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Hydrogen peroxide driven biocatalysis

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Cite this: Green Chem., 2019, 21,
3232
Received 20th February 2019,
Accepted 10th May 2019
DOI: 10.1039/c9gc00633h
rsc.li/greenchem
a
B. O. Burek, S. Bormann,b F. Hollmann, c
J. Z. Bloh a
and D. Holtmann *b
In general, hydrogen peroxide is a stable and relatively mild oxidant and it can be regarded as the ultimate
“green” reagent because water and oxygen are the only by-products. Besides the direct application of
H2O2 in chemical processes more and more enzymatic syntheses based on hydrogen peroxide were
developed. Different types of reactions can be addressed by using a hydrogen-peroxide driven biocataly-
sis (e.g. hydroxylations, epoxidations, sulfoxidations, halogenations, Baeyer–Villiger oxidations, decarboxy-
lations). H2O2-driven reactions can often be used to substitute NAD(P)H dependent reactions. Therefore,
laborious cofactor regeneration systems can be avoided by using H2O2-dependent enzymes. The tre-
mendous increase in the number of publications dealing with this type of reactions clearly demonstrates
the progress in this area in recent years. The described innovations range from new enzymes and types of
reaction to novel reaction engineering approaches. This review aims to give the scope of possible advan-
tageous applications of peroxyzymes and a critical discussion of their current limitations. The versatile
reactions, the ecological advantageous and the great progress in the discovery and engineering of novel
enzymes make a technical use feasible.

Introduction (wild-type and/or tailor-made variants)1 is highly valued. But

also the high reaction rates obtained with enzymes at mild
Biocatalysis is steadily gaining importance in industrial chem- reaction temperature (approx. 25–37 °C) are very beneficial for
istry. Particularly the outstanding selectivity of many enzymes many chemical processes. This could be regarded as one of
main advantages of enzymes compared to many hetero-
geneous and homogeneous catalysts. As a consequence,
a
DECHEMA Research Institute, Chemical Technology, Theodor-Heuss-Allee 25, savings in energy consumption are beneficial from an econ-
60486 Frankfurt am Main, Germany
b
omic but also from an environmental point-of-view.2,3
DECHEMA Research Institute, Industrial Biotechnology, Theodor-Heuss-Allee 25,
Furthermore, at lower temperatures less undesired thermally
60486 Frankfurt am Main, Germany. E-mail: holtmann@dechema.de
c
Delft University of Technology, Department of Biotechnology, van der Maasweg 9,
induced side reactions occur, resulting in higher quality pro-
2629 HZ Delft, The Netherlands ducts which require less downstream processing and product

Bastien Oliver Burek received his Sebastian Bormann received his

Bastien Oliver Burek
M. Sc. degree in Chemistry from
the University of Frankfurt
a. M. in 2015 where he investi-
gated biorepulsive and func-
tional SAMs. Currently he is a
PhD student in the Chemical
Technology group at DECHEMA
Research Institute in cooperation
with Prof. D. W. Bahnemann at
the Institute of Technical
Chemistry at the University of
Hannover. His research focuses
on photoenzymatic reactions,
photocatalysis and the scale-up
of photo reactions.
Sebastian Bormann
Bachelor’s degree in biotechnol-
ogy from University of Applied
Sciences Darmstadt and his
Master’s degree in biotechnology
from Braunschweig University of
Technology. Currently, he is a
PhD student in the industrial
biotechnology group at
DECHEMA Research Institute in
Frankfurt, Germany. His
research focuses on biocatalysis
with peroxygenases.

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Green Chemistry
purification efforts. Again, this translates into economic and
environmental benefits.4
Critically analyzing the use of enzymes in the chemical
industry5–7 it becomes clear that enzymes are used for only a
very limited range of reactions today. Simple hydrolytic reac-
tions are widespread ranging from the synthesis of chiral alco-
hols and amines, mild production of cosmetic products3 to
bulk chemicals such as acrylamide.8 More recently, alcohol
dehydrogenases, transaminases and imine reductases are
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receiving increased interest for the enantiospecific reduction
or reductive amination of prochiral ketones to yield chiral
pharmaceutical intermediates.9,10
Selective oxidation chemistry or even selective oxyfunctiona-
lisation of non-activated C–H-, C–C- or CvC-bonds remains a
white spot on the map of industrial biocatalysis with only a
few examples from the pharmaceutical industry.11 This is
astonishing as especially in this area the selectivity benefits of
enzymes can be fully exploited. Regio- and stereospecific
hydroxylation or halogenation reactions, frequently reported
with monooxygenases, are way beyond their chemical compe-
tition.12 Given this enormous potential of biocatalytic oxi-
dation reactions, the question of why these reactions are not
more widespread, especially on industrial scale, suggests itself.
We believe that a satisfactory answer involves several aspects
such as limited availability of suitable catalysts (only a limited
set of products is available today) and traditionally very low
substrate loadings (resulting in non-economical product load-
ings, which, by the way, are also far away from environmental
benignity!).4 Also the cofactor dependency of most oxidoreduc-
tases may negatively impact the economic viability of the
process. In case of dehydrogenases and likewise for many
other cofactor-dependent enzymes, however, decades of inten-
sive research have resulted in a broad portfolio of in situ regen-
eration systems, which allow the application of cofactors in
catalytic amounts and therefore satisfactorily solve the pre-
vious ‘cofactor issue’.13 Why then do these cofactor regener-
ation systems not solve the cofactor issue in case of monooxy-
Frank Hollmann studied
Chemistry at the University of
Bonn (Germany). After his PhD
at the Swiss Federal Institute of
Technology (ETH Zurich,
Switzerland supervised by Prof.
Andreas Schmid) and a postdoc at the University of
Postdoctoral stay with Prof.
Manfred T. Reetz at the Max-
Planck Institute for Coal
Research (Mülheim an der Ruhr,
Germany) he joined Evonik as
Frank Hollmann R&D manager. Since 2008 he is
member of the Biocatalysis group
at the Delft University of Technology. His research interest focus
around the application of redox enzymes for organic synthesis.
This journal is © The Royal Society of Chemistry 2019
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Critical Review
genases? We believe that the so-called Oxygen Dilemma14 may
account for this situation. In essence, monooxygenases cata-
lyse selective oxyfunctionalisation reactions by first partially
reducing molecular oxygen (O2); for this they generally depend
more or less directly on reduced nicotinamide cofactors to
supply the reducing equivalents needed for this reaction.
Often, these reducing equivalents are not directly delivered
from the reduced form of nicotinamide adenine dinucleotide
( phosphate) (NAD(P)H) to the monooxygenases’ prosthetic
groups but via rather complex, multi-component electron
transport chains. Especially in case of (non-)heme monooxy-
genases these electron transport chains are a mechanistic
requirement as the FeIII ion acts as obligate single electron
acceptor whereas NAD(P)H exclusively serves as hydride (two
electron) donor. The main function of the electron transport
chains, consequently, is to serve as relay system between two
incompatible redox systems. The main issue of these electron
transport chains is that they contain single electron mediators,
which usually react diffusion-controlled (i.e. very fast) with the
O2 present in the reaction medium. As a consequence, in
many cases the majority of reducing equivalents provided by
NAD(P)H are wasted in a futile cycle and do not reach the
monooxygenases’ active sites for productive catalysis
(Scheme 1). The final consequence thereof is that the reaction
schemes tend to be inefficient in terms of substrate turnover
rate and electron transfer yield. The latter is particularly pro-
blematic from an environmental point-of-view as it translates
into a higher than necessary molar surplus of the sacrificial
electron donor leading to increased consumption of resources
and additional waste generation.
In chemical synthesis hydrogen peroxide (H2O2) is often
regarded as a “green” oxidant.15 Hence, it is not very surpris-
ing that in recent years H2O2 has been receiving increased
interest as stoichiometric oxidant to also promote biocatalytic
oxidation and oxyfunctionalisation reactions.16,17 One major
advantage of H2O2 is that here the oxygen is already partially
reduced. Therefore, in situ reductive activation as described
Jonathan Zacharias Bloh studied
Life Science and later obtained
his Ph.D. in Technical Chemistry
in 2012 at the Leibniz University
Hannover. Thereafter he spent
one and a half years as a
Aberdeen in Scotland. Since
2014, he is the head of the
Chemical Technology group at
the DECHEMA Research Institute
in Frankfurt am Main, Germany.
Jonathan Z. Bloh His current research covers
heterogeneous photocatalysis,
reaction engineering and chemo-
enzymatic processes.
Green Chem., 2019, 21, 3232–3249 | 3233
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Critical Review Green Chemistry

