National Taiwan Ocean University Institutional Repository:Item 987654321/30286
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Title: 麥芽寡糖苷海藻糖生成酶基因之突變與突變後酵素之表現與活性分析
Mutation of maltooligosyltrehalose synthase genes and the expressions and activity assays of resulting mutant enzymes
Authors: Man-Ling Tham
譚曼凌
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 麥芽寡糖苷海藻糖生成酶;mRNA 二級結構
maltooligosyltrehalose synthase;mRNA secondary structure
Date: 2011
Issue Date: 2011-11-25T07:38:09Z
Abstract: 麥芽糖苷海藻糖生成酶 (Maltooligosyltrehalose synthase, MTSase) 能將麥芽寡糖進行分子內轉糖苷反應,使其還原端上的 α-1,4 糖苷鍵轉換成 α-1,1 鍵結,從而產生麥芽寡糖苷海藻糖 (maltooligosyltrehalose),產物隨後再以麥芽糖苷海藻糖水解酶 (Maltooligosyltrehalose trehalohydrolase, MTHase) 水解並產生海藻糖 (trehalose)。本實驗主要是針對 Sulfolobus solfataricus ATCC 35092 的 treY 進行靜默突變,而突變的部分主要是將之突變成與鄭 (2010) 的嵌合基因 (chimeric genes) SA21-G107W/G131E 序列相類似,此外,亦會對該片段進行 codon 最適化,並探討是否能提升其 MTSase 蛋白質表現量與活性。利用 megaprimed QCM 與 RF cloning 的方式,將原生型 S. solfataricus ATCC 35092 的 treY 基因進行靜默突變,並成功獲得基因突變株 SS1、SS2、SS3、SS4 以及 SS5。以 E. coli BL21(DE3)-CodonPlus-RIL 作為表現宿主的突變株,除了 SS1 不需添加 IPTG 之外,SS2~SS5 的最適 IPTG 皆為 0.05 mM;分別將低溫培養以及添加 0.05 mM IPTG 誘導後的菌株進行 SDS-PAGE 膠電泳分析後可發現,前者以 SS2 和 SS5 所產生 MTSase 較多,而後者則為 SS4 以及 SS5。此外,於 20℃ 低溫培養後,以 E. coli BL21(DE3)-CodonPlus-RIL 作為表現宿主的菌株進行小量破菌,於統計分析後,SS3 所產之 MTSase 蛋白質含量與 SS4 沒有顯著差異但其他組別則有,在活性方面,原生型與突變株則無顯著差異。而於添加 0.05 mM IPTG 的組別,SS2 以及 SS4 的蛋白質含量較原生型、SS1 以及 SS5 低;此外,SS3、SS4、SS5 的活性,皆較原生型與 SS1 高。將以 E. coli Rosetta(DE3) 作為表現宿主的菌株,於添加 0.05 mM IPTG 誘導並進行小量破菌後,除了 SS2 以外,原生型與靜默突變株所產之 MTSase 蛋白質含量在統計上並無顯著差異,但突變株的活性卻較原生型高。此外,以 E. coli Rosetta(DE3) 作為表現宿主的菌株,於不添加 IPTG 並以 20℃ 低溫培養以及進行大量破菌後,SS4 與 SS5 的粗酵素液之活性皆較原生型高,此外,經過 80℃、2 小時熱處理後,SS4 的活性較原生型高。於 mRNA 二級結構預測方面,以 SS5 所改變的構型與原生型 S. solfataricus ATCC 35092 treY 差異最大,這可能是因為改變了 27 個核苷酸,使其原本的結構受到破壞;此外,也可能因將其改變成宿主較常用的 codon,使得結構看起來較為鬆散。
The maltooligosyltrehalose synthase (MTSase) has glucosyltransferase activity and converts the first α-1,4-glycosidic linkage at the reducing end into α-1,1 linkage to produce maltooligosyltrehalose, and the maltooligosyltrehalose trehalohydrolase (MTHase) catalyses the hydrolytic release of trehalose from maltooligosyltrehalose. In order to improve the expression and activities of MTSase from S. solfataricus ATCC 33909, silent mutation had been used. Five silent-mutated were produced by mutating the SStreY genes sequence to be similar to chimeric gene of SA21-G107W/G131E, and displacing a partial sequence of SStreY gene to be codon which has higher usage frequencies in E. coli. The appropriate IPTG concentrations of silent-mutated MTSases in E. coli BL21(DE3)-CodonPlus-RIL were 0 mM for SS1 and 0.05 mM for SS2~SS5 respectively. According to the SDS-PAGE analysis, the expressions of MTSases of SS2 and SS5 were increased when cultivated at 20℃ for 20 hours, however, the expression of SS4 and SS5 were increased after induced by 0.05mM IPTG. After cultivated the silent-mutated at 20℃ for 20 hours, the MTSases expressions of SS3 and SS4 were decreased when the genes were expressed in E. coli BL21(DE3)-CodonPlus-RIL, but the activities of silent-mutated MTSases were significantly different to that of wild-type. When the genes were expressed in E. coli BL21(DE3)-CodonPlus-RIL, SS2 and SS4 are lower than wild type, SS1 and SS5, but the activities of SS3, SS4 and SS5 had higher than that of wild-type after induced by 0.05mM IPTG. The expression at 0.05mM IPTG for silent-mutated MTSases of SS4 and SS5 are increased when the gene were expressed in E. coli Rosetta(DE3), but the activities of SS1~SS5 were significantly different to that of wild-type. When SS4 and SS5 were expressed in E. coli Rosetta(DE3) without IPTG, the activities of SS4 and SS5 crude extract were higher than that of wild-type after treatment by French Press. After 2 hours heat treatment, the activities of SS5 was not significantly different to that of wild type excepted SS4. The SS5 mRNA secondary structure is different to that of wild-type, because SS5 had change 27 nucleic acids, which might destroy the secondary structure of wild-type, and might make the structure looser.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M98320066
http://ntour.ntou.edu.tw/handle/987654321/30286
Appears in Collections:[Department of Food Science] Dissertations and Theses

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