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研究生:方弘懿
研究生(外文):Fang, Hong-Yi
論文名稱:以蛋白質工程及固定化提昇Agrobacterium sp. ATCC 31750 來源重組 D-阿洛酮糖表異構酶之熱穩定性
論文名稱(外文):Protein engineering and immobilization of recombinant D-psicose 3-epimerase from Agrobacterium sp. ATCC 31750 to increase its thermostability
指導教授:方翠筠
指導教授(外文):Fang, Tsuei-Yun
口試委員:曾文祺林泓廷方翠筠
口試委員(外文):Tseng, Wen-ChiLin, Hong-TingFang, Tsuei-Yun
口試日期:2016-07-13
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:84
中文關鍵詞:D-阿洛酮糖表異構酶定位突變酵素熱穩定性固定化
外文關鍵詞:D-psicose 3-epimerasesite directed mutagenesisenzymatic thermostabilityimmobilization
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D-阿洛酮糖是 D-果糖三號碳上之表異構物 (epimer),為自然界中含量稀少的稀有醣類 (rare sugars) 之一,且具多種特性可應用於醫藥及食品產業。其生產可利用 DTE (D-tagatose 3-epimerase) 家族酶 (DTEase family enzymes) 轉換 D-果糖成 D-阿洛酮糖,DTE 家族酶之中又以 DPE (D-psicose 3-epimerase) 對於 D-果糖的活性較佳,較適合應用於生產,然而目前已研究之 DPE 都對溫度相當敏感,增加熱穩定性以提升工業利用性,成為當務之急,基於有學者比較高溫與中溫菌蛋白質之胺基酸組成以及探討蛋白質中弱交互作用 (weak interactions) 對於蛋白質熱穩定的影響後,發現 glycine 及 proline 可能有較大機率提供蛋白質熱穩定性以外,現今已有許多線上軟體可以預測蛋白質經單點突變後的熱力學穩定性 (thermodynamic stability),因此本研究以 Agrobacterium sp. ATCC 31750 來源 DPE (AsDPE),經 SWISS-MODEL 模擬結構後以線上軟體 PoPMuSic 及 ENCoM 評估單點突變,另外也以前人建構之雙突變及雙突變尾端融合 ATS 序列 (acidic tail of synuclein peptide) AsDPE 為模板突變 I66L,發現 I66L 影響了結構的彈性 (flexibility),可提升熱穩定性,其中 I33L/I66L/S213C AsDPE 將 65°C 半衰期延長至原生型之 20 倍。
本研究也選用常用於維持蛋白質穩定性且為生物可利用性的化學伴護子 (chemical chaperone)-arginine、sorbitol 及 trehalose 探討 AsDPE 之可溶性,發現三種物質皆沒提升可溶蛋白比例,而 ATS 序列能提升 20% 可溶蛋白比例。
由於固定化也是提升工業利用性的好方法之一,酵素依附於載體上使蛋白質更加穩定,且便於重複使用,另外也有直接固定細胞的方法,通常酵素在細胞內會更穩定,且省去純化酵素之步驟,因此本研究以海藻酸鈣的包埋法固定化有較佳特性的突變型 AsDPE,且以界面活性劑 hexadecyltrimethyl ammonium bromide (CTAB) 處理過之 ClearColi BL21 (DE3) 菌體,發現固定化菌體之熱穩定性較純化酵素佳,且能連續反應 10 次而沒有喪失活性,將其填入填充床反應器於 60°C 催化 15 mL 50% 果糖進行小量製備,發現反應 1.5 小時可達平衡,且轉換率為 31%。

D-Psicose, the C3-epimer of fructose, is one of the rare sugar, since it exists in trace amount in nature. It has several physiological functions can apply to food or pharmaceutical industry. D-Psicose can be produced by DTEase family enzymes (D-tagatose 3-epimerase family enzymes) which can convert D-fructose into D-psicose. Among the DTEase family enzymes, DPE (D-psicose 3-epimerase) which has better affinity to D-fructose, is more suitable for producing D-psicose. However, currently found DPEs have poor thermostability that discourages the process of D-psicose production. Therefore, it is necessary to improve thermostability of DPE for increasing its applicability to industry.
Gycine and proline have higher possibility to increase thermostability based on the studies on the weak interaction in proteins and comparing the amino acid compositions between mesophilic and thermophilic proteins. Furthermore, computational method also used to screen the thermostable mutants by predicting thermodynamic stability via amino acid sequence and protein structure. In this study, Agrobacterium sp. ATCC 31750 DPE (AsDPE) structure was modeled by SWISS-MODEL, and then online softwares ENCoM and PoPMuSiC were used to evaluate the possible mutations in order to increase its thermostability. Besides, I66L mutation which has improved the catalytic efficiency of A. tumefaciens ATCC 33970 DPE (AtDPE) was also combined to I33L/S213C and I33L/S213C/C’ATS AsDPEs. The results show the half life of I33L/I66L/S213C is about twenty times longer than that of wild-type at 65°C.
Chemical chaperones, the compounds keep proteins from aggregation are also used to study the solubility of AsDPE. The bioavailable chemical chaperones like arginine, sorbitol, and trehalose were added respectively during induction, but neither of them can enhance AsDPE solubility.
I33L/S213C/C’ATS AsDPE (LCATS) was then expressed in the non-endotoxin-producing strain ClearColi BL21 (DE3). These cells treated with hexadecyltrimethyl ammonium bromide were immobilized by calcium alginate in order to raise industrial applicability such as thermostability or reusability. As a result, the LCATS-containing immobilized cells can reach reaction equilibrium in 1.5 h at about 30% convertion rate. Moreover, the LCATS-containing immobilized cells can be used ten cycles in D-psicose production without losing enzyme activity at 60°C.

