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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/35025

Title: E. coli 植酸酶之選殖與在 Pichia pastoris 表現
Cloning and expression of E. coli phytase in Pichia pastoris
Authors: Wei-Chuan Chen
陳威全
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 植酸酶;大腸桿菌;嗜甲醇酵母菌
Phytase;E. coli;Pichia pastoris
Date: 2012
Issue Date: 2013-10-07T02:49:37Z
Abstract: 植酸酶大量的利用在飼料工業中,以水解位置為 6-phytase 和最適 pH 值在酸性的為最佳,因此找尋更符合工業需求之植酸酶。本實驗所研究的植酸酶是由 Escherichia coli genomic DNA 中,利用基因選殖方法得到完整的植酸酶基因片段,再依據 Pichia pastoris 密碼子使用偏好設計合成重組植酸酶基因列。首先將合成之 phytase 基因接到 pGAPZαC 表現載體上,在將此載體轉形到 Pichia pastoris 細胞內,經 72 小時培養,可於培養液中大量的表現出具功能性之重組植酸酶,最終產量可達 112.5 U/mL,培養第三天之菌液,以10,000 rpm 離心 30 分鐘後,利用過濾膜為 10 kD 之 Amicon 超過濾濃縮器將粗酵素液於 4oC 下進行超過濾濃縮,重組植酸酶可經由 nickel 親合性層析而純化。此階段酵素液之比活性為 42.6 kU/mg,純化倍率為 34.7 倍,回收率 69.0%。SDS-PAGE 分析其去醣基化後分子量大小為 46kDa,重組植酸酶之最適 pH 及溫度分別為 4.0-6.0 及 50oC,pH 與溫度安定性範圍為 pH 3.0-8.0 與 20-40oC。其活性會被 Cu2+、Zn2+、Hg2+、Fe2+、Fe3+、phenylmethylsulfonylfluoride (PMSF) 及N-tosyl-L-lysine chloromethyl ketone (TLCK) 等抑制;但受 Mg2+、Ca2+、Sr2+、Ba2+、glutathione (GSH)、ethylene diamine tetraacetic acid (EDTA) 及 N-ethylmaleimide (NEM) 等活化。此重組植酸酶對於植酸鈣有較高的親和力,且對於 trypsin 有良好的抵抗能力。凍乾之植酸酶分別儲藏於室溫、4oC 及 -20oC 八週,結果發現室溫下植酸酶活性可維持六週。
Large amount of phytase have been consumed in feed industry, especially for 6-p phytase with activity optimal at acidic pH. This study aimed to clone E. coli phytase and express in Pichia pastoris. A mature phytase cDNA of E. coli being altered according to the codons usage preference of Pichia pastoris was artificially synthesized and cloned into expression vector of pGAPZαC. The final extracellular phytase activity was 112.5 U/ml after 72 h high cell-density fermentation. The recombinant phytase was purified to electrophoretical homogeneity after Ultrafiltration and nickel affinity chromatography. At this stage, the yield, purification fold, and specific activity were 69.0%, 34.7 folds and 42.6 kU/mg, respectively. The recombinant phytase were deglycosylated to produce a homogeneous 46kDa protein. The optimal temperature and pH were at 50oC and 4.0-6.0, respectively. However, it was resistant to trypsin and stable at pH 3.0-8.0 and 20-40oC. Cu2+, Zn2+, Hg2+, Fe2+, Fe3+, phenylmethyl sulfonylfluoride (PMSF) and N-tosyl-L-lysine chloromethyl ketone (TLCK) strongly inhibited, while Mg2+, Ca2+, Sr2+, Ba2+, glutathione (GSH), ethylene diamine tetraacetic acid (EDTA) and N-ethylmaleimide (NEM) activated its activity. The purified recombinant phytase revealed higher affinity to calcium phytate than to other phosphate conjugates. Freeze-dried crud phytase was very stable during 6 week storage at room temperature, 4oC and -20oC.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0019932069
http://ntour.ntou.edu.tw/handle/987654321/35025
Appears in Collections:[食品科學系] 博碩士論文

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