本研究將使用已在本實驗室進行研究的 D-阿洛酮糖表異構酶以及 L-鼠李糖異構酶，生產 D-阿洛酮糖及 D-阿洛糖，首先以 D-果糖 (D-fructose) 為基質，使用 D-阿洛酮糖表異構酶 (D-psicose 3-epimerase, DPE)，將 D-果糖轉換成 D-阿洛酮糖 (D-psicose)，再利用 L-鼠李糖異構酶 (L-rhamnose isomerase, L-RhI)，將 D-阿洛酮糖進行醛酮醣之間的轉換，產出 D-阿洛糖，並使用反應曲面法 (response surface methodology, RSM) 進行生產條件的分析，以找出最佳生產條件。
首先探討單一因子對 D-阿洛糖產量之影響，同時利用兩種酵素，分別在不同溫度、D-果糖濃度及 Co2+ 離子濃度下，進行 D-阿洛糖產量試驗。在溫度方面，由結果得知酵素於 50℃ 下可生成較多的 D-阿洛糖；而在 D-果糖濃度方面，隨著 D-果糖濃度的提高，D-阿洛糖的產量隨之減少，於 5% D-果糖濃度下，具有最高的 D-阿洛糖產量。而在 D-果糖濃度 10-30% 之間，D-阿洛糖的產量並無明顯差異；接下來探討 Co2+ 離子濃度的影響，結果顯示添加 0.1 mM的 Co2+ 離子濃度下，具有較多的 D-阿洛糖產出。
接著以 50℃、30% D-果糖及 0.2 mM Co2+ 為反應曲面法 (response surface methodology, RSM) 的中心點，進行 D-阿洛酮糖生產條件最佳化試驗，統計分析結果顯示溫度及 D-果糖濃度對 D-阿洛酮糖的生產有顯著影響，隨著溫度及 D-果糖濃度的增加，D-阿洛酮糖的產量也會隨之增加，而 Co2+ 離子濃度則對 D-阿洛酮糖的生產無顯著影響。接著以最佳化條件 (65℃、46.8% D-果糖、0.3 mM Co2+) 進行生產，酵素反應約在 6 小時後達成平衡。而在酵素濃度分別為 7、14 及 21 U/mL 下反應 24 小時，約可生產出 26.81、28.91 及 28.56% 的 D-阿洛酮糖。
在 D-阿洛糖生產條件最佳化試驗中，以 50℃、L-RhI: DPE 比例為 15: 1 及 Co2+ 離子濃度為 0.2 mM 為反應曲面法的中心點，進行 D-阿洛糖的生產，由統計分析結果顯示出溫度及 Co2+ 離子濃度對 D-阿洛糖的生產有顯著影響，隨著溫度及 Co2+ 離子濃度的提高，可對 D-阿洛糖的生產具有正面的影響，而 L-RhI: DPE 比例則對 D-阿洛糖的生產沒有顯著影響，其生產最佳化條件為 64.8℃、L-RhI: DPE 比例為 7.1: 1、Co2+ 離子濃度為 0.25 mM。

關鍵詞: D-阿洛糖、D-阿洛酮糖、D-阿洛酮糖表異構酶、L-鼠李糖異構酶

In this study, D-psicose 3-epimerase (DPE) and L-rhamnose isomerase (L-RhI) were used to produce D-psicose and D-allose. D-Psicose was firstly converted from D-fructose by D-psicose 3-epimerase (DPE); then the isomerization of D-allose from D-psicose by L-rhamnose isomerase (L-RhI) was carried out, and finally response surface methodology (RSM) was used to optimize the productions of D-psicose and D-allose.
To investigate the effect of single factor on the production of D-allose, the experiments were carried out at different temperatures in the presence of different concentrations of D-fructose and Co2+ by using DPE and L-RhI to produce D-allose. The results had shown that the higher D-allose yield were obtained at 50℃ in the presence of 0.1 mM Co2+, and the D-allose yield would be reduced by the increasing concentrations of D-fructose. The highest D-allose yield was obtained from 5% D-fructose, and the D-allose yields have no significant differences using 10-30% D-fructose as substrate.
To optimize D-psicose production, 50℃, 30% D-fructose and 0.2 mM Co2+ were used as the center point of response surface methodology (RSM). The results had shown that temperature and D-fructose concentration significantly affected D-psicose yield, while the concentration of Co2+ made no significant difference to the D-psicose production. When the temperature and D-fructose concentration increased, the D-psicose yield was also increased. By using different concentrations of DPE (7, 14, and 21 U/mL), the D-psicose production reached equilibrium after 6 h and 26.81, 28.91, and 28.56% of D-psicose were produced after 24 h under the optimized conditions, which were 65℃, 46.8% D-fructose, and 0.3 mM Co2+.
The optimize D-allose production, 50℃, L-RhI: DPE = 15: 1 and 0.2 mM Co2+ were used as the center point of response surface methodology (RSM). The results had shown that temperature and Co2+ concentration significantly affected D-allose yield, while the L-RhI: DPE ratio made no significant difference to D-allose production. When the temperature and Co2+ concentration increased, the D-allose yield also increased. The optimized conditions were 64.8℃, L-RhI: DPE = 7.1: 1, and 0.25 mM Co2+.

Keywords: D-allose, D-psicose, D-psicose 3-epimerase, L-rhamnose isomerase

摘要.... I
Abstract II
目錄.... III
表目錄.. V
圖目錄.. VI
附錄目錄 VII
壹、 前言 1
貳、 文獻整理 2
一、 稀有醣類 2
二、 D-阿洛酮糖 2
三、 D-阿洛糖 4
四、 D-阿洛酮糖表異構酶 5
五、 L-鼠李糖異構酶 8
六、 反應曲面法 12
參、 實驗設計與流程 17
肆、 實驗材料與方法 18
一、 實驗材料 18
1. 實驗用基因、質體與菌株 18
2. 蛋白質標準品 18
3. 抗生素…………………………………………………………………18
4. 實驗材料與藥品 18
5. 實驗設備 20
6. 電腦分析軟體 21
二、 實驗方法 21
1. 菌種保存 21
2. 大腸桿菌培養及酵素表現 21
3. 重組型 DPE 純化 21
4. 重組型 L-RhI 純化 22
5. 蛋白質定量 23
6. 蛋白質電泳分析 23
7. 酵素活性測定 25
7.1 重組型 DPE 酵素活性測定 25
7.2 重組型 L-RhI 酵素活性測定 26
8. 單因子對 D-allose 產量之試驗 26
9. D-psicose 最佳生產條件試驗 27
10. D-allose 最佳生產條件試驗 28
11. 反應產物之 HPLC-ELSD 分析 29
伍、 結果與討論 30
一、 Agrobacterium sp. ATCC 31750來源之 D-阿洛酮糖表異構酶與 T. saccharolyticum NTOU1 來源的 L-鼠李糖異構酶特性比較 30
二、 單一因子對於 D-阿洛糖生產量之影響 30
三、 D-阿洛酮糖生產條件最佳化試驗 31
1. 反應曲面法設計與實驗結果 31
2. 反應曲面法之最佳化分析 31
3. 反應曲面法模型之迴歸及配適度分析 32
4. 最佳化條件下 D-psicose 之生物轉換率及最佳酵素添加濃度探討 32
四、 D-阿洛糖生產條件最佳化試驗 33
1. 反應曲面法設計與實驗結果 33
2. 反應曲面法之最佳化分析 33
3. 反應曲面法模型之迴歸及配適度分析 34
陸、 結論 35
柒、 參考文獻 36

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