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研究生:呂尚潔
研究生(外文):Shang-Chieh Lu
論文名稱:星天牛(Anoplophoramalasiaca)新型纖維酵素之選殖、桿狀病毒系統表達及其活性分析
論文名稱(外文):Molecular Cloning, Baculovirus Expression, and Characterization of Novel Cellulases Isolated from White Spotted Longicorn Beetle, Anoplophora malasiaca
指導教授:陳健尉陳健尉引用關係趙裕展
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
畢業學年度:96
語文別:中文
論文頁數:82
中文關鍵詞:纖維酵素星天牛桿狀病毒
外文關鍵詞:cellulasecDNA libraryAnoplophora malasiacaglycoside hydrolase family(GHF)baculovirus
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Cellulose is the most abundant nature resource for the generation of bio-energy. Although numerous cellulases have been discovered and characterized from microbes, such as bacteria and fungi, only very few of them were isolated from higher animals. Insects are a group of high efficient cellulose consumers, and we have cloned three novel endogenous cellulases, Am-EGase 1, 2, and 3 from longicorn, Anoplophora malasiaca, with low general sequence homology with other known cellulases. Am-EGase 1 encodes 239 aa residues and sequence alignment of this gene showed a 75.7 % identity to cellulase of another longicorn, Apriona germari, which belongs to the glycosyl hydrolase families (GHF) 45. The Am-EGase 2 gene encodes 284 aa residues and Am-EGase 3 gene encodes 329 aa residues. Both genes exhibit sequence similarity with the cellulase isolated from Psacothea hilaris which belongs to GHF 5; the first one showed a 80.4 % identity and the other showed only 58.3 % identity. Through assays of these newly-identified cellulases using baculovirus expression system, the unique functions of these three novel cellulase genes were characterized. Our results also showed that the silkworm can be used as a very cheap and efficient bio-reactor to replace the conventional expensive cell cultural fermenters for the production of these valuable enzymes for future practical applications. Since the use of cellulose does not consume for food sources, the isolation of new insect cellulolytic enzyme genes will be significant for the bio-renewable energy research using rice straw or wood chips for our bright future.
誌謝 I
中文摘要 II
Abstract III
目錄 IV
圖目錄 VII
附圖、表目錄 IX
第一章、前言 1
第二章、文獻探討 2
一、纖維素(cellulose) 2
