*2.5. Alkene Cleavage Activity of PsaPOX*

To prove the ability of PsaPOX to convert *trans*-anethole to *p*-anisaldehyde, biotransformation experiments were performed at optimal conditions using 1 U/mL peroxidase activity. Substrate cleavage was detected in the presence of hydrogen peroxide (Table S3), whereas no activity was observed in its absence as expected from the peroxidase activity measurement with ABTS (Figure 6a). Due to the fact that the semi-purified wild-type DyP showed an alkene cleavage activity without addition of hydrogen peroxide (see Table S2) a low amount of H2O2 must have been present in the analyzed IEX fraction. This was verified by incubation of the fraction with *o*-dianisidine and HRP in the presence of *trans*-anethole. Oxidation of *o*-dianisidine by HRP, which requires H2O2 as cosubstrate, and formation of a red-brown reaction product occurred (Figure S2). Further, analysis of the IEF fraction regarding a hydrogen peroxide producing enzyme revealed a hypothetical protein from *P. ostreatus* (KDQ29984.1). It belongs to the glucose-methanol-choline (GMC) oxidoreductase family as evident from the best hit for the protein band at 75 kDa (Figure 2, lane 1) according to protein sequencing (identified tryptic peptides: AADLIK, AIAVEFVR, ELGGVVDTELR, AQYDAWAELNR, VADASIIPIPVSAHTSSTVYMIGER, DLASGDPHGVGVSPESIDVTNYTR, VLGGSTTINAMLFPR, EVVVSAGTIGTPK) and homology search against the public database NCBI (Figure S3). The protein contained seven of the eight tryptic peptides identified for the 75 kDa band. The last one was found with an amino acid exchange (Arg instead of Lys), which is most likely a result of the different fungal strains the proteins originate from. The protein from *P. ostreatus* showed >92% identity to another hypothetical GMC oxidoreductase from *P. ostreatus* and ≥55% identity to a glucose oxidase from *Moniliophthora roreri* and other fungal alcohol oxidases (Figure S3), which belong to the GMC oxidoreductase family and are known for the production of hydrogen peroxide during substrate oxidation. Thus, the oxidase (75 kDa band) most likely produced the detected hydrogen peroxide, which was subsequently used as cosubstrate by the wild-type PsaPOX. Due to the fact that the formation of *p*-anisaldehyde by the oxidase under production of H2O2 seemed highly unlikely and as no further oxidation products of *trans*-anethole were detected, *trans*-anethole was excluded as substrate. Instead, the buffer component Bis-Tris, which contains several alcohol groups, or carbohydrate functionalities of other proteins in the IEX fraction were assumed to be used as substrate by the oxidase.

As described for the wild-type DyP the *p*-anisaldehyde concentration increased for the biotransformation with the recombinant enzyme in the presence of 25 mM Mn2<sup>+</sup> (Table S3). However, product formation in general was low. The residual peroxidase activity was determined during the biotransformation of *trans*-anethole (Figure S4). After 16 h, 62% of the activity remained, thus inactivation of the enzyme was not responsible for the relatively low product yields.

PsaPOX (1 U/mL) was further examined for alkene cleavage activity regarding other substrates in the presence of hydrogen peroxide and Mn2<sup>+</sup>. The aryl alkenes (*E*)-methyl isoeugenol as well as α-methylstyrene, which are derivatives of *trans*-anethole, were converted to the expected products (veratraldehyde and acetophenone), while piperine was not cleaved (Figure 7a). However, the resulting product concentration was fivefold lower for the biotransformation of (*E*)-methyl isoeugenol and more than tenfold lower for the conversion of α-methylstyrene than for *trans*-anethole. Different substrate specificities were also observed for the alkene cleavage by other peroxidases, such as HRP, *Coprinus cinereus* peroxidase, and a human myeloperoxidase [10,11], but a conversion of aryl alkenes using a DyP-type peroxidase has not been described before.

**Figure 7.** Alkene cleavage activity of PsaPOX on different substrates in the presence of 100 μM H2O2 and 25 mM MnSO4 at pH 3.5. (**a**) Product concentration after conversion of *trans*-anethole (6.7 mM) to *p*-anisaldehyde, (*E*)-methyl isoeugenol (6.7 mM) to veratraldehyde, and α-methylstyrene (6.7 mM) to acetophenone by PsaPOX (1 U/mL) at RT. The presented product concentrations are the differences between the values determined for the reaction with the active and heat inactivated enzyme (blank) (the original values are shown in Table S4). (**b**) Decolorization of 7% (*v*/*v*) β-carotene and 7% (*v*/*v*) annatto by PsaPOX (1 U/L) at 40 ◦C. Cleavage of carotenoids was shown as extinction decrease per min. Values are the average of triplicate experiments with standard deviations shown as error bars.

In addition to the described substrates, PsaPOX (1 U/L) also showed an alkene cleavage activity towards the natural dyes β-carotene and annatto (mixture of the xanthophylls bixin and norbixin), which was detected by substrate bleaching (Figure 7b). The activity for annatto was higher than for β-carotene. Cleavage of β-carotene and annatto is also known for other fungal DyPs [19,39–41]. For example, cleavage of β-carotene by a DyP from *Lepista irina* resulted in formation of the volatiles β-ionone, β-cyclocitral, dihydroactinidiolide, and 2-hydroy-2,6,6-trimethylcyclohexanone [41].
