Structure of the O-Specific polysaccharide from Shewanella japonica KMM 3601 containing 5,7-Diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulosonic acid

Structure of the O-specific polysaccharide chain of the lipopolysaccharide (LPS) of Shewanella japonica KMM 3601 was elucidated. The initial and O-deacylated LPS as well as a trisaccharide representing the O-deacetylated repeating unit of the O-specific polysaccharide were studied by sugar analysis along with ¹H and ¹³C NMR spectroscopy. The polysaccharide was found to contain a rare higher sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-d-talo-non-2-ulosonic acid (a derivative of 4-epilegionaminic acid, 4eLeg). The following structure of the trisaccharide repeating unit was established: →4)-α-4eLegp5Ac7Ac-(2→4)-β-d-GlcpA3Ac-(1→3)-β-d-GalpNAc-(1→.     

Structural analysis of the exopolysaccharide produced by <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> ST1 solely by NMR spectroscopy

The use of lactic acid bacteria in fermentation of milk results in favorable physical and rheological properties due to in situ exopolysaccharide (EPS) production. The EPS from S. thermophilus ST1 produces highly viscous aqueous solutions and its structure has been investigated by NMR spectroscopy. Notably, all aspects of the elucidation of its primary structure including component analysis and absolute configuration of the constituent monosaccharides were carried out by NMR spectroscopy. An array of techniques was utilized including, inter alia, PANSY and NOESY-HSQC TILT experiments. The EPS is composed of hexasaccharide repeating units with the following structure: → 3)[α-d-Glcp-(1 → 4)]-β-d-Galp-(1 → 4)-β-d-Glcp-(1 → 4)[β-d-Galf-(1 → 6)]-β-d-Glcp-(1 → 6)-β-d-Glcp-(1 →, in which the residues in square brackets are terminal groups substituting backbone sugar residues that consequently are branch-points in the repeating unit of the polymer. Thus, the EPS consists of a backbone of four sugar residues with two terminal sugar residues making up two side-chains of the repeating unit. The molecular mass of the polymer was determined using translational diffusion experiments which resulted in M_w = 62 kDa, corresponding to 64 repeating units in the EPS.     

Characterization of a novel ginsenoside-hydrolyzing α-l-arabinofuranosidase, AbfA, from Rhodanobacter ginsenosidimutans Gsoil 3054T

The gene encoding an α-l-arabinofuranosidase that could biotransform ginsenoside Rc {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-[α-l-arabinofuranosyl-(1–6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to ginsenoside Rd {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-β-d-glucopyranosyl-20(S)-protopanaxadiol} was cloned from a soil bacterium, Rhodanobacter ginsenosidimutans strain Gsoil 3054^T, and the recombinant enzyme was characterized. The enzyme (AbfA) hydrolyzed the arabinofuranosyl moiety from ginsenoside Rc and was classified as a family 51 glycoside hydrolase based on amino acid sequence analysis. Recombinant AbfA expressed in Escherichia coli hydrolyzed non-reducing arabinofuranoside moieties with apparent K _m values of 0.53 ± 0.07 and 0.30 ± 0.07 mM and V _max values of 27.1 ± 1.7 and 49.6 ± 4.1 μmol min⁻¹ mg⁻¹ of protein for p-nitrophenyl-α-l-arabinofuranoside and ginsenoside Rc, respectively. The enzyme exhibited preferential substrate specificity of the exo-type mode of action towards polyarabinosides or oligoarabinosides. AbfA demonstrated substrate-specific activity for the bioconversion of ginsenosides, as it hydrolyzed only arabinofuranoside moieties from ginsenoside Rc and its derivatives, and not other sugar groups. These results are the first report of a glycoside hydrolase family 51 α-l-arabinofuranosidase that can transform ginsenoside Rc to Rd.     

