α-半乳糖水解酶（α-galactosidase）是一種可以將末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-半乳糖水解酶參與醣類的代謝和運輸（source to sink transport）。在應用方面，α-半乳糖水解酶可以使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-半乳糖水解酶以進行血行轉換的測試和應用。
由每公斤的芋的塊莖中進行萃取可獲得約 83.3 Unit 的 α-半乳糖水解酶酵素活性，接著進行三次的管柱層析步驟使蛋白質純化 6019 倍，並使樣品最終純化至單一蛋白質片段，最後樣品的比活性為 54.18 Unit/mg。接著將片段送去進行蛋白質 N 端序列的鑑定和蛋白質中間部分片段序列的鑑定，並利用鑑定的序列和 GH27 family α-半乳糖水解酶的高度保留序列設計 degenerate primer dFP IA 和 dRP 2A 來夾出第一段的基因序列；利用已知的基因序列設計 forward 和 reverse gene specific primer 並經過數次的 PCR 之後夾出完整的基因序列，芋頭 α-半乳糖水解酶的基因由 1082 個 base 所組成，在經過轉譯後其蛋白質分子量為 40.01 kDa。最後將芋頭 α-半乳糖水解酶的基因接入 pPIC9k 質體中，並轉殖至 Pichia pastoris（GS115、SMD1168）中進行基因的誘導與表現。在 Pichia pastoris 中誘導基因表現六天後，GS115 和 SMD1168 的細胞內酵素活性最多分別為 0.5 和 0.45 Unit/mL，而分泌至細胞外的酵素活性則只有胞內的 1/10。

α-Galactosidase is capable of hydrolyzing terminal non-reducing galactosyl residues, and it is distributed in most organisms with many different biological funtions such as metabolism and transportation (source to sink transport) 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 produce large amount of taro α-galactosidase fastly to proceed the seroconversion of erythrocyte.
The crude taro α-galactosidase was extracted with 83.3 Unit/kg of enzyme activity from taro tubers. The crude extracts then purified to homogeneity by using column chromatography, and the specific activity was 54.18 Unit/mg after 6019 fold purification. Base on the results of N-terminal sequencing, inner peptide determination, and multiple alignment with GH27 family α-galactosidase, degenerated primers dFP IA and dRP 2A were designed to synthesis the first known sequence of taro α-galactosidase gene. Then forward and reverse gene specific primers were designed from the known sequence to obtain the whole gene sequence, which was consisted of 1082 bases, and the molecular mass of this enzyme was 40.01 kDa, by PCR techniques. The gene was cloned into pPIC9k, and then transformed into Pichia pastoris (GS115 and SMD1168). At last, Pichia pastoris was induced and expressed recombinant taro α-galactosidase. After the recombinant gene had been induced for six days, the intracellular enzyme activities of GS115 and SMD1168 were 0.5 and 0.45 Unit/mL(culture) at most, and the extracellular enzyme activities were only 1/10 fold of the intracellular activities.

目錄

第一章：α-半乳糖水解酶與其應用

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	4
1.2.3. 生物功能	8
	
1.3. α-半乳糖水解酶的應用	13
1.3.1. 工業應用	13
1.3.2. 紅血球的血型轉換	14

第二章：材料和儀器設備
2.1. 物種、菌種	18

2.2. 載體	18
2.3. 培養基	19

2.3.1. 大腸桿菌培養基	19
2.3.2. 酵母菌培養基	20
	
2.4. 耗材	22
2.5. 化學藥品	23
2.6. 儀器設備	26
	

第三章: 實驗方法和步驟

3.1. 基礎實驗	28
3.1.1. 蛋白質基礎實驗	29
3.1.2. 核酸基礎實驗	34
	
3.2. 芋頭α-半乳糖水解酶的純化	50
3.2.1. 芋頭粗萃取液的製備	50
3.2.2. 使用 DEAE SephadexTM A-50 管柱進行純化	51
3.2.3. 使用 SephadexTM G-100 管柱進行純	52


3.2.4. 使用 DEAE SepharoseTM Fast Flow FPLC 管柱進行純化	53
	
3.3. 芋頭α-半乳糖水解酶的蛋白質定序	55
3.3.1. N端胺基酸定序	55
3.3.2. 利用 ESI-MS-MS 進行胺基酸中間片段序列的鑑定	56
	
3.4. 芋頭α-半乳糖水解酶的基因序列鑑定	57
3.4.1. Total RNA 萃取	57
3.4.2. Degenerate primer 的設計	59
3.4.3. 芋頭α-半乳糖水解酶基因中間片段的序列鑑定	62
3.4.4. 3’RACE primer 的設計	63
3.4.5. 芋頭α-半乳糖水解酶 mRNA 之 3’端的序列鑑定	65
3.4.6. Gene specific reverse primer 和 N 端 degenerate
     primer 的設計	66
3.4.7. 芋頭α-半乳糖水解酶基因之 N 端的序列鑑定	68
	
