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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 施養信(Yang-Hsin Shih) | |
| dc.contributor.author | En-Sheng Hong | en |
| dc.contributor.author | 洪恩生 | zh_TW |
| dc.date.accessioned | 2021-06-16T05:09:54Z | - |
| dc.date.available | 2023-07-29 | |
| dc.date.copyright | 2020-08-03 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-07-29 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55873 | - |
| dc.description.abstract | 六溴環十二烷 (Hexabromocyclododecane, HBCD)為被廣泛使用之溴化阻燃劑，因其於環境中不易被降解之特性，使HBCD累積於環境與生物內，對食物鏈各階層之生物造成毒害。雖然近年來以微生物去除汙染物的概念廣為學界倡議，但目前對細菌如何進行HBCD生物轉化的所知仍有限。本研究於土壤中共篩選三株能降解HBCD之細菌，其中兩株細菌為不同品系之Bacillus cereus，另一種細菌為Pseudomonas plecoglossicida。前人已對Bacillus cereus做過HBCD之生物降解的研究，本研究以P. plecoglossicida做為研究目標菌株。P. plecoglossicida能於35°C、pH 8.0以及HBCD濃度為0.125 ppm之條件下，於降解試驗48小時後，去除70.9%之HBCD。培養於LB medium與PBS buffer的條件下，P. plecoglossicida皆能有效去除HBCD，但觀察細菌生長的狀態時，可以發現PBS buffer之降解試驗組，細菌族群密度未能增加，顯示P. plecoglossicida可能無法僅靠HBCD做為生長所需之碳源。此外，添加葡萄糖或蔗糖時，可以發現細菌在有額外之碳源時，能增加HBCD之降解效率。透過鑑定細菌降解HBCD之副產物，我們確認P. plecoglossicida能透過兩個途徑將HBCD轉化。第一個途徑為脫溴途徑，生物轉化之副產物為五溴環十二烯 (pentabromocyclododecene)、四溴環十二烯 (tetrabromocyclododecadiene)、三溴環十二烯 (tribromocyclododecadiene)、二溴環十二烯 (dibromocyclododecadiene)、一溴環十二烯 (bromocyclododecatriene)、環十二碳三烯 (cyclododecatriene)。第二個生物轉化之途徑為同時透過對HBCD進行脫溴與羥化的反應，生物轉化之副產物為七溴環十二烷 (heptabromocyclododecane)、七溴環十二碳二醇 (heptabromocyclododecanediol)、四溴環十二醇 (tetrabromocyclododecadienol)，以及四溴環十二碳三醇 (tetrabromocyclododecenetriol)。P. plecoglossicida之粗蛋白萃取液於pH 8.0、35°C以及HBCD濃度為50.0 ppb之條件下，能降解97.8%的HBCD。蛋白酶譜之活性染色，確認粗蛋白中含有能透過脫溴途徑降解HBCD之酵素。由P. plecoglossicida全基因體序列註解資訊與鑑定與HBCD作用之蛋白質，我們推測降解HBCD之可能酵素為脫氫酶(dehydrogenase)、谷胱甘肽S-轉移酶(glutahione S-transferase)以及細胞色素c (cytochrome c oxidase)。將P. plecoglossicida之粗蛋白以電紡之方式固定於聚乙烯醇 (polyvinyl alcohol, PVA)之奈米纖維上，能有效加強蛋白之穩定性，且使蛋白具有重複使用性。這個固定化酵素的方法可提供利用P. plecoglossicida之粗蛋白去除環境之HBCD的不同應用策略。本篇研究為HBCD之細菌與酵素的生物轉化提供新的觀點。 | zh_TW |
| dc.description.abstract | Hexabromocyclododecane (HBCD) is widely used as a brominated flame retardant (BFR) and is identified as an emerging and persistent comtaminant. It has been detected in various environment matrix and is dangerous to human health. However, limited information is available about bacterial biotransformation of HBCD. In this study, two different Bacillus cereus strains and one Pseudomonas plecoglossicida strain were isolated from BFR comtaminated soil with good HBCD biodegradation capability in aerobical