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研究生:黃智鴻
研究生(外文):Zhi-Hong Huang
論文名稱:發展高效率大腸桿菌生物觸媒系統生產聚麩胺酸分解酵素
論文名稱(外文):Development of an efficient recombinant E. coli whole-cell biocatalyst system for γ-PGA depolymerase production
指導教授:陳姍玗
指導教授(外文):Shan-Yu Chen
口試委員:施英隆趙雲鵬姜中人
口試委員(外文):Ing-Lung ShihYun-Peng ChaoChung-Jen Chiang
口試日期:2013-07-08
學位類別:碩士
校院名稱:元智大學
系所名稱:生物科技與工程研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:104
中文關鍵詞:聚麩胺酸分解酵素聚麩胺酸全細胞生物觸媒Lpp-OmpA混合表面表現系統幾丁質結合位置幾丁質球固定化細胞
外文關鍵詞:γ-PGAYwtDWhole cell biocatalystLpp-OmpA hybrid systemChitin binding domainChitin beads
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由微生物醱酵生產的聚麩胺酸分解酵素(γ-Poly glutamic acid depolymerase)為一種可將高分子量之聚麩胺酸分解成低分子量的水解酵素,聚麩胺酸分解酵素可於溫和的條件下進行不同程度的聚麩胺酸降解,使聚麩胺酸分子量的調控更具潛力。Bacillus licheniformis BCRC 12826為兼具聚麩胺酸與聚麩胺酸分解酵素之生產菌株,所產生的聚麩胺酸分解酵素為一胞外酵素,然而以該野生菌株生產聚麩胺酸分解酵素有產量少及產物回收時間難以掌控的問題。因此本研究利用全細胞生物觸媒(Whole cell biocatalyst)之開發,以Lpp-OmpA混合表面表現系統將聚麩胺酸分解酵素直接表達於細胞表面,使水解聚麩胺酸之生物程序更具可行性,同時亦可克服反應物之傳送屏障,促進聚麩胺酸之水解反應。研究結果顯示,基因重組大腸桿菌E. coli BL21/pOmp-YwtD在25oC下,以0.01 mM L-arabinose誘導6小時,可獲得較佳的聚麩胺酸分解酵素(YwtD)活性。以全細胞生物觸媒進行聚麩胺酸水解反應發現,以5%生物觸媒在37oC下水解20分鐘聚麩胺酸水解程度可達45%,水解24小時後更可達95%以上,並可將聚麩胺酸之分子量由大於5,900 kDa降低至9.5 kDa。為了簡化純化產物時所需的操作步驟以及生物觸媒的再回收使用,本研究亦開發幾丁質結合位置(chitin binding domain;ChBD)與聚麩胺酸分解酵素之表面共表達系統,運用幾丁質結合位置與幾丁質球(chitin bead)專一的結合特性使細胞固定於基丁質球上形成固定化細胞,研究結果發現,ChBD需融合於Lpp-OmpA-YwtD之C端,才具有結合於chitin beads之能力。利用幾丁質球固定化細胞進行聚麩胺酸水解反應也證實,該固定化細胞的確具有降低聚麩胺酸分子量的能力。
γ-PGA is water soluble, biodegradable, edible and non-toxic toward humans and the environment. Therefore, potential applications of γ-PGA and its derivatives have been of interest in the past few years in a broad range of industrial fields such as food, cosmetics, medicine and water treatment. The γ-PGA produced by Bacillus sp. generally has relatively high molecular weight, such a high molecular-weight polymer is useful as a viscosity-adding agent but not applicable to other use because it is too viscous, and difficult to be modified by chemical reagents. A few reports exist on the microbial and enzymatic biodegradation of microbial γ-PGA to lower molecular-weight polymer. In this study, we designed a molecular structure to display YwtD on the outer membrane of E. coli.
We fused YwtD with the first nine N-terminal residues of Lpp (major E. coli lipoprotein) and the 46–150 residues of OmpA (an outer membrane protein of E. coli ). It was found that the highest enzyme activities were detected when recombinant E. coli grow at 25oC and 0.01 M L-arabinose added to induce for 6 hours. The optimal temperature for enzyme catalysis was 37oC. The results demonstrated that a soluble protein, YwtD, can be anchored to the external surface of E.coli and successfully displayed YwtD on the surface of E. coli.
