臺灣博碩士論文加值系統

D-阿洛酮糖 (D-psicose) 為自然界中極少存在之稀有單醣，價格昂貴且具特殊生理活性，如應用於食品添加物取代蔗糖作為甜味劑及抑制肝臟脂肪合成酶以降低脂肪含量等功用，且美國食品藥物管理局 (Food and Drug Administration) 於 2012 年將其認可為一般安全性食品 (generally regarded as safe, GRAS)，因此具發展之潛力。D-阿洛酮糖表異構酶 (D-psicose 3-epimerase, DPE) 可針對 D-果糖與 D-阿洛酮糖 3 號碳進行表異構化反應而相互轉換。本實驗室先前已選殖找出 Agrobacterium sp. ATCC 31750 D-psicose 3-epimerase (AsDPE) dpe 基因並將其轉形至 E. coli BL21-CodonPlus (DE3)-RIL 表現。由於 AsDPE 與 Agrobacterium tumefaciens (AtDPE) 基因序列有 98% 相似度，本研究透過 AtDPE 的結構模擬突變，期能提升酵素活性，結果發現突變型 Y6R 沒有活性、F185K 比活性降為 68 U/mg、F185Y 比活性降為 23 U/mg，M181R 及 F185R 皆變為不可溶蛋白而沉澱。
後續實驗改用不會產生內毒素反應之 CleanColi BL21 (DE3) 為表現宿主，透過海藻酸鈉固定化菌體，以利酵素的重複使用與提升酵素熱穩定性。結果發現以 CleanColi 為表現宿主會使酵素活性由 80 U/mg 降為 48 U/mg，含原生型 AsDPE 之固定化菌體無法提升熱穩定性。將固定化 CleanColi BL21 (DE3) AsDPE 以填充床反應器生產 D-阿洛酮糖，填以含有 50% D-果糖、50 mM Tris-HCl 緩衝液 (pH 8.0) 及 0.1 mM Co2+ 反應液，於 55oC 下反應，約2 小時候達平衡，轉換率約 30%。

D-Psicose is a rare monosaccharide in the nature which has special physiological functions being used as a food additive or sweetener but expensive. It has been announced as generally recognized as safe (GRAS) by Food and Drug Administration (FDA) in 2012. D-Psicose 3-epimerase (DPE) can catalyze the epimerization of D-fructose and D-psicose. The Agrobacterium sp. ATCC 31750 D-psicose 3-epimerase (AsDPE) dpe gene had been previously cloned and expressed in Escherichia coli BL21 CodonPlus (DE3)-RIL. According to the 98% homology between AsDPE and Agrobacterium tumefaciens (AtDPE), molecular modeling of the mutations of AtDPE was performed to choose the candidates for site-directed mutagenesis. The results have shown that mutant F185K and F185Y AsDPEs has specific activity of 68 U/mg and 23 U/mg, respectively.
AsDPE was then expressed in the nonendotoxin producing strain CleanColi BL21 (DE3) and the AsDPE containing immobilized cells were prepared for the reuse of the enzyme and improve enzyme’s thermostability. However, immobilized cells didn’t improve AsDPE’s thermostability as expected. Continuous production of D-psicose from D-fructose using AsDPE containing immobilized cells has been performed in packed-bed bioreactor. Substrate of 50% D-fructose in 50 mM Tris-HCl buffer (pH 8.0) plus 0.1 mM Co2+ was passed through the bioreactor at 55oC, and the convertion rate was about 30%. Under the reaction conditions, the epimerization reaction between D-fructose and D-psicose reached equilibrium after 2 h.

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、 稀有醣類 3
1. 簡介 3
2. 稀有醣類應用 3
3. 製備及生產 3
4. D-阿洛酮糖 (D-Psicose) 3
5. D-阿洛糖 (D-Allose) 5
二、 DTE-家族酶之研究 5
1. 簡介 5
2. 來源及特性 6
3. D-阿洛酮糖表異構酶 (D-psicose 3-epimerase, DPE) 6
三、 氫鍵對酵素之影響 8
1. 酵素與基質間之氫鍵 8
四、 CleanColi BL21 (DE3) 9
五、 固定化菌體 9
1. 固定化種類 9
2. 固定化材料 9
3. 界面活性劑 11
4. 固定化菌體優缺點 12
參、 實驗設計與流程 13
1. AsDPE 定位突變 Y6R、M181R、F185K、F185R 及 F185Y 對酵素活性及特性之影響 13
2. AsDPE 於 CleanColi 宿主之表現 13
3. 利用固定化菌體生產 D-psicose 13
肆、 實驗材料與方法 15
一、 實驗材料 15
1. 菌株與載體 15
2. 抗生素 15
3. 標準品 15
4. 蛋白質標準品 16
5. 市售套組 16
6. 酵素 16
7. 化學藥品 16
8. 實驗設備 18
二、 藥品配製 20
1. Luria-Bertani (LB) 培養液 20
2. Terrific broth (TB) 培養液 20
3. SOB 培養液 21
4. SOC 培養液 21
5. Ampicillin (100 mg/mL) 22
6. Chloramphenicol (34 mg/mL) 22
7. Bacillus transformation buffer 22
8. Bacillus recovery medium 22
9. DNA 瓊脂膠體製備 23
10. 製備定量蛋白質之相關試劑 23
11. SDS-PAGE 相關藥劑製備 24
三、 實驗方法 26
1. 定位突變 26
2. 轉形作用及重組質體之篩選 29
3. 重組型 DPE 之蛋白質表現與純化 32
4. 重組型 DPE 之活性分析與特性探討 36
5. 固定化菌體之活性分析與特性探討 40
伍、 結果與討論 44
陸、 結論 54
柒、 參考文獻 56
捌、 附錄 93


