尼古丁降解菌Pusillimonas sp.T2的筛

发布时间：2018-03-04 12:11

  本文选题：尼古丁　切入点：代谢途径　出处：《浙江工业大学》2015年硕士论文　论文类型：学位论文

【摘要】：尼古丁是一种会使人上瘾或产生依赖性的生物碱,其进入人体会引起多种疾病,包括肺癌、口腔癌、食道癌、心血管病和胃功能障碍等。我国烟民数量之多,烟草生产及加工产业之庞大,产生了大量含高浓度尼古丁的烟草废弃物,给环境带来了极大的安全隐患。微生物作为环境污染物降解的主力军,其在环境修复中的应用一直被认为是高效、可靠和环境友好的。因此,发现具尼古丁降解功能的微生物,研究其降解特性及机理,探索其“变废为宝”的内在规律,是符合我国可持续性发展战略要求的,具有重大的理论意义和应用价值。本研究从杭州一农药厂的活性污泥中筛选得到两株尼古丁降解菌,并选取其中降解能力更强的菌株作为本次研究的对象,命名为T2。根据其生理生化特性及其16S rDNA基因序列同源性分析,鉴定为极小单胞菌(Pusillimonas)。这是在该菌属里首次发现具有尼古丁降解能力的菌株。对T2降解特性的研究结果显示菌株T2降解尼古丁的最佳条件为:pH 7.0左右,温度30℃左右,接种量100%左右,尼古丁初始浓度500 mg/L左右。本研究利用紫外扫描法(UV)、高效液相色谱法(HPLC)和高效液相色谱-质谱联用法(LC-MS)共检测到六种中间代谢产物,分别为6-羟基尼古丁(6HN),6-羟基-N-甲基麦斯明(6HMM),6-羟基假氧化尼古丁(6HPON),6-羟基-3-琥珀酸吡啶(HSP),2,5-二羟基吡啶(2,5-DHP)和2,6-二羟基吡啶(2,6-DHP)。之前未见报道有中间产物2,5-DHP(吡咯烷途径产物)和2,6-DHP(吡啶途径产物)同时出现在同一菌株代谢过程中,由此我们推测T2的尼古丁降解途径可能是一条吡啶-吡咯烷结合的新途径。进一步的降解实验显示,以尼古丁、6HPON或HSP为底物时降解液的颜色分别为蓝、灰蓝和绿。菌株T2以6HPON作为唯一碳源时,检测到产物HSP,2,5-DHP和2,6-DHP。在菌株T2的粗酶液中还分别检测到HSP水解酶和2,6-DHP氧化酶活性,酶活性大小分别为0.087和0.012 U/mg蛋白。这些结果进一步证明:T2先将尼古丁降解成6HN,继而6HN转变为6HMM,接着6HMM自发水解生成6HPON,然后6HPON分别通过吡啶途径和吡咯烷途径降解成2,6-DHP以及HSP和2,5-DHP。这是一条新型的尼古丁降解途径,即吡啶-吡咯烷结合途径。
[Abstract]:Nicotine is an alkaloid that can be addictive or dependent. Its entry into the body can cause many diseases, including lung cancer, oral cancer, esophageal cancer, cardiovascular disease and gastric dysfunction. The huge production and processing industry of tobacco has produced a large amount of tobacco waste containing high concentration of nicotine, which has brought a great security hazard to the environment. Microbes are the main force in the degradation of environmental pollutants. Its application in environmental remediation has always been considered to be highly efficient, reliable and environmentally friendly. Therefore, microbes with nicotine degradation function are found, their degradation characteristics and mechanisms are studied, and the inherent law of "turning waste into treasure" is explored. In this study, two strains of nicotine degrading bacteria were screened from activated sludge of an agricultural pharmaceutical factory in Hangzhou. The strain with stronger biodegradability was selected as the object of this study, named T2.According to its physiological and biochemical characteristics and the homology analysis of 16s rDNA gene sequence, The strain was identified as Pusillimonasus. This is the first strain with nicotine degradation ability found in the genus Pusillimonas.A study on the degradation of nicotine by T2 showed that the optimum conditions for the degradation of nicotine by strain T2 were about 7. 0 pH and 30 鈩，

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