A review of acacic acid-type saponins from Leguminosae-Mimosoideae as potent cytotoxic and apoptosis inducing agents

The aim of this review is to highlight updated results on the biologically active saponins from Leguminosae-Mimosoideae. Acacic acid-type saponins (AATS), is a class of very complex glycosides possessing a common aglycon unit of the oleanane-type (acacic acid = 3β, 16α, 21β trihydroxy-olean-12-en-28 oic acid), having various oligosaccharide moieties at C-3 and C-28 and an acyl group at C-21. About sixty molecules of this type have been actively explored in recent years from Leguminosae family, from a chemical point of view and some fifty were reported to possess cancer related activities. These include cytotoxic/antitumor, immunomodulatory, antimutagenic, and apoptosis inducing properties and appear to depend on the acylation and esterification by different moieties at C-21 and C-28 of the acacic acid-type aglycone. One can observe that the (6S) configuration of the outer monoterpenyl moiety (MT) seems more potent in mediating high cytotoxicity than its (6R) isomer. Furthermore, the trisaccharide moiety {β-d-Xylopyranosyl-(1→2)-β-d-Fucopyranosyl-(1→6)- N-Acetamido 2-β-d-Glucopyranosyl-} at C-3, the tetrasaccharide moiety {β-d-Glucopyranosyl-(1→3)-[α-L-Arabinofuranosyl-(1→4)]-α-l-Rhamnopyranosyl-(1→2)-β-d-Glucopyranosyl} at C-28 of the aglycone, and the inner MT hydroxylated at its C-9, having a (6S) configuration can be important substituent patterns for the induction of apoptosis of AATS. Because of their interesting cytotoxic/apoptosis inducing activity, some AATS can be useful in the search for new potential antitumor agents from Fabaceae. Furthermore, the sequence 28-O-{Glc-(1→3)-[Ara_f-(1→4)]-Rha-(1→2)-Glc-Acacic acid}, often encountered in the genera Acacia, Albizia, Archidendron, and Pithecellobium may represent a chemotaxonomic marker of the Mimosoideae subfamily.     

Temporal and spatial appearance of wall polysaccharides during cellularization of barley (Hordeum vulgare) endosperm

Barley endosperm begins development as a syncytium where numerous nuclei line the perimeter of a large vacuolated central cell. Between 3 and 6 days after pollination (DAP) the multinucleate syncytium is cellularized by the centripetal synthesis of cell walls at the interfaces of nuclear cytoplasmic domains between individual nuclei. Here we report the temporal and spatial appearance of key polysaccharides in the cell walls of early developing endosperm of barley, prior to aleurone differentiation. Flowering spikes of barley plants grown under controlled glasshouse conditions were hand-pollinated and the developing grains collected from 3 to 8 DAP. Barley endosperm development was followed at the light and electron microscope levels with monoclonal antibodies specific for (1→3)-β-d-glucan (callose), (1→3,1→4)-β-d-glucan, hetero-(1→4)-β-d-mannans, arabino-(1→4)-β-d-xylans, arabinogalactan-proteins (AGPs) and with the enzyme, cellobiohydrolase II, to detect (1→4)-β-d-glucan (cellulose). Callose and cellulose were present in the first formed cell walls between 3 and 4 DAP. However, the presence of callose in the endosperm walls was transient and at 6 DAP was only detected in collars surrounding plasmodesmata. (1→3,1→4)-β-d-Glucan was not deposited in the developing cell walls until approximately 5 DAP and hetero-(1→4)-β-d-mannans followed at 6 DAP. Deposition of AGPs and arabinoxylan in the wall began at 7 and 8 DAP, respectively. For arabinoxylans, there is a possibility that they are deposited earlier in a highly substituted form that is inaccessible to the antibody. Arabinoxylan and heteromannan were also detected in Golgi and associated vesicles in the cytoplasm. In contrast, (1→3,1→4)-β-d-glucan was not detected in the cytoplasm in endosperm cells; similar results were obtained for coleoptile and suspension cultured cells.     

