二苯醚及低溴代二苯醚好氧降解菌筛选及降解机制研究

发布时间：2018-04-03 15:11

本文选题：二苯醚　切入点：好氧细菌　出处：《浙江大学》2016年博士论文

【摘要】：多溴二苯醚作为阻燃剂,长期用于电器、玩具、服装等众多涉及到生产和生活的领域。该化合物与塑料本身不存在任何化学键的结合,随时间可从塑料制品中流失,进入周围环境。这类化合物的可降解性极差,在土壤中半衰期可达20年,一旦进入环境,便处于长期存留状态而日积月累。由于其具有亲脂性,一旦进入动物体内,容易在组织中积累,并可通过食物链的传递而逐步富集。多年的研究表明,多溴二苯醚可以对动物产生毒性,其作用包括影响正常的神经系统发育、干扰内分泌系统,甚至有可能具有潜在的致癌性。生物降解多溴二苯醚相较于采用其他理化方法,可操作更高,更容易应对土壤等自然环境的需要。其中,厌氧细菌修复可以有针对性地使取代溴从苯环骨架上脱落；而好氧细菌则有能力降解取代溴较少的二苯醚,使其分解为更小的分子结构。考虑到好氧细菌更易于应对环境原位修复,本研究将研究侧重点放在好氧的二苯醚以及低溴代二苯醚降解细菌的发掘之上。在本文中,还对发现的细菌降解二苯醚及低溴代二苯醚的机制进行了详尽的解析,以期为进一步阐明该菌株的降解性能做好理论上的准备。本文的主要研究结果如下：1)二苯醚降解菌的筛选及其降解性能分析采用二苯醚作为筛选底物,以可利用二苯醚作为碳源生长作为筛选指标,从杭州一处污水处理厂的活性污泥中获得可以二苯醚为碳源稳定存在并将其降解的混合细菌富集物。从该富集物中,分离得一株可利用二苯醚为碳源生长的革兰氏阴性菌,鉴定后命名为Cupriavidus basilensis WS。该菌株在6天内可以将1g/L的二苯醚完全降解,转化为自身的菌体,在整个过程中无任何中间产物检出。该菌株也可降解4-溴二苯醚以及4,4’-二溴二苯醚,在降解4-溴二苯醚的过程中,检测到中间产物苯酚。2)基于C. basilensis WS随机突变定位降解基因以mini-Tn5转座子作为工具,随机于基因组上打断Cupriavidus sp. WS的基因,构建了包含数千个菌株的突变库。对该突变库进行筛选后,获得了6株确定已经丧失二苯醚降解能力的菌株。通过染色体步移确定了这6个突变株被转座子打断的基因后,发现所有被打断的基因都位于bph基因簇,所编码蛋白都与联苯-2,3-双加氧酶相关。3)各bph基因在二苯醚降解中功能及降解途径的确定在将二苯醚降解能力与bph基因簇关联后,我们采用E. coli作为外源表达的平台,将多个候选的bph基因重新组合,一步步还原出了二苯醚的降解过程。在此期间,对每一次反应生成的产物作了详细的鉴定。结果表明,BphA (BphAl+BphA2+BphA3+BphA4)、BphB、BphC依次完成了二苯醚降解的1-3步,直至生成苯酚和PCA。结合前期对C. basilensis WS降解产物的分析,我们认为该菌株有能力矿化二苯醚。4)基于天然质粒pWS的C. basilensislE. coli穿梭载体构建及应用从C. basilensis WS中分离得到天然质粒pWS并测定其序列后,我们对pWS的序列进行多次分割并构建为不同的载体,确定了pWS所需的最小复制子。该复制子中,除了rep基因的编码区不可替代,其他原件包括rep的启动子均可以剔除或替换。以最小复制子为核心,我们构建了C. basilensislE. coli穿梭载体pCB5。有趣的是,通过调节pCB5中Rep的供应量,我们将该载体在C. basilensisWS中的拷贝数从1-3提高到16。在pCB5的基础上,我们构建了pBP-lacZ和pDMP-lacZ两个报告载体,并以此分析了dmp基因簇在二苯醚降解过程中的转录情况。5)基于比对的启动子预测方法开发该方法需要首先以PWM从基因组中预测启动子,获得PWM预测的启动子库,同时随机构建非启动子序列组成的库。任一需要分析的序列分别与前者和后者的每一条序列进行比对,以其差异判断作为启动子达到可能性。
[Abstract]:Two polybrominated diphenyl ether as a flame retardant, long used in electrical appliances, toys, clothing and many other related to production and life field. Combined with the compound and the plastic itself does not exist any chemical bond, with time can be lost from the plastic products, into the surrounding environment. This kind of compounds degradation in soil is poor. The half-life of up to 20 years, once in the environment, in the long-term retention state. Because of the days and months multiplying with a lipophilic, once in the animal body, easy to accumulate in the organization, and through the food chain and gradually enriched. Years of research shows that two polybrominated diphenyl ether can be toxic to the animal, including its role in effect the normal development of nervous system, interfere with the endocrine system and may even be potentially carcinogenic. Biodegradation of polybrominated diphenyl ether two compared with other physical and chemical methods, operation more easier to deal with soil To the soil natural environment. Among them, anaerobic bacteria repair can be targeted to the substitution of bromine off from benzene skeleton; two benzene ether and aerobic bacteria have the ability of degrading bromine replaces less, which is decomposed into smaller molecular structure. Considering the aerobic bacteria more easily cope with environmental remediation. This study will focus on the two phenyl ether aerobic and explore the low bromide two benzene ether degrading bacteria. In this paper, the bacterial degradation of two phenyl ether discovery and mechanism of low brominated diphenyl ether two for a detailed analysis, in order to further clarify the performance degradation strains to make a theoretical preparation. The main results are as follows: 1) analysis using two