茄子抗壞血酸過氧化酶之生化與生物物理特性的探討

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Title:	茄子抗壞血酸過氧化酶之生化與生物物理特性的探討 Biochemical and Biophysical Characterization of Ascorbate Peroxidase (APX) from Eggplant (Solanum melongena L.)
Authors:	楊世弘
Contributors:	生物科技研究所
Keywords:	抗壞血酸過氧化酶 Ascorbate peroxidase(APX) 茄子 eggplant 基因選殖 cloning
Date:	2013
Issue Date:	2013-03-21 11:26:46 (UTC+8)
Abstract:	抗壞血酸過氧化酶 (Ascorbate peroxidase, APX；EC 1.11.1.11)是一植物體內代謝過氧化氫且含有血基質 (heme) 之重要酵素，而其發現於高等植物、藻類、及藍綠藻中，並藉由抗壞血酸 (Ascorbic acid, AsA) 的氧化反應清除過氧化氫的危害。前人研究證實耐淹水之茄子栽培品種（Solanum melongena L.,var.Ping-tong Long Eggplant），其APX於淹水環境中扮演著調控因子的重要角色，且可有效的抵禦過氧化氫對於植物體的傷害。本研究利用聚合酶鏈鎖反應（polymerase chain reaction, PCR）於前後兩端引子上加入限制酶切位，大量複製後，將耐淹水之茄子的抗壞血酸基因構築於載體 pGEM® T easy-vector 上，接著進行次選殖至表現載體pQE30上，並轉殖至大腸桿菌DH5α中大量表現。在大腸桿菌表現的APX與載體pQE30上的序列融合形成具有六個histidine殘基的標誌 (Tag)，可利用鎳親和性管柱進行快速的單一步驟快速純化，接著以膠體過濾層析進一步純化即可獲得高純度的抗壞血酸過氧化酶，用以進行APX的酵素活性及特性分析。SDS-PAGE及膠體過濾分析可觀察到變性 (denature)及原態的(native)酵素分子量分別為29 kDa及41kDa，而結果顯示抗壞血酸過氧化酶是以二聚體(dimer)的形式存在。APX的氧化作用將過氧化氫(H2O2)轉換成水(H2O)和氧(O2)，而AsA於波長290 nm具有吸光特性，因此藉由偵測波長290 nm的吸收值的減少可以換算出AsA的消耗量，並計算出酵素的活性。於不同pH值下分析APX的活性，顯示酵素的最適催化活性位於pH 6.0；於不同的溫度下分析酵素活性，顯示其最適催化溫度約為35℃。 Ascorbate peroxidase (APX, EC 1.11.1.11) is an important heme-containing enzyme that has been found in higher plants, algae, and some cyanobacteria, which scavenges hydrogen peroxide by preferentially oxidizing ascorbate. APX of flood-tolerant eggplant cultivar (Solanum melongena L., var. Ping-tong Long Eggplant) has been demonstrated to play a key role under flood and regulated the antioxidant availability to resist H2O2.In this study, eggplant APX was cloned into pGEM® T easy-vector, and then subcloned into pQE30 for over-expressing APX. The recombinant eggplant APX was contructed by fusing its N-terminus with a 6× Histidine tag; therefore, the recombinant APX could be quickly purified by Ni-chelatin chromatography in one step, followed bypurifing by gelfiltration to obtain a pure APX with high homogeneity for activity analysis and enzyme characterization. Analyses of denature and native APX by SDS-PAGE and gel filtration showed a molecular mass of 29 kDa and 41 kDa, respectively. It suggested the eggplant APX had a dimeric structure in nature. APX catalyzes the oxidation reaction of H2O2coupled to reduction reaction of Ascorbic acid (AsA). AsA possesses absorbance at 290 nm, so measurement of the decrease at 290 nm could be used to calculate the enzyme activity. Analysis of enzyme activity in different pHs and in different temperatures showed that the eggplant APX had a pH optima at pH 6.0, and the best catalyzing temperature at 35℃.
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