A DyP-type peroxidase of pleurotus sapidus with alkene cleaving activity

Article abstract of DOI:10.3390/molecules25071536

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⁺-oxidation site on its surface. The gene was expressed in Komagataella pfaffii GS115 yielding activities up to 142 U/L using 2,2⁰-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⁺ 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⁺. 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.

Full text of DOI:10.3390/molecules25071536

molecules
Article
A DyP-Type Peroxidase of Pleurotus sapidus with
Alkene Cleaving Activity
Nina-Katharina Krahe * , Ralf G. Berger and Franziska Ersoy
Institut für Lebensmittelchemie, Gottfried Wilhelm Leibniz Universität Hannover, Callinstraße 5,
3
0167 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
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
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
2
+
non-canonical Mn -oxidation site on its surface. The gene was expressed in Komagataella pfaffii
0
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
2
+
the presence of 100
anthraquinone dyes, but oxidized Mn to Mn . In addition, bleaching of
was observed. Biotransformation experiments verified the alkene cleavage activity towards the aryl
alkenes (E)-methyl isoeugenol, -methylstyrene, and trans-anethole, which was increased almost
µM H O and 25 mM Mn . PsaPOX lacked the typical activity of DyPs towards
2 2
2
+
3+
β-carotene and annatto
α
+
2
twofold in the presence of Mn . 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 pfaffii; Pleurotus sapidus
1
. Introduction
Many small aromatic aldehydes and ketones are volatiles with olfactory properties and therefore
of high interest to the fragrance and flavor industry [
ketones is the oxidative cleavage of alkenes. Chemical options are ozonolysis, dihydroxylation
followed by oxidative glycol cleavage, or metal-based methods [ ]. 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 [ ].
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 [ ]. 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 [ ]. Different proteins of different
1]. One method to generate aldehydes and
2–4
2
3
“
5
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)
3+
and Trametes hirsuta (Mn -dependent proteinase A homologue) oxidatively cleaved the benzylic
double bond of different aryl alkenes, such as isoeugenol and trans-anethole to form the respective
Molecules 2020, 25, 1536; doi:10.3390/molecules25071536
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1536
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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
0
(
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 -helices and four-stranded
antiparallel -sheets, and a GXXGD motif [14 15]. The active site (heme pocket) including a catalytic
α
β
,
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
2
+
biochemical characterization of the DyP. The enzyme was able to oxidize Mn , 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.
2
. Results and Discussion
2
.1. Purification and Identification of the Alkene Cleavage Activity
Within a screening of 17 basidiomycetes for alkene cleavage activity using the substrate
trans-anethole (Table S1) P. sapidus turned out to be a promising candidate for the production of
the desired activity. The lyophilized mycelium as well as the culture supernatant was examined for
the ability to cleave trans-anethole after submerged cultivation. The culture supernatant showed no
activity, whereas the incubation in the presence of the mycelium resulted in formation of 5.36 mM
p-anisaldehyde (molar yield of 79.03%; Figure 1).

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Figure 1. Bioconversion of trans-anethole by P. sapidus. (a) Alkene cleavage of trans-anethole resulted
in the formation of p-anisaldehyde. ( ) GC-FID chromatogram of an n-hexane extract of the conversion
b
of the blank sample (blue) and after incubation with lyophilized mycelium of P. sapidus (orange).
Retention times: trans-anethole (11.53 min) and p-anisaldehyde (13.45 min).
For the identification of the enzyme catalyzing the trans-anethole cleavage, it was semi-purified
from the rehydrated mycelium by hydrophobic interaction and anion exchange chromatography (IEX).
During the purification, a high activity loss occurred, which resulted in low product concentrations
after conversion (Table S2). This was most likely a result of protein loss, enzyme degradation or
denaturation as described for other enzymes [22
–24]. Another possibility is the loss of cofactors or
]. Addition of Mn2 led
+
–substrates, such as metal ions or peroxides during the purification steps [6
to a 15-fold increase in p-anisaldehyde concentration, which was further increased by addition of
hydrogen peroxide, indicating a cosubstrate dependency (Table S2). Chemical conversion by Mn2
+
alone was excluded, while product formation was observed with hydrogen peroxide (18 µM), but with
a yield around 30-fold lower than the one for the enzymatic reaction (Table S2). Thus, the improved
bioconversion in the presence of Mn2 and hydrogen peroxide was verified to be the result of an
increased enzyme activity.
+
A class of fungal enzymes that requires hydrogen peroxide and some of which need Mn2+ for
catalysis are peroxidases [12,25,26]. The anion exchange fractions, which showed alkene cleavage
activity, also exhibited peroxidase activity, thus verifying the presence of a peroxidase. For visualization
of the activity, a semi-native PAGE was performed and stained with ABTS in the presence of hydrogen
peroxide (Figure 2). Two peroxidases running at 45 and 52 kDa were detected. The respective protein
bands stained with Coomassie Brilliant Blue were excised for electrospray ionization tandem mass
spectrometry. Due to the low protein concentration of the 52 kDa band, no meaningful peptides were
found (Figure 2, lane 1). This paper presents the data obtained for the protein running at 45 kDa
(Figure 2, arrow).
Three tryptic peptides (EGSELLGAR, DGSFLTFR, and SGAPIEITPLKDDPK) were identified by
ESI-MS/MS. Homology searches against the public database NCBI using the mascot search engine
Matrix Science, London, UK) identified a DyP-type peroxidase of P. ostreatus PC15 (Pleos-DyP4 [27];
GenBank accession no. KDQ22873.1), a close relative of P. sapidus, as the best hit.
(