benzene ether as the substrate screening screening two phenyl ether degrading bacteria and their degradation properties, to be used as a carbon source for growth as a screening index by two phenyl ether, a waste water from Hangzhou You can get two phenyl ether as the carbon source is stable and its degradation of the mixed bacteria enrichment of activated sludge treatment plant. From the enrichment, separation of gram negative bacteria strains can grow as carbon source by two phenyl ether, named after Cupriavidus basilensis WS. identified the strain in within 6 days. The 1g/L two phenyl ether completely degraded into their cell, in the whole process without any intermediate products detected. This strain could also degrade 4- bromide two phenyl ether and 4,4 '- dibromo two phenyl ether, in the degradation process of 4- bromo phenyl ether two, detected the intermediate product of phenol.2 C. basilensis) random mutations in the WS gene localization degradation by mini-Tn5 transposon in the genome as a tool based on the random Cupriavidus sp. interrupted WS gene to construct the mutation library contains thousands of strains. The mutant libraries were screened and obtained 6 strains has been determined to lose two Phenyl ether degrading strains. By chromosome walking to identify these 6 mutant strains were transposon interrupted, found that all interrupted genes are located in the BPH gene cluster, the encoding protein with -2,3- biphenyl dioxygenase.3) function in two benzene ether degradation and degradation of the BPH gene the way to determine the two phenyl ether degradation ability and BPH gene cluster Association, we use E. coli as the exogenous expression of BPH gene of the platform, a plurality of candidate new combinations, step by step to restore the degradation process of two benzene ether. During this period, on the formation of every reaction in detail identification. The results showed that BphA (BphAl+BphA2+BphA3+BphA4), BphB, BphC in order to complete the two phenyl ether degradation in the 1-3 step, until the formation of phenol and PCA. analysis of C. basilensis WS degradation products early, we believe that the two strains had the ability of mineralization of benzene ether.4 base) In the C. basilensislE. coli pWS plasmid shuttle vector construction and application of natural pWS plasmid and sequenced basilensis isolated from C. WS, the pWS sequence was fractionated and construct different vectors, determines the minimal replicon required for pWS. The complex system of child, in addition to rep gene encoding area can not be replaced, other components including Rep promoter can be removed or replaced. With minimal replicon as the core, we constructed C. basilensislE. coli shuttle vector pCB5. is interesting, the supply of Rep in the regulation of pCB5, we will be the carrier in the C. basilensisWS copy number increased from 1-3 to 16. on the basis of pCB5, we constructed pBP-lacZ and pDMP-lacZ two report vector, and then analyzed the DMP gene cluster of.5 transcription in two benzene ether degradation process) method is developed to predict the promoter based on comparison The method needs first to PWM from the genome predicted promoter, PWM predicted promoter library, and randomly constructed non promoter sequence library. Any need to analyze the sequence respectively each with the former and the latter sequence were compared to the difference judgment as a promoter to the possibility.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：X172

【参考文献】