Lemon Ascorbate Peroxidase: cDNA Cloning and Biochemical Characterization

檸檬抗壞血酸過氧化酶選殖及生化特性研究

戴亞涵(Ya-Han Dai) ； 黃智郁(Chich-Yu Huang) ； 溫麗莎(Lisa Wen) ； 許垤棊(Dey-Chyi Sheu) ； 林棋財(Chi-Tsai Lin)

《Botanical Studies》 53卷1期 (2012/01) Pp. 9-17

摘要

抗壞血酸過氧化酶（Ascorbate peroxidase, Apx）扮演氧化抗壞血酸（AsA）以清除過氧化氧（H2O2）。從檸檬（Citruslimon）cDNA 庫選殖出Apx cDNA 序列（1,068bp,GQ465430），全長共1,068個核苷酸，可轉譯出250個胺基酸。經序列比較ClApx 與其他物種的序列有很高的相似性，依據已知結構，建立一模擬立體結構（3-Dstructuralmodel）。在演化樹上屬於cs2（cytosolsoluble）。進一步將其轉譯區選殖入表現載體pYEX-S1，以酵母菌Saccharomycescerevisiae作為表現宿主，經親和性管柱純化可得到具有活性的ClApx，對AsA 和H2O2 其KM 值分別為0.40和0.11m,。其特性在45°C加熱活性降低一半的時間為6.5分鐘，在pH6.0~8.0 仍然具有相當的活性。

關鍵字

檸檬 ； 抗壞血酸過氧化酶 ； 模擬立體結構 ； 酵母菌

並列摘要

Ascorbate peroxidase (Apx) plays important roles both as a reductant and as a H2O2 scavenger via ascorbate (AsA). In this paper, we discuss how a ClApx cDNA (1,068 bp, GQ465430) encoding a putative Apx was cloned from lemon (Citrus limon). The deduced amino acid sequence is similar to the Apxes from other plant species. A 3-D structural model of ClApx was constructed based on the crystal structure of Pisum sativum Apx (PDB code 1APX). To characterize the ClApx protein, the coding region was subcloned into an expression vector pYEX-S1 and transformed into Saccharomyces cerevisiae. The recombinant His6-tagged ClApx was overexpressed and purified by Ni(superscript 2+) -nitrilotriacetic acid Sepharose. The purified enzyme showed two prominent bands on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Michaelis constant (K(subscript M)) values of the recombinant enzyme for AsA and H2O2 were 0.40 and 0.11 mm, respectively. The enzyme was active from pH range 6 to 8. The thermal inactivation of the enzyme showed a half-life of 6.5 min at 45°C, and its inactivation rate constant K(subscript i) was 1.1 × 10(superscript -1) min(superscript -1). The enzyme retained 35% activity after chymotrypsin digestion at pH 8 and 37°C for 40 min.

並列關鍵字

Ascorbate peroxidase ； Citrus limon ； Saccharomy cescerevisiae ； Three-dimension structural model