臺灣博碩士論文加值系統

本論文為從牛樟芝(Taiwanofungus camphorata)子實體中選殖出 2,3-丁二醇脫氫酶(2,3-butanediol dehydrogenase ; Bdh, E.C. 1.1.1.4) cDNA，全長 1395 個鹼基對，轉譯區為 1219 個鹼基對，可轉譯出408個胺基酸，其預測分子量大小為 49.3 KDa，命名為牛樟芝 2,3-丁二醇脫氫酶 ( TcBdh ) 。2,3-Bdh利用NAD(P)H作為輔因子提供電子催化將乙醯甲基甲醇 (acetoin)還原成2, 3-丁二醇 (2,3-Bd)，屬於中鏈脫氫酶家族，此胺基酸序列與其他物種的2,3-Bdh之間具有高度保留性，皆具有GXGXXG序列。
以pET-20b(+)做為表現載體，轉型至大腸桿菌 C43 (DE3)宿主與S. cerevisiae中進行表現帶有His6-taq的重組蛋白質，以Ni Sepharose TM 6 Fast Flow 親和性管柱進行純化。再以acetoin測其活性，並分析TcBdh之酵素特性。
最後，從酵素動力學分析得到，TcBdh對acetoin的KM值為8.46 mM，Vmax為0.02 mM/min ，kcat 為55 min-1。觀察2,3-Bdh之基本特性，於45℃之半衰期為5.3 min ；處在不同pH值的環境下發現，2,3-Bdh在pH 6下具有較高的活性。

The purpose of this thesis is to study NAD(P)H-dependent 2,3-butanediol dehydrogenase (2,3-Bdh) from Taiwanofungus camphorata , a medium chain dehydrogenase/reductase (MDR) is the main enzyme catalyzing the reduction of acetoin to 2,3-butanediol. It contains an open reading frame of 1219 bp which encodes a protein of 408 amino acid , molecular mass is 49.3 kDa and has the highly conserved GXGXXG sequence found in the MDR family and residues found in the coenzyme-binding pocket. To characterize the 2,3-Bdh , the coding region of Bdh was introduced into an expression vector pET-20b(+), and transform into Escherichia coli C43 (DE3) and S. cerevisiae.
The purified enzyme showed two bands on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited TcBdh activity via acetoin assay. The Michaelis constant (Km) values for acetoin was 8.46 mM. The enzyme has a half-life of 5.3 min at 45℃ with a thermal inactivation rate constant kd of 3.5 × 10-2 min-1. The enzyme was active in pH 6.

目錄
謝辭 I
摘要 II
Abstract III
縮寫表 IV
目錄 VI
壹、前言 1
一、 丁二醇生合成 (2,3-Bd biosynthesis pathway) 1
二、 牛樟芝丁二醇脫氫酶 (2,3-Butanediol dehydrogenase , 2,3-Bdh) 4
三、 牛樟芝 5
四、 研究動機 7
貳、實驗材料 8
一、材料 8
二、載體 8
三、宿主 8
四、培養基 9
五、套組 (kits) 10
六、標準品 10
七、儀器 11
八、實驗耗材 12
九、實驗藥品 12
十、引子 15
參、實驗方法 16
一、牛樟芝丁二醇脫氫酶基因之選殖 16
二、牛樟芝丁二醇脫氫酶基因之序列分析 20
三、牛樟芝丁二醇脫氫酶基因與表現載體之建構 20
四、牛樟芝丁二醇脫氫酶與表現載體pYEX-S1之建構 23
五、勝任細胞之製備 23
六、牛樟芝丁二醇脫氫酶重組蛋白質表現與純化 24
七、 利用聚丙烯醯胺膠體電泳進行蛋白質分析 27
八、 西方墨點轉漬 30
九、 牛樟芝丁二醇脫氫酶之活性測定 32
十、牛樟芝丁二醇脫氫酶基因之特性分析 33
肆、結果 35
一、牛樟芝丁二醇脫氫酶之基因選殖 35
二、樟芝丁二醇脫氫酶之3-D結構模擬 36
三、TcBdh的表現與純化 37
四、牛樟芝丁二醇脫氫酶之活性測定 39
五、牛樟芝丁二醇脫氫酶之酵素動力研究 40
六、牛樟芝丁二醇脫氫酶之特性分析 41
伍、討論 43
參考文獻 46
圖表 49
Appendix 61

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