本研究從嗜熱菌Thermus thermophilus中分離萃取熱穩定性高的葡萄糖去氫化酶，作為生物燃料電池的陽極觸媒，配合鐵氰化鉀(potassium ferrocyanide)為攜電分子，製作生物燃料電池的陽極系統，並測試操作條件中葡萄糖濃度、鹽濃度與溫度對所產生電流密度與功率的影響。利用鹽析法與phenyl-sepharose 液相層析，自Thermus thermophilus部分純化了葡萄糖去氫化酶，活性則是利用dye-reduction方法進行分析。在生物燃料電池之陽極系統測試中，使用自行分離萃取之葡萄糖去氫化酶，於相對Ag/AgCl參考電極之操作電壓為0.35 V 時與37 ℃的狀況下，電流密度達52 μA cm-2；當操作溫度升至70 ℃時，電流密度更提昇至127 μA cm-2。可知由嗜熱菌Thermus thermophilus所得之葡萄糖去氫化酶具廣泛的活性反應溫度，其活性會隨著溫度昇高而提昇。因此，以此一酵素所製作的生物燃料電池，在高溫的操作環境中，會有更好的表現。

Biofuel cells are miniature power-generating systems that convert chemical energy to electrical energy by using biocatalysts to catalyze the oxidation of organic substances. Because of its insensitivity to oxygen and high catalytic efficiency, glucose dehydrogenase is an ideal anodic catalyst for biofuel cells. However, the heat instability of this enzyme inherently limits its application in the bioanode. In this research, a thermostable glucose dehydrogenase was isolated from Thermus thermophilus. Utilizing the thermostable glucose dehydrogenase as the catalyst and potassium ferrocyanide as the mediator, we successfully produced a thermostable anodic system. The effects of glucose concentration, salt concentration and temperature on current density were evaluated.
The glucose dehydrogenase in Thermus thermophilus was partially purified by the ammonium sulfate fractionation and phenyl-sepharose chromatography. The activity of the glucose dehydrogenase at every step was determined by dye-reduction method.
In the study for anode of biofuel cell, the glucose dehydrogenase gave a current density of 52 μA cm-2 at 0.35 V with Ag/AgCl as a reference at 37oC. When the operating temperature was raised to 70oC , an anodic current density of 127 μA cm-2 at 0.35 V was obtained. The results show that the glucose dehydrogenase from Thermus thermophilus is a thermostable enzyme and has a wide reaction temperature. Its acitivity increased as the temperature was increased. The biofuel cell would get better efficiency by using the enzyme at higher operating temperatures.

中文摘要 Ⅰ
英文摘要 Ⅱ
目錄 Ⅲ
表目錄 Ⅶ
圖目錄 Ⅷ

第一章 緒論 1
1.1生物燃料電池 1
1.2嗜熱性微生物 3
1.3研究目的與範疇 3
第二章 文獻回顧 6
2.1生物燃料電池之類型 6
2.1.1微生物燃料電池(Microbial fuel cells) 7
2.1.2酵素燃料電池(Enzyme fuel cells) 8
2.2嗜熱菌(Thermophiles) 10
2.3電化學測定方法 10
2.3.1循環伏安法(Cyclic voltammetry) 10
2.3.2時間－安培法(Chronoaperommetry) 12
2.4燃料電池的過電壓(Overpotential) 13
2.4.1活性過電壓之活性極化 (Activation polarization) 14
2.4.2歐姆過電壓之歐姆極化 (Ohmic polarization) 14
2.4.3質傳過電壓之濃度極化(Concentration polarization) 15
第三章 材料與實驗方法 25
3.1實驗藥品 25
3.2實驗設備 27
3.3試藥配製 28
3.3.1培養液(Medium) 28
3.3.2磷酸緩衝液(Phosphate buffer solution, PBS) 28
3.4電極的準備 28
3.4.1玻璃化碳電極 28
3.4.2鉑電極 29
3.4.3銀/氯化銀參考電極 30
3.5粗萃取(Crude extract) 30
3.6製備嗜熱菌的葡萄糖去氫化酶萃取液(Purified GDH) 31
3.7蛋白質濃度的測定 31
3.8葡萄糖去氫化酶(GDH)活性測定 32
3.9電化學測定 33
3.9.1循環伏安量測 33
3.9.1.1簡介 33
3.9.2.2實驗步驟 33
3.9.2定電位量測 33
3.9.2.1簡介 33
3.9.2.2實驗步驟 34
3.9.3定電流量測 35
3.9.3.1簡介 35
3.9.3.2實驗步驟 35
3.10熱穩定性的測定 36
第四章 結果與討論 37
4.1葡萄糖去氫化酶的活性測試 37
4.2葡萄糖去氫化酶之純化 38
4.3循環伏安量測 38
4.4操作環境之影響 39
4.4.1葡萄糖濃度 39
4.4.2 NaCl濃度 40
4.4.3溫度 40
4.5陽極之電流-電壓曲線 41
4.6熱穩定性 42
第五章 結論 55
5.1 酵素最佳反應條件 55
5.2最佳純化步驟 55
5.3生物燃料電池之陽極效能 55
第六章 參考文獻 56

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