Polysaccharide Nanostructures

Polysaccharides are carbohydrate polymers where sugar units are linked together through glycosidic linkages. In living organisms polysaccharides are the structural polymers that provide support (e.g., cellulose in plants or chitin in arthropods) or the sources of energy for plant development (e.g., starch). Polysaccharides are routinely used in the food industry, most frequently as thickeners, stabilizers of dispersions (emulsions, foams) or structuring agents of water and air

Carbohydrate specificity of sea urchin sperm bindin: a cell surface lectin mediating sperm-egg adhesion.

We have examined the carbohydrate specificity of bindin, a sperm protein responsible for the adhesion of sea urchin sperm to eggs, by investigating the interaction of a number of polysaccharides and glycoconjugates with isolated bindin. Several of these polysaccharides inhibit the agglutination of eggs by bindin particles. An egg surface polysaccharide was found to be the most potent inhibitor of bindin-mediated egg agglutination. Fucoidin, a sulfated fucose heteropolysaccharide, was the next most potent inhibitor, followed by the egg jelly fucan, a sulfated fucose homopolysaccharide, and xylan, a beta(1 leads to 4) linked xylose polysaccharide. A wide variety of other polysaccharides and glycoconjugates were found to have no effect on egg agglutination. We also report that isolated bindin has a soluble lectinlike activity which is assayed by agglutination of erythrocytes. The bindin lectin activity is inhibited by the same polysaccharides that inhibit egg agglutination by particulate bindin. This suggests that the egg adhesion activity of bindin is directly related to its lectin activity. We have established that fucoidin binds specifically to bindin particles with a high apparent affinity (Kd = 5.5 X 10(-8) M). The other polysaccharides that inhibit egg agglutination also inhibit the binding of 125I-fucoidin to bindin particles, suggesting that they compete for the same site on bindin. The observation that polysaccharides of different composition and linkage type interact with bindin suggests that the critical structural features required for binding may reside at a higher level of organization. Together, these findings strengthen the hypothesis that sperm-egg adhesion in sea urchins is mediated by a lectin-polysaccharide type of interaction

Regulation of surface architecture by symbiotic bacteria mediates host colonization

Microbes occupy countless ecological niches in nature. Sometimes these environments may be on or within another organism, as is the case in both microbial infections and symbiosis of mammals. Unlike pathogens that establish opportunistic infections, hundreds of human commensal bacterial species establish a lifelong cohabitation with their hosts. Although many virulence factors of infectious bacteria have been described, the molecular mechanisms used during beneficial host–symbiont colonization remain almost entirely unknown. The novel identification of multiple surface polysaccharides in the important human symbiont Bacteroides fragilis raised the critical question of how these molecules contribute to commensalism. To understand the function of the bacterial capsule during symbiotic colonization of mammals, we generated B. fragilis strains deleted in the global regulator of polysaccharide expression and isolated mutants with defects in capsule expression. Surprisingly, attempts to completely eliminate capsule production are not tolerated by the microorganism, which displays growth deficits and subsequent reversion to express capsular polysaccharides. We identify an alternative pathway by which B. fragilis is able to reestablish capsule production and modulate expression of surface structures. Most importantly, mutants expressing single, defined surface polysaccharides are defective for intestinal colonization compared with bacteria expressing a complete polysaccharide repertoire. Restoring the expression of multiple capsular polysaccharides rescues the inability of mutants to compete for commensalism. These findings suggest a model whereby display of multiple capsular polysaccharides provides essential functions for bacterial colonization during host–symbiont mutualism

Chemoenzymatic elaboration of monosaccharides using engineered cytochrome P450_(BM3) demethylases

Polysaccharides comprise an extremely important class of biopolymers that play critical roles in a wide range of biological processes, but the synthesis of these compounds is challenging because of their complex structures. We have developed a chemoenzymatic method for regioselective deprotection of monosaccharide substrates using engineered Bacillus megaterium cytochrome P450 (P450_(BM3)) demethylases that provides a highly efficient means to access valuable intermediates, which can be converted to a wide range of substituted monosaccharides and polysaccharides. Demethylases displaying high levels of regioselectivity toward a number of protected monosaccharides were identified using a combination of protein and substrate engineering, suggesting that this approach ultimately could be used in the synthesis of a wide range of substituted mono- and polysaccharides for studies in chemistry, biology, and medicine

