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系統識別號 U0002-1608200711495100
中文論文名稱 重組稻米半乳糖水解酶之表現與鑑定
英文論文名稱 Expression and Characterization of Recombinant α-Galactosidase from Rice
校院名稱 淡江大學
系所名稱(中) 生命科學研究所碩士班
系所名稱(英) Graduate Institute of Life Sciences
學年度 95
學期 2
出版年 96
研究生中文姓名 陳詩慧
研究生英文姓名 Shi-Hui Chen
學號 694290197
學位類別 碩士
語文別 中文
口試日期 2007-07-17
論文頁數 80頁
口試委員 指導教授-簡素芳
委員-張可中
委員-莊子超
中文關鍵字 Pichia pastoris  α-galactosidase 
英文關鍵字 Pichia pastoris  α-galactosidase 
學科別分類 學科別醫學與生命科學生物學
中文摘要 半乳糖水解酶可分解多醣,水解B型紅血球表面抗原的半乳糖基,使B型紅血球轉換成O型紅血球,可用於臨床輸血。本研究將稻米半乳糖水解酶基因整合到pPIC9K質體,在轉殖到重組酵母菌(Pichia pastoris)的染色體DAN上,並以4 mg/ml G418篩選出具有multicopy的菌株,以表現稻米半乳糖水解酶。重組酵母菌之培養條件測試,分別針對培養基種類、誘導溫度、誘導起始A600值、每日甲醇量及時間間隔、誘導培養基添加額外碳源檢測其對產量的影響,得到最佳表現量,細胞內酵素單位為5.124 units/ml,細胞外酵素單位為1.127 units/ml。大量培養之細胞內及分泌酵素,經由陰離子交換樹脂(DEAE-Sepharose FF)與疏水性管柱層析(Phenyl-Sepharose FF)分離純化,純化倍率分別為74及94。純化後重組半乳糖水解酶經SDS-PAGE分析,有2種主要蛋白質大小分別為40及60 kDa。重組半乳糖水解酶特性分析,最適反應溫度為37℃、熱穩定性為50℃以下、最適反應pH為4、pH穩定性為3.0~5.0。以HPLC分析重組半乳糖水解酶水解α1→6連結的多糖特異性,Melibiose>Raffinose>Stachyose。在實驗特定條件下,以1.5 units的純化酵素在2小時內可將約50%的B型紅血球轉為O型紅血球。
英文摘要 α-Galactosidase hydrolyzed the terminal galactosyl residues from oligosaccharides including blood group B substance on the B red blood cell surface. For the blood conversion purpose, We had cloned the rice-α-galactosidase in pPIC9K vector and transformed into Pichia pastoris SMD1168. We intended to find a optimal culture condition for α-galactosidase to be expressed. We had tested the type of the medium , the start pH of the medium, the induce temperature, the starting A600 of the culture, the concentration of methanol to induce and the effects of carbon source concentration for the expression on α-galactosidase. The enzyme activity inside the cells was 5.124 units/ml; in the medium was 1.127 units/ml. Both ion exchange(DEAE-Sepharose) and hydrophobic interaction (Phenyl-Sepharose) column chromatographies were used to purify intracellular and secreted protein. They were purified 74 and 94 fold, respectively. The purified enzyme showed two major band on SDS-PAGE. The molecular weight of recombinant α-Galactosidase was estimated about 60 and 40 kDa. The maximum activity occurred at a temperature of 37℃;however, inactivation was observed at the temperature above 50℃.The enzyme showed maximal activity at pH 4.4 and was slowly inactivated above pH 5.0 and below pH 3.0. The substrate specificities of the enzyme for α1→6 linked galactose were investigated by using galactose-containing oligosaccharides: melibiose, raffinose and stachyose. The enzyme specificity of these oligosaccharides was in the decreasing order: melibiose> raffinose> stachyose. For the blood conversion test, in the experiment condition, 1.5 units of purified enzyme converted 50% B RBC into O RBC in 2 hours.
論文目次 中文摘要 Ⅰ
英文摘要 Ⅱ
目錄 Ⅲ
圖表目錄 Ⅴ

第一章 緒論
第一節 前言 1
第二節 α-半乳糖水解酶 3
第三節 嗜甲基酵母菌(P. pastoris) 7

第二章 實驗材料、設備
第一節 實驗材料 17
第二節 實驗設備 20
第三節 培養基配置 22

第三章 實驗方法
第一節 測試培養條件 25
第二節 400ml大量培養 29
第三節 酵素的萃取與純化 29
第四節 蛋白質的鑑定 32

第四章 結果與討論
第一節 不同培養條件對表現重組半乳糖水解酶的影響 40
第二節 400ml大量培養 45
第三節 酵素的萃取與純化 46
第四節 重組半乳糖水解酶的鑑定 48

第五章 結論與未來展望 73

第六章 參考文獻 76
圖表目錄
表1 利用P. pastoris 表現的異源蛋白質 16
表2 細胞內重組半乳糖水解酶純化表 63
表3 分泌重組半乳糖水解酶純化表 63
表4 以HPLC 分析重組稻米α-半乳糖水解酶水解Melibiose、
Raffinose 與Stachyose 的能力 70
圖1 α-半乳糖水解酶催化作用 12
圖2 稻米α-半乳糖水解酶結構 13
圖3 P. pastoris 利用甲醇的代謝路徑 14
圖4 異源蛋白質分泌至胞外示意圖 15
圖5 培養基種類測試 53
圖6 誘導溫度測試 54
圖7 誘導起始菌液濃度測試 55
圖8 誘導添加甲醇量測試 56
圖9 誘導添加碳源種類測試 57
圖10 誘導添加碳源量測試 58
圖11 誘導添加甲醇時間間隔測試 59
圖12 400ml 大量培養菌數及活性變化圖 60
圖13 FPLC DEAE-Sepharose Fast Flow 純化流程圖 61
圖14 FPLC Phenyl-Sepharose Fast Flow 純化流程圖 62
圖15 誘導後細胞及培養基蛋白質以SDS-PAGE 分析含量變化 64
圖16 酵素純化結果 65
圖17 酵素最適pH 值與最穩定pH 值 66
圖18 酵素熱穩定性 67
圖19 酵素產物p-nitrophenol 標準線 68
圖20 酵素動力學測試 69
圖21 測試血球所需凝血時間及量 71
圖22 B 型紅血球轉成O 型紅血球能力測試 72
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