Monodehydroascorbate Reductase cDNA from Sweet Potato: Expression and Kinetic Studies

單去氫抗壞血酸還原酶(monodehydroascorbate reductase, MDAR)在抗氧化中還原單去氫抗壞血酸(MDA)成抗壞血酸(AsA)。依據甘藷(Ipomoea batatas [L.] Lam)表現庫序列資訊(expressed sequence tag database)選殖出MDAR cDNA序列(1,548bp, EMBL accessionno. EF118034)，全長共1,548個核苷酸，內含轉譯區1302個核苷酸，可轉譯出434個胺基酸的還原酶。經序列比較IbMDAR與其他物種的序列有很高的相似性，依據已知結構，建立一相似性立體結構(3-Dhomology structure)。進一步將其轉譯區選殖入表現載體pET-20b(+)，以大腸桿菌E. coli BL21(DE3)pLysS作爲表現宿主，經親和性管柱純化可得到具有活性的IbMDAR，其特性在70℃加熱活性降低一半的時間爲12.4分鐘，在pH6.0~10.0仍然具有相當的活性。對MDA和NADH其K(下標 m)值分別爲21.1和39.7μM。

關鍵字

甘藷；相似性立體結構；基因表現；單去氫抗壞血酸還原酶

並列摘要

A cDNA encoding a putative monodehydroascorbate reductase (MDAR) was cloned from sweet potato. The deduced protein showed high level of sequence homology with MDARs from other plants or related family from bacteria (23~80%). A 3-D homology structure was created for this MDAR. Functional sweet potato MDAR was expressed and purified. The purified enzyme showed an active monomeric form on a 10% native PAGE. The protein's half-life of deactivation at 70℃ was 12.4 min, and its thermal inactivation rate constant K(subscript d) was 5.6×10^(-2) min^(-1). The enzyme was stable in a broad pH range from 6.0-10.0, and in the presence of 0.8 M imidazole. The K(subscript m) value for monodehydroascorbate (MDA) and NADH were 21.1 and 39.7 μM, respectively.

並列關鍵字

Sweet potato Ipomoea batatas L. Lam ； 3-D homology structure ； Expression ； Monodehydroascorbate MDAR

國際替代計量

Monodehydroascorbate Reductase cDNA from Sweet Potato: Expression and Kinetic Studies

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