铜绿假单胞菌NY3分泌绿脓杆菌螯铁蛋白及其对烃类降解特性的影响作用

发布时间：2018-06-05 06:52

本文选题：铜绿假单胞菌NY3 + 绿脓杆菌螯铁蛋白　；参考：《西安建筑科技大学》2017年硕士论文

【摘要】：铜绿假单胞菌可分泌包括鼠李糖酯(Rha)在内的各种小分子活性物和蛋白酶等丰富的胞外物,除Rha在疏水性化合物降解中的作用被充分研究外,其它胞外分泌物在该菌属降解烃类过程中的作用则知之甚少。PCH是铜绿假单胞菌分泌的一种铁载体—螯铁蛋白,对于菌体获取铁元素至关重要。铁是大多启动烷烃降解关键氧化酶的活性中心,缺铁可直接影响NY3菌降解烃的效率。基于NY3菌胞外分泌物对NY3菌降解石油烃污染物的促进作用,以及PCH对NY3菌生长的重要作用,本论文就此展开研究工作,对石油烃污染物的降解有重要的意义。本文研究铜绿假单胞菌NY3分泌铁载体PCH的特性,优化NY3菌分泌PCH的条件,研究PCH对NY3菌降解十六烷的影响作用及影响机理,以及Fe PCH对十六烷的降解作用及作用机理,获得以下成果:(1)NY3菌可分泌绿脓杆菌螯铁蛋白(PCH),且分泌的PCH是一对手性对映异构体的混合。纯化后混合物的水溶液在215 nm、252 nm、360 nm处有强的吸收峰。M9培养基适合该菌高产PCH,优化基础M9培养基可提高NY3菌分泌PCH量至240.240 mg/L,优化结果为:Fe Cl3合适浓度为0.02 mmol/L时;NH4Cl为氮源,适合浓度为5 g/L,M9培养基中以丙酮酸钠为最适合碳源。(2)PCH对于NY3降解烃是“双刃剑”。5.2 mg/L及低于5.2 mg/L的PCH可提高菌体生长量,提高降解烃酶活性,同时也使其胞外液以自由基途径降解转化部分烃;然而,分泌量过高则使细胞死亡。(3)NY3菌分泌于其培养液中PCH越多,培养液降解十六烷能力越高。但从胞外液中提取的PCH纯品对十六烷的降解能力却较低。当Fe Cl3或Fe Cl3+H2O2与之配合则提高对烃的降解效率,8 h最高十六烷降解率可达13.81%。PCH与铁螯合降解十六烷的机理是由其螯合过程所产生的羟基自由基引发的氧化转化反应,且反应体系中自由基强度与十六烷的降解效率呈正相关。(4)利用气质谱共鉴定了7种PCH与Fe Cl3或与H2O2或与Fe Cl3+H2O2配合等体外降解十六烷的转化产物,包括正丁醇、4,5-二羟基-2戊烯、1-甲基,3-羟基戊醇、1-羟基己酸、1-甲基,2-羟基己醇、己二酸、十六烷酸。鉴定了3种降解芘的转化产物,包括1-甲基-2丙烯酸苯、1,2-二羟基萘、4-醇羟基菲。
[Abstract]:Pseudomonas aeruginosa can secrete a variety of small molecular active compounds, including rhamnose, and protease, except for the role of Rha in the degradation of hydrophobic compounds. Little is known about the role of other extracellular secretions in the degradation of hydrocarbons by Pseudomonas aeruginosa. PCH is a ferritin carrier secreted by Pseudomonas aeruginosa. Iron is the active center that most of the alkane degrade key oxidase. Iron deficiency can directly affect the efficiency of hydrocarbon degradation by NY3 bacteria. Based on the effect of extracellular secretion of NY3 bacteria on the degradation of petroleum hydrocarbon pollutants by NY3 bacteria and the important role of PCH on the growth of NY3 bacteria, this paper has carried out research work, which is of great significance to the degradation of petroleum hydrocarbon pollutants. In this paper, the characteristics of NY3 ferric carrier PCH of Pseudomonas aeruginosa were studied, the conditions of PCH secretion by NY3 were optimized, the effect and mechanism of PCH on the degradation of cetane by NY3, and the degradation of cetane by Fe PCH were studied. The following results were obtained: P.aeruginosa chelate ferritin was secreted by NY3, and the PCH secreted was a mixture of chiral enantiomers. The purified aqueous solution had a strong absorption peak at 215nm ~ 252nm ~ (-1) ~ 360nm. M9 medium was suitable for high PCHs production. The optimized basic M9 medium could increase the PCH secretion of NY3 bacteria to 240.240 mg / L, and the optimized results were as follows: (1) NH _ 4Cl was used as nitrogen source when the appropriate concentration of NY3 was 0.02 mmol/L. Sodium pyruvate is the most suitable carbon source for NY3 degradation of hydrocarbon. 5.2 mg/L and PCH with less than 5.2 mg/L can increase the cell growth and increase the activity of hydrocarbon-degrading enzymes in 5 g / L M9 medium. At the same time, the extracellular solution could degrade some of the transformed hydrocarbons by free radical pathway. However, the higher the secretion, the higher the ability of degradation of hexadecane in the culture medium with more PCH secreted by the cell death strain. However, the degradation of cetane by purified PCH extracted from extracellular solution was relatively low. When Fe Cl3 or Fe Cl3 H2O2 were combined with it, the highest degradation efficiency of cetane for 8 h could reach that of 13.81%.PCH and Fe chelate to degrade cetane, which was caused by the hydroxyl radical induced by the chelating process. The free radical strength in the reaction system was positively correlated with the degradation efficiency of hexadecane. It includes n-butanol 4-dihydroxy-2-pentene, 1-methyl-3-hydroxypentanol, 1-hydroxy-caproic acid, adipic acid, hexadecanoic acid. Three kinds of pyrene degradation products were identified, including 1-methyl-2-benzene-2-dihydroxy-naphthalene 4-ethanol-phenanthrene.
【学位授予单位】：西安建筑科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X172

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