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題名: Biochemical Characterization of a Catalase from Antrodia Camphorata: Expression in Escherichia coli and Enzyme Properties
樟芝過氣化氫酶在大腸稈菌中的表現及其生化特性
作者: Ken, Chuian-Fu;Chen, Hsueh-Tai;Chang, Reny-Chang;Lin, Chi-Tsai
貢獻者: 生物學系
關鍵詞: Antrodia camphorata;Catalase;Expression
樟芝;過氧化氫酶;基因表現
日期: 2008-04
上傳時間: 2013-06-05T08:48:19Z
出版者: 中央研究院植物研究所
摘要: Catalase plays important roles in antioxidation and cell signaling. One cDNA (1794bp, DQ021914) encoding the putative catalase was cloned from Antrodia camphorata. The deduced amino acid sequence is conserved among the reported catalases. To characterize the A. camphorata catalase, the coding region was subcloned into a vector pET-20b(+) and transformed into E. coli. The recombinant 6His-tagged catalase was expressed and purified by Ni(superscript 2+)-nitrilotriacetic acid sepharose. The purified enzyme showed one band by 10% SDS-PAGE. The enzyme retained 50% activity at 60℃ for 14 min. The enzyme was active under a broad pH range from 7.8 to 11.2. The enzyme showed 67% activity after 4h of incubation at 37℃ with trypsin. It was also proven able to protect intact supercoiled plasmid DNA from •OH-induced nicking. Study of the enzyme's properties may prove beneficial for future applications in medicine or health food.
過氧化氫酶(catalase)在抗氧化與綑胞信息傳遞中扮演重要的角色。依據樟芝(Antrodia camphorata)表現庫序列資訊(expressed sequence tag database)選殖出過氧化氫酶cDNA序列(DQ0Z1914)，全長共1794個核苷酸，內含轉譯區1527個核苔酸，可轉譯出509個胺基酸。經序列比較樟芝過氧化氫嗨與其他物種的序列有很高的相似性。將其轉譯區選殖入表現載體pET-20b(+)，以大腸桿菌E. coli BL21(DE3)pLysS作為表現宿主，經親和性管柱純化可得到具有活性的過氧化氫酶。其特性在60℃加熱活性降低一半的時間為14分鐘，在鹼性環境下(H7.8~11.2)仍然具有相當的活性，以trypsin處理4小時後仍然有67%的活性。此酶具有保護DNA防止自由基(•○H)攻擊。此酶特性之研究有利於未來開發為醫學及健康食品之應用。
關聯: Botanical Studies, 49(2): 119-125
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