An acidic exopolysaccharide α-D-galacturono-ß-D-glucan produced by the cyanobacterium Scytonema sp.

Some cyanobacteria produce a wide range of secondary metabolites, some of which are of industrial interest. Exopolysaccharides, particularly interesting among them, represent relatively complex primary structures with interesting bioactivity, biodegradability and specific applications. Cultivation of the freshwater cyanobacterium Scytonema sp. provided a proteoglycan-type exopolysaccharide with a relatively low yield and a wide spectrum of molecular weights (Mw) ranging from 2.2 to 1313 × 103 g/mol. Chemical analyses detected the presence of carbohydrates (46 wt%), proteins (10 wt%) and uronic acids (8 wt%). Monosaccharide analysis revealed up to seven neutral sugars with a dominance of glucose (23.6 wt%), galactose (7.4 wt%) and fucose (5.0 wt%) residues, while the others had a much lower content (0.9-3.4 wt%). The presence of galacturonic acid (8.0 wt%) indicated the appearance of ionic type of exopolysaccharide. A preliminary structural study indicated that the α-D-galacturono-ß-D-glucan forms a dominant part of Scytonema sp. exopolymer. Its backbone is composed of two 1,6-linked and one 1,2-linked ß-D-Glcp residues, which is branched at O6 by side chains composed of α-D-GalAp(1 â†’ 2)-ß-D-Glcp(1→ dimer or monomeric ß-D-Glcp(1→ residue.

Dendrobium officinale polysaccharides-chemical properties and pharmacodynamic effects on the airways in experimental conditions.

The study aimed to analyze the effects of Dendrobium polysaccharides on the cough and airway reactivity and compare them with the effects of clinically used antitussives (codeine phosphate and butamirate citrate) and bronchodilators (salbutamol), using the guinea pig test system. Dendrobium officinale polysaccharides contained proteins (4.0 wt%) and phenolic compounds (1.7 wt%) with a molecular weight of 25,000 g/mol. The sugar analysis revealed a dominance of glucose (93.7 wt%) and a lesser amount of mannose (5.1 wt%) while other sugar quantities were negligible. Methylation analysis indicated the presence of highly branched polysaccharides. Glucose was found mainly as terminal, 1,4- and 1,6-linked. Furthermore, some 1,4- and 1,6-linked glucose units were found branched at O2, O3, and O6/O4. Mannose was terminal and 1,4-linked. NMR spectra signals indicate the presence of the (1→4)-linked α-d-glucan, (1→4)-linked ß-d-glucan branched at position O6, (1→6)-linked ß-d-glucan branched at position O3 and (1→4)-linked glucomannan. Pharmacological studies showed statistically significant antitussive activity of Dendrobium polysaccharides, exceeding the effect of clinically used antitussives, which may be partially associated with confirmed bronchodilation and the ability of polysaccharides to increase the threshold of cough receptor activation. Dendrobium polysaccharides may increase the possibility of symptomatic treatment of cough, especially in asthmatics.

The Application of HPLC-FLD and NMR in the Monitoring of Therapy Efficacy in Alpha-Mannosidosis.

BACKGROUND: Alpha-mannosidosis is a rare lysosomal storage disorder, caused by decreased activity of α-D-mannosidase. This enzyme is involved in the hydrolysis of mannosidic linkages in N-linked oligosaccharides. Due to the mannosidase defect, undigested mannose-rich oligosaccharides (Man2GlcNAc - Man9GlcNAc) accumulating in cells are excreted in large quantities in urine. METHODS: In this work, we determined the levels of urinary mannose-rich oligosaccharides in a patient subjected to novel enzyme replacement therapy. Urinary oligosaccharides were extracted using solid phase extraction (SPE), labeled by fluorescent tag 2-aminobenzamide, and quantified by high-performance liquid chromatography (HPLC) with fluorescence detector (FLD). The identity of peaks was determined by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. In addition, the levels of urinary mannose-rich oligosaccharides were also quantified by 1H nuclear magnetic resonance (NMR) spectroscopy. The data were analyzed using one-tailed paired t-test and Pearson's correlation tests. RESULTS: Compared to levels before the administration of therapy, an approximately two-folds decrease in total mannose-rich oligosaccharides after one month of treatment was observed by NMR and HPLC. After four months, an approximately ten-folds significant decrease in total urinary mannose-rich oligosaccharides was detected, suggesting therapy effectiveness. A significant decrease in the levels of oligosaccharides with 7-9 mannose units was detected by HPLC. CONCLUSIONS: The application of both HPLC-FLD and NMR in quantification of oligosaccharide biomarkers is a suitable approach for monitoring of therapy efficacy in alpha-mannosidosis patients.

