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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/27109

Title: Lemon ascorbate peroxidase: cDNA cloning and biochemical characterization
Authors: Ya-Han Dai;Chich-Yu Huang;Lisa Wen;Dey-Chyi Sheu;Chi-Tsai Lin
Contributors: NTOU:Institute of Bioscience and Biotechnology
國立臺灣海洋大學:生物科技研究所
Keywords: Ascorbate peroxidase;Citrus limon;Saccharomy cescerevisiae;Three-dimension structural model
Date: 2012-01-01
Issue Date: 2011-10-21T02:22:45Z
Publisher: Botanical Studies
Abstract: Abstract: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.
Relation: 53(1), pp.11-19
URI: http://ntour.ntou.edu.tw/handle/987654321/27109
Appears in Collections:[生命科學暨生物科技學系] 期刊論文

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