Expression, characterization and application of sucrose synthase from Pisum Satium

• Main Authors: Hsu, Chia-Chun, 許嘉郡
• Other Authors: Li, Yaw-Kuen
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/90013189218798002223

碩士 === 國立交通大學 === 應用化學系碩博士班 === 102 === Natural phenols, such as quercetin, rasveratrol and many others, have low solubility in water and low uptake rate in human. The strategy occurring in nature is to convert these phenols into a corresponding glycosylated compound. In the past year, our laboratory has successfully employed BcGT-1, a marcrolide glucosyltransferase from Bacillus cereus, for transforming the phenolic compounds into glycosides. The process requires the expensive UDP-glucose as the glucose donor. The aim of this study is trying to establish an enzymatic process to produce UDP-glucose by sucrose synthase and consequently cost down the price of glycosylation. Sucrose synthase （SUS） is an enzyme that catalyzes the reaction of producing UDP-glucose from sucrose and UDP. A synthetic gene of SUS from Pisum Sativum was cloned into pRSET A plasmid and overexpressed in E.coli BL21(DE3). The recombinant SUS was obtained as an inclusion body, which was further successfully refolded into an active enzyme with 80% purity. The characterization of SUS was performed by quantitatively analyzed the product, UDP-glucose, using HPLC. Our Study exhibited that enzyme is stable in the range of pH 7 ~ 9 at 25℃. The optimal reaction condition at 25℃ is within pH 6 ~ 8. When SUS was stored at -80℃ in the condition of 10% glycerol for 2 weeks, at least 60% activity can be retained. The examples of coupling the catalysis of SUS and BcGT-1, sequentially, to prepare the glucosylated harmalol, quercetin glucoside and 4-methylumbelliferyl-β-D-glucopyranoside were also successfully demonstrated.