臺灣博碩士論文加值系統

D-阿洛酮糖表異構酶 (D-psicose 3-epimerase, DPE) 能以非專一性的方式將酮糖上 3 號碳位置的氫氧根以及氫基團產生表異構化反應 (epimerization)，將 D-果糖 (D-fructose) 轉換成 D-阿洛酮糖 (D-piscose)。
Agrobacterium tumefaciens DPE (AtDPE) 和 Agrobacterium sp. ATCC 31750 之 DPE 胺基酸序列只有 6 個胺基酸殘基不同 ，但 DPE之比活性卻比 AtDPE 高出 10 倍，因此將以 Agrobacterium sp. ATCC 31750 為來源之 DPE 序列進行單點定位突變，結果發現這六個胺基酸殘基皆會降低酵素活性，其中，X、Y、Z 的活性分別為原生型的 39%、31% 以及 19%。因此，再利用定位突變改變 X、Y 以及Z 之胺基酸殘基，探討此 3 個胺基酸殘基對於酵素活性之影響，結果發現，A 的比活性為 112 U/mg，較原生型 DPE (76.4 U/mg) 高。
A 之最適作用溫度為 55-60℃，最適作用 pH 值為 pH 7.5-9，添加 Ni2+、Mg2+、Mn2+、Co2+ 皆可提升其酵素活性，其中又以添加 Co2+效果最好；若不添加金屬離子或是添加 Ca2+、Cu2+、Zn2+ 和 EDTA時，則不具有酵素活性。A 在 35℃ 以下保溫 2 小時以及在pH 7-9 的緩衝液中保存 24 小時並無明顯的活性喪失，表示在此環境下酵素具有良好的穩定性，55℃ 下的半衰期為3 分鐘，最適作用基質為 D-阿洛酮糖，且在 55℃ 下反應 30 分鐘即可到達 22% 的轉換率。
另外，不論是原生型或突變型 DPE，在經過鎳離子親合性管柱之純化過程中，皆會產生白色沉澱，因此將原生型 DPE 之 C 端加入一段由 22 個胺基酸殘基所組成之 B 序列，並將其命名為 B，發現 B 經鎳親合性管柱之純化的過程中並無明顯白色沉澱物，推測於序列中加入 B 序列能夠改善純化過程中蛋白質所產生之沉澱。並以SDS-PAGE 分析 B，分子量約為 34 kDa，純化後所得到的活性回收率為 54%，比活性為 69.2 (U/mg)，將經過鎳離子親和性管柱的酵素液計算產量，每公克的菌可得到 796 U/g 的活性，每公升培養基可以得到 9550 U/L 的酵素；而原生型 DPE 純化後所得到的活性回收率為 44%，比活性為 76.4 (U/mg)，將經過鎳離子親和性管柱的酵素液計算產量，每公克的菌可得到 449 U/g 的活性，每公升培養基可以得到 10467 U/L 的酵素。

D-Psicose 3-epimerase (DPE) catalyzes the C3 conversion between different ketohexoses, and is currently used to produce D-psicose from D-fructose.
There are 6 different amino acid residues between Agrobacterium tumefaciens DPE (AtDPE) and Agrobacterium sp. ATCC 31750 DPE , and the specific activity of Agrobacterium sp. ATCC 31750 DPE was 10 times higher than that of AtDPE. The mutant plasmids were obtained by PCR using mutation-designed primers. Then the mutant plasmids were transformed into E. coli BL21 (DE3) CodonPlus to express mutant DPEs. The specific activities of the six mutant DPEs were reduced. The specific activities of purified X, Y and Z were 39%, 31% and 19%, respectively, of wild-type. Therefore, site-directed mutagenesis was applied to change the side-chains on residues X,Y and Z to explore the effects of the residues on the enzyme activity .As a result, the specific activity of A is 112 U / mg, which is much higher than that of wild-type (76.4 U / mg).
The optimum temperature and pH of A were 55-60℃ and 7.5-9.0, respectively, and D-psicose was a better substrate. The presence of 1mM Ni2+, Mg2+, Mn2+ and Co2+ would enhanced the enzyme activity. In the absence of any metal ion and the presence of 1mM Ca2+, Cu2+, Zn2+, EDTA, the enzyme is inactive. In addition, A was quite stable at 35℃ and in the pH range of 7-9.The equilibrium ratio between D-psicose and D-fructose was 25:75 at 55℃.
Wild-type and mutant DPEs were mostly precipated during the purification by HisTrap HP affinity chromatography. In order to reduce the precipitation, DPE was fused with the B peptide at the C-terminus. Proteins were expressed in E. coli BL21(DE3) CodonPlus. The precipitation was then reduced during the purification by HisTrap HP affinity chromatography. The result showed that B fusion has reduced precipitation during purification. The molecular weight of the B is about 34 kDa. After purified by HisTrap HP affinity chromatography and dialysed overnight, B had a specific activity of 69.2 U/mg and recovery of 54%. After purification, the yield of active B expressed in E. coli was 9550 U/L and 796 U/g of wet cells. The wild-type DPE had a specific activity of 76.4 U/mg and recovery of 44%. After purification, the yield of active wild-type DPE expressed in E. coli was 10467 U/L and 449 U/g of wet cells.

