D-阿洛酮糖 (D-psicose, D-ribo-2-hexulose, or D-allulose) 為 D-果糖三號碳上的鏡像異構物，屬稀有醣類少量存在於自然界中，有研究顯示其具有高甜度但低熱量、提高食品凝膠性、維持血糖水平及降體脂等有利之生理功能，在食品及醫療上具有發展潛力。目前 D-阿洛酮糖多以 D-阿洛酮糖表異構酶進行酵素轉換法生產，然而，D-阿洛酮糖表異構酶對溫度相當敏感，不利於工業上大量生產。本研究利用蛋白質工程提昇 Agrobacterium sp. ATCC 31750 來源之 D-阿洛酮糖表異構酶 (AsDPE) 的熱穩定性及特性，並參考前人之突變位點進行組合突變，得單突變 F155Y/C’ATS (F155Y-ATS) 及三突變 I33L/F155Y/S213C/C’ATS (F155Y-LCATS)，與原生型 AsDPE (wild-type, WT) 相比，突變型酵素之最適作用溫度皆提升至 65°C，最適作用 pH 則與 WT 一致皆為 pH 8.0， F155Y-LCATS於 60°C 及 65°C 保溫 30 分鐘殘存活性分別為 73.0% 及 42.9%，半衰期則為 182 及 15.0 分鐘，與 WT 的 7.0 及 2.2 分鐘相比熱穩定性提升許多。酵素動力學方面，突變型之催化效率皆比 WT 好，分別為 WT 的2.4 及 1.8 倍。將不同分子伴護蛋白與 C’ATS 共表現，發現伴護蛋白質體 Gro7 能使 C’ATS 的可溶蛋白表現量由 50% 增加至 60%，使得酵素表現量增加，再將伴護蛋白質體 Gro7 與可表現 F155Y-LCATS 的質體共轉形，發現可以增加酵素整體表現量，也能使 F155Y-LCATS 的可溶蛋白表現量由 54.8% 增加至 56.4%，利用海藻酸鈣乳化法固定化 WT，得最適作用海藻酸鈉濃度為 1%，最適作用菌體濃度為 80 g/L，其固定化效率為 70%。以鎳離子親和性管柱固定化突變型酵素 F155Y-LCATS，並循環流入 D-果糖以生產 D-阿洛酮糖，其在反應10小時達到平衡，轉換率為 23%，管柱經過循環使用五次後，其轉換率剩 8%，表示部份酵素失活。

D-Psicose (D-ribo-2-hexulose, or D-allulose) is a C3-epimer of D-fructose, and is a rare sugar found in nature in small quantities. It has high sweetness but low calorie content, improved food gelation, maintenance of blood glucose level and body fat reduction. Therefore, it has potential for development in food and medicine. At present, D-psicose is mostly produced by the enzyme conversion method using D-psicose 3-epimerase (DPE). However, DPE is quite sensitive to temperature and is not conducive to industrial mass production.
In this study, protein engineering was used to improve the thermostability and characteristics of D-psicose 3-epimerase (AsDPE) from Agrobacterium sp. ATCC 31750. The mutation sites were chosen from previous studies, mutations F155Y/C'ATS (F155Y-ATS) and I33L/F155Y/S213C/C'ATS (F155Y-LCATS) were studied. The optimum temperature and pH of both mutants are 65°C and pH8.0, respectively. The thermostability of F155Y-LCATS is much higher than that of wild-type (WT). The residual activity of F155Y-LCATS was 73.0% and 42.9% at 60°C and 65°C , respectively , for 30 minutes, and the half-lives were 182 and 15.0 minutes, respectively. In terms of enzyme kinetics, the catalytic efficiencies of F155Y-ATS and F155Y-LCATS are better than WT, which are 2.4 and 1.8 times higher than WT, respectively. Co-expression of different molecular chaperones with C’ATS showed that chaperone expressed from pGro7 increased the amount of soluble protein from 50% to 60%, and the expression of C’ATS was also increased. AsDPE-containing cells were immobilized by calcium alginate by emulsification method. The optimal concentration of sodium alginate was 1%, the optimal cell mass was 80 g/L, and the immobilization efficiency was 70%. The mutant enzyme F155Y-LCATS was immobilized on a nickel-ion affinity column and D-psicose was produced by cyclic loading of D-fructose. The reaction reached equilibrium in 10 hours, the conversion rate was 23%. After five times reaction, the conversion rate is reduced to 8% due to partial enzymatic inactivation.

