Functional exploration of the glycoside hydrolase family GH113.

ß-Mannans are a heterogeneous group of polysaccharides with a common main chain of ß-1,4-linked mannopyranoside residues. The cleavage of ß-mannan chains is catalyzed by glycoside hydrolases called ß-mannanases. In the CAZy database, ß-mannanases are grouped by sequence similarity in families GH5, GH26, GH113 and GH134. Family GH113 has been under-explored so far with six enzymes characterized, all from the Firmicutes phylum. We undertook the functional characterization of 14 enzymes from a selection of 31 covering the diversity of the family GH113. Our observations suggest that GH113 is a family with specificity towards mannans, with variations in the product profiles and modes of action. We were able to assign mannanase and mannosidase activities to four out of the five clades of the family, increasing by 200% the number of characterized GH113 members, and expanding the toolbox for fine-tuning of mannooligosaccharides.

Structure and enzymatic degradation of the polysaccharide secreted by Nostoc commune.

Noctoc commune is a cyanobacterium living in various and extreme environments. Its ability to survive in desert, on ice or high altitude is explained by its exceptional metabolism and its capacity to resist to desiccation. N. commune cells are embedded in a gelatinous matrix made of polysaccharides which fixes water and participates in maintaining the cells in hydrated conditions. The structure of the polysaccharide of N. commune harvested in Saint Martin d'Uriage (France) and the oligosaccharides obtained after its enzymatic degradation were determined. The repeating unit of the main chain is a tetra-saccharide: [→4)-ß-D-Glcp-(1 â†’ 4)-ß-D-Xylp-(1 â†’ 4)-ß-D-Glcp-(1 â†’ 4)-α-D-Galp-(1→], branched at position 6 of a glucose residue by a ß-linked pyruvated glucuronic acid residue. About 30% of the Xylp residues were branched with a Xylf residue. Comparison of this structure with the polysaccharides secreted by other Nostoc species and strains suggest a strong selection pressure on the structure in agreement with its important biological role.

Optimizing Chitin Depolymerization by Lysozyme to Long-Chain Oligosaccharides.

Chitin oligosaccharides (COs) hold high promise as organic fertilizers in the ongoing agro-ecological transition. Short- and long-chain COs can contribute to the establishment of symbiotic associations between plants and microorganisms, facilitating the uptake of soil nutrients by host plants. Long-chain COs trigger plant innate immunity. A fine investigation of these different signaling pathways requires improving the access to high-purity COs. Here, we used the response surface methodology to optimize the production of COs by enzymatic hydrolysis of water-soluble chitin (WSC) with hen egg-white lysozyme. The influence of WSC concentration, its acetylation degree, and the reaction time course were modelled using a Box-Behnken design. Under optimized conditions, water-soluble COs up to the nonasaccharide were formed in 51% yield and purified to homogeneity. This straightforward approach opens new avenues to determine the complex roles of COs in plants.

Structure of the Polysaccharide Secreted by Vibrio alginolyticus CNCM I-5035 (Epidermist 4.0TM).

Vibrio alginolyticus (CNCM I-5035) secretes an exopolysaccharide used as ingredient in cosmetic industry under the trademark Epidermist 4.0TM. It is appreciated for its ability to improve the physical and chemical barrier functions of the skin by notably increasing the keratinocyte differentiation and epidermal renewal. Composition analyses and in depth characterization of the polysaccharides as well as oligosaccharides obtained by mild acid hydrolyses revealed that it was composed of a repetition unit of three residues: d-galactose (d-Gal), d-N-acetylglucosamine (GlcNAc) and l-N-acetylguluronic acid, of which 30% (M/M) was acetylated in position 3. The complete structure of the polysaccharide was resolved giving the repetition unit: [→3)-α-d-Gal-(1→4)-α-l-GulNAcA/α-l-3OAc-GulNAcA-(1→4)-ß-d-GlcNAc-(1→].

Structure of the Exopolysaccharide Secreted by a Marine Strain Vibrio alginolyticus.

