臺灣博碩士論文加值系統

岩藻糖基化是一種寡糖修飾，在多種生物和病理過程中發揮著重要作用。過去許多研究使用疊氮基(Azido group)或炔基(Alkynyl group)修飾岩藻糖進入癌細胞進行專一性聚糖標記，少有研究涉及岩藻糖衍生物(Fucose analogs)在細菌和蛋白質上的應用。
岩藻糖化糖體的應用仰賴生物合成途徑中的鳥苷二磷酸岩藻糖衍生物(GDP-Fucose analogs)。FKP 是由岩藻糖激酶(L-Fucokinase)及鳥苷二磷酸岩藻糖焦磷酸化酶(GDP-fucose pyrophosphorylase)組成的雙功能酶。FKP能將岩藻糖(L-Fucose)轉化成岩藻糖-1-磷酸 (Fuc-1-P)中間體後，進而轉化為鳥苷二磷酸岩藻糖(GDP-fucose)。FKP對於修飾後的Fucose analogs有廣泛的接受範圍。
Bf13FT和FucTa為α1,3/4-岩藻糖基轉移酶，可用於合成各種母乳岩藻糖苷，如Lewisa、Lweisx、LNFP V和LNnDFH II。我們透過基因序列比對發現Bf13FT、FucTa與Helicobacter pylori 26695 α1,3-岩藻糖轉移酶(H. pylori 26695 α1,3-FT)催化活性中心基因序列相似。H. pylori 26695 α1,3-FT能催化GDP-fucose analogs合成岩藻糖基化人乳寡糖衍生物。本論文實驗結果中證實Bf13FT及FucTa能催化GDP-fucose analogs合成岩藻糖基化人乳寡糖衍生物。
本研究利用3D結構比對推測FKP對於催化合成C-6官能基修飾GDP-fucose analogs之機制。此外我們也探討Bf13FT和 FucTa對於GDP-fucose analogs之受質耐受性及二者催化活性位置。

Fucosylation is an oligosaccharide modification that plays an essential role in a variety of biological and pathological processes. In the past, many studies utilized an azido or alkynyl substituted fucose in cancer cells for specific glycan labeling, rare researches focused on the application of fuocse analogs to bacteria and proteins.
The applications of fucose-containing glycans rely on the use of GDP-fucose analogs involved in the biosynthetic pathway. FKP is a bifunctional enzyme consisting of L-fucokinase and GDP-fucose pyrophosphorylase. FKP converts L-fucose into GDP-fucose via a fucose-1-phosphate (Fuc-1-P) intermediate. FKP is capable of accepting an even broader range of modified fucose analogs.
Bf13FT and FucTa were characterized as α1,3/4-fucosyltransferases that could be used for the synthesis of various human milk fucosides, such as Lewisa, Lweisx, LNFP V, and LNnDFH II. Through gene sequence alignments of Bf13FT, FucTa, and Helicobacter pylori 26695 α1,3- fucosyltransferase (H. pylori 26695 α1,3-FT), we discovered that the sequence of their catalytic active sites was similar. H. pylori 26695 α1,3-FT could catalyze the synthesis of fucosylated human oilgosacchsride derivatives using GDP-fucose analogs. The experiment results in this thesis confirmed that Bf13FT and FucTa could catalyze the synthesis of these derivatives.
In this work, we proposed a mechanism of FKP catalyzing the synthesis of C-6-modified GDP-fucose analogs through 3D structure alignments. In addition, we explored the substrate tolorance and catalytic activity positions of Bf13FT and FucTa for GDP-fucose analogs.

Abstract 1
摘要 2
Fucose analogs對照表 3
縮寫表 5
目錄 6
圖目錄 9
表目錄 14
流程目錄 15
第一章 緒論 16
1.1 官能基修飾糖體之生化應用 16
1.2 岩藻糖化糖體生物功能及重要性 19
1.2.1 岩藻糖化糖體對於腸道菌的作用 19
1.2.2 岩藻糖化糖體之生合成 20
1.3 L-Fucokinase/GDP-fucose pyrophosphorylase(FKP)合成GDP-fucose 22
1.3.1 FKP結構及預測催化活性位點 22
1.4 Fucosyltransferase 23
1.4.1 鬆脆桿菌α1,3/4-岩藻糖轉移酶(Bacteroides fragilis α1,3/4-fucosyltransferase , Bf13FT) 24
1.4.2 幽門螺旋桿菌α1,3/4-岩藻糖轉移酶(Helicobacter pylori UA948 α1,3/4-fucosyltransferase, FucTa) 26
1.5 研究動機 27
1.5.1 Fucose analogs之生化特性及應用 27
1.5.2 C-6修飾之Fucose analogs在酵素合成上的影響 29
1.5.3 FucTa及Bf13FT之耐受性探討 30
第二章 實驗結果與討論 31
2.1 大腸桿菌誘導表現目標蛋白質 31
2.1.1 目標基因產物之表現 31
2.1.2 酵素表達之分析 31
2.2 FKP與Fucose analogs之耐受性探討 42
2.2.1 Fucose analogs去乙醯基(O-acetyl)保護方式探討 42
2.2.2 GDP-fucose analogs之合成 54
2.2.3 FKP與Fucose analogs之機制探討 57
2.3 FucTa及Bf13FT合成Lewisx analogs及LNFP V analogs之探討 60
2.3.1 Lewisx analogs合成與鑑定 60
2.3.2 LNFP V analogs合成與鑑定 80
2.3.3 FucTa與Fucose analogs之機制探討 89
2.3.4 Bf13FT與Fucose analogs之機制探討 91
2.4 結論 94
2.4.1 FKP之酵素耐受性 94
2.4.2 FucTa及Bf13FT之酵素耐受性 95
2.5 未來展望 99
第三章 實驗材料與方法 100
3.1. Material and Methods 100
3.2. Overexpression of enzyme 101
3.2.1. Gene resource 101
3.2.2. Protein overexpression 101
3.3. Synthetic procedures and characterization 102
3.3.1. Chemical synthetic procedure of fucose analogs 102
3.3.2. Synthetic procedures of sugar nucleotides 102
3.3.3. Enzymatic synthetic of Lewisx analogs 104
3.3.4. Enzymatic synthetic of LNFP V analogs 104
3.3.5. Fucose analogs 105
3.3.6 GDP-fucose analogs 113
3.3.7 Lewisx analogs 117
3.3.8 LNFP V analogs 121
第四章 參考文獻 122
第五章 光譜及質譜附圖 130
5.1 光譜附圖目錄 130

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