本研究之目的在於探討 Aeromonas salmonicida MAEF108 所產的 Agarase AS-IIIb (AS-IIIb) 經純化、N 端胺基酸定序，比對 MAEF108 genomic DNA 序列以確認 AS-IIIb 的完整 DNA 序列，依序列設計專一性引子，以 RF cloning 進行基因轉殖至大腸桿菌及酵母菌的實驗，轉殖表現後萃取純化 recombinant Agarase AS-IIIb (rAS-IIIb)，分析其生化特性及其 agarose 水解液產物組成。AS-IIIb 的 N 端胺基酸定序結果比對 MAEF108 genomic DNA 序列，得知AS-IIIb 基因全長為 834 bp，可轉譯出 269 個胺基酸，分子質量約 29.6 kDa 的成熟型 AS-IIIb。使用 pET 表現系統以及無限制?剪切位基因選殖法 (RF cloning)，成功建立起二株 rAS-IIIb 轉殖株 agaI 以及 agaII。二株轉殖株同時具有胞內及胞外 agarase 酵素活性的表現，且不需添加誘導劑 isopropyl β-D-1-thiogalactopyranoside (IPTG) 即可表現活性，但添加 IPTG 可增加其表現量。胞外 agarase 產量低，需經過 80-100 倍濃縮後才可測得酵素活性，後續實驗皆以轉殖株 agaII 進行。rAS-IIIb 的最適誘導培養條件是在 30oC 下培養 3 小時，酵素活性表現 1.57 U/mg。AS-IIIb 與 rAS-IIIb 分別在 40oC 及 50oC 反應條件下具有最佳的活性表現，但二者的最適反應 pH 值均為 pH 6。AS-IIIb 及 rAS-IIIb 在反應基質中添加 5 mM Mn2+ 後皆可增加其活性表現，但添加 5 mM Fe3+ 可同時完全抑制兩者之活性表現。在反應基質中添加 5 mM Ca2+ 進行反應可增加 rAS-IIIb 之活性表現，但卻會抑制 AS-IIIb 27.6% 的活性表現。0.2% agarose 分別以 AS-IIIb 及 rAS-IIIb 進行水解，所得水解產物以 Thin-layer chromatography (TLC) 及 High-performance liquid chromatography (HPLC) 進行分析，二者皆可得到二種主要的水解產物，分子大小介於neoagarohexaose (NA6) 及 neoagarobiose (NA2) 之間，HPLC 分析結果顯示兩個水解產物分別與 neoagarotetraose (NA4) 及 NA6 具有近似的 retention time。將 Agarase AS-IIIb 基因以 RF cloning 模式轉殖到酵母菌表現系統的 pPICZ�� B 載體，並成功獲得一株 DNA 序列正確無誤之 E. coli 轉殖株 Y-aga。以電穿孔的方法將 Y-aga 質體轉形至酵母菌 X-33 Pichia strain 的實驗結果已初步觀察到電轉形株菌落形成，經過計算後其電轉形率為每 �慊 DNA 可得到 10.9 株電轉形株。

The study was aimed to purify Agarase AS-IIIb (AS-IIIb) produced from Aeromonas salmonicida MAEF108 and identified the N-terminal sequence of AS-IIIb. According to the full genomic DNA sequencing results of Aeromonas salmonicida MAEF108 and N-terminal amino acid sequencing result of AS-IIIb, the full DNA sequence of AS-IIIb gene was confirmed. Specific primers were designed to clone the AS-IIIb gene into E. coli and yeast by restriction-free (RF) cloning. The recombinant Agarase AS-IIIb (rAS-IIIb) was extracted from expression host to analyze the biochemical properties and the agarose hydrolytic products composition. The Agarase AS-IIIb gene consists of 834 bp, and encodes a protein of 269 amino acids in mature type with molecular weight about 27.6 kDa. By using pET expression system and RF cloning method, two rAS-IIIb clones, agaI and agaII were constructed; both have extra- and intracellular agarase activity. Further study was focused on the agaII clone. The agarase can be produced without adding IPTG as inducer, but its addition will improve the expression on agarase activity. The extracellular agarase activity is too low to be detected in culture broth before being concentrated into 80-100 folds. The optimal agarase expression induced condition is at 30oC for 3 hr, the enzyme activity is 1.57 U/mg. AS-IIIb and rAS-IIIb are optimally active at 40oC and 50oC, respectively; both are optimally active at pH 6. The activities of two agarases were both increased by 5 mM Mn2+, while decreased by 5m M Fe3+. Ca2+ ion of 5mM concentration increases the activity of rAS-IIIb, but decreased 27.6% activity of AS-IIIb. Hydrolytic products of agarose by either AS-IIIb or rAS-IIIb were analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Both of the two agarases have two main hydrolytic products, which have molecular size between neoagarohexaose (NA6) and neoagarobiose (NA2); and have the similar retention time of neoagarotetraose (NA4) and NA6 separately in HPLC analysis. The Agarase AS-IIIb gene was cloned into vector pPICZ�� B (for yeast expression system) by RF cloning, and obtained a successful clone Y-aga within E. coli with correct DNA sequence. The Y-aga clone vector was transformed into X-33 Pichia strain by electroporation, the electrotransformation frequency calculated in the experiment is 10.9 transformants obtained per �慊 DNA..

