南极嗜冷菌Psychrobacter sp.B-3胞外寡聚糖的研究

发布时间：2018-04-28 23:05

本文选题：南极菌Psychrobacter + sp.B-3　；参考：《青岛科技大学》2011年硕士论文

【摘要】：南极因其干燥、酷寒、强辐射等自然环境,生存于此的微生物有独特的分子生物学机制和生理生化特征,能产生新型的活性物质,如胞外多糖。胞外多糖在南极海冰中可以保护其生物体免受冰晶伤害,降低其它化学物质的伤害。南极微生物胞外多糖在南极生态系统中具有重要作用,但其潜在的生物生化活性物质应用研究报道还很少。本实验以中国第24次南极科学考察乌拉圭站(S62°11’50.52”W58°55’50.4")采集的海冰样品,经佐贝尔2216E培养基筛选,分离获得一株产高糖南极菌B-3,经16SRNA鉴定为嗜冷菌Psychrobacter,通过对该菌的培养条件优化,胞外多糖分离纯化获得高纯度样品；采用高效凝胶渗透色谱(HP-GPC)分析确定B-3胞外多糖的组成；对B-3多糖的植物诱导活性进行研究,以期为南极菌的应用开发提供科学依据。 1.对57株南极菌的外形观察、刚果红法和显微镜检测法筛选出一株产糖菌。16S rDNA鉴定结果表明,南极海洋菌B-3是一株嗜冷菌Psychrobacter sp.B-3。由于嗜冷菌是南极生态系统中常见的一类细菌,所以对其进行进一步的研究。 2.南极嗜冷菌B-3最佳的实验室产糖条件是：最佳碳源,1%的葡萄糖；最佳氮源,0.5%的黄豆粉；最佳NaCl浓度,3%；最佳温度,10℃；最佳pH值为7；培养时间,60 h。试验结果表明,南极海洋菌在4-25℃下均能正常生长,因此确定该菌为嗜冷菌,即南极嗜冷菌B-3。100L发酵罐结果表明,其条件与实验室培养条件大体相同,其胞外多糖产量比实验室发酵有所提高。 3.南极嗜冷菌B-3发酵液经过95%乙醇沉淀、sevege法去蛋白后获得粗多糖。获得的B-3多糖粗品经Sephadex G-50凝胶层析进一步纯化,获得纯品。以高效凝胶渗透色谱(HP-GPC)测定多糖分子量分别为：2112Da。水解后纯品的糖氰乙酸酯衍生物经气相色谱分析,确定其单糖组成为甘露糖和葡萄糖。 4.该实验用0.3%、0.5%和0.8%(或0.2%、0.4%、0.8%、1.()%)的B-3胞外寡聚糖对黄瓜幼苗分别喷施不同时间后,黄瓜体内的几丁质酶,β-1,3-葡聚糖酶,苯丙氨酸解氨酶(PAL),过氧化物酶(POD)、过氧化氢酶(CAT)、谷胱甘肽-S转移酶(GSH-ST)、谷胱甘肽过氧化物酶(GSH-PX)等活性均较对照有不同程度的提高,并且与0.5%的壳聚糖具有相似的诱导效果；同时,0.5%的B-3多糖能够显著的降低黄瓜白粉病的病情指数,防效达24.49%,B-3寡聚糖有望开发为新型抗病诱导子。 5对南极嗜冷菌B-3胞外多糖的低温保护作用的测定表明,不同的浓度南极嗜冷菌B-3胞外多糖,均可以降低溶液的冰点。研究还表明,重复的冻融循环后,胞外多糖保护菌体免受对其造成的伤害,使该菌保持较好的生长能力,所以推测该胞外多糖具有一定的低温保护作用。综上所述,南极嗜冷菌B-3的胞外多糖经气相色谱分析是由葡萄糖和甘露糖组成；该糖能够黄瓜幼苗的抗病性,降低黄瓜白粉病的发病率；同时,该菌最佳产糖温度为10℃、盐度为3%,其产生的胞外多糖还能够降低冰点,避免重复冻融对菌体的伤害作用。本结果为研究南极微生物的应用生产提供了依据,以及南极资源的开发利用提供了科学依据。
[Abstract]:Antarctica, because of its natural environment, such as dry, cold and strong radiation, has a unique molecular biological mechanism and physiological and biochemical characteristics, which can produce new active substances, such as extracellular polysaccharide. Extracellular polysaccharide can protect its organisms from ice crystals in the Antarctic sea ice and reduce the damage of other chemical substances. Antarctic microbes Extracellular polysaccharide (exo) plays an important role in the Antarctic ecosystem, but the potential application of biobioactive substances is rarely reported. In this experiment, the sea ice samples collected from the twenty-fourth Chinese Antarctic scientific survey Uruguay station (S62 11 '50.52 "W58 55' 50.4") were selected by the screening of the Zoar 2216E medium, and a high sugar South was obtained. B-3 was identified as eosinophilic bacteria Psychrobacter by 16SRNA, and high purity samples were obtained by optimizing the culture conditions of the bacteria and separation and purification of extracellular polysaccharide. The composition of B-3 extracellular polysaccharide was determined by high performance gel permeation chromatography (HP-GPC) analysis, and the plant induced viability of B-3 polysaccharide was studied in order to provide the application development of Antarctic bacteria. Scientific basis.
1. to observe the shape of 57 strains of Antarctic bacteria, the results of.16S rDNA identification of a strain of sugar producing bacteria by Congo red method and microscope detection show that the Antarctic marine bacteria B-3 is a cold strain Psychrobacter sp.B-3., which is a common type of bacteria in the Antarctic ecosystem because of the eosinophilia.
