\makeatletter
\@ifundefined{HCode}
{\documentclass[screen,CRCHIM,Unicode,biblatex]{cedram} 
\addbibresource{crchim20240751.bib}
\newenvironment{noXML}{}{}
\def\thead{\noalign{\relax}\hline}
\def\tbody{\noalign{\relax}}
\def\endthead{\noalign{\relax}\hline}
\def\tabnote#1{\vskip4pt\parbox{.65\linewidth}{#1}}
\usepackage[figuresright]{rotating}
\RequirePackage{etoolbox}
\def\jobid{crchim20240751}
\def\inlinefig#1{\includegraphics{#1}}
\newenvironment{xxcenter}{\begin{center}}{\end{center}}
\def\xsection#1{}
%\graphicspath{{/tmp/\jobid_figs/web/}}
\graphicspath{{./figures/}}
\usepackage[T1]{fontenc}
\usepackage{comment}
\newcounter{runlevel}
\usepackage{upgreek}
\let\MakeYrStrItalic\relax
\def\Smallskip{\smallskip}
\csdef{Seqnsplit}{\\}
\def\refinput#1{}
\def\back#1{}
\def\0{\phantom{0}}
\def\hyphen{\mbox{-}}
\newenvironment{inftab}{}{}
\def\xmorerows#1#2{#2}
\usepackage{multirow} 
\def\nrow#1{\@tempcnta #1\relax%
\advance\@tempcnta by 1\relax%
\xdef\lenrow{\the\@tempcnta}}
\def\morerows#1#2{\nrow{#1}\multirow{\lenrow}{*}{#2}}
\def\xmorerows#1#2{#2}
\def\botline{\\\hline}
\def\Lbreak{\newline}
\def\ubreak{\break}
\def\xvarnothing{\emptyset}
\def\mn{\phantom{$-$}}
\def\og{\guillemotleft}
\def\fg{\guillemotright}
\def\bgamma{\boldsymbol{\gamma}}
\makeatletter
\g@addto@macro{\UrlBreaks}{\UrlOrds}
\gappto{\UrlBreaks}{\UrlOrds}
\makeatother
\DOI{10.5802/crchim.375}
\datereceived{2024-09-17}
\dateaccepted{2024-12-10}
\ItHasTeXPublished
}{\documentclass[crchim]{article}
\def\CDRsupplementaryTwotypes#1#2{}
\def\CDRdoi{10.5802/crchim.375} 
\newenvironment{sidewaystable}{\begin{table*}}{\end{table*}}
\def\href#1#2{\url{#1}}
\def\xsection#1{}
\newenvironment{xxcenter}{}{}
\def\Smallskip{}
\let\newline\break
\def\ubreak{}
\let\xvarnothing\emptyset
\makeatletter
}
\makeatother

\usepackage[T1]{fontenc}
\usepackage{upgreek}

\begin{document}

\begin{noXML}

\CDRsetmeta{articletype}{research-article}

\title{From characterization to biocatalytic application of two
peroxygenases from  \textit{Collariella virescens} and \textit{Daldinia
caldariorum}}

\alttitle{De la caract\'{e}risation aux applications biocatalytiques de
deux peroxyg\'{e}nases de \textit{Collariella virescens} et 
\textit{Daldinia caldariorum}}

\author{\firstname{Angelique} \lastname{Pothuizen}\IsEqualContrib}
\address{Department of Biotechnology, Delft University of Technology,
Van der Maasweg 9, 2629 HX Delft, The Netherlands}

\author{\firstname{Rosalie I.} \lastname{Wouters}\IsEqualContrib}
\addressSameAs{1}{Department of Biotechnology, Delft University of Technology,
Van der Maasweg 9, 2629 HX Delft, The Netherlands}

\author{\firstname{Hugo} \lastname{Brasselet}}
\addressSameAs{1}{Department of Biotechnology, Delft University of Technology,
Van der Maasweg 9, 2629 HX Delft, The Netherlands}

\author{\firstname{Thomas} \lastname{Hilberath}}
\addressSameAs{1}{Department of Biotechnology, Delft University of Technology,
Van der Maasweg 9, 2629 HX Delft, The Netherlands}

\author{\firstname{Yinqi} \lastname{Wu}}
\addressSameAs{1}{Department of Biotechnology, Delft University of Technology,
Van der Maasweg 9, 2629 HX Delft, The Netherlands}

