本文選題：二苯醚　切入點(diǎn)：好氧細(xì)菌　出處：《浙江大學(xué)》2016年博士論文

【摘要】：多溴二苯醚作為阻燃劑,長(zhǎng)期用于電器、玩具、服裝等眾多涉及到生產(chǎn)和生活的領(lǐng)域。該化合物與塑料本身不存在任何化學(xué)鍵的結(jié)合,隨時(shí)間可從塑料制品中流失,進(jìn)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(dá)20年,一旦進(jìn)入環(huán)境,便處于長(zhǎng)期存留狀態(tài)而日積月累。由于其具有親脂性,一旦進(jìn)入動(dòng)物體內(nèi),容易在組織中積累,并可通過(guò)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(duì)動(dòng)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(yīng)對(duì)土壤等自然環(huán)境的需要。其中,厭氧細(xì)菌修復(fù)可以有針對(duì)性地使取代溴從苯環(huán)骨架上脫落；而好氧細(xì)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(jié)構(gòu)。考慮到好氧細(xì)菌更易于應(yīng)對(duì)環(huán)境原位修復(fù),本研究將研究側(cè)重點(diǎn)放在好氧的二苯醚以及低溴代二苯醚降解細(xì)菌的發(fā)掘之上。在本文中,還對(duì)發(fā)現(xiàn)的細(xì)菌降解二苯醚及低溴代二苯醚的機(jī)制進(jìn)行了詳盡的解析,以期為進(jìn)一步闡明該菌株的降解性能做好理論上的準(zhǔn)備。本文的主要研究結(jié)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(zhǎng)作為篩選指標(biāo),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(xì)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(zhǎng)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(zhuǎn)化為自身的菌體,在整個(gè)過(guò)程中無(wú)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(guò)程中,檢測(cè)到中間產(chǎn)物苯酚。2)基于C. basilensis WS隨機(jī)突變定位降解基因以mini-Tn5轉(zhuǎn)座子作為工具,隨機(jī)于基因組上打斷Cupriavidus sp. WS的基因,構(gòu)建了包含數(shù)千個(gè)菌株的突變庫(kù)。對(duì)該突變庫(kù)進(jìn)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(guò)染色體步移確定了這6個(gè)突變株被轉(zhuǎn)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(guān)。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(guān)聯(lián)后,我們采用E. coli作為外源表達(dá)的平臺(tái),將多個(gè)候選的bph基因重新組合,一步步還原出了二苯醚的降解過(guò)程。在此期間,對(duì)每一次反應(yīng)生成的產(chǎn)物作了詳細(xì)的鑒定。結(jié)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(jié)合前期對(duì)C. basilensis WS降解產(chǎn)物的分析,我們認(rèn)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(gòu)建及應(yīng)用從C. basilensis WS中分離得到天然質(zhì)粒pWS并測(cè)定其序列后,我們對(duì)pWS的序列進(jìn)行多次分割并構(gòu)建為不同的載體,確定了pWS所需的最小復(fù)制子。該復(fù)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(dòng)子均可以剔除或替換。以最小復(fù)制子為核心,我們構(gòu)建了C. basilensislE. coli穿梭載體pCB5。有趣的是,通過(guò)調(diào)節(jié)pCB5中Rep的供應(yīng)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(chǔ)上,我們構(gòu)建了pBP-lacZ和pDMP-lacZ兩個(gè)報(bào)告載體,并以此分析了dmp基因簇在二苯醚降解過(guò)程中的轉(zhuǎn)錄情況。5)基于比對(duì)的啟動(dòng)子預(yù)測(cè)方法開(kāi)發(fā)該方法需要首先以PWM從基因組中預(yù)測(cè)啟動(dòng)子,獲得PWM預(yù)測(cè)的啟動(dòng)子庫(kù),同時(shí)隨機(jī)構(gòu)建非啟動(dòng)子序列組成的庫(kù)。任一需要分析的序列分別與前者和后者的每一條序列進(jìn)行比對(duì),以其差異判斷作為啟動(dòng)子達(dá)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(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：X172

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 彭平安;盛國(guó)英;傅家謨;;電子垃圾的污染問(wèn)題[J];化學(xué)進(jìn)展;2009年Z1期

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本文編號(hào)：1705734