目錄
中文摘要 VIII
英文摘要 X
目錄 XII
表目錄 XVII
圖目錄 XIX
第一章 序論 1
1-1 前言 1
1-2 研究動機 2
1-3 論文架構 5
第二章 文獻回顧 6
2-1 聚麩胺酸之結構與生合成 6
2-2 聚麩胺酸之應用 12
2-3 聚麩胺酸水解酵素 14
2-4 細胞表面表現技術(CELL SURFACE DISPLAY) 16
2-4-1 載體蛋白(CARRIER ROTEIN) 17
2-4-2 目標蛋白(TARGET PROTEIN) 17
2-4-3 宿主細胞(HOST CELL) 18
2-4-4 細胞表面表現技術應用 19
第三章 材料與方法 20
3-1藥品 20
3-2儀器設備 23
3-3菌株儲存與培養 26
3-3-1 菌株與質體來源 26
3-3-1 菌種保存 26
3-3-2 菌種活化 26
3-4 DNA純化與PCR反應 27
3-4-1 染色體DNA純化 27
3-4-2 質體DNA製備 28
3-4-4 瓊脂膠體電泳(AGAROSE GEL ELECTROPHORESIS) 29
3-4-5 膠體DNA 回收 29
3-5 剪切反應、接合反應、與轉型作用 30
3-5-1 限制酶剪切反應(DIGESTION) 30
3-5-2 部分剪切(PARTIAL DIGESTION) 30
3-5-3 質體DNA接合反應(LIGATION) 32
3-5-4 製作勝任細胞(COMPETENT CELL) 32
3-5-5 轉型作用(TRANSFORMATION) 33
3-5-6 篩選轉型成功之基因重組大腸桿菌 33
3-6 聚合酵素鏈鎖反應(POLYMERASE CHAIN REACTION, PCR) 34
3-7質體建構 35
3-6-1 質體pOmp-ywtD之建構 35
3-6-2 質體pOmp-YC之建構 38
3-6-3 質體pOmp-CY之建構 38
3-8 融合蛋白(LPP-OMPA-YWTD)之分離萃取與分析 42
3-8-1 細胞膜蛋白分離 42
3-8-2 細胞膜蛋白分析(SDS-PAGE) 42
3-8-3 YwtD胺基酸序列比對 43
3-9 聚麩胺酸之生產 46
3-10 聚麩胺酸水解酵素之活性分析方法 48
3-10-1 SDS-PAGE分析法 48
3-10-2 TNBSA (2,4,6-TRINITROBENZENE SULFONIC ACID ASSAY)分析法 49
3-10-2-1 分析原理 49
3-10-2-1 分析方法 50
3-10-3 高效率液相層析 (HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY;HPLC)分析方法 51
3-10-3-1 HPLC分析條件 51
3-10-3-2 聚麩胺酸分子量之分析方法 51
3-10-3-3 HPLC分析聚麩胺酸水解酵素之活性 53
3-11 聚麩胺酸水解溶液之配置與水解方法 55
3-12 基因重組大腸桿菌膜融合蛋白之最佳誘導條件 55
3-12-1 最佳誘導劑濃度之測試方法 55
3-12-2 最佳誘導溫度之測試方法 56
3-12-3 最佳誘導時間之測試方法 56
3-13 聚麩胺酸水解條件之探討 57
3-13-1 最佳水解溫度之測試方法 57
3-13-2 最佳水解pH環境之測試方法 57
3-13-3 最佳水解轉速之測試方法 58
3-13-4 不同生物觸媒菌量對聚麩胺酸水解之影響 58
3-13-5 聚麩胺酸水解水解時間與分子量測試 59
3-13-6 不同來源之聚麩胺酸水解測試 59
3-14 細胞固定化 60
3-14-1 基因重組菌株培養方法 60
3-14-2 CHITIN BEAD細胞固定化條件 60
3-15 利用CHITIN BEAD固定化細胞進行聚麩胺酸之水解 60
3-16 固定化細胞分析方法 61
第四章 結果與討論 62
4-1-1 確認重組菌株之質體DNA 62
4-1-2 ywtD序列定序(SEQUENCING)和蛋白質序列比對(BLASTING) 64
4-1-3 SDS-PAGE分析膜融合蛋白 67
4-2 膜融合蛋白之最佳誘導條件 69
4-2-1 誘導劑濃度對於YwtD活性之影響 69
4-2-2 誘導溫度對於YwtD活性之影響 70
4-2-3 誘導時間對於YwtD活性之影響 71
4-3 YWTD最佳水解條件測試 72
4-3-1 溫度對於酵素活性之影響 72
4-3-2 水解環境pH值對於酵素活性之影響 73
4-3-3 轉速對於酵素活性之影響 76
4-4 不同生物觸媒之菌量對水解聚麩胺酸之影響 77
4-5 聚麩胺酸水解酵素之水解時間對分子量與水解程度之影響 80
4-6 不同來源之聚麩胺酸之水解影響 81
4-7 TNBSA分析方法 82
4-8 聚麩胺酸水解酵素之固定化細胞系統開發 85