condition. We focus on P. plecoglossicida for the sparse knowledgement on the involvement of HBCD biotransformation of this species. P. plecoglossicida is able to biotransform 70.9% of HBCD at the initial concentration of 0.125 ppm at 35°C and pH 8.0 within 48 hours. This metabolic capability is observed in LB medium as well as in PBS buffer, while the cell density generally decreased in minimum salt medium. In addition, P. plecoglossicida shows a higher biotransformation rate when glucose or sucrose is supplied to minimal salt medium, indicating P. plecoglossicida could require extra carbon source than HBCD. Based on the identification of metabolites, P. plecoglossicida may biotransform HBCD by two pathways. In the first, HBCD was sequentially debrominated to pentabromocyclododecene, tetrabromocyclododecadiene, tribromocyclododecadiene, dibromocyclododecadiene, bromocyclododecatriene and cyclododecatriene. The second pathway is a debrominating and hydroxylating process to form heptabromocyclododecane, heptabromocyclododecanediol, tetrabromocyclo- dodecadienol and tetrabromocyclododecantriol. Crude protein from P. plecoglossicida is able to efficiently degrade 50.0 ppb HBCD at 35°C and pH 8.0. Zymogram assay demonstrated that the ability of dehalogenating HBCD is contributed by proteins in particular electrophoretic character. Based on the assembled genome sequence of P. plecoglossicida and proteome analysis, glutathione S-transferase, dehydrogenase and cytochrome c oxidase were proposed to be the possible enzymes involved in biodegradation of HBCD via debromination. Immobilizing crude protein into polyvinyl alcohol nanofibers via electrospinning could enhance protein stability and allow the protein to be recovered and reused. To sum up, this study provides a novel insight into microbial and enzymatic biotransformation of HBCD. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T05:09:54Z (GMT). No. of bitstreams: 1 U0001-2807202017565700.pdf: 3853796 bytes, checksum: c28fb15283393dfb964b5ddf21cbf418 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 口試委員會審定書 i 誌謝 ii 中文摘要 iii Abstract iv 目錄 v 圖目錄 viii 表目錄 xv 第一章 前言 1 1.1研究背景 1 1.2 研究目的 3 1.3 研究策略 3 1.4 試驗設計 4 第二章 文獻回顧 5 2.1 HBCD物理化學特性 5 2.2 HBCD於環境與生物之累積 5 2.3 HBCD對於動植物之毒性 7 2.4 降解HBCD之方法 8 2.5微生物對 HBCD之降解 9 2.6 HBCD之生物降解機制 12 2.7降解HBCD之酵素與其降解機制 15 2.8 酶譜的應用 15 2.9 酵素固定化 16 第三章 材料與方法 18 3.1試劑與材料 18 3.1.1化學藥品與溶劑 18 3.1.2實驗所用培養基與緩衝溶液 19 3.1.3 實驗處理方法 21 3.2 菌種來源與菌種鑑定 21 