臺灣博碩士論文加值系統

三硝基甲苯(2, 4, 6-Trinitrotoluene，TNT)，俗稱黃色炸藥，為軍事及工業上常大量運用火炸藥之一，會造成土壤與地下水的污染，研究顯示人與動物長期暴露於三硝基甲苯環境罹患貧血症和肝功能失常的機會增加亦證明對男性生殖功能有不良影響，世界衛生組織將TNT列為可能致癌物，會損壞造血系統及影響免疫系統。因此TNT污染場域的整治與復育一直以來是刻不容緩的重要環境議題。目前已有物理、化學、生物等技術處理方式應用於TNT污染的整治，其中以生物處理的復育方法進行環境污染整治時對環境的影響較為溫和。實驗室先前研究成果已知適當的生物刺激可實際運用於TNT汙染土的整治──篩選菌株透過添加胺基氮源之生物刺激方式可以達到高效能分解TNT的效果，再進一步探討經微生物降解的效能及產物，卻發現TNT雖能以高效能分解，其代謝產物(亦可能為中間代謝產物)所產生的毒性更高。探究微生物行有氧代謝TNT的可能反應途徑得判斷，極可能係因TNT降解後的產物多數在其苯環結構上帶有羥基(-OH)形成苯酚類化合物所致，因此本研究將探討該類有毒中間產物與Laccase參與代謝的影響。
漆氧化酶(laccase)是一種很早就發現於植物、真菌及微生物的含銅共同氧化酵素，該酵素在大腸桿菌中的直向同源基因稱為CueO。由於此酵素具備將苯環上羥基氧化的潛力，因此本研究利用大腸桿菌K12 MG1655菌株為模式生物，將 CueO基因剔除處理(knockout)或是以乳糖誘導大量表現(overexpression)後，以含100ppm TNT培養基於37°C進行六天培養，並以HPLC檢測其TNT降解效能，並同時以水蚤靜水式檢驗法測量代謝產物毒性，以探討大腸桿菌CueO在減緩TNT降解產物毒性的機制。初步結果顯示大腸桿菌在達到一定的TNT降解效果時，其代謝產物較初始培養劑量的TNT毒性高，但比較K12野生型和CueO基因剔除菌株的毒性後顯示，失去CueO基因後的菌株其TNT代謝產物毒性較野生型菌株的高，可證實CueO的確在去除TNT代謝產物毒性上扮演某種角色。未來研究將繼續以Laccase (CueO)為重點，進一步分析其在大腸桿菌進行TNT中間產物代謝的角色，並進一步探討CueO是否能在銅離子的共同作用下，產生更好的降解效果並降低降解產物之毒性。
中文關鍵字：三硝基甲苯降解、生物復育、漆氧化酶

2, 4, 6-Trinitrotoluene, the yellow-colored solid, is a useful explosive dynamite best known as TNT and massive utilized for military and industry. After widely used, it caused environmental pollution both for soil and groundwater contaminated. The influence of repopulation is difficult and expensive to remedy. In addition, the literature indicates that human exposure to TNT waste will stand a high probability of getting anemia and liver dysfunction. TNT and related metabolites also listed as carcinogens, for a reason that TNT contamination of remediation is an important environment issue currently. There are physical, chemical and biological technical treatments applied to TNT pollution remediation which biological treatment of environmental pollution remediation more moderate nowadays. In our previously study, we used the microbial remediation technique and selected TNT-degradable bacteria strains exhibited a significantly higher TNT degradation capability by treated stimulate amino-peptide. However, the acute toxicity of effluents and receiving waters test showed the toxicity of TNT metabolites is higher than TNT. Due to the department of phenolic compounds after degradation, the structure of metabolites with hydroxyl (-OH) on its benzene ring from TNT aerobic metabolism pathway.
Laccase, are copper-containing oxidase enzymes that are found in many plants, fungi, and microorganisms. The enzyme orthologous genes in Escherichia coli called CueO and it has the potential to be oxidized hydroxyl groups on the benzene ring. Its strong oxidation widely used in dye contamination treatment. Because of the fact that we use biological model Escherichia coli K12 strains MG1655 as wild type and aim the CueO gene to treat as knockout and overexpression strains in this study. Accordingly we cultured these experiment strains in same nutrition with TNT and analysis results both degradation and metabolites toxicity. Preliminary results show that when E. coli reaching a level of TNT degradation, its metabolites toxicity higher than initial dose TNT. But comparison of wild type and knockout strains, the later has higher toxicity. Base on the results, we concluded that E.coli has TNT degradation capability and CueO indeed play a role in removing toxic metabolites of TNT. Our research continue to discuss and confirm the function of laccase (CueO) in TNT metabolism and how is it participate in degradation to decrease toxicity of TNT metabolites.
keywords: 2,4,6-Trinitrotoluene, TNT、TNT degradation、bioremediation、laccase、E.coli CueO