Molecules 2020, 25, 1536
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Figure 2. Semi-native PAGE of the active fraction after purification of the alkene cleavage enzyme from
P. sapidus by IEX. 1: gel stained with Coomassie Brilliant Blue; 2: gel stained with ABTS in the presence
of hydrogen peroxide, M: pre-stained molecular mass marker. An arrow and a box marks the protein
that was successfully identified by sequencing.
2
.2. Amplification and Expression of PsaPOX
Specific primers successfully amplified the 1512 bp coding region of the gene from P. sapidus.
The translated amino acid sequence of 504 aa contained the peptide fragments that were obtained by
ESI-MS/MS and showed highest identity (94%) to the sequence of Pleos-DyP4 (Figure 3), which has not
been investigated for its alkene cleavage activity against trans-anethole or other substrates before [27,28].
In our hands, P. ostreatus showed a weaker trans-anethole cleavage activity, too (Table S1). Identity of
the P. sapidus peroxidase (PsaPOX) to other DyPs and proteins was lower than 60%. A sequence
alignment with other DyPs (Figure 3) confirmed that PsaPOX exhibited the typical GXXGD motif and
all important residues known for the catalytic activity of DyPs, such as the proximal histidine (His-334)
(
(
fifth ligand of heme iron) and distal Asp-196 and Arg-360 involved in the activation of the enzyme
formation of compound I) by H O cleavage [14 15]. Furthermore, Trp-405 was identified as a homolog
,
2
2
to the surface exposed Trp-377 of AauDyP of Auricularia auricula-judae, which serves as an oxidation
site for bulky substrates such as Reactive blue 19 (RBB19) using a long-range electron transfer [29].
Comparison of the PsaPOX and Pleos-DyP4 sequence indicated that PsaPOX exhibited a non-canonical
2
+
Mn -oxidation site on its surface (Asp-215, Glu-345, Asp-352 and Asp-354; Trp-339 participates in the
electron transport from the oxidation site to the heme) like Pleos-DyP4 [28] and can oxidize Mn2 to
+
3
+
Mn , which is known for a few fungal DyPs only [24,27,30,31].
A structural homology model of PsaPOX (Figure S1), which was generated using the X-ray crystal
structure of Pleos-DyP4 (PDB-ID 6fsk) on the SWISS-MODEL server, possessed typical characteristics
of the DyP-type peroxidase family (N- and C-terminal ferredoxin-like domain, each formed by
four-stranded antiparallel β-sheets and several α-helices) [15] and supported the classification of
PsaPOX as DyP. Furthermore, the analysis of the amino acid sequence using PeroxiBase [32] related
the P. sapidus peroxidase (PsaPOX) to the class “DyP-type peroxidase D”. As known from literature,
DyPs differ significantly in amino acid sequence, tertiary structure, and catalytic residues from other
representatives of the heme peroxidases, such as HRP, human myeloperoxidase, lignin peroxidase,
or Coprinus cinereus peroxidase [12
,15,33], all of which are able to cleave trans-anethole or structurally
related alkenes [10,11]. So far no DyP is known to catalyze the mentioned reaction.

Molecules 2020, 25, 1536
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Figure 3. Alignment of alkene cleaving peroxidase from P. sapidus (PsaPOX) with the Pleos-DyP4
of P. ostreatus (PosDyP; KDQ22873.1) and other characterized DyPs. AgaDyP: Armillaria gallica
PBK80505.1), VvoDyP: Volvariella volvacea (AKU04643.1), CtrDyP: Coriolopsis trogii (AUW34346.1),
(
GluDyP: Ganoderma lucidum (ADN05763.1), and TveDyP: Trametes versicolor (XP_008039377.1).
Inverted triangles show amino acids important for heme binding (histidine (magenta) functions
as ligand for heme and the four other amino acid residues form a hydrogen peroxide binding pocket).
Aspartic acid, which forms a hydrogen bond with histidine to stabilize compound I (oxidized heme
after transfer of two electrons to H O ) is shown in grey. The black box indicates the GXXDG motif
2
2
containing the catalytic aspartic acid residue (yellow), which cleaves H O heterolytically with the
2
2
help of the neighboring arginine (green) to form compound I, and the circle presents an exposed
tryptophan potentially involved in an LRET (long range electron transfer). Important amino acids
2
+
for Mn -oxidation are highlighted in cyan; asterisks indicate conserved residues, colons equivalent
residues and dots partial residue conservation. Peptides identified by protein sequencing are underlined.
Alignment was performed with Clustal Omega (European Bioinformatics Institute, Hinxton, UK).

Molecules 2020, 25, 1536
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2
.3. Production and Purification of the Recombinant PsaPOX
The PsaPOX gene was amplified and cloned into the K. pfaffii expression vector pPIC9. The initial
expression of the gene yielded average peroxidase activities of 65 U/L after 72 h of cultivation. The best
performing colonies produced activities up to 142 U/L, indicating a multiple insertion of the expression
construct [34]. Similar results were obtained for the heterologous production of a DyP from Funalia
trogii in K. pfaffii previously [24]. Further experiments were performed using the clone with the highest
peroxidase activity for maximum protein production.
The recombinant peroxidase was purified by Ni-NTA affinity. Using SDS-PAGE, a molecular
mass of around 61 kDa was determined (Figure 4a), which is slightly higher than the calculated
molecular mass of 54.9 kDa (ExPASy). In addition, the native recombinant enzyme was detected
at 52 kDa after semi-native PAGE, while the native wild-type enzyme showed a band at 45 kDa
(
Figures 2 and 4b). Deglycosylation by endoglycosidase H (EndoH) showed that the higher molecular
mass was attributed to post-translational modifications by K. pfaffii, as has been described for other
proteins [35 36]. The wild-type peroxidase, on the contrary, was not glycosylated (see Figures 2 and
b). That is uncommon for DyPs, which usually exhibit a carbohydrate content of 9 to 30% [12].
The molecular mass of the monomeric PsaPOX was similar to other DyP-type peroxidases [12].
,
4
Figure 4. Purification of the recombinant PsaPOX by Ni-IMAC. (
Brilliant Blue. 1: flow through, 2: elution fraction, 3: elution fraction incubated with EndoH, 4: EndoH,
M1: molecular mass marker. ( ) Semi-native PAGE stained with ABTS in the presence of hydrogen
peroxide. 5: elution fraction, 6: elution fraction incubated with EndoH, M2: pre-stained molecular
mass marker. ( ) Isoelectric focusing gel. 7: elution fraction stained with phenylendiamine in the
a) SDS-PAGE stained with Coomassie
b
c
presence of urea peroxide. M3: standard protein marker for isoelectric focusing stained with Coomassie
Brilliant Blue.
Analysis of the purified recombinant peroxidase by isoelectric focusing indicated an isoelectric
point around pH 6.7 (Figure 4c), which differs slightly from the calculated value of 6.28 (ExPASy),
but was similar to the isoelectric point of another DyP-type peroxidase from P. sapidus [19]. Most other
proteins belonging to the DyP-type peroxidase family showed lower values (pI 3.5-4.3, [12]).
2
.4. Biochemical Characterization of PsaPOX
The influence of pH and temperature on PsaPOX activity and stability was determined using
ABTS in the presence of hydrogen peroxide as substrate (Figure 5). The enzyme showed a pH optimum
of 3.5 while more than 50% of activity was conserved between pH 3 and 5 (Figure 5a). At lower or
higher pH values of
enzyme. The results were consistent with the findings for other fungal DyPs, which had pH optima in
the range between pH 2 and 5 [13 19 27]. PsaPOX showed the highest pH stability with a residual
≤
25% of activity remained, most likely due to conformational changes of the
,
,

