Skip to Content
Merck
  • DNA-Linked Enzyme-Coupled Assay for Probing Glucosyltransferase Specificity.

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

ACS synthetic biology (2015-01-27)
David J Sukovich, Cyrus Modavi, Markus de Raad, Robin N Prince, J Christopher Anderson
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Acetonitrile, for chromatography
Sigma-Aldrich
Formic acid, SAJ first grade, 88.0-89.5%
Sigma-Aldrich
Acetonitrile, ≥99.8%, for residue analysis, JIS 300
Sigma-Aldrich
Acetonitrile, JIS special grade, ≥99.5%
Sigma-Aldrich
Acetonitrile, SAJ first grade, ≥99.0%
Sigma-Aldrich
Acetonitrile, ≥99.8%, for residue analysis, JIS 1000
Sigma-Aldrich
3,4-Dihydroxybenzoic acid, ≥97.0% (T)
Sigma-Aldrich
Acetonitrile, for residue analysis, JIS 5000
Sigma-Aldrich
Formic acid, JIS special grade, ≥98.0%
Sigma-Aldrich
Acetonitrile, ≥99.8%, suitable for HPLC
Sigma-Aldrich
Formic acid, ≥95%, FCC, FG
Sigma-Aldrich
Acetonitrile, anhydrous, 99.8%
Sigma-Aldrich
3-Hydroxybenzoic acid, ReagentPlus®, 99%
Sigma-Aldrich
4-Hydroxybenzoic acid, ReagentPlus®, 99%
Sigma-Aldrich
Acetonitrile, ≥99.5%, ACS reagent
Sigma-Aldrich
4-Hydroxybenzoic acid, ReagentPlus®, ≥99%
Sigma-Aldrich
Acetonitrile, electronic grade, 99.999% trace metals basis
Sigma-Aldrich
Glycerol, JIS special grade, ≥99.0%
Sigma-Aldrich
Glycerol, SAJ first grade, ≥98.0%
Sigma-Aldrich
Glycerol, FCC, FG
Sigma-Aldrich
Glycerol, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for electrophoresis, ≥99% (GC)
Sigma-Aldrich
Glycerol, ≥99.5%
Sigma-Aldrich
Glycerol, BioXtra, ≥99% (GC)
Sigma-Aldrich
Glycerol, for molecular biology, ≥99.0%
Sigma-Aldrich
Glycerol, BioUltra, for molecular biology, anhydrous, ≥99.5% (GC)
Sigma-Aldrich
Glycerin, meets USP testing specifications
Sigma-Aldrich
3-Hydroxyflavone, ≥98%
Sigma-Aldrich
7-Hydroxycoumarinyl-4-acetic acid, 97%
Sigma-Aldrich
Kaempferol, ≥97.0% (HPLC)
Sigma-Aldrich
L-(+)-Arabinose, 98%