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系統識別號 U0026-3108201909084900
論文名稱(中文) 以大腸桿菌異源表達碳酸酐酶及其特性分析與應用
論文名稱(英文) Cloning, Characterization and Application of Recombinant Carbonic Anhydrase in Escherichia coli
校院名稱 成功大學
系所名稱(中) 化學工程學系
系所名稱(英) Department of Chemical Engineering
學年度 107
學期 2
出版年 108
研究生(中文) 何太皓
研究生(英文) Tai-Hao He
電子信箱 2802478221@qq.com
學號 N36053017
學位類別 碩士
語文別 中文
論文頁數 85頁
口試委員 指導教授-吳意珣
口試委員-李思禹
口試委員-鄭宇伸
口試委員-陳柏庭
中文關鍵字 碳酸酐酶  二氧化碳封存  固定化酵素  冰核蛋白  表面展示 
英文關鍵字 Carbonic anhydrase  carbon capture and storage  immobilized enzyme  ice nucleation protein  surface display 
學科別分類
中文摘要 自工業革命以來,二氧化碳排放量已增加二十倍,地表溫度增加二度,造成嚴重的溫室效應,目前碳封存技術是解決溫室效應的途徑之一,綠色製程及永續發展策略已經成為國際間必須共同面對的議題。本研究選用碳酸酐酶 (CA, EC 4.2.1.1),藉由 CO2 溶入水中轉化為碳酸鹽來封存,利用大腸桿菌基因工程表達目標蛋白,由 pET28a (+)表達 hCAII (Human carbonic anhydrase II) 測得其全細胞的活性達 3047 WAU/mg,且表達蛋白量達1 g/L。hCAII熱穩定性較差,40 ℃下處理150分鐘活性下降至10 %;但在pH 6.0到pH 8.0之間活性能維持80%以上。除K+、Na+、Mg2+、Ca2+等離子對其活性略有提升外,其餘各種離子均對其活性具有不同程度的抑制作用。測試鹽度的影響,結果表明兩者對於鹽的穩定性良好,在鹽度3~4 %,活性仍有70%。本研究亦利用Agar進行固定化CA,以2 % (w/v) Agar 包埋hCAII全細胞具有殘存活性 500.4 WAU/mg,重複使用5次的活性剩餘62.16%;另外保存3週後,其活性維持不變。經過Agar固定化後,可以明顯提高hCAII的耐熱性,其他性狀影響較小。本研究亦分析了hCAII的動力學參數,其全細胞kcat/Km=1.66*108 s-1M-1,並求得CO2在大腸桿菌中擴散係數De = 157 (μm^2)/s,及其在Agar中擴散係數De = 0.413 (μm^2)/s。最後為了提高全細胞利用的效率,設計冰核蛋白 (Ice Nucleation Protein, INP) 作為媒介將hCAII展示在E. coli細胞表面。由SDS-PAGE及胰蛋白酶分析結果表明INP-hCAII能夠被大量表達於細胞表面,然而其活性卻不高,推測可能為蛋白結構受立體障礙,利用agar固定化INP-hCAII全細胞,其殘餘活性與未經表面展示之hCAII固定化產物相差不大,但因其初始活性較低故效果不如未經表面展示之hCAII固定化產物。
英文摘要 In this study, we use IDT g-block synthesis to obtain 2 CA gene from Helicobacter pylori (HpCA) and human beings (hCAII). Through genetic engineering in E. coli, the activity of HpCA and hCAII was achieved up to 93.87 WAU/mg 15743 WAU/mg in pET28a system with highest expression level. For hCAII, the thermostability was very poor. The pH stability was between pH6.0 and pH8.0, whereas the activity maintained more than 80%. In salinity effect, the activity kept at 70% in the salinity of 3% to 4%. Agar has been used for immobilization of hCAII. The residual activity is 500.4 WAU/mg when encapsulating whole cells in 2% (w/v) agar. It can be reused 5 times with the final activity of 62.16%. The immobilized CA kept 100% activity after 3 weeks of preservation. Surface display of hCAII mediated by ice nucleation protein (INP) on cell has been designed to enhance the utilized efficiency of whole cell. The results showed that INP-hCAII can be overexpressed on the outer membrane of E. coli through trypsin digestion. The kinetic parameters of hCAII were also calculated, and the diffusion coefficient of CO2 in E. coli was determined to be De = 157 μm2/s, and its diffusion coefficient in agar was De = 0.413 μm2/s.
