Laboratory of Industrial Microbiology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, 9000 Ghent, Belgium.
Anal Biochem. 2010 Jun 1;401(1):162-7. doi: 10.1016/j.ab.2010.02.028. Epub 2010 Feb 24.
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.
糖苷磷酸化酶（GPs）是用于化学分子糖基化的有趣酶。它们只需要一个糖基磷酸作为糖供体和一个带有游离羟基的受体分子。然而，它们狭窄的底物特异性限制了 GPs 在一般糖苷合成中的应用。尽管可以通过蛋白质工程和定向进化来改变和拓宽酶的底物特异性，但这需要合适的筛选测定。目前还没有针对 GPs 的这种筛选测定方法的报道。在这里，我们报告了一种基于糖苷合成方向上释放的无机磷酸盐测量的 GPs 筛选程序。似乎需要用钼酸盐抑制粗大肠杆菌细胞提取物中的内源性磷酸酶活性，并使用改良的磷钼酸盐法测量无机磷酸盐。该筛选系统是通用的，可以用于筛选具有新型供体和受体特异性的 GP 酶文库。它成功地应用于筛选纤维素单胞菌纤维二糖磷酸化酶（CP）的残基 E649 饱和突变文库，以获得新型受体特异性。发现了一种 E649C 酶变体，对烷基 β-葡萄糖苷和苯基 β-葡萄糖苷具有新型受体特异性。这是首例报道具有修饰的受体特异性的 CP 酶变体。
