited, lysosomal storage disease characterised by a marked deficiency in acid β-d-galactosidase, while the 2-acetamido-2-deoxy-β-d-galactose 6-sulphate sulphatase activity is normal. Urinary oligosaccharides were studied in order to evaluate the effect of the diminished β-d-galactosidase activity on the catabolism of glycoconjugates and to compare their structures with those excreted by patients with GM₁-gangliosidosis. The following oligosaccharides were isolated: β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→6)-β-d-Manp-(1→4)- d-GlcpNAc (1), β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→6)-[α-d-Manp- (1→3)]-β-d-Manp-(1→4)-d-GlcpNAc (2a), β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)- α-d-Manp-(1→3)-[α-d-Manp-(1→6)]-β-d-Manp-(1→4)-d-GlcpNAc (2b), β-d-Galp- (1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→3)-[β-d-Galp-(1→4)-β-d-GlcpNAc-(1→ 2)-α-d-Manp-(1→6)]-β-d-Manp-(1→4)-d-GlcpNAc (3), β-d-Galp-(1→4)-β-d-Glcp- NAc-(1→2)-α-d-Manp-(1→3)-{β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-[β-d-Galp- (1→4)-β-d-GlcpNAc-(1→6)]-α-d-Manp-(1→6)}-β-d-Manp-(1→4)-d-GlcpNAc (4), β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→3)-[β-d-GlcpNAc-(1→4)]-[β- d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→6)]-β-d-Manp-(1→4)-d-Glcp- NAc (5). Significant differences between Morquio syndrome type B and GM₁-gangliosidosis have been observed, with regard to the excretion rate and the specific structures of urinary oligosaccharides. Compounds 2a, 2b, and 5 are novel members of the series of oligosaccharides isolated from the urine of patients with inherited, lysosomal storage diseases.