臺灣博碩士論文加值系統

細菌纖維素生產菌株中以木質醋酸菌Gluconacetobacter xylinus 所產生之細菌纖維素量遠高於其它生產菌株，近年來有許多研究皆著重於降低發酵成本及提升細菌纖維素品質，而纖維素結合功能域 (Cellulose-binding domains)由文獻記載能促進植物纖維素生長，且能增加細菌纖維素之合成速率，本研究利用木質醋酸菌異源表現纖維素功能域與雙纖維素結合功能域 (Double cellulose-binding domains)以及外加纖維素功能域與雙纖維素結合功能域於培養基當中，期以提升細菌纖維素產量與提升細菌纖維素品質。研究結果顯示，以建構CBD與dCBD之表現質體轉殖入G. xylinus中，能成功表現出CBD，而基改G. xylinus與外加CBD實驗比較顯示以基改G. xylinus較好，在30℃靜置培養8天其細菌纖維素產量最高能達 2.78 g/l，相較於對照組能提升20.4 %；另在細菌纖維素品質方面結晶度從78.0 % 提升至 86.8 %，保水力從 83.4 % 提升至 94.7 %。因此木質醋酸菌異源表現CBD蛋白質不僅提升細菌纖維素產量更能提升品質。

Bacterial cellulose can be produced by Gram-negative bacterium, such as rod-shaped Gluconacetobacter xylinus. As the organism is obligate aerobic, bacterial cellulose is always produced at the air/liquid interface. In recent years there have been more reports about minimizing the cost of culture and improving the quality of bacterial cellulose. Cellulose-binding domains (CBD) have previously been shown to modulate the elongation of cellulose. In this work, the cellulose-binding domains gene and double cellulose-binding domains (dCBD) were cloned and successfully expressed in G. xylinus. In this study, the feasibility of using G. xylinus to drive the expression of heterologous proteins as well as the function of expressed CBD to enhance cell growth, cellulose productivity, crystallinity, water-holding capacity and at static culture was studied. After 8 days culturing at 30℃, the maximum cell density was 1.16 g/l while bacterial cellulose density was 2.78 g/l (20.4 % higher than control). In terms of bacterial cellulose (BC) quality, the crystallinity index increased from 78.0 % to 86.8 % while water-holding capacity increased from 83.4 % to 94.7 %. The expression system in this study is very successful in that it not only increased cell density and cellulose productivity, but also increased the BC quality.

目 錄

中文摘要 i
英文摘要 ii
誌謝 iii
目錄 iv
表目錄 v
圖目錄 vi
第一章 緒論 1
1.1 研究動機 1
1.2. 研究目的 2
第二章 文獻回顧 3
2.1 纖維素結合功能域 (Cellulose-binding domains) 3
2.1.1 纖維素功能域的簡介 3
2.1.2 纖維素功能域的發展簡史 3
2.1.3 纖維素功能域的分類 4
2.1.3.1 真菌之纖維素結合功能域 5
2.1.3.2 細菌之纖維素結合功能域 5
2.1.4 纖維素功能域的應用 6
2.2木質醋酸菌 (Gluconacetobacter xylinus) 7
2.2.1木質醋酸菌之生理特性 7
2.2.2影響木質醋酸菌生產細菌纖維素的因素 9
2.2.3基改木質醋酸菌 9
2.3細菌纖維素 (Bacterial cellulose) 10
2.3.1 細菌纖維素簡介 10
2.3.2 細菌纖維素之合成途徑 10
2.3.3 影響細菌纖維素生產之特性與應用 12
2.3.4 細菌纖維素結構與性質 13
2.3.6.1 細菌纖維素結晶度 13
2.3.6.2 細菌纖維素保水力 14
第三章 材料與方法 15
3.1 實驗流程 15
3.2 質體建構流程 16
3.2.1 纖維素結合功能域質體建構 16
3.2.2 雙纖維素結合功能域質體建構 17
3.3 實驗材料 18
3.3.1 菌株 18
3.3.2 載體 18
3.3.3 引子 18
3.3.4 操作試液套件組 (Kit) 18
3.3.5 標準分子量溶液 19
3.3.6 酵素 19
3.3.7 實驗藥品 19
3.3.8 實驗儀器 20
3.4 實驗方法 21
3.4.1 質體純化 21
3.4.2 質體建構 22
3.4.2.1 建構pET14b-CBD質體 22
3.4.2.2 建構pET14b-bla-CBD質體 22
3.4.2.3 建構pBBR-bla-CBD質體 22
3.4.2.4 建構pBBR-bla-dCBD質體 23
3.4.3 基因轉殖法 23
3.4.3.1 製備勝任細胞 23
3.4.3.2 基因轉殖 24
3.4.4 重組蛋白之純化 25
3.4.5 質體穩定度 25
3.4.6 靜置培養G. xylinus生產細菌纖維素 25
3.5 分析方法 26
3.5.1 蛋白質之濃度分析 26
3.5.2 乾菌重量測 26
3.5.3 細菌纖維素量測 27
3.5.4 掃描式電子顯微鏡 27
3.5.5 X光繞射分析（XRD） 27
3.5.6 保水力 27
3.5.7 復水率 28
3.5.8 水散失速率 28
3.5.9 CBD活性測試 28
第四章 結果與討論 29
4.1 質體建構 29
4.1.1 建構pET14b-CBD質體 29
4.1.2 建構pET14b-bla-CBD質體 30
4.1.3 建構pBBR-bla-CBD質體 31
4.1.4 建構pBBR-bla-dCBD質體 32
4.2 電轉殖G. xylinus 34
4.3 質體穩定度分析 35
4.4 以G. xylinus表現CBD蛋白質 36
4.5 測試CBD活性 37
4.6 培養基因改質G. xylinus 38
4.7 外加CBD與dCBD於培養基中培養G. xylinus 40
4.8 細菌纖維素性質分析 44
結論 50
參考文獻 51

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