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研究生:吳家榮
論文名稱:重組麥芽寡醣苷海藻糖生成酶之定位飽和突變與活性篩選
論文名稱(外文):The site saturation mutagenesis and activity screening of recombinant maltooligosylterhalose synthase
指導教授:方翠筠
指導教授(外文):Tsuei-Yun Fang
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:78
中文關鍵詞:麥芽寡糖苷海藻糖生成酶海藻糖
外文關鍵詞:Maltooligosyltrhalose synthasetrehalose
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麥芽寡糖苷海藻糖生成酶 (Maltooligosyltrhalose synthase) 為可進行分子內轉糖苷作用之酵素,它可將麥芽寡糖末端之 α,1-4 糖苷鍵結轉變成 α,1-1 糖苷鍵結,本實驗將已建構於 pET-21b 載體之 Sulfolobus acidocaldarius ATCC 33909 的 MTSase 進行突變,以試圖找出轉糖苷活性提昇之酵素。在實驗中,為了使麥芽寡糖苷海藻糖生成酶的純化能夠更快速的純化,因此先利用定位突變 (Site-directed mutagenesis) 的方法將將麥芽寡糖苷海藻糖生成酶之 C 端加上 His-tag,使得酵素能直接以鎳親和性管柱層析進行純化,在得到具有 His-tag 之麥芽寡糖苷海藻糖生成酶 (MTSase-His) 之後,以此基因為模板進行定位飽和突變 (Site saturation mutagensis),分別針對酵素的 Asn89、Leu199、Ala389 與 Val409 四個胺基酸殘基進行突變,於突變完成後再經過轉糖苷活性篩選的方式找出轉糖苷活性提高之突變酵素,然而在突變完成後,雖然在篩選過程中有找到數個轉糖苷活性提高之突變酵素,然而經過核酸序列分析比對後發現皆仍為原生型酵素。除了定位飽和突變的實驗之外,還以增加氫鍵為目的設計四個定位突變 (Leu199 → Arg、Leu199 → Lys、Pro385 → Thr 與 Ala389 → Ser),試圖直接得到轉糖苷活性提高之突變酵素,經過核酸序列分析比對確認四個突變皆完成後,對原生型及突變型之酵素進行酵素純化,得到純的酵素後進行酵素特性及活性分析,發現,具有 His-tag 之麥芽寡糖苷海藻糖其比活性較無 His-tag 者低了 53.47% ,而突變型的酵素的部分,除了 Pro385 → Thr 較原先所參考之 Pro385 → Ser 高 20% 之外,其它突變酵素皆低於所參考之突變,並且四個酵素之比活性皆比原生型酵素差,顯示四個突變皆未能提高轉糖苷活性。
壹、 前言………………………………………………………...1
貳、 文獻整理……………………………………………….......3
一、 海藻糖簡介……………………………………………3
1. 海藻糖的發現……………………………………………..3
2. 自然界中之海藻糖………….………...………………..3
3. 海藻糖之結構與特性………………………………......4
4. 海藻糖應用與發展……………………….………...…..4
5. 海藻糖之生產方式………………….………………….5
二、MTSase 之相關研究…………………………..…….6
1. MTSase 介紹………………………………..….……….6
2. MTSase 與 α-澱粉酶家族酵素之比較…………………..7
3. MTSase 之轉糖機制……………………………….…..8
4. MTSase 之水解作用…………………...……………….8
三、定位突變與定位飽和突變之簡介…………….…...............9
參、 實驗設計……………...…………...…………………………..10
肆、 材料方法…………………...……………………………..11
一、實驗材料…………………………….………………..11
1. 菌株與質體………………………………………….11
1.1 treY 基因來源………..………….....………..11
1.2 保存質體之菌株………………….…...………..11
1.3 蛋白質表現系統之生產菌株…………....……..11
2. 標準品…………………………...……………...…11
2.1 DNA 標準品……………………………………11
2.2 蛋白質標準品………………………………….11
3. 酵素………………………………………………...11
3.1 聚合酶………………………...………………..11
3.2 限制酶…………………………...……………..12
3.3 核酸酶…………………………...……………..12
4. 培養基材料…………………………...…………...12
5. 化學藥品…………………………………………...12
6. 市售快速套組………………………….………...14
7. 儀器設備…………………………………………...14
二、實驗方法……………………………………………..17
1. 重組 MTSase 之蛋白質工程………………………....17
1.1 宿主細胞與培養機……………..……………….17
1.2 質體 DNA 之製備…………………………....17
1.3 DNA 之定量……………………………….......18