Scheme 2 Hydroxylation based on a P450 BM3/NADPH in combination

with a glucose dehydrogenase (GDH) to regenerate the cofactor and on
an AaeUPO/H2O2-system (ttnNADP = 1000,18 ttnP450BM3 = 54 700,19
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ttnGDH = 10,7,20 ttnAaeUPO = 400 000 (ref. 21)).

Scheme 1 Comparison of the molecular architecture of monooxygen-
ases depending on electron transport chains (e.g.
P450 monooxygenase) and the Oxygen Dilemma originating from the
high reactivity of the mediator components with molecular oxygen with
H2O2-dependent ‘peroxyzymes’.
above for the monooxygenases is no longer necessary. This
also means that the issues of the Oxygen Dilemma in principle
do not apply to H2O2-driven oxidation/oxyfunctionalisation
reactions. The promise of this hydrogen peroxide driven bioca-
talysis is that the high oxidation power of H2O2 and its ecologi-
cal properties can be combined with the high selectivity and
further advantages of the enzymatic oxidation reactions. This
point motivates a lot of researchers to investigate H2O2-driven
enzymes for different reactions, e.g. hydroxylations, epoxida-
tions or halogenations. The possible advantages become par-
ticularly obvious by the comparison of a hydroxylation of a
substrate with P450 monooxygenases from Bacillus megaterium
(P450 BM3) and the unspecific peroxygenase from Agrocybe
aegerita (AaeUPO), (Scheme 2). Assuming in both cases 100%
conversion of the substrate S–H to the hydroxylated product
P-OH the produced waste per mol product can be calculated
based on the published total-turnovers (ttn) for the enzymes
and cofactor as well as the molecular masses of the com-
ponents. Albeit this is only a rough comparison it clearly
shows that when using the peroxygenase the amount of waste
produced is reduced to less than 10% versus using the P450
BM3. By using the AaeUPO, only water and the inactivated
enzyme are produced as waste, this has an additional positive
effect on the costs and complexity of the subsequent product
purification. It should also be mentioned that using the P450
BM3/glucose dehydrogenase (GDH) system requires the use of
many cost-intensive substances, in particular NADP+. Although
the applied amount of enzyme has only a small contribution
to the overall waste formation, it should be emphasized that
the higher ttn of peroxygenases also contributes to the poten-
tial advantages of these systems. This high stability is due to
the fact that peroxygenases as extracellular enzymes are gener-
ally more stable to environmental influences than intracellular
enzymes such as P450s. The use of extracellular enzymes also
facilitates their purification after production. This comparison
clearly shows the high potential of H2O2-driven biocatalysis.
Therefore, in this review the most important enzymes and
reactions are presented.
The catalysts – H2O2 utilizing
‘peroxizymes’
A range of enzymes can utilise hydrogen peroxide as oxidant.
Some examples are shown in Scheme 3, e.g., peroxidases

Dirk Holtmann has completed

his diploma in chemical engin-
eering/biotechnology in 1999. He
obtained his PhD at the Otto von
Guericke University of
Magdeburg on the electro-
chemical measurement of the
microbial activities. He is head
of the Industrial Biotechnology
Group at the DECHEMA
Research-Institute in Frankfurt,
Germany. His current research
Dirk Holtmann activities concentrate on biocata-
lysis and bioelectrochemical
synthesis. Scheme 3 H2O2 as oxidant for various biocatalytic oxidation reactions.

