Expression, characterization and application of sucrose synthase from Pisum Satium
|關鍵字:||蔗糖合成酶;尿嘧啶雙磷酸葡萄糖;蛋白質再摺疊;酵素耦合;sucrose synthase;UDP-glucose;protein refolding;enzyme coupling|
|摘要:||天然苯環化合藥物如黃酮素、白藜蘆醇等，水溶性不佳在人體吸收率低，天然界中的策略是將其醣基化以增加溶解度。本實驗室先前成功地利用醣基轉移酶BcGT-1將苯酚類化合物轉化成醣基化合物，然而過程中需使用昂貴的UDP-glucose作為醣基化的醣基提供者，故本實驗目的在於利用蔗糖合成酶（sucrose synthase, SUS）能催化蔗糖和UDP轉化為UDP-glucose的特性以生物合成法生產UDP-glucose，以降低醣基化反應之成本。
本研究選用Pisum Satium之Sucrose synthase（SUS），研究過程先利用基因工程的技術將人造基因建構在大腸桿菌表現系統BL21（DE3）中，透過蛋白質大量表現得到高濃度的重組蛋白質包含體（inclusion body），再透過清洗步驟及再摺疊（refolding）得到純度80%具有活性的蔗糖合成酶。
本研究室以高效能液相層析定量分析反應產物UDP-glucose對SUS做性質研究，其最佳酸鹼穩定度位於pH 7~ 9，熱穩定性在25℃最佳，最佳酸鹼反應性在pH 6~8，及最佳反應溫度為25℃，酵素可保存在10% 甘油中-80℃，其活性在兩星期後仍有60%以上。
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.
|Appears in Collections:||Thesis|