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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 蘇南維(Nan-Wei Su) | |
dc.contributor.author | Yu-Chuan Chang | en |
dc.contributor.author | 張又權 | zh_TW |
dc.date.accessioned | 2021-07-10T22:16:59Z | - |
dc.date.available | 2021-07-10T22:16:59Z | - |
dc.date.copyright | 2017-08-31 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-08-14 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77705 | - |
dc.description.abstract | 苯並吡喃酮(benzopyrone)類化合物為苯並吡喃(benzopyran)之酮基衍生物,涵蓋了許多類黃酮(flavonoids)。類黃酮為一類自然界中普遍存在之多酚類植物二次代謝物,具備多方面豐富及重要之生理活性。然而文獻指出,大多數類黃酮化合物的水溶性不佳,生物可利用率(bioavailability)極低,使其應用受到很大的限制。本研究室先前篩選出 Bacillus subtilis BCRC 80517,可對daidzein及genistein 進行磷酸酯化修飾,形成daidzein 7-O-phosphate (D7P)及genistein 7-O-phosphate (G7P),轉換後產物可大幅提升其水溶性及生物可利用率。研究室已自B. subtilis純化出該磷酸化酵素,完成蛋白質及基因定序,並建立基因轉殖大腸桿菌大量表現該酵素-苯並吡喃酮類化合物磷酸化酵素(benzopyrone phosphorylation enzyme, BPE)。本論文以此為基礎,進行後續研究。
本研究首先探討BPE對各種類黃酮化合物形成其類黃酮磷酸酯衍生物之最適化酵素反應系統,然後進行各衍生物的萃取與純化,再以LC-MS/MS和NMR鑑定其化學結構。結果顯示,BPE可催化異黃酮類如daidzein及genistein、黃酮類如apigenin及6-hydroxyflavone、黃酮醇類如quercetin及kaempferol、黃烷酮類如naringenin及hesperetin與二氫黃酮醇木脂素類如silibinin進行磷酸化反應。反應大多產生兩個產物,經LC-MS/MS和NMR鑑定後,確認為類黃酮磷酸酯。主產物為flavonoid 7-O-phosphate,副產物則為flavonoid 4’-O-phosphate。另外,除了7-OH、4'-OH位置之外,6-OH及3’-OH位置亦可被磷酸化。本研究之酵素動力學結果顯示,BPE對基質之催化效率如下:naringenin > hesperetin > silibinin > genistein > daidzein > apigenin。酵素之催化效率會受flavonoid上2號和3號碳之鍵結影響,當2號和3號碳為飽和鍵時,酵素之催化效率較好。 | zh_TW |
dc.description.abstract | Benzopyrone refers to either of two ketone derivatives of benzopyran which constitute the core skeleton of many flavonoids. Flavonoids are polyphenolic secondary metabolites that are ubiquitous in nature and possess numerous health benefits. Nevertheless, research indicated that flavonoids have low aqueous solubility and poor bioavailability, and therefore limit its use. Our previous study screened out Bacillus subtilis BCRC 80517, which could phosphorylate daidzein and genistein and form daidzein 7-O-phosphate (D7P) and genistein 7-O-phosphate (G7P). Thereby, these phosphate conjugates may be greatly improved in water solubility and bioavailability. In addition, the BCRC 80517 can also biotransform most of the flavonoids. In addition, we have also purified the enzyme and identified its protein and DNA sequence. The gene recombinant E. coli were already constructed to prepare the recombinant protein. Nevertheless, among the biotransformed products, only isoflavone-phosphate conjugates have been identified so far. Therefore, the objective of this research is focusing on the substrate specificity of benzopyrone phosphorylation enzyme (BPE), and identification of the biotransformed products.
In this thesis, the optimal condition for preparing flavonoid-phosphate conjugates by gene recombinant cells system was determined first. The biotransformed products were isolated and purified. Their structures were identified by LC-MS/MS and NMR. The results showed that BPE could catalyze the phosphorylation of isoflavone (daidzein and genistein), flavone (apigenin and 6-hydroxyflavone), flavonol (quercetin and kaempferol), flavanone (naringenin and hesperetin) and (silibinin), and yielded two kinds of products which were identified by LC-MS/MS and NMR to be flavonoid 7-O-phosphates as the major product,and flavonoid 4’-O-phosphates as by-product. Besides, 7-OH and 4’-OH, 6-OH and 3’-OH could be also phosphorylated. The catalytic efficiency of BPE on flavonoids was naringenin > hesperetin > silibinin > genistein > daidzein > apigenin in a decreasing order. The efficiency was affected by the binding of C-2 and C-3 in the flavonoids. Flavanone with single bond between C-2 and C-3 has higher efficiency. | en |
