**Nina-Katharina Krahe \*, Ralf G. Berger and Franziska Ersoy**

Institut für Lebensmittelchemie, Gottfried Wilhelm Leibniz Universität Hannover, Callinstraße 5, 30167 Hannover, Germany; rg.berger@lci.uni-hannover.de (R.G.B.); franziska.ersoy@lci.uni-hannover.de (F.E.)

**\*** Correspondence: nina.krahe@lci.uni-hannover.de; Tel.: +49-511-762-17257

Received: 20 February 2020; Accepted: 23 March 2020; Published: 27 March 2020

**Abstract:** Alkene cleavage is a possibility to generate aldehydes with olfactory properties for the fragrance and flavor industry. A dye-decolorizing peroxidase (DyP) of the basidiomycete *Pleurotus sapidus* (PsaPOX) cleaved the aryl alkene *trans*-anethole. The PsaPOX was semi-purified from the mycelium via FPLC, and the corresponding gene was identified. The amino acid sequence as well as the predicted tertiary structure showed typical characteristics of DyPs as well as a non-canonical Mn2<sup>+</sup>-oxidation site on its surface. The gene was expressed in *Komagataella pfa*ffi*i* GS115 yielding activities up to 142 U/L using 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) as substrate. PsaPOX exhibited optima at pH 3.5 and 40 ◦C and showed highest peroxidase activity in the presence of 100 μM H2O2 and 25 mM Mn2+. PsaPOX lacked the typical activity of DyPs towards anthraquinone dyes, but oxidized Mn2<sup>+</sup> to Mn3+. In addition, bleaching of β-carotene and annatto was observed. Biotransformation experiments verified the alkene cleavage activity towards the aryl alkenes (*E*)-methyl isoeugenol, α-methylstyrene, and *trans*-anethole, which was increased almost twofold in the presence of Mn2<sup>+</sup>. The resultant aldehydes are olfactants used in the fragrance and flavor industry. PsaPOX is the first described DyP with alkene cleavage activity towards aryl alkenes and showed potential as biocatalyst for flavor production.

**Keywords:** alkene cleavage; aryl alkenes; basidiomycota; biocatalysis; carotene degradation; dye-decolorizing peroxidase (DyP); manganese; *Komagataella pfa*ffi*i*; *Pleurotus sapidus*
