The molecular structure and solution conformation of an acidic heteropolysaccharide from Auricularia auricula-judae.

A water soluble acidic heteropolysaccharide named WAF was isolated from Auricularia auricula-judae by extracting with 0.9% NaCl solution. By using gas chromatography, gas chromatography-mass spectrometry, and NMR, its chemical structure was determined to be composed of a backbone of α-(1→3)-linked D-mannopyranose residues with pendant side groups of ß-D-xylose, ß-D-glucose, or ß-D-glucuronic acid at position O6 or O2. Six fractions prepared from WAF with a weight-average molecular mass (M(w)) between 5.9 × 104 and 64.7 × 104 g/mol were characterized with laser light scattering and viscometry in 0.1M NaCl at 25°C. The dependence of intrinsic viscosity ([η]) and radius of gyration (R(g)) on M(w) for this polysaccharide were found to be [η] = 1.79 × 10⁻³ M(w) °.96 cm³ g⁻¹ and R(g) = 6.99 × 10⁻² M(w) (0.54) nm. The molar mass per unit contour length (M(L)) and the persistence length (L(p)) were estimated to be 1124 nm⁻¹ and 11 nm, respectively. The WAF exhibited a semirigid character typical of linear polysaccharides. Molecular modeling was then used to predict the ordered and disordered states of WAF; the simulated M(L) and L(p) were however much smaller than the experimental values. Taken altogether, the results suggested that WAF formed a duplex in solution.

Homogeneous suspensions of individualized microfibrils from TEMPO-catalyzed oxidation of native cellulose.

Never-dried native celluloses (bleached sulfite wood pulp, cotton, tunicin, and bacterial cellulose) were disintegrated into individual microfibrils after oxidation mediated by the 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) radical followed by a homogenizing mechanical treatment. When oxidized with 3.6 mmol of NaClO per gram of cellulose, almost the totality of sulfite wood pulp and cotton were readily disintegrated into long individual microfibrils by a treatment with a Waring Blendor, yielding transparent and highly viscous suspensions. When observed by transmission electron microscopy, the wood pulp and cotton microfibrils exhibited a regular width of 3-5 nm. Tunicin and bacterial cellulose could be disintegrated by sonication. A bulk degree of oxidation of about 0.2 per one anhydroglucose unit of cellulose was necessary for a smooth disintegration of sulfite wood pulp, whereas only small amounts of independent microfibrils were obtained at lower oxidation levels. This limiting degree of oxidation decreased in the following order: sulfite wood pulp > cotton > bacterial cellulose, tunicin.

Chemoenzymatic synthesis of 6omega -modified maltooligosaccharides from cyclodextrin derivatives.

Regioselectively substituted maltooligosaccharides were prepared by enzymatic transformation of modified cyclodextrins by using simultaneously two different enzymes: cyclodextrin glucanotransferase (CGTase) and amyloglucosidase. Oligosaccharides were obtained in very good yields and their structures were identified by 1D and 2D NMR spectroscopy. These results provided new information about the specificity of the catalytic sites of CGTase and amyloglucosidase. They also offered new ways for the synthesis of regioselectively modified maltooligosaccharides.

Isolation and characterization of xylans from seed pericarp of Argania spinosa fruit.

Hemicellulose-type polysaccharides were isolated from the pericarp of seeds of Argania spinosa (L.) Skeels fruit by sequential alkaline extractions and fractionated by precipitation. Water soluble and water insoluble fractions were obtained, purified and characterized by sugar analysis and 1H and 13C NMR spectroscopy. The water soluble fractions were assumed to be (4-O-methyl-D-glucurono)-D-xylans, with 4-O-methyl-D-glucopyranosyluronic acid groups linked to C-2 of a (1-->4)-beta-D-xylan. The 1H NMR spectrum showed that the water soluble xylans have, on average, one non-reducing terminal residue of 4-O-methyl-D-glucuronic acid for every seven xylose units. The water insoluble fractions consisted of a neutral xylan with linear (1-->4)-beta-D-xylopyranosyl units.

Role of the two catalytic domains of DSR-E dextransucrase and their involvement in the formation of highly alpha-1,2 branched dextran.

