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An Ultrasensitive Fluorescence Assay for the Detection of Halides and Enzymatic Dehalogenation

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ASLAN-UZEL Askin S. BEIER Andy KOVÁŘ David CZIEGLER Clemens PADHI Santosh K. SCHUITEN Eva D. DORR Mark BOTTCHER Dominique HOLLMANN Frank RUDROFF Florian MIHOVILOVIC Marko D. BURYŠKA Tomáš DAMBORSKÝ Jiří PROKOP Zbyněk BADENHORST Christoffel P. S. BORNSCHEUER Uwe T.

Rok publikování 2020
Druh Článek v odborném periodiku
Časopis / Zdroj ChemCatChem
Fakulta / Pracoviště MU

Přírodovědecká fakulta

Citace
www https://onlinelibrary.wiley.com/doi/full/10.1002/cctc.201901891
Doi http://dx.doi.org/10.1002/cctc.201901891
Klíčová slova dehalogenase; fluorescence; halides; haloalkane; haloperoxidase
Popis Halide assays are important for the study of enzymatic dehalogenation, a topic of great industrial and scientific importance. Here we describe the development of a very sensitive halide assay that can detect less than a picomole of bromide ions, making it very useful for quantifying enzymatic dehalogenation products. Halides are oxidised under mild conditions using the vanadium-dependent chloroperoxidase from Curvularia inaequalis, forming hypohalous acids that are detected using aminophenyl fluorescein. The assay is up to three orders of magnitude more sensitive than currently available alternatives, with detection limits of 20 nM for bromide and 1 mu M for chloride and iodide. We demonstrate that the assay can be used to determine specific activities of dehalogenases and validate this by comparison to a well-established GC-MS method. This new assay will facilitate the identification and characterisation of novel dehalogenases and may also be of interest to those studying other halide-producing enzymes.
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