生物催化是未來在資源使用及環境保護上的重要課題之一。一般化學反應常需高溫條件，酵素的使用雖然可以改變其反應性及產率，但也常因溫度影響而失去活性。本論文是利用高溫菌的特性，使其產生不同性質的金屬酵素，以改善在高溫中對化學反應的催化活性。在台灣特有種嗜熱菌Meiothermus taiwanensis NTU30的培養基中，改變金屬離子成份，Mn(II)、Zn(II)、B(III)、Cu(II)、Mo(VI)及Co(II)，發現僅一種或缺少單一種金屬離子時，對其生長速率影響不大，但所誘導出之蛋白質，經由SDS-聚丙烯醯胺膠體電泳（SDS-PAGE）的分析結果，卻明顯地具有不同消長的差異性。經破菌、硫酸銨沈澱及透析後所得之整體蛋白質，先行測試磷酸根水解的催化反應（dephosphorylation）。根據紫外光-可見光分子吸收光譜及高效率液相層析資料顯示，M. taiwanensis NTU30整體蛋白質於60℃下仍具有催化磷酸根水解反應的活性。然而在相同條件下，Escherichia coli的整體蛋白質則因高溫變性而無法進行水解作用。更有趣的是，當缺乏Cu(II)、Mo(VI)及Co(II)這些金屬離子時，則此高溫催化水解反應即無法進行。本實驗結果顯示，由嗜熱菌所分離出之酵素在高溫下仍能維持催化活性，而金屬離子則扮演了嗜熱菌蛋白質催化反應成敗的重要角色。本論文的研究結果，不但提供了新的耐高溫金屬酵素的製備觀念，對未來發展耐高溫的生物催化劑也具有相當重要的指標意義。

Biocatalysis is an important topic on resource regeneration and environmental protection. The use of enzyme is capable of improving the reaction rate and product efficiency. However, the enzyme easily losses its activity at high temperature. In our laboratory the thermophilic micro-organism, Meiothermuc taiwanesis NTU30, produces a variety of metalloenzymes to catalyze the chemical reactions at high temperature. The growth rate of M. taiwanesis NTU30 did not alter significantly in the absence or presence of either one of metal ions, Mn(II), Zn(II), B(III), Cu(II), Mo(VI) and Co(II). However, the protein patterns differed obviously from each other revealed by the analysis of SDS-PAGE. After sequential procedures of cell disruption, ammonium sulfate precipitation, and dialysis, the total proteins were obtained and ready to analyze the dephosphorylation activities. Nitrophenyl phosphate was successfully hydrolyzed by the total proteins from M. taiwanesis NTU30 at 60°C based on the UV-visible electron spectrum and HPLC data. In contrast, the total protein Escherichia coli did not show such reactivity in 3 hours. The catalytic activity was suppressed in the absence of Cu(II), Mo(VI) or Co(II). Therefore, thermophilic metalloenzymes have been demonstrated to retain their activities at high temperature. As well, the specific metal ions play an important role in the hydrolysis of phosphate monoester. These results not only provide a novel method in the process of metalloenzymes in thermophilic micro-organisms, but also develop a new vision of
the high temperature-resistant biocatalysts.

目 錄
圖 目 錄 ...............................................................................................II
表 目 錄 ............................................................................................. IV
第一章 緒論 ................................................................................................1
1-1 前言 .......................................................................................................1
1-2 嗜熱菌簡介 ...........................................................................................2
1-3 金屬酵素在生理功能上所扮演的角色 ...............................................5
1-4 磷酸根分子的水解在生理功能上的重要性 ......................................8
1-5 研究動機與目的 .................................................................................13
第二章 實驗材料及方法 ..........................................................................14
2-1 儀器 .....................................................................................................14
2-2 實驗材料 .............................................................................................16
2-3 菌株之整體蛋白質的取得 ................................................................26
2-4 改變培養基中之金屬離子成份 ........................................................37
2-5 磷酸根分子的催化水解作用 ............................................................39
第三章 結果與討論 ....................................................................................42
3-1 嗜熱菌整體蛋白質的取得 ................................................................42
3-2 磷酸根催化水解反應之結果 ............................................................51
3-3 不同的金屬離子對磷酸根水解反應的影響 ....................................67
3-4 結論 .....................................................................................................75
參 考 資 料 ................................................................................................79