目錄


目錄 i
表目錄 vi
圖目錄 vii
附錄目錄 x
中文摘要 xi
英文摘要 (Abstract) xiii
壹、前言 01
貳、文獻整理 03
一、洋菜的構造與特性 03
二、洋菜?生產菌株及其分離來源 03
三、洋菜?對洋菜水解的作用機制 04
四、洋菜?之生化特性 06
4-1 溫度和 pH 值 06
4-2 分子質量 06
4-3 金屬離子 07
五、洋菜?的應用 08
5-1 細胞的分離 08
5-2 DNA的分離 09
5-3 藻類原生質體的製作 09
5-4 海藻多醣的生產與黏度降低 10
5-5 藻類組成的分析 10
5-6 融合瘤細胞的分離 11
六、洋菜?基因的選殖 11
6-1 大腸桿菌做為宿主之轉殖系統 11
6-2 無限制?剪切位基因選殖 (Restriction-free cloning) 12
七、酵母菌簡介 13
7-1 營養需求 13
7-2 菌種 13
7-3 發酵過程之生化反應 14
7-4 基因轉殖之應用 15
7-5 胞內重組蛋白之表現 17
7-6 胞外分泌重組蛋白之表現 17
八、海藻寡醣組成成分鑑定 18
8-1 薄層層析法 (Thin-layer chromatography, TLC) 18
8-2 高效能液相層析法 (High-performance liquid chromatography, HPLC) 19
8-3 磁核共振光譜法 (Nuclear magnetic resonance, NMR) 19
8-4 液相層析串連質譜分析法 (Liquid Chromatography/
Mass Spectrometry/Mass Spectrometry， LC-MS-MS) 20
參、實驗設計 22
肆、實驗材料與方法 23
一、實驗材料 23
1-1 實驗菌株 23
1-2 試驗藥品 23
　　　　　1-2-1 藥品 23
　　　　　1-2-2 載體 26
　　　　　1-2-3 引子 26
　　　　　1-2-4 聚合?鏈鎖反應試劑 26
　　　　　1-2-5 酵素及蛋白?抑制劑 27
　　　　　1-2-6 DNA 及蛋白質萃取純化套裝試劑 27
　　　　　1-2-7 電泳標準品 27
　　　　　1-2-8 培養基組成 28
　　　　　1-2-9 洋菜?反應基質 30
　　　　　1-2-10 DNS 溶液 30
　　　　　1-2-11 電泳膠片配製 30
　　　　　1-2-12 電泳溶液 31
　　　　　1-2-13 無菌過濾 32
　　　　　1-2-14 離心式超過濾濃縮 32
　　　　　1-2-15 薄層層析矽膠片 (TLC Silica gel) 32
　　　　1-3 儀器設備 32
二、實驗方法 33
　　　　2-1 菌種保存與活化 33
　　　　　2-1-1 菌株保存 34
　　　　　2-1-2 菌株活化 34
　　　　2-2 Agarase AS-IIIb的純化分離 35
　　　　　2-2-1 洋菜?之生產 35
　　　　　2-2-2 洋菜?之濃縮純化 35
　　　　　2-2-3 洋菜?的活性測定 36
　　　　　2-2-4 洋菜?活性單位定義 36
　　　　　2-2-5 蛋白質的定量 36
　　　　　2-2-6 分子量的鑑定 36
　　　　　2-2-7 洋菜? AS-IIIb N 端胺基酸定序 37
　　　　2-3 Agarase AS-IIIb 基因以大腸桿菌系統進行選殖 37
　　　　　2-3-1 MAEF108 染色體 DNA 的萃取純化 37
　　　　　2-3-2 Agarase AS-IIIb 完整基因序列比對 38
　　　　　2-3-3 引子設計 38
　　　　　2-3-4 無限制?剪切位基因選殖 (RF cloning) 及 DpnI 限制?剪切 38
　　　　　2-3-5 勝任細胞 (competent cell) 之製作 41
　　　　　2-3-6 熱休克 (heat shock) 轉形 41
　　　　　2-3-7 利用 PCR 確認插入之基因片段大小 42
　　　　　2-3-8 DNA 序列比對 43
　　　　2-4 Agarase 基因以酵母菌系統進行選殖 43
　　　　　2-4-1 質體接受細胞製作 43
　　　　　2-4-2 電穿孔轉形 44
　　　　2-5 Recombinant Agarase AS-IIIb的誘導表現 44
　　　　　2-5-1 Recombinant Agarase AS-IIIb的萃取純化 44
　　　　　2-5-2 最適誘導溫度及時間 45
　　　　　2-5-3 IPTG 最適誘導濃度 46
　　　　2-6 Agarase AS-IIIb 之生化性質測試 46
　　　　　2-6-1 最適作用溫度 46
　　　　　2-6-2 熱安定性 46
　　　　　2-6-3 最適作用 pH 值 47
　　　　　2-6-4 pH 值的安定性 47
　　　　　2-6-5 金屬離子的影響 47
　　　　2-7 Agarase AS-IIIb之水解產物分析 47
　　　　　2-7-1 TLC 鑑定 47
　　　　　2-7-2 HPLC 鑑定 48
伍、結果與討論 49
　　一、Agarase AS-IIIb 的純化分離 49
　　二、Agarase AS-IIIb 基因大腸桿菌系統轉殖與表現 49
　　三、Recombinant Agarase AS-IIIb 基因誘導與表現 51
　　四、洋菜?之生化性質 54
　　　　4-1 最適作用溫度及溫度安定性 54
　　　　4-2 最適作用 pH 值及 pH 值安定性 55
　　　　4-3 金屬離子之影響 57
　　　　4-4 水解產物分析 58
　　五、Agarase AS-IIIb 基因酵母菌系統轉殖與表現 58
陸、結論 60
柒、參考文獻 62
捌、附錄 112