摘要 I
Abstract II
目錄 III
表目錄 VI
圖目錄 VII
壹、研究背景與目的 1
貳、文獻整理 2
一、 稀有糖類簡介 (rare sugars) 2
二、 D-阿洛酮糖 (D-Psicose) 簡介 2
三、生產 D-阿洛酮糖相關酵素 3
1. DTE-家族酶 (DTEase-family) 簡介 3
2. DTE-家族酶之來源 3
3. DTE-家族酶之特性 3
4. DTE-家族酶之結構 4
5. DTE-家族酶之蛋白質工程 4
四、源自 Agrobacterium sp. ATCC 31750 之 DPE 介紹 5
五、ATS 序列 (acidic tails of synulein peptide) 介紹 5
参、實驗流程設計 6
一、經由基因突變後所產 D-阿洛酮糖表異構酶對於活性及特性之影響 6
二、ATS 序列對於 DPE 純化時沉澱情形的影響 6
肆、實驗材料方法 7
一、實驗材料 7
1. 菌株與載體 7
1.1 dpe 基因來源 7
1.2 質體來源 7
1.3 菌株來源 7
2. 抗生素 7
3. 標準品 7
3.1 DNA 標準品 7
3.2 蛋白質標準品 7
4. 市售套組 7
5. 酵素 7
6. 化學藥品 8
7. 實驗設備 9
二、實驗方法 10
1.基因操作 10
1.1 點突變 10
1.1.1 點突變引子設計 10
1.1.2 點突變之引子合成 10
1.1.3點突變 PCR 溶液組成及反應條件 10
1.1.4. dsDNA template 之剪切 11
1.2將 Agrobacterium sp. ATCC 31750 之 C 端加入一段 ATS 序列 11
2. 轉形作用及重組質體之篩選 11
2.1 製備 E. coli DH5α 電穿孔勝任細胞 11
2.2 製備 E. coli DH5α 熱休克勝任細胞 12
2.3 電穿孔轉形作用 12
2.4 熱休克轉形作用 12
2.5 Colony PCR 篩選轉形株 12
2.6 膠電泳分析 13
2.7 序列分析 13
2.8 菌種保存 13
3. 原生型及突變型 DPEs 之蛋白質表現與純化 13
3.1 製備 E. coli BL21(DE3)-CodonPlus-RIL 電穿孔勝任細胞 13
3.2 原生型及突變型 DPEs 轉形至大腸桿菌中之表現 14
3.3 原生型及突變型 DPEs 之純化 14
3.4 蛋白質定量 14
3.4.1 製備定量蛋白質之相關試劑 15
3.4.2 測定蛋白質濃度 15
3.5 蛋白質電泳分析 15
3.5.1 膠體的製備 15
3.5.2 試劑配法 16
3.6 膠電泳之操作方法 16
3.6.1 2X SDS sampling buffer 製備 16
3.6.2 膠片的染色與脫色 17
3.6.3 染色液的製備 17
3.6.4 脫色液的製備 17
4. 原生型與突變型 DPEs 之活性分析與特性探討 17
4.1 酵素活性分析 17
4.1.1 酵素反應 17
4.1.2 D-阿洛酮糖定量 17
4.1.3 活性定義 17
4.1.4 HPLC-ELSD 分析 17
4.2 酵素特性探討 18
4.2.1 最適作用溫度 18
4.2.2 最適作用 pH 值 18
4.2.3 熱穩定性及半衰期 18
4.2.4 pH 穩定性 18
4.2.5 金屬離子影響 18
4.2.6 基質特異性 19
4.2.7 轉換率 19
伍、結果與討論 20
一、定位突變對於所產 D-阿洛酮糖表異構酶之活性影響 20
二、Q53A DPE 特性之探討 21
1.離子之影響 21
2.最適作用溫度 22
3.最適作用 pH 值 22
4. 熱穩定性及半衰期 22
5. pH 值穩定性 23
6. 最適作用基質 23
7. 轉換率 23
三、ATS 序列對於 D-阿洛酮糖表異構酶之可溶性以及活性之探討 23
1. CATS DPE 定序確認 23
2. CATS DPE 之純化 23
陸、結論 25
柒、參考文獻 26

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