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研究生:陳逸華
研究生(外文):Yi-Hua Chen
論文名稱:解澱粉芽孢桿菌重組幾丁聚醣酶基因之表現及其生化特性
論文名稱(外文):Expression and Characterization of Recombinant Chitosanase Gene from Bacillus amyloliquefaciens
指導教授:林富邦
指導教授(外文):Fu-Pang Lin
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
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:125
中文關鍵詞:解澱粉芽孢桿菌幾丁聚醣酶生化特性
外文關鍵詞:Bacillus amyloliquefacienschitosanasecharacterization
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The chitosanase gene from B. amyloliquefaciens was cloned by PCR techniques using the specific 5’ and 3’-end primers based on the DNA sequence of the chitosanase from B. amyloliquefaciens csn-UTK (Accession number AB0051574).
The full-length chitosanase gene of B. amyloliquefaciens was 858 base pairs and encoded 286 amino acid residues containing the signal peptide of 36 amino acid residues on the N-terminals. The mature BamyCSN was calculated to have a molecular weight of 27.5 kDa. The recombinant chitosanase gene was expressed in E. coli BL21(DE3)pLysS. It was efficiently purified from the host cell lysate by His-tag affinity chromatography and was found enzymatically active.
The optimal pH and temperature of BamyCSN was 7.0 and 45 ℃, respectively. Kinetic parameters Km and kcat using the water soluble chitosan as a substrate was 2.12 μg / μl and 2.25x10-5 s-1, respectively. Tween 20 at 1% concentration could stimulate BamyCSN slightly, while most metal ions inhibited the chitosanase activity except 1mM NH4+ activated BamyCSN. By TLC analysis, GlcN, (GlcN)2, (GlcN)3 and (GlcN)4 could be found in the soluble chitosan hydrolyzed by BamyCSN at 37 ℃for 16 h. The major product was (GlcN)4 .
以Bacillus amyloliquefaciens csn-UTK (Accession number AB0051574) 幾丁聚醣酶的基因序列設計B. amyloliquefaciens幾丁聚醣酶基因的5’端及3’端引子,以聚合酶鏈反應 (Polymerase Chain Reaction) 複製出B. amyloliquefaciens幾丁聚醣酶基因全長。
B. amyloliquefaciens幾丁聚醣酶基因全長858個鹼基對,可轉譯出286個胺基酸的蛋白質,其在氮端有36個胺基酸訊息胜肽(signal peptide),分子量為27.5 kDa。此基因於E. coli BL21(DE3)pLysS宿主中表現,以6xHis標記親合性層析可純化具有幾丁聚醣酶活性的重組幾丁聚醣酶。
B. amyloliquefaciens幾丁聚醣酶最適pH為7.0,最適作用溫度為45℃。以水溶性幾丁聚醣為受質,幾丁聚醣酶BamyCSN酵素動力學的Km值為2.12 μg / μl,kcat為2.25x10-5 s-1。1% Tween 20對BamyCSN有些微促進活性,金屬離子大都會抑制,但1 mM NH4+ 促進BamyCSN活性。以水溶性幾丁聚醣為受質,於37℃水解16小時,以薄層色層分析其水解產物有GlcN、(GlcN)2、(GlcN)3與(GlcN)4,主要產物為(GlcN)4。
Abstract
摘要
第壹章 緒論……………………………………………… 1
第貳章 文獻回顧………………………………………….. 3
第一節 解澱粉芽孢桿菌(Bacillus amyloliquefaciens)………3
1. 芽孢桿菌屬 (Bacillus spp.) 簡介………………………….3
2. B. amyloliquefaciens 的演進……………………………….4
3. B. amyloliquefaciens 特性………………………………….5
4. B. amyloliquefaciens 的研究和發展……………………….6
第二節 幾丁質與幾丁聚醣…………………………………………8
1. 幾丁質的發現………………………………………………. 8
