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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48932

Title: 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
Protein Engineering of Recombinant D-Psicose 3-Epimerase from Agrobacterium sp. ATCC 31750 and the Production of D-Psicose by Immobilized Cells
Authors: Wu, Yi-Huang
吳易皇
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;D-阿洛酮糖表異構酶;定位突變
D-psicose;D-psicose 3-epimerase;Site-directed mutation
Date: 2015
Issue Date: 2018-08-22T06:39:27Z
Abstract: D-阿洛酮糖 (D-psicose) 為自然界中極少存在之稀有單醣,價格昂貴且具特殊生理活性,如應用於食品添加物取代蔗糖作為甜味劑及抑制肝臟脂肪合成酶以降低脂肪含量等功用,且美國食品藥物管理局 (Food and Drug Administration) 於 2012 年將其認可為一般安全性食品 (generally regarded as safe, GRAS),因此具發展之潛力。D-阿洛酮糖表異構酶 (D-psicose 3-epimerase, DPE) 可針對 D-果糖與 D-阿洛酮糖 3 號碳進行表異構化反應而相互轉換。本實驗室先前已選殖找出 Agrobacterium sp. ATCC 31750 D-psicose 3-epimerase (AsDPE) dpe 基因並將其轉形至 E. coli BL21-CodonPlus (DE3)-RIL 表現。由於 AsDPE 與 Agrobacterium tumefaciens (AtDPE) 基因序列有 98% 相似度,本研究透過 AtDPE 的結構模擬突變,期能提升酵素活性,結果發現突變型 Y6R 沒有活性、F185K 比活性降為 68 U/mg、F185Y 比活性降為 23 U/mg,M181R 及 F185R 皆變為不可溶蛋白而沉澱。 後續實驗改用不會產生內毒素反應之 CleanColi BL21 (DE3) 為表現宿主,透過海藻酸鈉固定化菌體,以利酵素的重複使用與提升酵素熱穩定性。結果發現以 CleanColi 為表現宿主會使酵素活性由 80 U/mg 降為 48 U/mg,含原生型 AsDPE 之固定化菌體無法提升熱穩定性。將固定化 CleanColi BL21 (DE3) AsDPE 以填充床反應器生產 D-阿洛酮糖,填以含有 50% D-果糖、50 mM Tris-HCl 緩衝液 (pH 8.0) 及 0.1 mM Co2+ 反應液,於 55oC 下反應,約2 小時候達平衡,轉換率約 30%。
D-Psicose is a rare monosaccharide in the nature which has special physiological functions being used as a food additive or sweetener but expensive. 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 Agrobacterium sp. ATCC 31750 D-psicose 3-epimerase (AsDPE) dpe gene had been previously cloned and expressed in Escherichia coli BL21 CodonPlus (DE3)-RIL. According to the 98% homology between AsDPE and Agrobacterium tumefaciens (AtDPE), molecular modeling of the mutations of AtDPE was performed to choose the candidates for site-directed mutagenesis. The results have shown that mutant F185K and F185Y AsDPEs has specific activity of 68 U/mg and 23 U/mg, respectively. AsDPE was then expressed in the nonendotoxin producing strain CleanColi BL21 (DE3) and the AsDPE containing immobilized cells were prepared for the reuse of the enzyme and improve enzyme’s thermostability. However, immobilized cells didn’t improve AsDPE’s thermostability as expected. Continuous production of D-psicose from D-fructose using AsDPE containing immobilized cells has been performed in packed-bed bioreactor. Substrate of 50% D-fructose in 50 mM Tris-HCl buffer (pH 8.0) plus 0.1 mM Co2+ was passed through the bioreactor at 55oC, and the convertion rate was about 30%. Under the reaction conditions, the epimerization reaction between D-fructose and D-psicose reached equilibrium after 2 h.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010232031.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48932
Appears in Collections:[食品科學系] 博碩士論文

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