α-半乳糖水解酵素（α-galactosidase）是一種可以將非還原端半乳糖水解的酵素，在各種生物體中都存在，並且擁有許多不同的生物功能，例如在植物中α-半乳糖水解酵素參與醣類的代謝和運輸。在應用方面，α-半乳糖水解酵素可以使B型紅血球表面抗原上的半乳糖水解而變成O型紅血球，因此藉由基因工程的方法取得基因並利用 Pichia pastoris 的蛋白質表現系統來產生大量的芋頭α-半乳糖水解酵素以進行血型轉換的測試和應用。
本研究將已轉殖入α-半乳糖水解酵素基因的酵母菌大量培養，並進行誘導一天產生蛋白質，細胞內酵素活性單位最高達到3.6 units/mL。利用水解酵母菌細胞壁的方式，將酵母菌打破取出α-半乳糖水解酵素。大量的萃取液經過濃縮之後，分別進行四步純化步驟，依序是分子篩 SephadexTM G-100管柱、陰離子交換樹脂Q SepharoseTM Fast flow管柱、疏水性吸附 HiTrap Phenyl FF (high sub), 1mL管柱，分子篩SepharoseTM 6 10/300 GL。純化後的酵素樣品經過十二烷基硫酸鈉-聚丙烯醯胺凝膠電泳與基質輔助雷射脫附離子化－飛行時間質譜儀的分析，確認是α-半乳糖水解酵素。進行一系列酵素的定性分析，包含紅血球轉型實驗，2 units的純化酵素可於2小時將實驗中總體積為120 μL的B型紅血球懸浮液轉型為O型紅血球，其轉型百分比約為80％。

α-Galactosidase is capable of hydrolyzing terminal non-reducing galactosyl residues, and it is distributed in most organisms with many different biological functions such as metabolism and transportation of photoassimilates in plants. In application, α-galactosidase can be used to hydrolyze the galactosyl group of type B antigen on the red blood cell surface, and covert the type B red blood cell into type O. Thus, the Pichia patoris expression system was used to fastly produce large amount of taro α-galactosidase to proceed the seroconversion of erythrocyte.
In this work, the Pichia patoris cells harboring the taro α-galactosidase gene on the expression vector were grown to large scale. After the recombinant gene had been induced for one day, the highest intracellular enzyme activities were 3.6 units/mL. Lysis of yeasts was achieved by using Lyticase to extract the crude taro α-galactosidase.     
From the crude extractsα-galactosidase was then purified to homogeneity by using four different types of column chromatography, include SephadexTM G-100, Q SepharoseTM Fast flow, HiTrap Phenyl FF (high sub), and SepharoseTM 6 10/300 GL. The purified enzyme showed single band on SDS-PAGE, and then identified by by MALDI-TOF MS analysis. 
The purified α-galactosidase was characterized in terms of blood conversion, and a conversion rate of 80％ type B red blood cell into type O with use 2 units of purified  α-galactosidase in 2h was achieved.

目錄

第一章：序論
1.1. 前言                                                  1
1.2. GH27家族α-半乳糖水解酶                               3
    1.2.1. 性質與分類                                      3
    1.2.2. 催化機制                                        5
    1.2.3. 生物功能                                        8
1.3. α-半乳糖水解酵素的應用                                13
    1.3.1. 食品添加應用                                   13
    1.3.2. 製糖工業應用                                   13
    1.3.3. 疾病醫療應用                                   14
1.4. 人類血型系統的介紹與應用                             14
    1.4.1. 紅血球的血型分類                               14
    1.4.2. ABO血型系統                                   15
    1.4.3. 紅血球的血型轉換                               17
1.5. 嗜甲基酵母菌（P.pastoris）                           20
    1.5.1. P. pastoris蛋白質表現系統                         20
    1.5.2. P. pastoris的甲醇代謝路徑                         21
    1.5.3. 酒精氧化酵素之啟動子（AOX promoter）           23
    1.5.4. P. pastoris表現異源蛋白質                         23
    1.5.5. 使用P. pastoris表現系統的優點                    24






























第二章：實驗材料與儀器設備
2.1. 物種與菌種                                           26
2.2. 培養基製備                                           28
2.3. 實驗耗材                                             30
2.4. 化學藥品                                             31
2.5. 儀器設備                                             35












第三章: 實驗方法與步驟
3.1. 蛋白質實驗                                           37
    3.1.1. 蛋白質的定量：Bradford method                    37
    3.1.2. 酵素活性測定                                   38
    3.1.3. 十二烷基硫酸鈉-聚丙烯醯胺凝膠電泳（SDS-PAGE） 40
    3.1.4. 基質輔助雷射脫附離子化－飛行時間質譜儀         44
3.2. 酵母菌培養條件測試                                   47
    3.2.1. 誘導時間測試                                   47
    3.2.2. 誘導起始菌液濃度測試                           48
3.3. 酵母菌大量培養與蛋白質誘導表現                      49
3.4. α-半乳糖水解酵素的萃取與純化                         50
    3.4.1. 酵母菌細胞內外的酵素濃度測試                   50
    3.4.2. 酵母菌細胞內酵素的萃取                         50
    3.4.3. 酵素濃縮                                       52
    3.4.4. 酵素的純化方法                                 53
3.5重組α-半乳糖水解酵素的定性分析                       58
    3.5.1. 酵素最適pH值與最穩定pH值                    58
    3.5.2. 酵素熱穩定性                                   59
    3.5.3. 酵素動力學測試                                 60
    3.5.4. 酵素水解Melibiose、Raffinose 與Stachyose 的能力  61
    3.5.5. 人類紅血球轉型測試                             63





















第四章：實驗結果與討論
4.1. 不同培養條件對表現重組α-半乳糖水解酵素的影響         65
4.2. 酵母菌大量培養與蛋白質誘導表現                       66
4.3. α-半乳糖水解酶的萃取與純化                            67
4.4. 純化後重組α-半乳糖水解酵素的電泳與質譜分析           73
4.5. 重組α-半乳糖水解酵素的定性分析結果                   75
















第五章：結論與未來展望
5.1. 表現重組芋頭α-半乳糖水解酵素的培養條件修正          102
5.2. 酵母菌大量培養與蛋白質誘導表現的產量                103
5.3. α-半乳糖水解酵素的萃取方法選擇與純化結果分析         104
5.4. 純化後重組α-半乳糖水解酵素的電泳與質譜分析          105
5.5. 重組α-半乳糖水解酵素的定性分析結果                  106
5.6. 未來展望                                            107


第六章：參考文獻                                        109

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