\author{\firstname{Frank} \lastname{Hollmann}\CDRorcid{0000-0003-4821-756X}\IsCorresp}
\addressSameAs{1}{Department of Biotechnology, Delft University of Technology,
Van der Maasweg 9, 2629 HX Delft, The Netherlands}
\email[F. Hollmann]{f.hollmann@tudelft.nl}

\keywords{\kwd{Peroxygenase}
\kwd{Oxidation}
\kwd{Hydroxylation}
\kwd{Epoxidation}
\kwd{Sulfoxidation}
\kwd{\textit{Collariella virescens}}
\kwd{\textit{Daldinia caldariorum}}}

\altkeywords{\kwd{Peroxyg\'{e}nase}
\kwd{Oxydation}
\kwd{Hydroxylation}
\kwd{\'{E}poxydation}
\kwd{Sulfoxydation}
\kwd{\textit{Collariella virescens}}
\kwd{\textit{Daldinia caldariorum}}}

\thanks{European Union (ERC, PeroxyZyme, No. 101054658)}

\begin{abstract}
Peroxygenases are promising biocatalysts for selective
oxyfunctionalization reactions including hydroxylation, epoxidation,
and sulfoxidation. In this study, we explore the activity of two
recently reported peroxygenases from \textit{Collariella virescens}
(\textit{Cvi}UPO) and \textit{Daldinia caldariorum} (\textit{Dca}UPO)
in a range of synthetically relevant transformations. Both enzymes were
heterologously expressed in \textit{Escherichia coli} and tested for
various oxidative reactions. \textit{Dca}UPO generally demonstrated
higher activity compared to \textit{Cvi}UPO across several substrates,
showing significant conversions in alcohol and arene oxidations as
well as enantioselective epoxidations of styrene derivatives. Notably,
the enzymes exhibited complementary selectivities in several reactions
including allylic hydroxylation and benzylic oxidation. These results
broaden the substrate scope of \textit{Cvi}UPO and \textit{Dca}UPO and
highlight their potential for industrial applications. However,
challenges with enzyme expression in \textit{E. coli} remain,
necessitating future work on alternative expression systems such as
\textit{Pichia pastoris} to improve yields.
\end{abstract}

\begin{altabstract}
Les peroxyg\'{e}nases sont des biocatalyseurs prometteurs pour des
r\'{e}actions d'oxyfonctionnalisation s\'{e}lective, incluant
l'hydroxylation, l'\'{e}poxydation et la sulfoxydation. Dans cette
\'{e}tude, nous explorons l'activit\'{e} de deux peroxyg\'{e}nases
r\'{e}cemment d\'{e}couvertes, provenant de {\textit{Collariella
virescens}} ({\textit{Cvi}}{UPO) et de} {\textit{Daldinia caldariorum}}
({\textit{Dca}}{UPO), dans une gamme de transformations
d'int\'{e}r\^{e}t synth\'{e}tique. Les deux enzymes ont \'{e}t\'{e}
exprim\'{e}es de mani\`{e}re h\'{e}t\'{e}rologue dans}
{\textit{Escherichia coli}} {et test\'{e}es dans diverses r\'{e}actions
d'oxydation.} {\textit{Dca}}{UPO a g\'{e}n\'{e}ralement montr\'{e} une
activit\'{e} sup\'{e}rieure \`{a} celle de} {\textit{Cvi}}{UPO sur
plusieurs substrats, avec des conversions significatives lors des
oxydations d'alcools et d'ar\`{e}nes, ainsi que des \'{e}poxydations
\'{e}nantios\'{e}lectives de d\'{e}riv\'{e}s du styr\`{e}ne. Notamment,
les enzymes ont montr\'{e} des s\'{e}lectivit\'{e}s compl\'{e}mentaires
dans plusieurs r\'{e}actions, notamment l'hydroxylation allylique et
l'oxydation benzylique. Ces r\'{e}sultats \'{e}largissent le spectre
des substrats de} {\textit{Cvi}}{UPO et} {\textit{Dca}}{UPO et
soulignent leur potentiel pour des applications industrielles.
Cependant, des d\'{e}fis subsistent concernant l'expression enzymatique
dans} {\textit{E. coli}}{, n\'{e}cessitant des travaux futurs sur des
syst\`{e}mes d'expression alternatifs comme} {\textit{Pichia pastoris}}
{pour am\'{e}liorer les rendements.}
\end{altabstract}

