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標題: | 苯並吡喃酮類化合物磷酸化酵素基質特異性之研究 Studies on the Substrate Specificity of Benzopyrone Phosphorylation Enzyme |
作者: | Yu-Chuan Chang 張又權 |
指導教授: | 蘇南維(Nan-Wei Su) |
關鍵字: | 酵素轉化,磷酸化,苯並?喃酮,類黃酮,基質特異性, enzyme-transformation,phosphorylation,benzopyrone,flavonoid,substrate specificity, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 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。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77705 |
DOI: | 10.6342/NTU201703272 |
全文授權: | 未授權 |
顯示於系所單位: | 農業化學系 |
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