*3.13. Alkene Cleavage Activity of PsaPOX*

The purified recombinant PsaPOX was used for transformation of *trans*-anethole as mentioned above ("4.10") to confirm alkene cleavage activity. For this, 1 U/mL (0.25 mg/mL) of enzyme was used for biotransformation. Biotransformation was performed with 100 mM sodium acetate buffer pH 3.5 in the presence of 100 μM hydrogen peroxide and 25 mM manganese sulfate at RT for 16 h. Biotransformation of the alkenes methyl isoeugenol (6.7 mM), α-methylstyrene (6.7 mM), and piperine (0.7 mM) was tested accordingly. Blanks were performed without enzyme (chemical blank) or with heat inactivated enzyme (1 h at 95 ◦C, biological blank). The determined product concentrations for the blanks were subtracted from the concentrations yielded for the reaction with the active enzyme to calculate the enzymatically generated product concentration. For carotene degradation, a β-carotene emulsion was prepared according to Linke et al. [43]. 7% (*v*/*v*) of β-carotene emulsion or annatto (Chr. Hansen, Nienburg, Germany, Prod. No. 240569), 100 μM hydrogen peroxide, 25 mM manganese sulfate, 100 mM sodium acetate pH 3.5, and 1 U/L (0.25 mg/L) PsaPOX in a total volume of 300 μL was incubated at 40 ◦C for 20 min. Alkene cleavage of both substrates was measured photometrically as extinction decrease at 455 nm.

## *3.14. Detection of Hydrogen Peroxide*

For the detection of H2O2, 75 μL IEX fraction, 50 mM Bis-Tris pH 6.0, 6.7 mM *trans*-anethole, 10 U/mL HRP (Sigma Aldrich), and 0.5 mM *o*-dianisidine in a total volume of 300 μL were incubated at

RT for 1 h. In the presence of hydrogen peroxide, formation of a red-brown reaction product occurred. Blanks were performed with 50 mM Bis-Tris pH 6.0 instead of IEX fraction.
