博碩士論文 973204015 詳細資訊




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姓名 陳光澤(Guang-ze Chen)  查詢紙本館藏   畢業系所 化學工程與材料工程學系
論文名稱 位向性固定化葡萄糖氧化酶之新方法
(A new strategy for oriented immobilization of glucose oxidase)
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摘要(中) 近年來,蛋白質晶片儼然已成為生物檢測以及疾病診斷的重要工具,但常因
為蛋白質不適當的接合位向而使蛋白質失去功能,進而影響檢測的精確性。在本
研究中,我們提出一種將蛋白位向固定化的新方法,其中包含了兩個步驟,第一,
在遠離蛋白活性中心的地方尋找一個對接(docking)位置;第二,試著找出一個能
與此位置有很強的親和力的配體(ligand),為了避免ligand 會與蛋白的活性中心
結合,因此希望所設定的docking 位置與活性中心附近帶有相反的電荷,又為了
增強ligand 與蛋白間的結合,於是設計的ligand 同時結合了疏水與靜電的作用力
與蛋白結合,由此可知,擬定的docking 位置附近需有疏水性的胺基酸以提供疏
水作用力,而ligand 則是透過molecular docking 的模擬所得到。
在實驗中,目標蛋白為來自於黑曲黴菌(Aspergillus niger)的葡萄糖氧化酶
(glucose oxidase,GOX),此蛋白的活性中心以親水性胺基酸居多,因此我們以
一疏水性分子萘對GOX 作docking,藉此找出所有疏水性的區塊,再針對一結
合自由能較低的位置作性質上的分析,並發現周圍以帶正電荷的胺基酸居多,最
後根據此位置設計出有較好結合能的胺基酸LLGEG。隨後將此胜肽接枝在矽膠
體(Silica Gel,SiGel)上,並利用蛋白質的等溫吸附實驗來得到親和力的大小及活
性測試來驗證蛋白質的吸附位向。由GOX 在LLGEG-SiGel 上的等溫吸附曲線得
知GOX 與LLGEG-SiGel 的解離常數只有1.69×10-6 M。當外在離子強度下降時,
即會造成些許的GOX 因而脫附,由此可知,蛋白與ligand 間的作用,確實包含
了靜電及疏水的作用力。此外,由比活性的比較看來,位向固定化的GOX 相較
於隨機吸附於一級胺表面的GOX,比活性高了將近5 倍,因此推論GOX 可能以
活性位置曝露於外的位向吸附於LLGEG-SiGel。因此證實本研究所設計的ligand
與GOX 間具有高親和力,並且可使GOX 以合適的位向吸附於材料表面展現蛋
白質的活性,所以藉由這種研究策略可以提供一個良好的方式加強蛋白質晶片檢
測的精確性。
摘要(英) In the past decades, protein chips are an important tool for applying in the
examinations of bioassay and disease, but the examination efficiency of protein chips
usually decrease as immobilized protein adopting random orientation. In this study, a
new strategy for oriented immobilization of proteins was proposed. The strategy
contains two steps. The first step is to search for a docking site away from the active
site on the protein surface. The second step is trying to find a ligand that is able to
grasp the targeted site of the protein. To avoid ligand binding to the active site of
protein, the targeted docking site is selected to own opposite charges to those near the
active site. To enhance the ligand-protein binding, both hydrophobic and electrostatic
interactions need to be included. The targeted docking site should therefore contain
hydrophobic amino acids. The ligand is then selected through the help of molecular
docking simulations.
The enzyme glucose oxidase (GOX) derived from Aspergillus niger was taken
as an example for oriented immobilization. The active site of GOX is surrounded by
hydrophilic amino acids. All the possible hydrophobic sites on the surface of GOX
were evaluated by the free energy estimation through naphthalene docking. A
hydrophobic site on the opposite side of GOX’s active site was found to be positive in
net charges. A possible peptide ligand, LLGEG, was found to catch GOX by the
designated docking site. Then, the LLGEG molecules were grafted onto silica gels
and measured the affinity of GOX adsorption and the specific activity of thereby
immobilized enzymes. It was found that GOX had a dissociation constant as low as
1.69×10-6 M toward the ligand LLGEG on silica gel. The decrease in ionic strength
has little effect on desorption of GOX, which indicated the existence of hydrophobic
and electrostatic interactions between ligands and proteins. The specific activity of the
III
immobilized GOX was compared with the randomly adsorbed GOX on primary
amine containing silica gel. It was found that the orderly immobilized GOX owns a
specific activity with about five-folds as high as the one randomly adsorbed by ionic
interaction. Consequently, this new strategy for protein oriented immobilization by
designing the proper peptide ligand through the help of molecular docking is
executable.