Structural properties of the extracellular biopolymer (ß-D-xylo-α-D-mannan) produced by the green microalga Gloeocystis vesiculosa Nägeli.

Many species of microalgae produce a relatively diverse range of metabolites that are interesting for biotechnological applications, and among them exopolysaccharides attract attention due to their structural complexity, biological activities, biodegradability or biocompatibility. An exopolysaccharide of high molecular weight (Mp) of 6.8 × 105 g/mol was obtained by cultivation of the freshwater green coccal microalga Gloeocystis vesiculosa Nägeli 1849 (Chlorophyta). Chemical analyses revealed a dominance of Manp (63.4 wt%), Xylp and its 3-O-Me-derivative (22.4 wt%), and Glcp (11.5 wt%) residues. The results of the chemical and NMR analyses showed an alternating branched 1,2- and 1,3-linked α-D-Manp backbone terminated by a single ß-D-Xylp and its 3-O-methyl derivative at O2 of the 1,3-linked α-D-Manp residues. The α-D-Glcp residues were found mainly as 1,4-linked and to a lesser extent as the terminal sugar, indicating partial contamination of ß-D-xylo-α-D-mannan with amylose (∼10 wt%) in G. vesiculosa exopolysaccharide.

An arabinan isolated from the antioxidant active fraction of wild blackthorn fruits (Prunus spinosa L.).

Despite the many health benefits of wild blackthorn fruits, their use is negligible and little explored. Preliminary results suggest that this fruit is a rich source of natural antioxidants. Antioxidant active fraction, extracted from wild blackthorn fruits by cold water extraction (Cw), was subjected to ion-exchange chromatography, which gave ten fractions after a successive elution with water, sodium chloride and sodium hydroxide solutions. Fraction eluted by water contained an arabinan of a low molecular weight (Mw = 5000). Its chemical and spectroscopic studies have revealed a highly branched structure with backbone composed of 1,5-linked α-l-arabinofuranose residues; about 76% of them are branched dominantly through O3 and much less through O2, however, some arabinose units (∼8%) were fully branched. In addition to terminal α-L-Araf, the presence of ß-L-Araf unit was also identified in blackthorn L-arabinan. Antioxidant activity tests of Cw ion exchange fractions revealed significant effects only of fractions eluted with 1 M sodium chloride (6F) and sodium hydroxide (7F) solutions, while phenolic free arabinan did not possess this effect. Phenolic compounds linked via α-l-arabinosyl side chains appear to be responsible for the antioxidant effect of two Cw fractions.

Lactylated acidic exopolysaccharide produced by the cyanobacterium Nostoc cf. linckia.

Cyanobacteria produce a wide range of metabolites of interest for industrial or medical use. The cultivation of freshwater Nostoc cf. linckia yielded 5.4 g/L of a crude exopolysaccharide (cEPS) with a molecular weight of 1.31 × 105 g/mol. Ion-exchange chromatography of cEPS yielded two dominant fractions, EPS-1 and EPS-2, differing in molecular weight. The lower molecular weight fraction (EPS-1) was subjected to structural studies. Results of chemical and spectroscopic analyses showed that three of the four dominant sugars, glucose, galactose and xylose are 1,4-linked in the backbone in the following order: [→4)-ß-D-Xylp-(1 â†’ 4)-ß-D-Glcp-(1 â†’ 4)-α-D-Galp-(1 â†’ 4)-ß-D-Glcp-(1→]n. Terminal mannose residues were identified as side chains linked at C3 of every third backbone xylose and every second glucose is branched at C6 by 3-O-lactyl-ß-D-glucuronic acid (nosturonic acid). Antioxidant properties of EPS were tested using two in vitro methods. Both assays showed that the cEPS was more active than purified EPS-1 and EPS-2 fractions and deproteinized EPS.

Optimizing acid hydrolysis for monosaccharide compositional analysis of Nostoc cf. linckia acidic exopolysaccharide.