Structural modification of polysaccharides: A biochemical-genetic approach

Polysaccharides have a wide range of industrial and biomedical applications. An industry trend is underway towards the increased use of bacteria to produce polysaccharides. Long term goals of this work are the adaptation and enhancement of saccharide properties for electronic and optic applications. In this report we illustrate the application of enzyme-bearing bacteriophage on strains of the enteric bacterium Klebsiella pneumoniae, which produces a polysaccharide with the relatively rare rheological property of drag-reduction. This has resulted in the production of new polysaccharides with enhanced rheological properties. Our laboratory is developing techniques for processing and structurally modifying bacterial polysaccharides and oligosaccharides which comprise their basic polymeric repeat units. Our research has focused on bacteriophage which produce specific polysaccharide degrading enzymes. This has lead to the development of enzymes generated by bacteriophage as tools for polysaccharide modification and purification. These enzymes were used to efficiently convert the native material to uniform-sized high molecular weight polymers, or alternatively into high-purity oligosaccharides. Enzyme-bearing bacteriophage also serve as genetic selection tools for bacteria that produce new families of polysaccharides with modified structures

Site-Specific Conditions Change the Response of Bacterial Producers of Soil Structure-Stabilizing Agents Such as Exopolysaccarides and Lipopolysaccarides to Tillage Intensity

Agro-ecosystems experience huge losses of land every year due to soil erosion induced by poor agricultural practices such as intensive tillage. Erosion can be minimized by the presence of stable soil aggregates, the formation of which can be promoted by bacteria. Some of these microorganisms have the ability to produce exopolysaccharides and lipopolysaccharides that "glue" soil particles together. However, little is known about the influence of tillage intensity on the bacterial potential to produce these polysaccharides, even though more stable soil aggregates are usually observed under less intense tillage. As the effects of tillage intensity on soil aggregate stability may vary between sites, we hypothesized that the response of polysaccharide-producing bacteria to tillage intensity is also determined by site-specific conditions. To investigate this, we performed a high-throughput shotgun sequencing of DNA extracted from conventionally and reduced tilled soils from three tillage system field trials characterized by different soil parameters. While we confirmed that the impact of tillage intensity on soil aggregates is site-specific, we could connect improved aggregate stability with increased absolute abundance of genes involved in the production of exopolysaccharides and lipopolysaccharides. The potential to produce polysaccharides was generally promoted under reduced tillage due to the increased microbial biomass. We also found that the response of most potential producers of polysaccharides to tillage was site-specific, e.g., Oxalobacteraceae had higher potential to produce polysaccharides under reduced tillage at one site, and showed the opposite response at another site. However, the response of some potential producers of polysaccharides to tillage did not depend on site characteristics, but rather on their taxonomic affiliation, i.e., all members of Actinobacteria that responded to tillage intensity had higher potential for exopolysaccharide and lipopolysaccharide production specifically under reduced tillage. This could be especially crucial for aggregate stability, as polysaccharides produced by different taxa have different "gluing" efficiency. Overall, our data indicate that tillage intensity could affect aggregate stability by both influencing the absolute abundance of genes involved in the production of exopolysaccharides and lipopolysaccharides, as well as by inducing shifts in the community of potential polysaccharide producers. The effects of tillage intensity depend mostly on site-specific conditions

Immunomodulatory, antioxidant and anti-tumor capacity of acidic polysaccharides from Euglena gracilis.