Synthesis of p-nitrophenyl sulfated disaccharides with beta-D-(6-sulfo)-GlcNAc units using beta-N-acetylhexosaminidase from Aspergillus oryzae in a transglycosylation reaction

When α-d-GlcNAc-OC₆H₄NO₂ -p and β-d-(6-sulfo)-GlcNAc-OC₆H₄NO₂-p (2) were used as substrates, β-N-acetylhexosaminidase from Aspergillus oryzae transferred the β-d-(6-sulfo)-GlcNAc(unit from 2 to α-d-GlcNAc-OC₆H₄NO₂ -p to afford β-d-(6-sulfo)-GlcNAc-(1→4)-α-d-GlcNAc-OC₆H₄NO₂-p (3) in a yield of 94% based on the amount of donor, 2, added. β-d-(6-sulfo)-GlcNAc-(1→4)-α-d-Glc-OC₆H₄NO₂-p (4) was obtained with α-d-Glc-OC₆H₄NO₂ -p as acceptor in a similar manner. With a reaction mixture of 2 and β-d-GlcNAc-OC₆H₄NO₂-p (1) in a molar ratio of 6:1, the enzyme mediated the transfer of β-d-GlcNAc from 1 to 2, affording disaccharide β-d-GlcNAc-(1→4)-β-(6-sulfo)-d-GlcNAc-OC₆H₄NO₂-p (5) in a yield of 13% based on the amount of 1 added.     

Production, purification and structural characterization of an exopolysaccharide produced by a probiotic Lactobacillus plantarum MTCC 9510

Exopolysaccharides (EPS) from lactic acid bacteria contribute to specific rheology and texture of fermented milk products and finds applications even in non-dairy foods and in therapeutics. Box-Behnken model of response surface methodology (RSM) was employed to formulate the production medium for exopolysaccharide (EPS). FT-IR spectral analysis of the purified EPS from Lactobacillus plantarum MTCC 9510 revealed prominent characteristic groups corresponding to polyhydric alcohols. The degradation temperature (Td) of the polysaccharide was found to be 260°C with the help of thermo gravimetric analysis (TGA). Structure elucidation of the EPS showed that it consists of a trisaccharide repeating unit of α-d-glucose, β-d-glucose and α-d-mannose.     

Microbial transformation of ginsenoside Rb<Subscript>1</Subscript> by <Emphasis Type="Italic">Acremonium strictum</Emphasis>

Preparative-scale fermentation of ginsenoside Rb₁ (1) with Acremonium strictum AS 3.2058 gave three new compounds, 12β-hydroxydammar-3-one-20 (S)-O-β-d-glucopyranoside (7), 12β, 25-dihydroxydammar-(E)-20(22)-ene-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (8), and 12β, 20 (R), 25-trihydroxydammar-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (9), along with five known compounds, ginsenoside Rd (2), gypenoside XVII (3), ginsenoside Rg₃ (4), ginsenoside F₂ (5), and compound K (6). The structural elucidation of these metabolites was based primarily on one- and two-dimensional nuclear magnetic resonance and high-resolution electron spray ionization mass spectra analyses. Among these compounds, 2–6 are also the metabolites of ginsenoside Rb₁ in mammals. This result demonstrated that microbial culture parallels mammalian metabolism; therefore, A. strictum might be a useful tool for generating mammalian metabolites of related analogs of ginsenosides for complete structural identification and for further use in pharmaceutical research in this series of compounds. In addition, the biotransformation kinetics was also investigated.     

Separation of β-d-galactosidases in rabbit tissues: Genetics of neutral β-d-galactosidase

Three different types of β-d-galactosidase (EC 3.2.1.23) could be distinguished in rabbit tissues using electrophoretic procedures. (1) Acid β-d-galactosidase with a low mobility and maximal activity atpH 3–5 was found in the particulate fraction of various tissue homogenates. This enzyme hydrolyzed 4-methylumbelliferyl-d-galactoside, but no activity against other glycoside substrates could be demonstrated. The enzyme was inhibited by galactono-(1 → 4)-lactone. (2) Lactose-hydrolyzing β-d-galactosidase with an intermediate mobility was found only in juvenile small intestine. Most of the activity was found in the particulate fraction of the cell. The enzyme hydrolyzed several other synthetic glycoside substrates besides lactose. It was most active atpH 5–6 and strongly inhibited by glucono-(1 → 5)-lactone but not much affected by galactono-(1 → 4)-lactone. (3) Neutral β-d-galactosidase with a fast mobility and maximal activity atpH 6–8 was found in the soluble fraction of homogenates from liver, kidney, and small intestine. This enzyme also showed a broad substrate specificity; it possessed activity against aryl-β-d-glucoside, -fucoside, and -galactoside substrates but not against lactose. The enzyme was strongly inhibited by glucono-(1 → 5)-lactone and (less) by galactone-(1 → 4)-lactone. Neutral β-d-galactosidase and neutral β-d-glucosidase (EC 3.2.1.21) are probably identical enzymes in the rabbit. Individual variation, in both electrophoretic mobility and activity, was found for neutral β-d-galactosidase. Genetic analysis of the electrophoretic variants revealed that two alleles at an autosomal locus are responsible for this variation. This investigation was supported in part by Public Health Service Grant RR-00251 from the Division of Research Resources and by funds of the University of Utrecht.     

