淡江大學機構典藏:Item 987654321/32597
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    題名: Bacillus subtilis TKU007納豆激酶基因之選殖、異源表現與活性
    其他題名: Cloning, heterologous expression, and activity of nattokinase from bacillus subtilis TKU007
    作者: 陳柏帆;Chen, Po-fan
    貢獻者: 淡江大學生命科學研究所碩士班
    陳銘凱;Chern, Ming-kai
    關鍵詞: 納豆激酶;異源表現;酵母菌;大腸桿菌;Bacillus subtilis TKU007;nattokinase;heterologous expression;yeast;E. coli
    日期: 2008
    上傳時間: 2010-01-11 02:25:13 (UTC+8)
    摘要: 本研究目的是針對Bacillus subtilis TKU007納豆激酶(nattokinase),利用比對、選殖的方式去找出全長基因且研究其中與穩定性相關的基因序列。將TKU007納豆激酶基因轉殖入其他異源表現宿主中偵測其表現活性。首先利用聚合酶連鎖反應進行基因的放大,並轉型入pGEM-T easy vecetor做TA-cloning,將其質體DNA定序與B. subtilis 比對確認序列的正確性。發現TKU007納豆激酶基因有幾個胺基酸與一般B. subtilis的納豆激酶基因不同,顯示出這TKU007與B. subtilis之間這幾個胺基酸之差異,可能因此造成物化特性或是其他酵素活性表現的差異。也試著將TKU007所分泌的納豆激酶表現在異源的酵母菌細胞中,藉此試驗是否在較複雜的真核生物體中也同樣能有其蛋白質活性的表現。將序列重組於pYES2 表現載體,其載體中含有半乳糖的promoter,可用半乳糖去誘導,在啤酒酵母菌(BJ2168)中去試著表現。也將序列重組於pET-26大腸桿菌的表現載體在原核大腸桿菌BL21(DE3)中,以乳糖誘導表現。
    This research was first to find the complete gene sequence of nattokinase of Bacillus subtilis TKU007. We are also interested in the correlation of the gene sequence and enzyme stability. The B. .subtilis TKU007’s gene was transformed into heterologous hosts to over-express the protein for activity detection. First, the gene was amplified using the polymerization chain-reaction and ligated to pGEM-T easy vecetor by TA-cloning. Sequence comparison between TKU007 and common B.. subtilis discovered that the TKU007 nattokinase have 2 amino acid substitutions.These differences perhaps create the different enzyme properties in the TKU 007 nattokinase.
    We first tired to over-express TKU007 nattokinase heterologously in the Saccharomyces cerevisiae BJ2168 cells using pYES2 expression vector, containing the galactose promoter which was thus induced by galactose. We also over-expressed this nattokinase in E. coli BL21(DE3) expression using pET-26 vector, which was induced by lactose.
    DOI: 10.6846%2fTKU.2008.00312
    顯示於類別:[生命科學研究所] 學位論文

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