二、纖維酵素水解之前處理 3
1. 物理方法 3
2. 化學方法 4
3. 生物法 5
三、纖維酵素(cellulase) 5
1. 內切型纖維酵素(Endo-β-1,4-glucanase, E.C. 3.2.1.4.) 6
2. 外切型纖維酵素(Exo-β-1,4-glucanase, E.C. 3.2.1.91.) 6
3. β-葡萄糖苷酵素(β-1,4-glucosidase, E.C. 3.2.1.21.) 6
四、纖維酵素協同作用機制 6
五、可分解纖維素的物種 7
六、星天牛(Anoplophora macularia) 8
七、桿狀病毒和家蠶表達系統 9
1. 桿狀病毒的構造 9
2. 分類 9
3. 生活史 10
4. 桿狀病毒的應用優勢 11
5. 家蠶表現系統 12
八、纖維酵素的應用 13
九、研究目的 14
第三章、實驗材料與方法 15
一、昆蟲 15
二、純化訊息核糖核酸(mRNA) 15
三、建構互補去氧核醣核酸基因庫(cDNA library) 16
四、設計消退引子組(degenerate primer sets)和探針(probe)的製備 18
五、菌落雜合(colony hybridization) 18
六、互補去氧核醣核酸末端快速擴增技術(rapid amplification of cDNA ends , RACE) 19
七、重疊聚合酵素鏈鎖反應 (overlapping PCR) 20
八、序列分析及比對(sequence analysis and alignment) 21
九、細胞培養 21
十、建構轉移載體(transfer vector) 21
十一、構築重組病毒(recombinant virus) 22
十二、純化重組桿狀病毒 23
十三、感染家蠶並收集體液 23
十四、西方式墨點法(Western blotting) 24
十五、酵素活性測定 25
十六、酵素特性分析 26
十七、衣黴素(Tunicamycin) 抑制蛋白質醣化之研究 27
第四章、結果 28
一、從去氧核醣核酸基因庫(cDNA library)選殖(cloning)全長纖維素酶基因 28
二、纖維素酶基因的序列比對(sequence alignment) 及譜系分析(phylogenetic analysis) 29
三、利用桿狀病毒表現載體系統於細胞中表達纖維素酶基因 31
四、細胞內纖維素酶活性分析 31
五、家蠶過表現纖維素酶並分析其特性 32
六、纖維酵素間與β-葡萄糖苷酵素之協同作用 33
七、酵素N-醣基化分析 35
第五章、實驗討論 37
第六章、結論 42
第七章、圖表 43
第八章、附圖表 69
第九章、參考文獻 78

圖目錄
圖一、基因載體pD-hxpaR2圖示 43
圖二、轉移載體pBpXhE圖示 44
圖三、星天牛纖維素酶基因篩選流程圖 45
圖四、以菌落雜合法篩選去氧核醣核酸基因庫 46
圖五、Am-EGase 1之蛋白質序列和目前已知的纖維酵素進行多重序列比對 47
圖六、醣苷水解酶家族45之演化樹狀圖 48
圖七、比較Am-EGase 1 和其它物種纖維酵素的相似度百分比 49
圖八、多重序列比對Am-EGase 2和Am-EGase 3之蛋白質序列與目前已知的纖維酵素 50
圖九、醣苷水解酶家族5之演化樹狀圖 51
圖十、Am-EGase 2 和目前已知之纖維素酶間的相似度百分比 52
圖十一、Am-EGase 3和不同物種的纖維素酶之相似度比較 53
圖十二、纖維酵素表達於桿狀病毒感染之細胞株 54
圖十三、測試由Bm細胞所生產的纖維酵素活性 55
圖十四、經家蠶表現系統生產之纖維酵素活性比較 56
圖十五、纖維酵素於不同pH值下的特性分析 57
圖十六、纖維酵素最適合反應溫度測定 58
圖十七、纖維酵間與β-葡萄糖苷酵素分解羧甲基纖維素之協同作用 59
圖十八、纖維酵素與β-葡萄糖苷酵素分解微晶纖維素之協同作用 60
圖十九、纖維酵素與β-葡萄糖苷酵素分解濾紙之協同作用 61
圖二十、於昆蟲細胞內分析Am-EGase 1的N-醣基化 62
圖二十一、於昆蟲細胞內分析N-醣基化對於Am-EGase 1酵素活性之影響 63
圖二十二、分析Am-EGase 2於昆蟲細胞內的N-醣基化現象 64
圖二十三、於昆蟲細胞內分析N-醣基化對於Am-EGase 2酵素活性之影響 65
圖二十四、測試Am-EGase 3 的N-醣基化修飾 66
圖二十五、於昆蟲細胞內分析N-醣基化對於Am-EGase 3酵素活性之影響 67
圖二十六、細胞株及家蠶生產的纖維酵素活性比較圖 68

附圖、表目錄
附圖一、由木質纖維素(lignocellulose)轉為乙醇的多步驟整理 69
附表一、木質纖維素於不同植物及來源中纖維素、半纖維素和木質素的平均含量百分比 70
附圖二、纖維素的基本構造 71
附圖三、纖維素Ⅰ(a)及纖維素Ⅱ(b)的結構圖 72
附表二、總結目前生物法、物理法和化學法三大類纖維素前處理方法的主要作用來源與機制 73
附圖四、纖維素酶協同作用機制 74
附圖五、圖示主要類型的包涵體病毒 75
附圖六、桿狀病毒感染週期 76
附圖七、藉著重疊聚合酵素鏈鎖反應的方式將兩段基因融合 77
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