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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 羅翊禎(Yi-Chen Lo) | |
| dc.contributor.author | Szu-Yu Kuo | en |
| dc.contributor.author | 郭思妤 | zh_TW |
| dc.date.accessioned | 2022-11-23T09:05:42Z | - |
| dc.date.available | 2022-02-21 | |
| dc.date.available | 2022-11-23T09:05:42Z | - |
| dc.date.copyright | 2022-02-21 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-02-08 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79628 | - |
| dc.description.abstract | "轉醣是醣基水解酶 (glycosyl hydrolase, GH) 的功能之一，它可以透過將醣基從受質供體 (substrate donor) 轉移到目標受體 (acceptor donor) 來形成新的醣苷鍵。用此方式可以合成複雜或新型的寡醣，也可能是滿足當今市場對功能性寡醣的高度需求的一種替代生產方法。先前研究發現酵母菌 Saccharomyces cerevisiae中的 EXG1蛋白 (ScEXG1) 具有轉醣而合成昆布寡醣的能力。但對於ScEXG1產生的寡醣結構組成或產量相關資訊仍有許多未知。因此，我們將進一步的了解在不同反應時間、受質濃度、酵素濃度條件下，ScEXG1 in vitro合成昆布寡醣的能力，以及其產物的結構表徵。在我們的研究中以Laminaribiose為受質，利用ScEXG1 新合成了三種不同的寡糖 (DP3、DP4-1、DP4-2)，其中最主要的寡醣 DP3鑑定為β-Glc-(1 → 6)-β-Glc-(1→ 3)-β-Glc。另外，當使用不同的酵素濃度時，原始受質和新合成的寡醣水解成葡萄糖的速率會有所不同。在固定的酵素濃度下，所有測試的Laminaribiose受質濃度 (4.38 – 32.85 mM) 都可以合成 DP3、DP4-1 和 DP4-2。但在 4.38 mM, 75 min 和 21.9 mM, 180 min下，分別合成了最低 (12.24 ± 0.18 µg/mL, p < 0.05) 和最高 (136.55 ± 5.21 µg/mL, p < 0.05) 的DP3。DP4-1 和 DP4-2 的含量均低於定量極限。為了提高ScEXG1合成能力仍需要進一步研究如何降低 ScEXG1 的水解活性，同時提高合成能力，以便能夠充分利用其酵素進行大規模寡醣合成。" | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-23T09:05:42Z (GMT). No. of bitstreams: 1 U0001-0702202215163400.pdf: 20772126 bytes, checksum: 19e23ac55beea7487e33fd6bfa6b7bf4 (MD5) Previous issue date: 2022 | en |
| dc.description.tableofcontents | Acknowledgments.........................................................................................................................................i 摘要..............................................................................................................................................................iii Abstract.......................................................................................................................................................iv Table of Contents........................................................................................................................................vi List of Figures...........................................................................................................................................ix List of Tables............................................................................................................................................xi Supplementary........................................................................................................................................xii CHAPTER ONE INTRODUCTION.................................................................................................................1 CHAPTER TWO LITERATURE REVIEW.........................................................................................................2 2.1 Functional oligosaccharides.............................................................................................................2 2.2 Brown algae (Phaeophyta)................................................................................................................3 2.3 Laminari-oligosaccharides................................................................................................................7 2.3.1 Origin and natural source(s).......................................................................................................7 2.3.2 Health benefits of laminarin and its oligosaccharide.................................................................11 2.3.3 Laminari-oligosaccharides synthesis........................................................................................12 2.4 Glycosyl hydrolase family 5 (GH5).................................................................................................15 2.4.1 GH5 family protein structure.....................................................................................................15 2.4.2 Catalytic mechanism.................................................................................................................15 2.5 Saccharomyces cerevisiae BY4741.................................................................................................18 2.5.1 S. cerevisiae cell wall.................................................................................................................18 2.5.2 S. cerevisiae EXG1 (ScEXG1).....................................................................................................21 2.6 Analysis of carbohydrates by High-Performance Liquid Chromatography Porous Graphitic Carbon coupled with Tandem Mass Spectrometry (HPLC-PGC-ESI-MS/MS)...........................................23 2.6.1 Porous graphitic carbon (PGC)................................................................................................26 CHAPTER THREE RESEARCH OBJECTIVES AND EXPERIMENTAL DESIGN.............................................29 CHAPTER FOUR MATERIALS AND METHODS.........................................................................................31 4.1 Materials..........................................................................................................................................31 4.1.1 Chemicals..................................................................................................................................31 4.1.2 Reagents and Kits.....................................................................................................................33 4.1.3 Biological medium.....................................................................................................................33 4.1.4 Enzymes....................................................................................................................................34 4.1.5 Equipment.................................................................................................................................34 