臺灣博碩士論文加值系統

烷烴氧化酶存在於許多細菌中，利用烷類作為碳源及能量的來源，因此，其於烷類衍生物進行生物降解與能量利用上，扮演非常重要的角色。烷烴氧化酵素系統中烷烴氧化酶 ( alkane hydroxylase, AlkB )含有非紫質二鐵活性中心，是具有中等直鏈烷類(C5~C12)羥基化功能的膜蛋白；此外，此系統中的另外兩個還原酵素rubredoxin-2 (AlkG) 和 NADH-dependent rubredoxin reductase (AlkT)，負責將電子從NADH傳遞至膜上的AlkB，使中等直鏈烷類做羥基化反應。於此，我們使用來自於Pseudomonas putida GPo1的烷烴氧化酵素系統，並將此系統表現於大腸桿菌。我們利用系統的羥基化性質，將純化後的蛋白質與一系列受質包含烷類及含鹵素烷類衍生物進行反應，並藉由氣相層析儀鑑定產物。結果顯示隨著碳鏈增加，AlkB活性也隨之降低；針對含氟受質來看，相等鏈長受質含有氟原子數量多寡也會影響其活性。此研究顯示AlkB 具有獨特的位向選擇性反應，含鹵素受質中，具高選擇性地以遠離鹵素的一端與活性中心進行羥基化，推測主要是受質上的鹵素原子，尤其是氟原子，與疏水口袋間產生特定的凡德瓦親和作用力及(或)立體障礙等兩項可能因素，進而影響受質進入疏水口袋後與活性中心反應的位置。
除此之外，藉由X-ray 吸收光譜分析，純化後的烷烴氧化酶之活性中心含有三價鐵離子，且其配位環境可能為五配位結構。
利用生化合成的技術，由細菌內蛋白質就可以將烷類化合物轉變成醇類化合物，不僅可以簡單合成並有效縮短反應時間，並可以再將醇類化合物進行官能基修飾，應用於藥物化學上。並且細菌反應的合成方式，可以避免使用大量有機溶劑，由此可達到綠色化學的要求。

Alkane hydroxylases play important roles in the conversion of alkanes to alcohols.
Herein, Herein, a recombinant alkane hydroxylase (AlkB) system from Pseudomonas putida Gpo1 expressed in E. coli was established, including a membrane-bound non-heme di-iron monooxygenase, AlkB, and its two other soluble redox cofactors, rubredoxin and rubredoxin reductase. Proteins were purified to allow the hydroxylation of medium chain length alkanes (C6-C12) upon the supplement of NADH. A series of alkanes, including fluorinated alkanes, were prepared or synthesized to test the selectivity of purified AlkB. First, the identification of hydroxylation products was made by GC and compared with authentic standards. The activity is calculated based on the formation of ω-alcohol. Rcaesults show that the AlkB activity for linear alkanes decreases with increasing chain-length. Also, for terminally fluorinated substrates, the number of fluorine atom(s) on a particular alkane may affect the AlkB activity. This study demonstrates the preference of AlkB toward unique regio-selectivity in distal control by its hydrophobic pocket and implies encompassed amino acid residues can be used to tune the orientations of substrates for the controlled oxidation of aliphatics by the transition metal center. The effect caused by the replacement of fluorine atom(s) might be rooted from the unique van der waals interaction in between the substrate and the wall of the hydrophibic pocket. Second, using X-ray absorption spectroscopy, the properties of the iron center can be inferred. The best fit from EXAFS (Extended X-ray Absorption Fine Structure) results suggest that the diiron are penta-coordinated with Fe-Fe distance is 4.0 Å. Results here shed lights on applications of AlkB in future bioremediation and biocatalysis.

目錄
謝誌 I
中文摘要 III
ABSTRACT V
圖目錄 IX
表目錄 XI
第一章 緒論 1
一、假單胞桿菌與辛烷氧化酵素 1
1. 烷烴氧化菌的發現 1
2. Pseudomonas putida GPo1反應機構 1
3. 氫氧化酵素系統中三大不可或缺之酵素 5
4. AlkB, AlkT, AlkG 三個酵素之電子傳遞關係 8
二、單加氧酶的羥基化反應和環氧化反應之反應機構 9
1.羥基化反應之反應機構 9
2. 環氧化反應之反應機構 13
3. Pseudomonas putida GPo1之氫氧化反應形成自由基中間態 14
三、有機氟化學的發展與其重要性 15
四、表現載體 17
五、X光吸收近邊緣結構與延伸X光吸收近邊緣細微結構 18
1. X光之概論 18
2. X 光吸收光譜主要有兩大應用-XANES與EXAFS 18
六、研究目的 20
第二章 實驗 24
一、蛋白質來源 24
二、宿主細胞 24
三、藥品 24
四、儀器 26
五、研究方法 30
1. ω-hydroxylase的轉殖與大量表現 30
2. 重組ω-hydroxylase的純化 32
3. 重組rubredoxin reductase( AlkT蛋白質)的轉殖與表現 34
4. 重組rubredoxin reductase蛋白質的純化 34
5. 細胞膜(membrane portion)活性分析 35
6. 純化後重組 AlkB活性分析 36
7. 細胞膜(membrane portion)與各種受質反應 37
8. 重組AlkB蛋白質與各種受質反應 38
9. 氣相層析儀之條件設定 38
10.含氟化合物之合成 41
11.產物之標準品之合成 47
12. X光吸收光譜實驗( X-ray Absorption Spectroscopy) 57
第三章 結果與討論 58
一、 重組ALKB及ALKT蛋白質純化 58
1.蛋白質純化---AlkB 58
2.蛋白質純化---AlkT 59
二、利用有機合成方法製備受質 60
1.利用Dexo-Fluor reagent合成1,1-difluorooctane,1-fluorododecane及1,1-difluorododecane 之合成 60
2. 1,1,1-Trifluorooctane之合成 61
三、重組ALKB蛋白質與各種受質之活性反應測試 61
1.烷類受質與酵素之反應式 61
2. 一系列烷類受質與酵素反應之活性總表 63
3.烷類受質與AlkB蛋白質作用後之氣相層析圖 64
4. AlkB活性之推算 75
5.受質與酵素間活性探討 80
四、X-RAY吸收光譜分析 87
1. XANES(X-ray absorption near edge structure)光譜 87
2. EXAFS ( extended X-ray absorption fine structure )光譜 88
第四章 結論 91
第五章 參考文獻 93
附錄 96
NMR 圖譜 96

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