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Green Chemistry
which utilise H2O2 for hydrogen atom abstraction reactions
from phenols and anilines, resulting in radicals which
undergo spontaneous polymerisation reactions.22,23 More
recently, specific oxyfunctionalization reactions using peroxy-
genases have moved into the focus.24–26 Also, haloperoxidases,
despite catalysing halide oxidations only, are enjoying
increased interest due to the many follow-up reactions poss-
ible.27 Finally, exploiting the catalytic promiscuity of many
hydrolases in terms of using H2O2 as nucleophile ( perhydro-
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lase activity) opens up a range of synthetic possibilities.28–30
Peroxidases
First of all, the classical peroxidases (E.C. 1.11) have to be
mentioned here. This diverse class of enzymes comprises a
large diversity of prosthetic groups ranging from Mn3+ to Fe-
protoporphyrin IX and likewise acts on a broad variety of
different substrates ranging from metal ions via halides to
lignin. Two catalytic mechanisms, i.e. the mechanism of the
heme-dependent peroxidases and of the vanadium-dependent
peroxidases are worth being discussed in some more detail.
The key-intermediate of heme-peroxidases, -peroxygenases
and -haloperoxidases is the so-called compound I (Scheme 4).
It is formed from the resting (heme-FeIII) state of the enzyme
in a sequence of H2O2-addition to the FeIII-cation and dehydra-
tisation, which oxidizes it to an FeIV-radical cation. Depending
on the enzyme, compound I has different possibilities of
Scheme 4 Compound I as the central reactive intermediate of heme-
dependent haloperoxidases, -peroxidases and -peroxygenases.
Depiction of the completion of the catalytic cycle (return to resting
state) is omitted for the sake of brevity.
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returning into its resting state. In case of haloperoxidases,
upon binding of a halide ion an FeIII-coordinated hypohalite is
formed (compound X), which eventually hydrolyses yielding
free hypohalites as product. Attention should be directed at
the difference between the peroxidase and peroxygenase reac-
tion mechanisms. In case of peroxidases, substrates exhibiting
labile O–H-, N–H- or C–H-bonds (such as phenols, anilines or
β-diketones, respectively) bind undergoing a H-atom abstrac-
tion followed by release of a radical. Upon binding of a second
substrate, another H-atom abstraction takes place. In the per-
oxygenation pathway, the substrate radical is hydroxylated by
compound II in a rebound step.31–34 Many enzymes with
namesake halogenase or peroxidase activity also exhibit perox-
ygenase activity, such as horseradish peroxidase,35 myeloperox-
idase36 or even non-peroxidase heme-enzymes such as hemo-
globin.37 Yet most of these examples only present oxygenation
of weaker bonds such as allylic bonds or sulfoxidation of thio-
esters. The oxygenation of non-activated C–H bonds on the
other hand has only been reported for P450 monooxygenases
and more recently for unspecific peroxygenases.38–40 P450 and
UPOs share the characteristic proximal heme ligand cystein,
which is also present in chloroperoxidase from Caldariomyces
fumago (CfuCPO), in contrast to other peroxidases like horse-
radish peroxidase (HRP), dye-decolorizing peroxidase (DyP),
lignin peroxidase (LiP), manganese peroxidase (MnP) and cyto-
chrome c peroxidase (CcP) which share histidine as a proximal
ligand. The thiolate-ligand of these enzymes is crucial since
thiolate electron ‘push’ results in high basicity of compound II
which in turns allows the oxygenation of high energy bonds
such as C–H at a reasonable one electron reduction potential
of compound I.41–44 Other than the proximal ligand, UPOs
share little structural similarity with P450 but high similarity
with CfuCPO.45,46
Vanadium-dependent haloperoxidases contain, as suggested
by their name, a Vanadium(V) ion, more specifically a vanadate
prosthetic group responsible for H2O2-activation and oxidation
of the halide substrates (Scheme 5).27 In the presence of H2O2
the vanadate group forms a peroxo complex, in which one of
the peroxo-oxygen-atoms is attacked by a nucleophilic halide
ion leading to the corresponding hypohalous acid, being
released from the enzyme active site and restoring the resting
state of the enzyme.
One major advantage of V-dependent haloperoxidases com-
pared to the aforementioned heme-dependent enzymes clearly
lies in their much higher robustness against H2O2.47 On the
other hand, a major disadvantage of these enzymes is that
hypohalous acids (the products of V-dependent peroxidases)
diffuse out of the enzymes’ active sites and undergo unspecific
chemical transformations. As a consequence, any influence
exerted by the enzyme can be excluded and the selectivity of
the reactions is dictated by the chemical reactivity of the
reagents.
Perhydrolases
In the 1990s researchers from Novo Nordisk reported that
hydrolases are capable of reacting carboxylic acid (esters) with
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Scheme 5 Simplified mechanism of vanadium-dependent haloperoxi-
dase-catalysed formation of hypohalites from halides and H2O2.27 For
reasons of clarity not all stabilising interactions are shown.
Scheme 6 Hydrolase-catalysed formation of peracids from carboxylic
acids (R’ = H) or -esters (R’ = alkyl) and H2O2.
H2O2 forming peracids (Scheme 6).48,49 The latter then can
undergo spontaneous chemical transformations such as
Baeyer–Villiger oxidations of ketones or Prilezhaev oxidations
of CvC-double bonds.12
The chemoenzymatic nature of the perhydrolase reaction
again precludes any selectivity advantages by the biocatalyst.
The peracid formed reacts spontaneously with the starting
material hence, the selectivity is again controlled by the
reagent and not by the catalyst.
Reactions catalysed by peroxizymes
Hydroxylation reactions
Aromatic hydroxylations. Especially UPOs have been
reported for the hydroxylation of aromatic systems. A selection
of products reported is shown in Scheme 7.
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Scheme 7 Examples of UPO-catalysed aromatic hydroxylation pro-
ducts. The OH-group introduced is shown in red. (a),50 (b),53 (c),54 (d),55
(e).56
Mechanistically, the hydroxylation of many aromatic com-
pounds comprises an epoxidation reaction of an aromatic
CvC-double bond followed by a fast rearrangement of the
unstable intermediate arene oxide (NIH shift).50 A major chal-
lenge of aromatic hydroxylations using UPOs is to avoid the
peroxidase activity of peroxygenases. As shown in Scheme 7
peroxygenases can also catalyse the H-atom abstraction from
the phenol products generated in the first step. The resulting
phenoxy radicals generally undergo radical polymerisation
reactions lowering the yield of the desired product. This may
be overcome by using radical scavengers or by protein
engineering.51,52
Even more challenging than aromatic hydroxylation is the
hydroxylation of sp3-C–H-bonds. As early as the 1990s hydroxy-
lations catalysed by CfuCPO were reported, especially on
allylic, propargylic and benzylic C–H bonds.57–65
The turnover numbers of this enzyme, however, were disap-
