FOx News: Towards Methanol-driven Biocatalytic Oxyfunctionalisation Reactions

Article abstract of DOI:10.1002/cctc.202000197

The novel formate oxidase from Aspergillus oryzae (AoFOx) is a useful catalyst to promote H₂O₂-dependent oxyfunctionalisation reactions. In this contribution we exploit the substrate promiscuity of AoFOx to fully oxidise methanol and formaldehyde to CO₂ and drive peroxygenase-catalysed stereoselective oxyfunctionalisation reactions. The highly atom efficient H₂O₂ generation system also enabled high catalytic turnover of the peroxygenase production enzyme.

Full text of DOI:10.1002/cctc.202000197

Accepted Article
Title: FOx News: Towards methanol-driven biocatalytic
oxyfunctionalisation reactions
Authors: Sébastien J.-P. Willot, Manh Dat Hoang, Caroline E.
Paul, Miguel Alcalde, Isabel W.C.E. Arends, Andreas S.
Bommarius, Bettina Bommarius, and Frank Hollmann
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10.1002/cctc.202000197
ChemCatChem
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FOx News: Towards methanol-driven biocatalytic
oxyfunctionalisation reactions
Sébastien J.-P. Willot,[a] Manh Dat Hoang, [b] Caroline E. Paul,[a] Miguel Alcalde,[c] Isabel W.C.E.
Arends,[d] Andreas S. Bommarius,[e] Bettina Bommarius,[e] Frank Hollmann*[a]
Abstract: The novel formate oxidase from Aspergillus oryzae
(AoFOx) is useful catalyst to promote H2O2-dependent
also necessitates a sacrificial co-substrate to provide the reducing
equivalents needed for the reductive activation of O2. Envisioning
large-scale preparative applications, H2O2 generation systems
producing no or innocuous wastes are preferred. Electrochemical
generation of H2O2, for example, is an attractive means to drive
a
oxyfunctionalisation reactions. In this contribution we exploit the
substrate promiscuity of AoFOx to fully oxidise methanol and
formaldehyde to CO2 and drive peroxygenase-catalysed
stereoselective oxyfunctionalisation reactions. The highly atom
efficient H2O2 generation system also enabled high catalytic turnover
of the peroxygenase production enzyme.
[5]
peroxygenase reactions.[4] Also, simple reductants such as H2
or H2O[6] appear promising. Methanol would be a very suitable
sacrificial electron donor as it is readily available, easy to handle
and, in principle, can be fully oxidised to CO2 to provide reducing
equivalents for 3 eq. H2O2 per eq. MeOH.[7] Similarly, complete
oxidation of methnol to regenerate reduced nicotinamide
cofactors has been reported.[8]
Recently, we reported that the formate oxidase from
Aspergillus oryzae (AoFOx) is an efficient catalyst for the in situ
generation of H2O2 to drive peroxygenase-catalysed
oxyfunctionalisation reactions.[9] Encouraged by the very
promising results, we originally aimed at a further characterisation
of AoFOx. To our surprise, while evaluating methanol as potential
co-solvent, we found that AoFOx also exhibited a methanol
oxidase activity. We therefore further investigated AoFOx to drive
peroxygenase catalysed oxyfunctionalisation reactions using
either methanol, formaldehyde or formic acid as sacrificial
electron donor (Figure 1). As production enzyme we chose the
recombinant, evolved peroxygenase from Agrocybe aegerita
(rAaeUPO).[10]
Selective oxyfunctionalisation of C-H-bonds is one of the most
challenging reactions in organic chemistry. In recent years,
peroxygenases have emerged as promising catalysts for this
reaction enabling a broad range of regio- and stereoselective
oxyfunctionalisation reactions.[1] Like the well-known cytochrome
P450 monooxygenases[2] peroxygenases utilise a highly oxidised
heme Fe-oxo complex (Compound I) to electrophilically insert an
oxygen atom into the starting material. While P450
monooxygenases depend on molecular oxygen and rather
complicated electron transport chains to attain the reductive
activation of O2 (yielding Compound I), peroxygenases use
already reduced H2O2 for the same goal. Hence, peroxygenases
appear as simpler, easier applicable alternatives to P450
monooxygenases. However, such as all heme enzymes,
peroxygenases are rapidly inactivated by H2O2, necessitating
controlled provision with H2O2 to balance the H2O2-dependent
catalytic activity and the (likewise H2O2-dependent) inactivation
reaction.[3] In situ generation of H2O2 through catalytic reduction
of O2 is one of the most promising approaches, which, however,
Using 250 mM of either MeOH, HCHO or HCO2H as sacrificial
reductant, hydroxylation of ethyl benzene or cyclohexane as well