目錄
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. 應用 3
3. 生產 3
4. D-阿洛酮糖 3
二、 DTE 家族酶 4
1. 簡介 4
2. 來源及特性 4
3. D-阿洛酮糖表異構酶 (D-psicose 3-epimerase, DPE) 5
三、 電腦軟體輔助之蛋白質工程 7
1. 同源結構模擬 7
2. 線上軟體評估單點突變 7
四、 重組蛋白在細菌中之蛋白質表現 8
1. 蛋白質之凝集 8
2. 化學伴護子 8
3. ATS 序列 (acidic tail of synuclein peptide) 10
五、 重組蛋白表現宿主 10
1. ClearColi BL21 (DE3) 10
2. E. coli BL21-CodonPlus (DE3)-RIL 11
六、 固定化 11
1. 簡介 11
2. 海藻酸 11
3. 界面活性劑 12
參、 實驗設計與流程 13
一、 AsDPE 突變對酵素特性之影響 13
1. I66L 突變對 LCDPE 及 LCATS 之影響 13
2. 電腦軟體評估單點突變之穩定性 14
二、 AsDPE 於 ClearColi 宿主之表現 14
三、 固定化菌體並生產D-阿洛酮糖 14
肆、 實驗材料與方法 16
一、 實驗材料 16
1. 菌株與載體 16
2. 抗生素 16
3. 標準品 16
4. 市售套組 16
5. 酵素 17
6. 化學藥品 17
7. 實驗設備 19
8. 軟體 20
二、 藥品配置 20
1. Luria-Bertani (LB) 培養液 20
2. Terrific broth (TB) 培養液 20
3. Super Optimal Broth (SOB) 培養液 21
4. Salt-optimized carbon broth (SOC) 培養液 21
5. Ampicillin (100 mg/mL) 21
6. Chloramphenicol (34 mg/mL) 21
7. DNA 瓊脂膠體製備 21
8. 定量蛋白質之相關試劑 22
9. SDS-PAGE 相關藥劑製備 22
三、 實驗方法 23
1. 預測提升穩定性突變 23
2. 定位突變 24
3. 轉形作用及重組質體之篩選 26
4. 重組型 DPE 之蛋白質表現與純化 27
5. 重組型 DPE 之活性分析與特性探討 30
6. 固定化/未固定化菌體條件與特性探討 33
7. D-阿洛酮糖生產 33
伍、 結果與討論 35
一、 AsDPE 之結構模擬 35
二、 AsDPE 之定位突變 35
三、 原生型與突變型 AsDPE 之表現與純化 36
四、 原生型與突變型 AsDPE 之活性分析 36
1. 酵素反應後之產物分析 36
2. 酵素活性分析 36
五、 原生型與突變型 AsDPE 的特性探討 37
1. 最適作用溫度 37
2. 最適作用 pH 值 37
3. 熱穩定性及半衰期 37
4. 酵素動力學參數 38
六、 AsDPE 之可溶性 38
1. LCDPE 與 LCATS 於 CleanColi 之表現 38
2. 誘導溫度對於 LCDPE 之可溶性 38
3. 化學伴護子對於 LCDPE 之可溶性 39
4. ATS 序列對於 LLCDPE 之可溶性 39
七、 固定化菌體探討 39
1. 固定化菌體之菌體濃度探討 39
2. 固定化菌體之熱穩定性 40
3. 固定化菌體生產 D-阿洛酮糖 40
陸、 結論 41
柒、 參考文獻 42
捌、 圖表 50
玖、 附錄 76


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吳易皇,2014,Agrobacterium sp. ATCC 31750 D-阿洛酮糖表異構酶之蛋白質工程及利用固定化菌體生產 D-阿洛酮糖,國立臺灣海洋大學食品科學系碩士論文,基隆。
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