pointingly low and only activated benzylic or propargylic C–H-
bonds are converted (Scheme 8), which is why no serious
attempt has been undertaken so far to use this enzyme for pre-
parative application.
Since approx. 2004, peroxygenases are experiencing a
revival with several new candidate enzymes filling the gap. The
peroxygenase from A. aegerita for example has been shown by
Hofrichter and coworkers to hydroxylate a range of (cyclo)
alkanes.34,66–68 Fatty acids are predominantly hydroxylated by
AaeUPO in ω-1 or ω-2-position.68 Selective ω-hydroxylation can
be achieved using the unspecific peroxygenase from
Marasmius rotula (MroUPO).69 However, also enzymes catalys-
ing α- or β-hydroxylation of fatty acids have been reported.70,71
Also more complex starting materials such as Vitamin D
can be converted with different regioselectivities by different
UPOs.72,73
Quite interestingly UPOs from different origins exhibit
different, sometimes complementary selectivities. The
hydroxylation of cyclohexane, for example, using AaeUPO
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Scheme 8 Selection of aliphatic hydroxylation catalysed by peroxy-
genases. (a),57,58 (b)59–61 (c),66 (d),74 (e),34,68 (f),66 (g).69
Scheme 9 UPO-catalysed O- or N-dealkylation.
rather selectively yields cyclohexanol whereas the same reac-
tion using MroUPO predominantly yields the overoxidation
product cyclohexanone.66
Another interesting application of UPOs is the oxidative
dealkylation of O- or N-alkyl groups (Scheme 9). These reac-
tions may be useful for diverse applications ranging from
selective deprotection and synthesis of drug metabolites to
degradation of PEG polymers or EPA priority pollutants.11,56,75–78
Epoxidation reactions
Two peroxizyme classes can be used for epoxidation reactions.
Peroxygenases catalyze the direct epoxidation of a broad range
of CvC-double bonds (Scheme 10). Similar to the case of
hydroxylation, the first examples have been reported early on
using CfuCPO but the upcoming new peroxygenases tend to
have higher activities and a broader substrate scope.
One issue that all UPO-catalyzed epoxidation reactions have
in common is the sometimes-poor selectivity. This is true both
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Scheme 10 Examples of peroxygenase-catalysed epoxidation reac-
tions. (a),79 (b),80 (c).80
Scheme 11 Selected examples of the perhydrolase-catalysed
Prilezhaev reaction. (a),81 (b),82 (c),83–85 (d),86 (e).30
in terms of enantio- and chemoselectivity. For example,
AaeUPO catalyzes the highly enantiospecific epoxidation of cis-
methyl-styrene while e.g. styrene is transformed into the race-
mate.74 Also in the presence of allylic C–H bonds frequently a
mixture of epoxidation and hydroxylation products is
observed.
The perhydrolase-catalyzed epoxidation of CvC-double
bonds generally does not exhibit the chemoselectivity issues
mentioned before. Peracids are sufficiently reactive for the
desired Prilezhaev reaction but not reactive enough to attack
the allylic C–H-bond (Scheme 11). Furthermore, chemoenzy-
matic epoxidation reactions are frequently performed under
non-aqueous reaction conditions thereby principally allowing
for high product titres.
Baeyer–Villiger oxidations
Biocatalytic Baeyer–Villiger oxidations are usually associated
with flavin-dependent monooxygenases.87–89 The perhydrolase
reaction of hydrolases, however, also opens up this possibility,
albeit at the expense of lacking enzyme-derived selectivity.
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Scheme 12 Selection of chemoenzymatic Baeyer–Villiger oxidations
utilising the perhydrolysis activity of hydrolases. (a),90 (b),91 (c).91,92
A range of chemoenzymatic Baeyer–Villiger oxidations have
been reported (Scheme 12).89
Especially the resulting lactones are attractive building
blocks for the synthesis of poly esters. Furthermore, the hydro-
lase catalysts used for the Baeyer–Villiger oxidation in principle
could also serve as polymerisation catalysts. However, the
hydrolytic activity of the hydrolases used necessitates strictly
water-free reaction conditions as otherwise the undesired
hydrolysis of the lactone products prevails over the desired
poly condensation reaction. Some protein engineering
approaches have been proposed to address this issue.93,94
Sulfoxidation reactions
The asymmetric oxidation of thioanisole and other organic
thioethers is the model reaction for many UPOs (Scheme 13).
Compared to the well-established chemical sulfoxidation reac-
tions, the enantiospecificity of peroxygenases as well as their
chemoselectivity ( predominantly forming the desired sulfoxide
instead of the sulfone product) is very appealing.95
It is worth mentioning here that CfuCPO, despite generally
being inferior as compared to the ‘new’ peroxygenases, per-
forms very well as sulfoxidation catalyst.
Flavin-dependent monooxygenases catalyse some pharma-
ceutically relevant sulfoxidations.12 Comparable examples
using peroxizymes are currently scarce, again, engineered UPO
variants are highly desirable!
An interesting alternative route was described by Fraaije
and coworkers.103 Using chemically modified (N-alkylated)
Scheme 13 Selection of UPO-catalysed sulfoxidation
(a)96–101 (b).102
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Scheme 14 N5-Alkylated flavins can serve as peroxizymes e.g. in the
asymmetric sulfoxidaton reaction.
flavins, they have demonstrated that flavoenzymes, in prin-
ciple, can be transformed into peroxizymes. An N5-alkylated
riboflavin derivate can react directly with H2O2 forming a per-
oxoflavin species similar to the oxygenating species of flavo-
monooxygenases. When bound to the chiral environment of a
riboflavin-binding protein, the N-alkylated riboflavin mediated
the H2O2-driven, enantiospecific sulfoxidation of a range of
thioethers (Scheme 14).
The enantiospecificities observed with the wild-type protein
were rather low but give rise to the hope that engineered ver-
sions may become preparatively relevant catalysts.
Halogenation reactions
Today, H2O2-dependent halogenation reactions are limited to
the above-described haloperoxidases. Haloperoxidases catalyse
the H2O2-dependent oxidation of halides to the corresponding
hypohalites. It is generally accepted that within their natural
environment, this reaction serves as defence
mechanism.27,104,105 Nevertheless, the hypohalites released
from the enzymes’ active sites can undergo a range of chemi-
cally relevant reactions (Scheme 15).
Miscellaneous
In addition to the reactions discussed before, some ‘miscella-
neous’ applications of peroxizymes have been reported in the
literature and will be briefly introduced in the following
section.
Pioneered by Deska and coworkers, the haloperoxidase- or
lipase-initiated Achmatoviz reaction113–116 may play some role
in natural product synthesis in the future (Scheme 16). An aza-
variant of this reaction was reported as well.114
Another interesting application of haloperoxidases is the
halolactonisation of γ-unsaturated carboxylic acids
(Scheme 17). Compared to the state-of-the-art of chemical
equivalents, this method does not rely on elementary halogens
formed from large precursors (such as NBS) and thereby pro-
duces much less waste.
