臺灣博碩士論文加值系統

NAD+依賴型酒精脫氫酶( NAD+- dependent alcohol dehydrogenase)可以分三大家族，分別是短鏈酒精脫氫酶、長鏈酒精脫氫酶、鐵依賴型酒精脫氫酶，而NAD+依賴型甲醇脫氫酶( NAD+- dependent methanol dehydrogenase；MDH)(EC 1.1.1.244)屬於鐵依賴型酒精脫氫酶的一員，這個家族成員具有一段高度保留性胺基酸序列GGGSX2DX2K，為NAD(H)結合的區域。其MDH的作用是利用NAD(H)做為輔因子催化甲醇與甲醛之間互相轉換。
本研究是利用PCR技術從牛樟芝( Taiwanofungus camphorata )中選殖出MDH之cDNA基因序列，全長1640 bp，轉譯區1517 bp，可以轉譯出496個胺基酸，分子量約為53 kDa。以pYEX-S1作為表現載體，將牛樟芝MDH基因去除掉含Eco RⅠ序列的胺基酸( tTcMDH )，接上6個 His後於酵母菌中表現，並純化測定酵素活性。因酵素會快速的失去氧化甲醇的能力，改以乙醇做為受質，測定酵素動力與特性分析。結果顯示tTcMDH 的KM 為 8.49 mM，在75C半衰期為0.43 min，熱失活常數 ( kd) 為1.44×10-1 min-1。在 pH 6~8時，酵素仍具活性，但於pH 9~10有最佳活性 。

摘要 I
Abstract II
縮寫表 III
目錄 IV
壹、前言 1
一、牛樟芝 1
二、NAD+依賴型甲醇脫氫酶酵素 2
三、甲醇 4
四、乙醇 4
五、酒精代謝 5
貳、實驗材料 8
一、物種 8
二、載體 8
三、宿主 8
四、藥品試劑 8
五、套組 12
六、儀器設備 12
七、實驗耗材 13
八、引子 14
參、實驗方法 15
一、TcMDH之基因選殖 15
二、表現載體之建構 19
三、重組蛋白質表現與純化 22
四、西方墨點轉漬 25
五、蛋白質透析與濃度測定 27
六、酵素活性測定 28
七、酵素之特性分析 30
肆、結果 31
一、TcMDH 之 cDNA 選殖 31
二、tTcMDH重組蛋白質之表現與純化 33
三、tTcMDH重組蛋白質之酵素動力學研究 36
四、tTcMDH重組蛋白質之特性分析 37
伍、討論 39
參考文獻 43
圖 50
Appendix 63

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