\shortrunauthors

\maketitle
\vspace*{3pt}

\twocolumngrid
\end{noXML}

\section{Introduction}\label{sec1}

Selective oxyfunctionalization, that is, the regio- and enantioselective
insertion of an oxygen atom into (nonactivated) C--H bonds still
represents one of the major challenges for organic synthesis. Among
the biocatalytic methods (striking by their high selectivity),
peroxygenase-catalyzed oxyfunctionalization reactions have been receiving
particular interest~\cite{1,2,3}. Particularly, heme-containing enzymes
catalyze a broad range of synthetically relevant oxyfunctionalization
reactions. The catalytically active species in these enzymes is
the so-called Compound I (CpdI, a highly reactive oxyferryl heme species).
Compared to P450 monooxygenase-catalyzed oxyfunctionalization
reactions~\cite{4}, peroxygenases are marked by their simplicity owing to the
following reason. Instead
of relying on complex and weak electron transport chains to
generate CpdI via reductive activation of O$_2$ as in the case of P450
monooxygenases, unspecific peroxygenases (UPOs) form CpdI directly from
H$_2$O$_{2}$ (Scheme~\ref{sch1}).

\begin{scheme*}
{\vspace*{-2pt}}
\includegraphics{sc01}
{\vspace*{4pt}}
\caption{\label{sch1}Comparison of Compound I (CpdI) formation in the case
of P450 monooxygenases and peroxygenases (UPOs).}
{\vspace*{-6pt}}
\end{scheme*}

The enormous synthetic potential of UPOs is exemplified by their
outstanding performance in terms of total turnover numbers~\cite{5,6}
and productivity achievable~\cite{7}. Frequently, UPOs outperform
their P450 monooxygenase counterparts~\cite{8}.

However, one current drawback of UPOs is the rather narrow substrate
scope, which can be assigned to the yet low number of practical UPOs
and UPO-mutants reported so far~\cite{9,10,11,12,13,14,15}. Hence,
broadening the scope of UPOs available is mandatory to address this
limitation. 

Recently, two new UPOs from the ascomycetes \textit{Collariella
virescens} (\textit{Cvi}UPO) and \textit{Daldinia caldariorum}
(\textit{Dca}UPO) have been reported for the oxyfunctionalization of
fatty acids~\cite{16,17,18,19,20,21,22}. Both enzymes have been
expressed in \textit{Escherichia coli} and preliminarily evaluated for
alkane hydroxylation. Though alkane hydroxylation (e.g., on renewable
fatty acids) is of tremendous interest for the synthesis of polyester
precursors, it represents only a fraction of chemically relevant
oxyfunctionalization reactions.\looseness=-1

We therefore set out to further explore the scope of \textit{Cvi}UPO
and \textit{Dca}UPO for more oxyfunctionalization reactions.

\section{Materials and methods}
\subsection{Expression of UPO genes in \textit{E.~coli}}

The pET-28a plasmids containing gene sequences for  the UPOs from
\textit{C.~virescens} {or} \textit{D.~caldariorum} (SI 1)
were transformed into chemically competent \textit{E.~coli} BL21 (DE3).
Recombinant \textit{E.~coli} strains were grown in autoinduction
media (12~g${\cdot}$L$^{-1}$ peptone, 24~g${\cdot}$L$^{-1}$ yeast
extract, 15~mg${\cdot}$L$^{-1}$ glucose, 220~mg${\cdot}$L$^{-1}$
{lactose, 6.3~g${\cdot}$L$^{-1}$ {glycerol, 90~mM KPi buffer, pH 7.0) for
4~days at 16~\textdegree C and 140~rpm~\cite{23}. Production cultures
were inoculated to an optical density at 600~nm (OD$_{600}$) of 0.05
from overnight precultures. When the cells reached an OD$_{600}$ of
0.5, 5-aminolevulinic acid (5-Ala) and FeSO$_{4}$ {were added to final
concentrations of 500 ${\upmu}$M and 0.1 M, respectively.