reactions. Another, potentially useful application of peroxizymes may
lie in the radical polymerisation of phenols or anilines.22
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Scheme 15 Chemoenzymatic halogenation reactions. (a),106 (b),106
(c),107–110 (d),111 (e).112
Scheme 16 Peroxizyme-initiated Achmatoviz reaction. (a),113 (b).114
Scheme 17 Haloperoxidase-initiated halolactonisation reaction.
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Critical Review
Scheme 18 Terminal alkenes from carboxylates using OleT.
Finally, the oxidative decarboxylation of carboxylic acids to
terminal alkenes using a recently described peroxizyme from
Jeotgalicoccus sp. (OleT) is worth being discussed here
(Scheme 18).117–120
OleT appears to be a heme enzyme in between a
P450 monooxygenase and a peroxygenase as it is capable
of both NADPH/O2-121,122 and H2O2-driven118,123 reaction
schemes.
Issues of H2O2-related inactivation of the biocatalysts
The hydrogen peroxide mediated inactivation of enzymes is
one of the major issues on their way towards industrial appli-
cations. Therefore, it is important to understand the under-
lying mechanisms of the inactivation to be able to identify
suitable solutions overcoming this issue e.g. via enzyme engin-
eering, immobilization or in situ H2O2 production methods
(vide infra). H2O2 is a strong oxidant, therefore, it is not very
astonishing that in particular labile amino acids can be oxi-
dized by H2O2 16 or reactive oxygen species derived thereof.124
While oxidative modification of one or few surface-exposed
amino acids generally has little effect on the stability of the
enzyme unless the oxidation leads to chemical follow up reac-
tions such as polymerization,125 the situation becomes more
critical if the amino acid modified is within the enzyme’s
active site or even involved in its catalytic mechanism. In case
of heme-dependent enzymes yet another inactivation mecha-
nism comes into play, the oxidative inactivation of the catalytic
heme group. This has been studied in detail for various heme-
enzymes such as catalase,126 CfuCPO,127 AaeUPO128 or
HRP.129–131 It is often assumed that a reaction of compound
III is responsible for the inactivation.132,133 In particular it is
possible that compound III reacts in a Haber–Weiss like reac-
tion with an additional H2O2 molecule. The thereby generated
hydroxyl radicals are able to oxidize for example the C5 of the
porphyrin.
The resulting α-meso-hydroxy-heme can react with oxygen to
form verdo-heme. Further oxidation yields biliverdin under the
release of iron (heme-bleaching) which is tantamount with an
irreversible inactivation of the enzyme (cf. Scheme 19).133
Furthermore, an oxidation of a vinyl side chain effecting a clea-
vage of the heme iron could be observed in HRP-catalyzed
phenol oxidation.129 Beside the direct destruction of the heme,
oxidation of the methionine next to the active site has also
been described for CfuCPO, indicating that oxidation of amino
acids in the vicinity of the active site might also effect enzyme
activity.127
In most cases, the exact mechanisms of H2O2 mediated
enzyme inactivation remains not fully understood. However, a
deeper understanding of the inactivation mechanism and
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Critical Review
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Scheme 19 Proposed H2O2-mediated inactivation of peroxygenases
via malfunction within the catalase catalytic cycle.
kinetics is highly important to be able to design more sustain-
able enzymes as well as enzymatic processes.
There are several approaches to prevent or at least minimize
inactivation by H2O2. It is possible to modify the enzyme, e.g.
via exchange of amino acids prone to oxidize to gain robust-
ness against radicals which could be successfully shown for
peroxidase form Coprinus cinereus.134 Furthermore, the immo-
bilization of the enzyme can also reduce the H2O2-mediated
inactivation.135 (vide infra). Obviously, lowering the concen-
tration of peroxide present during the reaction by employing
in situ generation techniques reduces enzyme inactivation.
However, as the inactivation appears to mainly occur through
the more reactive •OH radicals it is equally important to mini-
mize formation of those during H2O2 generation, for instance
by employing radical scavengers.136 Presumably, a combi-
nation of an advanced and immobilized enzyme with an
in situ H2O2 generation method might be an approach to yield
very stable processes since all three methods already showed
great improvement on their own.
Reaction engineering
Reagent solubility
The majority of reagents of interest are rather hydrophobic in
nature.137,138 Therefore, their solubility in aqueous media is
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Green Chemistry
rather limited. Such reaction conditions are not attractive for
preparative applications from a practical, an economic and an
environmental point of view. Practically, such dilute reagent
mixtures necessitate the handling of large volumes and
tedious extraction of the desired products from them.
Economically, the low reagent titres result in poor space–time-
yields translating into additional production costs. From an
environmental point-of-view, large streams of contaminated
waste water have to be dealt with meaning further input of
reagents and energy for the clean-up.
Similarly, in case of in situ generation of H2O2, the poor
solubility of O2 in aqueous media (approx. 0.25 mM, 8.3 mg
L−1 at 25 °C) poses a practical challenge. Since the dissolved
O2 is consumed rapidly, external input of O2 is necessary. To
avoid O2 diffusion becoming the overall rate-limitation, hetero-
geneous intake of O2 is necessary. The rate of O2-transfer
thereby correlates with the interfacial area (gas–liquid inter-
face) and can be maximized by bubbling of O2 in fine bubbles.
Unfortunately, however, dissolved proteins also tend to
denature at such interfaces.
A common approach to increase the reagents’ solubility is
to use water-miscible co-solvents such as acetonitrile or
DMSO. This approach, however, only gradually increases the
solubility, often leads to decreased stability of the biocatalysts
and complicates the downstream processing.
More elegantly, the so-called two liquid phase system
approach (2LPS, Scheme 20) allows for overall increased
reagent loadings. Here, a hydrophobic organic phase (ideally
the starting material itself ) serves as reservoir for the hydro-
phobic starting material and as product sink for the (usually
only slightly less hydrophobic) product.
For example, in the CfuCPO-catalysed sulfoxidation of thio-
anisole, using thioanisole itself as second organic phase could
improve the initial rate of the sulfoxidation reaction by several
orders of magnitude as compared to the ‘aqueous only’
system.139 Maximizing the surface area was crucial to attain
these improvements. The addition of surface-active ingredients
helped to stabilise the emulsion and achieve high dispersity at
minimal mechanical stress.
Scheme 20 The two liquid phase system (2LPS) approach to achieve
overall high reagent loadings.
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Green Chemistry Critical Review