Vibrio alginolyticus (CNCM I-4151) secretes an exopolysaccharide whose carbohydrate backbone is decorated with amino acids, likely conferring its properties that are appreciated in cosmetics. Here, the secreted polysaccharide of another strain of V. alginolyticus (CNCM I-5034) was characterized by chromatography and one- and two-dimensional NMR spectroscopy experiments. The structure was resolved and shows that the carbohydrate backbone is made of four residues: D-galactose (Gal), D-galacturonic acid (GalA) D-N-acetylglucosamine (GlcNAc) and D-glucuronic acid (GlcA), forming a tetrasaccharide repetition unit [→4)-ß-d-GlcA-(1→3)-α-d-Gal-(1→3)-α-d-GalA-(1→3)-ß-GlcNAc(1→]. GlcA is derivatized with a lactate group giving 'nosturonic acid', and GalA is decorated with the amino acid alanine.

Depolymerization of Pseudomonas stutzeri exopolysaccharide upon fermentation as a promising production process of antibacterial compounds.

Many researchers have focused on high molecular weight (Mw) exopolysaccharides (EPS) as a source of potentially bioactive lower Mw derivatives. Therefore, it is of interest to find means for efficient and safe production of depolymerized-polymer derivatives. Exopolysaccharide-depolymerization products (EDP) varying in molecular weight were recovered from fermentative depolymerization of a native EPS produced by Pseudomonas stutzeri AS22. Following their purification and physicochemical characterization, the antibacterial activity of EDP on food spoilage and food poisoning microorganisms was evaluated through the measurement of the inhibition zone diameter, the half maximal (IC50) and the minimal (MIC) inhibitory concentrations. Our results indicate that the lower the Mw, the higher will be the effectiveness of EDP on reducing Gram-negative bacteria growth and the opposite trend was observed in the case of Gram-positive bacteria. EDP bioactivities may provide novel insights into the potentiality of P. stutzeri EPS and its derivatives to be used as functional-food components.

Bacterial synthesis of polysialic acid lactosides in recombinant Escherichia coli K-12.

Bacterial polysialyltransferases (PSTs) are processive enzymes involved in the synthesis of polysialic capsular polysaccharides. They can also synthesize polysialic acid in vitro from disialylated and trisialylated lactoside acceptors, which are the carbohydrate moieties of GD3 and GT3 gangliosides, respectively. Here, we engineered a non-pathogenic Escherichia coli strain that overexpresses recombinant sialyltransferases and sialic acid synthesis genes and can convert an exogenous lactoside into polysialyl lactosides. Several PSTs were assayed for their ability to synthesize polysialyl lactosides in the recombinant strains. Fed-batch cultures produced α-2,8 polysialic acid or alternate α-2,8-2,9 polysialic acid in quantities reaching several grams per liter. Bacterial culture in the presence of propargyl-ß-lactoside as the exogenous acceptor led to the production of conjugatable polysaccharides by means of copper-assisted click chemistry.

Structure of an Amino Acid-Decorated Exopolysaccharide Secreted by a Vibrio alginolyticus Strain.

Vibrio alginolyticus (CNCM I-4994) secretes an exopolysaccharide that can be used as an ingredient in cosmetic applications. The structure was resolved using chromatography and one- and two-dimensional NMR spectroscopy experiments. The results show that the carbohydrate backbone is made of two residues: d-galacturonic acid and N-acetyl-d-glucosamine (GlcNac), which together constitute a tetrasaccharide repetition unit: [→3)-α-d-GalA-(1→4)-α-d-GalA-(1→3)-α-d-GalA-(1→3)-ß-GlcNAc(1→]. Two amino acids, alanine and serine, are linked to GalA residues via amido linkages. The position and the distribution of the amino acids were characterized by two-dimensional NMR spectroscopy. To our knowledge, this is the first description of a structure for a marine exopolysaccharide decorated with an amino acid.

Characterization and structure of the polysaccharide produced by Pseudomonas fluorescens strain TF7 isolated from an arid region of Algeria.

Many bacteria possess a natural ability to synthesize and excrete exopolysaccharides which are widely varied in structure and function. These bacteria have the ability to solubilize inorganic phosphorus, which is important to promote growth and increase crop yields. The objective of this study is to select an adaptive strain to the constraints of erratic rainfall and large temperature variations and to determine the possible synergistic effects of its EPS and organic acid on tricalcium phosphate (TCP) solubilization. The strain TF7 isolated from an arid region of Algeria was characterized on the basis of its morphological and physiological traits. Polysaccharide production and the phosphate-solubilizing activity of the strain were evaluated using sucrose and tricalcium phosphate. This EPS was studied by sugar analysis as well as proton NMR spectra. The 16S rRNA gene sequence of this strain shared a similarity of more than 96% with Pseudomonas fluorescens. The maximum polysaccharide productivity was estimated at 4.5g·L(-1) after 5 days. The analyzed sugar was comprised of fructose, glucose, and mannose in a ratio of 4:1:0.6. NMR spectra indicated that the polysaccharide produced by the strain was levan with ß-(2→6)-linked fructose units in accordance with the generally accepted structure. The strain TF7 solubilizes phosphate and forms a clear halo around the colony. The phosphate-solubilizing index is 2.33.

