硝酸還原酶(narB)來自於海洋藍藻聚球藻(marine cyanobacterium)中，硝酸還原酶包含著 Mo 做為輔助因子和一個鐵硫簇[4Fe4S]，Mo 由鉬蝶呤鳥嘌呤雙核苷酸 (bis-MGD)提供，硝酸還原酶將硝酸還原成亞硝酸，亞硝酸會還原成一氧化氮，是植物和微生物重要的氮來源。
本實驗增加了誘導劑 IPTG(異丙基-β-D-硫代半乳糖苷)的量來提升蛋白質濃度並用 UV-vis 來觀測反應的進行，並且利用 EPR 來觀測硝酸還原酶中的 MO 輔酶訊號。

Nitrate reductase (narB) comes from the marine cyanobacterium Synechococcus
(marine cyanobacterium). Nitrate reductase contains Mo as a cofactor and an ironsulfur cluster [4Fe4S]. -MGD) provides that nitrate reductase will reduce nitrate to
nitrous acid, which will be reduced to nitric oxide, which is an important source of
nitrogen for plants and microorganisms.
In this experiment, the amount of inducer IPTG (isopropyl-β-D-thiogalactoside)
was increased to increase the protein concentration and UV-vis was used to
observe the progress of the reaction, and EPR was used to observe the MO
coenzyme in nitrate reductase signal

謝誌 I
摘要 III
ABSTACT V
目錄 VII
圖目錄 IX
第一章 緒論 1
1.1 研究背景 1
1.2 研究目的 3
第二章 文獻回顧 5
2-1 蛋白質介紹 5
2.1.1 酶介紹 6
2.1.2 輔酶 6
2.2 硝酸還原酶 7
2.2.1 藍藻硝酸還原酶(NarB) 7
2.3 輔酶鉬蝶呤鳥嘌呤雙核苷酸 (Mo-bis-MGD) 8
2.4 硝酸還原酶反應機制 10
第三章 實驗儀器與原理 11
3.1 層析法 11
3.1.1 快速液相層析儀( Fast performance liquid chromatography, FPLC) 11
3.1.2 固定金屬離子親合層析 12
3.1.3 分子大小層析 13
3.2 電泳膠片分析法 13
3.3 紫外光-可見光分光光度法 15
3.4 電子順磁共振儀(EPR) 16
3.4.1 離曼效應 16
3.4.2 精細結構交互作用(Hyperfine Interactions) 18
第四章 材料與方法 21
4.1 養菌 21
4.1.1 小量培養 21
4.1.2 大量培養 21
4.1.3 收菌 & 洗菌 23
4.2 破菌 24
4.3 純化 24
4.4 SDS-PAGE 分析 28
第五章 實驗結果與討論 33
5.1 純化 33
5.1.1 Ni-NTA 組氨酸標籤親和性層析 33
5.1.2 S200 分子顆粒層析 34
5.2 UV-VIS 光吸收 36
5.3 EPR 訊號 38
第六章 結論與總結 41
參考文獻 43

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