Reducing the aqueous layer to nil is possible using peroxi-
zymes as well. Lipase-catalyzed perhydrolysis reactions, for
example, are routinely performed in organic reaction
systems.81,82 But also peroxygenase-catalyzed reactions are
possible in non-aqueous media.140 The dependence on H2O2
or organic-solvent-soluble organic hydroperoxides enables this
application, which with P450 monooxygenases is practically
impossible due to the exclusive water solubility of the nicotina-
mide cofactors and the redox mediators needed for P450
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lead to enzyme inactivation, rather dilute H2O2 stock solutions
together with vigorous mixing of the reaction mixture are
necessary. As a consequence, large volume increases and con-
comitantly significant dilution of the reaction mixtures are
inevitable. Also, the mechanically demanding mixing con-
ditions may negatively influence the biocatalyst stability.
Nevertheless, this method is widely applied on lab-scale
reactions.141–143 Also organic peroxides as somewhat ‘weaker’
oxidants are regularly used.144,145

catalysis. In case of lipase reactions in organic media, the use of urea-

For example, stereoselective hydroxylation of ethyl benzene
was achieved using an immobilized peroxygenase and tert-Bu-
OOH as stoichiometric oxidant. Product titres of up to
100 mM were achieved in this system, albeit at the expense of
drastically decreased enzyme activity, which was attributed to
the loss of catalytic activity of the enzyme upon immobiliz-
ation.140 We expect that optimized enzyme immobilization
procedures will yield practical production systems.
In situ H2O2 generation to alleviate oxidative inactivation
As mentioned above, particularly the heme-dependent peroxi-
dases are prone to irreversible oxidative inactivation by H2O2.
The major challenge en route to practical reaction systems is to
control the H2O2 concentration at levels which enable efficient
catalytic turnover of the enzyme while simultaneously mini-
mising the undesired inactivation reaction.
One obvious approach is based on the slow continuous
portion-wise addition of H2O2. To avoid ‘hot spots’, i.e. locally
high concentrations of H2O2 due to the external dosage which
H2O2 as a slow release agent is rather popular, albeit bearing the
disadvantage of quite significant solid (urea) wastes.90,92,146,147
More elegantly, H2O2 is generated in situ through a catalytic
reduction of molecular oxygen (Table 1). Hence, by carefully
adjusting the H2O2 generation rate with the rate of the (H2O2-
consuming) oxidation reaction, stable reaction systems can be
achieved.
The past years have seen the development of a broad range
of such in situ H2O2 generation systems,148 which will be
briefly discussed in the following.
Traditionally, glucose oxidase (GOx) is the system of choice
for in situ generation of H2O2 to promote peroxidase-catalysed
oxidation reactions. GOx is commercially available at reason-
able prices, it is a rather robust enzyme and glucose as cosub-
strate does not represent a major cost-driver either. Hence, the
majority of the recent applications are based on the GOx-
system. There are, however, a range of disadvantages that, to
our mind, disqualify GOx as in situ H2O2 generation system for
future (large-scale) applications. First of all, glucose is edible,

Table 1 Selection of in situ H2O2 generation systems to promote biocatalytic oxidation reactions (the calculation of the minimal waste which orig-
inates from H2O2 generation is based on the assumptions that 100% of the produced H2O2 is used in the productive catalytic cycle and the co-sub-
strate is used in the most efficient way)

Cosubstrate Coproduct Catalyst(s) Min. waste/g mol−1 Ref.

Enzymatic methods
Glucose Glucuronic acid GOx 196 149
MeOH CO2 AOx/FDM/FDH/3HB6H/NAD 15 150
Alanine Ammonium pyruvate AAOx 105 151
Choline chloride Betaine hydrochloride ChOx 154 85
HCO2H CO2 FDH/NAD/YqjM 44 152
HCO2H CO2 FOx 44 153
Electrochemical, photochemical and miscellaneous methods
Cathode — Flavin-SWCNT/450 nm — 154
Cathode — — — 21, 108 and 155–158
EDTA EDTriA/CH2O/CO2 FMN/450 nm 308 67, 139 and 159
H2O — Au-TiO2/>300 nm — 160
MeOH CO2 Au-TiO2/>300 nm 15 136
H2 — Pd — 161

GOx: glucose oxidase; AOx: alcohol oxidase; FDM: formaldehyde dismutase; FDH: formate dehydrogenase; 3HB6H: 3-Hydroxybenzoate-6-
hydroxylase; NAD: nicotinamide cofactor; AAOx: amino acid oxidase, ChOx: Choline Oxidase, YqjM: ene-reductase from Bacillus subtilis; FOx –
formate oxidase; Flavin-SWCNT: flavin-modified carbon nanotube-functionalized cathodes; Ethylene diamine tetraacetate; EDTriA: ethylene
diamine triacetate; FMN: flavin mononucleotide.

