DNA-Linked Enzyme-Coupled Assay for Probing Glucosyltransferase Specificity

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• 作者：David J. Sukovich ; Cyrus Modavi ; Markus de Raad ; Robin N. Prince ; J. Christopher Anderson
• 刊名：ACS Synthetic Biology
• 出版年：2015
• 出版时间：July 17, 2015
• 年：2015
• 卷：4
• 期：7
• 页码：833-841
• 全文大小：431K
• ISSN：2161-5063

文摘

Traditional enzyme characterization methods are low-throughput and therefore limit engineering efforts in synthetic biology and biotechnology. Here, we propose a DNA-linked enzyme-coupled assay (DLEnCA) to monitor enzyme reactions in a high-throughput manner. Throughput is improved by removing the need for protein purification and by limiting the need for liquid chromatography mass spectrometry (LCMS) product detection by linking enzymatic function to DNA modification. We demonstrate the DLEnCA methodology using glucosyltransferases as an illustration. The assay utilizes cell free transcription/translation systems to produce enzymes of interest, while UDP-glucose and T4-尾-glucosyltransferase are used to modify DNA, which is detected postreaction using qPCR or a similar means of DNA analysis. OleD and two glucosyltransferases from Arabidopsis were used to verify the assay鈥檚 generality toward glucosyltransferases. We further show DLEnCA鈥檚 utility by mapping out the substrate specificity for these enzymes.