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

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