dc.description.provenance | Made available in DSpace on 2021-07-10T22:16:59Z (GMT). No. of bitstreams: 1 ntu-106-R04623013-1.pdf: 7640753 bytes, checksum: 327155821297605c29abefc4b1794732 (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | 口試委員審定書
誌謝 I 中文摘要 III Abstract IV 目錄 VI 圖目錄 X 表目錄 XIII 縮寫對照表 XIV 第一章 前言 1 第二章 文獻回顧 2 第一節 類黃酮 2 1. 類黃酮簡介 2 2. 類黃酮之生理活性 2 3. 類黃酮之生物可利用率 4 3-1. BCS分類系統 4 3-2. 類黃酮於人體之代謝與吸收 5 3-3. 異黃酮於人體之口服生物可利用率 6 第二節 類黃酮之微生物轉換 16 第三節 前驅藥物 17 1. 前驅藥物定義與介紹 17 2. 磷酸酯前驅藥物 17 第四節 磷酸化酵素 24 1. Pyruvate, phosphate dikinase, PPDK (EC 2.7.9.1) 25 2. Phosphoenolpyruvate synthase , PEPS (EC 2.7.9.2) 25 3. 磷酸苯酯合成酶 (phenylphosphate synthase, PPS) 25 第五節 酵素動力學 30 第三章 材料與方法 33 第一節 實驗架構 33 第二節 實驗材料 34 1. 菌株 34 2. 培養基 34 2-1 固態培養基: Lysogeny broth (LB)/ Km agar 34 2-2 液態培養基: Lysogeny broth (LB) /Km 34 3. 緩衝液 35 3-1 Enzyme buffer 35 3-2 Cell lysis buffer 35 3-3 Binding buffer 35 3-4 Washing buffer 36 3-5 Elution buffer 36 4. 試藥與溶劑 37 5. 儀器設備 38 第三節 實驗方法 39 1. BPE之表現與分離純化 39 1-1菌株活化與種菌培養 39 1-2 以 IPTG誘導重組蛋白基因大量表現 39 1-3 BPE粗酵素液之製備 40 1-4 BPE之純化 40 1-4-1 Ni-NTA鎳離子親和性管柱 40 1-4-2 Superdex 200膠體過濾層析 40 1-4-3 BPE之冷凍保存 40 1-5 蛋白質電泳分析 41 1-5-1 SDS-PAGE電泳分析 41 1-5-2 膠體染色 41 1-6 蛋白質定量方法 41 1-7 酵素活性測定 41 2. 不同種類類黃酮之酵素轉換 44 2-1 酵素反應 44 2-2 分析級高效液相層析儀分析條件 45 3. 分離純化反應後之類黃酮磷酸酯衍生物 45 3-1 酵素反應 45 3-2 類黃酮磷酸酯衍生物之萃取 45 3-3 以半製備級高效液相層析儀純化類黃酮磷酸酯衍生物 46 3-4 半製備級高效液相層析儀分離類黃酮磷酸酯衍生物之條件 46 4. 類黃酮磷酸酯衍生物結構鑑定 47 4-1核磁共振光譜分析類黃酮衍生物之條件 47 4-2高效液相層析串聯式質譜儀分析類黃酮衍生物之條件 48 5. 酵素動力學分析 48 6. 統計分析 48 第四章 結果與討論 49 第一節 BPE對各基質之轉換與衍生物結構鑑定 49 1. BPE之分離 49 2. BPE之純化 50 2-1 Ni-NTA鎳離子親和性管柱 50 2-2 Superdex 200膠體過濾層析 52 3. 建立BPE活性測定方法 52 4. BPE之基質特異性 53 4-1 BPE對異黃酮(isoflavone)之反應與其衍生物結構鑑定 53 4-1-1 BPE對daidzein之反應 53 4-1-2 BPE對genistein之反應 60 4-2 BPE對黃酮(flavone)之反應與其衍生物結構鑑定 65 4-2-1 BPE對apigenin之反應 65 4-2-2 BPE對6-hydroxyflavone之反應 73 4-3 BPE對黃酮醇(flavonol)之反應與其衍生物結構鑑定 78 4-3-1 BPE對quercetin之反應 78 4-3-2 BPE對kaempferol之反應 84 4-4 BPE對黃烷酮(flavanone)之反應與其衍生物結構鑑定 93 4-4-1 BPE對naringenin之反應 93 4-4-2 BPE對hesperetin之反應 101 4-5 BPE對水飛薊素(silibinin)之反應與其衍生物結構鑑定 109 4-5-1 以分析級高效液相層析儀分析silibinin酵素反應液 109 4-5-2 以半製備級高效液相層析儀分離S7P-1與S7P-2 109 4-5-3 Spectral data of silibinin 7-O-phosphate-1 109 4-5-4 Spectral data of silibinin 7-O-phosphate-2 110 4-6 BPE磷酸化反應與類黃酮化學結構之關係 117 第二節 酵素動力學 119 第五章 結論 122 第六章 參考文獻 123 第七章 附錄 131 | |
dc.language.iso | zh-TW | |
dc.title | 苯並吡喃酮類化合物磷酸化酵素基質特異性之研究 | zh_TW |
dc.title | Studies on the Substrate Specificity of
Benzopyrone Phosphorylation Enzyme | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李敏雄,林雲蓮(Yun-Lian Lin),古國隆,許庭禎 | |
dc.subject.keyword | 酵素轉化,磷酸化,苯並?喃酮,類黃酮,基質特異性, | zh_TW |
dc.subject.keyword | enzyme-transformation,phosphorylation,benzopyrone,flavonoid,substrate specificity, | en |
dc.relation.page | 133 | |
dc.identifier.doi | 10.6342/NTU201703272 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2017-08-15 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 農業化學研究所 | zh_TW |
顯示於系所單位: | 農業化學系 |
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