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4’-去甲基表鬼臼毒素(4’-demethylepipodophyllotoxin,DMEP)的糖苷衍生物具有多种药理活性,但其化学合成面临位置及立体选择性和基团的保护与脱保护等诸多挑战。该研究从库拉索芦荟Aloe barbadensis中克隆得到1个新颖糖基转移酶(glycosyltransferase,GT)基因Ab GT5,并成功进行了外源表达及蛋白纯化。重组Ab GT5能够催化4’-去甲基表鬼臼毒素进行糖基化,获得的产物经MS,~1H-NMR,~(13)C-NMR,HSQC以及HMBC等波谱技术鉴定为4’-去甲基表鬼臼毒素-4’-O-β-D-葡萄糖苷。酶学性质研究发现AbGT5的最适反应温度为20℃,最适pH 9.0,且不依赖金属离子。在最适反应条件下,4’-去甲基表鬼臼毒素的转化率可达80%。该研究表明利用新颖糖基转移酶AbGT5可实现4’-去甲基表鬼臼毒素的高效酶法糖基化,为其糖基化提供新方法。
Glycoside derivatives of 4’-demethylepipodophyllotoxin (DMEP) have a variety of pharmacological activities, but their chemical synthesis are confronted with many challenges, such as their position and stereoselectivity, as well as the protection and deprotection of groups. One novel glycosyltransferase (GT) gene, Ab GT5, was cloned from Aloe barbadensis, and the exogenous expression and protein purification were successfully performed. Recombinant Ab GT5 was able to catalyze the glycosylation of 4’-demethylepipodophyllotoxin. The obtained product was identified as 4’-demethylase by spectroscopic techniques such as MS, 1H-NMR, 13C-NMR, HSQC and HMBC, Methyl epipodophyllotoxin-4’-O-β-D-glucoside. The enzymatic properties of AbGT5 found that the optimum reaction temperature was 20 ℃, the optimum pH 9.0, and does not depend on metal ions. Under optimal reaction conditions, 4’-demethylated epipodophyllotoxins can reach 80% conversion. This study shows that the use of novel glycosyltransferase AbGT5 achieves efficient enzymatic glycosylation of 4’-demethylated epipodophyllotoxin and provides a new method for its glycosylation.