Study of In-vivo One-pot Sterol β-monosaccharide Biosynthesis

• Main Authors: Lyu, Jason Wenjay, 呂汶杰
• Other Authors: 吳東昆
• Format: Others
• Language: en_US
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/5ad3rb

碩士 === 國立交通大學 === 生物科技學系 === 105 === 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.