**4. Conclusions**

A DyP-type peroxidase of *P. sapidus* with alkene cleavage activity as well as the corresponding gene were identified and the gene was heterologously expressed in *Komagataella pfa*ffi*i*. The PsaPOX possessed typical sequence motifs, structural topology, and catalytic residues as described for DyPs, even though the decolorization of the anthraquinone Reactive blue 19, a common reaction for DyPs, was not observed. A non-canonical Mn2+-oxidation site on the protein surface was detected, which allows PsaPOX to oxidize Mn2+. After biochemical characterization, the alkene cleavage activity of PsaPOX towards different aryl alkenes was confirmed by biotransformation. PsaPOX is the first described DyP-type peroxidase with such an activity. In addition, bleaching of β-carotene and annatto was determined. The results for the alkene cleavage underline the potential of the PsaPOX as biocatalyst for the generation of aromatic aldehydes with olfactory properties, such as *p*-anisaldehyde, veratraldehyde, or acetophenone, which are used in the fragrance and flavor industry [1]. Improvement of the conversions and product yields may be accomplished by protein engineering, as has been shown for the alkene cleaving manganese-dependent Cupin TM1459 from *Thermotoga maritima* [53]. Another application beyond aroma production could be carotene bleaching of whey or wheat dough.

**Supplementary Materials:** The following are available online, Figure S1: Structural homology model of PsaPOX, Figure S2: Detection of hydrogen peroxide in the IEX fraction with *o*-dianisidine and HRP in the presence of *trans*-anethole after 1 h of incubation at pH 6.0 and RT, Figure S3: Alignment of the hypothetical protein (KDQ29984.1) from *P. ostreatus*, which was the best hit for the 75 kDa band of the IEX fraction by a homology search against the public database NCBI, and other members of the GMC oxidoreductase family, Figure S4: Stability of PsaPOX during biotransformation of *trans*-anethole over 16 h, Table S1: *p*-Anisaldehyde concentration after biotransformation of *trans*-anethole with different basidiomycetes, Table S2: *p*-Anisaldehyde concentration after biotransformation of *trans*-anethole with the active IEX fraction in the presence or absence of Mn2<sup>+</sup> and/or H2O2 for 16 h at RT, Table S3: *p*-Anisaldehyde concentration after bioconversion of *trans*-anethole by recombinant PsaPOX with and without addition of H2O2 and Mn2<sup>+</sup> for 16 h at RT, Table S4: *p*-Anisaldehyde concentration after biotransformation of *trans*-anethole, (*E*)-methyl isoeugenol, and α-methylstyrene by recombinant PsaPOX (1 U/mL) in the presence of 100 μM H2O2 and 25 mM MnSO4 for 16 h at pH 3.5 and RT.

**Author Contributions:** Conceptualization, N.-K.K. and R.G.B.; methodology, N.-K.K.; validation, N.-K.K., R.G.B. and F.E.; formal analysis, N.-K.K.; investigation, N.K.K; writing—original draft preparation, N.-K.K.; writing—review and editing, R.G.B. and F.E.; visualization, N.-K.K.; supervision, F.E.; project administration, R.G.B.; funding acquisition, R.G.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded the BMBF cluster Bioeconomy International 2015, grant number 031B0307A. The APC was funded by the Open Access fund of the Gottfried Wilhelm Leibniz Universität Hannover.

**Acknowledgments:** B. Fuchs and A. Nieter are thanked for detecting the cleavage reaction and the peroxidase activity during the screenings.

**Conflicts of Interest:** The authors declare no conflict of interest.
