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研究生:陳姵如
研究生(外文):Pei-Ju Chen
論文名稱:腸炎弧菌 Vibrio parahaemolyticus α 澱粉酶基因選殖、表現及其蛋白質工程
論文名稱(外文):Cloning, Expression and Protein Engineering of α-amylase Gene from Vibrio parahaemolyticus
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:2010
畢業學年度:98
語文別:中文
論文頁數:130
中文關鍵詞:腸炎弧菌α 澱粉酶
外文關鍵詞:Vibrio parahaemolyticusα-amylase
相關次數:
  • 被引用被引用:1
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  • 下載下載:62
  • 收藏至我的研究室書目清單書目收藏:0
本研究自腸炎弧菌 (Vibrio parahaemolyticus) BCRC13026的染色體 DNA 選殖出 α 澱粉酶基因。專一性 PCR 引子設計,參考 NCBI 基因庫中腸炎弧菌 AQ3810 染色體核苷酸序列,檢索號 NZ_AAWQ01000007 之核苷酸位置 8896 至 10419,註解為 α 澱粉酶之基因。將基因構築於 pET20b(+) 後轉形至大腸桿菌表現系統,成功純化出分子大小約 53 kDa,具有 α 澱粉酶活性的重組澱粉酶 (VpAmy)。重組 VpAmy 之最適反應 pH 值為 6,最適反應溫度為 60 ℃,耐熱性可達 60 ℃,鈣離子可提高其耐熱性至 70 ℃。為擴大 VpAmy 的受質範圍及改變水解產物為麥芽寡糖,故將VpAmy與自 Thermoanaerobacterium saccharolyticum NTOU1 選殖出之澱粉普魯南糖酶 (TsaNTOU1Apu) 的催化活性區置換,所得之嵌合酵素 (VpAmy-Chimera) 於 pET20b(+) 大腸桿菌表現系統表現,結果目標蛋白表現於內涵體,且不具酵素活性,但經胜肽質量指紋 (PMF) 分析確認。VpAmy 之 Aamy C 功能區於基質之結合或水解能力所扮演的角色,可由碳端定位修剪觀察。Aamy C 功能區完全去除之 VpAmyK424 仍具有酵素活性,但蛋白質產量遠低於 VpAmy,難以進行生化特性之比較。嘗試以 pET15b 做過量表現,仍多表現於不溶性內涵體內,可溶部分產量並未提高。
The α-amylase gene from Vibrio parahaemolyticus BCRC13026 (VpAmy) was cloned and verified by sequencing. The cloning primers were derived from the putative α-amylase (EC 3.2.1.1) of V. parahaemolyticus AQ3810 genome, according to the nucleotides from 8896 to 10419 of GenBank accession number NZ_AAWQ01000007. The VpAmy gene encoding a 53 kDa protein with 507 amino acids was expressed using pET20b(+) vector. The recombinant VpAmy had α-amylase activity with the optimal pH and temperature, pH 6 and 60 ℃, respectively. The thermostability of VpAmy was enhanced to 70 ℃ in the presence of 5 mM Ca2+. In order to expand the substrate specificity and alter the catalytic product to malto-oligosaccharides, the catalytic domain of VpAmy was substituted with a homologous region of TsaNTOU1Apu, isolated from Thermoanaerobacterium saccharolyticum NTOU1. The chimeric enzyme, VpAmy- Chimera, was constructed and expressed in E. coli system. However, the expressed VpAmy-Chimera protein, through confirmed by PMF analysis, was found in the inclusion body without any catalytic activity. To explore the functional necessity of Aamy C domain in VpAmy, four truncated enzymes, including VpAmyK424 whose Aamy C domain was completely removed, with different length was generated from the C-terminal delection mutagenesis. The expression level of truncated enzymes was too low to compare the biochemical characteristics with full-length VpAmy, but they showed α-amylase activities. The smallest truncated protein, VpAmyK424, was cloned into pET15b and was attempted to resolve the expression problem. Again, most protein products were found in the inclusion body, confirmed by PMF analysis, and the soluble enzyme was not increased. Our results showed that the Aamy C deletion mutants still have α-amylase activity, but the detail influence of Aamy C truncation on the enzyme’s biochemical characteristics remained unclear.
