臺灣博碩士論文加值系統

硝基還原酶利用NAD(P)H作為輔因子提供電子催化還原硝基芳香化合物，可分為氧不敏感型 (type I) 和氧敏感型 (type II)。從樟芝選殖出硝基還原酶全長cDNA基因序列，大小為1040 bp，而轉譯區為747 bp，可轉譯出248個胺基酸，預測分子量大小為27.8 kDa，命名為樟芝硝基還原酶 (TcNr)，屬於type I。利用EuK-mPLoc軟體分析TcNr，發現其胺基端有將酵素運送至粒線體的訊號序列，長度為47個胺基酸。建構無訊號序列之TcNr，轉形至E. coli C43 (DE3) 進行表現。大量表現帶His6-tag的重組蛋白質，再利用鎳離子親合管住進行純化。利用鐵氰化鉀與重組蛋白質進行酵素動力與特性分析。所得KM為0.86 mM。在45C半衰期為12.3 min，熱失活常數 (kd) 為4.9 × 10-2 min-1。在 pH 6~10時，酵素仍具有活性。目前發現樟芝硝基還原酶對二硝基苯的活性最好，另外還可還原其他硝基芳香化合物，如：氯黴素、鄰硝基苯--D-吡喃葡萄糖苷、磷酸硝基苯及硝基苯甲酸。

The nitroreductase family has tow types: oxygen-insensitive (type I) and oxygen-sensitive (type II). A Nr cDNA (1040 bp) encoding a putative Nr was cloned from Taiwanofungus camphoratus (formerly named Antrodia camphorata). The deduced amino acid sequence is conserved among the reported Nrs. The coding region of Nr cDNA was 747 bp that encodes a protein of 248 amino acid residues with calculated molecular mass of 27.8 kDa. Based on EuK-mPLoc software from ExPASy, the Nr has a signal peptide containing 47 animo acid residues in N-terminus. To characterize the Nr protein, the coding region was subcloned into an expression vector pET-20b(+) and transformed into E. coli C43 (DE3). The recombinant His6-tag Nr was overexpressed and purified by Ni2+-nitrilotriacetic acid agarose. The purified enzyme showed two bands on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited Nr activity via potassium ferricyanide assay. The Michaelis constant (KM) values for ferricyanide was 0.86 mM. The enzyme has a half-life of 12.3 min at 45C with a thermal inactivation rate constant kd of 4.9 × 10-2 min-1. The enzyme was active in a broad pH range from 6 to 10. In this study, the enzyme showed its best activity for nitroaromatic compounds was 1-chloro-2,4-dinitrobenzene and could catalyzed NADH-
dependent reductions of chloramphenicol, o-nitrophenyl--D-
glucopyranoside, p-nitrophenyl phosphate and 2-nitrobenzoic acid.

謝誌 I
摘要 II
Abstract III
縮寫表 V
目 錄 VII
壹、 前言 1
一、 硝基芳香化合物 1
二、 樟芝 2
三、 硝基還原酶 4
四、 研究動機 6
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 9
六、 標準品 9
七、 儀器 9
八、 實驗藥品 11
九、 實驗耗材 13
十、 引子 13
參、 實驗方法 14
一、 樟芝硝基還原酶基因之選殖 14
二、 樟芝硝基還原酶基因之序列分析 17
三、 樟芝硝基還原酶基因之建構 18
四、 勝任細胞的製備 20
五、 樟芝硝基還原酶重組蛋白質表現與純化 20
六、 製備12% 聚丙烯醯胺膠體 23
七、 西方墨點轉漬 24
八、 樟芝硝基還原酶之活性測定 26
九、 樟芝硝基還原酶基因之特性分析 28
十、 mTcNr對不同電子接受者的比活性 29
十一、 mTcNr對硝基芳香化合物的活性 29
肆、 結果 31
一、 樟芝硝基還原酶之基因選殖 31
二、 樟芝硝基還原酶之生物資訊分析與3-D結構模擬 32
三、 TcNr與mTcNr的表現與純化 33
四、 mTcNr之吸光光譜分析 35
五、 mTcNr之活性測定 35
六、 mTcNr對不同基質的活性比較 36
七、 不同緩衝溶液對mTcNr活性的影響 36
八、 mTcNr之酵素動力研究 36
九、 mTcNr之特性分析 38
十、 mTcNr對電子接受者之比活性 39
十一、 mTcNr對硝基芳香化合物之比活性 39
伍、 討論 40
陸、 參考文獻 45
附圖 50
Appendix 68

廖怡珍，2006，樟芝 2-Cys peroxiredoxin 之基因選殖及其特性分析，國立臺灣海洋大學 生物科技研究所 碩士學位論文

楊喬筑，2006，以細胞培養模式評估固態培養牛樟芝菌絲體萃取物之抗肝癌生物活性及其機制，國立臺灣海洋大學 食品科學系碩士學位論文

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