The exact estimation of monosaccharide composition is important in the primary structure elucidation of polysaccharides. An acid hydrolysis is usually performed for glycosidic bonds cleavage and releasing of monosaccharides. In this study, optimal conditions of total acid hydrolysis using trifluoroacetic acid (TFA) of acidic lactylated Nostoc cf. linckia exopolysaccharide (EPS) were investigated by NMR spectroscopy. Results of a series of experiments with modified acid concentration, temperature and time of hydrolysis, have shown 2 M TFA, 110 °C, 3 h as the most optimal. The stability of EPS monosaccharide components was also explored. Low stability was found at all tested conditions already during the first hour of hydrolysis; all neutral monosaccharides were degraded from 25% to 40% and glucuronic acid to 75%. NMR, contrary to standard techniques used in monosaccharide compositional analysis (HPLC, HPAEC), allowed simultaneous quantification of all GlcA forms; the free one, that one linked in oligosaccharides, as well as GlcA degradation product γ-lactone. NMR as detection method improves information about uronic acid content in EPS.

Structural features of the bioactive cyanobacterium Nostoc sp. exopolysaccharide.

Microalgal biopolymers are studied mainly in terms of physico-chemical characterization, biological effects as well as possible biotechnological applications. Due to the significant antitussive, bronchodilator, anti-inflammatory and immunomodulatory effects of the previously isolated crude extracellular polysaccharide (EPS) produced by the cyanobacterium Nostoc sp., the purified biopolymer and its oligosaccharides, obtained after partial acid hydrolysis, were subjected to an in-depth NMR structural study. Analyses of the data obtained by chemical methods and NMR showed that the EPS backbone is composed of the repeating unit [→4)-ß-D-Xylp-(1 → 4)-ß-D-Glcp-(1 → 4)-α-L-Arap-(1 → 3)-ß-D-Manp-(1→]n, in which about 60% of glucose units are substituted at C6 by uronic acids, in particular by the unusual unsaturated 3-O-lactyl-4-deoxy-α-erythro-hex-4-enopyranuronic acid, and to a lesser extent by ß-D-glucuronic acid and 3-O-lactyl-ß-D-glucuronic acid. These findings, structural features and identified biological effects, suggest the potential use of this biopolymer in the medical-pharmaceutical field.

Structural characteristics and biological effects of exopolysaccharide produced by cyanobacterium Nostoc sp.

Complex structure of cyanobacterium Nostoc sp. exopolysaccharide (EPS), with apparent molecular weight 214 × 103 g/mol, can be deduced from its composition. Chemical and NMR analyses found four dominant sugar monomers, namely (1 → 4)-linked α-l-arabinopyranose, ß-d-glucopyranose, ß-d-xylopyranose and (1 → 3)-linked ß-d-mannopyranose, two different uronic acids and a lactyl group, with (1 → 4,6)-linked ß-d-glucopyranose as the only branch point suggest a complex structure of this polymer. The dominant uronic acid is α-linked, but it remained unidentified. ß-d-Glucuronic acid was present in lower amount. Their position as well as that of lactyl remained undetermined too. Different doses of orally administered EPS in guinea pigs evoked a significant decrease in cough effort and a decrease in airway reactivity. The antitussive efficacy and bronchodilator effect of higher doses of EPS were found to be similar to that of the antitussive drug codeine and the antiasthmatic salbutamol. Without significant cytotoxicity on the RAW 264.7 cells, EPS stimulated the macrophage cells to produce pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and prostaglandins (PGs) and nitric oxide (NO) via induction of COX-2 and iNOS expression, respectively, suggesting that this biopolymer potentiates an early innate immune response and can therefore be used as a new immune modulator.

A Structural Analysis of the Angucycline-Like Antibiotic Auricin from Streptomyces lavendulae Subsp. Lavendulae CCM 3239 Revealed Its High Similarity to Griseusins.