謝誌 ............................................................................................................ I
中文摘要 ................................................................................................. IV
Abstract ................................................................................................... VI
目錄 ...................................................................................................... VIII
表目錄 ..................................................................................................... XI
圖目錄 ................................................................................................... XII
附錄 ...................................................................................................... XIV
一、緒論 ................................................................................................... 1
1.1研究背景與動機 ......................................................................... 1
1.2研究目的 ..................................................................................... 2
1.3研究範圍與限制 ......................................................................... 2
二、文獻回顧 ........................................................................................... 4
三、研究材料及方法 ............................................................................... 7
3.1研究架構與程序 ......................................................................... 7
3.2菌株與質體 ................................................................................. 7
3.2.1 菌株 ................................................................................. 7
3.2.2 質體 ................................................................................. 8
3.3 K12勝任細胞之製備 ................................................................. 8
3.4 引子對設計與製備 .................................................................... 8
3.5 Laccase-null實驗菌株建構：K12ΔCueO ................................ 9
3.5.1 同源序列及抗-抗生素基因之構築 ............................... 9
3.5.2 線性DNA接合作用(TA cloning)與熱休克之轉型作用(Heat shock transformation) ........................................... 9
3.5.3 K12同源序列重組及K12ΔCueO::cat菌株篩選 ........ 10
3.5.4 PCR確認K12ΔCueO::cat ............................................ 11
3.5.5 K12ΔCueO建構 ............................................................ 12
3.5.6 PCR確認K12ΔCueO ................................................... 13
3.6 蛋白表現質體pET21b-laccase之建構 .................................. 13
3.7 重組Laccase蛋白的大量表達和純化 ................................... 13
3.8 三硝基甲苯(TNT)降解實驗 ................................................... 14

3.8.1 培養基 ........................................................................... 14
3.8.2 K12衍生菌種培養瓶條件配置 .................................... 15
3.8.3 菌株生長測試 ............................................................... 16
3.9 三硝基甲苯(TNT)殘留量檢定 ............................................... 16
3.9.1 樣本採集 ....................................................................... 16
3.9.2 高效能液相層析儀(HPLC)分析 .................................. 16
3.10 降解產物之生物急毒性試驗方法 ........................................ 18
3.10.1 試驗方法 ..................................................................... 18
3.10.2 大腸桿菌降解TNT代謝產物樣本與試驗動物觀察....................................................................................... 18
四、結果 ................................................................................................. 19
4.1 逐步確認K12 MG1655菌株剔除CueO基因 ...................... 19
4.2 大腸桿菌K12衍生菌株生長試驗 ......................................... 20
4.3 大腸桿菌K12衍生菌株代謝TNT生長曲線記錄 ............... 20
4.4 大腸桿菌K12衍生菌株代謝TNT效能 ............................... 22
4.5 大腸桿菌K12衍生菌株降解TNT效能比較 ....................... 23
4.6 TNT降解後之代謝物以生物急毒性檢驗 .............................. 24
4.7 SDS-PAGE檢驗CueO蛋白誘導表現情形 ........................... 25
4.8 CueO蛋白之訊號胜肽(signal peptide)處理 ........................... 26
4.8.1新建構pET21b-no signal peptide laccase確認 ........... 26
4.8.2新建構21b-nospLCC質體誘導測試與確認 ............... 26
4.9 以SDS-PAGE與Western blot確認誘導Laccase蛋白表現情形 ............................................................................................. 27
4.10 改以BL21 pCueO-21b菌株進行誘導測試 ......................... 27
五、討論 ................................................................................................. 28
5.1 比較實驗A、B組利用E.coli降解TNT效能 ..................... 28
5.1.1 生長曲線探討 ............................................................... 28
5.1.2 TNT代謝情形探討 ....................................................... 29
5.1.3 HPLC偵測到其他分析條件層析圖（2D） ............... 29
5.2 水蚤靜水式檢驗法測量代謝產物毒性 .................................. 30
5.3 誘導大腸桿菌Laccase大量表現問題探討....................... 31
5.4 訊號胜肽處理 ........................................... 31

