An Ultrasensitive Fluorescence Assay for the Detection of Halides and Enzymatic Dehalogenation. Issue 7 (31st January 2020)

• Record Type: Journal Article
• Authors: Aslan‐Üzel, Aşkın S.; Beier, Andy; Kovář, David; Cziegler, Clemens; Padhi, Santosh K.; Schuiten, Eva D.; Dörr, Mark; Böttcher, Dominique; Hollmann, Frank; Rudroff, Florian; Mihovilovic, Marko D.; Buryška, Tomáš; Damborský, Jiří; Prokop, Zbyněk; Badenhorst, Christoffel P. S.; Bornscheuer, Uwe T.
• Is Part Of: ChemCatChem. Volume 12:Issue 7(2020)
• Page Start: 2032
• Page End: 2039
• Publication Date: 2020-01-31
• Subjects: dehalogenase -- fluorescence -- halides -- haloalkane -- haloperoxidase; Catalysis -- Periodicals; 541.39505
• DOI: 10.1002/cctc.201901891
• Languages: English
• ISSNs: 1867-3880

Abstract

Abstract: 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 μM 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. Abstract : How sensitive ! A simple, safe, and very sensitive assay for enzymatic dehalogenation is presented. The halides produced by dehalogenases are generally very unreactive and thus hard to detect. We use hydrogen peroxide and a haloperoxidase to activate halides to hypohalous acids, which are easily detected using the sensitive fluorogenic probe aminophenyl fluorescein.