Development and application of a screening assay for glycoside phosphorylases
Development and application of a screening assay for glycoside phosphorylases
复制标题
DOI：
10.1016/j.ab.2010.02.028
复制
发表时间：
2010-06-01
影响因子：
2.9
通讯作者：
Soetaert, W.
中科院分区：
文献类型：
--
作者：
De Groeve, M. R. M.;Tran, G. H.;Soetaert, W.
糖苷磷酸化酶(GP)是化学分子糖基化的重要酶。它们只需要糖基磷酸作为糖供体和带有游离羟基的受体分子。然而，由于其较窄的底物专一性，限制了其在糖苷合成中的应用。虽然一种酶的底物专一性可以通过蛋白质工程和定向进化来改变和扩大，但这需要一个合适的筛选实验。这样的筛查试验还没有被描述用于全科医生。在这里，我们报告了一种基于在糖苷合成方向上释放的无机磷的测量来筛选GP的程序。用钼酸盐抑制大肠杆菌细胞粗提液中的内源性磷酸酶活性似乎是必要的，无机磷是用改良的磷钼酸盐法测定的。该筛选系统具有通用性，可用于筛选新的供体和受体特异性的GP酶文库。该方法已成功应用于纤维单胞菌UDA纤维二糖磷酸化酶(CP)E649残基饱和突变文库的筛选。发现E649C酶变异体对烷基-β-葡萄糖苷和苯基-β-葡萄糖苷具有新的受体特异性。这是首次报道具有修饰受体特异性的CP酶变异体。(C)2010 Elsevier Inc.保留所有权利。
Glycoside phosphorylases (GPs) are interesting enzymes for the glycosylation of chemical molecules. They require only a glycosyl phosphate as sugar donor and an acceptor molecule with a free hydroxyl group. Their narrow substrate specificity, however, limits the application of GPs for general glycoside synthesis. Although an enzyme's substrate specificity can be altered and broadened by protein engineering and directed evolution, this requires a suitable screening assay. Such a screening assay has not yet been described for GPs. Here we report a screening procedure for GPs based on the measurement of released inorganic phosphate in the direction of glycoside synthesis. It appeared necessary to inhibit endogenous phosphatase activity in crude Escherichia coli cell extracts with molybdate, and inorganic phosphate was measured with a modified phosphomolybdate method. The screening system is general and can be used to screen GP enzyme libraries for novel donor and acceptor specificities. It was successfully applied to screen a residue E649 saturation mutagenesis library of Cellulomonas uda cellobiose phosphorylase (CP) for novel acceptor specificity. An E649C enzyme variant was found with novel acceptor specificity toward alkyl beta-glucosides and phenyl beta-glucoside. This is the first report of a CP enzyme variant with modified acceptor specificity. (C) 2010 Elsevier Inc. All rights reserved.