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研究生:熊柏凱
研究生(外文):Bill Bor-Kai Hsiung
論文名稱:酵母菌表現之葡糖澱粉酵素之功能及穩定性分析─連接片段長度及醣基化之影響
論文名稱(外文):Stability and functional analysis of glucoamylase expressed by Saccharomyces cerevisiae - The effects of linker length and glycosylation
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:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
論文頁數:61
中文關鍵詞:葡糖澱粉酵素醣基化穩定性功能
外文關鍵詞:glucoamylaseglycosylationstabilityfunction
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葡糖澱粉酵素可以從多醣類的非還原端經由一個反轉構形之反應水解出b構形之葡萄糖。 真菌中的葡糖澱粉酵素被廣泛的使用在葡萄糖及果糖糖漿的製作。 然而,一些真菌類對人體有毒,因而無法被安全的使用在食品及製酒工業上。 為了在產業上之應用,我們使用Saccharomyces cerevisiae異體表現重組之葡糖澱粉酵素。 我們建構了一系列在澱粉結合區及連接片段縮短了的Rhizopus oryzae葡糖澱粉酵素之突變株,並將之置於具有不同醣基化行為之宿主中表現。首先,我們分別最佳化葡糖澱粉酵素在各不同宿主中之表現條件,接著大量表現並純化不同長度之重組葡糖澱粉酵素,然後觀察它們的功能及穩定度之變化。 我們發現當葡糖澱粉酵素在高醣基化行為之宿主中表現時,其酵素之熱穩定度會增加,但酵素的活性會受到些微的影響。 這顯示了醣基化與酵素的熱穩定性質高度的相關,而在異種宿主中非自然的高度醣基化行為可能會些微的抑制酵素催化反應的進行。 我們也發現醣基化會增加重組葡糖澱粉酵素在低pH環境下之pH穩定度但卻會降低其在高(中性)pH環境下之穩定度,同時使得高醣基化葡糖澱粉酵素的最適反應pH範圍往酸性區域移動。 實驗結果同時顯示了連接片段和結構穩定性的相關性,全長的連接片段會穩定葡糖澱粉酵素催化區的二級結構。

Glucoamylases (GAs) hydrolyse both a-1,4 and a-1,6 glucosidic bonds from the non-reducing end of polysaccharides and releases b-D-glucose with inversion of the anomeric configuration. Fungal GA is widely used in the manufacture of glucose and fructose syrups, however, some fungi are toxic to humans and can not be safely used in the food and wine industries. For the application purpose, we express recombinant GA in yeast, Saccharomyces cerevisiae. We constructed a series of truncated mutants in the starch binding domain and linker region of GA from Rhizopus oryzae and expressed in different hosts performing different degrees of glycosylation, then we assayed their functional activity. We found that when GA is expressed in the yeast that performing hyperglycosylation, its stability increased with slightly decreased in its enzyme activity. It suggested that glycosylation was heavily related to the enzyme’s stability, but unnatural hyperglycosylation in the heteroorganisms may slightly hold back the catalyzed reaction to proceed. We also found that glycosylation increased rGAs’ pH stability at the low pH range but decreased rGAs’ stability at the high (neutral) pH range. The linker was also indicated to be related to the structural stability, full length linker region stabilized the secondary structures of the catalytic domain of GA.

Abstract (Chinese) .......................................... i
Abstract (English) ......................................... ii
Acknowledgement ........................................... iii
Table of Contents .......................................... iv
List of Tables .............................................. v
List of Figures ............................................ vi
List of Appendices ........................................ vii
Abbreviations ............................................ viii
Introduction ................................................ 1
Materials and Methods ....................................... 6
Results .................................................... 12
Discussion ................................................ 18
Tables ..................................................... 21
Figures and Legends ........................................ 25
Appendices ................................................. 43
References ................................................. 57

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