4-8-1 融合蛋白ChBD-YwtD與YwtD-ChBD之酵素水解活性 85
4-8-2 以CHITIN BEAD 固定化細胞進行聚麩胺酸水解 87
4-8-3 SEM分析固定化細胞 88
第五章 結論 90
未來展望 92
參考文獻 93
附錄一 ywtD 基因序列 100
附錄二 ChBD 基因序列 104


表目錄
表2-1 由微生物合成之聚麩胺酸組成與分子量 11
表2-2 聚麩胺酸與其衍生物之應用 (Shih and Van, 2001) 13
表2-3 聚麩胺酸水解酶 (Kimura and Fujimoto, 2010) 16
表3-1 TE buffer配方 27
表3-2 聚合酵素鏈鎖反應條件 28
表3-3 限制酶剪切反應之成分 30
表3-4 CPG solution 32
表3-5 SOB medium 33
表3-6 聚合酵素鏈鎖反應條件 34
表3-7 聚合酵素鏈鎖反應成分 34
表3-8 本研究中所使用之引子 35
表3-9 12% SDS-PAGE Separation Gel配方 43
表3-10 5% SDS-PAGE Separation Gel配方 44
表3-11 SDS-PAGE 5X Sample Buffer 配方 44
表3-12 SDS-PAGE 1X Running Buffer配方 44
表3-13 Coomassie Stain配方 45
表3-14 De-Stain for Coomassie Blue 配方 45
表3-15 10X Methylene Blue 配方 45
表3-16 6% SDS-PAGE Separation Gel 配方 46
表3-17 Medium E配方 (王相懿, 2010) 47
表4-1 酵素水解後之聚麩胺酸產物分子量 79
表4-2 水解不同來源之聚麩胺酸 81


圖目錄
圖1-1 聚麩酸水解酶之膜融合蛋白示意圖 3
圖1-2 大腸桿菌利用ChBD與幾丁質球進行固定化 4
圖1-3 幾丁質球(CHITIN BEAD)細胞固定化之示意圖 4
圖2-1 聚麩胺酸之化學結構 8
圖2-2 聚麩胺酸結構 (Sung et al., 2005) 8
圖2-3 合成聚麩胺酸之基因群 (Bajaj and Singhal, 2011) 9
圖2-4 pgs基因群轉譯之胺基酸序列 (Sung et al., 2005) 9
圖2-5 聚麩胺酸生合成路徑圖 (Shih and Van, 2001) 10
圖2-6 聚麩胺酸合成複合酶示意圖 (Candela and Fouet, 2006) 11
圖2-7 B. licheniformis 水解D-form聚麩胺酸之作用機制 (King et al., 2000) 15
圖3-1 限制酶部分剪切反應 31
圖3-2 pOmp-ywtD建構圖 37
圖3-3 於pBluescript中建構ChBD-ywtD基因與ywtD-ChBD基因 40
圖3-4 pOmp-YC與pOmp-CY之建構 41
圖3-5 使用6% SDS-PAGE 可初步確認聚麩胺酸分解酵素之活性 48
圖3-6 TNBSA與胺基化學反應圖 (Fields, 1972) 49
圖3-7 TNBSA 檢量線 50
圖3-8 分子量與滯留時間之關係圖 52
圖3-9 分子量標準曲線 52
圖3-10 不同分子量Dextran之HPLC分析圖譜 54
圖4-1 確認建構質體之瓊脂膠體電泳圖 63
圖4-2 (A) 蛋白水解酶之多重序列比對 65
圖4-2 (B) 蛋白水解酶之多重序列比對 66
圖4-3 以12% SDS-PAGE分析膜融合蛋白 68
圖4-4 不同誘導劑濃度對重組菌株膜蛋白活性之影響 69
圖4-5 誘導溫度對於YwtD活性之影響 70
圖4-6 誘導時間對於YwtD活性之影響 71
圖4-7 溫度對於酵素活性之影響 72
圖4-8 pH對於酵素活性之影響 74
圖4-9 以SDS-PAGE分析水解環境PH值對於酵素活性之影響 75
圖4-10 轉速對於酵素活性之影響 76
圖4-11 不同生物觸媒之菌量對水解聚麩胺酸之影響 78
圖4-12 水解時間與對分子量與水解程度之影響 80
圖4-13 以TNBSA分析菌株不同誘導時間對於水解之影響 83
圖4-14 以TNBSA檢測聚麩胺酸水解溶液之胺基濃度聚麩酸水解酶 之膜融合蛋白示意圖 84
圖4-15 於室溫下靜置5分鐘後之CHITIN BEAD 86
圖4-16 融合蛋白ChBD-YwtD與YwtD-ChBD之酵素水解活性 86
圖4-17 以CHITIN BEAD進行聚麩胺酸酵素之水解 87
圖4-18 全細胞生物觸酶、固定化酵素與固定化細胞之相對活性 88
圖4-19 CHITIN BEAD分析(SEM) 89
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