Structure of the O-antigen and characterization of the O-antigen gene cluster of Escherichia coli O108 containing 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic (8-epilegionaminic) acid

On mild acid degradation of the lipopolysaccharide of Escherichia coli O108, the O-polysaccharide was isolated and studied by sugar analysis and one- and two-dimensional ¹H- and ¹³C-NMR spectroscopy. The polysaccharide was found to contain an unusual higher sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic acid (di-N-acetyl-8-epilegionaminic acid, 8eLeg5Ac7Ac). The following structure of the tetrasaccharide repeating unit of the polysac-charide was established: →4)-α-8eLegp5Ac7Ac-(2→6)-α-D-Galp-(1→3)-α-L-FucpNAc-(1→3)-α-D-GlcpNAc-(1→. Functions of the E. coli O108 antigen biosynthetic genes, including seven putative genes for synthesis of 8eLeg5Ac7Ac, were assigned by sequencing the O-antigen gene cluster along with comparison with gene databases and known biosynthetic pathways for related nonulosonic acids.     

Discovery of nigerose phosphorylase from Clostridium phytofermentans

A novel phosphorylase from Clostridium phytofermentans belonging to the glycoside hydrolase family (GH) 65 (Cphy1874) was characterized. The recombinant Cphy1874 protein produced in Escherichia coli showed phosphorolytic activity on nigerose in the presence of inorganic phosphate, resulting in the release of d-glucose and β-d-glucose 1-phosphate (β-G1P) with the inversion of the anomeric configuration. Kinetic parameters of the phosphorolytic activity on nigerose were k _cat = 67 s⁻¹ and K _m = 1.7 mM. This enzyme did not phosphorolyze substrates for the typical GH65 enzymes such as trehalose, maltose, and trehalose 6-phosphate except for a weak phosphorolytic activity on kojibiose. It showed the highest reverse phosphorolytic activity in the reverse reaction using d-glucose as the acceptor and β-G1P as the donor, and the product was mostly nigerose at the early stage of the reaction. The enzyme also showed reverse phosphorolytic activity, in a decreasing order, on d-xylose, 1,5-anhydro-d-glucitol, d-galactose, and methyl-α-d-glucoside. All major products were α-1,3-glucosyl disaccharides, although the reaction with d-xylose and methyl-α-d-glucoside produced significant amounts of α-1,2-glucosides as by-products. We propose 3-α-d-glucosyl-d-glucose:phosphate β-d-glucosyltransferase as the systematic name and nigerose phosphorylase as the short name for this Cphy1874 protein.     

Transglycosidase activity of Bifidobacterium adolescentis DSM 20083 α-galactosidase