2. 幾丁質的分布………………………………………………. 8
3. 幾丁質與幾丁聚醣的分子結構……………………………. 9
4. 幾丁質的理化特性…………………………………………. 10
5. 幾丁寡醣的製備……………………………………………. 11
6. 幾丁寡醣在醫學上的應用…………………………………. 11
第三節 幾丁質水解酵素……………………………………………15
1. 幾丁質水解酵素的天然分布………………………………. 15
2. 幾丁質水解酵素的分類……………………………………. 15
3. 幾丁質酶與幾丁聚醣酶的比較……………………………. 20
4. 幾丁聚醣酶活性測定方法…………………………………. 21
5. 幾丁聚醣酶家族……………………………………………. 22
6. 幾丁聚醣酶的水解模式……………………………………24
7. 幾丁聚醣酶水解的反應機制………………………………25
8. 細菌幾丁聚醣酶的結晶分析………………………………26
第參章 實驗材料與方法………………………………….. 34
第一節 實驗材料與實驗藥品………………………………………34
1. 實驗材料……………………………………………………. 34
2. 藥品…………………………………………………………. 39
第二節 解澱粉芽孢桿菌幾丁聚醣酶基因之選殖…………………40
1. 菌株培養及染色體DNA的製備…………………………….. 40
2. 小量製備質體DNA…………………………………………. 42
3. 專一性引子的設計………………………………………….. 43
4. 聚合酶鏈反應……………………………………………….. 44
5. 洋菜膠體電泳……………………………………………….. 44
6. 由洋菜膠體回收PCR產物DNA……………………………. 45
7. 核苷酸自動定序……………………………………………... 46
8. 重組幾丁聚醣酶表現載體之構築…………………………… 46
9. 限制酶之剪切..………………………………………………. 46
10. 表現載體 pET-20b(+) 與 BamyCsn 的接合反應…….…… 47
11. E. coli Novablue 受容細胞的製備…………………………47
12. 細菌的轉形………………………………………………… 48
13. 快速篩選…………………………………………………… 48
14. 核苷酸自動定序……………………………………………. 49
第三節 B. amyloliquefaciens幾丁聚醣酶重組蛋白質之表現與純化…………………………………………………………. 50
1. 表現寄主大腸桿菌 BL21(DE3)pLysS 的轉形……………… 50
2. 重組幾丁聚醣酶基因表現之誘導及粗重組蛋白質的製備…. 50
3. 蛋白質的定量……………………………………………….. 51
4. 聚丙烯醯胺膠蛋白質電泳…………………………………... 52
5. 蛋白質染色………………………………………………….. 53
6. 蛋白質的六個組胺酸標記染色………………………………. 53
7. 膠片乾燥…………………………………………………….. 54
8. 重組幾丁聚醣酶蛋白質的純化……………………………… 54
9. 蛋白質去鹽………………………………………………….. 55
10. 蛋白質濃縮………………………………………………… 55
第四節 幾丁聚醣酶之生化活性測試………………………………56
1. 幾丁聚醣酶活性測定………………………………………... 56
2. 最適pH 的測定…………………………………………….. 57
3. 最適溫度的測定……………………………………………... 58
4. 酵素動力學的分析…………………………………………... 58
5. 化學藥劑對酵素活性的影響………………………………... 58
6. 金屬離子對酵素活性的影響………………………………... 59
7. 溫度對 BamyCSN 活性的影響……………………………. 59
8. pH對 BamyCSN 活性的影響……………………………… 60
9. 水解產物的分析……………………………………………... 60
第肆章 實驗結果…………………………………………. 62
第一節 解澱粉芽孢桿菌 B. amyloliquefaciens 幾丁聚醣酶基因之選殖與表現……………………………………………………. 62
1. B. amyloliquefaciens 幾丁聚醣酶基因全長之選殖……….62
2. B. amyloliquefaciens 幾丁聚醣酶重組蛋白質的表現及其純化………………………………………………………….. 63
第二節 重組幾丁聚醣酶的生化特性………………………………89
1. D(+)-Glucosamine螢光衍生物螢光強度波長掃描……….. 89
2. GlcN之標準曲線…………………………………………… 89
3. BamyCSN之最適作用溫度與最適作用pH……………….. 89
4. 酵素動力學分析……………………………………………. 89
5. 化學藥劑對BamyCSN的影響…………………………….. 90
6. 金屬離子對BamyCSN的影響…………………………….. 90
7. 溫度對BamyCSN活性的影響…………………………….. 91
8. pH對BamyCSN活性的影響………………………………. 91
9. 水解產物的分析……………………………………………. 91
第伍章 討論與結論……………………………………….. 105
第一節 討論…………………………………………………………105
1. B. amyloliquefaciens幾丁聚醣酶基因的選殖……………...105
2. BamyCSN之生化特性……………………………………… 105
第二節 結論…………………………………………………………109
第陸章 未來展望………………………………………….. 110
第柒章 參考文獻………………………………………….. 112
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