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研究生:陳世訓
研究生(外文):Shi-Xun Chen
論文名稱:豚鼠氣單胞菌幾丁質酶之蛋白質工程
論文名稱(外文):Protein engineering of chitinase from aeromonas caviae D1
指導教授:陳幸臣陳幸臣引用關係林富邦
指導教授(外文):Hsing-Chen ChenFu-Pon Lin
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:2004
畢業學年度:92
語文別:中文
論文頁數:95
中文關鍵詞:豚鼠氣單胞菌幾丁質酶蛋白質工程
外文關鍵詞:Aeromonas caviaechitinaseProtein engineering
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摘要

利用聚合酶鏈鎖反應,將Aeromonas caviae幾丁質酶基因Ac ChiAG561做定位修剪,保留其主要的催化活性區,切除A131以前的胺基酸,基因大小由1.6 kb剪切為1.3 kb。將此基因構築於表現載體pET 20b(+)之NdeⅠ及XhoⅠ選殖位置,所得之重組質體為pETAc chiAA131,再將pETAc chiAA131轉形於E. coli BL21(DE3) pLysS中表現,其重組蛋白質Ac ChiAA131具幾丁質酶的活性。利用His-tag親和性層析純化重組幾丁質酶Ac ChiAA131,於SDS-PAGE上檢視其分子量約為45 kDa。Ac ChiAA131的最適作用溫度及pH分別為40℃及7.0。酵素活性可被K+及Ni2+促進,而被Fe2+、Fe3+及SDS等所抑制。酵素動力學分析,以4-MU-(NAG)2為受質,ChiAA131的Km及kcat 分別為10 μM 及1.3 × 10-3 s-1 ;而以4-MU-(NAG)3為受質時之Km及kcat分別為9 μM 及8.1 × 10-2 s-1。對於不溶性幾丁質的吸附性,以ChiA的能力最高,ChiAG561次之,而ChiAA131最低。
目錄
目錄..........................................................................................................Ⅰ
圖目錄………………………………………………………..…………Ⅴ
表目錄………………………………………………………..…………Ⅶ
中文摘要……………………………………………………..…………Ⅷ
英文摘要……………………………………………………...…...........Ⅸ
第一章、前言………………………………………………….………….1
第二章、文獻整理…………………………………………….………….2
一、幾丁質酶之特性……………………………………….………..2
1. 幾丁質酶的天然分佈與作用……………………...………..2
2. 幾丁質之特性與分類……………………………...………..2
3. 幾丁質酶對幾丁質之水解作用…………………...………..4
4. 細菌幾丁質酶之結構與功能部位………………...………..5
4-1.受質結合區……………………………………...………...5
4-2.催化活性區……………………………………...………...6
4-3.第三型纖維網蛋白區……………..……………...…….…7
5. 幾丁質的後轉譯修飾作用…………………………..……...8
二、 Aeromonas sp.之幾丁質酶……………………………...…….9
1. Aeromonas sp.之特性………………………………….…….9
2. Aeromonas sp.之幾丁質酶…………………………………10
2-1. Aeromonas sp.10S-24…………………………………...10
2-2. Aeromonas hydrophila…………………………………..11
2-3. Aeromonas caviae D1…………………………………...11
2-3-1. Aeromonas caviae D1 ChiA………………………11
2-3-2. Aeromonas caviae D1 ChiAG561…………….......12
2-4. Aeromonas caviae Chi 1…………………………....…...12
2-5. Aeromonas caviae WS7b………………………………..13
第三章、材料與方法………………………………………………..…..14
一、實驗設計…………………………………………...………..…14
二、材料………………………………………………...………..…17
1.培養基……………………………………………………….17
2.菌種………………………...…………………...………….17
3.質體…...……………………………………………..…….…18
4.His-bind resin…………………..…………………………….18
5.藥品………………………………………………..………....18
6.酵素…………………………………………………………..20
7.試劑及緩衝液………………………………………………......20
三、方法…………………………………………………….........…21
1.Aeromonas caviae幾丁質酶蛋白質工程…………………...21
1-1.蛋白酶切割………………………………………..……….21
1-2.聚丙烯醯胺膠蛋白質電泳分析…………………….……...21
1-2-1.膠體製備與電泳分析……………………..………....21
1-3.電泳膠體之染色…………….……………………………...23
1-3-1.蛋白質染色……………………….………………….23
1-3-2.幾丁質酶活性染色…………………..………….........23
1-4.電泳溶離…………………….……………………………..23
1-5.蛋白質轉漬…………..………………………………….....23
1-6.氮端胺基酸定序…………………………………………...24
1-7.LC/ MASS/ MASS……………………………………….24
2. A. caviae幾丁質酶基因的剪切、表現與純化…....…………....24
2-1.菌株及質體…………………………………………..…...24
2-2.染色體DNA的抽取……………………………………...25
2-3.小量質體DNA的製備………………………..……….…25
2-4.洋菜膠體電泳………………………………………..….....25
2-5. A. caviae幾丁質酶基因的定位剪切設計………….…...…26
2-5-1.引子的設計……………………………………......….26
2-5-2.聚合酶鏈反應……………………………….……......27
2-5-3. PCR DNA回收…………………………………...….27
2-6.限制酶 之剪切作用…………………….………………..…28
2-7.表現載體pET20b(+)與Ac ChiA A131接合反應……….…..28
2-8.重組質體的轉形…………………………………………...28
2-8-1.受容細胞的製備………………….…………….......28
2-8-2.轉形………………………..……………………...….29
2-9.重組質體的快速篩選…………………………………........29
2-9-1.洋菜膠電泳檢視……………………………..……….29
2-9-2.限制 酶切割檢視………………………..…………….30
2-10.聚合 酶 鏈反應檢視…………………………………….…30
2-11.自動核苷酸序列分析……………………………………..31
2-12.幾丁質酶重組基因的表現與純化………………………..31
2-12-1.表現宿主大腸桿菌BL21(DE3)pLysS的轉形…..........31
2-12-2.酵素之誘導表現……………………………………..31
2-12-3.蛋白質的定量………………………………………..32
2-12-4.聚丙烯醯胺膠蛋白質電泳分析………………...........32
2-12-5.電泳膠體之染色……………………………………..32
2-12-5-1.蛋白質染色………………………………………..32
2-12-5-2.幾丁質酶活性染色……………………..………….32
2-12-6.幾丁質酶重組蛋白質的純化……………………….33
2-12-7.蛋白質濃縮…………………………………………..33
2-12-8.濾紙快速檢視幾丁質酶活性………………….........33
3.幾丁質酶生化特性……………………………………..34
3-1.幾丁質酶活性測定…………………………………..........34
3-2.最適pH的測定……………………………………………34
3-3.最適溫度的測定…………………………………………..35
3-4.金屬離子對幾丁質酶活性之影響………………………..35
3-5.化學試劑對幾丁質酶活性之影響………………………..35
3-6.水解產物的分析…………………………………..………35
3-6-1.水解反應………………….………………….……..35
3-7.酵素動力學的分析…………………………………..……36
3-8.不溶性受質吸附性試驗……………………………..……37
3-9.結構模擬……………………………………………..……37
第三章、結果…………………………………………………………....38
第四章、討論……………………………………………………………79
第五章、結論…………………………………………………………....83
第六章、參考文獻………………………………………………………84



