枯草桿菌UDP-glycosyltransferase之受質專一性及應用研究

Abstract

醣基化化合物是植物藥用成分中常見之現象，其醣類分子會提高化合物的親水性及溶解度；當藥物之水溶性提高，亦可增加其口服之吸收率，則可降低劑量以達到治療目標。 在本研究中，利用UDP-glycosyltransferase，可將UDP-glucose之葡萄醣基，轉移至受質上，形成具有醣基化之化合物。利用基因工程的方式，將UDP-glycosyltransferase之人造基因建構在大腸桿菌BL21（DE3）表現系統，可得到大量表現之酵素。將純化後的UDP-glycosyltransferase對多種受質進行轉醣反應，可成功在受質接上葡萄醣基。 UDP-glycosyltransferase可以單醣基化合物為受質基底，進行醣基化反應，形成多醣基化合物。UDP-glycosyltransferase在pH值7到9之間較為穩定，而受質之最適反應pH < 7，因此將反應活性控制在pH值7為最佳。 利用UDP-glycosyltransferase進行轉醣反應之生物合成法，可產生許多新穎之醣基化合物，作為新藥開發的基石。

Natural pharmaceutical compounds often contain many kinds of glycoside modification. Glycosylation will enhance the hydrophilicity and water solubility of compounds, therefore increase the oral absorption and decrease the dosages. UDP-glycosyltransferase（MGT）from Bacillus cereus converted aglycones to glycosides. An artificial gene of MGT was cloned into pRSET A plasmid and the protein was successfully expressed in E.coli BL21（DE3）. Using the purified MGT to catalyze the glycosylation with different glucose acceptors and UDP-glucose, it could convert phenolic aglycones to phenolic glucosides successfully, but it couldn’t convert alcohols to glycosides. The phenolic oligo-glucosides could be converted from phenolic mono-glucosides by MGT. The enzyme showed optimum pH between 7 and 9. However, some phenolic compounds underwent hydrolytic degradation at pH8-9, therefore the optimum reaction pH values was 7. Biosynthesis of glycosides by using MGT was proved to be possible in many kinds of phenolic compounds. And some products might have unnatural structure and novel bioactivity.