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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/15291

Title: 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
Authors: Chih-Wei Hsu
許志維
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 洋菜酶;基因轉殖;大腸桿菌
agarase;RF cloning;Escherichia coli
Date: 2008
Issue Date: 2011-06-30T08:10:16Z
Abstract:   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..
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M95320059
http://ntour.ntou.edu.tw/ir/handle/987654321/15291
Appears in Collections:[食品科學系] 博碩士論文

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