Journal Article

FZJ-2020-03186

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png A Fluorescence‐Based Assay System for the Determination of Haloperoxidase‐Activity Using a Two‐Dimensional Calibration Ap‐proach

Fejzagić, A. V.FZJ* ; Myllek, S.FZJ* ; Hogenkamp, F.FZJ* ; Greb, J.FZJ* ; Pietruszka, J.FZJ* ; Classen, T. (Corresponding author)FZJ*

2020
Wiley-VCH-Verl. Weinheim

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Please use a persistent id in citations: http://hdl.handle.net/2128/25835  doi:10.1002/open.202000184

Abstract: Screening for an interesting biocatalyst and its subsequent kinetic characterization depends on a reliable activity assay. In this work, a fluorometric assay based on the halogenation of 4‐methyl‐7‐diethylamino‐coumarin was established to monitor haloperoxidase‐activity. Since haloperoxidases utilize hydrogen peroxide and halide ions to halogenate a broad range of substrates by releasing hypohalous acids, a direct quantification of haloperoxidase‐activity remains difficult. With the system presented here, 3‐bromo‐4‐methyl‐7‐diethylaminocoumarin is preferentially formed and monitored by fluorescence measurements. As starting material and product share similar spectroscopical properties, a two‐dimensional calibration ap‐proach was utilized to allow for quantification of each compound within a single measurement. To validate the system, the two‐dimensional Michaelis‐Menten kinetics of a vanadium‐dependent chloroperoxidase from Curvularia inaequalis were recorded, yielding the first overall kinetic parameters for this enzyme. With limits of detection and quantification in the low μm range, this assay may provide a reliable alternative system for the quantification of haloperoxidase‐activity.

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Contributing Institute(s):

1. Biotechnologie (IBG-1)
2. Institut für Bioorganische Chemie (HHUD) (IBOC)

Research Program(s):

1. 581 - Biotechnology (POF3-581) (POF3-581)

Appears in the scientific report 2020

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