誌謝 I
中文摘要 II
英文摘要 III
目 錄 IV
圖目錄 VIII
表目錄 IX
第一章 前言 1
1.1 腸炎弧菌 1
1.2 澱粉水解酵素在工業之應用 1
1.3 澱粉與普魯南糖組成 2
1.4 醣類水解酵素 2
1.4.1 澱粉酶 4
1.4.1.1 α澱粉酶 4
1.4.2 普魯南糖酶 5
1.4.2.1 澱粉普魯南糖酶 6
1.4.3 醣類水解酵素胺基酸序列分析 7
1.4.3.1 活性部位分析 7
1.4.3.2 功能部位分析 9
1.4.3.3 GH 13分子結構 11
1.4.4 水解機制 12
1.5 實驗緣起 12
第二章 材料與方法 22
2.1 實驗材料 22
2.1.1 菌株與質體 22
2.1.2 酵素 26
2.1.3 純化套組與試劑材料 26
2.1.3.1 純化套組 26
2.1.3.2 試劑材料 27
2.1.4 MW Marker 27
2.1.4.1 DNA marker製備 27
2.1.4.2 Protein marker 28
2.1.5 濃縮器及過濾器 28
2.1.6 SDS-PAGE 28
2.1.7 藥品 30
2.1.8 試劑 31
2.1.9 緩衝液配製 32
2.1.10 儀器 33
2.1.11 引子 34
2.1.12 定序 34
2.1.13 胜肽質量指紋分析 34
2.2 實驗方法 35
2.2.1 染色體DNA製備 35
2.2.2 專一性引子設計 36
2.2.3 VpAmy基因之選殖 40
2.2.4 VpAmy-Chimera澱粉酶/普魯南糖酶嵌合基因之構築 40
2.2.5 VpAmy-Chimera澱粉普魯南糖酶嵌合基因定點突變之磷酸化引子
設計 44
2.2.6 VpAmy基因之碳端Aamy C定位修剪引子設計 44
2.2.7 聚合酶鏈鎖反應 46
2.2.8 洋菜膠體電泳 47
2.2.9 質體的小量製備 48
2.2.10 限制酶剪切 48
2.2.11 載體與目標DNA接合反應 49
2.2.12 重組基因表現載體之構築 49
2.2.13 受容細胞的製備 49
2.2.14 轉形 50
2.2.15 快速篩選 50
2.2.16 重組質體DNA之檢視 51
2.2.16.1 Direct lysate PCR analysis 51
2.2.16.2 重組質體的製備 51
2.2.16.3 限制酶剪切檢視 51
2.2.16.4 聚合酶鏈鎖反應檢視 51
2.2.17 核苷酸定序 52
2.2.18 表現宿主大腸桿菌Rosetta(DE3)pLysS的轉形 52
2.2.19 重組澱粉酶基因表現之誘導及粗酵素液的製備 52
2.2.20 受質液的配製與粗酵素液活性檢測 53
2.2.20.1 澱粉受質液的配製 53
2.2.20.2 普魯南糖受質液的配製 53
2.2.20.3 澱粉酶與普魯南糖酶粗酵素液活性初步檢視 53
2.2.21 蛋白質的定量 53
2.2.21.1 蛋白質標準檢量線 53
2.2.21.2 樣品蛋白質定量 54
2.2.22 蛋白質電泳 54
2.2.22.1 聚丙烯醯胺膠蛋白質電泳 54
2.2.22.2 α 澱粉酶之活性染色 55
2.2.22.3 蛋白質染色 55
2.2.23 重組VpAmy蛋白質純化 55
2.2.24 蛋白質濃縮 56
2.2.25 酵素活性檢測 56
2.2.25.1 α澱粉酶之活性測定 56
2.2.25.2 普魯南糖酶之活性檢測 56
2.2.26 重組α澱粉酶之生化特性分析 56
2.2.26.1 最適pH 56
2.2.26.2 最適溫度 57
2.2.26.3 熱安定性與鈣離子之影響 57
2.2.26.4 基質特異性 57
2.2.26.5 水解產物薄層色層分析 57
2.2.27 基因定點突變 58
2.2.28 胜肽質量指紋分析 59
第三章 結果 60
3.1 VpAmy基因選殖與蛋白質表現 60
3.1.1 Vp Amy基因選殖 60
3.1.2 重組VpAmy蛋白質表現與純化 60
3.2 VpAmy重組蛋白質之生化特性 61
3.2.1 最適pH與最適溫度 61
3.2.2 熱安定性與鈣離子對熱安定性之影響 61
3.2.3 VpAmy對不同受質水解產物的分析 61
3.3 VpAmy與TsaNTOU1ApuM957之催化活性區置換 62
3.3.1 VpAmy-Chimera構築與選殖 62
3.3.2 VpAmy-Chimera之定點突變 62
3.3.3 VpAmy-Chimera重組蛋白質表現與純化 62
3.3.4 VpAmy-Chimera胜肽質量指紋分析 63
3.4 VpAmy碳端Aamy C定位修剪 63
3.4.1 VpamyK424、VpamyG459、VpamyG477與VpamyG498 之選殖 63
3.4.2 VpAmy全長與碳端Aamy C定位修剪後重組蛋白質之表現與純化 64
3.4.3 VpAmyK424構築於載體pET15b 64
3.4.4 VpAmyK424-15b重組蛋白質之表現與純化 64
3.4.5 VpAmyK424-15b胜肽質量指紋分析 65
第四章 討論 66
4.1 VpAmy基因選殖,蛋白質表現及其生化特性 66
4.2 VpAmy與TsaNTOU1Apu之催化活性區置換67
4.3 VpAmy碳端Aamy C功能區定位修剪69
4.4 未來展望 70
第五章 結論 71
第六章 參考文獻 113




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