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研究生： 吳哲緯
Wu, Jhe-Wei
論文名稱： 希瓦氏菌鐵還原酶動力學和生物製造奈米金
Biofabrication of gold nanoparticles and the ferric reductase kinetic study in Shewanella
指導教授： 吳意珣
Ng, I-Son
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程學系
Department of Chemical Engineering
論文出版年： 2017
畢業學年度： 105
語文別： 中文
論文頁數： 73
中文關鍵詞： 希瓦氏菌 、鐵還原酶 、奈米金 、休眠細胞
外文關鍵詞： Shewanella, Ferric reductase, gold nanoparticles, resting cell
相關次數： 點閱：129  下載：6 
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    • 希瓦氏菌是一種桿狀的兼性厭氧菌，對於鐵和錳具有極強的還原能力，廣泛應用於生物復育、微生物燃料電池及金屬污水整治中。本研究利用奧奈達希瓦氏菌 (MR-1) 與廈門希瓦氏菌 (SXM)，藉由 Ferrozine 法來測試細胞濃度、培養時間和轉速對鐵還原酶活性的影響。結果顯示，鐵還原酶活性隨著細胞濃度上升而升高，但細胞活性在 24小時後或靜置培養條件下均大幅下降，故選擇培養條件在轉速 150 rpm、30oC 培養 12 h 下濃縮至細胞量 3.6 g/L，奧奈達希瓦氏菌 (MR-1) 與廈門希瓦氏菌 (SXM) 的最佳鐵還原酶分別為 79 U/g-DCW 和 110 U/g-DCW。採用 Michaelis–Menten 動力學模型， 計算得出 SXM 之 Vmax 和 km 分別是 114.94 U/g-DCW 與 2.2 mM ，MR-1 則是 86.21 U/g-DCW與 6.33 mM，顯示 SXM 有較大之反應速率和親和力。經由基因工程導入 Mtr 通道蛋白後發現 MtrC 顯著提昇了活性 (141.6 U/g-DCW)，為原菌 MR-1 的 1.5 倍 (92.6 U/g-DCW)。
    希瓦氏菌已被報導具有生成奈米金的能力，但最佳化與機制的探討仍不清楚。本研究探討 pH、細胞濃度、氯金酸濃度與光照效應等因素來對生成之奈米金的影響。找出最適化條件為 pH 5、2.4 g/L 細胞、300 ppm 氯金酸下光照 24 h，SXM 與 MR-1 所生成奈米金量分別為 108 ppm和 62 ppm， 更首次發現光照效應對奈米金生成的影響。由 SEM 與 Zeta potential的電位分析，我們假設希瓦氏菌奈米化金的反應機制為金離子先吸附在細胞表面上，藉由乳酸鈉提供電子經由希瓦氏菌的電子通道把電子傳遞到細胞表面，並將金酸還原成奈米金。文中最後完成了希瓦氏菌的休眠細胞應用測試，結果顯示放置 25 天之休眠細胞依然具備奈米金生成的能力。

    In this study, we used S. oneidensis MR-1 (MR-1) and S. xiamenensis BC01 (SXM), to analyze ferric (Fe) reductase by using the Ferrozine assay to detect the Fe(II) concentration. Finally the optimal Fe reductase were determined with 3.6 g/L of biomass which cultured at 150 rpm, 30oC for 24 h. The best ferric reductase was 110 U/g-DCW and 79 U/g-DCW for SXM and MR-1, respectively. Under estimation of the kinetic parameters of Fe reductase were 114.9 U/g-DCWand 2.2 mM for SXM while 86.2 U/g-DCW and 6.33 mM for MR-1. It means that SXM has higher maximum velocity and affinity than MR-1. The genetic strains harboring of genes of mtrA, mtrC, and mtrCAB showed the highest enzymatic activity of 141.6 U/g-DCW obtained by the mtrC strain, which is 1.53 times of wild strain.
    To discover the optimal condition for Au@NPs production from SXM and MR-1 was our purpose. Herein, the pH, biomass, concentration of gold ion, and photo effect are evaluated to optimize Au@NPs production by Shewanella. As a result, the highest biofabrication of Au@NPs was at pH 5 immersed in 300 ppm Au3+ and 50 mM sodium lactate at 2.4 g/L biomass after 24 h under light intensity of 100 µmole photons/m2s, which the yield of 108 ppm for SXM and 59 ppm for MR-1.The supposed mechanism of Au@NPs formation was Shewanella used sodium lactate as electron donor and followed by nucleation on cell membrane. Finally, the resting cells remained their ability for production of Au@NPs after 25 days.