表目錄


表一、 A. salmonicida MAEF108 所產洋菜?純化表 73
表二、 RF cloning 所使用之引子及其序列 74
表三、不同培養溫度對 rAS-IIIb 其表現於胞內、胞外酵素活性之
影響 75
表四、不同培養溫度及添加 IPTG 對 rAS-IIIb 其表現於胞內酵素
活性之影響 76
表五、不同培養溫度對 rAS-IIIb 其表現於胞內酵素活性之影響 77
表六、不同 IPTG 添加濃度對 rAS-IIIb 其表現於胞內酵素活性之
影響 78
表七、金屬離子對 Agarase AS-IIIb 酵素活性之影響 79
表八、金屬離子對 recombinant Agarase AS-IIIb 酵素活性之
影響 80
表九、洋菜? AS-IIIb 及 rAS-IIIb 之生化特性表 81


圖目錄


圖一、 A. salmonicida MAEF108 所產洋菜?純化過程之
12.5% SDS-PAGE 電泳分析 82
圖二、 A. salmonicida MAEF108 所產洋菜? AS-IIIb 之
12.5% SDS-PAGE 電泳分析 83
圖三、 MAEF108 agarase AS-IIIb DNA 序列所演繹之胺基酸
序列 84
圖四、 Agarase AS-IIIb 的完整 DNA 序列 85
圖五、 RF cloning 實驗過程中各階段之 PCR產物其 1%
洋菜膠電泳分析 86
圖六、 MAEF108 agarase AS-IIIb 轉殖株 (agaI clone) 基因定
序序列 87
圖七、 Agarase AS-IIIb 轉殖株 (agaI clone) 與 MAEF108
agarase AS-IIIb 基因序列比對 88
圖八、 MAEF108 agarase AS-IIIb、Pseudoalteromonas
atlantica ��-agarase 與 Aeromonas sp. ��-agarase
胺基酸序列比對 89
圖九、 轉殖株 agaI 及 agaII 之 Agarase AS-IIIb DNA 之
序列比對 90
圖十、 成熟型 Agarase AS-IIIb 基因之 E. coli BL21 (DE3)
轉殖株 91
圖十一、 成熟型 agarase AS-IIIb 基因之 E. coli BL21 (DE3)
轉殖株 (agaII clone) agarase 活性表現確認 92
圖十二、 成熟型 agarase AS-IIIb 基因之 E. coli BL21 (DE3)
轉殖株不具 agarase 活性表現之菌落轉劃培養確認 93
圖十三、 AgaII clone 及 pET21b 上 T7 promoter 及 lac
operator 區域 DNA 序列比對 94
圖十四、 AgaI clone 其胞外 agarase 酵素活性表現確認及
IPTG 對生長之影響 95
圖十五、 MAEF 108 agarase AS-IIIb 及以鎳親和管柱純化之重
組 agarase AS-IIIb其 12.5% SDS-PAGE 電泳分析圖 96
圖十六、 AgaII clone 及帶有 vector pET21b E. coli BL21 (DE3)