Molecules 2020, 25, 1536
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peroxidase activity of
≥
90% between pH 2.0 and 5.5 after 1 h of incubation (Figure 5c). At pH values
higher than six, near the isoelectric point, the stability decreased drastically, probably due to a reduced
solubility and changes of the protein structure, which may have resulted in protein aggregation.
Figure 5. Influence of pH and temperature on activity and stability of PsaPOX. The pH optimum
◦
(
[
a
) was determined to be 3.5 and the temperature optimum (
%] was defined as the percentage of activity detected with respect to the highest activity in each
) was determined after incubation of PsaPOX in Britton Robinson buffer
b) 40 C. Relative peroxidase activity
experiment. pH stability (
ranging from pH 2.0 to 9.5 for 1 h at RT and temperature stability (
c
◦
d
) after incubation at 20 to 90 C
◦
and pH 3.5 for 1 h. Residual activities were determined at pH 3.5 and 40 C. Values are the average of
triplicate experiments with standard deviations shown as error bars.
◦
Peroxidase activity of PsaPOX increased with rising temperature, reaching its maximum at 40 C
◦
(
Figure 5b), which was similar to the optimum (30–40 C) of a recombinant DyP from P. ostreatus [13],
but higher than the optimum (RT) of another DyP-type peroxidase of P. sapidus produced heterologously
in Escherichia coli [37]. With further temperature increase, the peroxidase activity of PsaPOX decreased
continuously. The temperature stability of PsaPOX was determined after an incubation for 1 h at
◦
different temperatures (Figure 5d). The enzyme was relatively stable at temperatures from 20 to 60 C
with a residual activity 80%. At higher temperatures, a high loss of activity was observed due to
≥
protein denaturation, resulting in residual activities <5%. The temperature stability of PsaPOX was
higher than the stabilities of DyPs from Bjerkandara adusta and Auricularia auricular-judae, which were
◦
◦
produced heterologously in E. coli (residual activity
≥
80% and <5% after 1 h at 20–50 C and 60 C,
respectively [13]), and of a DyP from P. sapidus produced in Trichoderma reesei (residual activity
≥
80%
◦
◦
and <65% after 5 min at 15-45 C and 50 C, respectively [19]).
As mentioned above, the addition of hydrogen peroxide as well as Mn2+ led to an increase of the
product concentration for the biotransformation of trans-anethole using the lyophilized mycelium of
P. sapidus containing the wild-type PsaPOX (Table S2). For this reason, the hydrogen peroxide and
Mn dependencies were examined for the recombinant enzyme using ABTS as substrate at optimal
pH and temperature (Figure 6). As expected, no peroxidase activity was detectable without hydrogen
peroxide. The activity rose with increasing peroxide concentration and reached its optimum in the
2
+
presence of 100 µM H O (Figure 6a). An increase of the hydrogen peroxide concentration led to a
2 2
continuous activity decrease. Suicide inhibition in the presence of excess hydrogen peroxide is well
known for classical peroxidases as a result of the formation of an inactive oxidative state (Compound
III) by reaction of H O and Compound II [20,21], even if the existence of Compound II has not
2
2

Molecules 2020, 25, 1536
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been confirmed for DyP-type peroxidases universally [17]. However, inhibition of other DyPs in the
presence of higher hydrogen peroxide concentrations has been reported [18,19].
) and Mn2 concentration (
+
b) on the activity of PsaPOX.
Figure 6. Effect of hydrogen peroxide (
a
Relative peroxidase activity [%] was defined as the percentage of activity detected with respect to the
highest activity obtained in each experiment. Values are the average of triplicate experiments with
standard deviations shown as error bars.
Investigation of the Mn2 dependency (Figure 6b) showed that PsaPOX activity rose with
+
increasing Mn2 concentration, but was not completely dependent on the addition of Mn . 30% of
+
2+
2+
peroxidase activity were detected without addition of Mn . PsaPOX reached the maximal activity in
the presence of 25 mM Mn . Evaluation of Mn formation by Mn oxidation revealed a manganese
peroxidase activity of 0.4 U compared to 1 U of peroxidase activity using ABTS as substrate. This result
2
+
3+
2+
2
+
fits the prediction of a Mn oxidation site. Only a few fungal DyPs are known to catalyze the oxidation
of Mn2
+
[
24
,
27
,
30
,
31]. Calculation of kinetic constants (Table 1) showed that the catalytic efficiency of
2
+
PsaPOX towards Mn was similar to the one of Pleos-DyP1 from P. ostreatus and Ftr-DyP from Funalia
trogii [24,27]. However, the catalytic efficiency of Pleos-DyP4 was higher [27].
Table 1. Michaelis constants (K
m
), catalytic constants (kcat), and catalytic efficiencies (kcat/K
m
) for
PsaPOX using ABTS, Mn2 , Reactive blue 19 (RB19), and Reactive black 5 (RB5) as substrate. Values are
+
the average of triplicate experiments with indication of standard deviations.
kcat (s 1)
−
−1
−1
)
Substrate
Km (µM)
kcat/Km (s mM
ABTS
Mn
RB19
RB5
37 ± 4
1025 ± 79
n. d.
6.8 ± 0.2
184 ± 5
2
+
7.2 ± 0.1
7 ± 0.1
-
-
-
-
n. d.
n. d.: no activity was detected.
Kinetic parameters were also calculated for the oxidation of ABTS at optimal conditions (Table 1).
The affinity of PsaPOX to ABTS (37 M) was similar to a DyP from Irpex lacteus (28 M), but higher in
comparison to the FtrDyP from F. trogii (182 M) and the Pleos-DyP2 from P. ostreatus (787 M) [18 24 27].
In contrast, the catalytic efficiency of PsaPOX (184 s mM ) was lower than the efficiency of the DyP
µ
µ
µ
µ
,
,
−
1
−1
−1
−1
−1
−1
from Irpex lacteus (8000 s mM ) and Pleos-DyP4 (352 s mM ), but higher than the efficiency of the
−
1
−1
FtrDyP (54 s mM ).
It is known that DyP-type peroxidases typically oxidize anthraquinones and other dyes. Exemplary,
decolorization of Reactive blue 19 (anthraquinone dye) and Reactive black 5 (recalcitrant azo dye)
by recombinant PsaPOX (1 U/L) was tested. Unexpectedly, PsaPOX showed activity for neither of
the substrates (Table 1), although the protein sequence and tertiary structure as well as the presence
of typical catalytic residues and the GXXDG motif identified the enzyme as a DyP-type peroxidase.
However, Pleos-DyP1 from P. ostreatus and TvDyP1 from Trametes versicolor also did not oxidize the