圖 目 錄
圖1-1 Meiothermus taiwanensis sp. nov.型態 .............................................4
圖1-2 聚合酶I 的簡單示意圖 ...................................................................6
圖1-3 Mg(II) 金屬離子加速催化RNA 的水解反應 ...............................6
圖1-4 可逆的蛋白質磷酸化-去磷酸化作用示意圖 ................................8
圖1-5 利用Na+K+-ATPase 完成Na+和K+的基本轉換路徑示意圖 .....10
圖1-6 ABC transporter 中兩個高度保守的ATP 結合區 .......................11
圖1-7 維生素B12 利用ABC transporter 進入細胞內部圖 ....................12
圖2-1 形成聚丙烯醯胺凝膠簡單示意圖 ................................................24
圖2-2 正十二烷基硫酸鈉（SDS）之化學結構 ....................................24
圖2-3 溴酚藍（Bromophenol Blue）之化學結構 .................................25
圖2-4 Coomassie® Brilliant blue G-250 及R-250 之結構 .....................25
圖2-5 取得整體蛋白質之實驗流程圖 ....................................................26
圖2-6 分離培養M. taiwanensis NTU30 單一菌落之方法 ....................28
圖2-7 分離培養BL21(DE3)單一菌落之方法 ........................................29
圖2-8 典型之細菌生長曲線 ....................................................................31
圖2-9 改變培養基中金屬離子成份之實驗流程圖 ................................37
圖2-10 磷酸根分子的催化水解作用之實驗流程圖 ..............................39
圖3-1 M. taiwanensis NTU30 之生長曲線圖 .........................................46
圖3-2 M. taiwanensis NTU30 整體蛋白質取得之流程圖 .....................48
圖3-3 NTU30 及BL12(DE3)之SDS-PAGE 比較 ..................................50
圖3-4 4-NPP 去磷酸根反應示意圖 ........................................................52
圖3-5 HEPES 化學結構 ...........................................................................54
圖3-6 室溫時對4-NPP 催化水解反應之影響 .......................................57
圖3-7 50 OC 時對4-NPP 催化水解反應之影響 .....................................58
圖3-8 60 OC 時對4-NPP 催化水解反應之影響 .....................................59
圖3-9 不同溫度下對4-NPP 催化水解之比較 .......................................60
圖3-10 HEPES buffer pH 5.80 時對4-NPP 催化水解反應之影響 .......62
圖3-11 HEPES buffer pH 7.01 時對4-NPP 催化水解反應之影響 .......63
圖3-12 不同緩衝液pH 值對4-NPP 催化水解反應之比較 ..................64
圖3-13 HPLC 鑑定圖 ...............................................................................66
圖3-14 改變培養基所含金屬離子成份對嗜熱菌生長之影響 ..............69
圖3-15 缺少單一金屬離子對4-NPP 催化水解反應之影響 .................71
圖3-16 缺少單一金屬離子對4-NPP 催化水解反應之結果比較 .........72
圖3-17 只含單一金屬離子對4-NPP 催化水解反應之影響 .................73
圖3-18 只含單一金屬離子對4-NPP 催化水解反應之結果比較 .........74


表 目 錄
表1-1 金屬酵素中所含有之金屬離子成份 ................................................7
表2-1 典型之細菌生長曲線的各期別特徵 ..............................................31
表3-1 烏來地熱溫泉基本化學組成分析表 ..............................................44
表3-2 本實驗所使用菌株之基本資料一覽表 ..........................................44
表3-3 各種常用之緩衝溶液及其使用範圍 ..............................................54

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