Comparative Analysis of High-Throughput Assays of Family-1 Plant Glycosyltransferases
Comparative Analysis of High-Throughput Assays of Family-1 Plant Glycosyltransferases
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DOI：
10.3390/ijms21062208
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发表时间：
2020-03-01
影响因子：
5.6
通讯作者：
Schwab, Wilfried
中科院分区：
文献类型：
--
作者：
McGraphery, Kate;Schwab, Wilfried
糖基转移酶(GTS)通过糖基化降低挥发性，增加溶解度，从而改变小分子的生物利用度的能力在制药、营养食品和化妆品工业中引起了极大的关注。已知的GT的缺乏和高通量(HTP)可用方法的稀缺阻碍了进一步的新应用的推断。在这项研究中，测试了适用于HTP筛查的新的GT检测方法的适用性，并从无害性、稳健性、成本效益和重复性方面进行了比较。UDP-Glo GT法、磷酸GT活性法、pH敏感型GT法和UDP2-TRRET法适用于植物UDP GT(UGT)。葡萄(UGT72B27)GT与多种酚类物质发生糖基化反应。对底物筛选和动力学参数进行了评价。PH敏感法和UDP2-TRRET法不具有可比性，不适用于HTP植物GT-1家族的UGT筛选。此外，UDP-Glo GT测定法和磷酸GT活性测定法得到的K-M、v(Max)和k(CAT)值非常相似且可重复性。因此，这两种方法具有实验装置简单、读数快的特点，适用于植物超氧化物歧化酶的筛选和定量动力学分析。这项研究揭示了新的和新兴的HTP分析方法，这将允许分析新的家族1植物GT，并将发现进一步的应用。
The ability of glycosyltransferases (GTs) to reduce volatility, increase solubility, and thus alter the bioavailability of small molecules through glycosylation has attracted immense attention in pharmaceutical, nutraceutical, and cosmeceutical industries. The lack of GTs known and the scarcity of high-throughput (HTP) available methods, hinders the extrapolation of further novel applications. In this study, the applicability of new GT-assays suitable for HTP screening was tested and compared with regard to harmlessness, robustness, cost-effectiveness and reproducibility. The UDP-Glo GT-assay, Phosphate GT Activity assay, pH-sensitive GT-assay, and UDP2-TR-FRET assay were applied and tailored to plant UDP GTs (UGTs). Vitis vinifera (UGT72B27) GT was subjected to glycosylation reaction with various phenolics. Substrate screening and kinetic parameters were evaluated. The pH-sensitive assay and the UDP2-TR-FRET assay were incomparable and unsuitable for HTP plant GT-1 family UGT screening. Furthermore, the UDP-Glo GT-assay and the Phosphate GT Activity assay yielded closely similar and reproducible K-M, v(max), and k(cat) values. Therefore, with the easy experimental set-up and rapid readout, the two assays are suitable for HTP screening and quantitative kinetic analysis of plant UGTs. This research sheds light on new and emerging HTP assays, which will allow for analysis of novel family-1 plant GTs and will uncover further applications.