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研究生： 許高榜
Hsu, Kao-Pang
論文名稱： 建立基因重組大腸桿菌碳酸酐酶之定向進化與篩選平台
Establishment of Screening Platform and Direct Evolution of Recombinant Carbonic Anhydrase in E. coli
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： 2019
畢業學年度： 107
語文別： 中文
論文頁數： 104
中文關鍵詞： 碳酸酐酶 、定向進化 、二氧化碳封存 、篩選平台
外文關鍵詞： Carbonic anhydrase, Direct evolution, Carbon Capture and Storage, Screening Platform
相關次數： 點閱：103  下載：4 
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    • 碳酸酐酶 ( Carbonic Anhydrase, CA ) 廣泛存在於大自然的各種生物中，是一種以鋅離子結合在活性中心的酵素，能夠催化二氧化碳與水反應形成碳酸氫根離子。本研究其初選用來自Sulfurihydrogenibium yellowstonense YO3AOP1的CA (簡稱 SyCA)，在大腸桿菌中進行異源表達、比較不同質體的酵素活性，同時建立了Arduino-pH tracker (ART) 硬體系統，應用於 SyCA 酵素的動力學及特性分析。接者以此數據為基礎，進行活性高但耐熱性不足的 MlCA 及表達量佳但活性及穩定性不足的 hCAII 的定向進化實驗，期待打造出具耐熱性、高穩定性、高活性的碳酸酐酶，且應用於工業的二氧化碳封存製程中。
    實驗結果顯示，透過pET28a及pET32a 生產的 SyCA 粗蛋白活性分別為 20558 WAU/mg及22773 WAU/mg，在80 oC加熱 100 分鐘後，粗蛋白殘餘活性為 37.47 %，全細胞殘餘活性為 79.91%。SyCA 在pH 4 的環境下活性最好，在 9 種不同的金屬離子的環境中 ( 包含 IA、IIA，及過度金屬 ) 進行試驗，發現 Cu2+ 以及 Zn2+ 會大幅度降低 SyCA 活性。由 ART 系統分析 SyCA 粗蛋白，獲得kcat 及 kcat/KM 為 5.98.x106 s-1 和 37.9x107 s-1 M-1，全細胞則為 3.37.x106 s-1 和 8.62x107 s-1 M-1。
    在定向進化的篩選實驗中，架設了兩套不同的篩選平台，分別是酚紅指示劑篩選平台及菌落尺度篩選，以利用於定向進化突變後的篩選實驗。而在突變實驗中，設計了3種不同的突變策略：(一) 電腦設計的雙硫鍵添加技術，(二) 隨機突變的RECODE，及 (三) 易錯 (error-prone) PCR ( 即 EP-PCR ) 技術。在 EP-PCR 突變實驗中，成功的生產超過 1400 個單菌落，挑選其中 576 個單菌落進行篩選實驗，完成突變技術展示及 CA 定向進化篩選平台成效的驗證。

    Carbonic Anhydrase (CA) catalyzes the chemical reaction in which carbon dioxide react with water to form bicarbonate ions. Due to its high catalytic efficiency and its eco-friendliness, scientists look insight to apply CA for capturing carbon dioxide in the industrial process. However, there exist technical difficulties to industrialize this process, such as high cost, limited heat tolerance and poor in stability. In this study, we chose CA from Sulfurihydrogenibium yellowstonense YO3AOP1 (denoted as SyCA) as candidate for enzyme characterics analysis. Furthermore, based on the results, MlCA, which owns the highest activity but is low heat tolerance, and hCAII, which can be highly expressed by pET system in E. coli but poor in stability, are chosen as candidates for direct evolution. We expected to create a heat tolerance and stability CA through direct evolution, thus applied it into industrial Carbon Capture and Storage (CCS) process.
    At first, the SyCA is successfully heterologous expressed in E. coli BL21(DE3), and the characterics, including enzyme activity, heat stability, pH effect, ion effect and kinetic parameters were further tested. In direct evolution experiment, two different types of screening platfom and three differen stategies of mutations have been established. As a result, total 576 candidates were screened in EP-PCR experiment on hCAII, but no candidates showed imporovments. The amount of diverse mutation candidates have to raise to the level of 105 ~ 107 in order to pick up the desirable candidates with significant improvement in characteristics. We have setted up the screening platform to selected CA with higher activity but further investigation are needed in the future.

