臺灣博碩士論文加值系統

甲苯類化合物之催化為現今社會重要的課題，不論是在工業應用亦或是環境污染物分解皆與其相關，本實驗以具有此特性的二甲苯單加氧酵素為研究目標。
二甲苯單加氧酵素為一具有非血基質雙鐵反應中心的膜結合型去飽和酶超級家族蛋白(membrane-bound desaturase superfamily)，由於此類蛋白如何對反應物進行催化的相關研究相對少，此研究除了常見的全細胞催化反應外，我們也使用電化學的催化模式進行探討，進而發現二甲苯單甲氧酵素可以由電極進行直接電子轉移接受電子，在最後的催化結果可以認定二甲苯單加氧酵素為一具有苯環上甲基選擇性的的蛋白，對於甲苯衍生物有相當大的發展性。
再加上至今世界上僅有少數膜蛋白的立體結構被解析出,因此在現階段之研究只能夠透過 X 光吸收光譜或電化學分析其反應中心的金屬配位。經由數據分析後的結果可以推測出二甲苯單加氧酵素雙鐵中心鍵結的氨基酸，以及初步了解反應物與蛋白之間作用的關聯性。

Catalytic oxidation of toluene derivatives exhibits application for fine chemical supply in chemical industry. The corresponding transformation is essential to the remediation of environmental pollutants. XylMA system encoded by TOL plasmid for the protein expression of xylene monooxygenase (XylM) and its reductase (XylA) from Pseudomonas putida mt-2 was selected for an in-depth study. E. coli was employed as a host for their heterogeneously expression and purification. The catalytic conversions of alkyl benzenes were not only carried out by the whole cell catalysis mediated by the co-transformation of XylMA in E. coli system. To simplify the catalytic reaction without imposing expensive cofactor protein(s) or coenzymes, i.e. NAD(P)H, we herein also developed a facile electrochemical transformation system to conduct the direct electron transfer (DET) either directly to the intracytoplasmic membranes enriched with recombinant XylM or mediated by the iron-sulfur cofactor proteins such as XylA towards XylM protein. The X-ray absorption spectroscopy (XAS) studies of purified XylM was performed and displayed the core structure of active center in XylM protein is non-heme diiron center. Similar to recent discovery of the crystal structure of a mammalian stearoyl-CoA desaturase (Mouse SCD1), His-box (8-histidine motif) is essential for the coordination of the iron core complexes.
Keywords: Xylene monooxygenase (XylM); XANES, EXAFS; Electrocatalysis