3.5. 芋頭α-半乳糖水解酶基因的轉殖	69



3.5.1. 在芋頭α-半乳糖水解酶基因兩端增加限制酶切位	69
3.5.2. 基因的轉殖	70
3.5.3. 基因的表現	71

第四章：實驗結果

4.1.芋頭α-半乳糖水解酶的純化	73
4.2. 純化後的芋頭α-半乳糖水解酶進行電泳分析	74
4.3. 純化後的芋頭α-半乳糖水解酶蛋白質序列鑑定	75
4.4. 芋頭α-半乳糖水解酶基因序列的鑑定	76
4.4.1. Total RNA 萃取	76
4.4.2. 芋頭α-半乳糖水解酶基因（中間片段）的序列鑑定	76
4.4.3. 芋頭α-半乳糖水解酶 mRNA 之 3’ 端的序列鑑定	79
4.4.4. 芋頭α-半乳糖水解酶基因之 N 端的序列鑑定	81
4.4.5. 芋頭α-半乳糖水解酶之完整基因序列	82
	
4.5. 芋頭α-半乳糖水解酶基因的轉殖與表現	83
4.5.1. 轉殖基因序列的鑑	83
4.5.2. 轉殖基因的表現
	83
第五章：實驗討論和總結

5.1. 實驗討論	113
5.1.1. 芋頭中的α-半乳糖水解酶	113
5.1.2. 芋頭α-半乳糖水解酶的純化	114
5.1.3. 芋頭α-半乳糖水解酶之 RT-PCR 的表現	115
5.1.4. Degenerate Primer 的設計和使用	116
5.1.5. 芋頭α-半乳糖水解酶基因之 N 端 的序列鑑定	117
5.1.6. 重組的芋頭α-半乳糖水解酶基因在酵母菌中表現	118
	
5.2. 總結與未來展望	120
	
	
	
	


 
圖片索引

圖 1.1. 瑞/里氏木霉α-半乳糖水解酶的3D模型圖和比較圖	6
圖 1.2. 維持型酵素的反應機制圖示	8
圖 1.3. 棉子糖家族寡糖結構	9
圖 1.4. Galactomannans 的結構	10
圖 1.5. α-半乳糖水解酶 A 在人類溶小體中的水解GL3	11
圖 1.6. A、B、O型紅血球所帶的血型表面抗原	12
圖 3.1. PNPG的呈色機制	30
圖 4.1. DEAE A-50　純化圖	86
圖 4.2. G-100　純化圖	87
圖 4.3. DEAE SepharoseTM Fast Flow FPLC column 純化圖	88
圖 4.4. 芋頭α-半乳糖水解酶的 SDS-PAGE 結果	89
圖 4.5. 蕃茄α-半乳糖水解酶的胺基酸序列	90
圖 4.6.芋頭α-半乳糖水解酶蛋白質中間片段和其他物種比對	91
圖 4.7. Total RNA 進行 agarose 電泳的結果	92
圖 4.8. 利用 degenerate primer 進行 PCR 後的電泳結果	93
圖 4.9.抽取質體利用 EcoR I 和 Xba I 水解	94

圖 4.10.利用 PCR 來進行 double check	95
圖 4.11. 3’RACE 第一次 PCR 之電泳結果	96
圖 4.12. 3’RACE 第二次 PCR 之電泳結果	97
圖4.13. 3’RACE 所得到的序列跟 680 bp 已知序列進行比對	98
圖 4.14. N 端序列的 PCR 之電泳結果	99
圖4.15. N 端 PCR 所得到的7多個序列進行比對	100
圖4.16. 芋頭α-半乳糖水解酶已鑑定之序列	101
圖4.17. 芋頭α-半乳糖水解酶和其他物種胺基酸序列比對	102
圖4.18. 芋頭α-半乳糖水解酶和其他物種核酸序列比對結果	103
圖4.19. 芋頭α-半乳糖水解酶基因接入 pPIC9k 後鑑定結果	104
圖4.20. 少量誘導六天的菌數變化	105
圖4.21. 少量誘導六天的細胞內酵素活性變化	106
圖4.22. 少量誘導六天的細胞外酵素活性變化	107
圖4.23. 大量誘導三天的菌數變化	108
圖4.24. 大量誘導三天的細胞內酵素活性變化	109
圖4.25. 大量誘導三天的細胞外酵素活性變化	110
	


 
表格索引

表 1.1. 咖啡豆、稻米和芋頭α-半乳糖水解酶的比較	2
表 3.1. Dgenerate primers	60
表 3.2. 簡併符號對照表	61
表 3.3. 3’RACE primers	64
表 3.4. N 端定序使用的 primer	67
表 4.1. 由 9.84 kg 的芋頭塊莖萃取純化流程一覽表	111
表 4.2. 680 bp 序列和NCBI的資料庫進行比對	112

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