Study on the Biosynthesis of Sterol/Steroid α-Glycosides

• Main Authors: Guo, Ting-Wei, 郭庭瑋
• Other Authors: Wu, Tung-Kung
• Format: Others
• Language: en_US
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/2jf5y2

碩士 === 國立交通大學 === 生物科技學系 === 105 === Saponins are widespread in our surroundings. They are a group of secondary metabolites expressed in plants, fungi and lower marine animals. Saponins can be divided into tetracyclic triterpenoids and pentacyclic triterpenoids based on their carbon skeletons. The whole structure is classified into two parts: hydrophobic sapogenin and hydrophilic sugar moiety. Saponins are divided into monosaccharide saponins, disaccharides saponins and trisaccharides saponins and have great potential for the pharmaceutical industry, they can be used as anti-cancer and anti-microbe agent as well as biochemical inhibitors. The biosynthesis of saponins are mainly achieved through the following three steps: (1) initial sapogenin backbones are formed by epoxidation of oxidosqualene cyclase; (2) hydroxylation of specific sapogenin carbon; and (3) glycosyltransferase catalyzes saccharides to form glycosidic bonds with sapogenins; glycosylation will bond hydrophobic sapogenin and hydrophilic sugar moieties. Moreover, glycosylation is particularly important for biological activity in several saponins. Furthermore, glycosylation can also improve solubility and stability. In my research, not only does this have an impact on biosynthesis of nucleotide diphosphate sugars, but it also catalyzes different sterols/steroids glycosides. Using this mechanism, development and research of glycosides would be much easier and diversified in the future. In this thesis, the following five genes were successfully cloned and expressed in an E. coli system: NahK (Bifidobacterium infantis), GalK (Escherichia coli), BLUSP (Bifidobacterium longum), PmPpA (Pasteurella Multocida) and HP0421 (Helicobacter pylori). All of these proteins were successfully purified using Ni-NTA chromatography. The glycosyltransferase HP0421 is of most interest due to its unique α-glycosylaion. According to previous studies, we focused on the activities of α-glycosyltransferase on five compounds: trans-androsterone, cis-androsterone, dehydroepiandrosterone, pregnenolone and cholesterol. They were determined and characterized using thin layer chromatography and high performance liquid chromatography.