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研究生:溫智民
研究生(外文):Chih-Min Wen
論文名稱:仙人掌桿菌中幾丁質酵素的選殖與表現之研究
論文名稱(外文):Cloning and Expression of the Chitinase from Bacillus cereus NCTU2
指導教授:李耀坤李耀坤引用關係
指導教授(外文):Yaw-Kuen Li
學位類別:碩士
校院名稱:國立交通大學
系所名稱:應用化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:69
中文關鍵詞:仙人掌桿菌幾丁質幾丁質酵素選殖表現包涵體
外文關鍵詞:Bacillus cereuschitinchitinasecloningexpressioninclusion bodiesBacillus megaterium
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本研究由Bacillus cereus NCTU2的染色體DNA選殖得幾丁質酵素(ChiNCTU2)之基因,並成功的接進載體pPCR上。經由定序,幾丁質酵素(ChiNCTU2)之基因共有1083個核苷酸,相當於360個胺基酸。經與Genebank基因資料庫上B. cereus ChiA比對,發現兩段基因有27個核苷酸不一樣,導致7個胺基酸的差異。經與先前純化酵素之N端序列比較推測,其前27個胺基酸為訊息胜肽,剩餘之333個胺基酸形成胞外酵素,分子量為36184 Da。
此酵素經胺基酸序列比對,在醣類水解酵素中,屬於家族18,其蛋白質結構應為(α/β)8的結構,且僅具有單一催化區域(catalytic domain),而不具備其他的輔助區域。
此基因被建構於pRSET A載體上並以大腸桿菌[BL21(DE3)]表達之。其中含訊息胜肽之基因,(p)chi/21,無法大量表現幾丁質酵素,但去除訊息胜肽之基因,(m)chi/21,則可表現出蛋白質,可惜所得到之酵素為包涵體而不具活性。嘗試利用透析的方式對包涵體進行蛋白質再摺疊,尚未找到可以使幾丁質酵素恢復活性之條件。
另外,ChiNCTU2此刻正被建構於Bacillus megaterium之表現系統,本報告亦將涵蓋此系統之先期研究成果。
A chitin-degrading Bacillus strain, designated as NCTU2, was screened from soil and identified. An extra-cellular chitinase was purified to > 90% homogeneity from the culture filtrate. chitobiose is the predominant product through out the enzymatic hydrolysis of the colloidal chitin, indicating that the purified chitinase is an exo-chitinase. The first 15 N-terminal amino acid sequence of the enzyme is determined to be ANNLGSKLLVGYWHN, which is highly homologous to that of ChiA from Bacillus cereus. PCR cloning technique was employed for obtaining the corresponding gene from the Bacillus NCTU2. The gene sequence was determined to be 1080 bp encoding a polypeptide of 360 amino acids. By inspecting the N-terminal sequence of the purified enzyme and the amino acid sequence deduced from the gene, the signal peptide is identified as the first 27 amino acids of the enzyme. As compared with the gene of Chi36 and that of NCTU2, though both enzymes are similar in molecular size, 70 nucleotides resulting in 16 amino acids are different. Since the recombinant NCTU2 produced in E. coli was exhibited as an inclusion body, a Bacillus megaterium strain was attempted to be used as the expression host. The preliminary study on construction of the NCTU2 in this system was also included in this report.
目錄
中文摘要………………………………………………………………….I
英文摘要…………………………………………………………………II
謝誌………………………………………………………………………III
目錄………………………………………………………………………Ⅳ
圖表目錄…………………………………………………………………Ⅶ
第一章 緒論
1-1 前言……..………………………………………………..………..1
1-2 幾丁質(chitin)………………...…..……………….….…………...1
1-3 幾丁質酵素(chitinase)…….………………..……………………..3
1-3.1幾丁質酵素(chitinase)的概述及其種類……………………3
1-3.2幾丁質酵素在演化上的分類………………….……………3
1-3.3幾丁質酵素的水解反應機制………………….…………....8
1-3.4幾丁質酵素的活性測定方法……………………………...11
1-4 幾丁質酵素與幾丁寡醣之應用………..…………………..……12
1-4.1幾丁質酵素的應用…………………………………….… .12
1-4.2 N-乙醯幾丁質寡醣的性質與應用………………………..12
1-5枯草桿菌屬(Bacillus spp. )重組基因表現系統……….………..13
1-5.1枯草桿菌屬之簡介………………………………………..13