之胞內萃取蛋白的 12.5% SDS-PAGE 電泳分析圖 97
圖十七、 Agarase AS-IIIb 的最適作用溫度 98
圖十八、 Recombinant Agarase AS-IIIb 的最適作用溫度 99
圖十九、 溫度對 Agarase AS-IIIb 及 recombinant Agarase
AS-IIIb 酵素活性安定性的影響 100
圖二十、 Agarase AS-IIIb 的最適作用 pH 值 101
圖二十一、Recombinant agarase AS-IIIb 的最適作用 pH 值 102
圖二十二、pH 值對 Agarase AS-IIIb 及 recombinant Agarase
AS-IIIb 酵素安定性之影響 103
圖二十三、Agarase AS-IIIb 水解 agarose 0-24 小時之薄層色層
分析圖譜 104
圖二十四、Agarase rAS-IIIb 水解 agarose 0-24 小時之薄層色層
分析圖譜 105
圖二十五、Agarase AS-IIIb 及 rAS-IIIb水解 agarose 之薄層
色層分析圖譜 106
圖二十六、Agarase AS-IIIb 與 0.2% agarose 反應 0-24 hr 之
高效能液相層析圖 107
圖二十七、Agarase rAS-IIIb 與 0.2% agarose 反應 0-24 hr 之
高效能液相層析圖 108
圖二十八、RF cloning 實驗過程中各階段之 PCR產物之 1%
洋菜膠電泳分析 109
圖二十九、轉殖株 agaI 及 Y-aga 之 Agarase AS-IIIb DNA 序列
比對 110
圖三十、 以電穿孔法將 Y-aga 轉殖株載體電轉形至酵母菌
X-33 Pichia strain 111


附錄目錄


附錄一之一、 Agarose 的基本構造及洋菜?作用位置 112
附錄ㄧ之二、 Agaropectin可能組成基本單位構造 112
附錄二、 Agarases 的生產微生物、種類及分離來源 113
附錄三、 微生物所產洋菜?分解洋菜之產物 115
附錄四、 洋菜?的生化特性 117
附錄五、 RF cloning 的實驗原理流程圖 119
附錄六、 pET-21a(+) 的限制?切位及基因圖譜 120
附錄七、 pET-21a-d (+) 上基因片段插入位區域的 DNA
序列 121
附錄八、 pET 重組蛋白表現系統原理圖 122
附錄九、 Agarase AS-IIIb 基因大腸桿菌表現系統選殖反應
結果彙整表 123
附錄十、 新洋菜四糖 (RT = 26.32 min) 與新洋菜二糖
(RT = 28.16) 之高效能液相層析圖譜 124
附錄十一、 新洋菜六糖 (RT = 24.81 min) 之高效能液相層析
圖譜 125
附錄十二、 pPICZ�� B 的限制?切位及部份基因圖譜 126
附錄十三、 pPICZ�� B上基因片段插入位區域的 DNA 序列 127
附錄十四、 Agarase AS-IIIb 基因酵母菌表現系統選殖反應結
果彙整表 128

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