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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/35168

Title: Agrobacterium sp. ATCC 31750 菌株經基因重組後所產阿洛酮糖表異構酶對於活性及特性之影響
Effects of genetic recombination on the activity and properties of D-psicose 3-epimerase from Agrobacterium sp. ATCC 31750
Authors: Ming-Jun Wang
王銘駿
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: D-阿洛酮糖表異構酶;D-阿洛酮糖;定位突變;比活性;D-果糖;沉澱
D-Psicose 3-epimerase;D-psicose;site directed mutagenesis);specific activity;D-fructose;precipitation
Date: 2013
Issue Date: 2013-10-07T02:51:13Z
Abstract: D-阿洛酮糖表異構酶 (D-psicose 3-epimerase, DPE) 能以非專一性的方式將酮糖上 3 號碳位置的氫氧根以及氫基團產生表異構化反應 (epimerization),將 D-果糖 (D-fructose) 轉換成 D-阿洛酮糖 (D-piscose)。 Agrobacterium tumefaciens DPE (AtDPE) 和 Agrobacterium sp. ATCC 31750 之 DPE 胺基酸序列只有 6 個胺基酸殘基不同 ,但 DPE之比活性卻比 AtDPE 高出 10 倍,因此將以 Agrobacterium sp. ATCC 31750 為來源之 DPE 序列進行單點定位突變,結果發現這六個胺基酸殘基皆會降低酵素活性,其中,X、Y、Z 的活性分別為原生型的 39%、31% 以及 19%。因此,再利用定位突變改變 X、Y 以及Z 之胺基酸殘基,探討此 3 個胺基酸殘基對於酵素活性之影響,結果發現,A 的比活性為 112 U/mg,較原生型 DPE (76.4 U/mg) 高。 A 之最適作用溫度為 55-60℃,最適作用 pH 值為 pH 7.5-9,添加 Ni2+、Mg2+、Mn2+、Co2+ 皆可提升其酵素活性,其中又以添加 Co2+效果最好;若不添加金屬離子或是添加 Ca2+、Cu2+、Zn2+ 和 EDTA時,則不具有酵素活性。A 在 35℃ 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,55℃ 下的半衰期為3 分鐘,最適作用基質為 D-阿洛酮糖,且在 55℃ 下反應 30 分鐘即可到達 22% 的轉換率。 另外,不論是原生型或突變型 DPE,在經過鎳離子親合性管柱之純化過程中,皆會產生白色沉澱,因此將原生型 DPE 之 C 端加入一段由 22 個胺基酸殘基所組成之 B 序列,並將其命名為 B,發現 B 經鎳親合性管柱之純化的過程中並無明顯白色沉澱物,推測於序列中加入 B 序列能夠改善純化過程中蛋白質所產生之沉澱。並以SDS-PAGE 分析 B,分子量約為 34 kDa,純化後所得到的活性回收率為 54%,比活性為 69.2 (U/mg),將經過鎳離子親和性管柱的酵素液計算產量,每公克的菌可得到 796 U/g 的活性,每公升培養基可以得到 9550 U/L 的酵素;而原生型 DPE 純化後所得到的活性回收率為 44%,比活性為 76.4 (U/mg),將經過鎳離子親和性管柱的酵素液計算產量,每公克的菌可得到 449 U/g 的活性,每公升培養基可以得到 10467 U/L 的酵素。
D-Psicose 3-epimerase (DPE) catalyzes the C3 conversion between different ketohexoses, and is currently used to produce D-psicose from D-fructose. There are 6 different amino acid residues between Agrobacterium tumefaciens DPE (AtDPE) and Agrobacterium sp. ATCC 31750 DPE , and the specific activity of Agrobacterium sp. ATCC 31750 DPE was 10 times higher than that of AtDPE. The mutant plasmids were obtained by PCR using mutation-designed primers. Then the mutant plasmids were transformed into E. coli BL21 (DE3) CodonPlus to express mutant DPEs. The specific activities of the six mutant DPEs were reduced. The specific activities of purified X, Y and Z were 39%, 31% and 19%, respectively, of wild-type. Therefore, site-directed mutagenesis was applied to change the side-chains on residues X,Y and Z to explore the effects of the residues on the enzyme activity .As a result, the specific activity of A is 112 U / mg, which is much higher than that of wild-type (76.4 U / mg). The optimum temperature and pH of A were 55-60℃ and 7.5-9.0, respectively, and D-psicose was a better substrate. The presence of 1mM Ni2+, Mg2+, Mn2+ and Co2+ would enhanced the enzyme activity. In the absence of any metal ion and the presence of 1mM Ca2+, Cu2+, Zn2+, EDTA, the enzyme is inactive. In addition, A was quite stable at 35℃ and in the pH range of 7-9.The equilibrium ratio between D-psicose and D-fructose was 25:75 at 55℃. Wild-type and mutant DPEs were mostly precipated during the purification by HisTrap HP affinity chromatography. In order to reduce the precipitation, DPE was fused with the B peptide at the C-terminus. Proteins were expressed in E. coli BL21(DE3) CodonPlus. The precipitation was then reduced during the purification by HisTrap HP affinity chromatography. The result showed that B fusion has reduced precipitation during purification. The molecular weight of the B is about 34 kDa. After purified by HisTrap HP affinity chromatography and dialysed overnight, B had a specific activity of 69.2 U/mg and recovery of 54%. After purification, the yield of active B expressed in E. coli was 9550 U/L and 796 U/g of wet cells. The wild-type DPE had a specific activity of 76.4 U/mg and recovery of 44%. After purification, the yield of active wild-type DPE expressed in E. coli was 10467 U/L and 449 U/g of wet cells.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0010032012
http://ntour.ntou.edu.tw/handle/987654321/35168
Appears in Collections:[食品科學系] 博碩士論文

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