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  1. NTU Theses and Dissertations Repository
  2. 生物資源暨農學院
  3. 食品科技研究所
Please use this identifier to cite or link to this item: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/81014
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???org.dspace.app.webui.jsptag.ItemTag.dcfield???ValueLanguage
dc.contributor.advisor羅翊禎
dc.contributor.authorTzu-Chun Linen
dc.contributor.author林子淳zh_TW
dc.date.accessioned2022-11-24T03:26:12Z-
dc.date.available2021-09-02
dc.date.available2022-11-24T03:26:12Z-
dc.date.copyright2021-09-02
dc.date.issued2021
dc.date.submitted2021-08-31
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dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/81014-
dc.description.abstract"羅漢果 (Luo Han Kuo, LHK)中主要的甜味物質為Mogroside V (MG V),為一帶有五個葡萄糖的皂苷結構。而iso-mogroisde V (IMV)是較MG V甜度更高的皂苷同分異構物。兩者差異是在皂苷元三號碳上雙醣之間的鍵結不同。MG V為β-1,6鍵結,而IMV為β-1,4鍵結。因為自然界中IMV含量非常稀少,且無法透過化學合成產生,因此希望透過微生物或轉醣酵素產生IMV。實驗中Saccharomyces cerevisiae野生型菌株會將羅漢果皂苷萃取物中所含微量的IMV水解,無法達到產生IMV的目的,但產生未知的MG V 同分異構物 (compound ),為了瞭解compound 的甜度、結構和功效,需要產生大量的compound 。首先,透過基因缺陷菌株轉換羅漢果皂苷,並且與S. cerevisiae野生型菌株比較發酵結果的差異,篩選出可能的轉醣基因。將可能的轉醣基因放回原本的缺陷菌株中進行確認,證實 compound 的生成與外切β-1,3葡聚醣酶Exg1有關。之後利用大量表現EXG1基因的菌株和純化的ScExg1酵素轉換羅漢果希望得到大量的compound ,但產量仍然很少。實驗中也利用Dekkera bruxellensis的外切葡聚醣酶 (DbExg1)進行相同反應,然而DbExg1則沒有產生compound 和IMV的活性。因此在本研究中雖未能找到可轉換IMV的酵素,但也證實S. cerevisiae Exg1具有微弱的compound 轉換能力。未來也期望利用其他β-1,4葡聚醣酶轉換出IMV。"zh_TW
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dc.description.tableofcontents謝誌 i 摘要 ii Abstract iii Table of contents iv List of Figures viii List of Tables xi List of Appendix xiii Chapter 1 Introduction 1 Chapter 2 Literature review 2 2.1 Sweeteners 2 2.1.1 Sugar alcohols 3 2.1.2 Protein sweeteners 4 2.1.3 Terpenoid glycosides 5 2.2 Siraitia grosvenorii (Swingle) 7 2.2.1 Chemical structures of mogrosides 7 2.2.2 Mogroside V isomers 8 2.2.3 Sweetness of mogrosides 9 2.2.4 Conversion of mogrosides 10 2.3 Biotransformation 11 2.3.1 Biotransformation by whole cells 11 2.3.2 Biotransformation by enzymes 12 2.4 Enzymatic glycosylation 13 2.4.1 Glycosidase 13 2.4.2 Glycosyltransferase 18 2.4.3 Glycosylation of mogrosides 19 2.5 Saccharomyces cerevisiae 21 2.5.1 EXG1 22 2.5.2 ATG26 22 2.5.3 KRE6 23 Chapter 3 Research objectives and experimental design 25 Chapter 4 Material and methods 27 4.1 Materials 27 4.1.1 Luo Han Kuo extract 27 4.1.2 Strains 27 4.1.3 Plasmids 27 4.1.4 Primers 27 4.1.5 Chemicals and reagents 31 4.1.6 Device and instrument 33 4.2 Methods 36 4.2.1 Production of quality control sample 36 4.2.2 Construction of strains 36 4.2.3 Examination of the strain’s basic characterization 41 4.2.4 Bioconversion of Luo Han Kuo with strains 44 4.2.5 Bioconversion of Luo Han Kuo by purified ScExg1 46 Chapter 5 Results and Discussion 49 5.1 HPLC-MS/MS analysis of mogrosides 49 5.1.1 Characterization of IMV 49 5.1.2 Characterization of compound 57 5.1.3 Preparation of quality control (QC) sample 64 5.2 Bioconversion of Luo Han Kuo with S. cerevisiae 66 5.2.1 Bioconversion of Luo Han Kuo with wild-type strain 66 5.2.2 Identification of the gene responsible for the biosynthesis of compound in S. cerevisiae 70 5.3 Bioconversion of Luo Han Kuo with ScEXG1 expression strains 74 5.3.1 Construction of ScEXG1 expression strains 74 5.3.2 Comparing growth curves of ScEXG1 expression strains with wild-type strain 81 5.3.3 Protein concentration and β-glucosidase activity of ScEXG1 expression strains’ extracellular proteins 84 5.3.4 Western blot of extracellular ScExg1 from ScEXG1 expression strains 87 5.3.5 Bioconversion of Luo Han Kuo with ScEXG1 expression strains 89 5.4 Bioconversion of Luo Han Kuo with purified ScExg1 protein 91 5.5 Effect of the strains with EXG1 from S. cerevisiae and D. bruxellensis on bioconversion of Luo Han Kuo 94 5.6 Bioconversion of Luo Han Kuo with DbEXG1 expression strains 98 5.6.1 Construction of DbEXG1 expression strains 98 5.6.2 Comparing growth curves of DbEXG1 expression strains with wild-type strain 101 5.6.3 Protein concentration and β-glucosidase activity of DbEXG1 expression strains’ extracellular proteins 104 5.6.4 Western blot of extracellular DbExg1 from DbEXG1 expression strains 107 5.6.5 Bioconversion of Luo Han Kuo with DbEXG1 expression strains 109 5.7 Transglycosylation of strains with LAS21 deletion in high and low concentration of Luo Han Kuo 111 Chapter 6 Conclusion 114 Chapter 7 Supplementary materials 115 Chapter 8 References 123 Chapter 9 Appendix 132
dc.language.isoen
dc.subjectDekkera bruxellensiszh_TW
dc.subjectSaccharomyces cerevisiaezh_TW
dc.subjectiso-mogroside Vzh_TW
dc.subject轉醣zh_TW
dc.subjectExg1zh_TW
dc.subjectExg1en
dc.subjecttransglycosylationen
dc.subjectSaccharomyces cerevisiaeen
dc.subjectiso-mogroside Ven
dc.subjectDekkera bruxellensisen
dc.title探討不同β-葡聚醣酶進行羅漢果皂苷轉醣作用zh_TW
dc.titleEffect of β-glucanases on transglycosylation of mogrosidesen
dc.date.schoolyear109-2
dc.description.degree碩士
dc.contributor.oralexamcommittee高承福(Hsin-Tsai Liu),呂廷璋(Chih-Yang Tseng),陳宏彰,王如邦
dc.subject.keywordiso-mogroside V,轉醣,Saccharomyces cerevisiae,Exg1,Dekkera bruxellensis,zh_TW
dc.subject.keywordiso-mogroside V,Saccharomyces cerevisiae,transglycosylation,Exg1,Dekkera bruxellensis,en
dc.relation.page138
dc.identifier.doi10.6342/NTU202102869
dc.rights.note同意授權(限校園內公開)
dc.date.accepted2021-09-01
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
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