\begin{table*}
\caption{\label{tab1}ABTS activity determination of  
\textit{E.~coli} cell extracts}
\begin{tabular}{lccc}
\thead
Enzyme & \textit{E.~coli} BL21 empty vector & \textit{Dca}UPO & 
\textit{Cvi}UPO \\
\endthead
Activity (U${\cdot}$mg$^{-1}$) & 0.006 ${\pm}$ 0.0004 
& 0.034 ${\pm}$ 0.002 & 0.662 ${\pm}$ 0.167
\botline
\end{tabular}
\end{table*}

\subsection{Preparation of crude cell extract}\label{sec-22}

After cultivation, cells were harvested by centrifuging at  
17,500${\times}g$
for 30~min at 4~\textdegree C. The supernatant was discarded, and the
remaining cell pellets were washed with 50~mM phosphate buffer (pH
7.0). The remaining cells were resuspended in the same buffer at a
concentration of 50 g/L after which the cells were disrupted during
three consecutive cycles using a CF1 Cell Disruptor from Constant
Systems at 1.5~kbar. The remaining solution after cell disruption was
spun down for 30~min at 36,635${\times}g$ and 4~\textdegree C. The
supernatant containing the enzymes was stored at ${-}$20~\textdegree C
until further use.

\subsection{ABTS activity assays}\label{sec23}

To determine the peroxidase activity of the UPOs, a photometric assay
using 2,2$'$-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as
the substrate was performed. Citric acid buffer of 850 ${\upmu}$L 100~mM  (pH
4.4) was mixed with 50 ${\upmu}$L 10~mM ABTS and 50 ${\upmu}$L CFE
(diluted if necessary). The reaction was started with the addition of 2
mM H$_{2}$O$_{2}$  (final concentration). The
increase in absorption at 420~nm was tracked for 90~s in a
Cary 60 UV--Vis Spectrophotometer (Agilent Technologies). The linear
slope between 20 and 80~s was used to calculate the volumetric
activity (U${\cdot}$ml$^{-1}$) using the extinction coefficient for ABTS
(${\upvarepsilon}_{420} = 36.0$~mM$^{-1}\cdot$cm$^{-1}$). 1~U is defined as
the amount of enzyme required to convert 1 ${\upmu}$mol substrate in 
1~min under assay conditions.



\subsection{CO-difference spectra}\label{sec24}

The amount of UPO present in the CFE was determined using CO-difference
spectra. 950~${\upmu}$L of the enzyme was diluted in 50~mM phosphate
buffer (pH~7). A baseline spectrum was recorded between 400 and 500~nm,
after which the sample was shortly exposed to CO. Next, 50~${\upmu}$L
of 1~M Na$_{2}$S$_{2}$O$_{4}$ was added and the difference spectrum
between 400 and 500~nm was recorded. Per sample, 10 spectra with 30~s
intervals were recorded to obtain the maximum difference
spectra. Measurements were carried out in duplicate. The UPO
concentration was calculated using the difference in absorption at 
490~nm and the maximum absorption value at 446~nm (\textit{Dca}UPO) and 
442~nm (\textit{Cvi}UPO). Because the extinction coefficients for these
enzymes are so far unknown, the general P450 extinction coefficient in
CO-difference spectra of 91~mM$^{-1}\cdot$cm$^{-1}$ was used~\cite{24}.

\subsection{Substrate screening assays}\label{sec25}
Substrate screening was performed under the same reaction conditions
with a total reaction volume of 500~${\upmu}$L. Reactions were
performed in 50~mM phosphate buffer (pH 7) with 1~${\upmu}$M UPO and 5~mM
of the substrate in 10 vol\% ACN using a feeding rate of 2~mM
H$_2$O$_{2}$ per hour. As the negative control, CFE of \textit{E.~coli}
transformed with an empty vector containing no peroxygenase gene was
used. Reactions were continued for 3~h at 25~\textdegree C and
600~rpm in an Eppendorf Thermomixer. Reaction mixtures were analyzed using 
gas chromatography (GC); detailed GC protocols can be found in  supplementary
information (SI 2).

