The role of the glucuronoxylan carboxyl groups in the action of endoxylanases of three glycoside hydrolase families: A study with two substrate mutants.

BACKGROUND: Bacterial appendage-dependent GH30 glucuronoxylan hydrolases recognize the substrate through an ionic interaction of a conserved positively charged arginine with the carboxyl group of 4-O-methyl-d-glucuronic acid. One of the options to verify this interaction is preparation of enzyme mutants. An alternative approach is a chemical modification of the substrate, glucuronoxylan, in which the free carboxyl group in all residues of MeGlcA is eliminated. METHODS: In this work the carboxyl groups of 4-O-methyl-d-glucuronic acid residues of an alkali extracted beechwood xylan were esterified with methanol. A water-soluble fraction of the polysaccharide methyl ester was converted by NaBH4 reduction to the second soluble derivative, 4-O-methylglucoxylan. Specific activities of several endoxylanases (EXs) of GH families 10, 11 and 30 were determined on glucuronoxylan, and its two new uncharged derivatives. RESULTS: Elimination of the free carboxyl group from the polysaccharide did not influence activities of GH10 EXs, but resulted in 50% decrease of specific activity of GH11 EXs, and led to more than 300-fold reduction of specific activity of Erwinia chrysanthemi GH30 xylanase. CONCLUSIONS: These results confirm the crucial role of the interactions between GH30 xylanases and the MeGlcA carboxyl group for efficient cleavage of the polysaccharide. Analysis of the hydrolysis products by TLC and MS confirmed that all three types of xylanases hydrolyzed uncharged glucuronoxylans similarly as the original one. SIGNIFICANCE: The uncharged glucuronoxylan derivatives will be useful to differentiate GH30 xylanases with various degree of selectivity for glucuronoxylan, including fungal enzymes without the conserved arginine.

Comparison of conventional and ultrasound-assisted extraction of phenolics-rich heteroxylans from wheat bran.

Various two-step extraction procedures with and without a short application of ultrasound in the first step at various reaction conditions (extracting agent, temperature and time), and constant conditions in the second step (5% NaOH, room temperature, 60 min) were used to isolate the hemicellulose component of industrial wheat bran. The polysaccharides recovered from the extracts were characterised by yield, chemical composition and radical scavenging activity. Similar total yields of polysaccharides were achieved by a short ultrasound-assisted extraction (up to 10 min) in the first step using 0.5% or 2% NaOH, when compared to the classical extraction lasting 60 min, what represents a shortening of the process by about 60% and lower consumption of the NaOH. The extracted polysaccharides comprised a complex of phenolics-rich heteroxylans contaminated to various extents with co-extracted amylose-rich starch as well as protein, which can be in part governed by the extraction conditions. The water-soluble fractions from both the classical and ultrasonics experiments were shown to exhibit significant radical scavenging activity in vitro, indicating their potential application as polysaccharide-based antioxidants.

In vitro cytostatic and immunomodulatory properties of the medicinal mushroom Lentinula edodes.

Lentinula edodes, known as "shiitake" is one of the widely used medicinal mushrooms in the Orient. Antitumour activity of extracts of this mushroom has been widely demonstrated in animals and humans. However, this activity was shown to be host mediated and not by direct cytotoxic activity to cancer cells. This study demonstrates cytotoxic and cell growth inhibitory (cytostatic) effect of aqueous extracts of the mushroom on MCF-7 human breast adenocarcinoma cell line using an MTT cytotoxicity assay. Such effect was demonstrated with fruit body and mycelial extracts, the difference being that there was no significant suppression on normal cells with the latter. Furthermore mycelial extracts did not induce any cytostatic effect in both cancer and normal cell lines based on a DNA synthesis assay. The significant suppression of the proliferation of cancer cells was reflected by the comparatively low IC(50) values and the simultaneous higher respective values on normal fibroblast cells. The immunostimulatory activity of both fruit body and mycelial extracts was tested by the lymphocyte transformation test (LTT), which is based on the capacity of active immunomodulators to augment the proliferative response of rat thymocytes to T mitogens in vitro. Both fruit body and mycelial preparations were able to enhance the proliferation of rat thymocytes directly and act as co-stimulators in the presence of the T-mitogen PHA. Interestingly both extracts, similarly to zymosan showed SI(comit)/SI(mit) ratios of about 2, indicating adjuvant properties. Overall L. edodes aqueous extracts have demonstrated direct inhibition of the proliferation of human breast cancer cells in vitro and immunostimulatory properties in terms of mitogenic and co-mitogenic activity in vitro.