Bifidobacterium adolescentis, a gram-positive saccharolytic bacterium found in the human colon, can, alongside other bacteria, utilise stachyose in vitro thanks to the production of an α-galactosidase. The enzyme was purified from the cell-free extract of Bi. adolescentis DSM 20083^T. It was found to act with retention of configuration (α→α), releasing α-galactose from p-nitrophenyl galactoside. This hydrolysis probably operates with a double-displacement mechanism, and is consistent with the observed glycosyltransferase activity. As α-galactosides are interesting substrates for bifidobacteria, we focused on the production of new types of α-galactosides using the transgalactosylation activity of Bi. adolescentisα-galactosides. Starting from melibiose, raffinose and stachyose oligosaccharides could be formed. The transferase activity was highest at pH 7 and 40 °C. Starting from 300 mM melibiose a maximum yield of 33% oligosaccharides was obtained. The oligosaccharides formed from melibiose were purified by size-exclusion chromatography and their structure was elucidated by NMR spectroscopy in combination with enzymatic degradation and sugar linkage analysis. The trisaccharide α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp and tetrasaccharide α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp were identified, and this indicates that the transgalactosylation to melibiose occurred selectively at the C-6 hydroxyl group of the galactosyl residue. The trisaccaride α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp formed could be utilised by various intestinal bacteria, including various bifidobacteria, and might be an interesting pre- and synbiotic substrate. Received: 15 March 1999 / Received revision: 8 June 1999 / Accepted: 11 June 1999     

Structural characterisation and biological activities of a unique type β-<Emphasis Type="SmallCaps">d</Emphasis>-glucan obtained from <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

A β-d-glucan obtained from Aureobasidium pullulans (AP-FBG) exhibits various biological activities: it exhibits antitumour and antiosteoporotic effects and prevents food allergies. An unambiguous structural characterisation of AP-FBG is still awaited. The biological effects of β-d-glucan are known to depend on its primary structures, conformation, and molecular weight. Here, we elucidate the primary structure of AP-FBG by NMR spectroscopy, and evaluate its biological activities. Its structure was shown to comprise a mixture of a 1-3-β-d-glucan backbone with single 1-6-β-d-glucopyranosyl side-branching units every two residues (major structure) and a 1-3-β-d-glucan backbone with single 1-6-β-d-glucopyranosyl side-branching units every three residues (minor structure). Furthermore, this β-d-glucan exhibited immunostimulatory effects such as the accumulation of immune cells and priming effects against enterobacterium. To our knowledge, 1-3-β-glucans like AP-FBG with such a high number of 1-6-β-glucopyranosyl side branching have a unique structure; nevertheless, many 1-3-β-glucans were isolated from various sources, e.g. fungi, bacteria, and plants.     

Adhesion forces in the self-recognition of oligosaccharide epitopes of the proteoglycan aggregation factor of the marine sponge <Emphasis Type="Italic">Microciona prolifera</Emphasis>

Cell aggregation in the marine sponge Microciona prolifera is mediated by a multimillion molecular-mass aggregation factor, termed MAF. Earlier investigations revealed that the cell aggregation activity of MAF depends on two functional domains: (i) a Ca²⁺-independent cell-binding domain and (ii) a Ca²⁺-dependent proteoglycan self-interaction domain. Structural analysis of involved carbohydrate fragments of the proteoglycan in the self-association established a sulfated disaccharide β-d-GlcpNAc3S-(1→3)-α-l-Fucp and a pyruvated trisaccharide β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp. Recent UV, SPR, and TEM studies, using BSA conjugates and gold nanoparticles of the synthetic sulfated disaccharide, clearly demonstrated self-recognition on the disaccharide level in the presence of Ca²⁺-ions. To determine binding forces of the carbohydrate–carbohydrate interactions for both synthetic MAF oligosaccharides, atomic force microscopy (AFM) studies were carried out. It turned out that, in the presence of Ca²⁺-ions, the force required to separate the tip and sample coated with a self-assembling monolayer of thiol-spacer-containing β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH₂)₃S(CH₂)₆S- was found to be quantized in integer multiples of 30 ± 6 pN. No binding was observed between the two monolayers in the absence of Ca²⁺-ions. Cd²⁺-ions could partially induce the self-interaction. In contrast, similar AFM experiments with thiol-spacer-containing β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH₂)₃S(CH₂)₆S- did not show a binding in the presence of Ca²⁺-ions. Also TEM experiments of gold nanoparticles coated with the pyruvated trisaccharide could not make visible aggregation in the presence of Ca²⁺-ions. It is suggested that the self-interaction between the sulfated disaccharide fragments is stronger than that between the pyruvated trisaccharide.     