Structural data and biological properties of almond gum oligosaccharide: application to beef meat preservation.

Enzymatic hydrolysis of almond gum generates low molecular weight oligosaccharides (OAG) with a yield of 33.5%. The generated oligosaccharides were purified and identified. OAG analyses show that the most prominent residues were galactose and arabinose with traces of xylose, rhamnose, glucose and mannose. The glycosyl linkage positions were analyzed using gas chromatography-mass spectrometry showing a main chain composed of galactose units [ → 3)-Gal-(1 → ] branched mainly with arabinose residues [Ara-(1 → ]. The antioxidant and antimicrobial activities of OAG were investigated. As regards the in vitro antioxidant activities, the OAG showed a high total antioxidant activity (347 µg ascorbic acid equivalent/mL), an important DPPH (1,1-diphenyl-2-picrylhydrazyl) scavenging activity (IC50 = 0.64 mg/mL) and a high reducing capacity (RP0.5AU = 3.6 mg/mL). Furthermore, OAG had a high antimicrobial activity against Salmonella thyphimirium, Bacillus cereus, Actinomycetes sp, Klebsiella pneumoniae, Escherichia coli, Alternaria alternate and Candidat albicans. Finally, OAG efficiency was tested using 0.5%; 0.75% and 1% concentrations in beef meat preservation. Microbial growth and lipid oxidation were monitored during 9 days at 4 °C. The results showed significant inhibitions (p < 0.05) of lipid oxidation and microbial growth in ground beef meat containing OAG.

Healing efficiency of oligosaccharides generated from almond gum (Prunus amygdalus) on dermal wounds of adult rats.

Almond gum is a naturally occurring polymer produced by almond trees and shrubs. Its abundance, as well as its low cost production makes it a potential feedstock for use in food and pharmaceuticals. In this regard, almond gum oligosaccharides were enzymatically generated, purified and their monosaccharide composition assessed using gas chromatography-flame ionization detector. Oligosaccharide analyses show that the most prominent residues were galactose and arabinose with traces of xylose, rhamnose, glucose and mannose. The glycosyl linkage positions were analyzed using gas chromatography - mass spectrometry showing a main chain composed of galactose units [→3)-Gal-(1→] branched mainly with arabinose residues [Ara-(1→]. The potent role of the generated oligosaccharides on rats wound healing was investigated. They have been applied either alone or supplemented, as active substance, with cream formulation, on full-thickness wound created on the dorsum of the rats. The effect of oligosaccharides was assessed by measuring the wound closure percentage, reaching an average of around 100% when applied alone or supplemented to cream formulation. The healing percentage for the control group was only 74.3% at the same day. The histological evaluation of skin sections visualized by light microscopy revealed an improved collagen deposition and an increased fibroblast and vascular densities.

Purification and structural data of a highly substituted exopolysaccharide from Pseudomonas stutzeri AS22.

Pseudomonas stutzeri AS22, when grown on media containing starch and yeast extract and incubated at 30 °C and 200 rpm for 24h, was found to produce an acidic and high-molecular mass exopolysaccharide (EPS22). The EPS22 was purified and a yield of 1.3g/l was achieved. The average molecular mass of the EPS22 was determined by high-performance size-exclusion chromatography (HPSEC) and showed an average molecular mass of 9.9 × 10(5)Da and a polydispersity index Mw/Mn (Mw, weight-average and Mn, number-average) of 1.197 ± 0.015. Structural data of this EPS22 were determined using a combination approach including monosaccharide composition (HPAEC-PAD and GLC), methylation analysis (GC-MS) and NMR spectroscopy analysis. EPS22 was found to be a complex heteropolysaccharide with a repeating unit mainly composed of glucose, mannose and lactyl rhamnose in a molar ratio of 1:1.1:0.7. The acidic nature of the polysaccharide is due to the presence of three non-osidic substituents consisting of a lactyl, acetyl, and pyruvyl groups.