臺灣博碩士論文加值系統

本研究的主旨是運用大腸桿菌來生產一種酵素複合物。我們建構了一個具有結合性的蛋白質，經實驗顯示，這個蛋白質在試管中可吸附水解酵素。當在大腸桿菌中同時生產此蛋白質和水解酵素，可輕易由細胞獲得酵素複合物。

The subjective of this study was to produce an enzyme complex in Escherichia coli. To this end,we constructed a protein with an ability of protein affinity. As a result,the constructed protein was able to absorb hydrolysis enzymes in vitro. Moreover,an enzyme complex could be easily obtained when the affinity protein and hydrolysis enzymes were co-expressed in E.coli.

目錄
第一章 緒論
1.1 前言 1
1.2 文獻回顧 2
1.3 研究動機 8
第二章 實驗方法
2.1 菌種之儲存與馴養 9
2.1.1 菌種儲存 9
2.1.2 菌種培養 9
2.2 DNA (Deoxyribonucleic AciD)的純化與回收 10
2.2.1 染色體純化 (Chromosome Purification) 10
2.2.2 質體純化(Plasmid Purification) 12
2.2.3 瓊脂凝膠萃取DNA 片段（DNA Extraction From Agarose Gels） 13
2.2.4 PCR 產物純化（Purification of PCR Product） 14
2.2.5 DNA濃度測量 15
2.3 剪切反應（Digestion）、凝膠電泳法（Gel Electrophoresis）、連接反應（Ligation） 16
2.3.1 剪切反應(Digestion) 16
2.3.2 凝膠電泳法(Gel Electrophoresis) 16
2.3.3 連接反應（Ligation） 17
2.4 勝任細胞的準備 18
2.4.1 化學法 18
2.5 轉殖作用(Transformation) 19
2.5.1 以化學法製備勝任細胞的轉殖 19
2.6 聚合酵素連鎖反應（Polymerase Chain Reaction） 19
2.7 重組菌種製備 20
2.8 重組質體之建構 21
2.8.1 建構 pET-CelK 21
2.8.2 建構 pTac-RCelAD2 21
2.8.3 建構 pAC-GlsD 21
2.8.4 建構 pTr-Coh3Ch 21
2.8.5 建構 pETD-CelKAD2 21
2.9 重組蛋白生產檢測 26
2.9.1 蛋白質定量(total protein) 26
2.9.2 蛋白質電泳（SDS-PAGE） 26
2.9.3 目標蛋白定量(Target protein) 29
2.10 DNS試劑與反應基質製備 29
2.10.1 DNS試劑製備 29
2.10.2 0.2% phosphoric acid swollen cellulose(PASC)製備 30
2.10.3 2% Carboxymethylcellulose sodium salt(CMC)製備 30
2.10.4 1% Salicin製備 31
2.11 纖維分解酵素活性測試與分析 31
2.11.1 還原糖測定法(DNS呈色法) 31
2.11.2 高效液相層析法定量 33
2.12 酵素吸附反應 33
2.13 重組微纖維素酶體之活性分析 34
第三章 實驗結果與討論
3.1 系統敘述 36
3.2 表達質體pET-CelK、pTac-RCelAD2、pAC-GlsD和pTr-Coh3Ch目標蛋白生產測試 37
3.3 質體pTr-Coh3Ch測試 38
3.3.1 測試Coh3Ch之multiple cohesin peptide能否吸附在幾丁質球上 38
3.3.2 測試multiple cohesin peptide上的CohI與CelK上的DocI之結合情形 39
3.3.3 估計Coh3Ch蛋白對酵素CelK的吸附量 40
3.3.4 吸附後CelK酵素與吸附前CelK酵素的活性分析 41
3.3.5 測試multiple cohesin peptide上的CohII與RCelAD2上的DocII之結合情形 41
3.3.6 估計Coh3Ch蛋白對酵素RCelAD2的吸附量 42
3.3.7 吸附後RCelAD2酵素與吸附前RCelAD2酵素的活性分析
43
3.3.8 測試multiple cohesin peptide上的CCoh與GlsD上的CDoc之結合情形 44
3.3.9 估計Coh3Ch蛋白對酵素GlsD的吸附量 44
3.3.10 吸附後GlsD酵素與吸附前GlsD酵素的活性分析 45
3.3.11 測試multiple cohesin peptide能否同時吸附CelK和RCelAD2酵素 46
3.3.12 估計Coh3Ch蛋白對酵素CelK和RCelAD2的吸附量 47
3.3.13 吸附後CelK和RCelAD2酵素與吸附前CelK和RCelAD2酵素的活性分析 47
3.3.14 測試multiple cohesin peptide能否同時吸附CelK、RCelAD2和GlsD酵素 48
3.3.15 估計Coh3Ch蛋白對酵素CelK、RCelAD2和GlsD的吸附量 49
3.3.16 吸附後CelK、RCelAD2和GlsD酵素與吸附前CelK、RCelAD2和GlsD酵素的活性分析 50
3.4 比較multiple cohesin peptide的存在是否能增加分解效率 51
3.4.1 以PASC為基質比較multiple cohesin peptide對活性的影響
51
3.4.2 以PASC為基質測得的還原糖濃度對時間做圖 53
3.4.3 以Avicel為基質比較multiple cohesin peptide對活性的影響
54
3.4.4 以Avicel為基質測得的還原糖濃度對時間做圖 55
3.5 表達質體pETD-CelKAD2和pTr-Coh3Ch目標蛋白生產測試 56
3.6 改善質體pETD-CelKAD2生產CelK酵素的量 58
3.7 胞內生產微纖維素酶體 59
3.7.1 胞內生產微纖維素酶體的個別吸附結果 61
3.7.2 胞內生產微纖維素酶體的個別活性分析 64
第四章 結論與未來展望
參考文獻
附錄

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