3.2.1篩選細菌方法 21 3.2.2 DNA萃取方式 21 3.2.3聚合酶連鎖反應(PCR) 22 3.2.4定序與親源分析 23 3.3 HBCD生物降解研究 24 3.3.1 HBCD萃取方法 24 3.3.2 HBCD分析方法 24 3.3.3細菌對HBCD之生物降解試驗 25 3.3.4 於降解試驗培養基的溶氧量測定 26 3.3.5碳源對細菌降解HBCD影響之試驗 26 3.4 HBCD生物降解機制 27 3.4.1檢測脫溴反應之方法 27 3.4.2檢測副產物之方法利用氣相層析質譜法 27 3.5 酵素對HBCD之生物降解試驗 29 3.5.1破菌之方法 29 3.5.2粗蛋白純化之方法 30 3.5.3粗蛋白降解HBCD之試驗 30 3.5.4 HBCD誘導細菌產生目標酵素試驗 30 3.5.5 酶譜實驗條件與方法 31 3.6 酵素固定化試驗 33 3.6.1 蛋白質固定於聚乙烯醇(Polyvinyl alcohol, PVA)奈米纖維的方法 33 3.6.2 含粗蛋白之PVA奈米纖維對HBCD之降解試驗方法 33 3.7蛋白質鑑定試驗 34 3.7.1 UHPLC-MS/MS之樣本處理方法 34 3.7.2 UHPLC-MS/MS分析方法 34 3.8 HBCD之生物性降解效率的比較方法 35 3.8.1 HBCD降解反應之化學動力常數之計算 35 3.8.2 HBCD降解反應之效率 35 3.8.3 HBCD脫溴反應之效率 36 3.8.4 微生物對HBCD之吸附比例與降解比例 36 3.8.5 蛋白質相對活性的計算方法 37 3.9 以反應曲面法分析環境因子對細菌降解HBCD之影響的方法 37 第四章 結果與討論 38 4.1 HBCD不同菌種降解HBCD之能力 38 4.2 HBCD降解菌種鑑定 40 4.2.1.菌種鑑定 40 4.2.2. P. plecoglossicida的親緣分析 41 4.3 P. plecoglossicida對HBCD的生物降解 42 4.3.1溫度對P. plecoglossicida降解HBCD之影響 42 4.3.2 HBCD濃度對P. plecoglossicida降解HBCD之影響 45 4.3.3 pH值對P. plecoglossicida降解HBCD之影響 49 4.4培養基對P. plecoglossicida降解HBCD之影響 53 4.4.1溫度對P. plecoglossicida降解HBCD之影響 53 4.4.2 pH值對P. plecoglossicida降解HBCD之影響 55 4.4.3 HBCD濃度對P. plecoglossicida降解HBCD之影響 58 4.5以反應曲面法分析環境因子對細菌降解HBCD之影響 64 4.5.1 反應曲面法模型的擬合與確認 64 4.5.2 反應曲面法的分析結果 65 4.6 碳源對P. plecoglossicida降解HBCD之影響 69 4.7 P. plecoglossicida降解HBCD的機制 70 4.7.1 P. plecoglossicida對HBCD之脫溴反應 70 4.7.2 P. plecoglossicida降解HBCD之副產物鑑定 73 4.8 P. plecoglossicida降解HBCD之酵素 89 4.8.1.不同粗蛋白濃度降解之差異 89 4.8.2.溫度對粗蛋白降解HBCD效率之影響 92 4.8.3 粗蛋白之區分與其降解能力之分析試驗 94 4.8.4 HBCD的誘導對粗蛋白降解HBCD效率之影響 95 4.8.5 pH值對粗蛋白降解HBCD效率之影響 97 4.8.6粗蛋白降解HBCD的脫溴反應 99 4.8.7 粗蛋白酶譜分析 100 4.8.8 酵素鑑定試驗 105 4.8.9 酵素固定化試驗 108 第五章 結論 117 參考文獻 119 附錄 130 | |
| dc.language.iso | zh-TW | |
| dc.title | 土壤中篩選出之假單孢菌屬對六溴環十二烷之有氧生物轉化與其功能性酵素的鑑定 | zh_TW |
| dc.title | Aerobic biotransformation of hexabromocyclododecane by Pseudomonas strain isolated from soil and identification of its functional enzyme | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王如邦(Ru-Bang Wang),陳淑華(Shu-Hua Chen),陳玟伶(Wen-Ling Chen),林仲彥(Zhong-Yan Lin) | |
| dc.subject.keyword | 六溴環十二烷,有氧生物轉化,假單孢菌,生物整治, | zh_TW |
| dc.subject.keyword | hexabromocyclododecane,Aerobic biotransformation,Pseudomonas strain,Bioremediation, | en |
| dc.relation.page | 139 | |
| dc.identifier.doi | 10.6342/NTU202001990 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-07-30 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 農業化學研究所 | zh_TW |
| 顯示於系所單位： | 農業化學系 | |
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