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. 生產方式 ...........................................................................................3
3. D-阿洛酮糖 ........................................................................................ 3
二、D-阿洛酮糖表異構酶 (D-psicose 3-epimerase, DPE).........................4
1.簡介 .....................................................................................................4
2.特性及來源 .......................................................................................4
3. 源自 A. tumefaciens ATCC33970 D-阿洛酮糖表異構酶 ...................5
4. 源自Agrobacteriumsp.ATCC31750D-阿洛酮糖表異構酶.............5
三、定位突變.................................................................................................... 6
四、重組蛋白在細菌中之蛋白質表現............................................................ 7
1. 蛋白質之凝集 .....................................................................................7
2. ATS 序列 (acidic tail of synuclein peptide) .......................................7
3. 分子伴護蛋白 .....................................................................................7
五、ClearColi BL21 (DE3)...............................................................................8
六、固定化........................................................................................................ 8
1. 簡介 ..................................................................................................... 8
2. 固定化菌體介紹 ................................................................................. 9
3. 利用鎳離子親和性管柱固定化酵素 ............................................... 10
參、 實驗設計與流程............................................................................... 11
一、F155Y、F185K 突變對 21-C’ATS 及 LCATS 之影響.....................11
二、含 ATS 序列之 AsDPE 與 chaperone 共表現於 ClearColi BL21(DE......... 11
三、利用乳化液相法固定化菌體.................................................................. 12
四、利用鎳離子親和性管柱固定化酵素並生產 D-阿洛酮糖 ....................12
肆、 實驗材料與方法........................................................................... 14
一、實驗材料............................................................................... 14
1. 菌株與質體 ....................................................................................... 14
2.抗生素 ............................................................................................... 14
3.標準品 ............................................................................................... 14
4.市售套組 ........................................................................................... 15
5.酵素 ................................................................................................... 15
6.化學藥品 .........................................................................................15
7.實驗設備 .........................................................................................17
二、藥品配置................................................................................ 18
1. Luria-Bertani (LB) 培養液.............................................................18
2. Terrific broth (TB) 培養液.............................................................18
3. Super Optimal Broth (SOB) 培養液 ..............................................18
4. salt-optimized carbon broth (SOC) 培養液 ...................................18
5. Ampicillin (100 mg/mL) .................................................................18
6. Chloramphenicol (34 mg/mL).........................................................18
7. DNA 瓊脂膠體製備 ....................................................................... 18
8. 定量蛋白質之相關試劑 ................................................................. 18
9. SDS-PAGE 相關藥劑製備 ............................................................ 19
三、實驗方法.................................................................................... 22
1. 定位突變 ......................................................................................... 22
2. 轉形作用及重組質體之篩選 ......................................................... 24
3. AsDPE之蛋白質表現與純化 ....................................................... 25
4. AsDPE之活性分析與特性探討 ................................................... 27
5. AsDPE與分子伴護蛋白共表現之可溶性影響 ........................... 29
