菌內一鍋化合成β相位固醇糖苷之研究

Abstract

皂苷是一種廣泛分布於植物及真菌當中的二級代謝物。從結構上來看，皂苷分別由疏水性的苷元以及親水性的糖基所組成。而由苷元的碳骨架來看，又可以分為四環三萜類和五環三萜類。皂苷具有多種的生物活性，包括抗發炎，抗真菌，抗微生物甚至是抗癌，也因此被許多科學家注意到而開始廣泛地研究，認為具有發展成醫療用藥的潛力。而皂苷的多樣性源自於他合成時的三大步驟: (1)經氧化鯊烯酵素催化所產生的皂苷碳骨架，此反應也被認為是生物合成中最複雜的反應之一 (2) 氫氧化酶對於碳骨架上之特定的碳原子進行氫氧化作用，使之攜帶不同的官能基 (3) 醣基轉移酶將醣類與皂苷上的氫氧基進行作用，並藉由共價的方式形成醣苷鍵。由於每一步的受質，酵素，以及醣基都是可以改變的，也因此皂苷才擁有廣大的多樣性。其中，最後的醣化作用更是改變了苷元許多的特性。皂苷上的醣基深深地影響了其生物活性，而許多研究也指出醣基不僅能改變活性，更增加了溶解性及穩定性，讓皂苷能更容易被生物所利用。 本論文分為兩個部分。第一部份是針對比菲德氏龍根菌(Bifidobacterium longum)中的尿苷二磷酸醣焦磷酸化酶(UDP-sugar pyrophosphorylase, USP)、以及抗菌素鏈黴菌的轉醣酵素變異體 OleD-ASP 兩種酵素做體外反應的研究。藉由尿苷二磷酸醣焦磷酸化酶，我們可以得到轉醣作用所必須的醣基，再來將其使用在轉醣酵素上，達到生產皂苷的目標。根據前人的研究結果，我主要針對五種四環固醇作為轉醣酵素之反應對象，並進行一定規模的量產。 第二部份，則是建立起體內一鍋化皂苷制備系統。我們先將個別帶有這兩種基因的質體植入勝任細胞中，再一起培養跟誘導。相較於體外反應使用純化蛋白，我們直接使用回溶的菌液進行反應。由於體內反應有可能增加酵素的活性，我們挑了18種不同的苷元來試驗其活性的增減。最後則發現有新產物誕生。 不論是第一或第二部份，其產物都先由薄層層析法來做初步確認，而後經過高效液相層析法，電噴霧離子化質譜和核磁共振儀來鑑定其分子量跟結構。未來這些產物將會被用於活性分析，以開發此類人工皂苷在藥物上的潛力。

Saponins are a secondary metabolite widely distributed in plants and fungi. Structurally, saponins are composed of hydrophobic aglycones and hydrophilic glycosyl groups. According to the difference of carbon skeleton, saponins can also be divided into tetracyclic triterpenoids and pentacyclic triterpenes. Saponins have a variety of biological activities, including anti-inflammatory, anti-fungal, anti-microbial and even anti-cancer. Therefore many scientists have begun to study on saponins and believe they have potential for developing into medical drugs. The diversity of saponins is derived from three major steps in its synthesis: (1) the saponin carbon skeleton produced by the oxidation of the oxidosqualene cylase, which is also considered to be one of the most complex reactions in biosynthesis. (2) Cytochrome hydroxylates specific carbon atoms on the carbon skeleton to carry different functional groups. (3) The glycosyltransferase catalyzes the saccharides and the hydroxyl groups on aglycones. They are connected with glycosidic bonds. Due to the fact that substrates, enzymes, and sugar can be distinct in every step, saponins possess a wide range of diversity. Among synthesis, the glycosylation is critical to many characteristics of aglycone. Saccharides on saponins significantly affect their biological activity. Many studies have pointed out that the glycosyl can not only change the activity, but also increase the solubility and stability, so that saponins can be more easily utilized in plants and animals.

Our thesis is divided into two parts. The first part is studying on in-vitro reaction of UDP-sugar pyrophosphorylase (USP) from Bifidobacterium longum and Oleandomycin glycosyltransferase OleD-ASP from Streptomyces antibioticus. By uridine diphosphate pyrophosphorylase, we can get the essential UDP-sugar for glycosylation, and then use it on glycosyltransferase to achieve the aims of synthesis of saponins. According to previous research results, I mainly use five kinds of sterols/steroids as the reaction object of glycosylation, and carry out large-scale production of saponins. The second part is to establish in-vivo one pot saponin synthesis. We first implant plasmids which contain OleD-ASP and BLUSP genes into competent cells, and perform culture and induction together. Unlike in-vitro reaction of using purified protein, we directly use the cell solution in the reaction without disrupting cells. As the in-vivo glycosylation is likely to increase the activity of the enzyme, we pick 18 different aglycones to test for the activity. The results reveal that new product has been diccovered. The products are first confirmed by thin layer chromatography, followed by high performance liquid chromatography, electrospray ionization mass spectrometry, and NMP spectrum to identify their molecular weight and structures.