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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/17712

Title: 甘藷去氫抗壞血酸還原酶與樟芝榖氧還蛋白之基因選殖、表現及酵素特性分析
Cloning, Expression and Enzyme Properties of Ipomoea batatas (L.) Lam Dehydroascorbate Reductase and Antrodia camphorata Glutaredoxin
Authors: Yu-Chi Jiang
姜郁琦
Contributors: NTOU:Institute of Bioscience and Biotechnology
國立臺灣海洋大學:生物科技研究所
Keywords: 去氫抗壞血酸還原酶;穀氧還蛋白;維生素C;穀胱甘肽;樟芝;甘藷
Sweet potato (Ipomoea batatas [L.] Lam),;Antrodia camphorata;dehydroascorbate reductase;DHAR;glutaredoxin;Grx;ascorbate;glutathione
Date: 2008
Issue Date: 2011-07-04
Abstract: 摘要 本論文分為兩部份,分別針對甘藷去氫抗壞血酸還原酶與樟芝穀氧還蛋白進行基因選殖、表現、特性分析以及酵素動力學研究。 去氫抗壞血酸還原酶 (dehydroascorbate reductase, DHAR) 為普遍存在於動植物中之抗氧化酵素,可利用穀胱甘肽 (glutathione, GSH) 將去氫抗壞血酸還原。本實驗由台農 57 號甘藷 (Ipomoea batatas (L.) Lam) 塊根中選殖出 DHAR 之全長 cDNA,並轉殖至大腸桿菌使其大量表現此酵素。藉由電泳分析,具有活性之 DHAR 為單體 (monomeric form)。分析 DHAR 的特性:加熱至 50 ℃ 時其半衰期約為 10.1 分鐘;以 pH 6.0-11.0 處理 0.5 小時後仍有 78 % 以上之活性,以 0.8 M 咪唑 (imidazole) 處理後仍然具有88 % 的活性。以去氫抗壞血酸 (DHA) 與穀胱甘肽 (GSH) 之不同受質濃度為變因測出之米氏常數 (Km) 分別為 0.19 mM 與 2.38 mM。 穀氧還蛋白 (glutaredoxin, Grx) 為一耐熱性高之小分子蛋白質,其主要功能為還原過氧化物或 DHA,藉由與 GSH mixed disulfides 作用來還原被氧化的雙硫鍵並抵擋氧化壓力造成的傷害,或使某些酵素 (如 Prx) 的雙硫鍵還原以維持這些酵素活性。本實驗成功自樟芝子實體選殖 Grx 之 cDNA 並以基因重組技術轉殖至大腸桿菌使其大量表現此酵素。藉由 HED assay 可證明 Grx 除本身具有之穀氧還蛋白活性外,亦具有去氫抗壞血酸還原酶之活性。當受質為 HED 時,其米氏常數為1.29 mM;當受質為 DHA 時,其米氏常數為 2.03 mM。經由 VII 特性分析可知Grx 在 100 ℃ 時其半衰期為 8.5 分鐘,藉由電泳分析可推知 Grx 在加熱後會形成雙體結構並失去活性。Grx 如同大多數抗氧化酵素般,在鹼性環境下具有較高的活性。將 Grx 以 pH 6.0-10.0 的緩衝溶液處理半小時,其活性仍有 95 % 以上;以 0.8 M imidazole 處理 Grx 時仍有 67 % 以上的活性;以 SDS 作用後無活性;加入 protease 作用後迅速失活。
The study of this thesis falls into two parts. One is the studying of sweet potato dehydroascorbate reductase (DHAR) and the other is the studying of Antrodia camphorata glutaredoxin (Grx). A cDNA encoding a putative DHAR was cloned from sweet potato. The deduced protein showed high level of sequence homology when aligned with DHARs from other organisms. Functional sweet potato DHAR was overexpressed and purified. The purified enzyme showed an active monomeric form on a 12 % native PAGE. The protein’s half-life of deactivation at 50 ℃ was 10.1 min, and its thermal inactivation rate constant Kd was 6.4 x 10-2 min-1. The enzyme was stable in a broad pH range of 6.0-11.0 in the presence of 0.8 M imidazole. The Km values for DHA and GSH were 0.19 mM and 2.38 mM, respectively. A cDNA encoding a putative Grx was cloned from Antrodia camphorata. The deduced protein showed high level of sequence homology when aligned with Grxs from other organisms. The recombined Grx was transformed to E.coli and overexpressed. After purified, It showed an active monomeric form on a 10 % native PAGE. The purified enzyme demonstrated that it has both Grx and DHAR activity by HED assay. This protein’s half-life of deactivation at 100 ℃ was 8.5 min. The enzyme was stable in a broad pH range of 6.0-11.0 in the presence of 0.8 M imidazole. When treated with protease, the enzyme was cleaved immediately.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M95360048
http://ntour.ntou.edu.tw/ir/handle/987654321/17712
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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