**1. Introduction**

Many small aromatic aldehydes and ketones are volatiles with olfactory properties and therefore of high interest to the fragrance and flavor industry [1]. One method to generate aldehydes and ketones is the oxidative cleavage of alkenes. Chemical options are ozonolysis, dihydroxylation followed by oxidative glycol cleavage, or metal-based methods [2–4]. However, all of these methods have disadvantages, such as the generation of explosive intermediates, the use of environmentally unfriendly and/or toxic oxidants and metal catalysts, or low yield and low chemoselectivity [2]. An alternative is the application of enzymes due to their high chemo-, regio-, and stereospecificity as well as the possibility to use mild reaction conditions [3]. Another advantage is the generation of "natural" flavors according to effective legislation in Europe and the US. This becomes more and more important considering the rising popularity of natural products [5]. Different proteins of different enzyme classes, which are heme, non-heme iron, or non-iron metal dependent and have different protein structures as well as different reaction mechanism are known to catalyze alkene cleavage reactions [3]. Specifically, an isoeugnol and *trans*-anethole oxygenase from *Pseudomonas putida* and two manganese dependent enzymes from *Thermotoga maritima* (manganese-dependent Cupin TM1459) and *Trametes hirsuta* (Mn3+-dependent proteinase A homologue) oxidatively cleaved the benzylic double bond of different aryl alkenes, such as isoeugenol and *trans*-anethole to form the respective

aldehydes [6–9]. In addition, alkene cleavage activity towards aryl alkenes was also detected for several peroxidases. Cleavage of different styrene derivatives was described for *Coprinus cinereus* peroxidase and a human myeloperoxidase as minor side reaction [10], while horseradish peroxidase (HRP) showed a chemoselectivity of 92% for the conversion of*trans*-anethole (90%) to *p*-anisaldehyde [11]. Furthermore, transformations of *o*-ethylisoeugenol and *trans*-anethole to the corresponding benzaldehyde derivatives by lignin peroxidases were described [3,11]. However, to our best knowledge no alkene cleavage activity of a dye-decolorizing peroxidase (DyP) is known.

DyP-type peroxidases (EC: 1.11.1.19) are a new superfamily of heme peroxidases that oxidize various dyes, in particular xenobiotic anthraquinone dyes, which are hardly oxidized by other peroxidases [12]. Furthermore, typical peroxidase substrates, such as ABTS (2,2 -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and phenolic compounds are also substrates for DyPs [13,14]. However, amino acid sequences, protein structures, and catalytic residues differ highly between DyPs and other classes of heme peroxidases [15]. Typical structural characteristics of DyPs are the ferredoxin-like fold, which is formed by two domains containing α-helices and four-stranded antiparallel β-sheets, and a GXXGD motif [14,15]. The active site (heme pocket) including a catalytic aspartic acid and arginine over the heme plane (distal) and proximal histidine is structurally similar to other heme peroxidases, even though other peroxidases contain histidine instead of aspartic acid [16]. The proximal histidine in the heme pocket functions as the fifth ligand of the heme iron, while the distal aspartic acid and arginine are involved in the activation of the enzyme [14,15]. The deprotonated aspartic acid (or asparagine) mediates the rearrangement of a proton from hydrogen peroxide after it enters the heme pocket in the resting state. This results in the heterolytic cleavage of hydrogen peroxide to water and oxidation of the heme to the radicalic-cationic oxoferryl species Compound I by two-fold single electron transfer [16]. Even though the distal arginine is not directly involved in the rearrangement of hydrogen peroxide, it is essential for the coordination of hydrogen peroxide to the heme iron and the stabilization of Compound I [16]. During the following reaction cycle, Compound I is reduced by oxidation of two substrate molecules to the state during enzyme resting state in two sequential steps with Compound II as intermediate. However, the existence of Compound II has not been confirmed universally for all DyP-type peroxidases [17]. In the presence of excessive hydrogen peroxide suicide inhibition was observed for different DyPs [18,19]. This is also well known for classical peroxidases as a result of an inactive oxidative state (Compound III) and results from reaction of hydrogen peroxide with Compound II [20,21].

The objective of the present study was to identify new enzymes of basidiomycetes with alkene cleavage activity towards aryl alkenes. A screening was performed using *trans*-anethole as model alkene. A new DyP-type peroxidase from *P. sapidus* was semi-purified and the coding gene was identified. Heterologous expression resulted in the production of soluble protein and allowed the biochemical characterization of the DyP. The enzyme was able to oxidize Mn2+, but did not catalyze the degradation of anthraquinone dyes, which is typical for other DyPs. Biotransformation experiments verified the cleavage activity towards different alkenes. This is the first study describing a DyP with alkene cleavage activity towards aryl alkenes.