Molecules 2020, 25, 1536
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high redox-potential Reactive black 5, although they degraded Reactive blue 19 [27
,30]. A missing
activity against Reactive blue 19 or another anthraquinone has not been described for a fungal or class
D type DyP before, but one bacterial DyP of Pseudomonas fluorescens (DyP2B, DyP typ class B) is known
not to oxidize the anthraquinone dye Reactive blue 4 [38].
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 H O must have been present in the analyzed IEX
2
2
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 H O as cosubstrate, and formation
2
2
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 H O seemed
2
2
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 (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
+
2
+
in the presence of hydrogen peroxide and Mn . The aryl alkenes (E)-methyl isoeugenol as well as
-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 -methylstyrene than for trans-anethole. Different
substrate specificities were also observed for the alkene cleavage by other peroxidases, such as HRP,
α
(
α
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.

Molecules 2020, 25, 1536
10 of 17
Figure 7. Alkene cleavage activity of PsaPOX on different substrates in the presence of 100
and 25 mM MnSO at pH 3.5. ( ) Product concentration after conversion of trans-anethole (6.7 mM) to
p-anisaldehyde, (E)-methyl isoeugenol (6.7 mM) to veratraldehyde, and -methylstyrene (6.7 mM) to
µM H O
2 2
a
4
α
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
which was detected by substrate bleaching (Figure 7b). The activity for annatto was higher than
for -carotene. Cleavage of -carotene and annatto is also known for other fungal DyPs [19 39 41].
For example, cleavage of -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].
β-carotene and annatto (mixture of the xanthophylls bixin and norbixin),
β
β
, –
β
3
. Materials and Methods
3
.1. Chemicals and Materials
Chemicals were obtained from Sigma-Aldrich (Seelze, Germany), Carl-Roth (Karlsruhe, Germany),
or Merck (Darmstadt, Germany) in P. a. quality. Enzymes were from Thermo Fisher Scientific
Braunschweig, Germany), if not stated otherwise. PCR primers were obtained from Eurofins MWG
Operon (Ebersberg, Germany).
(
3
.2. Cultivation of P. sapidus
P. sapidus (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ,
strain no. 2866) was pre-grown on 1.5% (w/v) agar plates with standard nutrient liquid (SNL) medium
◦
2
and maintained at 4 C until use [39]. For pre-cultivation, 1 cm of grown agar was transferred to 100 mL
SNL medium and homogenized using an Ultraturrax homogenizer (ART Prozess- & Labortechnik,
◦
Müllheim, Germany). The pre-cultures were incubated for 5 days at 150 rpm and 24 C. Afterwards,
6
.5 g of pre-grown mycelium was used to inoculate 250 mL SNL. The main culture was incubated
◦
at 150 rpm and 24 C. After six days, the mycelium was separated from the culture supernatant by
centrifugation (5000× g, 4 C, 15 min) and lyophilized as described elsewhere [42].
◦
3
.3. Purification Strategy
Ten g of lyophilized mycelium were re-suspended in 400 mL buffer A (50 mM Bis-Tris, pH 6.0,
◦
1
M (NH ) SO ) and extracted for 1 h at 4 C in horizontal position in an orbital shaker. Insoluble
4
2
4
◦
components were removed by centrifugation (5000
×
g, 4 C, 15 min) followed by filtration (PES filter,

Molecules 2020, 25, 1536
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0
.45 µm, Merck). Subsequently, 80 mL filtered supernatant were applied on a Phenyl Sepharose fast
flow column (20 mL, GE Healthcare Bio-Sciences AB, Uppsala, Sweden) pre-equilibrated with buffer A.
After the column was washed with buffer A, the active enzyme was eluted with a linear gradient (130 mL,
1
00–0% buffer A) with 100% distilled water at a constant flow rate of 2 mL/min. Active fractions were
pooled, desalted and concentrated by ultrafiltration (3 kDa cut off, polyethersulfone (PES), Sartorius,
Göttingen, Germany). Concentrate (20 mL) was diluted two times with 20 mM sodium acetate pH
4.0 (buffer B) and loaded onto three linked HiTrap SP Sepharose columns (1 mL, GE Healthcare
Bio-Sciences AB) pre-equilibrated with buffer B. Proteins were eluted with a stepwise ionic strength
gradient (0, 20, 100% buffer C: 20 mM sodium acetate pH 4.0, 1 M NaCl) with 100% buffer C at a
constant flow rate of 1 mL/min.
3
.4. Gel Electrophoresis
SDS-PAGE analysis was performed as described elsewhere [43]. Semi-native PAGE was performed
under non-denaturing conditions using 12% gels. For this, samples were prepared with a native
loading buffer (without DTT and without 2% (w/v; 6.9 mM) SDS) and gel electrophoresis was performed
◦
at 10 mA per gel and 4 C. Gels were stained with 0.5 mM ABTS (dissolved in 100 mM sodium
acetate buffer pH 3.5 or 4.5) in the presence of 100
For deglycosylation, samples were treated with 1
BioLabs, Ipswich, MA, USA) in 20 µL for 2 h at 37 C before gel electrophoresis.
µM hydrogen peroxide for detection of peroxidases.
µL (500 U) endoglycosidase H (EndoH, New England
◦
3
.5. Isoelectric Focussing
TM
Analytical isoelectric focusing polyacrylamide gel electrophoresis was performed on a HPE
TM
system (Serva Electrophoresis GmbH, Heidelberg, Germany) using ServalytTM
Precast Gels (Serva Electrophoresis GmbH) with an immobilized pH gradient of pH
BlueHorizon
TM
Precotes
3
to 10. To determine the isoelectric points of the enzymes, marker proteins (IEF-Marker 3-10,
Serva Electrophoresis GmbH) were used. Gels were stained with Coomassie Brilliant Blue, or for
specific visualization of peroxidases, with 1% (w/v; 9.2 mM) phenylendiamine and 1% (w/v; 10.6 mM)
urea peroxide (dissolved in 100 mM sodium acetate buffer pH 3.5) at RT until a yellow band
was observed.
3
.6. Peptide Mass Fingerprinting
Protein bands were excised from SDS gels, dried, and tryptically hydrolysed. The resulting
peptides were extracted and purified according to standard protocols and the amino acid sequence
was analyzed with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) using a maXis
quadrupole time of flight (QTOF) mass spectrometer (Bruker, Bremen, Germany) as described
previously [43
,44]. The obtained partial sequences of PsaPOX and of the oxidase were used for a
similarity search against public databases (NCBI BlastP).
3
.7. cDNA Synthesis and Gene Amplification
Isolation of total RNA from mycelium of P. sapidus at culture day six and cDNA synthesis were
0
performed as described previously [13] using the primer 5 -AAGCAGTGGTATCAACGCAGAGT
0
ACGCTTTTTTTTTTTTTTTTTTT-3 for reverse transcription. Specific primers for gene amplification
were deduced from the ORF-start (P1: 5 -ATGACTACACCTGCACCACCCCTCGACCTC-3 ) and -stop
P2: 5 -TCAAGCAGAGATTGGAGCTTGGGTSWGAGGA-3 ) region of the homologous peroxidase of
0
0
0
0
(
Pleurotus ostreatus PC15 (GenBank accession no. KDQ22873.1). PCRs were performed with Phusion
High-Fidelity DNA Polymerase and the Master Cycler gradient (Eppendorf, Hamburg, Germany)
◦
as described elsewhere [45]. The cycler program was as follows: denaturation for 2 min at 98 C,
3
◦
◦
◦
◦
5 cycles at 98 C for 1 min, 62 C for 30 s and 72 C for 90 s, and a final elongation at 72 C for 10 min.
Analysis of PCR products, ligation, transformation in Escherichia coli, colony PCR, and sequencing
were performed as described by Behrens et al. [13]. Translation of DNA sequences was performed