1-5.2以枯草桿菌屬作為生產重組蛋白質的宿主細胞………..14
1-5.3 Bacillus megaterium表現系統之簡介……………………15
1- 6研究目的……………………………………………………….. 16
第二章 實驗方法與步驟
2-1 概述 ……………………………………………………………17
2-1.1一般敘述…………………………………………………..17
2-1.2藥品、器材與儀器總覽……………………………….….17
2-1.3幾丁質酵素活性之測定…………………………………..18
2-2幾丁質酵素的基因選殖及定序………………………………...18
2-2.1 B. cereus NCTU2染色體DNA的純化………………….18
2-2.2幾丁質酵素基因的選殖……………………………… ….19
2-2.3幾丁質酵素基因與pPCR載體的接合(ligation)………...19
2-2.4基因片段之定序(cycle-sequencing)……………………...20
2-3 E. coli系統中幾丁質酵素之表現…………………………....…21
2-3.1 chi gene與表現型載體(expression vector)pRSET A
的接合(ligation)…………………………………………...21
2-3.2質體DNA之轉型………………………………………....23
2-3.3重組幾丁質酵素誘導表現之測試………………….……..23
2-3.4重組幾丁質酵素之活性測試……………………………...24
2-3.5重組蛋白質在細胞內的分布情形………………………...25
2-4重組幾丁質酵素的再折疊(Refolding)………………………….25
2-4.1概述--文獻回顧……………………………….………….....25
2-4.2重組幾丁質酵素的再折疊………………………………....27
2-5 Bacillus megaterium系統中幾丁質酵素之表現………………28
2-5.1質體DNA 之建構……………………………………….......28
2-5.2 B. megaterium WH320原生質體(protoplasts)的製備…........29
2-5.3質體DNA的轉型……………………………………….......30
2-5.4 B. megaterium質體DNA的純化………………………........30
2-5.5蛋白質表現之測試………………………………………...31
第三章 結果與討論
3-1幾丁質酵素的選殖與序列分析………..………………………33
3-1.1 Bacillus cereus NCTU2幾丁質酵素的純化與性質
之回顧…………………………………………………….33
3-1.2幾丁質酵素基因之選殖………………………………….33
3-1.3幾丁質酵素基因(chi gene)與pPCR載體的接合……….34
3-1.4幾丁質酵素基因與胺基酸序列………………………….35
3-1.5 ChiNCTU2之胺基酸序列分析與比對………………….39
3-1.6 ChiNCTU2之胺基酸序列特徵………………………….39
3-1.7 ChiNCTU2蛋白質立體結構的摹擬…………………….44
3-2幾丁質酵素(ChiNCTU2)在E. coli中的表現………………..47
3-2.1表現質體的建構…………………………………………47
3-2.2幾丁質酵素的表現情形……………………………..…..48
3-2.3幾丁質酵素的活性測試……………………………..…..50
3-2.4重組蛋白質在細胞中的分布情形………………………51
3-2.5其他途徑回復幾丁質酵素活性的嘗試………………....52
3-3重組幾丁質酵素的再折疊……………………..……………..52
3-4在Bacillus megaterium系統表現的情形………….………....54
3-4.1表現質體的建構………………………………….……..54
3-4.2原生質體的製備、質體轉型、質體純化
與誘導表現之討論…………………………….………55
第四章 結論………………………………....….....57
第五章 參考文獻………….…………..……..…....58
附錄一………………………………………………......65
附錄二………………………………………………......68
圖表目錄
圖1-1 幾丁質,幾丁聚醣以及纖維素的化學結構……………………2
圖1-2 三種幾丁質水解酵素水解反應的形式…………………………4
圖1-3 家族18 hevamine幾丁質酵素的3D結構圖………………...6
圖1-4 家族19 Hordeum vulgare幾丁質酵素的3D結構圖………...7
圖1-5 一步置換水解反應機制…………………………………………8
圖1-6 兩步置換的水解反應機制……………………………………....9
圖1-7 形成中間產物(oxazoline ion)的反應機制示意圖……………. 10
圖3-1 chi gene PCR反應放大後之DNA電泳圖……………………34
圖3-2 幾丁質酵素基因在pPCR載體(chi/pPCR)上的方向示意圖…35
圖3-3 B. cereus NCTU2幾丁質酵素之基因與胺基酸序列…………36
圖3-4 ChiNCTU2與其他幾丁質酵素之胺基酸序列比對圖………..40
圖3-5 ChiNCTU2與細菌幾丁質酵素的區域(domain)組成示意圖...43
圖3-6 ChiNCTU2與hevamine chitinase之胺基酸序列比對圖…….45
圖3-7、ChiNCTU2與hevamine chitinase之區域組成示意圖……….45
圖3-8 (A)以Hevamine chitinase為基礎摹擬出的ChiNCTU2
的3D結構 (B) Hevamine chitinase的3D結構……………....46
圖3-9 質體(p)chi/pRSET A與(m)chi/pRSET A的示意圖…………..47
圖3-10 (p)chi/21及(m)chi/21的誘導表現情形(whole cell)……..….....48
圖3-11 (p)chi/21與(m)chi/21誘導活性追蹤圖……………………….....50
圖3-12 重組蛋白質在細胞內分布的情形……………………………….51
圖3-13 (m)chi/21之包涵體處理過程的電泳分析圖…………………….53
圖3-14 (p)chi/pWH1520及(m)chi/pWH1520的示意圖…………….......55
表一 利用Vector NTI 軟體程式分析ChiNCTU2氨基酸組成之統計表
..………….....38
表二 包涵體在不同緩衝液中再折疊後的活性測試結果………………54
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