    摘要 i Extended Abstract ii 致謝 vi 總目錄 vii 表目錄 x 圖目錄 xi 第一章 緒論 1 1.1 前言 1 1.2 研究目的與架構 1 第二章 文獻回顧 3 2.1 希瓦氏菌簡介 3 2.1.1 希瓦氏菌簡史 3 2.1.2 奧奈達希瓦氏菌 Shewanella oneidensis MR-1 4 2.1.3 廈門希瓦氏菌 Shewanella xiamenensis 6 2.2 希瓦氏菌特徵行為研究 7 2.2.1 電子傳遞方式 7 2.2.2 希瓦氏菌 Mtr 通道 8 2.2.3 希瓦氏菌鐵還原能力研究 10 2.2.4 希瓦氏菌還原奈米金屬顆粒 11 2.2.5 希瓦氏菌的其他應用 12 第三章 材料與方法 14 3.1 實驗藥品 14 3.2 實驗儀器 15 3.3 實驗材料 17 3.3.1 實驗菌種 17 3.3.2 培養基配方 17 3.3.3 常用溶液配制 17 3.3.4 培養條件 18 3.4 實驗分析方法 19 3.4.1 生長曲線測定 19 3.4.2 休眠細胞 (Resting cells) 製作方法 20 3.4.3 一維蛋白電泳分析 20 3.4.4 掃描電子顯微鏡 (SEM) 分析 21 3.4.5 穿透式電子顯微鏡 (TEM) 分析 22 3.4.6 菌體介面電位 (Zeta potential) 分析 22 3.4.7 感應耦合電漿放射光譜儀 (ICP-OES) 測定 23 3.4.8 鐵還原酶活性分析 23 3.4.9 生物法製造奈米金量測定 23 3.4.10 2,6-DMP過氧化氫酶的酶活測定 24 第四章 結果與討論 25 4.1 希瓦氏菌鐵還原酶動力學研究分析 25 4.1.1 不同培養時間的鐵還原酶 25 4.1.2 不同細胞濃度的鐵還原酶 27 4.1.3不同培養轉速的鐵還原酶值 29 4.1.4 鐵還原酶動力參數分析 30 4.1.5 Mtr 通道蛋白對鐵還原酶的影響 32 4.2 希瓦氏菌生物製造奈米金粒子 34 4.2.1 不同 pH 對奈米金形成的影響 34 4.2.2 金濃度對奈米金製備的影響 37 4.2.3 乳酸鈉對製備奈米金的影響 39 4.2.4 不同菌體濃度對奈米金形成的影響 41 4.2.5 光與溫度對奈米金形成的影響 44 4.3 希瓦氏菌的其他應用 47 4.3.1 希瓦氏菌還原奈米金的假設機制 47 4.3.2 希瓦氏菌還原奈米金的回收 49 4.3.3 休眠細胞 (Resting cells) 生成奈米金測試 51 4.3.4 休眠細胞 (Resting cells) 鐵還原酶測試 52 4.3.5 希瓦氏菌對金銀銅的選擇率測定 52 4.3.6 酶活測定 56 第五章 結論與展望 57 5.1 結論 57 5.2 未來展望 63 第六章 參考文獻 64 表4-1、在不同細胞濃度下SXM和MR-1的鐵還原酶活性 28 表 4-2、添加與未添加 50 mM 乳酸鈉於300 ppm的氯金酸溶液4 h 後之上清金離子濃度比較 40 表5-1、希瓦氏菌鐵還原酶與文獻的比較 61 表5-2、希瓦氏菌還原奈米金與其他菌株的比較 62 圖 1-1、研究架構 2 圖2-1、奧奈達希瓦氏菌 (Shewanella oneidensis MR-1) 奈米導線圖[26] 5 圖2-2、原子力顯微鏡 (AFM) 和利用外膜染色所拍出奧奈達希瓦氏菌 (Shewanella oneidensis MR-1) 生成之奈米導線圖[28] 6 圖2-3、掃描式電子顯微鏡 (SEM) 所拍攝出廈門希瓦氏菌 (Shewanella xiamenensis) 7 圖 2-4、希瓦氏菌電子傳遞方式 (a) 直接接觸傳遞，(b) 電子中介體傳遞，(c) 奈米導線傳遞 8 圖 2-5、希瓦氏菌 MR-1 的電子傳遞機制 [36] 10 圖 2-6、以希瓦氏菌為例的微生物燃料電池組成結構 13 圖3-1、希瓦氏菌 SXM 和 MR-1 乾重和光學密度 (OD600) 的檢量線 19 圖3-2、光學密度 (OD530nm) 對應奈米金濃度 (mg/L) 的檢量線 24 圖 4-1、SXM和MR-1在LB培養基培養下的生長狀況 26 圖 4-2、在不同培養時間下SXM和MR-1的鐵還原酶值 27 圖 4-3、動態分析SXM和MR-1在細胞濃度1.2、2.4、3.6及6.0 g/L下之吸收波長(OD 562 nm) 變化 29 圖 4-4、SXM和MR-1在不同培養轉速下的鐵還原酶值 30 圖 4-5、檸檬酸鐵濃度(S)對SXM和MR-1的鐵還原酶活性(V)影響。