Molecules 2020, 25, 1536
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using SnapGene® (GSL Biotech LLC, Chicago, IL, USA). Sequence homology was examined using
BLAST [46]. Alignments were produced by ClustalOmega [47].
3
.8. Heterologous Expression of PsaPOX in Komagataella pfaffii
The gene of PsaPOX was amplified with a C-terminal 6x His tag using the
0
0
primers PsaPOX_fw 5 -AAAAAGAATTCatgactacacctgcaccacccctcgacctcaacaa-3 and PsaPOX_rev
5
0
0
-atatatGCGGCCGC tcaGTGGTGATGGTGATGATGggtagagatcggagcctgggcctg-3 (underlined are
the EcoRI and NotI restriction sites, respectively; lower cases represent parts of the coding PsaPOX).
In addition, it was inserted in frame with the Saccharomyces cerevisiae -factor secretion signal
α
sequence into the K. pfaffii pPIC9 expression vector (Invitrogen, Karlsruhe, Germany). The resulting
expression construct pPIC-PsaPOX-His was transformed into E. coli TOP10 for vector propagation,
isolated (NucleoSpin, Macherey-Nagel, Düren, Germany), linearized with PmeI, and used for
transformation of K. pfaffii GS115 according to a standard protocol [48]. The linearized empty
vector was transformed in the same way and served as negative control. Forty-eight transformants
were tested for peroxidase activity after selection according to their ability to grow on histidine-deficient
agar plates in 96-well plates for 120 h, as described elsewhere [49]. Gene expression was induced by
daily addition of 1% (v/v) methanol.
3
.9. His-Tag Purification of Recombinant PsaPOX
For purification of the His-tag labelled recombinant enzyme from K. pfaffii culture supernatant,
Ni-NTA affinity chromatography was used according to Nieter et al. [50].
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 L 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 L (6.7 mM) trans-anethole. Blanks
chemical: without lyophilisate or liquid sample; biological: with heat inactivated mycelium (1 h at
µ
µ
(
9
◦
5 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
00 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,
.25 m, Agilent, Santa Clara, CA, USA), a split/splitless injector port (1:5) and a flame ionization
1
×
0
µ
detection (FID) system. Hydrogen was used as carrier gas at a constant flow rate of 2.1 mL per minute.
◦
One
(
µ
L 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
4
−1 −1
oxidation of ABTS in the presence of hydrogen peroxide at 420 nm (
3
ε
= 3.6
420
×
10 M cm ) and
◦
0 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.

Molecules 2020, 25, 1536
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Mn3 formation was monitored photometrically at 270 nm ( 2
3
+
ε
= 1.16
×
10 M cm ) and 30 C for
4
−1
−1
◦
70
3
+
0 min. One unit of enzyme activity was defined as 1 µmol Mn 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
pH 3.5 in a total volume of 300
at 595 nm (RB19;
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.
µ
M hydrogen peroxide, 25 mM manganese sulfate, and 100 mM sodium acetate buffer
◦
µ
4
L at 40 C for 20 min. Decolorization was monitored photometrically
−1
−1
4
−1 −1
ε
= 1.0
×
10 M cm ) or 598 nm (RB5; ε =3.0
598
×
10 M cm ). One unit of
595
3
.12. Biochemical Characterization of PsaPOX
Effects of pH and temperature on peroxidase activity of PsaPOX (0.25 mg/L) were analyzed
with ABTS as substrate as described above (see “4.11”). Relative activities were normalized to the
highest activity and residual activities to the initial activity prior incubation. The pH optimum was
determined using Britton-Robinson buffer [51] in a range of pH 2.0–9.5 instead of sodium acetate
buffer. For determination of the temperature optimum the activity assay was performed at different
◦
temperatures (20–90 C) at pH 3.5, whilst for analysis of the temperature stability the enzyme was
◦
◦
incubated for 1 h at 20–90 C prior enzyme activity measurement at pH 3.5 and 40 C. For the analysis
of pH-stability PsaPOX was incubated in Britton-Robinson buffer from pH 2.0 to 9.5 for 1 h at RT before
◦
the peroxidase activity was examined at pH 3.5 and 40 C.
Hydrogen peroxide as well as Mn2 dependency of PsaPOX were determined for PsaPOX by
+
evaluation of peroxidase activity as described above (“4.11”) with changing hydrogen peroxide
2
+
and manganese sulfate concentrations (H O : 0–1 mM H O , without addition of MnSO ; Mn :
2
2
2
2
4
1
00
PsaPOX were calculated for Mn and ABTS (0–300
5 mM MnSO ) by SigmaPlot 12.5 (Systat Software Inc., Chicago, IL, USA) with nonlinear regression.
µM H O , 0–100 mM MnSO ) at optimal pH and thermal conditions. Kinetic constants of
2 2 4
2
+
µ
M ABTS in the presence of 100 µM H O and
2 2
2
4
Protein concentrations were determined according to Lowry et al. [52] using bovine serum albumin
as standard.
3
.13. Alkene Cleavage Activity of PsaPOX
The purified recombinant PsaPOX was used for transformation of trans-anethole as mentioned
above (“4.10”) to confirm alkene cleavage activity. For this, 1 U/mL (0.25 mg/mL) of enzyme was
used for biotransformation. Biotransformation was performed with 100 mM sodium acetate buffer
pH 3.5 in the presence of 100
Biotransformation of the alkenes methyl isoeugenol (6.7 mM),
0.7 mM) was tested accordingly. Blanks were performed without enzyme (chemical blank) or with
µM hydrogen peroxide and 25 mM manganese sulfate at RT for 16 h.
α-methylstyrene (6.7 mM), and piperine
(
◦
heat inactivated enzyme (1 h at 95 C, biological blank). The determined product concentrations for
the blanks were subtracted from the concentrations yielded for the reaction with the active enzyme to
calculate the enzymatically generated product concentration. For carotene degradation, a
emulsion was prepared according to Linke et al. [43]. 7% (v/v) of -carotene emulsion or annatto
Chr. Hansen, Nienburg, Germany, Prod. No. 240569), 100 M hydrogen peroxide, 25 mM manganese
β-carotene
β
(
µ
sulfate, 100 mM sodium acetate pH 3.5, and 1 U/L (0.25 mg/L) PsaPOX in a total volume of 300
incubated at 40 C for 20 min. Alkene cleavage of both substrates was measured photometrically as
extinction decrease at 455 nm.
µ
L was
◦
3
.14. Detection of Hydrogen Peroxide
For the detection of H O , 75
µL IEX fraction, 50 mM Bis-Tris pH 6.0, 6.7 mM trans-anethole,
2
2
1
0 U/mL HRP (Sigma Aldrich), and 0.5 mM o-dianisidine in a total volume of 300 µL were incubated at