Dynamics of ginsenoside biosynthesis in suspension culture of <Emphasis Type="Italic">Panax quinquefolium</Emphasis>

Biosynthesis of six saponins (ginsenosides) in suspension culture of P. quinquefolium Z₅ was investigated. Ginsenoside content in biomass reached the highest level, nearly 30 mg g⁻¹ d.w., between 25 and 30 days of the culture. Saponins were synthesized simultaneously with cell growth but their synthesis rate was not proportional to the growth rate. During the phase of rapid biomass multiplication, after which biomass reached 90% of its maximum yield, only half examined ginsenosides was produced. The second half of the final saponins yield was produced during the slow growth phase, in which only 10% of biomass was grown. During the intensive growth phase the productivity of six saponins examined per biomass (dry weight) unit was 3.4 μg mg⁻¹ d.w. day⁻¹, however, this parameter calculated for slow growth phase reached nearly 30 μg mg⁻¹ d.w. day⁻¹. There were differences in increase of the contents of six saponins determined in biomass, and it was the highest for saponins Re (20(S)-protopanaxatriol-6-[O-α-l-rhamnopyranosyl(1 → 2)-β-d-glucopyranoside]-20-O-β-d-glucopyranoside) and Rg₁ (20(S)-protopanaxatriol-6,20-di-O-β-d-glucoside).     

α-(4-O-Methyl)-d-glucuronidase activity produced by the rumen anaerobic fungusPiromonas communis: A study of selected properties

The rumen anaerobic fungusPiromonas communis, unlike the rumen anaerobic fungiNeocallimastix frontalis andNeocallimastix patriciarum, produced extracellular α-(4-O-methyl)-d-glucuronidase when grown in cultures containing filter-paper, barley straw, birchwood xylan or birchwood sawdust as carbon source. The highest concentration of enzyme was produced in cultures containing birchwood sawdust. The aldobiouronic acidO-α-(4-O-methyl-d-glucopyran-osyluronic acid)-(1 → 2)-d-xylopyranose (MeGlcAXyl) was the best substrate of those tested: the aldotriouronic acidO-α-(4-O-methyl-d-glucopyranosyluronic acid (1 → 2)-O-\-d-xylopyranosyl-(1 → 4)-d-xylopyranose (MeGlcAXyl₂) and the aldotetraouronic acidO-α-(4-O-methyl-d-glucopyranosyluronic acid)-(1 → 2)-O-\-d-xylopyranosyl-(1 → 4)-O-\-d-xylopyranosyl-(1 → 4)-d-xylopyranose (MeGlcAXyl₃) were also attacked but the rate fell as the degree of polymerisation increased. When the same substituted xylooligosaccharides were reduced to the corresponding alditols the enzyme activity disappeared. Similarly,p-nitrophenyl-α-d-glucuronide was not a substrate. Remarkably, the relative rates of attack shown by the α-(4-O-methyl)-d-glucuronidase on the aldouronic acids and on xylans extracted from birchwood, oat spelts and oat straw differed according to the carbon source used to produce the enzyme. The α-(4-O-methyl)-d-glucuronidase had a pH optimum of 5.5 and a temperature optimum of 50°C. On gel filtration the enzyme was shown to be associated with proteins covering the range 100–300 kDa, but a major peak of activity in the column effluent appeared to have a molecular mass of 103 kDa.     

Anionic derivatives of xyloglucan function as acceptor but not donor substrates for xyloglucan endotransglucosylase activity

Tamarind xyloglucan was oxidised by reaction with sodium hypochlorite in the presence of 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO). Galactose residues and non-xylosylated glucose residues were thus converted into galacturonic and glucuronic acid residues, respectively, producing an anionic polysaccharide. Acid hydrolysis of oxidised xyloglucan yielded two aldobiouronic acids, deduced to be β-d-GalpA-(1→2)-d-Xyl and β-d-GlcpA-(1→4)-d-Glc. Anionic xyloglucan had a decreased ability to hydrogen-bond to cellulose and to complex with iodine. It was almost totally resistant to digestion by cellulase [endo-(1→4)-β-glucanase] and did not serve as a donor substrate for xyloglucan endotransglucosylase (XET) activity. Like several other anionic polysaccharides, it promoted XET activity when unmodified (non-ionic) xyloglucan was used as donor substrate. Anionic xyloglucan may mimic polyanions whose presence in the plant cell wall promotes the action of endogenous XTH proteins. NaOCl with TEMPO oxidised the heptasaccharide, XXXG, to form XXX-glucarate, which did serve as an acceptor substrate although at a rate approximately fourfold less than XXXG itself. Anionic derivatives of xyloglucan, acting as acceptor but not donor substrates, may be valuable tools for exploring the biological roles of XTHs in the integration versus the re-structuring of xyloglucan in the plant cell wall.     

