High overexpression of dye decolorizing peroxidase TfuDyP leads to the incorporation of heme precursor protoporphyrin IX

•Dye decolorizing peroxidase TfuDyP binds heme and protoporphyrin IX in vivo.•The activity of TfuDyP is dependent on the expression level in E. coli.•Expression of fully functional DyPs can be tuned by the type of expression host and expression conditions.

The heterologous overexpression level of the bacterial dye decolorizing peroxidase TfuDyP in Escherichia coli was increased sixty fold to approximately 200 mg of purified enzyme per liter culture broth by fusing the enzyme to the small ubiquitin-related modifier protein (SUMO). The highly overexpressed SUMO-TfuDyP was, however, almost inactive. Analysis of the enzyme by UV-vis absorption spectroscopy and high-resolution mass spectrometry showed that a large fraction of the highly overexpressed enzyme contained the iron deficient heme precursor protoporphyrin IX (PPIX) instead of heme. Here we show that the activity of the enzyme was dependent on the expression level of the protein.

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