Molecules 2020, 25, 1536
14 of 17
RT for 1 h. In the presence of hydrogen peroxide, formation of a red-brown reaction product occurred.
Blanks were performed with 50 mM Bis-Tris pH 6.0 instead of IEX fraction.
3
.15. Sequence Accession Numbers
The nucleotide sequence of the PsaPOX gene has been deposited in the GenBank database under
accession number MT043310.
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 pfaffii. 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
2
+
DyPs, was not observed. A non-canonical Mn -oxidation site on the protein surface was detected,
2+
which allows PsaPOX to oxidize Mn . 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
2
+
biotransformation of trans-anethole with the active IEX fraction in the presence or absence of Mn and/or H O2
for 16 h at RT, Table S3: p-Anisaldehyde concentration after bioconversion of trans-anethole by recombinant
2
2
+
PsaPOX with and without addition of H O and Mn for 16 h at RT, Table S4: p-Anisaldehyde concentration
2
2
after biotransformation of trans-anethole, (E)-methyl isoeugenol, and
α-methylstyrene by recombinant PsaPOX
(
1 U/mL) in the presence of 100 µM H O and 25 mM MnSO for 16 h at pH 3.5 and RT.
2
2
4
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.
References
1
.
Fahlbusch, K.-G.; Hammerschmidt, F.-J.; Panten, J.; Pickenhagen, W.; Schatkowski, D.; Bauer, K.; Garbe, D.;
Surburg, H. Flavors and Fragrances. In Ullmann’s Encyclopedia of Industrial Chemistry; Wiley-VCH Verlag
GmbH & Co. KGaA: Weinheim, Germany, 2003; pp. 73–140.
2
.
Spannring, P.; Bruijnincx, P.C.A.; Weckhuysen, B.M.; Klein Gebbink, R.J.M. Transition metal-catalyzed
oxidative double bond cleavage of simple and bio-derived alkenes and unsaturated fatty acids.
Catal. Sci. Technol. 2014, 4, 2182–2209. [CrossRef]