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Critical Review
essentially leading to a food-or-fuel conflict, which from an
ethical point of view is difficult to resolve. Secondly, future
large-scale applications will have to produce significant (hun-
dreds of grams per liter) product titres, which, will at least
require equivalent concentrations of glucose (and/or of its oxi-
dation product glucuronic acid). This will inevitably lead to
dramatic increases of the viscosity of the reaction medium and
consequent increases in energy consumption for the mixing
and downstream processing. Finally, from an atom economy
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point-of-view, oxidation of glucose (i.e. 6 carbon atoms at an
average oxidation state of elementary carbon, which at full oxi-
dation would enable formation of 12 equivalents of H2O2) to
glucuronic acid (i.e. generating one equivalent of H2O2 instead
of 12 theoretical ones) represents a very inefficient system.
This is also reflected by the rather high ‘E-factor’ of the GOx-
system (Table 1). Early attempts of circumventing these issues
e.g. by using amino acid oxidases151 or by photochemical oxi-
dation of synthetic sacrificial electron donors such as
EDTA67,139,159 do not satisfactorily solve this issue as also here,
large amounts of undesired (and sometimes toxic) by-products
are formed. An elegant solution may be the complete oxidation
of methanol or formate to CO2 resulting in the formation of
three or one H2O2 respectively from a small sacrificial electron
donor.136,150,153,162,163 Ideally, water could serve as sacrificial
electron donor essentially leaving no by-product from the
H2O2 generation reaction.160 However, significant improve-
ments in the performance of the individual catalysts and
reactor engineering are required to achieve preparative
feasibility.164–166 Finally, electrochemical reduction of O2 rep-
resents an interesting alternative for in situ generation of
H2O2.21,108,155–158 Especially if the electrical power is derived
from renewable energy sources, this represents an interesting
and potentially environmentally benign way of generating
H2O2 to promote peroxymatic reactions.
The major challenge of those combined systems remains in
the adjustment of both components to find conditions where
they fit optimally together. The H2O2-generating component
cannot just be used in excess as the resulting high concen-
trations of peroxide would lead to enzyme deactivation. Even
slightly over-tuning the generation rate will lead to a runaway
process as the peroxide will accumulate and inactivate the per-
oxide-dependent enzyme which in turn will lead to a lower
consumption rate and even higher peroxide excess. Therefore,
the generation rate needs to be adjusted precisely to the con-
sumption rate or below it and to operate in peroxide-limited
reaction regime in order to have a respective safety margin.
Unfortunately, operating in a peroxide-limited regime
means that its concentration is usually far below the enzyme’s
Km-value and consequently the turnover rate is dramatically
reduced. Nevertheless, the most efficient systems to date in
terms of ttn reached employed this strategy of slowing down
the reaction in favour of enzyme stability.155
Enzyme immobilization
The general procedures for enzyme immobilization have
already been summarized in several excellent reviews.167–174
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Immobilization of enzymes in hydrogen peroxide-driven reac-
tions is often described to improve their performance by
enhancing their overall toughness. It has also been evaluated
as a good strategy to minimize the H2O2-caused enzyme inacti-
vation, mainly two effects are held responsible for the stability
enhancement. The local concentration of H2O2 in the enzyme
environment is decreased, e.g. via generating suitable nano-
environments,175 and through the covalent attachment expo-
sition of susceptible regions is reduced, e.g. via keeping critical
groups inside the enzyme core. Both lead to less chemical
modification, either of the active centre or of structure-lending
side-chain groups, which are responsible for inactivation.16
Different supports have been evaluated for various enzymes
and enzyme classes. For example peroxygenases such as
CfuCPO have been immobilized onto glass,176 titanium
dioxide based supports,177 inorganic mesoporous silica178,179
and titania,180 hydrophilic polymers,181 magnetic beads,182
chitosan membranes,183,184 on agarose gels,62 as cross-linked
enzyme aggregates,185 or encapsulated in PEG-doped silica
matrices.186 Immobilization of unspecific peroxygenases (EC
1.11.2.1) in PVA/PEG gel and hollow fiber modules,187 and
AaeUPO on methacrylate based polymers140 has been
described. Furthermore, a UPO mutant (S221C mutation at the
surface of the enzyme) enabled disulfide bridging to previously
activated carriers like sepabeads.188 Also immobilization of
P450’s onto mesoporous silica has been described in reactions
with H2O2 as co-substrate.189
On top of the improved stability against H2O2-caused de-
activation, higher temperatures and additive solvents could be
used with a smaller impact on enzyme inactivation. Also, a re-
use of the immobilized enzymes could often be described, for
example the CfuCPO encapsulated in polysaccharide-silica
composites could be used for up to nineteen reaction
cycles.186 Furthermore, those enzymes are not soluble in non-
aqueous media, so utilization in pure organic solvents is only
possible via immobilization.140
Another enzyme class which has shown enhanced stability
against H2O2 through immobilization are lipases which are
used together with H2O2 in chemo-enzymatic epoxidation or
Baeyer–Villiger reactions. One of the most studied enzymes
here is the lipase B from Candida Antarctica (CALB). In
addition to the commercially available variants (e.g. Novozyme
435) CALB has also been immobilized on clays,190 silica sup-
ports,146 octadecyl sepabeads191 or multiwalled carbon nano-
tubes.192 Generally, immobilization of CALB enhanced its
stability against H2O2 and elevated temperature. Hydrophobic
supports seem to be very efficient since they reduce the local
H2O2 concentration in the enzyme environment and therefore
even H2O2 concentrations as high as 10 M could be used while
the enzyme still retains high activity.191
A promising strategy is to combine enzyme immobilization
and in situ H2O2-production via co-immobilization of peroxi-
dases and a H2O2 production system which in this case can be
an oxidase. In this context, CfuCPO and GOx were co-immobi-
lized on mesoporous molecular sieves,193,194 Au@Fe3O4 nano-
particles195 or polyurethane supports196 which induced
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Green Chemistry
enhanced stability in comparison to solely immobilizing
CfuCPO on the same support. Also, NADH oxidase has been
used together with HRP for the oxidation of phenolic com-
pounds which yielded higher turnovers of the peroxidase.197
For this system a third enzyme for the regeneration of NADH
was necessary, in this case formate dehydrogenase (FDH). The
three enzymes could successfully be co-immobilized on an
agarose-type carrier. Since there are many different possibili-
ties for co-immobilization, understanding of the influence of
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bioarchitecture in those systems seems to be the most challen-
ging part.
The dismutation of H2O2 through catalases, which yields
water and molecular oxygen, is also utilized in a co-immobiliz-
ation of catalase and oxidase on porous solid supports. The
catalase is responsible for liberation of oxygen from hydrogen
peroxide directly at the oxidase, therefore overcoming oxygen
solubility and diffusion limitations. This system has for
instance been reported for the oxidation of amino acids.198,199
All in all, the benefits of immobilization have to be balanced
against the accompanying disadvantages from practical and
economic perspectives. It requires additional steps in the cata-
lyst preparation which results in increasing costs200 and often
leads in a lower activity due to kinetic limitations. However, we
think immobilization is a powerful tool that may bring
enzymes closer to an industrial application but further devel-
opment is essential.
Protein engineering
In general, enzyme optimization through directed evolution
and enzyme production at high levels often require heter-
ologous expression in hosts that are genetically available and
well established at industrial scale. Even though CfuCPO is by
far the longest-known peroxygenase, its heterologous
expression has to-date only been possible in Aspergillus niger at
relatively low levels of 10 mg L−1 (ref. 201) compared to the
native host C. fumago (up to over 2000 mg L−1).202,203
Unfortunately, the successful genetic engineering of the latter
has only been presented once204 and is made more difficult by
the presence of a recently described second CfuCPO gene205 so
that only few CfuCPO mutants have been reported.206–209 In
contrast, especially given the relatively recent discovery of
UPOs, great advancements in heterologous production of this
enzyme have been made. An evolved form of AaeUPO can be
expressed as a secreted protein at moderate levels (8 mg L−1)
in Saccharomyces cerevisiae210,211 which allows high-through-
put screening of enzyme variants. Moreover, the evolved
enzyme can be expressed in the industrially established
protein production host Pichia pastoris at significantly higher
levels (217 mg L−1).212 Advances in enzyme engineering for the
production of enhanced enzyme variants have already been
made, producing variants that show an improved peroxygenase
– peroxidation ratio,213 increased solvent tolerance214 and
improved enzyme activity for the production of 1-naphtol from
naphthalene while reducing the amount of side products.52
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Critical Review
Unfortunately, the native enzyme can hardly be expressed in
these organisms and the only example of the heterologous
expression of another UPO was in a proprietary, industrial
Aspergillus oryzae strain ( protein concentration not
provided).215
P450 are ubiquitous to all kingdoms, yet the successful het-
erologous expression of animal and plant P450 has proven
difficult. Directed evolution of bacterial P450s such as those
from BM3 and Pseudomonas putida (“P450 cam”) has been uti-
lized to greatly enhance the activity of hydrogen peroxide
driven P450 biocatalysis via the peroxide shunt pathway (20×
fold improvement) and increase substrate and product scope,
yet turnover rates are still too low for preparative use of these
enzymes (ttn < 1000).216–218
In some cases, even new reactivities could be introduced
through protein engineering that were previously not present
in the enzyme. Such was the case with rational mutants of a
DyP which was shown to then catalyze the sulfoxidation of
methyl phenyl sulfoxide and a derivative to the corresponding
sulfoxide with high enantiomeric excess.219
Examples for improving peroxidase properties by directed
evolution include enhancement of the enzyme stability and
production of versatile peroxidase,220–222 the enhancement of
stability and activity of an esterase in the presence of elevated
hydrogen peroxide concentrations223 and the increase of the
catalytic activity of a vanadium peroxidase.224 Often, the
mutations beneficial for increased oxidative stability replace
easily oxidizable amino acids which has led to successful
rational approaches of exchanging such residues (e.g. methion-
ine) to improve peroxide stability.225–227 Such approaches are
valid to improve the oxidative stability for both hydrogen per-
oxide dependent and independent enzymes. In contrast to this
intuitive strategy, no clear protein engineering principles that
alleviate reaction mechanism dependant inactivation have
been proposed thus far.
Conclusions and outlook
The controlled, highly regio-selective and straight-forward
functionalization of C–H bonds remains a dream-reaction for
organic chemists. Nature offers a great toolbox of enzymes for
this type of reactions. In this review, we have presented H2O2
utilizing peroxizymes as catalyst of choice for these reactions.
The presented advantages of H2O2-driven biocatalysis com-
pared to cofactor-depended systems as well as the multiple
types of catalyzed reactions show the broad application poten-
tial of peroxizymes. However, the most important thing to
mention here is that peroxizymes can be used to overcome the
so-called Oxygen Dilemma. Therefore, the application of the
peroxizymes will lead to processes with a reduced demand of
resources (chemicals and energy).
Nonetheless, the current state of research leaves some
essential questions unanswered. The mechanisms of H2O2
mediated enzyme inactivation remain unclear in most of the
cases. Yet, a deeper understanding of the inactivation mecha-
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Critical Review Green Chemistry

