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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/35052

Title: Agrobacterium sp. ATCC 31750菌株所產阿洛酮糖表異構酶之基因選殖、表現、純化及特性探討
Cloning, Expression, Purification, Characterization of D-psicose 3-epimerase from Agrobacterium sp. ATCC 31750
Authors: 陳昭南
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 阿洛酮糖表異構酶;阿洛酮糖;果糖
Agrobacterium sp. ATCC 31750;D-psicose 3-epimerase
Date: 2012
Issue Date: 2013-10-07T02:49:55Z
Abstract: D-阿洛酮糖 (D-psicose, D-ribo-2-hexulose or D-allulose) 為一種具有特殊生理活性且昂貴的稀有醣類。目前已知用來生產 D-阿洛酮糖的酵素皆為 DTE 家族酶 (EC 5.1.3),其催化機制為將六碳酮醣 3 號碳上並進行酮醣-酮醣間轉換。D-阿洛酮糖表異構酶 (D-psicose 3-epimerase, DPE) 屬於 DTE-家族酶的其中一員,即為一種能催化 D-果糖 (D-fructose) 與 D-阿洛酮糖 (D-psicose) 之間轉換的酵素,此外,亦能將 D-塔格糖 (D-tagatose) 轉換成 D-山梨糖 (D-sorbose)。 本實驗選用中溫菌 Agrobacterium sp. ATCC 31750 作為來源,將其基因 dpe 藉由選殖的方式建構於載體中形成重組型載體 (pET-21b-dpe-6H),並將終止密碼子刪除,使其可表現出 C 端帶有六個組胺酸 (His-tag) 的蛋白以利後續純化,將此重組型載體 pET-21b-dpe-6H 轉形至宿主 E. coli BL21(DE3)-CodonPlus-RIL 表現,經由 20 小時培養並添加 0.05 mM IPTG 下進行誘導,有助於大量表現具有活性之目標蛋白。重組型 DPE 的單體分子量約為 32 kDa,且經過鎳離子親和性管柱進行純化,並以緩衝液透析可得到純化之酵素,其比活性為 116 U/mg,活性回收率為 194%,純化倍率則為 20.9。重組型 DPE 之最適作用溫度為 60-65℃,最適作用 pH 值為 pH 7-9 之間,在不添加金屬離子、Ca2+、Cu2+、Zn2+ 和 EDTA時,DPE 不具有酵素活性,添加 Ni2+、Mg2+、Mn2+、Co2+、Fe2+ 皆可提升其酵素活性,重組型 DPE 在 30℃ 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,半衰期為 55℃,10 分鐘,最適作用基質為 D-阿洛酮糖,且在 55℃ 下反應 40 分鐘即可到達最大轉換率 30%。
D-Psicose also known as D-ribo-2-hexulose or D-allulose is one of rare sugars that exhibit unique bioactivity but only exists in trace amount in nature. The of DTEase family enzymes (EC 5.1.3) catalyze the C3 conversion between different ketohexoses, and are currently used to produce D-Psicose from D-Fructose. D-Psicose 3-epimerase (DPE), a DTEase family enzyme that catalyzes the epimerization of D-fructose and D-psicose to form two epimers, but also reacted on D-tagatose and D-sorbose. In this study, the mesophilic bacteria Agrobacterium sp. ATCC 31750 was used as the source strain. The gene dpe was successfully cloned into vector pET-21b, and the stop codon of the dpe gene was deleted, then expressed recombinant DPE containing the His-tag at the C-terminal for further purification. The recombinant pET-21b-dpe-6H was transformed into E. coli BL21(DE3)-CodonPlus-RIL, and the target protein was overexpressed after 20 hour incubation in the presence of 0.05 mM IPTG. The molecular weight of the recombinant DPE is about 32 kDa. After purified by nickel-chelating chromatography and dialysed overnight, recombinant DPE had a specific activity of 116 U/mg and purified 20.9 fold with a yield of 194%. The optimum temperature and pH of recombinant DPE were 60-65℃ and pH 7.0-9.0. In the absence of any metal ion and the presence of 1 mM Ca2+, Cu2+, Zn2+, EDTA, the enzyme is inactive. The presence of 1 mM Ni2+, Mg2+, Mn2+, Co2+ and Fe2+ would enhanced the enzyme activity. In addition, recombinant DPE were quite stable at 30℃ and in the pH range of 7-9.The equilibrium ratio between D-Psicose and D-fructose was 30:70 at 55℃.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0019932015
http://ntour.ntou.edu.tw/handle/987654321/35052
Appears in Collections:[食品科學系] 博碩士論文

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