\section{Results and discussion}
\subsection{Cloning and expression of \textit{Cvi}UPO and
\textit{Dca}UPO} 

\textit{Dca}UPO and \textit{Cvi}UPO were produced in recombinant}
\textit{E.~coli} BL21 (DE3) containing the respective pET28a-expression
plasmids. Typically cells were cultivated in Studier's autoinduction
medium~\cite{23} for 4 days at  16~\textdegree C and 140~rpm. After
cultivation, \textit{E.~coli} cells were harvested and disrupted
resulting in crude cell extract containing the UPOs of interest. An
SDS-PAGE analysis revealed that both genes were overexpressed upon
induction but mostly yielded the desired UPOs as an insoluble fraction
(Figure~\ref{fig1}).

\begin{figure*}
\vspace*{-2pt}
\includegraphics{fig01}
\vspace*{-3pt}
\caption{\label{fig1}SDS-PAGE analysis of the  \textit{E.~coli}
production of \textit{Dca}UPO and \textit{Cvi}UPO. 
Lanes marked with B.I.\ and A.I.\ are
whole-cell SDS samples before and after induction of protein
expression, respectively. CFE samples show proteins in the supernatant
after cell disruption. CFE SDS samples have been normalized to a
protein concentration of 1~mg/mL based on BC assay. The Bio-Rad
Precision Plus Protein{\texttrademark} Unstained Protein Standards
ladder was used for size reference.}
\vspace*{-3pt}
\end{figure*}

Nevertheless, correct folding of the UPOs in the soluble lysate was
confirmed though recording of CO-difference spectra, showing the
characteristic shift of the Soret peak from 420 to 442
(\textit{Cvi}UPO) and 446~nm (\textit{Dca}UPO) when CO is bound to the
reduced heme cofactor (}Figure~\ref{fig2}). Based on the abundance of
the 450~nm peak, we estimated the UPO titers to be 
8.4~mg${\cdot}$L$^{-1}$ and 5.8~mg${\cdot}$L$^{-1}$ for \textit{Cvi}UPO
and \textit{Dca}UPO, respectively.

\begin{figure}
\vspace*{-1pt}
\includegraphics{fig02}
\vspace*{-2pt}
\caption{\label{fig2}CO-difference spectra of  
\textit{E. coli} cell crude extracts expressing \textit{Cvi}UPO (---, red)
and \textit{Dca}UPO (---, green) compared to the negative control (empty
vector, $\cdots$).}
\vspace*{-4pt}
\end{figure}

Soluble production of the UPOs was further confirmed by measuring the
peroxidase activity. The peroxidase activity of the produced UPOs was
tested with an ABTS assay using crude cell extract (Table~\ref{tab1}). 

\subsection{Oxidation reactions}\label{sec32}

Having \textit{Cvi}UPO and \textit{Dca}UPO in hand, we investigated
their applicability for several chemical transformations of interest.
Among them, we investigated C--H bond hydroxylation reactions, alcohol
oxidations, epoxidation reactions, and sulfoxidation reactions.

\subsubsection{Oxidation of alcohols}\label{sec321}

A selection of primary and secondary alcohols was subjected to
\textit{Cvi}UPO- and \textit{Dca}UPO-catalyzed conversion. Already, the
semi-quantitative screening revealed some interesting observations
(Figure~\ref{fig3}). First, \textit{Dca}UPO generally was more active
than \textit{Cvi}UPO. Second, both enzymes exhibited a clear preference
for secondary alcohols over primary alcohols. The latter observation is
in line with the lower C--H bond dissociation energy of secondary
alcohols compared to primary alcohols. Rhododendrol was not converted
by any of the UPOs, possibly due to steric hindrance.

\begin{figure}
\includegraphics{fig03}
\caption{\label{fig3}Substrate screening of selected alcohols using
\textit{Dca}UPO (red) and \textit{Cvi}UPO (black). Conditions:  $c$(UPO)
${=}$ 1~${\upmu}$M, $c$(substrate) ${=}$ 5~mM, buffer: 50~mM NaPi buffer,  pH
${=}$ 7.0, containing 10\% ($v/v$) of acetonitrile. H$_2$O$_{2}$ addition:
2~mM per hour (from 100~mM stock). $T = 25$~\textdegree C, orbital shaking
at 600~rpm, reaction time ${=}$ 3~h. Red/black: estimated conversion into
the envisaged carbonyl product (aldehyde or ketone), light red/gray:
yet undefined side \mbox{product}.}
\end{figure}

