DNA-Linked Enzyme-Coupled Assay for Probing Glucosyltransferase Specificity.

Abstract:

: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-β-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's generality toward glucosyltransferases. We further show DLEnCA's utility by mapping out the substrate specificity for these enzymes.

journal_name

ACS Synth Biol

journal_title

ACS synthetic biology

authors

Sukovich DJ,Modavi C,de Raad M,Prince RN,Anderson JC

doi

10.1021/sb500341a

subject

Has Abstract

pub_date

2015-07-17 00:00:00

pages

833-41

issue

7

issn

2161-5063

journal_volume

4

pub_type

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