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研究生:江盛國
研究生(外文):Sheng-kuo Chiang
論文名稱:醯亞胺水解酶的結晶與X光晶體學分析
論文名稱(外文):Crystallization and Preliminary X-ray Crystallographic Analysis of Imidase from Pig Liver and Agrobacterium radiobacter
指導教授:孫玉珠
指導教授(外文):Yuh-ju Sun
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:103
中文關鍵詞:醯亞胺水解酶
外文關鍵詞:imidase 
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醯亞胺水解酶 (imidase, E.C 3.5.2.2),又名為dihydropyrimidinase, hydantoinase, dihydropyrimidine hydrase, 及dihydropyrimidine amidohydrolase,其生理上為代謝uracil和thymine的第二個酵素,即可以催化可逆的水解反應使5,6-dihydrouracil被水解為3-ureidopropionate,和許多種含imide官能基的化合物。 In vitro的資料指出, 醯亞胺水解酶的受質選擇性不高,所催化的大部分受質都是外來物 (xenobiotics)。在細菌的和哺乳動物的醯亞胺水解酶其在受質的選擇性上、金屬的喜好上和胺基酸的序列上均很不相同。 在我們的研究中,自豬肝和Agrobacterium radiobacter中純化出具有嗜熱性的醯亞胺水解酶, 並且可以被結晶。 在豬肝的醯亞胺水解酶 ( pig liver imidase) , 藉著懸滴式擴散法 ( handing-drop vapor diffusion method ), 使用polyethylene glycol MME 5000和 異丙醇 (2-propanol)為沉澱劑, 可得到兩種醯亞胺水解酶的晶體, 一種為triclinic P1 空間群,而其單位晶胞為a = 96.35Å , b = 96.87Å , c = 154.87Å,  = 82.10o,  = 72.54o,  = 77.19o, 另一個為orthorhombic C2221 空間群,其單位晶胞為a = 113.92Å , b =157.22Å , c =156.21Å, 在Agrobacterium radiobacter醯亞胺水解酶中, 藉由懸滴式擴散法,以硫酸氨 (ammonium sulfate)為沉澱劑,亦得到兩種醯亞胺水解酶的晶體,一種為cubic F23 (or rhombohedral R3)空間群,其單位晶胞為a = b = c =179.73Å ( a = b = 127.09Å , c = 311.29Å,  = 120o), 而另外一種為tetragonal I4空間群,其單位晶胞為a = b =129.39Å , c = 173.31Å。

Imidase also known as dihydropyrimidinase (E.C 3.5.2.2), hydantoinase, dihydropyrimidine hydrase or dihydropyrimidine amidohydrolase, that catalyzes the reversible hydrolysis of 5,6-dihydrouracil to 3-ureidopropionate and many other imides. The degradation of pyrimidine (uracil and thymine) is the only known physiological function of imidase. In vitro data indicate that imidase has a broad substrate specificity and prefer xenobiotics over natural substrates. Substrate specificity, metal content and amino-acid sequence all differ significantly between bacterial and mannalian imide-hydrolyzing enzymes. In our study, a thermophilic imidase were purified from pig liver and Agrobacterium radiobacter and crystallized. In pig liver imidase, two kinds of imidase crystals were grown by the hanging-drop vapor-diffusion method using polyethylene glycol MME 5000 and 2-propanol as precipitants. One belongs to the triclinic P1 space group,with unit-cell parameters a = 96.35Å , b = 96.87Å , c = 154.87Å,  = 82.10o,  = 72.54o,  = 77.19o, and the other belongs to the orthorhombic C2221 space group, with unit-cell parameters a = 113.92Å , b =157.22Å , c =156.21Å. In the Agrobacterium radiobacter imidase (A. radio.- D-hydantoinase), two kinds of imidase crystals were grown by the hanging-drop vapor-diffusion method using ammonium sulfate as precipitant. One belongs to cubic F23 (or rhombohedral R3) space group, with unit-cell parameters a = b = c =179.73Å (a = b = 127.09Å , c = 311.29Å ,  = 120o), and the other belongs to tetragonal I4 space group, with unit-cell parameters a = b =129.39Å , c = 173.31Å.

第一章 緒論
第一節: 醯亞胺水解酶 (imidase)………………………….1
第二節: 豬肝的醯亞胺水解酶
(一) 生理意義 - 代謝嘧啶 (pyrimidine)………………………. 2
(二) 哺乳動物醯亞胺水解酶的重要性……………………………3
(三) 對受質的選擇性 ( substrate specificity )…………………...4
(四) 嗜熱性和熱穩定性……………………………………………5
第三節 : 細菌的醯亞胺水解酶 (bacterial imidase)
(一) D型胺基酸與其在抗生素領域上的應用……………………..6
(二) 受質的選擇性……………………………………………………8
(三) 晶體結構…………………………………………………………9
第二章 材料、原理及方法
第一節 材料
(一) 蛋白質的來源……………………………………………………11
(二) 試劑藥品及工具…………………………………………………11
(三) 儀器設備………………………………………………………...13
第二節 X光結晶學決定大分子結構的原理
(一) 蛋白質的結晶…………………………………………………...14
(二) 繞射圖資料的收集……………………………………………...16
(三) 相位 (phase) 的決定…………………………………………...16
(四) 分子模型的建立與修正 (Model building & Refinement)…...17
第三節 實驗方法
(一) 樣品的取得……………………………………………………..18
(二) 樣品的處理……………………………………………………..24
(三) 二級結構的鑑定………………………………………………..25
(四) 醯亞胺水解酶的晶體培養方法................................................26
(五) 繞射數據的收集及處理………………………………………27
(六) Matthews coefficient (Vm) 的決定…………………………...29
(七) 溶劑比例 (solvent content) 的決定………………………….29
(八) 晶體相位之決定........................................................................29
第三章 結果
第一節 二級結構鑑定之結果.......................................................31
第二節 晶體培養
(一) 豬肝的醯亞胺水解酶 (pig liver imidase)…………………….31
(二) Agrobacterium radiobacter醯亞胺水解酶……………………..33
(三) Selenomethionyl-A. radio.-醯亞胺水解酶……………………...34
第三節 X光繞射分析
(一) 抗凍劑 (cryoprotectant) 的尋找 …………………………… 36
(二) X光繞射數據的分析及晶胞單位的判定……………………... 38
第四節 相位之決定
(一) 分子置換法……………………………………………………...41
(二) MAD (multiwavelength anomalous dispersion)………………41
第四章 討論
第一節 豬肝的醯亞胺水解酶……………………………………….43
第二節 Agrobacterium radiobacter醯亞胺水解酶………………...44
第五章 參考資料………………………...............................46
附錄 (一)…………………………………………….50
附錄 (二)…………………………………………….100

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