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論文名稱: | Aeromonas salmonicida MAEF108 所產 Agarase AS-IIIb 基因轉殖至大腸桿菌與酵母菌及其表現
| Molecular Cloning and Expression of Aeromonas salmonicida MAEF108 Agarase AS-IIIb in Escherichia coli and Yeast
研究生: | 許志維 Chih-Wei Hsu
指導教授: | 潘崇良 Chorng-Liang Pan
學位類別: | 碩士
學校名稱: | 國立臺灣海洋大學
系所名稱: | 食品科學系
學號: | M95320059
畢業學年度: | 96
語文別: | 中文
關鍵字: | 洋菜酶 agarase
| 基因轉殖 RF cloning
| 大腸桿菌 Escherichia coli
全文影像連結: | | (本論文20110729對校內公開)
| 論文全文(pdf)

論文頁數: | 144
摘要: | 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酶剪切位基因選殖法 (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 g 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 g DNA..
論文目次: | 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、洋菜酶生產菌株及其分離來源    03
| 三、洋菜酶對洋菜水解的作用機制    04
| 四、洋菜酶之生化特性    06
| 4-1 溫度和 pH 值    06
| 4-2 分子質量    06
| 4-3 金屬離子    07
| 五、洋菜酶的應用    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、洋菜酶基因的選殖    11
| 6-1 大腸桿菌做為宿主之轉殖系統    11
| 6-2 無限制酶剪切位基因選殖 (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 聚合酶鏈鎖反應試劑    26
| 1-2-5 酵素及蛋白酶抑制劑    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 洋菜酶反應基質    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 洋菜酶之生產    35
| 2-2-2 洋菜酶之濃縮純化    35
| 2-2-3 洋菜酶的活性測定    36
| 2-2-4 洋菜酶活性單位定義    36
| 2-2-5 蛋白質的定量    36
| 2-2-6 分子量的鑑定    36
| 2-2-7 洋菜酶 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    無限制酶剪切位基因選殖 (RF cloning) 及 DpnI 限制酶剪切    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、洋菜酶之生化性質    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 所產洋菜酶純化表    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、洋菜酶 AS-IIIb 及 rAS-IIIb 之生化特性表    81
|
|
| 圖目錄
|
|
| 圖一、    A. salmonicida MAEF108 所產洋菜酶純化過程之
| 12.5% SDS-PAGE 電泳分析    82
| 圖二、    A. salmonicida MAEF108 所產洋菜酶 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 的基本構造及洋菜酶作用位置    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、      微生物所產洋菜酶分解洋菜之產物    115
| 附錄四、      洋菜酶的生化特性    117
| 附錄五、      RF cloning 的實驗原理流程圖    119
| 附錄六、      pET-21a(+) 的限制酶切位及基因圖譜    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 的限制酶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
|
參考文獻: | 柒、參考文獻
| 吳紹祺。1999。海洋細菌所產 agarases 生產條件與海藻多醣經 agarase 分解所得寡醣類組成之探討。國立臺灣海洋大學食品科學系碩士學位論文，基隆，臺灣。
| 吳紹祺。2005。海藻多醣洋菜酶水解物及其發酵產物之生理活性研究。國立臺灣海洋大學食品科學系博士學位論文，基隆，臺灣。
| 許文浩。2002。臺灣酒類發酵微生物之研究。食品工業。34(1): 20-26。
| 莊志仁。2005。食用膠之技術與應用。初版。華香園出版社，台北，台灣。pp. 48-50。
| 陳正賢。2002。發酵李子酒製程之研究。輔仁大學食品營養學系研究所碩士學位論文。臺北。
| 陳君如。2006a。海菜酒之製備及其生理活性功能探討。國立臺灣海洋大學食品科學系碩士學位論文，基隆，臺灣。
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