The transformations of 2-heptanol, 2-octanol, oct-1-en-3-ol, and
1-phenyl ethanol were analyzed in more detail based on calibration
curves with {authentic} standards (Table~\ref{tab2}). Chiral GC analysis
of the \textit{Dca}UPO-catalyzed oxidation reactions showed that both
enantiomers of, for example, 2-octanol or 1-phenyl ethanol were converted at
roughly the same rate with a slight preference for the
($R$)-enantiomers (SI~3).\looseness=-1

\begin{table*}
\caption{\label{tab2}Quantitative analysis of some alcohol oxidations
catalyzed by \textit{Dca}UPO and \textit{Cvi}UPO}
\begin{tabular}{cccc}
\thead
Product$^{[\mathrm{a}]}$ & UPO & Concentration [mM]$^{[\mathrm{a}]}$ &
TN (UPO)$^{[\mathrm{b}]}$ \\
\endthead
\morerows{1}{\inlinefig{fx01}} & \textit{Dca}UPO & 1.42 & 1417 \\
& \textit{Cvi}UPO & 0.36 & 358 \vspace*{1.6pc}\\
\morerows{1}{\inlinefig{fx02}} & \textit{Dca}UPO & 1.43 & 1425 \\
& \textit{Cvi}UPO & 0.44 & \0437 \vspace*{1.6pc}\\
\inlinefig{fx03}\vspace*{.6pc} & \raisebox{1.5pc}{\textit{Dca}UPO} & \raisebox{1.5pc}{0.9} &
\raisebox{1.5pc}{899} \\
\morerows{1}{\inlinefig{fx04}} & \textit{Dca}UPO & 0.62 & \0618\\
& \textit{Cvi}UPO & 0.24 & \0243\vspace*{2.5pc}
\botline
\end{tabular}
\tabnote{$^{[\mathrm{a}]}$Based on authentic standards;
$^{[\mathrm{b}]}$TN $=\ c$(product) $\times\ c$(UPO)$^{-1}$.}
\vspace*{.6pc}
\end{table*}



\subsubsection{Oxidation of arenes}\label{sec322}

Next, a range of substituted arenes were evaluated (Figure~\ref{fig4}).
Like the oxidation of alcohols, \textit{Dca}UPO generally exhibited a
significantly higher activity as compared to \textit{Cvi}UPO. Toluene
was not converted by either UPO whereas ethyl benzene,
4-phenyl-2-butanone (containing elongated alkyl substituents), and
pseudocumene (1,2,4-trimethyl benzene) as well as p-cymene (4-isopropyl
toluene) and thymol (2-isopropyl-5-methylphenol or 3-hydroxy
p-cymene) were converted at least by \textit{Dca}UPO. 

\begin{figure}
\vspace*{-3pt}
\includegraphics{fig04}
\vspace*{-10pt}
\caption{\label{fig4}Substrate screening of some substituted arenes
using  \textit{Dca}UPO (top) and \textit{Cvi}UPO (bottom). Conditions: 
$c$(UPO) ${=}$ 1~${\upmu}$M, $c$(substrate) ${=}$ 5~mM, buffer: 50~mM NaPi buffer, pH =
7.0, containing 10\% ($v/v$) of acetonitrile. H$_2$O$_{2}$ addition: 2
mM per hour (from 100~mM stock). $T = 25$~\textdegree C, orbital
shaking at 600~rpm, reaction time ${=}$ 3~h. Blue: estimated conversion into
the envisaged carbonyl product (aldehyde or ketone), red: yet undefined
side product.}
\vspace*{-10pt}
\end{figure}

\textit{Dca}UPO-catalyzed conversion of ethyl benzene yielded
(\textit{R})-1-phenyl ethanol in very modest enantiomeric excess (ee)
of 55\%. Furthermore, some overoxidation to acetophenone was observed. With
toluene, no apparent transformation was observed, which may be
explained by the higher C--H bond strength of benzylic CH$_{3}$ group
compared to the benzylic CH$_{2}$ group in the case of ethyl benzene.
Interestingly, however, pseudocumene (1,3,4-trimethyl benzene) was
converted comparably well. Possibly the electron-donating effect of the
additional CH$_{3}$ groups already sufficed to activate the CH$_{3}$
group converted. Preliminary GC- and GC/MS data suggest selective
CH$_{3}$ hydroxylation (SI). Future, preparative-scale
transformations will yield sufficient amounts for more detailed
structure elucidation. \textit{p}-Cymene, containing a highly activated
tertiary C--H group, however, was almost not converted. 
Possibly steric
effects impeding the interaction of the bulky CH(CH$_{3}$)$_{2}$ with
CpdI dominated the electronic activation. \looseness=-1

