Study On Structure And Immunomodulatory Activity Of Extracellular Polysaccharide Of Antarctic Psychrotrophs Pseudoaltermonas Sp.S-5

The Antarctic is the coldest and driest region on the Earth. It is covered by ice permanently. Antarctic microorganisms possess special physiological and biochemical characteristics. Extreme environments are proving to be a valuable source of microorganisms that secrete interesting novel molecules, including extracellular-polysaccharides (EPS). Microbial EPSs are abundant in the Antarctic. The EPSs present in very high concentrations in the brine of these microhabitats appear to play important buffering and cryoprotectant roles for microorganisms against harsh winter conditions. However, few studies focusing on the biotechnological potential, especially on the macrophages immunemodulating ability of EPSs produced by bacteria from the Antarctic marine environment have been reported in the literature to date. Therefore a wide search for microorganisms to produce good yields of new polysaccharides has been undertaken. Bacteria collected by CTD during Chinese19th Antarctic Scientific Expedition in the Southern Ocean in2002were studied. Out of43strains of Antarctic marine bacteria,15were found to produce EPSs according to the staining technique with congo red and trypan blue. Phylogenetic tree analysis by neighbor-joining method demonstrated that Antarctic marine bacteria S-5was more closely related to the genus Pseudoalteromonas sp.(P.sp). The conditions of fermentation have been optimized and purified EPS (PEP) have been obtained after separation and purification. Then the structure of PEP from P.sp.S-5was determined by using IR spectroscopy, methylation analysis, IR spectroscopy, one-dimensional and two-dimensional NMR spectroscopy. And the macrophage immunemodulating effects of EPS produced by P.sp.S-5in vitro and vivo were carried out. This study will provide scientific data for the biotechnological application of Antarctic resource.1. Out of43strains of Antarctic marine bacteria,15were found to produce EPS according to morphological characterization and the staining techniques. Phylogenetic tree analysis by neighbor-joining method demonstrated that Antarctic marine bacteria S-5was more closely related to the genus Pseudoalteromonas. Pseudoalteromonas sp. is one of frequent and important population in Antarctic ecological system, so Antarctic marine bacteria S-5was selected for further study. The results of morphological identification showed that P.sp.S-5is oval bacteria. It was detected that P.sp.S-5is a Gram-negative bacterium.2. The effects of major environmental factors on the growth and EPS production of Antarctic marine bacteria S-5were investigated. The results showed that Antarctic marine bacteria S-5could grow normally between4-25℃, so it is belong to psychrotrophs. The results showed that the optimal conditions for EPS production were culture period,56h; growth temperature,9℃; carbon source,1%glucose; pH7.0.3. After removal of the cells by centrifugation, the supernatant fermentation was concentrated, CEP was precipitated by adding alcohol and then the protein was removed. Two EPS fractions N-CEP and A-CEP were obtained after purification by DEAE-Fast Flow ion exchange column, then N-CEP were further purified by Sephadex G-75, and a single eluant peak was detected. The results of iodine-iodide reaction, biuret reaction, Molish reaction, and ultraviolet spectrophotometer, showed that single peak was observed at200nm, no nucleic acid, no protein, no coloring matter were determined. There were symmetrical peak determined by high performance gel permeation chromatography, which showed the purity and homogeneity were suitable for analysis. The molecular weight of PEP was397kDa, which was determined by high performance gel permeation chromatography.4. GC analysis of PEP hydrolysis products indicated that polysaccharide is composed of galactose, glucose and mannose. However, the molecular weight of PEP was too big to operate methylation analysis and NMR spectroscopy. Then PEP was hydrolyzed by0.01M TFA for1h to obtain a oligosaccharide termed as H-PEP. molecular weight of H-PEP is4023Da. The structure of H-PEP was determined by using methylation analysis, IR spectroscopy, one-dimensional and two-dimensional NMR spectroscopy as follow.5. The immunity activity of extracellular polysaccharide from Antarctic psychrotrophs P.sp.S-5was studied. PEP produced by the P.sp.S-5obviously promoted proliferation of lymphocytes.Morphological alterations were observed in macrophages treated with PEP. In vitro exposure to PEP increased the occurrence of activated macrophages and endocytic index in a dose-dependent pattern (2.5-50μg/ml) after24h of incubation, since increase of136%and133%were detected in the activated macrophages percentage and endocytic index respectively compared to untreated cells. PEP at200μg/ml caused a greatest increase (44.5%) in NO production when compared to the control group; however, this polysaccharide did not affect respiratory burst in the absence of PMA. Furthermore, it was demonstrated PEP induce macrophages to secrete tumor necrosis factor (TNF)-α and interleukin (IL)-1β.6. The modulation of extracellular polysaccharide from Antarctic psychrotrophs P.sp.S-5, PEP on the immune system in normal mice was investigated. The mice were treated with PEP (10,25,50mg/kg/day) orally for10days. Our study showed that PEP could significantly increase the phagocytosis, the release of NO and the acid phosphatase enzyme activity in macrophage, while the serum hemolysin concentration and spleen lymphocyte proliferation activity were unchanged in normal mice. Serum IL-1β and TNF-α were significantly increased in the mice treated with PEP. PEP can significantly increase delayed type hypersensitivity of normal mice and by the thickness of the toe. The results demonstrated that the EPS from Antarctic psychrotrophs P.sp.S-5PEP was an immunomodulator performed well in enhancing non-specific immunity via macrophage but did not affect the humoral immunity and spleen lymphocyte proliferation.In conclusion, PEP was a heteropolysaccharide composed of mannose, galactose and glucose. Hydrolyzed PEP was H-PEP. The structure of H-PEP was complicated. PEP has an macrophage immunostimulating function and exerts strong immunomodulatory effects on mice in vivo. This research may prove Antarctic microorganisms extracellular polysaccharides as new Biological response modifiers (BRM) and make contribution to the theoretic basis for the development of Antarctic microorganisms metabolite.