論文目次 中文摘要 I
Extended Abstract II
誌謝 V
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. 固碳技術簡介 3
2.2. 大腸桿菌基因工程簡介 3
2.2.1. pET表達系統簡介 4
2.2.2. Lac operon 誘導機制 5
2.3. 碳酸酐酶簡介 7
2.4. HpCA 及 hCAII 簡介 9
2.5.固定化酵素簡介 11
2.5.1 固定化酵素的優缺點 13
2.5.2 固定化酵素在工業上之應用歷程 14
2.5.3 碳酸酐酶固定化研究進展 15
2.5.4 洋菜膠簡介 18
第三章 實驗方法與材料 20
3.1. 實驗藥品 20
3.2. 實驗儀器 23
3.3. 菌株、質體、引物材料 24
3.4. 常用試劑配製 25
3.5. 實驗步驟 27
3.5.1. 菌株培養與保存 27
3.5.2. 生長曲線測定 28
3.6. 基因重組構建 29
3.6.1. DNA 電泳分析與膠體回收 (DNA gel extraction) 29
3.6.2. 聚合酵素酵素連鎖反應 (Polymerase Chain Reaction) 31
3.6.3. 質體抽取 (Plasmid DNA extraction) 31
3.6.4. 限制酵素酵素切反應 (Restriction enzyme digestion) 33
3.6.5. 目標質體與基因之接合反應 (Ligation) 33
3.6.6. 轉化 (Transformation) 33
3.6.7. 驗證 (Confirmation) 34
3.7. 蛋白樣品收集與分離 35
3.7.1. 高壓破碎獲取胞內蛋白 35
3.7.2. 蛋白質濃度測定 36
3.8. 一維蛋白電泳分析 (SDS-PAGE) 38
3.8.1. 上下膠的配方 38
3.8.2. 蛋白質電泳分析步驟 38
3.8.3. 胰蛋白酶驗證表面展示步驟 39
3.9. 蛋白活性分析 (Wilbur-Anderson assay) 39
3.10. Agar包埋固定化 40
3.11. 碳酸酐酶特性分析 41
3.11.1. 熱穩定性 41
3.11.2. 酸鹼穩定性 41
3.11.3. 鹽度穩定性 42
3.11.4. 離子穩定性 42
3.11.5. 動力學參數分析 42
3.11.6. CO2擴散係數計算 44
3.11.7. CO2固定實驗 47
第四章 結果與討論 48
4.1. pET系统表達目標蛋白 48
4.1.1. 基因序列 48
4.1.2. pET28a-HpCA 及 pET28a-hCAII 質體構建 50
4.1.3. 誘導表達及活性測試 53
4.2. pET28a-HpCA 及 pET28a-hCAII 表徵 57
4.2.1. pET28a-HpCA 及 pET28a-hCAII 熱穩定性測試 57
4.2.2. pET28a-HpCA 及 pET28a-hCAII pH穩定性測試 58
4.2.3. pET28a-HpCA 及 pET28a-hCAII 鹽度穩定性測試 59
4.2.4. pET28a-HpCA 及 pET28a-hCAII 離子穩定性測試 60
4.3. pET28a-HpCA 及 pET28a-hCAII 的固定化及其表徵 61
4.3.1. pET28a-HpCA 及 pET28a-hCAII 的固定化 62
4.3.2. pET28a-hCAII 的固定化產物的表徵 65
4.4. hCAII 的表面展示 66
4.4.1. pET28a-INP-hCAII 及pET22b-INP-hCAII 質體構建 68
4.4.2. pET28a-INP-hCAII 及 pET22b-INP-hCAII 誘導表達 71
4.4.3. pET28a-INP-hCAII 及 pET22b-INP-hCAII 固定化 72
4.4.4. hCAII 及 INP-hCAII 動力學研究 73
4.4.5. CO2擴散係數計算 74
4.5. CO2固定實驗 76
第五章 結果與討論 78
5.1. 結論 78
5.2. 未來展望 79
第六章 參考文獻 80
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