Biochemical and Biophysical Characterization of Ascorbate Peroxidase (APX) from Eggplant (Solanum melongena L.)

• Main Authors: Shih-Hung Yang, 楊世弘
• Other Authors: Tzann-Shun Hwang, Ph.D.
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/28021944751785264158

碩士 === 中國文化大學 === 生物科技研究所 === 101 === 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℃.