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    標題: 樟芝漆氧化酶的酵素活性分析及其基因選殖之研究
    Characterization and gene cloning of a novel lacca...
    作者: 林秋燕
    Chiu-Yen Lin
    貢獻者: 呂敏勇
    嘉南藥理科技大學:生物科技研究所
    關鍵字: 漆氧化酶
    木質素
    樟芝
    ABTS 活性染色
    光誘導
    Laccase
    lignin
    Taiwanofungus camphoratus
    ABTS
    日期: 2007
    上傳時間: 2008-12-03 11:17:34 (UTC+8)
    摘要:  自然界中存量最多的大分子物質分別為纖維素、半纖維素、木質素,其中又以結構複雜的木質素最難以分解。現今已知主要分佈在白腐型真菌中,有三種酵素具有分解木質素之弁遄A分別為漆氧化酶(laccases)、含錳過氧化酶(manganese-dependent peroxidases)和木質素過氧化酶(lignin peroxidases)。
    樟芝屬於褐腐型真菌的一種,實驗中無意間發現其具有漆氧化酶的活性,故針對樟芝BCRC 35398所含有的漆氧化酶及其對應的基因進行一系列的探討。一方面以探討漆氧化酶的酵素活性為主, 利用ABTS「(2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)」做為活性測定的基質,以吸光値415 nm偵測,發現樟芝BCRC 35398漆氧化酶在pH 3.0、反應溫度40℃的情況下,具有最高的相對活性。利用Native SDS-PAGE蛋白質電泳分析,發現漆氧化酶的分子量約60 kDa,而且發現添加銅離子、錳離子或鈣離子於培養基的情況下,活性有被誘導而增加的情形,特別在添加50 μM銅離子作用2小時後,展現最大的酵素活性。另一方面,本文亦對樟芝BCRC 35398的漆氧化酶進行基因選殖及分析。以白腐型真菌的漆氧化酶基因之保守性序列設計一對漆氧化酶專一性的引子對,以基因體DNA
    (genomic DNA)為模板進行聚合酶連鎖反應,可得到一段1714 bp的基因片段,其中具有9個插入子(intron)。由其所對應的胺基酸序列與其他已知的漆氧化酶胺基酸序列比對,發現具有高的相似性。南方墨點法的結果顯示,樟芝BCRC 35398的漆氧化酶基因僅由一個基因進行調控;另外,以限制酶SacⅡ截切基因體DNA所得到的一段3066 bp的DNA片段,含有完整的漆氧化酶基因,其中包含12個插入子,刪除插入子後的open reading frame共含有1572 bp,對應523個胺基酸。
    另一個令人驚訝的實驗,我們利用光線當成一個調控因子,將樟芝BCRC 35398分別培養在照光和避光的環境下,再利用ABTS的活性染色法以及反轉錄聚合酵素連鎖反應與北方墨點法分析,結果發現,在避光的情況下,無發偵測到漆氧化酶的活性,在照光的情況下,持續照光120天,在去除菌絲的固態培養基中發現漆氧化酶的活性,而且反轉錄聚合連鎖反應和北方墨點法,可發現漆氧化酶轉錄隨照光天數而增加,故證明樟芝中的漆氧化酶可經由照光而被誘導,而且與基因轉錄的增加有關。
    Three classes of lignin-modifying enzymes, including laccases, lignin
    peroxidases, and manganese-dependent peroxidases, are involved in the fungal
    degradation of lignin in white rot fungi. Moreover, laccase is absolutely essential for lignin degradation in white rot fungi. Laccases can decompose lignin in the absence of lignin peroxidase and manganese-dependent peroxidase. In contrast, very little is known about the occurrence of ligninolytic systems in brown rot fungi because there are only few instances to show that these microorganisms can produce laccases. In this study we demonstrate that a novel laccase found in the brown rot fungus Taiwanofungus camphoratus (BCRC 35398).
    Extracellular laccase activity showed a lasting increase when glucose in
    culture medium was completely exhausted. The optimum pH for enzyme
    activity was around 3.0 against ABTS and it was most active at 40℃. The native
    SDS-PAGE analysis showed that the molecular weight was approximately 60 kDa. Exogenous addition of Cu2+, Mn2+, and Co2+ respectively to culture medium resulted in a dramatic increase in laccase activity, as well as copper exhibited the stimulatory effect with the highest level. The addition of 50 μM of CuSO4 could enhance the extracellular laccase production after 2 h. On the other hand, a 3066 bp DNA fragment containing full length of laccase gene was obtained, and its sequence comprised 12 introns. The open reading frame of
    laccase gene is 1572 bp corresponding to 523 residues. Surprisingly, the
    expression of laccase gene was upregulated by constant light.
    關聯: 校內校外均不公開
    Appears in Collections:[生物科技系(所)] 博碩士論文

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