Xyloglucan degradation using different radiation sources: a comparative study.

Tamarind seed xyloglucan was subjected to different radiation sources-ultrasound, gamma-radiation, and microwave heating, and the effects of these energies upon its molecular and structural properties were characterised by gel permeation chromatography, viscometry, sugar analysis, FT-IR and NMR spectroscopic techniques. In dependence on the degradation methods and experimental conditions used, the decrease of the relative molecular mass (RMM) was accompanied with alteration of the primary structure. Depolymerisation by ultrasound at a frequency of 20 kHz yielded after 120 min products with RMM of about 131 x 10(3) without significant alteration of the primary structure of the polysaccharide. Intense degradation of XG started by microwave heating at pH 1.5 yielding polymers with RMM in the range of higher oligosaccharides, however, with changed sugar composition due to cleavage of the glycosyl side chains. At gamma-irradiation doses >40 kGy, next to chain cleavage, very high-molecular mass components exhibiting UV(254)-absorption were formed, and the RMM decreased to about 50 x 10(3) at the highest applied dose (100 kGy). The results of the comparative study suggest that ultrasonication was the most convenient procedure to decrease the RMM of xyloglucan to 130 x 10(3) and preserve the primary structure of the polysaccharide.

Characterization of immunomodulatory polysaccharides from Salvia officinalis L.

Crude polysaccharide fractions, rich mainly in arabinogalactans (A), pectin (B) and glucuronoxylan-related polymers (D), have been obtained from aerial parts of sage (Salvia officinalis L.) by sequential extraction with various reagents. Arabinogalactans displayed on HPLC a dominance of lower molecular-mass polymers (MW < 10,000), while pectin and glucuronoxylan-related polysaccharides showed predominance of polymers with MW > 50,000. Individual polysaccharide fractions were examined for their immunomodulatory activity in the in vitro comitogenic thymocyte test. The polysaccharide fractions tested possessed the capacity to induce rat thymocyte proliferation in the order D>B>A. Besides, fraction D possessed a significant comitogenic effect, and the SIcomit/SImit ratio 3-4 indicates potential adjuvant properties of this glucuronoxylan-rich material.

A biologically active fructan from the roots of Arctium lappa L., var. Herkules.

From the roots of Arctium lappa L., var. Herkules a low-molecular-weight fructofuranan of the inulin-type has been isolated by water extraction and ethanol precipitation, followed by ion-exchange chromatography and gel filtration of the crude precipitate. The methods employed in structural determination were methylation analysis and 1H and 13C NMR spectral measurements. In tests for antitussive activity in cats the fructan was found to be equally active as some non-narcotic, synthetic preparations used in clinical practice to treat coughing, and in mitogenic and comitogenic tests its biological response was comparable to that of the commercial Zymosan immunomodulator.

Ultrasonic extraction of plant materials--investigation of hemicellulose release from buckwheat hulls.

Various one- and two-step extraction procedures with and without a short application of ultrasound at the beginning of the extraction were used to examine the effect of sonication on the extractibility of the hemicellulose components of buckwheat hulls. The polysaccharides recovered from the extracts were characterised by yield as well as composition determined by chemical methods and spectroscopic techniques. They comprised a complex of glucuronoxylan and co-extracted amylose-rich starch in various proportions contaminated with other cell wall components (protein, pectic polysaccharides). The hemicellulose fractions obtained by classical and ultrasound-assisted extraction exhibited significant immunomodulatory activities. The increased yield of ultrasonically extracted hemicelluloses, which have preserved their structural and molecular properties as well as immunological activity, confirmed the importance and great potential of ultrasound-assisted extraction of industrially important polysaccharides from different tissues of plant materials.