We previously identified the aur1 gene cluster in Streptomyces lavendulae subsp. lavendulae CCM 3239 (formerly Streptomyces aureofaciens CCM 3239), which is responsible for the production of the angucycline-like antibiotic auricin (1). Preliminary characterization of 1 revealed that it possesses an aminodeoxyhexose d-forosamine and is active against Gram-positive bacteria. Here we determined the structure of 1, finding that it possesses intriguing structural features, which distinguish it from other known angucyclines. In addition to d-forosamine, compound 1 also contains a unique, highly oxygenated aglycone similar to those of spiroketal pyranonaphthoquinones griseusins. Like several other griseusins, 1 also undergoes methanolysis and displays modest cytotoxicity against several human tumor cell lines. Moreover, the central core of the aur1 cluster is highly similar to the partial gris gene cluster responsible for the biosynthesis of griseusin A and B in both the nature of the encoded proteins and the gene organization.

Structural characterization of hemicellulose released from corn cob in continuous flow type hydrothermal reactor.

Hydrothermal reaction is known to be one of the most efficient procedures to extract hemicelluloses from lignocellulosic biomass. We investigated the molecular structure of xylooligosaccharides released from corn cob in a continuous flow type hydrothermal reactor designed in our group. The fraction precipitable from the extract with four volumes of ethanol was examined by 1H-NMR spectroscopy and MALDI-TOF MS before and after enzymatic treatment with different purified enzymes. The released water-soluble hemicellulose was found to correspond to a mixture of wide degree of polymerization range of acetylarabinoglucuronoxylan fragments (further as corn cob xylan abbreviated CX). Analysis of enzymatic hydrolyzates of CX with an acetylxylan esterase, GH3 ß-xylosidase, GH10 and GH11 xylanases revealed that the main chain contains unsubstituted regions mixed with regions of xylopyranosyl residues partially acetylated and occasionally substituted by 4-O-methyl-d-glucuronic acid and arabinofuranose esterified with ferulic or coumaric acid. Single 2- and 3-O-acetylation was accompanied by 2,3-di-O-acetylation and 3-O-acetylation of Xylp residues substituted with MeGlcA. Most of the non-esterified arabinofuranose side residues were lost during the hydrodynamic process. Despite reduced branching, the acetylation and ferulic acid modification of pentose residues contribute to high yields and high solubility of the extracted CX. It is also shown that different enzyme treatments of CX may lead to various types of xylooligosaccharides of different biomedical potential.

Reaction mechanism of ß-apiosidase from Aspergillus aculeatus.

Apiosidases are glycosidases relevant for aroma development during fermentation of wines and black tea. Reaction mechanism of apiosidase from Aspergillus aculeatus in commercial glycanase Viscozyme L was studied by 1H NMR technique. Study of hydrolysis of 4-nitrophenyl ß-D-apiofuranoside revealed that this reaction proceeds with inversion of hydroxyl group in the anomeric center, which confirms inverting mechanism of the enzyme and its inability to catalyze transapiosylation in syntheses of apiosides.

Glucuronoyl esterases are active on the polymeric substrate methyl esterified glucuronoxylan.

Alkali extracted beechwood glucuronoxylan methyl ester prepared by esterification of 4-O-methyl-D-glucuronic acid side residues by methanol was found to serve as substrate of microbial glucuronoyl esterases from Ruminococcus flavefaciens, Schizophyllum commune and Trichoderma reesei. The enzymatic deesterification was monitored by (1)H NMR spectroscopy and evaluated on the basis of the decrease of the signal of the ester methyl group and increase of the signal of methanol. The results show for the first time the action of enzymes on polymeric substrate, which imitates more closely the natural substrate in plant cell walls than the low molecular mass artificial substrates used up to present.

Carboxymethylated-, hydroxypropylsulfonated- and quaternized xylan derivative films.

Under alkaline/water conditions carboxymethyl, 2-hydroxypropylsulfonate and trimethylammonium-2-hydroxypropyl groups were introduced into xylan in one step with the goal to prepare film specimens. The materials were characterized by NMR, SEC-MALS, TG/DTG/DTA, AFM and mechanical testing. The properties of triple, double and mono-substituted materials were compared. The numerical molar masses of the specimens were from 12.3 to 17.6 kg/mol with Mw/Mn from 1.27 to 1.34. The elastic modulus values are decreasing in order: xylan (X; 7354 MPa)>carboxymethyl xylan (CX; 6090MPa)>2-hydroxypropylsulfonate xylan (SX; 6000 MPa)>carboxymethyl/2-hydroxypropylsulfonate xylan (CSX; 4490 MPa)>quaternized xylan (QX; 3600 MPa)>carboxymethyl/quaternary/2-hydroxypropylsulfonate xylan (CQSX; 3380 MPa)>carboxymethyl/quaternary xylan (CQX; 2805 MPa). The onset temperatures of SX (214°C), CQSX (212°C), QSX (211°C) and CQX (207°C) were higher than for X (205°C). The roughness values of the film surfaces (3.634-18.667 nm) are higher on top than on the bottom of the specimen.