5.5 未來研究方向 ........................................... 32
六、結論 ........................................................... 33
參考文獻 ............................................................................................... 34
附錄 ....................................................................................................... 65

表目錄
表 1、(A)組培養基：TNT+NFG+Y+P+lactose(lac) ........................... 21
表 2、(B)組培養基：TNT+NFG+Y+P+lactose(lac)+CuCl2 ............... 21
表 3、實驗菌株與質體特性及來源 ..................................................... 40
表 4、實驗之核酸引子序列表 ............................................................. 41
表 5、同源序列及抗-抗生素基因之構築 ........................................... 42

圖目錄
圖 1、研究架構與程序圖示 ................................................................... 7
圖 2 使用引子對確認檢查抗性基因的置換 ................................... 43
圖 3、不含TNT培養基(A)組生長曲線 ......................................... 44
圖 4、不含TNT培養基(B)組生長曲線...................................... 44
圖 5、不含TNT之(A)、(B)兩組生長曲線疊合比較 ........................ 45
圖 6、實際觀測照相記錄含TNT之菌液培養瓶 ............................... 46
圖 7、含TNT(B)組4號瓶(KO菌株)中沉澱物 ................................. 46
圖 8、含TNT(A)組生長曲線 .............................................................. 47
圖 9、含TNT (B)組生長曲線 .............................................................. 47
圖 10、wildtype菌株K12MG1655在(A)、(B)組的生長差異 ......... 48
圖 11、knockout菌株K12ΔCueO在(A)、(B)組的生長差異 ........... 48
圖 12、菌株K12-pET21b-Laccase在(A)、(B)組的生長差異 .......... 48
圖 13、TNT含量(殘留量)層析圖─24小時樣本 .............................. 49
圖 14、TNT含量(殘留量)層析圖─48小時樣本 .............................. 50
圖 15、TNT含量(殘留量)層析圖─72小時樣本 .............................. 51
圖 16、TNT含量(殘留量)層析圖─96小時樣本 .............................. 52
圖 17、TNT含量(殘留量)層析圖─144小時樣本 ............................ 53
圖 18、含TNT(A)組接種菌株代謝TNT情形 .................................. 54
圖 19、含TNT(B)組接種菌株代謝TNT情形 ................................... 54
圖 20、比較wildtype菌株K12MG1655在(A)、(B)組降解差異 .... 55
圖 21、比較knockout菌株K12ΔCueO在(A)、(B)組降解差異 ...... 55
圖 22、比較菌株K12-pET21b-Laccase在(A)、(B)組降解差異 ...... 55
圖 23、三種接種菌株於(A)、(B)培養條件下降解TNT效率 .......... 56
圖 24、虎斑猛水蚤48小時生物急毒性試驗 ..................................... 57
圖 25、虎斑猛水蚤96小時生物急毒性試驗 ..................................... 58
圖 26、以SDS-PAGE檢驗pET21b質體誘導情形(左)及BL21(DE3) pET21b-laccase誘導情形(右) ................................................. 59
圖 27、新建構pET21b-no signal peptide laccase定序確認 .............. 60
圖 28、SDS-PAGE檢驗新建構質體在BL21(DE3)、K12菌株內誘導情形 ............................................................ 61
圖 29、以SDS-PAGE檢驗新建構質體誘導表現並純化後樣本 ..... 62
圖 30、SDS-PAGE與Western blot確認Laccase蛋白表現 ............. 63
圖 31、BL21 pCueO-21b菌株誘導後Laccase蛋白表現情形 .......... 64

附錄
附錄 1、以質體pKD3為建構cat基因段模板 .................................. 65
附錄 2、Knockout(KO) strain：K12 ΔCueO 構築機制圖 ................. 66
附錄 3、蛋白表現表達質體pET21b-Laccase建構示意圖 ............... 67
附錄 4、目前三硝基甲苯被生物行有氧代謝降解的可能反映方式 68
附錄 5、使用HPLC以Thermo Acclaim Explosives E2 column分析TNT以及相關火炸藥與硝基芳香族化合物之圖譜 ........... 69
附錄 6、SDS-PAGE 配方(適用0.75mm /piece) ................................ 70

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