關鍵字(中) ★ 分子對接
★ 葡萄糖氧化酶
★ 位向性固定化
關鍵字(英) ★ oriented immobilization
★ glucose oxidase
★ molecular docking
論文目次 摘要............................................................................................................................... I
Abstract......…………………………………………………………………………………………………...…II
誌謝......……………………………………………………………………..………………..………………......IV
目錄............................................................................................................................. ..V
圖目錄 ....................................................................................................................... VII
表目錄 ...................................................................................................................... VIII
第一章 緒論 ........................................................................................................... 1
1.1 研究動機 .................................................................................................................... 1
1.2 研究目的 .................................................................................................................... 3
第二章 文獻回顧 ................................................................................................... 4
2.1 固定化技術簡介 ....................................................................................................... 4
2.1.1 固定化技術之定義 ..................................................................................... 4
2.1.2 固定化方法 ................................................................................................ 6
2.1.2.1 共價鍵結(covalent binding) ........................................................ 6
2.1.2.2 物理吸附(physical adsorption) .................................................... 8
2.1.2.3 物理包埋(physical entrapping) …...……………………………………….9
2.1.2.3.1 格子型(lattice type) ……………………………………………….9
2.1.2.3.2 微膠囊包埋法(microencapsulation) .………………………..9
2.1.2.3.2.1 界面聚合法 .……………………..…………………..9
2.1.2.3.2.2 液體乾燥法.……………………..…………………..9
2.1.3 蛋白質的位向 ..................................................................................... 10
2.1.3.1 隨機固定化(random immobilization) ...................................... 10
2.1.3.2 位向固定化(oriented immobilization) ................................... 10
2.1.3.2.1 生物親和性(bioaffinity) ………………………………………10
2.1.3.2.1.1 親和素-生物素系統(avidin-biotin system).11
2.1.3.2.1.2 組氨酸-標籤系統(His-tagged system) …….11
2.1.3.2.1.3 醣鏈分子辨識 …………………………………..…13
2.1.3.2.1.4 proteinA or protein G ……………………………. 14
2.1.3.2.2 捕捉配體(capture ligand) ……………………………………. 15
2.1.3.2.2.1 捕捉胜肽(capture peptides) …………………...15
2.2 葡萄糖氧化酶介紹……………………………………. ……………………………………….17
2.2.1 葡萄糖氧化酶的性質…………………………………………………………… 17
2.2.2 葡萄糖氧化酶的反應機制 …..………………………………………………. 18
VI
2.2.3 葡萄糖氧化酶的應用…………………………………………………………… 19
2.2.3.1 葡萄糖氧化酶在醫藥上的應用 …………………………..…19
2.2.3.2 葡萄糖氧化酶在食品加工上的應用 …………………….…19
2.3 分子對接…………………..………………………………. ……………………………………….20
2.3.1 分子對接(molecular docking)介紹……………..………………………...…20
2.3.2 Autodock…..………………………………. …………………………………….….23
第三章 實驗設備、藥品、策略、方法 ............................................................. 25
3.1 實驗藥品 .................................................................................................................. 25
3.2 實驗設備 .................................................................................................................. 26
3.3 實驗策略 .................................................................................................................. 27
3.3.1 Peptide ligand 的設計………………………………………………………….….27
3.3.1.1 葡萄糖氧化酶表面性質分析……………………………...….27
3.3.1.2 萘分子對葡萄糖氧化酶的分子對接分析…................….27
3.3.1.3 針對配體可能結合位置的分析…......................................….27
3.3.1.4 胜肽配體的設計……………………………………………….…..28
3.4 實驗方法 .................................................................................................................. 29
3.4.1 基材表面的改質……………………………………………………………….…..29
3.4.1.1 表面胺基改質 ............................................................... 30
3.4.1.2 乙醇胺(ethanol amine)阻隔.......................................... 30
3.4.1.3 Peptide ligand 接枝 ..................................................... 31
3.4.2 蛋白質吸附曲線……………………………………………………………….…..31
3.4.3 蛋白質脫附……………………………………………………………….……….....32
3.4.4 蛋白質活性測 ..……………………………………….……………….……….....32
3.4.4.1 測量原理…….…………………………….……………….……….....32
3.4.4.2 操作方法…….…………………………….……………….……….....33
3.4.5 蛋白質濃度測試-Bradford method………….….……………….……….....33
3.4.5.1 Bradford 方法………………...……….….……………….……….....33
3.4.5.2 測量方法………..……………...……….….……………….……….....34
第四章 結果與討論 ............................................................................................. 35
4.1 Peptide ligand 的設計 ............................................................................................ 35
4.1.1 葡萄糖氧化酶表面性質分析……………………...…….............................….36
4.1.2 萘對葡萄糖氧化酶的分子對接分析…………………............................….37
4.1.3 針對配體可能結合位置的分析………………………..............................….39
4.1.4 配體胜肽的設計……………………………………………..............................….40
4.2 胜肽配體共價接枝於矽膠體表面 ...................................................................... 44
4.3 葡萄糖氧化酶於不同基材表面的等溫吸附 ..................................................... 48
VII
4.4 葡萄糖氧化酶於低濃度下的穩定性 .................................................................. 51
4.5 蛋白質吸附在基材上的活性測試 ...................................................................... 54
第五章 結論 ......................................................................................................... 55
參考文獻……………………………………………………………………………………………………...56
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指導教授 阮若屈(Ruoh-chyu Ruaan) 審核日期 2010-7-29
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