    總目錄 摘要 I Extended abstract II 誌謝 VIII 總目錄 IX 表目錄 XIII 圖目錄 XV 第一章 緒論 1 1-1 前言 1 1-2 研究目的與架構 2 第二章 文獻回顧 4 2-1二氧化碳捕捉與儲存 4 2-1-1 二氧化碳捕捉技術發展與現況 4 2-1-2 生物法碳捕捉技術 6 2-2 碳酸酐酶 ( Carbonic Anhydrase ) 7 2-2-1 hCAII 8 2-3-2 SyCA 9 2-3 定向進化技術初探 11 2-3-1 隨機演化 (Random Mutation) 12 2-3-1-1 易錯PCR (EP-PCR) 12 2-3-1-2 重組定向進化技術 13 2-3-1-3 Rapidly Efficient Combinatorial Oligonucleotides for Directed Evolution (RECODE) 14 2-3-2 理性設計 15 2-3-2-1雙硫鍵 (disulfide bond) 設計 16 2-3-3定向進化篩選平台 16 2-3-3-1 平板篩選 17 2-3-3-2 細胞篩選 18 2-3-3-3 In vitro compartmentalisation 18 2-4 ART (Arduino-Based pH Tracker) 19 2-4-1 Arduino 軟硬體操作介紹 19 2-4-2 ART技術規格 20 2-4-3 藍牙無線傳輸技術 21 2-4-4 pH 電極 21 第三章 材料與方法 24 3-1 實驗藥品與材料 24 3-2 實驗儀器 29 3-3 溶液配製 30 3-3-1培養基溶液配製 30 3-3-2 DNA電泳分析試劑 31 3-3-3 SDS-PAGE用試劑 31 3-3-4 His-trap 純化用 buffer 32 3-3-5 化學法勝任細胞 ( Component Cell ) 製備用溶液 32 3-3-6 碳酸酐酶活性測定用溶液 32 3-4 實驗方法 33 3-4-1 基因質體構建 33 3-4-1-1 聚合酶酵素連鎖反應 33 3-4-1-2 DNA電泳分析與膠體回收 34 3-4-1-3 質體抽取 35 3-4-1-4 限制酶酶切 36 3-4-1-5目標質體與基因之接合連接 36 3-4-1-6 熱基因轉殖於大腸桿菌 37 3-4-2 異源表達蛋白分析 37 3-4-2-1 重組大腸桿菌培養與異源蛋白轉譯 37 3-4-2-2 一維蛋白電泳分析 (SDS-PAGE) 38 3-4-2-3 蛋白樣品純化 40 3-4-2-4 蛋白濃度測定 41 3-4-3 碳酸酐酶酵素分析 42 3-4-3-1 碳酸酐酶活性測定 ( Walbur-Anderson法 ) 42 3-4-3-2碳酸酐酶特性分析 43 3-4-4 定向進化策略 45 3-4-4-1 雙硫鍵定向進化設計 45 3-4-4-2 Error-prone PCR ( EP-PCR ) 45 3-4-4-3 RECODE 定向進化突變技術 47 3-4-4-4 平板突變初步篩選平台 49 3-4-4-5 酚紅指示劑篩選平台 50 3-5 ART 51 3-5-1儀器操作與軟體操作流程 51 3-5-2 ART程式碼 52 第四章 結果與討論 53 4-1 SyCA異源表達與分析 53 4-1-1構建E. coli異源表達 SyCA 之菌株 53 4-1-2 SyCA 表達及活性分析 57 4-1-2-1 SyCA 蛋白質電泳及其生長曲線分析 57 4-1-2-2 SyCA 酵素活性及特性分析 58 4-2 ART 65 4-2-1 ART 系統架構 65 4-2-2 將 ART 應用於 SyCA 酵素特性及動力學分析 67 4-3 定向進化突變技術 - 雙硫鍵設計 71 4-3-1 電腦輔助 CA 雙硫鍵突變位點設計 71 4-3-2 構建 E. coli 異源表現突變 CA 菌株 73 4-3-3 突變後 CA 之酵素分析 74 4-4 定向進化突變技術 – RECODE 75 4-4-1 pSB1C3-J23100 為骨架之 CA 質體 75 4-4-2 以 pSB1C3-J23100 為骨架之 CA 表達及活性分析 78 4-4-3 以 pET28a-hCAII 為樣板進行 RECODE 定向進化突變實驗 79 4-4-4 RECODE 突變後篩選 80 4-5 碳酸酐酶定向進化篩選平台 81 4-5-1 酚紅指示劑定向進化平台改良 82 4-5-2 菌落尺寸篩選平台 85 4-6 定向進化突變技術 – EP-PCR 87 4-6-1 以 pET28a-hCAII 為樣板進行 EP-PCR 定向進化突變實驗 87 4-6-2 EP-PCR 突變後篩選 88 第五章 結論與展望 90 5-1 結論 90 5-2 未來展望 93 第六章 參考文獻 94 附錄一 101

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