臺灣博碩士論文加值系統

壹、 研究背景與目的 1
一、 研究背景 1
二、 研究目的 2
貳、 文獻整理 3
一、 單醣 3
二、 稀有醣類 (rare sugars) 3
三、 D-阿洛酮糖 (D-allulose) 3
四、 D-阿洛糖 (D-allose) 4
五、 稀有醣類的酵素法生產 4
六、 L-鼠李糖異構酶 (L-rhamnose isomerase, L-RhI) 5
七、 E. coli BL21-CodonPlus (DE3) 6
八、 固定化 7
九、 離子交換樹脂 8
參、 實驗設計與流程 9
1. 表現並純化 L-鼠李糖異構酶 9
2. 探討固定化條件及其特性 9
肆、 實驗材料與設備 10
一、 實驗材料 10
二、 實驗設備 12
伍、 實驗方法 14
1. Terrific broth (TB) 培養液 14
2. TB salt 14
3. 電穿孔轉形 14
4. 菌種保存 15
5. L-鼠李糖異構酶之表現與純化 16
6. 蛋白質定量 17
7. SDS-PAGE 18
8. TsRhI 活性測定 19
9. 酵素固定化 20
陸、 結果與討論 23
一、 TsRhI 大量表現與純化 23
二、 不同離子交換樹脂之篩選探討 23
三、 最適 TsRhI 添加濃度之探討 23
四、 最適作用溫度 24
五、 最適作用 pH 值 24
六、 熱穩定性 24
七、 儲藏性試驗 25
八、 產物平衡趨勢 25
柒、 結論 26
捌、 參考文獻 27
圖目錄
圖一、SDS-PAGE 分析 TsRhI 粗酵素液經不同熱處理後離心之上清液的蛋白質分布。 32
圖二、SDS-PAGE 分析 70℃ 再次熱處理不同時間對 TsRhI 之純度的影響。 33
圖三、不同酵素濃度對固定化 TsRhI 之固定化效率影響。 34
圖四、不同溫度對固定化 TsRhI 之活性影響。 35
圖五、不同 pH 值對固定化 TsRhI 之活性影響。 36
圖六、溫度對固定化 TsRhI 之穩定性影響。 37
圖七、儲存時間對固定化 TsRhI 活性之影響。 38
圖八、以固定在Q Sepharose 之 TsRhI 生產 L-鼠李酮糖之產物平衡趨勢。 39
表目錄
表一、TsRhI 固定於不同離子交換樹脂之結合率 40
附錄目錄
Appendix 1. Izumori 策略。………………………………………………………...41
Appendix 2. 以 L-RhI 生產 D-阿洛糖之反應圖。…………………………………42

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