Thymol was accepted by both UPOs with a notable difference with respect
to selectivity; while the \textit{Dca}UPO-catalyzed transformation was
apparently highly selective, with \textit{Cvi}UPO at least four
different products were observed. The occurrence of multiple products
may be attributed to the peroxidase activity of UPOs with activated
arenes such as phenols resulting in radical-coupling
products~\cite{19,20,21}. We therefore compared the
\textit{Dca}UPO-catalyzed conversion of thymol in the presence and
absence of ascorbic acid, a well-established radical scavenger
(Figure~\ref{fig5}). Indeed, the product spectrum completely changed;
GC-MS analysis suggested thymoquinone being the main product in the
absence of ascorbic acid and the corresponding thymohydroquinone.

\begin{figure*}
{\vspace*{-2pt}}
\includegraphics{fig05}
%\vspace*{5pt}
\caption{\label{fig5}(A) GC chromatograms of thymol without the
addition of ascorbic acid (black) and with the addition of 20~mM
ascorbic acid (pink). (B) Suggested reaction pathway.}
%\vspace*{5pt}
\end{figure*}

Interestingly, 4-phenyl-2-butanone was converted by \textit{Dca}UPO
while in the case of structurally related rhododendrol, no conversion was
detectable. It is also interesting to note that three different
products were observed exhibiting M+ peaks of 164, 146, and 106. This
may be rationalized by assuming that \textit{Dca}UPO catalyzed the
benzylic hydroxylation of the starting \mbox{material} (resulting in the main
product with a putative molar mass of 164~g${\cdot}$mol$^{-1}$). The
latter may spontaneously undergo either dehydration (yielding the
product with a putative molar mass of 146~g${\cdot}$mol$^{-1}$) or a
retro-aldol reaction resulting in benzaldehyde  (${M}_W =
106~\mathrm{g}{\cdot}\mathrm{mol}^{-1}$  and acetone, which under 
current analytical conditions was not detectable) (Scheme~\ref{sch2}).
\vspace*{-5pt}

\begin{scheme*}
\vspace*{-4pt}
\includegraphics{sc02}
\vspace*{4pt}
\caption{\label{sch2}Hypothesized benzylic hydroxylation of
4-phenyl-2-butanone followed by (spontaneous) dehydration and/or
retro-aldol reaction.}
\vspace*{-16pt}
\end{scheme*}

\subsubsection{Epoxidation reactions}\label{sec323}

Next to hydroxylation reactions, epoxidations are of significant
interest for organic synthesis. Therefore, we evaluated the activity of
\textit{Dca}UPO and \textit{Cvi}UPO with some styrene derivatives
(Figure~\ref{fig6}).

\begin{figure}
\vspace*{-4pt}
\includegraphics{fig06}
\vspace*{-8pt}
\caption{\label{fig6}Product distribution of the  \textit{Dca}UPO- and
\textit{Cvi}UPO-catalyzed epoxidation of some styrene derivatives.
Conditions: [UPO] ${=}$ 1~${\upmu}$M, [substrate] ${=}$ 5~mM, buffer:
50~mM NaPi buffer, pH ${=}$ 7, containing 10\% ($v/v$) of acetonitrile.
H$_2$O$_{2}$ addition: 2~mM per hour (from 100~mM stock).  $T =
25$~\textdegree C, orbital shaking at 600~rpm, reaction time ${=}$
3~h.}
\vspace*{-18pt}
\end{figure}

First, the activity difference between \textit{Dca}UPO and
\textit{Cvi}UPO was not as pronounced as for the \mbox{transformations}
discussed before. The enantioselectivity of the epoxidation reactions
was highly dependent on the alkene substitution pattern. Styrene oxide
was obtained with rather low (\textit{S})-selectivity (24 and 34\% ee)
with \textit{Dca}UPO and \textit{Cvi}UPO, respectively. In contrast,
epoxides obtained from ${\upalpha}$-methyl styrene were essentially
optically pure. Interestingly, \textit{Dca}UPO yielded the
(\textit{R})-enantiomer whereas \textit{Cvi}UPO produced the
(\textit{S})-enantiomer exclusively. Moreover, in the case of
\textit{cis}-${\upbeta}$-methyl styrene both enzymes were
enantiocomplementary albeit again with rather low enantioselectivities.
Expectedly, the epoxides obtained from
\textit{trans}-${\upbeta}$-methyl styrene were enantiomeric to those
obtained from the \textit{cis}-starting \mbox{material}, again with poor
enantioselectivity. It is worth noting that with
\textit{trans}-${\upbeta}$-methyl styrene, the main products observed
with \textit{Cvi}UPO were allylic hydroxylation products (alcohol
and\break aldehyde).