Molecules 2020, 25, 1536
15 of 17
3
.
Rajagopalan, A.; Lara, M.; Kroutil, W. Oxidative Alkene Cleavage by Chemical and Enzymatic Methods.
Adv. Synth. Catal. 2013, 355, 3321–3335. [CrossRef]
4
5
.
.
Criegee, R. Mechanism of Ozonolysis. Angew. Chemie Int. Ed. Engl. 1975, 14, 745–752. [CrossRef]
Schrader, J.; Etschmann, M.M.W.; Sell, D.; Hilmer, J.-M.; Rabenhorst, J. Applied biocatalysis for the synthesis
of natural flavour compounds – current industrial processes and future prospects. Biotechnol. Lett. 2004
6, 463–472. [CrossRef] [PubMed]
,
2
6
.
.
Rajagopalan, A.; Schober, M.; Emmerstorfer, A.; Hammerer, L.; Migglautsch, A.; Seisser, B.; Glueck, S.M.;
Niehaus, F.; Eck, J.; Pichler, H.; et al. Enzymatic Aerobic Alkene Cleavage Catalyzed by a Mn3+-Dependent
Proteinase A Homologue. ChemBioChem 2013, 14, 2427–2430. [CrossRef] [PubMed]
Han, D.; Ryu, J.-Y.; Kanaly, R.A.; Hur, H.-G. Isolation of a Gene Responsible for the Oxidation of
trans-Anethole to para-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli.
Appl. Environ. Microbiol. 2012, 78, 5238–5246. [CrossRef] [PubMed]
7
8
.
.
Yamada, M.; Okada, Y.; Yoshida, T.; Nagasawa, T. Purification, characterization and gene cloning of
isoeugenol-degrading enzyme from Pseudomonas putida IE27. Arch. Microbiol. 2007, 187, 511–517. [CrossRef]
Hajnal, I.; Faber, K.; Schwab, H.; Hall, M.; Steiner, K. Oxidative Alkene Cleavage Catalysed by
Manganese-Dependent Cupin TM1459 from Thermotoga maritima. Adv. Synth. Catal. 2015, 357, 3309–3316.
9
[CrossRef]
1
0. Tuynman, A.; Spelberg, J.L.; Kooter, I.M.; Schoemaker, H.E.; Wever, R. Enantioselective Epoxidation
and Carbon–Carbon Bond Cleavage Catalyzed by Coprinus cinereus Peroxidase and Myeloperoxidase.
J. Biol. Chem. 2000, 275, 3025–3030. [CrossRef]
1
1
1
1
1
1. Mutti, F.G.; Lara, M.; Kroutil, M.; Kroutil, W. Ostensible Enzyme Promiscuity: Alkene Cleavage by
Peroxidases. Chem. Eur. J. 2010, 16, 14142–14148. [CrossRef]
2. Hofrichter, M.; Ullrich, R.; Pecyna, M.J.; Liers, C.; Lundell, T. New and classic families of secreted fungal
heme peroxidases. Appl. Microbiol. Biotechnol. 2010, 87, 871–897. [CrossRef] [PubMed]
3. Behrens, C.J.; Zelena, K.; Berger, R.G. Comparative Cold Shock Expression and Characterization of Fungal
Dye-Decolorizing Peroxidases. Appl. Biochem. Biotechnol. 2016, 179, 1404–1417. [CrossRef] [PubMed]
4. Sugano, Y. DyP-type peroxidases comprise a novel heme peroxidase family. Cell. Mol. Life Sci. 2009
6, 1387–1403. [CrossRef] [PubMed]
5. Linde, D.; Ruiz-Dueñas, F.J.; Fern ndez-Fueyo, E.; Guallar, V.; Hammel, K.E.; Pogni, R.; Mart
,
6
á
í
nez, A.T.
Basidiomycete DyPs: Genomic diversity, structural–functional aspects, reaction mechanism and
environmental significance. Arch. Biochem. Biophys. 2015, 574, 66–74. [CrossRef]
1
6. Strittmatter, E.; Liers, C.; Ullrich, R.; Wachter, S.; Hofrichter, M.; Plattner, D.A.; Piontek, K. First Crystal
Structure of a Fungal High-redox Potential Dye-decolorizing Peroxidase. J. Biol. Chem. 2013, 288, 4095–4102.
[CrossRef]
1
1
7. Rais, D.; Zibek, S. Biotechnological and Biochemical Utilization of Lignin. In Biorefineries; Wagemann, K.,
Tippkötter, N., Eds.; Springer International Publishing: Cham, Switzerland, 2017; pp. 469–518.
8. Salvachua, D.; Prieto, A.; Martinez, A.T.; Martinez, M.J. Characterization of a Novel Dye-Decolorizing
Peroxidase (DyP)-Type Enzyme from Irpex lacteus and Its Application in Enzymatic Hydrolysis of Wheat
Straw. Appl. Environ. Microbiol. 2013, 79, 4316–4324. [CrossRef]
1
9. Lauber, C.; Schwarz, T.; Nguyen, Q.K.; Lorenz, P.; Lochnit, G.; Zorn, H. Identification, heterologous
expression and characterization of a dye-decolorizing peroxidase of Pleurotus sapidus. AMB Express 2017
, 164. [CrossRef]
,
7
2
0. Busse, N.; Wagner, D.; Kraume, M.; Czermak, P. Reaction Kinetics of Versatile Peroxidase for the Degradation
of Lignin Compounds. Am. J. Biochem. Biotechnol. 2013, 9, 365–394. [CrossRef]
2
1. Arnao, M.B.; Acosta, M.; del Rio, J.A.; Var
of peroxidase by hydrogen peroxide. Biochim. Biophys. Acta Protein Struct. Mol. Enzymol. 1990, 1041, 43–47.
CrossRef]
ón, R.; García-Cánovas, F. A kinetic study on the suicide inactivation
[
2
2. Dako, E.; Bernier, A.-M.; Thomas, A.; Jankowski, C.K. The Problems Associated with Enzyme Purification.
In Chemical Biology; Ekinci, D., Ed.; InTech: Rijeka, Croatia, 2012; pp. 19–40.
2
3. Schulz, K.; Nieter, A.; Scheu, A.-K.; Copa-Patiño, J.L.; Thiesing, D.; Popper, L.; Berger, R.G. A type D ferulic acid
esterase from Streptomyces werraensis affects the volume of wheat dough pastries. Appl. Microbiol. Biotechnol.
2
018, 102, 1269–1279. [CrossRef]

