臺灣博碩士論文加值系統

D-阿洛酮糖 (D-psicose) 為 D-果糖 C3 鏡像異構物，在自然界中稀少且價格昂貴之單醣，其具有特殊生理活性，可應用於人工代糖或是食品添加物，在 2012 年已被美國食品及藥物管理局 (Food and Drug Administration, FDA) 認可為 GRAS (generally regarded as safe) 之食品甜味劑。D-阿洛酮糖表異構酶 (D-psicose 3-epimerase, DPE) 能針對 D-阿洛酮糖和 D-果糖之間的轉換，將六碳酮醣 3 號碳位置進行表異構化反應。本實驗室先前已選殖出中溫菌 Agrobacterium sp. ATCC 31750 之 dpe 基因，並表現於大腸桿菌而可獲得 AsDPE。由於發現突變型 Q53A AsDPE會因酵素表現量較差，造成總酵素活性回收降低，因此以緘默突變 (silent mutation) 方式探討其基因密碼子是否會影響酵素活性回收量，將 Alanine 原密碼子 GCG 改為 GCA、GCC、GCT 之每克菌體 AsDPE 活性分別為 949 U/g、836 U/g、751 U/g 及 822 U/g，而每公升培養液之總活性分別為 18,800 U/L、19,500 U/L、16,800 U/L 及 14,200 U/L，其比活性並無改變，顯示單位菌體之總活性會因基因密碼子不同而改變，進而造成酵素活性回收量也不同。
本實驗室先前已將 AsDPE 之羧基加入一 acidic tail of synuclein peptide 序列 (C’ATS AsDPE)，發現能減少酵素純化後所產生之白色沉澱，因此也探討其熱穩定性及特性。而由於 A. tumefaciens DPE (AtDPE) 已被發現雙突變 I33L/S213C 能增加熱穩定性，因此將雙突變位置應用於 AsDPE 並探討是否改善熱穩定性。結果發現添加 0.1 mM Co2+ 即能維持原生型、C’ATS 及 I33L/S213C AsDPE 在 50°C 及55°C之熱穩定性，且 21C’ATS 及 I33L/S213C AsDPE 在 60°C、65°C 的熱穩性皆優於原生型。C’ATS 及 I33L/S213C AsDPE 之最適作用溫度及 pH 值皆為 60°C、pH 7.5-8.5，而原生型為 55°C、pH 7.5-8.5。將 21C’ATS AsDPE進行 I33L/S213C 突變而得 LCATS AsDPE，其最適作用溫度及 pH 值為 65°C、pH 7.5-9.0，於 65°C 半衰期為 73.7 分鐘，較原生型提高 19.2 倍，因此 LCATS AsDPE 有潛力應用於工業上生產 D-阿洛酮糖。

D-Psicose, the C3 epimer of D-fructose, is a rare monosaccharide in the nature. This rare sugar has special physiological functions and can be used as a food additive or sweetener. 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 dpe gene was previously cloned from the genomic DNA of Agrobacterium sp. ATCC 31750 and expressed in Escherichia coli. The poor expression of mutant Q53A Agrobacterium sp. ATCC 31750 DPE (AsDPE) caused the decrease of the total activity recovery. To increase the expression of AsDPE, the original alanine codon of GCG was replaced by the synonymous codon, GCA, GCC and GCT, respectively. The total activities for per gram of wet cells of Q53A-G, Q53A-A, Q53A-C and Q53A-T were 949 U/g, 836 U/g, 751 U/g and 822 U/g, respectively. The total activities for per liter of culture of Q53A-G, Q53A-A, Q53A-C and Q53A-T were 18,800 U/L、19,500 U/L、16,800 U/L 及 14,200 U/L, respectively, while the specific activities were not affected. Hence, the synonymous codons caused the changes on their total activities and the following total activity recoveries. In addition, AsDPE has been previously fused with the ATS peptide at the C-terminus (C’ATS AsDPE) and its precipitation during purification has been reduced. Besides, the mutant I33L/S213C A. tumefaciens DPE (AtDPE) showed significant increases in thermal stability, compared with wild-type. Therefore, this study has investigated the thermal stabilities and characteristics of C’ATS and I33L/S213C AsDPEs. In the presence of 0.1 mM Co2+, C’ATS and I33L/S213C AsDPEs can retain their stability at 50-55°C, and the thermal stability of C’ATS and I33L/S213C AsDPEs were better than that of wild-type AsDPE. The optimum temperature and pH for C’ATS and I33L/S213C AsDPEs were 60°C and pH 7.5-8.5, respectively, while those for wild-type AsDPE were 55°C and pH 7.5-8.5, respectively. LCATS AsDPE was constructed by carrying out I33L/S213C mutation on C’ATS AsDPE. The optimum temperature and pH for LCATS AsDPE were 65°C and pH 7.5-9.0. The half-life of LCATS AsDPE (73.7 min) at 65°C was 19.2-fold higher than that of wild-type. These results suggest that LCATS AsDPE has the potential to be used industrially for D-psicose production.

目錄 I
表目錄 III
圖目錄 IV
壹、研究背景與目的 1
貳、文獻整理 3
一、稀有醣類 (rare sugars) 3
1. 簡介 3
2. 來源及生產 3
3. D-阿洛酮糖 (D-psicose, D-ribo-2-hexulose, or D-allulose) 3
4. D-阿洛糖 (D-allose) 4
二、DTE-家族酶之相關研究 5
1. 簡介 5
2. 來源與特性 5
3. 源自 A. tumefaciens 之 D-阿洛酮糖表異構酶 (AtDPE) 6
4. 源自 Agrobacterium sp. ATCC 31750 D-阿洛酮糖表異構酶 7
三、ATS 序列 (acidic tail of synuclein peptide) 7
參、實驗設計與流程 9
一、實驗設計 9
1. AsDPE 之 Q53A 經緘默突變對於活性回收之影響 9
2. AsDPE 經定位突變與 ATS 融合對熱穩定性及特性之影響 9
二、實驗流程 9
1. AsDPE 之 Q53A 經緘默突變對於活性回收之影響 9
2. AsDPE 經定位突變及 ATS 融合對於熱穩定性及特性之影響 10
肆、實驗材料方法 11
一、實驗材料 11
1. 菌株與載體 11
2. 抗生素 11
3. 標準品 12
4. 市售套組 12
5. 酵素 12
6. 化學藥品 12
7. 實驗設備 14
二、藥品配製 15
1. Terrific broth (TB) 培養液 15
2. DNA 瓊脂膠體製備 16
3. 製備定量蛋白質之相關試劑 16
4. SDS-PAGE 相關藥劑製備 16
三、實驗方法 18
1. 定位突變 18
2. 轉形作用及重組質體之篩選 21
3. 重組型 DPE 之蛋白質表現與純化 23
4. 重組型 DPE 之活性分析與特性探討 26
伍、結果與討論 30
一、突變型 AsDPE 之定位突變結果 30
二、重組型 AsDPE 之表現與純化 30
三、重組型 AsDPE 之活性影響 31
1. 酵素反應後之產物分析 31
2. 突變型 Q53A 活性分析 31
3. 原生型、融合型及突變型活性分析 32
四、重組型 AsDPE 之特性探討 32
1. 最適作用溫度 32
2. 最適作用 pH 33
3. 熱穩定性及半衰期 34
4. 酵素動力學參數 36
陸、結論 37
柒、參考文獻 38
捌、附錄 66

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