本文選題：二苯醚　切入點：好氧細菌　出處：《浙江大學》2016年博士論文

【摘要】：多溴二苯醚作為阻燃劑,長期用于電器、玩具、服裝等眾多涉及到生產(chǎn)和生活的領域。該化合物與塑料本身不存在任何化學鍵的結合,隨時間可從塑料制品中流失,進入周圍環(huán)境。這類化合物的可降解性極差,在土壤中半衰期可達20年,一旦進入環(huán)境,便處于長期存留狀態(tài)而日積月累。由于其具有親脂性,一旦進入動物體內(nèi),容易在組織中積累,并可通過食物鏈的傳遞而逐步富集。多年的研究表明,多溴二苯醚可以對動物產(chǎn)生毒性,其作用包括影響正常的神經(jīng)系統(tǒng)發(fā)育、干擾內(nèi)分泌系統(tǒng),甚至有可能具有潛在的致癌性。生物降解多溴二苯醚相較于采用其他理化方法,可操作更高,更容易應對土壤等自然環(huán)境的需要。其中,厭氧細菌修復可以有針對性地使取代溴從苯環(huán)骨架上脫落；而好氧細菌則有能力降解取代溴較少的二苯醚,使其分解為更小的分子結構�？紤]到好氧細菌更易于應對環(huán)境原位修復,本研究將研究側重點放在好氧的二苯醚以及低溴代二苯醚降解細菌的發(fā)掘之上。在本文中,還對發(fā)現(xiàn)的細菌降解二苯醚及低溴代二苯醚的機制進行了詳盡的解析,以期為進一步闡明該菌株的降解性能做好理論上的準備。本文的主要研究結果如下：1)二苯醚降解菌的篩選及其降解性能分析采用二苯醚作為篩選底物,以可利用二苯醚作為碳源生長作為篩選指標,從杭州一處污水處理廠的活性污泥中獲得可以二苯醚為碳源穩(wěn)定存在并將其降解的混合細菌富集物。從該富集物中,分離得一株可利用二苯醚為碳源生長的革蘭氏陰性菌,鑒定后命名為Cupriavidus basilensis WS。該菌株在6天內(nèi)可以將1g/L的二苯醚完全降解,轉化為自身的菌體,在整個過程中無任何中間產(chǎn)物檢出。該菌株也可降解4-溴二苯醚以及4,4’-二溴二苯醚,在降解4-溴二苯醚的過程中,檢測到中間產(chǎn)物苯酚。2)基于C. basilensis WS隨機突變定位降解基因以mini-Tn5轉座子作為工具,隨機于基因組上打斷Cupriavidus sp. WS的基因,構建了包含數(shù)千個菌株的突變庫。對該突變庫進行篩選后,獲得了6株確定已經(jīng)喪失二苯醚降解能力的菌株。通過染色體步移確定了這6個突變株被轉座子打斷的基因后,發(fā)現(xiàn)所有被打斷的基因都位于bph基因簇,所編碼蛋白都與聯(lián)苯-2,3-雙加氧酶相關。3)各bph基因在二苯醚降解中功能及降解途徑的確定在將二苯醚降解能力與bph基因簇關聯(lián)后,我們采用E. coli作為外源表達的平臺,將多個候選的bph基因重新組合,一步步還原出了二苯醚的降解過程。在此期間,對每一次反應生成的產(chǎn)物作了詳細的鑒定。結果表明,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。結合前期對C. basilensis WS降解產(chǎn)物的分析,我們認為該菌株有能力礦化二苯醚。4)基于天然質(zhì)粒pWS的C. basilensislE. coli穿梭載體構建及應用從C. basilensis WS中分離得到天然質(zhì)粒pWS并測定其序列后,我們對pWS的序列進行多次分割并構建為不同的載體,確定了pWS所需的最小復制子。該復制子中,除了rep基因的編碼區(qū)不可替代,其他原件包括rep的啟動子均可以剔除或替換。以最小復制子為核心,我們構建了C. basilensislE. coli穿梭載體pCB5。有趣的是,通過調(diào)節(jié)pCB5中Rep的供應量,我們將該載體在C. basilensisWS中的拷貝數(shù)從1-3提高到16。在pCB5的基礎上,我們構建了pBP-lacZ和pDMP-lacZ兩個報告載體,并以此分析了dmp基因簇在二苯醚降解過程中的轉錄情況。5)基于比對的啟動子預測方法開發(fā)該方法需要首先以PWM從基因組中預測啟動子,獲得PWM預測的啟動子庫,同時隨機構建非啟動子序列組成的庫。任一需要分析的序列分別與前者和后者的每一條序列進行比對,以其差異判斷作為啟動子達到可能性。
[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.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：X172

【參考文獻】

相關期刊論文 前1條

1 彭平安;盛國英;傅家謨;;電子垃圾的污染問題[J];化學進展;2009年Z1期

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本文編號：1705734