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論文中文名稱:二胺基庚二酸去羧基酶之基因選殖與特性探討 [以論文名稱查詢館藏系統]
論文英文名稱:Molecular cloning and characterization of coenzyme B6-dependent diaminopimelate decarboxylase from Propionibacterium acnes ATCC 6922 [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生物科技研究所
畢業學年度:97
出版年度:98
中文姓名:黃瑞鑫
英文姓名:Jui-Hsin Huang
研究生學號:96688014
學位類別:碩士
語文別:中文
口試日期:2009-06-15
論文頁數:109
指導教授中文名:黃志宏
口試委員中文名:徐駿森;蔡麗珠
中文關鍵詞:輔酶B6二胺基庚二酸去羧基酶
英文關鍵詞:diaminopimelate decarboxylasePropionibacterium acnes
論文中文摘要:自Propionibacterium acnes ATCC 6922菌中選殖出二胺基庚二酸去羧基酶(diaminopimelate decarboxylase,DAPDC)的基因,並在E. coli中建立蛋白表現系統。DAPDC蛋白的分子量為51,000 Da,屬於輔酶B6必需型酵素,對於反應受質有極高的專一性。本篇論文開發一種全新的DAPDC酵素活性分析方法,利用毛細管電泳分析技術來測量DAPDC之酵素動力學,此分析方法能更方便且快速地測量DAPDC酵素的活性。DAPDC酵素對反應受質及輔酶B6之Km值分別為1.6±0.3 mM與1.3±0.1 μM。另外,在測量DAPDC酵素與輔酶B6結合能力(Kd)上,本篇論文比較螢光光譜及微量透析兩種方法,螢光光譜法測得之Kd值為0.833 μM;微量透析法測得之Kd值為0.61±0.13 μM,兩種方法最後求得之Kd值也相當地接近。本論文的另一個主題,在探討Clostridium sticklandii菌株的離胺酸異構酶與輔酶B12 、B6的交互作用。實驗結果顯示,銨根離子會降低酵素與輔酶B12之Kd値,而輔酶B12結構中的ribonucleotide tail,為穩定結合輔酶B12所必須;另一方面,無論輔酶B6與反應受質是否存在,輔酶B12與其類似物對酵素之結合能力並未有明顯差異。
論文英文摘要:A gene encoding diaminopimelate decarboxylase was cloned from Propionibacterium acnes ATCC 6922 and over-expressed in Escherichia coli. It encodes a protein of 476 amino acid residues with Mr 51,000. Diaminopimelate decarboxylase is a pyridoxal-5'-phosphate-dependent enzyme and is the only amino acid decarboxylase known to act on a carbon atom with D configuration in the substrate. A novel capillary electrophoresis-based enzyme assay method was established in this study. The Km for diaminopimelate and pyridoxal-5'-phosphate is 1.6±0.3 mM and 1.3±0.3 μM, respectively. The binding of pyridoxal-5'-phosphate to diaminopimelate decarboxylase was investigated by equilibrium dialysis and fluorescence spectroscopy. Similar results were obtained by both methods. The interactions between coenzymes and lysine aminomutase from Clostridium sticklandii was also investigated in this study. My results show that ammonia ion can stimulate the binding of coenzyme B12 to lysine aminomutase. The ribonucleotide tail of AdoCbl plays an important role in the binding of the cofactor. No significant difference in the binding of coenzyme B12 was observed, no matter whether coenzyme B6 or substrate analog is present or not.
論文目次:中文摘要 i
英文摘要 ii
致謝 iv
目錄 v
表目錄 viii
圖目錄 ix
簡字表 xii
第一章 緒論 1
1.1 Propionibacterium acnes 1
1.2 二胺基庚二酸去羧基酶Diaminopimelate decarboxylase 1
1.2.1 酵素功能 1
1.2.2 DAPDC之應用潛能 3
1.2.3 DAPDC蛋白之結構 5
1.2.4 DAPDC酵素分析方法 8
1.3 輔酶B6與B12催化角色及特性 9
1.3.1 輔酶B6 9
1.3.2 輔酶B12 12
第二章 Diaminopimelate decarboxylase基因的選殖、表現及純化 17
2.1 前言 17
2.2 實驗藥品與器材 21
2.2.1 實驗藥品 21
2.2.2 實驗器材 26
2.3 實驗步驟 27
2.3.1 基因選殖 27
2.3.2 DAPDC蛋白的小量表現 33
2.3.3 DAPDC蛋白的純化 33
2.3.3.1 菌種培養 33
2.3.3.2 破碎菌體 34
2.3.3.3 陰離子交換管柱 35
2.3.3.4 疏水性交換管柱 36
2.4 結果與討論 38
第三章 DAPDC酵素的活性測試 44
3.1 前言 44
3.2 實驗藥品與器材 45
3.2.1 實驗藥品 45
3.2.2 實驗器材 46
3.3 實驗步驟 47
3.3.1 DAPDC蛋白消光係數的測量 47
3.3.1.1 樣品的製備 47
3.3.1.2 蛋白質水解分析 48
3.3.1.3 吸光系數計算 48
3.3.2 薄層色層分析 49
3.3.3 酵素反應條件測試 49
3.3.4 製作DAPDC酵素純化表 50
3.4 結果與討論 51
第四章 DAPDC對輔酶B6結合能力與催化活性的探討 56
4.1 前言 56
4.1.1 DAPDC與輔酶B6結合能力之測量方法 56
4.1.2 酵素動力學之量測方法 59
4.2 實驗藥品與器材 61
4.2.1 實驗藥品 61
4.2.2 實驗器材 63
4.3 實驗步驟 64
4.3.1 DAPDC對輔酶B6結合能力的探討 64
4.3.1.1 利用螢光光譜方式測量DAPDC對輔酶B6的Kd值 64
4.3.1.2 利用微量透析方式測量DAPDC對輔酶B6的Kd值 65
4.3.2 DAPDC酵素動力學常數(Km)之測量 67
4.3.2.1 酵素反應條件 67
4.3.2.2 胺基酸衍生化反應 68
4.3.2.3 毛細管電泳 68
4.4 結果與討論 71
4.4.1 DAPDC對輔酶B6結合能力之分析 71
4.4.2 DAPDC之酵素動力學分析 72
第五章 KamDE-OraS複合體與輔酶B12類似物結合能力之探討 79
5.1 前言 79
5.2 實驗藥品與器材 83
5.2.1 實驗藥品 83
5.2.2 實驗器材 84
5.3 實驗步驟 84
5.3.1 KamDE及OraS蛋白的製備 84
5.3.2 輔酶B6及反應受質對KamDE與輔酶B12類似物結合能力的影響 84
5.3.3 銨根離子對KamDE-OraS複合體與輔酶B12類似物結合能力的影
響 85
5.4 結果與討論 87
第六章 總結與討論 102
參考文獻 104
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