Molecules 2020, 25, 1536
16 of 17
2
4. Kolwek, J.; Behrens, C.; Linke, D.; Krings, U.; Berger, R.G. Cell-free one-pot conversion of (+)-valencene to
+)-nootkatone by a unique dye-decolorizing peroxidase combined with a laccase from Funalia trogii. J. Ind.
(
Microbiol. Biotechnol. 2018, 45, 89–101. [CrossRef]
2
2
2
5. Hofrichter, M. Review: Lignin conversion by manganese peroxidase (MnP). Enzyme Microb. Technol. 2002,
3
0, 454–466. [CrossRef]
6. Nakayama, T.; Amachi, T. Fungal peroxidase: Its structure, function, and application. J. Mol. Catal. B Enzym.
999, 6, 185–198. [CrossRef]
7. Fern ndez-Fueyo, E.; Linde, D.; Almendral, D.;
Mart nez, A.T. Description of the first fungal dye-decolorizing peroxidase oxidizing manganese(II).
Appl. Microbiol. Biotechnol. 2015, 99, 8927–8942. [CrossRef] [PubMed]
8. Fern ndez-Fueyo, E.; Dav -Siguero, I.; Almendral, D.; Linde, D.; Baratto, M.C.; Pogni, R.; Romero, A.;
Guallar, V.; Mart nez, A.T. Description of a Non-Canonical Mn(II)-Oxidation Site in Peroxidases. ACS Catal.
018, 8, 8386–8395. [CrossRef]
9. Linde, D.; Pogni, R.; Cañellas, M.; Lucas, F.; Guallar, V.; Baratto, M.C.; Sinicropi, A.; S
Coscol n, C.; Romero, A.; et al. Catalytic surface radical in dye-decolorizing peroxidase: a computational,
spectroscopic and site-directed mutagenesis study. Biochem. J. 2015, 466, 253–262. [CrossRef]
1
á
López-Lucendo, M.F.; Ruiz-Dueñas, F.J.;
í
2
2
3
á
ó
í
2
á
ez-Jiménez, V.;
í
0. Amara, S.; Perrot, T.; Navarro, D.; Deroy, A.; Benkhelfallah, A.; Chalak, A.; Daou, M.; Chevret, D.; Faulds, C.B.;
Berrin, J.-G.; et al. Enzyme Activities of Two Recombinant Heme-Containing Peroxidases, Tv DyP1 and
Tv VP2, Identified from the Secretome of Trametes versicolor. Appl. Environ. Microbiol. 2018, 84, e02826-17.
[
CrossRef]
1. Duan, Z.; Shen, R.; Liu, B.; Yao, M.; Jia, R. Comprehensive investigation of a dye-decolorizing peroxidase
and a manganese peroxidase from Irpex lacteus F17, a lignin-degrading basidiomycete. AMB Express 2018
, 119. [CrossRef]
3
3
,
8
2. Passardi, F.; Theiler, G.; Zamocky, M.; Cosio, C.; Rouhier, N.; Teixera, F.; Margis-Pinheiro, M.; Ioannidis, V.;
Penel, C.; Falquet, L.; et al. PeroxiBase: The peroxidase database. Phytochemistry 2007, 68, 1605–1611.
[CrossRef]
3
3
3
3. Colpa, D.I.; Fraaije, M.W.; van Bloois, E. DyP-type peroxidases: a promising and versatile class of enzymes.
J. Ind. Microbiol. Biotechnol. 2014, 41, 1–7. [CrossRef]
4. Romanos, M. Advances in the use of Pichia pastoris for high-level gene expression. Curr. Opin. Biotechnol.
1
995, 6, 527–533. [CrossRef]
5. Ogawa, S.; Shimizu, T.; Ohki, H.; Araya, T.; Okuno, T.; Miyairi, K. Expression, purification, and analyses
of glycosylation and disulfide bonds of Stereum purpureum endopolygalacturonase I in Pichia pastoris.
Protein Expr. Purif. 2009, 65, 15–22. [CrossRef] [PubMed]
3
6. Behrens, C.J.; Linke, D.; Allister, A.B.; Zelena, K.; Berger, R.G. Variants of PpuLcc, a multi-dye decolorizing
laccase from Pleurotus pulmonarius expressed in Pichia pastoris. Protein Expr. Purif. 2017, 137, 34–42. [CrossRef]
[PubMed]
3
7. Avram, A.; Sengupta, A.; Pfromm, P.H.; Zorn, H.; Lorenz, P.; Schwarz, T.; Nguyen, K.Q.; Czermak, P. Novel
DyP from the basidiomycete Pleurotus sapidus: substrate screening and kinetics. Biocatalysis 2018, 4, 1–13.
[CrossRef]
3
8. Rahmanpour, R.; Bugg, T.D.H. Characterisation of Dyp-type peroxidases from Pseudomonas fluorescens Pf-5:
Oxidation of Mn(II) and polymeric lignin by Dyp1B. Arch. Biochem. Biophys. 2015, 574, 93–98. [CrossRef]
9. Linke, D.; Leonhardt, R.; Eisele, N.; Petersen, L.M.; Riemer, S.; Nimtz, M.; Berger, R.G. Carotene-degrading
3
activities from Bjerkandera adusta possess an application in detergent industries. Bioprocess Biosyst. Eng. 2015
8, 1191–1199. [CrossRef]
,
3
4
4
4
0. Scheibner, M.; Hülsdau, B.; Zelena, K.; Nimtz, M.; de Boer, L.; Berger, R.G.; Zorn, H. Novel peroxidases of
Marasmius scorodonius degrade β-carotene. Appl. Microbiol. Biotechnol. 2008, 77, 1241–1250. [CrossRef]
1. Zorn, H.; Langhoff, S.; Scheibner, M.; Nimtz, M.; Berger, R.G. A Peroxidase from Lepista irina Cleaves
β-Carotene to Flavor Compounds. Biol. Chem. 2003, 384, 1049–1056. [CrossRef]
β,
2. Krings, U.; Lehnert, N.; Fraatz, M.A.; Hardebusch, B.; Zorn, H.; Berger, R.G. Autoxidation versus
biotransformation of -pinene to flavors with Pleurotus sapidus: Regioselective hydroperoxidation of
-pinene and stereoselective dehydrogenation of verbenol. J. Agric. Food Chem. 2009, 57, 9944–9950.
CrossRef]
α
α
[

Molecules 2020, 25, 1536
17 of 17
4
4
3. Linke, D.; Omarini, A.B.; Takenberg, M.; Kelle, S.; Berger, R.G. Long-Term Monokaryotic Cultures of
Pleurotus ostreatus var. florida Produce High and Stable Laccase Activity Capable to Degrade ß-Carotene.
Appl. Biochem. Biotechnol. 2019, 187, 894–912. [CrossRef]
4. Schulz, K.; Giesler, L.; Linke, D.; Berger, R.G. A prolyl endopeptidase from Flammulina velutipes for the
possible degradation of celiac disease provoking toxic peptides in cereal proteins. Process Biochem. 2018
3, 47–55. [CrossRef]
,
7
4
4
4
5. Nieter, A.; Haase-Aschoff, P.; Linke, D.; Nimtz, M.; Berger, R.G. A halotolerant type A feruloyl esterase from
Pleurotus eryngii. Fungal Biol. 2014, 118, 348–357. [CrossRef] [PubMed]
6. Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic local alignment search tool. J. Mol. Biol.
1
990, 215, 403–410. [CrossRef]
7. Sievers, F.; Wilm, A.; Dineen, D.; Gibson, T.J.; Karplus, K.; Li, W.; Lopez, R.; McWilliam, H.; Remmert, M.;
Söding, J.; et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal
Omega. Mol. Syst. Biol. 2011, 7, 539. [CrossRef] [PubMed]
4
4
5
8. Lin-Cereghino, J.; Wong, W.W.; Xiong, S.; Giang, W.; Luong, L.T.; Vu, J.; Johnson, S.D.; Lin-Cereghino, G.P.
Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia
pastoris. Biotechniques 2005, 38, 44–48. [CrossRef]
9. Sygmund, C.; Gutmann, A.; Krondorfer, I.; Kujawa, M.; Glieder, A.; Pscheidt, B.; Haltrich, D.; Peterbauer, C.;
Kittl, R. Simple and efficient expression of Agaricus meleagris pyranose dehydrogenase in Pichia pastoris.
Appl. Microbiol. Biotechnol. 2012, 94, 695–704. [CrossRef]
0. Nieter, A.; Kelle, S.; Takenberg, M.; Linke, D.; Bunzel, M.; Popper, L.; Berger, R.G. Heterologous production
and characterization of a chlorogenic acid esterase from Ustilago maydis with a potential use in baking.
Food Chem. 2016, 209, 1–9. [CrossRef]
5
5
5
1. Britton, H.T.S.; Robinson, R.A. CXCVIII.—Universal buffer solutions and the dissociation constant of veronal.
J. Chem. Soc. 1931, 1456–1462. [CrossRef]
2. Lowry, O.H.; Rosebrpugh, N.J.; Randall, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem.
1
951, 193, 265–275.
3. Fink, M.; Trunk, S.; Hall, M.; Schwab, H.; Steiner, K. Engineering of TM1459 from Thermotoga maritima for
increased oxidative alkene cleavage activity. Front. Microbiol. 2016, 7, 1511. [CrossRef]
Sample Availability: Samples of the pPIC9 vector containing the PsaPOX gene are available from the authors.
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