6. 探討固定化菌體條件 ..................................................................... 32
7. 探討固定化菌體之特性 ................................................................. 33
8. D-阿洛酮糖的生產 .......................................................................... 34
伍、結果與討論...................................................................................35
一、AsDPE 之定位突變及結果....................................................................36
二、突變型 AsDPE 之初步篩選..................................................................36
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
五、原生型與突變型 AsDPE 之特性探討..................................................36
1. 最適作用溫度 ................................................................................. 36
2. 最適作用 pH 值 ............................................................................ 36
3. 熱穩定性及半衰期 ......................................................................... 37
4. 酵素動力學參數 ............................................................................. 37
六、AsDPE 之可溶性蛋白討論....................................................................37
1. 不同分子伴護蛋白質體對 AsDPE 可溶性蛋白之影響 .............38
2. 分子伴護蛋白質體之誘導濃度對 AsDPE 可溶性蛋白之影響 ..38
七、固定化菌體之固定化條件及特性探討.................................................. 38
1. 固定化菌體之最適海藻酸鈉濃度 ................................................. 38
2. 固定化菌體之最適菌體濃度 ......................................................... 38
3. 不同溫度對固定化菌體之影響 ..................................................... 39
4. 不同 pH 值對固定化菌體之影響 ................................................ 39
5. 固定化菌體之熱穩定性 ................................................................. 40
八、以固定化酵素生產 D-阿洛酮糖 ............................................................40
1. 固定化酵素生產 D-阿洛酮糖之轉換率 ........................................ 40
2. 固定化酵素之重複使用率 .............................................................40
陸、結論 ..................................................................................................41
柒、參考文獻 ...................................................................................42
捌、圖表 ...........................................................................................46
表一、原生型及突變型 D-阿洛酮糖表異構酶於 E. coli ClearColi 表現之酵素純化表 ...........................................................................................................46
表二、原生型及突變型 D-阿洛酮糖表異構酶於 60 oC 及 65oC 之半衰期 .......47
表三、原生型及突變型對 D-果糖之動力學參數 .............................................48
圖一、洋菜糖膠體電泳分析 MEGAPRIMED-QCM 二階段定位突變產物 ........49
圖二、AsDPE 不同突變點之部分核苷酸序列定序圖譜........................................50 圖三、60OC 保溫對於原生型與突變型 AsDPE 之粗酵素活性影響....................51
圖四、以 SDS-PAGE 分析分析 C’ATS、F155Y-ATS、F155Y-LCATS ASDPES 及
wild-type 純化之結果........................................................................................52 圖五、溫度對原生型及突變型 AsDPE 活性之影響..............................................53 圖六、pH 值對原生型及突變型 AsDPE 活性之影響............................................54 圖七、原生型及突變型 AsDPE 於 60oC 之熱穩定性............................................55 圖八、原生型及突變型 AsDPE 於 65oC 之熱穩定性............................................56 圖九、以 SDS-PAGE 分析 C’ATS AsDPE 與不同伴護蛋白共表現於 E. coli
ClearColi 之結果................................................................................................57 圖十、C’ATSAsDPE 與不同伴護蛋白共表現對粗酵素活性之影響....................58
圖十一、以 SDS-PAGE 分析突變型 AsDPE 與由 pGro7 所表現的分子伴護蛋白
共表現於 E. coli ClearColi 之影響 ...................................................................59
圖十二、以 SDS-PAGE 分析 L-arabinose 誘導濃度對於 C’ATS AsDPE 與 pGro7 所表現之分子伴護蛋白共表現於 E. coli ClearColi 之影響 ...........................60
圖十三、以 SDS-PAGE 分析 L-arabinose 誘導濃度對於 F155Y-LCATS AsDPE 與 pGro7 所表現之分子伴護蛋白共表現於 E. coli ClearColi 之影響................61
圖十四、菌體濃度對含原生型 AsDPE 固定化菌體之固定化效率及活性影響..62
圖十五、溫度對含原生型 AsDPE 固定化菌體之活性影響..................................63
圖十六、pH 值對含原生型 AsDPE 固定化菌體之活性影響................................64
圖十七、含原生型 AsDPE 固定化菌體於 60oC 之熱穩定性.................................65 圖十八、以固定於鎳離子管柱之 F155Y-LCATS 酵素生產 D-阿洛酮糖............66
圖十九、固定化酵素管柱之重複使用性.......................................................... 67
玖、附錄 ............................................................................................68

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