麩胺酸轉化酶(Glutamate mutase, GM)，為輔酵酵素B12的相依酵素之一，關於受質的催化反應變化分類，歸類於第一類酵素碳-碳骨架重新排列，其催化反應為L-麩胺酸又異構化成3-甲基天冬胺酸的可逆反應，此催化作用在近年來已經被確認。
改良三點純化方式使純化後純度上升，以及使用線上程式Gel analyzer 對其電泳來測定其純度，並使用UV-Vis進行活性測定及進行EPR電子自旋共振進行測量。
為了探討與其具有相同受質但其輔酵素不同的麩胺酸 2,3 胺基異位酶(Glutamate 2,3-Aminomutase,G2,3-AM)其反應機制，以已經被確定的麩胺酸轉化酶來去比較兩者的差異性，本實驗室將分別植有GlmE和MutS兩個次單元基因的大腸桿菌，利用層析、濃縮等方法分別GlmE和MutS純化完成，測量其酵素動力學活性參數， 並且改良過去的純化方法使酵素的活性足以測量到良好的電子順磁共振光譜(EPR)，並對其譜線進行模擬。

Glutamate mutase is one of the coenzyme B 12 -dependent enzymes, that can catalyze the reversible carbon skeleton rearrangement from L-glutamate to (2S,3S)-3-methylaspartate. The catalysis mechanism has been confirmed in recent years.
In order to explore the reaction mechanism of glutamate 2,3-Aminomutase, the following experiments were done: First, the genes of the two subunits GlmE and MutS were cloned and over-expressed in E. coli individually. Second, the components, GlmE and MutS, have been purified by column chromatography, dialysis, and concentrate. Thirdly, the purification method makes the enzyme active enough to measure a good electron paramagnetic resonance (EPR) spectrum and simulate its spectral lines.
In the future, we will add unnatural substrates to the reaction and use electron paramagnetic resonance spectroscopy to Glutamate 2,3-Aminomutase. And compare the differences between Glutamate 2,3-Aminomutase and Glutamate mutase to understand that the two substrates are the same, but the coenzymes are different, Glutamate 2,3-Aminomutase’s coenzyme is SAM. In recent years, SAM has found that many enzymes require its to react.

謝誌 I
摘要 II
ABSTRACT III
目錄 IV
圖目錄 VI
表目錄 VIII
第一章緒論 1
1.1研究背景 1
1.2研究動機 1
1.3研究目的與方法 2
第二章 文獻回顧 3
2.1 蛋白質簡介 3
2.1.1 酶簡介 6
2.1.2 輔酶 6
2.2輔酶B12簡介 7
2.2.1腺苷鈷胺(Adenosylcobalamin)簡介 9
2.2.2 Adenosylcobalamin的相依酵素 9
2.3麩胺酸轉換酶(GLUTAMATE MUTASE)簡介 10
2.3.2 麩胺酸轉換酶之結構 11
2.3.1麩胺酸轉換酶反應機制 12
第三章 材料與製備 15
3.1養菌 15
3.2破菌 16
3.2.1破菌方法Α 16
3.2.2破菌方法Β 17
3.3 MUTS純化 18
3.3.1 MUTS純化方法Α 18
3.4 GLME純化 23
3.4.1 GLME純化方式Α 23
3.4.2 GLME純化方法Β 26
3.5 SDS-PAGE分析 29
3.6麩胺酸轉換酶活性測定 32
3.7 Β-METHYLASPARTASE培養與純化 32
第四章 結果與討論 35
4.2.2改良後的純化 38
4.2.3 MutS純化方式α、β差異比較 40
4.3.2 GlmE純化方法β 44
4.3.3 GlmE純化方式α、β差異比較 47
4.4酵素動力學參數分析 48
4.5 EPR光譜分析 50
第五章 結論與總結 55
參考文獻 57

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