*3.10. Biotransformation*

Transformation of *trans*-anethole was carried out in 4 mL gas tight glass vials in horizontal position at a shaking rate of 200 rpm for 16 h at RT in the absence of light. Reaction mixtures contained 30 mg *P. sapidus* lyophilisate or 100 μL liquid sample buffered in Bis-Tris (50 mM, pH 6) with or without addition of 1 mM manganese sulfate in a total volume of 1 mL and 1 μL (6.7 mM) *trans*-anethole. Blanks (chemical: without lyophilisate or liquid sample; biological: with heat inactivated mycelium (1 h at 95 ◦C)) were performed the same way. All experiments were performed as duplicates. After incubation, *trans*-anethole and its conversion product *p*-anisaldehyde were extracted with 1 mL hexane containing 100 mg/L (1 mM) cyclohexanol as internal standard (IS). The organic phase was dried with anhydrous sodium sulfate and subsequently analyzed by gas chromatography (GC). GC measurements were performed with an Agilent 7890 instrument equipped with a DB-WAX UI column (30 m × 0.32 mm, 0.25 μm, Agilent, Santa Clara, CA, USA), a split/splitless injector port (1:5) and a flame ionization detection (FID) system. Hydrogen was used as carrier gas at a constant flow rate of 2.1 mL per minute. One μL sample was injected via an autosampler and measured using the following method: 40 ◦C (3 min), a temperature increase of 10 ◦C per minute until 230 ◦C and a final hold time of 10 min. The *trans*-anethole and *p*-anisaldehyde were semi-quantified referring to the area of the internal standard. Biotransformation products were identified using standards and comparison of retention indices with literature.

#### *3.11. Enzyme Activities*

Total peroxidase activity was determined photometrically (EONTM High Performance Microplate Spectrophotometer, BioTek Instruments GmbH, Bad Friedrichshall, Germany) by monitoring the oxidation of ABTS in the presence of hydrogen peroxide at 420 nm (ε<sup>420</sup> = 3.6 <sup>×</sup> 104 M−<sup>1</sup> cm−1) and 30 ◦C for 10 min. For this, the samples were mixed with sodium acetate buffer (100 mM, pH 4.0 or pH 3.5), 0.1 mM hydrogen peroxide, and 0.5 mM ABTS in a total volume of 300 μL. One unit of enzyme activity was defined as 1 μmol substrate oxidized per minute under the experimental conditions.

To determine manganese peroxidase activity, samples were mixed with manganese sulfate (1 mM), malonate buffer (100 mM, pH 3.5), and hydrogen peroxide (0.1 mM) in a total volume of 300 μL. Mn3<sup>+</sup> formation was monitored photometrically at 270 nm (ε<sup>270</sup> <sup>=</sup> 1.16 <sup>×</sup> 104 <sup>M</sup>−<sup>1</sup> cm<sup>−</sup>1) and 30 ◦C for 30 min. One unit of enzyme activity was defined as 1 μmol Mn3<sup>+</sup> per minute released by manganese peroxidases at the given conditions.

Decolorization of Reactive blue 19 (RB19, 150 μM) and Reactive black 5 (RB5, 80 μM) by PsaPOX (1 U/L; 0.25 mg/L) was tested. The respective anthraquinone dye and the enzyme was incubated in the presence of 100 μM hydrogen peroxide, 25 mM manganese sulfate, and 100 mM sodium acetate buffer pH 3.5 in a total volume of 300 μL at 40 ◦C for 20 min. Decolorization was monitored photometrically at 595 nm (RB19; <sup>ε</sup><sup>595</sup> = 1.0 <sup>×</sup> 10<sup>4</sup> M−<sup>1</sup> cm−1) or 598 nm (RB5; <sup>ε</sup><sup>598</sup> =3.0 <sup>×</sup> 10<sup>4</sup> M−<sup>1</sup> cm−1). One unit of enzyme activity was defined as 1 μmol dye degraded per minute at the given conditions.

All enzyme assays were performed as triplicates. Blanks were carried out with water instead of enzyme and by omission of hydrogen peroxide.
