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研究生:劉威廷
研究生(外文):Wei-Ting Liu
論文名稱:根黴菌之葡糖澱粉酵素的連接片段區域對於吸附澱粉區域的功能和結構之影響
論文名稱(外文):The Linker Region of Glucoamylase from Rhizopus oryzae Affects Function and Structure of the Starch-Binding Domain
指導教授:張大慈
指導教授(外文):Margaret Dah-Tsyr Chang
學位類別:碩士
校院名稱:國立清華大學
系所名稱:分子與細胞生物研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:50
中文關鍵詞:根黴菌葡糖澱粉酵素連接片段區域吸附澱粉區域
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根黴菌 (Rhizopus oryzae) 之葡糖澱粉酵素 (glucoamylase) 包含N端的吸附澱粉區域 (SBD,106個氨基酸),o-醣基化的連接片段 (L,36個氨基酸) 和 C端的催化區域 (CD,437個氨基酸)。吸附澱粉區域具有吸附生澱粉的結合能力,而催化區域則能完全地水解澱粉成葡萄糖。至於連接片段,它的功能尚未被充分的描述。我們使用大腸桿菌 (E. coli) 和麵包酵母 (S. cerevisiae) 表現兩種重組蛋白:重組的吸附澱粉區域 (rSBD) 和重組含連接片段的吸附澱粉區域(rSBD-L)。大腸桿菌表現的胞內重組蛋白及麵包酵母分泌表現至培養液的重組蛋白利用快速蛋白質液相層析法直接純化。飽和結合分析和使澱粉分解的分析證明了這些蛋白質不同地特性。另外,圓二色光譜儀 (Circular Dichroism) 分析顯示來自於大腸桿菌和麵包酵母的重組吸附澱粉區域擁有β-摺板的構造,而且在吸附澱粉區域C端的連接片段的存在可能會影響其結構穩定性。在高溫時,藉由圓二色光譜儀、剛果紅分析和穿透式電子顯微鏡鑑定發現重組含連接片段的吸附澱粉區域能形成似類澱粉的結構。然而,在相同的條件下,單獨的重組吸附澱粉區域僅能形成不可溶的聚集體。我們證明連接片段強烈地影響吸附澱粉區域的生物化學的功能和物理的結構。除此之外,我們更進一步地表現 rSBD-s,rSBD-sk和rSBD-skptttta,研究需要多少C端的氨基酸殘基才能影響吸附澱粉區域的構造。結果顯示rSBD-s和rSBD有相似的結構性質,而rSBD-sk和rSBD-skptttta能和rSBD-L一樣形成β-似類澱粉的構造。因此,我們確定連接片段在影響吸附澱粉區域的結構和功能方面扮演重要的角色。
The mature glucoamylase from Rhizopus oryzae consists of an N-terminal starch-binding domain (SBD, 106 aa), an o-glycosylated linker (L, 36 aa), and a C-terminal catalytic domain (CD, 437 aa). The SBD processes raw-starch binding ability and the CD can hydrolyze starch completely to glucose. As for the linker region, its function is not well characterized. We have used both E. coli and S. cerevisiae strains to express two recombinant clones, rSBD and rSBD-L. Each protein was directly purified by fast protein liquid chromatography. Saturation binding and amylolytic assays demonstrated that these recombinant proteins behave differently. In addition, Circular Dichroism analysis revealed that rSBD from E. coli and S. cerevisiae possesses a β-strand conformation and the existence of a linker region at the C-terminal end of SBD may influence the structural stability of rSBD. At high temperature, the rSBD-L was found to form amyloid-like structures as determined by Circular Dichroism, Congo Red assay and transmission electron microscopy. However, rSBD alone could only form insoluble aggregates under the same condition. We have demonstrated that the linker region strongly influenced the biochemical function and physical structure of the starch-binding domain. We have further expressed rSBD-s, rSBD-sk and rSBD-skptttta to investigate how many C-terminal residues are required to influence the conformation of starch-binding domain. The results showed that rSBD-s had similar structural properties as rSBD, whereas rSBD-sk and rSBD-skptttta formed β-amyloid-like conformation as rSBD-L. Therefore, we have confirmed that the linker region plays an important role in the structure and function of the starch-binding domain.
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