Mitogenic and comitogenic activities of polysaccharides from some European herbaceous plants.

From the leaves of Plantago lanceolata, the green parts of Rudbeckia fulgida, the aerial parts of Salvia officinalis and the roots of Valeriana officinalis, crude polysaccharides have been isolated by extraction with water and further purified and fractionated by various techniques. The water-soluble polysaccharides obtained were examined for their immunomodulatory activities using the in vitro mitogenic and comitogenic rat thymocyte tests. The results indicate that in spite of the considerable differences in chemical composition and structural properties, the tested polysaccharides exhibited similar significant immunomodulatory properties with a particularly high adjuvans activity in the case of the Rudbeckia and Salvia polysaccharides. The pectic polysaccharide-rich complex from Valeriana was shown to also stimulate the immune function of bone marrow cells.

Effect of ultrasound on the extractibility of corn bran hemicelluloses.

Various two-step extraction procedures with and without a short application of ultrasound in the first step were used to examine the effect of sonication on the extractibility and properties of the non-cellulose components of industrial corn bran (CB). The polysaccharides recovered from the extracts of the first (FI) and second (FII) extraction step were characterised by yield and composition. Using water as extractant in the first step, similar yields of total extracted polysaccharides (FI + FII) were obtained by the short sonication treatment when compared to the classical procedures using H2O2-containing alkaline media in the first step. The yields of FI + FII showed a substantial increase (10-40%) after application of ultrasound in 1% and 5% NaOH in comparison to those of the classical procedures. The content of the neutral sugar components constituting the CB xylan varied from 65 to 88 mol% in dependence on the conditions used in the first extraction step.

Immunomodulatory activity of acidic xylans in relation to their structural and molecular properties.

The structure/function relationship of two acidic heteroxylan types, the arabino-(glucurono)xylan from corn cobs (AGX) and 4-O-methylglucuronoxylans (GXs) from beechwood and three medicinal herbs (Rudbeckia, Altheae, and Mahonia), has been studied. The effect of the molecular mass of AGX, as well as the content and distribution of the 4-O-methylglucuronic acid side chains in GXs on the immunological activity of these xylans was characterized by their biological response in the mitogenic and comitogenic thymocyte in vitro tests. Depolymerization of AGX by ultrasonication resulted in unequivocal decrease of the immunomodulatory activity, whereas already a short treatment by endo-beta-1,4-xylanase brought about a significant increase in its activity when applied in the highest dose. In the case of the GX samples, neither the uronic acid content nor the distribution pattern of the uronic acid side chains was found to be determinant for the expression of their immunomodulatory activity.

Ultrasound-assisted extraction of water-soluble polysaccharides from the roots of valerian (Valeriana officinalis L.).

The insoluble plant residues, obtained after preparation of medicinal tinctures from the roots of valerian (Valeriana officinalis L.) by classical and ultrasound-assisted extraction with aqueous ethanol in a pilot plant, were subsequently treated with hot water to isolate the accessible polysaccharide cell wall components. At almost equal amounts of the hot-water extractable material, the yields of the recovered polysaccharides were lower in the ultrasonical experiment. This is due to the fact that a part of accessible polysaccharides were already solubilised by the aqueous ethanol and recoverable from the medicinal tincture. Therefore, the net yield of extracted polysaccharides was enhanced in the ultrasonical procedure. This fact as well as the sugar composition and structural features of the isolated polysaccharides suggest that ultrasonication have attacked the integrity of cell walls, released and degraded its most accessible polysaccharides (pectic polysaccharides and starch) and increased also the extractibility of its less accessible components--xylan, mannan and glucan. The water-soluble polysaccharide fractions from both the conventional and ultrasonical experiments exhibit significant immunostimulatory activities in mitogenic and comitogenic thymocyte tests.

Comparison of classical and ultrasound-assisted extraction of polysaccharides from Salvia officinalis L.