表目錄
表一、定位突變 PCR 引子序列…………………………………………………..65
表二、比較原生型及突變型 D-阿洛酮糖表異構酶於 E. coli 或 CleanColi 表現之酵素純化表………………………………………………………………………..66
表三、比較原生型及突變型 D-阿洛酮糖表異構酶於 E. coli 或 CleanColi 表現之酵素活性回收……………………………………………………………………..67
表四、原生型與突變型對 D-果糖之動力學參數………………………………….68
表五、含 AsDPE 固定化 CleanColi 及未固定化之 D-阿洛酮糖表異構酶活性及固定化效率………………………………………………………………………………………….69
 
圖目錄
圖一、模擬 Agrobacterium tumefaciens D-阿洛酮糖表異構酶距離基質 4 Å、5 Å 及 6 Å 之殘基………………………………………………………………………70
圖二、分子模擬基質與 AtDPE 突變殘基 (Y6R、M181R) 之結合……………71
圖三、分子模擬基質與 AtDPE 突變殘基 (F185Y、F185K) 之結合…………..72
圖四、分子模擬基質與 AtDPE 突變殘基 (F185Y、F185K) 之結合…………..73
圖五、DNE 膠電泳分析 Megaprimed-QCM 三階段定位突變產物…………….74
圖六、pET-21b-dpe 不同點突變部分核甘酸序列分析之圖譜…………………...75
圖七、以 SDS-PAGE 分析突變型 AsDPE 於不同濃度 IPTG 誘導在 E. coli BL21-CodonPlus (DE3)-RIL 表現之結果……………………………………….....76
圖八、以 SDS-PAGE 分析原生型及突變型 AsDPE 於 E. coli BL21-CodonPlus (DE3)-RIL 之各純化過程蛋白質分布與表現……………………………………..77
圖九、以液相層析串連低溫蒸發光散射檢測器分析 D-果糖經酵素反應後之產物組成…………………………………………………………………………………..78
圖十、溫度對原生型及突變型 F185K AsDPE 活性之影響……………………..79
圖十一、pH 值對原生型及突變型 F185K AsDPE 活性之影響………………...80
圖十二、原生型 AsDPE 對溫度之穩定性………………………………………..81
圖十三、計算原生型及突變型 F185K AsDPE 之活化能………………………..82
圖十四、以 SDS-PAGE 分析原生型 AsDPE 於不同濃度 IPTG 誘導在 CleanColi BL21 (DE3) 表現之結果………………………………………………...83
圖十五、以 SDS-PAGE 分析原生型 AsDPE 於 CleanColi BL21 (DE3) 之純化過程蛋白質分布與表現……………………………………………………………..84
圖十六、海藻酸鈉濃度對含有 AsDPE 固定化菌體活性之影響………………..85
圖十七、不同界面活性劑在不同濃度下對含 AsDPE 固定化菌體活性之影響..86
圖十八、不同細胞濃度對含 AsDPE 固定化菌體活性之影響…………………..87
圖十九、不同氯化鈣濃度對含 AsDPE 固定化菌體活性之影響………………..88
圖二十、溫度對含原生型 AsDPE 之固定化菌體及純化之原生型 AsDPE 活性比較…………………………………………………………………………………..89
圖二十一、pH 值對含原生型 AsDPE 之固定化菌體及純化之原生型 AsDPE 活性比較……………………………………………………………………………..90
圖二十二、含 AsDPE 之固定化菌體之熱穩定性………………………………..91
圖二十三、填充床反應器填充含 AsDPE 固定化菌體生產 D-阿洛酮糖之情形.92

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