Abstract
Agrocybe aegerita peroxidase (AaP) is a versatile heme-thiolate protein that can act as a peroxygenase and catalyzes, among other reactions, the hydroxylation of aromatic rings. This paper reports a rapid and selective spectrophotometric method for directly detecting aromatic hydroxylation by AaP. The weakly activated aromatic compound naphthalene served as the substrate that was regioselectively converted into 1-naphthol in the presence of the co-substrate hydrogen peroxide. Formation of 1-naphthol was followed at 303 nm (ɛ 303 = 2,010 M−1 cm−1), and the apparent Michaelis–Menten (K m) and catalytic (k cat) constants for the reaction were estimated to be 320 μM and 166 s−1, respectively. This method will be useful in screening of fungi and other microorganisms for extracellular peroxygenase activities and in comparing and assessing different catalytic activities of haloperoxidase–peroxygenases.
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Acknowledgements
Financial support by the European Union (integrated project Biorenew), the “Bundesministerium für Bildung, Wissenschaft und Forschung” (BMBF; project 0313433D) and the German Academic Exchange Service (DAAD; projects A/04/20213 and D/05/11714) is gratefully acknowledged. We thank C. Liers, M. Kinne (Konrad), M. Brandt, and U. Schneider for technical and scientific assistance and K. Steffen (University of Helsinki) for providing fungal strains.
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Kluge, M.G., Ullrich, R., Scheibner, K. et al. Spectrophotometric assay for detection of aromatic hydroxylation catalyzed by fungal haloperoxidase–peroxygenase. Appl Microbiol Biotechnol 75, 1473–1478 (2007). https://doi.org/10.1007/s00253-007-0942-8
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DOI: https://doi.org/10.1007/s00253-007-0942-8