檸檬抗壞血酸過氧化酶：表現及酵素特性分析

Citrus limon Ascorbate Peroxidase: Expression and Characterization

戴亞涵(Ya-han Tai)

指導教授 ： 許垤棋

大同大學/工程學院/生物工程學系所/碩士(2011年)

摘要

抗壞血酸過氧化酶 (Ascorbate peroxidase , Apx)扮演氧化抗壞血酸(AsA)以清除過氧化氫(H2O2)。從檸檬(Citrus limon)cDNA庫選殖出Apx cDNA 序列(1068 bp, GQ465430)，全長共1068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-D structural model)。進一步將其轉譯區選殖入表現載體 pYEX-S1，以酵母菌Saccharomyces cerevisiae 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 0.11 mM。其特性在45℃加熱活性降低一半的時間為 6.5 分鐘，在pH 6.0 ~ 8.0仍然具有相當的活性。

關鍵字

檸檬抗壞血酸過氧化酶

並列摘要

Ascorbate peroxidase (Apx) plays important roles in scavenging H2O2 via ascorbate (AsA) as a reductant. Here, a ClApx cDNA (1068 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 has been constructed based on the crystallographic 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 Ni2+-nitrilotriacetic acid Sepharose. The purified enzyme showed two prominent bands on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Michaelis constant (KM) values of the recombinant enzyme for AsA and H2O2 were 0.40 and 0.11 mM, respectively. The enzyme was active under a broad pH range from 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 kd was 1.06 x 10-1 min-1. The enzyme retained 35% activity after chymotrypsin digestion at pH 8 and 37 C for 40 min.

並列關鍵字

Citrus limon Ascorbate Peroxidase

參考文獻

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