目錄
摘要……………………………………………………..…I
Extended Abstract………………………...II
謝誌………………………………………….………VIII
目錄…………………………………………………………X
表目錄…………………………………………....XVI
圖目錄…………………………………....XVII
第一章 緒論…………………………………………………….……1
1.1、BTX苯環類化合物的催化……………….……………….1
1.2、二甲苯單甲氧酵素複合體……………………………………………………….2
1.3、XylM催化活性的研究………………………..…..…………………………….15
1.4、表現載體………………………………………… ………………………………...16
1.5、X光吸收近邊緣結構(XANES)與
延伸X光吸收近邊緣細細微結構(EXAFS)分析法............17
1.6、西方墨點法(Western blot)..........................19
1.7、電化學分析（electrochemical analysis）................20
1.7.1、循環伏安法(cyclic voltammetry, CV) ..............20
1.7.2、安培法(amperometry, i-t curve) .....................22
1.7.3、網版印刷碳電極......................................23
第二章 實驗…………………………………………………………………..……........25
2.1、實驗材料…………............……………….…………..25
2.1.1、實驗菌種..........…….......…………………......……..…..25
2.1.2、載體DNA.............………………………….......25
2.1.3、實驗藥品.............………………….…….………….....25
2.2、實驗儀器…….……..…………........…..................28
2.2.1、旋轉式恆溫震盪培養箱(Orbital Shanker Incubator) ……...28
2.2.2、發酵槽(fermentor) ……….………….............…......28
2.2.3、CH, Instruments……….…………..........………..........28
2.2.4、落地型冷凍高速離心機(Ultra Centrifuge) …….…...…....28
2.2.5、超高速冷凍離心機(Ultra Centrifuge) …….....……….......29
2.2.6、照膠系統(UVP) …….………….....................………....29
2.2.7、高壓均質機(French press) …….………….........…........29
2.2.8、氣相層析質譜儀
(Gas Chromatography Mass Spectrometer GC-MS) ..…......29
2.2.9、氣相層析儀(Gas Chromatography, GC) ………............29
2.3、實驗流程…….…………...............................29
2.4、實驗方法…….………….....................………..............30
2.4.1、基因選殖、基因分析與共同表現質體構築................…..30
2.4.1.1、二甲苯單加氧酶複合體蛋白(xylene monooxygenase complex)
之基因選殖、基因分析與共同表現質體構築...................30
2.4.1.2、二甲苯單加氧酶(Xylene monooxygenase, XylM)
之基因選殖與共同表現質體構築...............................31
2.4.1.3、甲苯單加氧酶其電子傳遞蛋白(Xylene monooxygenase electron transfer component, XylA)之基因選殖與共同表現質體構築..................................................................................................32
2.4.2、二甲苯單加氧酶其電子傳遞蛋白(Xylene monooxygenase electron transfer component, XylA)質體基因表現..................................................................................................33
2.4.3、二甲苯單加氧酶其電子傳遞蛋白(Xylene monooxygenase electron transfer component, XylA)蛋白質純化............................................33
2.4.3.1、進Nickel-column前處理....................................................33
2.4.3.2、Nickel-column 純化….......................................................33
2.4.4、二甲苯單加氧酶其電子傳遞蛋白(Xylene monooxygenase electron transfer component, XylA) 西方墨點分析 (Western blotting)..........34
2.4.5、二甲苯單加氧酶其電子傳遞蛋白(Xylene monooxygenase electron transfer component, XylA)含鐵離子定量(ICP_O..............................34
2.4.6、二甲苯單加氧酶其電子傳遞蛋白(Xylene monooxygenase electron transfer component, XylA) X光吸收近邊緣結構(XANES)
與延伸X光吸收近邊緣細細微結構(EXAFS)分析法..............................35
2.4.6.1、樣品製備...........................................................................35
2.4.6.2、XANES與EXAFS數據處理.................................................35
2.4.7、二甲苯單加氧酶(Xylene monooxygenase, XylM)
質體基因表現.................................................................................35
2.4.8、二甲苯單加氧酶(Xylene monooxygenase, XylM)
蛋白質純化....................................................................................35
2.4.8.1、進Strep-Tactin sepharose column 前處理........................35
2.4.8.2、Strep-Tactin sepharose column純化...............................36
2.4.9、二甲苯單加氧酶(Xylene monooxygenase, XylM)之西方墨點分析 (Western blotting) .......................................................................36
2.4.10、二甲苯單加氧酶(Xylene monooxygenase, XylM)
含鐵離子定量(ICP_OES) ................................................................36
2.4.11、二甲苯單加氧酶(Xylene monooxygenase, XylM) X光吸收近邊緣結構(XANES)與延伸X光吸收近邊緣細細微結構(EXAFS)分析法.............37
2.4.11.1、樣品製備.......................................................................37
2.4.11.2、XANES與EXAFS數據處理.............................................37
2.4.12、二甲苯單加氧酶複合體蛋白(xylene monooxygenase complex)
質體基因表現與活性分析..............................................................37
2.4.13、二甲苯單加氧酶(Xylene monooxygenase, XylM)
活性分析.....................................................................................38
第三章 結果與討論.......................................................................40
3.1、二甲苯單加氧酶其電子傳遞蛋白(Xylene monooxygenase electron transfer component, XylA) 蛋白質誘導表現分析........................................40
3.2、二甲苯單加氧酶其電子傳遞蛋白(Xylene monooxygenase electron transfer component, XylA)蛋白質純化.........................................40
3.3、二甲苯單加氧酶其電子傳遞蛋白(Xylene monooxygenase electron transfer component, XylA) 胜肽質量指紋鑑定(Peptide mass Fingerprinting)...........................................................................42
3.4、二甲苯單加氧酶其電子傳遞蛋白(Xylene monooxygenase electron transfer component, XylA) 含鐵離子定量(ICP_OES)..................................43
3.5、二甲苯單加氧酶其電子傳遞蛋白(Xylene monooxygenase electron transfer component, XylA) X光吸收近邊緣結(XANES)..................45
3.6、二甲苯單加氧酶(Xylene monooxygenase, XylM)蛋白質誘導表現分析...............................................................................................45
3.7、二甲苯單加氧酶(Xylene monooxygenase, XylM)
蛋白質純化..................................................................................46
3.8、二甲苯單加氧酶(Xylene monooxygenase, XylM)
定量............................................................................................48
3.9、二甲苯單加氧酶(Xylene monooxygenase, XylM)
含鐵離子定量(ICP_OES)...............................................................49
3.10、二甲苯單加氧酶(Xylene monooxygenase, XylM)
X光吸收近邊緣結構(XANES)
與延伸X光吸收近邊緣細細微結構(EXAFS)分析法...............................................................................................51
3.11、二甲苯單甲氧酵素的活性催化..............................................53
3.11.1、二甲苯單加氧酶複合體蛋白(xylene monooxygenase complex)
之二甲苯單加氧酶(Xylene monooxygenase, XylM)定量................53
3.11.2、二甲苯單加氧酶複合體蛋白(xylene monooxygenase complex)
活性分析.....................................................................................55
3.11.3、二甲苯單加氧酶(Xylene monooxygenase, XylM)有無二甲苯單加氧還原酶 (xylene monooxygenase electron transfer component, XylA)
比較...........................................................................................58
3.11.4、二甲苯單加氧酶(Xylene monooxygenase, XylM)
活性分析....................................................................................62
3.11.4.1、純化XylM活性..............................................................62
3.11.4.2、二甲苯單加氧酶(Xylene monooxygenase, XylM)
萃膜活性....................................................................................63
3.11.5二甲苯單加氧酶(Xylene monooxygenase, XylM)
與AlkB比較................................................................................65
3.12、討論.................................................................................66
3.12.1、二甲苯單加氧酶其電子傳遞蛋白(Xylene monooxygenase electron transfer component, XylA) .......................................................66
3.12.2、二甲苯單加氧酶(Xylene monooxygenase, XylM) ............67
3.12.3、二甲苯單加氧酶(Xylene monooxygenase, XylM)
電化學活性催化..........................................................................70
第四章 結論................................................................................71
參考文獻....................................................................................72
附錄一、pACYC-Duet-strep-XylM序列..............................................................................................81
附錄二、pET21b-XylA-His序列.............................................................................................84

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