The dsrE gene from Leuconostoc mesenteroides NRRL B-1299 was shown to encode a very large protein with two potentially active catalytic domains (CD1 and CD2) separated by a glucan binding domain (GBD). From sequence analysis, DSR-E was classified in glucoside hydrolase family 70, where it is the only enzyme to have two catalytic domains. The recombinant protein DSR-E synthesizes both alpha-1,6 and alpha-1,2 glucosidic linkages in transglucosylation reactions using sucrose as the donor and maltose as the acceptor. To investigate the specific roles of CD1 and CD2 in the catalytic mechanism, truncated forms of dsrE were cloned and expressed in Escherichia coli. Gene products were then small-scale purified to isolate the various corresponding enzymes. Dextran and oligosaccharide syntheses were performed. Structural characterization by (13)C nuclear magnetic resonance and/or high-performance liquid chromatography showed that enzymes devoid of CD2 synthesized products containing only alpha-1,6 linkages. On the other hand, enzymes devoid of CD1 modified alpha-1,6 linear oligosaccharides and dextran acceptors through the formation of alpha-1,2 linkages. Therefore, each domain is highly regiospecific, CD1 being specific for the synthesis of alpha-1,6 glucosidic bonds and CD2 only catalyzing the formation of alpha-1,2 linkages. This finding permitted us to elucidate the mechanism of alpha-1,2 branching formation and to engineer a novel transglucosidase specific for the formation of alpha-1,2 linkages. This enzyme will be very useful to control the rate of alpha-1,2 linkage synthesis in dextran or oligosaccharide production.

An arabinogalactan from the skin of Opuntia ficus-indica prickly pear fruits.

The cold-water extract from the skin of Opuntia ficus-indica fruits was fractionated by anion-exchange chromatography. The major fraction, which was purified by size exclusion chromatography, consisted of a polysaccharide composed of galactose and arabinose residues in the ratio 6.3:3.3, with traces of rhamnose, xylose and glucose, but no uronic acid. The results of methylation analysis, supported by (13)C NMR spectroscopy, indicated that this polysaccharide corresponded to an arabinogalactan having a backbone of (1-->4)-linked beta-D-galactopyranosyl residues with 39.5% of these units branched at O-3. The side-groups consisted either of single L-arabinofuranosyl units or L-arabinofuranosyl alpha-(1-->5)-linked disaccharides. This polysaccharide is thus an arabinogalactan that can be classified in the type I of the arabinogalactan family.

Preparation of cellouronic acids and partially acetylated cellouronic acids by TEMPO/NaClO oxidation of water-soluble cellulose acetate.

Water-soluble cellulose acetates with a degree of substitution (DS) of 0.5, prepared by partial deacetylation of cellulose acetate of DS=2.5, were oxidized with catalytic amount of 2,2,6,6,-tetramethyl-1-piperidinyloxy radical (TEMPO), sodium hypochlorite, and sodium bromide to provide useful cellouronic acids. The oxidation was conducted at a constant pH of 10 and at 2 degrees C to avoid the occurrence of side products. Whereas only the primary hydroxyl groups of cellulose acetate were oxidized, a variable degree of oxidation (DO) resulted in a range of 0.33 to 1.0, depending on the concentration in sodium hypochlorite. Thus, polyglucuronic acid as well as partially acetylated cellouronic acid, having a range of DO were obtained.

TEMPO-mediated oxidation of cellulose III.

Various cellulose samples converted into cellulose III by two different ammonia treatments, either liquid or gaseous, were reacted with catalytic amounts of 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO), sodium hypochlorite, and sodium bromide in water. A substantial increase in the reactivity of cellulose III samples was observed in comparison to those in cellulose I, and a relationship between oxidation conditions and cellulose primary hydroxyl groups accessibility was directly established. For the characterization, we have used several methods, mainly (13)C NMR, methylene blue adsorption, FTIR, and conductometric titration. In all samples, the primary alcohol groups were selectively oxidized into carboxyl groups, provided the sodium hypochlorite is added dropwise and the reaction is performed at constant pH 10.

First evidence for the presence of weddellite crystallites in Opuntia ficus indica parenchyma.

Calcium oxalate crystallites occur very often in the plants tissues and their role is still poorly known. We report here the experimental protocol leading to the isolation of two forms of calcium oxalate crystallites differing in their hydration level in the parenchymal tissues of Opuntia ficus indica (Miller). Whereas the whewellite crystallites are habitual in all Opuntia species, the weddellite form has never been isolated from these species before, which is probably due to their small size (about 1 microm). We have identified these forms using X-ray diffraction and scanning electron microscopy.

Isolation and structure of D-xylans from pericarp seeds of Opuntia ficus-indica prickly pear fruits.

Xylans were isolated from the pericarp of prickly pear seeds of Opuntia ficus-indica (OFI) by alkaline extraction, fractionated by precipitation and purified. Six fractions were obtained and characterized by sugar analysis and NMR spectroscopy. They were assumed to be (4-O-methyl-D-glucurono)-D-xylans, with 4-O-alpha-D-glucopyranosyluronic acid groups linked at C-2 of a (1-->4)-beta-D-xylan. The sugar composition and the 1H and 13C NMR spectra showed that their chemical structures were very similar, but with different proportions of D-Xyl and 4-O-Me-D-GlcA. Our results showed that, on average, the water soluble xylans have one nonreducing terminal residue of 4-O-methyl-D-glucuronic acid for every 11 to 14 xylose units, whereas in the water non-soluble xylans, xylose units can varied from 18 to 65 residues for one nonreducing terminal residue of 4-O-methyl-D-glucuronic acid.