Terpenes such as limonene, ${\upalpha}$-pinene, and ${\upbeta}$-ionone
were converted at low selectivity (}Figure~\ref{fig7}). One notable
exception was the rather selective hydroxylation of ${\upbeta}$-ionone
to the 4-hydroxy product.


\begin{figure*}
\vspace*{-3pt}
\includegraphics{fig07}
\vspace*{-5pt}
\caption{\label{fig7}Selection of terpene hydroxylation reactions catalyzed by 
\textit{Dca}UPO and \textit{Cvi}UPO. Conditions: [UPO] ${=}$
1~${\upmu}$M, [substrate] ${=}$ 5~mM, H$_2$O$_{2}$ feeding rate ${=}$
2~mM${\cdot}$h$^{-1}$, performed in phosphate buffer (50~mM, pH ${=}$ 7,
NaPO$_{4}$) with 10\% ($v/v$) acetonitrile for 3~h
(25~\textdegree C, 600~rpm).}
\vspace*{-3pt}
\end{figure*}

\subsubsection{Sulfoxidation reactions}\label{sec324}

Finally, we evaluated (\textit{p}-Me-)thioanisole as the substrate for
sulfoxidation (Figure~\ref{fig8}). Both UPOs exhibited significant
activity but were not completely selective for the sulfoxide products
as in both cases  sulfone formation was also observable.

\begin{figure*}
\includegraphics{fig08}
\vspace*{-5pt}
\caption{\label{fig8}Sulfoxidation of  $p$-methylthioanisole.
Reaction conditions: [UPO] ${=}$ 1~${\upmu}$M, [substrate] ${=}$ 5~mM, 
H$_2$O$_{2}$ feeding rate of 2~mM${\cdot}$h$^{-1}$, performed in
phosphate buffer (50~mM, pH ${=}$ 7, NaPO$_{4}$) with 10\% ($v/v$)
acetonitrile for 3~h (25~\textdegree C, 600~rpm). Unfortunately, our
current analytical setup does not permit baseline separation of 
sulfoxide enantiomers, which is why no information about the optical
purity of the sulfoxide can be given.}
\vspace*{-5pt}
\end{figure*}

\section{Conclusions}\label{sec4}

In conclusion, with this contribution we have broadened the known
substrate scope of \textit{Cvi}UPO and \textit{Dca}UPO. In addition to
the previously reported ${\upomega}$-1 hydroxylation activity, both
enzymes are also capable of other typical peroxygenase reactions. 
Some reactivities such as the formation of 
${\upbeta}$-hydroxy ketones and
selective allylic hydroxylation, however, stand out from the regular
portfolio and certainly deserve further investigation and
rationalization. Currently, the poor performance of the 
\textit{E.~coli} based expression system presents the major hurdle 
en route to further investigation and upscaling. Our focus in further
investigations will be on producing these enzymes via \textit{Pichia
pastoris} based expression\break systems. 

\section*{Declaration of interests} 

The authors do not work for, advise, own shares in, or receive funds
from any organization that could \mbox{benefit} from this article, and
have declared no affiliations other than their research organizations.

\section*{Funding}

This work was funded by the European Union (European Research Council [ERC], 
PeroxyZyme, No. 101054658). The views and
opinions expressed are however those of the authors only and do not
\mbox{necessarily} reflect those of the European Union or the ERC.
Neither the European Union nor the granting authority
can be held responsible for them.

\CDRGrant[ERC]{101054658}

\section*{Supplementary data}
Supporting information for this article is available on the journal's
website under \printDOI\ or from the author.  

\CDRsupplementaryTwotypes{supplementary-material}{\cdrattach{crchim-375-suppl.pdf}}

\back{}

\printbibliography
\refinput{crchim20240751-reference.tex}

\end{document}