1.4 定位突變及定位飽和突變………...………….18
1.4.1 突變引子之設計………………...………18
1.4.2 突變引子之合成………………………...18
1.4.3 Megaprimed QCM 單管模式之 PCR 反
應………………………………………....19
1.4.4 Megaprimed QCM 雙管模式之 PCR 反應………………………...……………………20
1.5以 Dpn I 限制酶剪切 PCR 反應液中之 dsDNA
template………………………………….…….…..21
1.6 勝任細胞之製備……………………….………21
1.7 突變 DNA 之轉形作用………………..……...22
1.8膠體電泳分析……………….…………………22
2. 進行 MTSase 轉糖苷活性篩選……………..……22
2.1 於 96-well ELISA plate 中小量培養轉形
株……………………………………………...……...22
2.2 進行小量細胞破碎……………………………23
2.3以 DNS 法初步篩選具有轉糖苷活性提昇之突變株...23
2.4 於微量離心管中小量培養初步篩選重組 MTSase
活性提升之菌株………...……….………………24
2.4.1 於微量離心管中小量培養菌體並釋出酵素
液……………………………………………....24
2.4.2 於微量離心管中以 DNS 法測 MTSase 轉糖
苷活性………………………………………...24
2.5 序列分析………………………………………25
2.6 菌種保存………………………………………25
3. 重組 MTSase 的表現與純化…………………..……25
3.1 IPTG 最佳濃度之試驗……………………………25
3.2重組 MTSase 在大腸桿菌中之表現……………..26
3.3 MTSase 之純化………………….……………..26
3.3.1 細胞破碎……………….………………...26
3.3.2 熱處理……………………….…………...27
3.3.3 His-tag 親和性管柱層析………………...27
3.4 蛋白質電泳分析……………………………..27
3.4.1 膠體之製備……………………………...28
3.4.2 膠體組成成分…………………………...28
3.5 膠電泳之操作方法…………………………..29
3.5.1 2X SDS sampling buffer 之製備………..30
3.5.2 膠片染色與脫色…………………...……30
3.6 蛋白質定量…………………………………..30
3.6.1 蛋白質定量相關試劑之製備…………...31
4. 重組 MTSase之活性分析………………………….…31
4.1以 DNS 法測重組 MTSase 之活性……………...31
4.2重組 MTSase之特性分析……………..32
4.2.1重組 MTSase 之最適作用溫度………....32
4.2.2重組 MTSase 之最適作用 pH 值……...32
4.2.3重組 MTSase 之熱穩定性……….……...33
伍、 結果與討論……………………………………………….34
一、 以點突變方式建構 MTSase-His……………………34
1. 點突變之設計…………………………………...34
2. 點突變引子組之設計及分析…………………...34
3. SAtreY-His 之建構…………………………………..34
二、 對 MTSase-His 進行突變………………………….35
1. 點突變之設計………………………………….35
2. 突變引子組之設計與分析……………………36
2.1 定位飽和突變引子組之設計與分析………36
2.2 定位突變引子組之設計與分析……………37
3. SAtreY-His 之突變…………………………………37
3.1 定位飽和突變…………………………..………37
3.2 定位突變…………………………………..38
三、 MTSase-His 轉糖苷能力之活性篩選………………39
四、 原生型與突變形 MTSase-His 之表現與純化……..39
五、 原生型與突變型 MTSase-His 之酵素特性……….41
5.1 最適作用溫度………………………………41
5.2 最適作用 pH……………………………..41
5.3 熱穩定性……………………………………42
陸、 結論………………………………………………………44
柒、 參考文獻…...……………………………………………..45

柒、參考文獻
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林嘉銳,2006,以電腦模擬麥芽寡糖苷海藻糖生成酶與麥芽寡糖之結合部位並探討基質結合部位胺基酸殘基突變後對其糖基活性之影響,國立台灣海洋大學食品科學系碩士學位論文,基隆
魏岑芸,2007,增加基質結合部位殘基與基質間氫鍵對於 Sulfolobus solfataricus ATCC35092 麥芽寡糖苷海藻糖生成酶轉糖苷及水解作用之影響,國立台灣海洋大學食品科學系碩士學位論文,基隆
林敬瑋,2008,新型單點及多點定位突變方法之開發與利用定向演化進行麥芽寡糖苷海藻糖水解酶的基因重組,國立台灣海洋大學食品科學系碩士學位論文,基隆
郭家宏,2008,基質次結合部位殘基多點突變後對於麥芽寡糖苷海藻糖水解酶之活性與基質選擇性的影響國立台灣海洋大學食品科學系碩士學位論文,基隆
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