烟碱降解菌的分离鉴定、降解特性及其降解途径的初步研究

【作者】 孔雯；

【导师】 李晓华；

【作者基本信息】 中南民族大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 烟碱俗称尼古丁,是烟草中的主要生物碱,过量吸入烟碱则会抑制中枢系统,麻痹心脏,甚至有生命危险;同时烟草加工过程中产生了含高浓度烟碱的废弃物,严重污染了环境;而且我国烟草上部烟叶烟碱含量普遍偏高,不能用于烟组配方造成了资源浪费和经济损失,因此有效地控制烟草和环境中的烟碱含量,对于维护人类健康和环境有着深远的意义。在工业上利用微生物及其酶来降解烟草中的烟碱,比起传统的物理化学法,不仅可以产生更少有害副产品,而且可以改进烟气品质,提高烟叶资源利用率,有重大的经济效益。本研究以烟碱为唯一碳源,从湖北襄樊烟草种植基地的土壤根际土和烟叶废弃物中分离得到29株具有烟碱降解能力的菌株,降解率都在70%以上,其中19株烟碱降解率能达到90%以上。其中12株烟碱降解菌经常规的形态观察、生理生化分析和16S rDNA序列同源性分析,这12株烟碱降解菌分别属于节杆菌属、假单胞菌属、苍白杆菌属和根癌农杆菌属。通过一系列实验,初步确定根癌农杆菌BKCER11菌株在烟碱浓度0.5g/L-5g/L内生长良好,培养基烟碱浓度大于5g/L时,BKCER11菌株生长受到严重抑制,也不能降解烟碱;该菌在温度20℃-37℃范围内可以生长良好并能降解烟碱,其最适生长温度为30℃;在pH5.5-8.0范围内BKCER11菌株可以生长良好并能降解烟碱,其最适pH为5.5-6.0;经过正交优化,BKCER11菌株烟碱降解的最适条件为:烟碱初始浓度为2g/L、温度为30℃、初始pH为5.5-6.0、以蛋白胨和牛肉膏各0.5%作为复合氮源、微量元素的浓度为5mL/L,降解率能达到96.63%。利用GC-MS初步分析了根癌农杆菌BKCER11菌株降解烟碱的中间代谢产物,检测到有3-(2,3,4-三氢-5-吡咯基)-吡啶和可替宁两种新物质的积累。这与已知的微生物代谢烟碱的三种途径中所涉及到的物质均不一样。更多还原

【Abstract】 Nicotine, is a major alkaloid in tobacco Plants. Excessively taking into nicotine will restrain central nervous system, paralyse the heart, and even cause death; In manuafeutring tobacco products, wastes with high content of nicotine as the main toxic compound are produced, which seriouly contaminate the environment; and the nicotine content in the upper tobacco leaf from our country are generally high so that unable to be used as tobacco blend, which caused waste of resource and economic losses. Thus exploring an effective way to reduce the nicotine content in cigarette and environment is of great significance to human health. Interestingly, microbe and its metabolized enzyme can degrade nicotine, which is not only helpful for human health, but also improve the quality of smoke and utility of tobacco leaves and bring a lot of economic benefits.This study utilized nicotine as the sole source of carbon, 29 bacterial strains was isolated from soil and tobacco waste. The degradation efficiency test showed that all of the stains’degradation efficiency were more than 70%, and 19 strains among the isolates whose degradation efficiency were more than 90%. Further study was basing on 12 strains which have been isolated, by the experimental results of the morphological,physiological and biochemical analysis and the comparison of their 16S rDNA analysis, these 12 strains were identified as Arthrobacter sp., Pseudomonas sp., Ochrobactrum intermedium and Agrobacterium tumefaciens.Studies on the Agrobacterium tumefaciens BKCER11 strain’s degradation capability and optimization of fermentation conditions were carried out. Finaly the results showed that the BKCER11 strain could tolerate high concentration of nicotine up to 5g/L, when the concentration of nicotine in the culture medium was greater than 5g/L, the BKCER11 strain’s growth was highly inhibited, and the nicotine in the medium could not be degraded. The BKCER11 strain not only has a wide tolerance to pH, but also possesses the ability to tolerate temperature. Lots of experiments showed that the BKCER11 strain was stable at pH5.5-8 and growed well, and the optimum pH for the strain’s growing and degradating nicotine was 5.5; moreover the BKCER11 strain was stable at 20℃-37℃and growed well, and the optimum temperature was 30℃. In order to define the strain’s optimum degradation condition orthogonal test was used in the next study. The results indicated that the BKCER11 strain degradation efficiency could reach to 96.63%, when the peptone and beef extract simultaneously used as the complex nitrogen sources of the medium and their content were 0.5% respectively; besides some microelement was at the content of 5mL/L and the medium’s pH was at the range of 5.5-6.0, moreover the most important is the original content of nicotine was 2g/L and kept a temperature at 30℃.The intermediate metabolite was detected by using the method of GC-MS. Two novel intermediate metabolites, 3-(2,3,4-dihydro-3H-pyrrol-5-y1)-pyridine and cotinine were detected in the culture medium. They were both different from the substances involed in the three everkown pathways of microbial metabolism of nicotine.更多还原