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    題名: 乳酸菌α-半乳糖水解酵素之純化與鑑定
    其他題名: Purification and characterization of α-D-galactosidase from lactobacillus sporogenes
    作者: 陳奕任;Chen, Yi-jen
    貢獻者: 淡江大學化學學系碩士班
    簡素芳;Chien, Su-fang
    關鍵詞: α-半乳糖水解酵素;芽胞乳酸菌;快速蛋白質液相層析法;血型轉換;紅血球細胞;α-D-Galactosidase;Lactobacillus sporogenes;FPLC;Conversion;erythrocyte
    日期: 2005
    上傳時間: 2010-01-11 02:36:29 (UTC+8)
    摘要: 本實驗以基因工程的方法,將稻米α-galactosidase基因接到質
    體上(pLDHEB),並轉殖入乳酸菌(Lactobacillus sporogenes)表現出較多的α-半乳糖水解酵素。經由大量製備純化後予以定性和定量分析,並測試B型紅血球轉變為O型紅血球之情形。
    在最佳條件下培養乳酸菌,並利用超音波細胞破碎後,1升的菌液可以得到1.2 g、40單位的粗製蛋白質。再由液相層析方法使用分子篩管柱層析(Sephadex G-150、Sephacryl S-100)、陰離子交換樹脂(DEAE Sepharose FF)與疏水性管柱層析(Phenyl Sepharose FF)等
    過程純化,經由這4種管柱之總純化倍率為955倍,回收率為69%,比
    活性為44.9 units/mg。
    在定性分析方面,由SDS-PAGE與Native PAGE判斷α-半乳糖水解酵素
    之分子量為60 KDa;其最適合的pH值為7.7;對pH值之穩定性在6.4 ~ 8.7;在45°C以下對熱較為穩定。
    在一般測試的情況下,以0.3 unit的純化酵素在1小時可將70%的B型紅血球轉為O型紅血球。我們的最終目標是要能有更好的酵素將紅血球完全的轉型才可以用於輸血。
    By way of gene cloning method, we have cloned the rice α-Galactosidase gene into a vector(pLDHEB) and transformed into Lactobacillus sporogenes.The transformant which contains a gene encoded forα-Galactosidase was cultured at 30°C in TSB medium. α-Galactosidase was expressed intracellularly. We obtained 40-60 units per liter of culture, without secreted protein was observed. Six-hour culture was centrifuged andsonicated in order to break the cell. The enzyme was extracted and concentrated.
    We use the AKTA FPLC system to purify the enzyme. This apparatus was used for gel filtration(Sephacryl S-100)、ion exchange(DEAE Sepharose) and hydrophobic interaction(Phenyl Sepharose)column chromatographies. It results in 955 folds of purification with 69% recovery and 44.9 unit/mg of specific activity.
    From SDS-PAGE, enzyme protein shows as a major band.Its molecular weight is estimated about 60KDa. For native gel,a yellow band shows that the enzyme activity truly exist. The pH optimum of enzyme is at pH7.7; it is stable at pH between 6.4-8.7 and rather stable at the temperature below 45°C.
    For enzyme conversion of B red blood cells into O red blood cells, under the standard testing condition, it reveals that it could convert about 70% of B erythrocytes into group O cells. Our goal is to make that total conversion in order to make transfusion in human.
    顯示於類別:[化學學系暨研究所] 學位論文

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