After preparation of medicine tinctures from the herbal plant Salvia officinalis by classical and ultrasound-assisted extraction with aqueous ethanol, the insoluble plant residues were subsequently treated with hot water and dilute alkali to isolate polysaccharide cell wall components. The yields of the hot water extract as well as total extracted polysaccharides were higher in the case of the ultrasound-treated plant in both laboratory and pilot plant experiments. The water-extractable polysaccharide fractions, in all cases, contained glucose, galactose and arabinose as main sugar components, whereas the alkali-extractable fractions were rich in xylans. The fractions also contained considerable amounts of proteins. The water-soluble polysaccharides may contribute to the biological activity of the plant decoction. The results indicate that the addition of a subsequent extraction step during the preparation of the herbal tincture might contribute to a better exploitation of the raw material.

The effect of ultrasound on the structure and properties of the water-soluble corn hull heteroxylan.

Ultrasonic irradiation of a water-soluble corn hull xylan fraction in neutral and alkaline aqueous medium has been found to produce significant changes in its molecular properties. Degradation is first manifested by a decrease in the large molar mass component under generation of polymer chains with about the same size as those of the main molar mass component. The latter is slightly shifted to the lower molar mass region only at stronger irradiation conditions. Ultrasonication of the xylan in neutral aqueous medium at high ultrasound power and/or long irradiation caused no significant changes in its sugar composition, primary structure and viscoelastic properties.

Effect of ultrasound on the immunogenic corn cob xylan.

Changes in the structural, molecular and functional properties of the immunogenic corn cob xylan evoked by ultrasonication in water, 1% NaOH and 5% NaOH were investigated. The reduction of the high molar mass (MM) fraction was more intense than that of the medium MM fraction, depending on the sonic power, sonication time, and alkali concentration. The chain degradation was more effective in the alkaline media. The UV-absorbing component, accompanying the xylan polymers in the whole MM range, showed an accumulation in the high MM region as well as shiftening to higher sizes, particularly in 5% NaOH. The sugar composition and primary structure of the xylan was almost retained under all irradiation conditions studied. Although the biological activity of the xylan was affected by the ultrasound, no significant decrease of the biological response was found at short irradiation time and low sonic power.

Structure and mitogenic activities of corn cob heteroxylans.

Various structurally different water-soluble (ws) and water-insoluble (wis) heteroxylans have been tested for mitogenic and co-mitogenic activity and shown to differ in their stimulating potency. The ws arabinoglucuronoxylan (ws-AGX) from corn cobs exhibited the highest potency which was comparable to that of the immunomodulator zymosan, whereas the wis-AGX from corn cobs was inactive. Water-soluble derivatives of wis-AGX as well as hydrolytically modified ws-AGX fractions were prepared and tested for their mitogenic and co-mitogenic activity. The results indicate that the disaccharide side chains present only in ws-AGX might be important for expression of the immunostimulatory activity of this xylan.

Antimicrobial activity of quaternized heteroxylans.

A series of quaternized D-xylan polysaccharides, differing in the structural features of their macromolecular backbone, were tested for antimicrobial activity against Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens and Saccharomyces cerevisiae. Activity was comparable with that of the cationic surfactant, cetyltrimethylammonium bromide, and depended on the degree of quaternization and the structural backbone of the derivatives.

Structural features of a water-soluble L-arabino-D-xylan from rye bran.

A water-soluble L-arabino-D-xylan, obtained from the chlorite holocellulose of de-lipidated, de-starched, and de-pectinated rye bran by ammoniacal extraction, was composed of L-Ara and D-Xyl in the molar ratio 7.8:10 and had Mw 36,500 and Mn 26,950. The backbone of the polysaccharide was shown to comprise (1----4)-linked beta-D-Xylp residues, with approximately 41% unsubstituted, approximately 33% 2- or 3-substituted, and approximately 26% disubstituted. Single alpha-L-Araf groups were attached to the xylan core; only a small proportion was 2-, 3-, or 5-linked.