Quaternized and sulfated xylan derivative films.

Xylan quaternized sulfate films were prepared from beech xylan (X) and compared with fully sulfated xylan films (XS). When quaternized xylan (QX) was prepared in the first step (DS(Q)=0.55), than by sulfation of QX in the second step the fully substituted ampholytic derivative (QXS; DS(Q)=0.33; DS(S)=1.67) could be obtained. By sulfation in first step, xylan sulphate (SX, DS(S)=0.70) was obtained and by subsequent quaternization, SXQ film with DSQ=0.55 and DSS=0.33 which contained partially unsubstituted hydroxyls. The molar masses (M(n)) of the film's soluble parts were increasing in order X

Mode of action of acetylxylan esterases on acetyl glucuronoxylan and acetylated oligosaccharides generated by a GH10 endoxylanase.

BACKGROUND: Substitutions on the xylan main chain are widely accepted to limit plant cell wall degradability and acetylations are considered as one of the most important obstacles. Hence, understanding the modes of action of a range of acetylxylan esterases (AcXEs) is of ample importance not only to increase the understanding of the enzymology of plant decay/bioremediation but also to enable efficient bioconversion of plant biomass. METHODS: In this study, the modes of action of acetylxylan esterases (AcXEs) belonging to carbohydrate esterase (CE) families 1, 4, 5 and 6 on xylooligosaccharides generated from hardwood acetyl glucuronoxylan were compared using MALDI ToF MS. Supporting data were obtained by following enzymatic deacetylation by (1)H NMR spectroscopy. CONCLUSIONS: None of the used enzymes were capable of complete deacetylation, except from linear xylooligosaccharides which were completely deacetylated by some of the esterases in the presence of endoxylanase. A clear difference was observed between the performance of the serine-type esterases of CE families 1, 5 and 6, and the aspartate-metalloesterases of family CE4. The difference is mainly due to the inability of CE4 AcXEs to catalyze deacetylation of 2,3-di-O-acetylated xylopyranosyl residues. Complete deacetylation of a hardwood acetyl glucuronoxylan requires additional deacetylating enzyme(s). GENERAL SIGNIFICANCE: The results contribute to the understanding of microbial degradation of plant biomass and outline the way to achieve complete saccharification of plant hemicelluloses which did not undergo alkaline pretreatment.

Positional specifity of acetylxylan esterases on natural polysaccharide: an NMR study.

BACKGROUND: Microbial degradation of acetylated plant hemicelluloses involves besides enzymes cleaving the glycosidic linkages also deacetylating enzymes. A detailed knowledge of the mode of action of these enzymes is important in view of the development of efficient bioconversion of plant materials that did not undergo alkaline pretreatment leading to hydrolysis of ester linkages. METHODS: In this work deacetylation of hardwood acetylglucuronoxylan by acetylxylan esterases from Streptomyces lividans (carbohydrate esterase family 4) and Orpinomyces sp. (carbohydrate esterase family 6) was monitored by (1)H-NMR spectroscopy. RESULTS: The (1)H-NMR resonances of all acetyl groups in the polysaccharide were fully assigned. The targets of both enzymes are 2- and 3-monoacetylated xylopyranosyl residues and, in the case of the Orpinomyces sp. enzyme, also the 2,3-di-O-acetylated xylopyranosyl residues. Both enzymes do not recognize as a substrate the 3-O-acetyl group on xylopyranosyl residues α-1,2-substituted with 4-O-methyl-d-glucuronic acid. CONCLUSIONS: The (1)H-NMR spectroscopy approach to study positional and substrate specificity of AcXEs outlined in this work appears to be a simple way to characterize catalytic properties of enzymes belonging to various CE families. SIGNIFICANCE: The results contribute to development of efficient and environmentally friendly procedures for enzymatic degradation of plant biomass.