Inhibitory effects of arbutin-β-glycosides synthesized from enzymatic transglycosylation for melanogenesis

To develop a new skin whitening agent, arbutin-β-glycosides were synthesized and evaluated for their melanogenesis inhibitory activities. Three active compounds were synthesized via the transglycosylation reaction of Thermotoga neapolitana β-glucosidase and purified by recycling preparative HPLC. As compared with arbutin (IC₅₀ = 6 mM), the IC₅₀ values of these compounds were 8, 10, and 5 mM for β-d-glucopyranosyl-(1→6)-arbutin, β-d-glucopyranosyl-(1→4)-arbutin, and β-d-glucopyranosyl-(1→3)-arbutin, respectively. β-d-Glucosyl-(1→3)-arbutin also exerted the most profound inhibitory effects on melanin synthesis in B16F10 melanoma cells. Melanin synthesis was inhibited to a significant degree at 5 mM, at which concentration the melanin content was reduced to below 70% of that observed in the untreated cells. Consequently, β-d-glucopyranosyl-(1→3)-arbutin is a more effective depigmentation agent and is also less cytotoxic than the known melanogenesis inhibitor, arbutin.     

Characterization and antimicrobial activity of 4-(β-D-glucopyranosyl-1→4-α-L-rhamnopyranosyloxy)-benzyl thiocarboxamide; a novel bioactive compound from Moringa oleifera seed extract

Antimicrobial activity of crude seed extract of Moringa oleifera was investigated by thin layer chromatography bioassay against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Cladosporium cladosporioides, and Penicillium sclerotigenum; most of them were prominently inhibited by an isolate with R _F 0.92–0.96. Characterization and identification of the extract revealed the occurrence of three bioactive compounds: 4-(α-l-rhamnopyranosyloxy)benzyl isothiocyanate, methyl N-4-(α-l-rhamnopyranosyloxy) benzyl carbamate (both known compounds), and 4-(β-d-glucopyranosyl-1→4-α-l-rhamnopyranosyloxy)-benzyl thiocarboxamide, existence of which in any Moringa spp. or plant is reported for the first time. The UV spectrum of the novel compound showed maximum absorption at 273 and 225 nm in MeOH while the IR spectrum revealed several characteristic bands at 3100, 2900, 1700, 1500, 1300, 1100 and 1000 cm⁻¹. The ¹H-NMR showed signals at 1.2 and 3.77 ppm and the ¹³C-NMR presented signals at 155, 122, 91.7 and 98.4 ppm. All the compounds at 5 mg/L had very high bactericidal activity against some of test pathogens even at contact period 1–2 h. 4-(β-d-Glucopyranosyl-1→4-α-l-rhamnopyranosyloxy)benzyl thiocarboxamide was the most potent, with 99.2 % inhibition toward Shigella dysenteriae and 100 % toward Bacillus cereus, E. coli and Salmonella typhi within 4 h of contact.     

Induction of xylanase in Aspergillus tamarii by methyl β-d-xyloside

Aspergillus tamarii produced extracellular xylanase and intracellular β-xylosidase inductively in washed glucose-grown mycelia incubated with xylan and methyl β-d-xyloside, a synthetic glycoside. Methyl β-d-xyloside was a more effective inducer than xylan at the same concentration for both enzymes. Glucose and cycloheximide were found to inhibit xylanase production by methyl β-d-xyloside. Methyl β-d-xyloside was hydrolyzed to xylose by mycelial extract in vitro. Received: 23 May 1996 / Received revision: 5 September 1996 / Accepted: 13 October 1996