硝酸還原酶(Nitrate Reductates,NRs)屬於二甲基亞碸還原酶家族並含有雙鉬蝶呤鳥嘌呤二核苷酸(Mo-bisMGD)輔酵素，此輔酵素在催化反應中可藉由外部提供電子經過一連串的反應使硝酸鹽還原為亞硝酸鹽以解決環境中過多的硝酸鹽所產生的危害。
為了純化出硝酸還原酶未來能夠有更完善的反應機制，本論文利用將含有此蛋白質基因轉殖至BL21，經過培養、破菌並著重在利用快速液相層析儀中更改不同純化洗脫梯度進行層析使蛋白質能被分離出來，最後濃縮出硝酸還原酶。
由實驗結果顯示，在利用純化結果(三)的兩個洗脫梯度依序經過Ni-NTA組氨酸標籤親合性層析和S200分子顆粒層析能成功地純化出硝酸還原酶。

Nitrate reductates (NRs) belong to the dimethyl sulfoxide reductase family and contain the coenzyme Mo-bisMGD, which can be supplied by external sources in the catalytic reaction. The electrons go through a series of reactions to reduce nitrate to nitrite to solve the harm caused by too much nitrate in the environment.
In order to purify nitrate reductase and have a more complete reaction mechanism in the future, this paper uses the gene containing this protein to be transfected into BL21, cultured, sterilized, and focused on changing different purification and elution gradients in the use of fast liquid chromatography. Chromatography is performed so that the protein can be separated, and finally the nitrate reductase is concentrated.
The experimental results show that the nitrate reductase can be successfully purified by Ni-NTA histidine tag affinity chromatography and S200 molecular particle chromatography using the two elution gradients of the purification result (3).

謝誌 I
摘要 II
ABSTACT III
目錄 IV
圖目錄 VI
第一章 緒論 1
1-1 研究背景 1
1-2 研究目的 1
第二章 文獻回顧 3
2-1 蛋白質介紹 3
2-1.1 酶介紹 3
2-1.2 輔酶 4
2-2 硝酸還原酶 5
2-2.1 藍藻硝酸還原酶(NarB) 5
2-2.2 硝酸還原酶之輔酵素 6
第三章 實驗儀器與原理 9
3-1層析法 9
3-1.1快速液相層析儀( Fast performance liquid chromatography, FPLC) 9
3-2 電泳膠片分析法 12
3-3 紫外光-可見光分光光度法 14
第四章 材料與方法 17
4-1 養菌 17
4-1.1 小量培養 17
4-1.2 大量培養 19
4-1.3 收菌 & 洗菌 20
4-2 破菌 21
4-3 純化 23
4-4 SDS-PAGE分析 27
第五章 實驗結果與討論 31
5-1 純化結果(一) 31
5-1.1 Ni-NTA 組氨酸標籤親和性層析 31
5-1.2 S200分子顆粒層析 32
5-2 純化結果(二) 34
5-2.1 Ni-NTA 組氨酸標籤親和性層析 34
5-2.2 S200分子顆粒層析 36
5-3 純化結果(三) 37
5-3.1 Ni-NTA 組氨酸標籤親和性層析 37
5-3.2 S200分子顆粒層析 39
第六章 結論與總結 43
參考文獻 45

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