圖目錄

圖一、Ac ChiA重組幾丁質酶的活性染色……………..………………..43
圖二、SDS-PAGE分析Ac ChiA重組幾丁質酶……………..…………..44
圖三、P45涵蓋重組幾丁質酶Ac ChiA胺基酸序列的部分………...…..45
圖四、P60涵蓋重組幾丁質酶Ac ChiA胺基酸序列的部分………...…..46
圖五、活性染色分析Ac ChiA重組幾丁質酶受chymotrypsin分
  解之情形…………………………………………………………..47
圖六、SDS-PAGE分析Ac ChiA重組幾丁質酶受chymotrypsin
  分解之情形……………………………………………….……….48
圖七、活性染色分析Ac ChiA重組幾丁質酶受trypsin分解之情
  形……………………………………………………….………….49
圖八、SDS-PAGE分析Ac ChiA重組幾丁質酶受trypsin分解之
  情形………………………………………………………….….…50
圖九、Ac chiAA131的核苷序列…………………………...…………...51
圖十、以洋菜膠電泳分析A. caviae ChiAA131基因……………….........52
圖十一、快速篩選重組質體pETAc chiA A131………………...…...........53
圖十二、以洋菜膠電泳分析pETAc chiA A131之基因構築…….............54
圖十三、重組質體Ac chiAA131的核苷酸自動定序……...……………..55
圖十四、Ac chiA A131基因全長之核苷酸及胺基酸序列…………….…57
圖十五、SDS-PAGE分析Ac ChiA A131重組幾丁質酶純化的情
  形…………………………………………………………………..58
圖十六、溫度對Ac ChiA A131重組幾丁質酶的影響……...……….......59
圖十七、pH對Ac ChiA A131重組幾丁質酶的影響…...………………..60
圖十八、不同濃度的4-MU-(NAG)2對Ac ChiAA131催化速率之
影響……………………………………………………………….61
圖十九、不同濃度的4-MU-(NAG)3對Ac ChiAA131催化速率之
影響………………………………………………………….……61
圖二十、Ac ChiAA131水解4-MU-(NAG)2的雙倒數圖….......…....……62
圖二十一、Ac ChiAA131水解4-MU-(NAG)3的雙倒數圖………..…….63
圖二十二、不同濃度的4-MU-(NAG)2對Ac ChiA催化速率之影
響………………………………………………………….………64
圖二十三、不同濃度的4-MU-(NAG)3對Ac ChiA催化速率之影響………………………………………………………………….64
圖二十四、不同濃度的4-MU-(NAG)2對Ac ChiAG561催化速率
之影響……………………………………………….…………....65
圖二十五、不同濃度的4-MU-(NAG)3對Ac ChiAG561催化速率
之影響…………………………………………………....….........65
圖二十六、Ac ChiA水解4-MU-(NAG)2的雙倒數圖……….…………...66
圖二十七、Ac ChiA水解4-MU-(NAG)3的雙倒數圖…………...…….…67
圖二十八、Ac ChiAG561水解4-MU-(NAG)2的雙倒數圖……….….….68
圖二十九、Ac ChiAG561水解4-MU-(NAG)3的雙倒數圖…………..….69
圖三十、Ac ChiAA131重組幾丁質酶於40℃不同時間下水解
N-乙醯幾丁六糖之產物量分析圖譜…K………………….……70
圖三十一、Ac ChiA , Ac ChiAG561與Ac ChiAA131對於不溶
  性幾丁質之吸附性分析………………………………….…........71
圖三十二、Ac ChiAA131的結構模擬圖…………..……………….........72
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