由1/V與1/S的關係求取SXM和MR-1的Vmax，Km。 31 圖 4-6、pETSXM2-placI-mtrA、pETSXM2-placI-mtrC、pETSXM2-placI-mtrCAB質體圖譜。 33 圖 4-7、Mtr通道蛋白對鐵還原酶的影響。pETSXM2-placI-mtrA/MR-1、pETSXM2-placI-mtrC/MR-1、pETSXM2-placI-mtrCAB/MR-1的鐵還原酶值 33 圖 4-8、SXM和MR-1不同pH下之 Zeta potential 35 圖4-10、不同pH值對SXM和MR-1在300 ppm的氯金酸溶液所生成的奈米金的影響 37 圖 4-11、SXM 與 MR-1 在不同氯金酸濃度生成奈米金的線性關係 38 圖 4-12、未泡金酸的SXM (a) 和MR-1 (c) 與泡完金酸後之SXM (b) 和MR-1 (d) 38 圖 4-13、SXM 和MR-1浸泡在 (a) 100 ppm 與 (b) 500 ppm氯金酸之 SDS-PAGE 39 圖 4-15、SXM 和 MR-1 在乾重 0.6、1.2、 2.4、 3.6 和6.0 g/L的條件下浸泡 300 ppm 的金酸溶液 24 h 後的奈米金濃度 42 圖 4-17、SXM、MR-1、pETSXM2-placI-mtrC/MR-1、pETSXM2-placI-mtrCAB /MR-1在25oC、光照和37 oC的環境浸泡300 ppm氯金酸24 h 下的奈米金濃度 45 圖 4-18 (a) SXM與 (b) MR-1再浸泡300 ppm 氯金酸 24 h後之TEM分析 46 圖 4-19、SXM、MR-1 在光照的環境浸泡300 ppm 氯金酸24 h 內之變化圖 46 圖 4-20、SXM、MR-1 在 RT (25oC)的環境浸泡 300 ppm 氯金酸 24 h 內之變化圖。 46 圖 4-21、不同細胞量對SXM和MR-1在300 ppm 金酸溶液 24 h 後的 zeta potential 分析 48 圖 4-22、希瓦氏菌將金酸還原成奈米金的假設機制 49 圖 4-23、SEM 分析希瓦氏菌SXM和 MR-1經超聲波震盪前 (a, b) 及後(c, d) 釋放奈米金的結果。 50 圖 4-24、希瓦氏菌還原奈米金後在 200oC 下鍛燒 5 h 的結果 50 圖4-25、希瓦氏菌的休眠細胞 51 圖 4-26、希瓦氏菌休眠細胞放置 5、10、15、20 天後細胞所生成奈米金量的結果 51 圖 4-27、休眠細胞的鐵還原酶測試 52 圖4-28、希瓦氏菌浸泡在 100 ppm 硝酸銀下 0 h 和 12 h 後的掃瞄吸光波長 53 圖4-29、希瓦氏菌浸泡在 100 ppm 硫酸銅下 0 h 和 12 h 後的掃瞄吸光波長 54 圖4-30、SXM (a) 與 MR-1 (a) 在浸泡 100 ppm 硝酸銀 24 h 後之 SEM 與 EDS 分析 55 圖4-31、希瓦氏菌同時浸泡在100 ppm 金酸和 100 ppm 硝酸銀下 0 h 和 24 h 後的掃瞄吸光波長 55 圖 4-32、SXM 和 MR-1 浸泡 300 ppm 的 HAuCl4 和 100 ppm 的 AgNO3 12 h 後的過氧化氫酶活性。SXM及MR-1代表原菌活性，Au及Ag代表金屬離子的活性，SAu、MAu、 Sag、 Mag分別代表加入金及銀的活性影響。 56

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