Euglena sp. is a microalga producer of important molecules for the Biotechnology industry, since it is a producer of substances such as vitamins A, C and E, essential amino acids, polyunsaturated fatty acids, β-carotenes and paramilon (β-1,3-glucan). It has a modulating effect on the immune system, moderates blood glucose and the response to insulin, has anti-tumor activity and a cholesterol-lowering effect. In addition, its sulphated derivatives have anti-HIV activity. The present study was carried out with the objective of determining the immunomodulatory, antioxidant and anticancer activity of the acid polysaccharides extracted from Euglena gracilis. MTT colorimeter tests were carried out for the analysis of cytotoxicity on healthy cell lines murine macrophages (RAW 264.7) and human gingival fibroblasts (HGF-1) and for the anticancer activity cell lines were used colon cancer (HCT-116), breast cancer (MCF-7) and human leukemia (U-937) .The polysaccharide concentration at which cell survival was reduced by half (IC50) was estimated with these assays, showing that these polysaccharides have antitumor activity mainly on U-937 cells. (IC50 = 0.027 mg ml-1) against HCT-116 cells (IC50 = 0.036 mg ml-1) and MCF-7 (IC50 = 0.11 mg ml-1) An immunological test was performed to see the immunomodulatory capacity of the polysaccharides with which the production of proinflammatory cytokines IL-6 and TNF-α was determined by macrophages RAW 264.7 It was observed that these polysaccharides have a great stimulating capacity in the synthesis of these interleukins. Antioxidant capacity was (7.19 μmol TE g-1). In agreement with these results, it is suggested that E. gracilis polysaccharides could be considered for future studies as potential nutraceuticals that require their application when the activation of macrophages is needed.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Cell-wall polysaccharides play an important role in decay resistance of Sphagnum and actively depressed decomposition in vitro

Sphagnum-dominated peatlands head the list of ecosystems with the largest known reservoirs of organic carbon (C). The bulk of this C is stored in decomposition-resistant litter of one bryophyte genus: Sphagnum. Understanding how Sphagnum litter chemistry controls C mineralization is essential for understanding potential interactions between environmental changes and C mineralization in peatlands. We aimed to separate the effects of phenolics from structural polysaccharides on decay of Sphagnum. Wemeasured aerobic microbial respiration of different moss litter types in a lab. We used chemical treatments to step-wise remove the chemical compounds thought to be important in decay-resistance in three taxonomically distant moss genera. We also focused on the effect of Sphagnum-specific cell-wall pectin-like polysaccharides (sphagnan) on C and N mineralization. Removing polymeric lignin-like phenolics had only negligible effects on C mineralization of Sphagnum litter, but increased mineralization of two other bryophyte genera, suggesting a minor role of these phenolics in decay resistance of Sphagnum but a major role of cell-wall polysaccharides. Carboxyl groups of pectin-like polysaccharides represented a C-source in non-Sphagnum litters but resisted decay in Sphagnum. Finally, isolated sphagnan did not serve as C-source but inhibited C and N mineralization instead, reminiscent of the effects reported for phenolics in other ecosystems. Our results emphasize the role of polysaccharides in resistance to, and active inhibition of, microbial mineralization in Sphagnum-dominated litter. As the polysaccharides displayed decay-inhibiting properties hitherto associated with phenolics (lignin, polyphenols), it raises the question if polysaccharide- dominated litter also shares similar environmental controls on decomposition, such as temperature or nutrient and water availabilit

Aldehyde-containing urea-absorbing polysaccharides

A novel aldehyde containing polymer (ACP) is prepared by reaction of a polysaccharide with periodate to introduce aldehyde groups onto the C2 - C3 carbon atoms. By introduction of ether and ester groups onto the pendant primary hydroxyl solubility characteristics are modified. The ACP is utilized to absorb nitrogen bases such as urea in vitro or in vivo

Automated glycan assembly of arabinomannan oligosaccharides from Mycobacterium tuberculosis

Arabinomannan (AM) polysaccharides are clinical biomarkers for Mycobacterium tuberculosis (MTB) infections due to their roles in the interaction with host cells and interference with macrophage activation. Collections of defined AM oligosaccharides can help to improve the understanding of these polysaccharides and the development of novel therapeutical and diagnostic agents. Automated glycan assembly (AGA) was employed to prepare the core structure of AM from MTB, containing α-(1,6)-Man, α-(1,5)-Ara, and α-(1,2)-Man linkages. The introduction of a capping step after each glycosylation and further optimized reaction conditions allowed for the synthesis of a series of oligosaccharides, ranging from hexa- to branched dodecasaccharides