枯草桿菌BCRC 80517 qdoI基因對黃酮醇化合物微生物轉換之影響

Abstract

類黃酮化合物為自然界中普遍存在的多酚類植物二物代謝物，許多研究指出類黃酮具有良好的生理活性，在動物、人體試驗部分功效都已獲得證實，然而多數類黃酮水溶性低的特性，是造成生物可利用率(bioavailability)極低的原因之一，因而限制了諸多應用的可能性，目前許多研究致力於改善低溶解度的缺點以提高其生物可利用性。本實驗室先前篩選出的Bacillus subtilis BCRC 80517可將大豆異黃酮的daidzein和genistein進行磷酸化，轉為daidzein-7-O-phosphate (D7P)和genistein-7-O-phosphate (G7P)，讓水溶性大幅的提升，生物可利用率也顯著的增加，前人已自B. subtilis純化出該磷酸酯合成酶，完成蛋白質及基因定序，並建立基因載體轉殖到大腸桿菌大量表現，針對此酵素的基質特異性進行分析與酵素動力學的研究。本論文建立於磷酸酯合成酶 (FPS)可轉換的黃酮醇類，進行後續之研究。 本研究先利用磷酸酯合成酶轉換黃酮醇類的fisetin，再挑選三種不同黃酮醇進行微生物轉換，並與磷酸化酵素轉換之產物做比較，結果顯示fisetin可被B. subtilis 轉換成磷酸酯衍生物，而quercetin和kaempferol的衍生物並非磷酸酯產物，因此以LC-MS/MS和NMR對quercetin衍生物進行結構鑑定。發現此衍生物為protocatechuoyl-phloroglucinolcarboxylic acid (PCPGCA)的相關產物，文獻查閱知道進行此反應的是quercetinase，比較三種黃酮醇類轉化結果，5號位置的hydroxyl group是影響黃酮醇類轉為PCPGCA產物的重要位點。另一方面，麴酸是quercetinase的抑制劑，然而添加麴酸進行共培養時，雖然增加了磷酸化產物，卻無法完全抑制開環途徑，因此透過同源重組突變開環基因qdoI為根本解決辦法。定序實驗室B. subtilis 菌株的qdoI，並做抗生素抗性測試挑選抗性基因，組裝質體各片段，以E. coli DH5α為質體複製宿主，成功組裝質體pUC-qdoNeo。

Flavonoids are polyphenolic secondary metabolites that are ubiquitous in plants and their processed products. According to many research results, flavonoids possess numerous health benefits. Some of effects have been proved in animals or humans. Nevertheless, flavonoids have low aqueous prosperity and poor bioavailability. These disadvantages substantially limit their possibility of application. Our previous study screened Bacillus subtilis BCRC 80517, which could phosphorylate some types of isoflavone, daidzein and genistein, and biotransform them into daidzein 7-O-phosphate (D7P) and genistein 7-O-phosphate (G7P). Besides, these phosphate conjugates bioabailability are greatly improved. In addition, we have purified the enzyme, identified its protein、DNA sequence and utilized recombinant E. coli to perform the flavonoids phosphate synthetase (FPS) by a constructed plasmid. We discovered that this FPS not only can transform isoflavone, but also can phosphorylate other types of flavonoids, like flavonols. Therefore, the objective of this research is utilizing B. subtilis to transform flavonols and identify what the pathway Bacillus subtilis will go when they come from flavonols. In this thesis, transforming fisetin, one type of flavonols would be done first. Next, I chose three types of flavonol, fisetin, kaempferol, quercetin to be biotransformed by B. subtilis. The product was isolated, purified and identified by LC-MS/MS and NMR. The results showed that quercetin and kaempferol were not biotransformed into phosphate conjugates. Depending on the results, we know that the hydroxyl group on the 5th position carbon is the critical point for flavonols to be phosphorylated or not. According to the metabolites, the double bond between the 2nd and 3rd carbon of quercetin and kaempferol were cleavaged and became protocatechuoyl-phloroglucinolcarboxylic acid. Although little of quercetin-O-phosphate were produced, there was no obvious effect by adding quercetinase inhibitor, kojic acid. Therefore, constructing a plasmid and mutating the qdoI gene by homologous recombination will be the fundamental method if we want to get flavonol-O-phosphate products. Hence, sequencing B. subtilis BCRC 80517 qdoI sequence and choosing what antibiotic to select successfully mutated colony should be done. The method chosen to assembly plasmid inserts and vector was Gibson Assembly. At last, the plasmid was transformed in E. coli DH5α and I successfully got assembling plasmid.