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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。

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?aC, 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 ?aC for 40 min.

謝誌 i
Abstract ii
中文摘要 iii
目錄 iv
縮寫表 vii
第一章 前言 1
1.1 檸檬 1
1.2 自由基 (free radical) 4
1.3 植物的抗氧化機制 9
1.4 抗壞血酸過氧化酶 13
1.5 研究動機 13
第二章 實驗材料 14
2.1 材料 14
2.2 培養基 15
2.3 實驗耗材 16
2.4 儀器設備 17
2.5 實驗藥品 18
2.6 引子 20
第三章 實驗方法 21
3.1 檸檬mRNA之萃取 21
3.2 檸檬cDNA之合成 22
3.3 檸檬抗壞血酸過氧化酶之基因選殖 23
3.4 檸檬抗壞血酸過氧化酶之蛋白質表現與純化 27
3.5 檸檬抗壞血酸過氧化酶之活性測定 33
3.6 檸檬抗壞血酸過氧化酶之特性分析 34
第四章 結果與討論 36
4.1 檸檬抗壞血酸過氧化酶之基因選殖 36
4.2 檸檬抗壞血酸過氧化酶之蛋白質表現與純化 38
4.3 檸檬抗壞血酸過氧化酶之酵素動力學 39
4.4 檸檬抗壞血酸過氧化酶之特性分析 41
4.5 檸檬抗壞血酸過氧化酶之親緣演化數分析 43
參考文獻 55
附錄一 58
附錄二 59
附錄三 60

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