nism and kinetics is paramount for the design of more sus- 2LPS Two liquid phase system
tainable enzymes as well as enzymatic processes. Besides this MeOH Methanol
basic-orientated question there is still – to the best of our MnP Manganese peroxidase
knowledge – no industrial synthesis processes based on peroxi- MroUPO Unspecific peroxygenase from Marasmius
zymes. Here, studies on the scalability of the processes are rec- rotula
ommended. This includes the production of enzymes, the NADP+ Nicotinamide adenine dinucleotide
(in situ) generation of H2O2 as well as the actual biocatalytic phosphate
process. We hope that this review will motivate further aca- NADPH Reduced form of NADP+
demic and industrial researchers to investigate H2O2 depend- NBS N-Bromosuccinimide
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ing enzymes. The presented advantages and possibilities NIH shift 1,2-Hydride shift; rearrangement
clearly show the high technical potential of these enzymes. where a hydrogen atom on an aro-
Finally we predict a prosperous future for H2O2-driven biocata- matic ring undergoes an intra-
lysis based on advances in enzyme engineering and adapted molecular migration during a hydroxy-
reaction engineering. lation reaction
OleT Fatty acid decarboxylase from
Jeotgalicoccus species
Abbreviations P450 BM3 P450 monooxygenase from Bacillus
megaterium
AaeUPO Unspecific peroxygenase from Agrocybe P450 cam P450 monooxygenase from Pseudomonas
aegerita putida
AAOx Amino acid oxidase Pd Palladium
AOx Alcohol oxidase PEG Polyethylene glycol
Au-TiO2 Gold loaded titanium dioxide nano- PVA Polyvinylalkohol
particles tert-BuOOH tert-Butyl hydroperoxide
Au@Fe2O3 Gold loaded iron(III)oxide nanoparticles ttn Total turnover number; the amount
CALB Lipase B from Candida Antarctica of produced product divided by the
CcP Cytochrome c peroxidase amount of enzyme
CfuCPO Chloroperoxidase from Caldariomyces V Vanadium
fumago Vmax Maximum rate achieved by the
ChOx Choline oxidase enzyme at saturating substrate con-
CiP Peroxidase form Coprinus cinereus centration
CO2 Carbon dioxide YqjM Ene-reductase from Bacillus subtilis
DMSO Dimethyl sulfoxide
DyP Dye-decolorizing peroxidase
EDTA Ethylenediaminetetraacetic acid
Conflicts of interest
EDTriA Ethylenediamine triacetate
EPA priority pollutants List of priority pollutants from the There are no conflicts to declare.
United States environmental protec-
tion agency
FDH Formate dehydrogenase Acknowledgements
FDM Formaldehyde dismutase
Flavin-SWCNT Flavin-modified carbon nanotube- JZB and BOB gratefully acknowledge German Research
functionalized cathodes Foundation (DFG, Grant No: BL 1425/1-1) for financial
FMN Flavin mononucleotide support.
Fox Formate oxidase F. H. gratefully acknowledge funding by the European
GDH Glucose dehydrogenase Research Commission (ERC consolidator grant, No. 648026),
GOx Glucose oxidase the European Union (H2020-BBI-PPP-2015-2-1-720297) and the
3HB6H 3-Hydroxybenzoate-6-hydroxylase Netherlands Organisation for Scientific Research (VICI grant
H2 Hydrogen No.724.014.003).
H2 O Water
H 2 O2 Hydrogen peroxide
HRP Horseradish peroxidase Notes and references
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