4.1.5.1 Consumables........................................................................................................................34 4.1.5.2 Instrumentation....................................................................................................................35 4.2 Methods.........................................................................................................................................37 4.2.1 Recombinant pET21a-ScEXG1-6HIS protein............................................................................37 4.2.2 YCL1497 (BY4741 exg1::pGPD-pEXG1-ScEXG1-tCYC1 las21) for ScEXG1 expression......41 4.2.3 SDS-PAGE................................................................................................................................42 4.2.4 Immunoblotting........................................................................................................................42 4.2.5 Determination of protein concentration...................................................................................43 4.2.6 Enzyme activity........................................................................................................................43 4.2.7 Enzymatic transglycosylation assay........................................................................................43 4.2.8 Thin layer chromatography (TLC)............................................................................................44 4.2.9 Porous graphitic carbon solid phase extraction (PGC-SPE) sample purification....................44 4.2.10 Structural identification with HPLC-PGC-ESI-MS/MS analysis...............................................45 4.2.11 Calibration curve.....................................................................................................................48 CHAPTER FIVE RESULTS AND DISCUSSION..........................................................................................49 5.1 Recombinant pET21a-ScEXG1-6HIS plasmid construction and heterologous protein expression in E. coli........................................................................................................................................................49 5.1.1 Plasmid construction...............................................................................................................49 5.1.2 Heterologous protein expression of ScEXG1 in E. coli BL21....................................................49 5.1.3 Codon optimized pET21a-ScEXG1-6HIS expression...............................................................50 5.2 YCL1497 (BY4741 exg1::pGPD-pEXG1-ScEXG1-tCYC1 las21) expressing ScEXG1................52 5.2.1 Protein purification with Ni-NTA affinity column......................................................................52 5.3 Enzymatic transglycosylation with purified ScEXG1......................................................................55 5.3.1 Qualitative analysis of ScEXG1 transglycosylation – Time optimization...................................55 5.3.2 Enzyme amount optimization for the transglycosylation of ScEXG1........................................62 5.3.3 Enzyme/substrate ratio optimization........................................................................................65 5.4 Analytical platform set-up..............................................................................................................72 5.4.1 Analytical recoveries of different oligosaccharides (DP2 – 4)..................................................72 5.5 Quantitative analysis of ScEXG1 transglycosylation product..........................................................76 5.5.1 Quantification of the synthesized DP3 and DP4s with ScEXG1................................................78 5.5.2 Glucose content as a side-product from the transglycosylation reactions of ScEXG1............82 5.6 TLC analysis of the ScEXG1 transglycosylation under different laminaribiose concentrations and reaction times............................................................................................................................................85 5.7 Structural characterization of the transglycosylation products of ScEXG1...................................87 5.7.1 DP3...........................................................................................................................................87 5.7.2 DP4-1........................................................................................................................................90 5.7.3 DP4-2........................................................................................................................................93 CHAPTER SIX CONCLUSIONS AND FUTURE PERSPECTIVES...............................................................102 CHAPTER SEVEN REFERENCES.............................................................................................................105 CHAPTER EIGHT SUPPLEMENTARY.......................................................................................................108 | |
| dc.language.iso | en | |
| dc.title | 探討酵母菌中 EXG1 轉醣合成昆布寡醣 | zh_TW |
| dc.title | Enzymatic transglycosylation of Saccharomyces cerevisiae EXG1 to synthesize laminarin-derived oligosaccharides | en |
| dc.date.schoolyear | 110-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 呂廷璋(Fan-Ren Chang),陳勁初(Dah-Jing Jwo),陳冠翰(He-Sheng Wang) | |
| dc.subject.keyword | 轉醣,醣基水解酶 (glycosyl hydrolase, GH),Saccharomyces cerevisiae EXG1,昆布寡醣,結構表徵, | zh_TW |
| dc.subject.keyword | Transglycosylation,glycosyl hydrolases (GHs),Saccharomyces cerevisiae EXG1,laminari-oligosaccharide,structural characterization, | en |
| dc.relation.page | 117 | |
| dc.identifier.doi | 10.6342/NTU202200331 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2022-02-09 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 食品科技研究所 | zh_TW |
| 顯示於系所單位： | 食品科技研究所 | |
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