2. the best laboratory sugar producing conditions for Antarctic eosinophilic bacteria B-3 are: the best carbon source, 1% glucose, the best nitrogen source, 0.5% soybean powder, the best NaCl concentration, 3%, the optimum temperature, 10 centigrade, and the optimum pH value of 7; the incubation time, the 60 h. test results show that the Antarctic marine bacteria can grow normally at 4-25 C, thus determining that the bacterium is eophilia, that is, The results showed that the conditions of the Antarctic cold B-3.100L bacteria fermentation tank were similar to those of the laboratory culture conditions, and the yield of extracellular polysaccharides was higher than that of laboratory fermentation.
3. the fermentation broth of Antarctic eosinophilic bacteria B-3 was precipitated through 95% ethanol, and crude polysaccharides were obtained by sevege method. The purified B-3 polysaccharide was purified by Sephadex G-50 gel chromatography and obtained pure products. The molecular weight of polysaccharide was determined by high performance gel permeation chromatography (HP-GPC). The monosaccharide composition was identified as mannose and glucose by spectral analysis.
4. in this experiment, the chitinase, beta -1,3- glucanase, phenylalanine ammonia lyase (PAL), peroxidase (POD), peroxidase (CAT), glutathione -S transferase (GSH-ST), glutathione peroxidase (GSH), glutathione peroxidase (GSH), and glutathione peroxidase (GSH) were applied to cucumber seedlings with 0.3%, 0.5% and 0.8% (or 0.2%, 0.2%, 0.4%, 0.8%, 1. ()%). -PX) and other activities were improved in varying degrees and similar to 0.5% of chitosan. At the same time, 0.5% B-3 polysaccharide could significantly reduce the disease index of cucumber powdery mildew, and the control effect was 24.49%. The B-3 oligosaccharide was expected to be a new type of disease resistant inducer.
5 the determination of the low temperature protection of the Antarctic eosinophilic B-3 extracellular polysaccharide showed that the different concentrations of the Antarctic eosinophilic B-3 extracellular polysaccharide could reduce the freezing point of the solution. The study also showed that after the repeated freezing and thawing cycle, the extracellular polysaccharide protected the mycelium from the damage caused by the bacteria, so that the bacteria maintained good growth ability, so that the extracellular polysaccharide could be maintained. Polysaccharide has a certain protective effect on low temperature.
To sum up, the GC analysis of the extracellular polysaccharide of Antarctic cold strain B-3 is composed of glucose and mannose, which can resist the disease of cucumber seedlings and reduce the incidence of cucumber powdery mildew. At the same time, the optimum temperature is 10 C and the salinity is 3%. The production of extracellular polysaccharide can also reduce freezing point and avoid repeated freezing and thawing bacteria. This result provides a scientific basis for the study of the application and production of Antarctic microorganism and the exploitation and utilization of Antarctic resources.

【学位授予单位】：青岛科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

【参考文献】