as epoxidation of cis-ß-methyl styrene to (R)-1-phenyl ethanol,
cyclohexanol
and
(1R,2S)-cis-ß-methyl
styrene
oxide,
respectively was observed (Figure 1). AoFOx exhibited a low, but
[a]
Sébastien J.-P. Willot, Dr. Caroline E. Paul, Prof. Dr. Frank
Hollmann
detectable methanol oxidase activity.
Department of Biotechnology
Delft University of Technology
van der Maasweg 9, 2629 HZ Delft, The Netherlands
E-mail: f.hollmann@tudelft.nl
Twitter: @BiocatTUD
Interestingly, AoFOx appeared to be promiscuous with
respect to the oxidation state of the C1-substrate but accepted no
other starting material such as ethanol or propanol (Supporting
Information, Figure S8).
[b]
[c]
[d]
[e]
Manh Dat Hoang
Institute of Biochemical Engineering
Technical University of Munich
Boltzmannstr. 15, 85748 Garching, Germany
Prof. Dr. Miguel Alcalde
Department of Biocatalysis
Institute of Catalysis, CSIC
Madrid Spain
Prof. Dr. Isabel W.C.E. Arends
Faculty of Science
University of Utrecht
The Netherlands
Prof. Dr. Andreas S. Bommarius, Dr. Bettina Bommarius
School of Chemical and Biomolecular Engineering
Georgia Institute of Technology
311 Ferst Drive, N.W., Atlanta, GA 30332 (USA)
Supporting information for this article is given via a link at the end of
the document.
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we rather see the advantage of this relative activity as it, in
principle, results in low in situ concentrations of harmful (HCHO)
or acidifying (HCO2H) intermediate oxidation states of the
sacrificial cosubstrate. However, even the comparably low
methanol oxidase activity under saturation conditions
corresponded to a specific activity of 0.43 U mg-1.
Robust and efficient reaction schemes involving
peroxygenases need a balancing of the H2O2 generation reaction
rate and the (H2O2-consuming) peroxygenase reaction rate. On
the one hand, high H2O2-generation rates are desirable to attain
high space-time-yield. On the other hand, accumulation of H2O2
must be avoided in order to minimise the oxidative inactivation of
the prosthetic heme group. We therefore investigated the
influence of the methanol concentration on the overall reaction
system (Figure 2). As expected from the kinetic measurements
(Table 1), increasing the methanol concentration increased the
overall product formation (Figure 2 A). A methanol concentration
around 10 % (vol/vol, approx. 2.5 M) appeared optimal. We
attribute the decreasing product formation at higher methanol
concentrations to a decreasing long-term stability of either AoFOx
or rAaeUPO (or both) in aqueous methanol solutions. We
therefore used a methanol concentration of 2.5 M for further
experiments. Under these conditions, AoFOx-catalysed H2O2
generation appeared to be overall rate-limiting as up to at least
1000 nM AoFOx (equimolar to rAaeUPO) the productivity of the
system increased linearly with the increasing AoFOx
concentration (Figure 2B, Figure S12). Higher productivities may
be accessible upon further increase of the AoFOx concentration.
However, to avoid possible H2O2 accumulation, we continued with
the more conservative equimolar ratio of AoFOx and rAaeUPO.
Figure 1. Comparison of rAaeUPO product formation when either 250 mM
methanol, formaldehyde or formate is applied. Conditions : c(substrate) = 100
mM, c(rAaeUPO) = 1 µM, c(AoFOx) = 1 µM, 25 °C, 100 mM KPi buffer (pH 6.0),
600 rpm, 24 h.
We suspected a poor affinity of wild-type AoFOx for MeOH to
account for the low product formation observed in these
preliminary experiments (Figure 1). We therefore determined the
kinetic parameters of AoFOx for the oxidation of methanol and
formaldehyde (Table 1).
Table 1. Kinetic parameters for the AoFOx-catalysed oxidation of methanol,
formaldehyde and formic acid.
CH3OH
oxidation
H2CO
oxidation
HCO2H
oxidation[11]
kcat [s-1]
0.46 ± 0.02
3300 ± 400
8.28 ± 0.12
380 ± 16
82
KM [mM]
160
Experimental conditions: c(AoFOx) = 42 nM, 25 °C, 50 mM KPi buffer (pH 6.0),
c(O2) = 0.25 mM (1 atm air), 10 U horseradish peroxidase, 1 mM ABTS (see SI
for further details).
Figure 2. Influence of (A) methanol concentration on product formation over 24
h and (B) AoFOx: rAaeUPO ratio on product formation rate. Conditions (unless
mention otherwise): 10 %(v/v) methanol, c(ethylbenzene)
= 100 mM,
c(rAaeUPO) = 1 µM, c(AoFOx) = 200 nM, 25 °C, 100 mM Kpi buffer (pH 6.0),
600 rpm.
Indeed, the catalytic performance of AoFOx on formaldehyde
and especially methanol falls back behind its natural formate
oxidation activity. Instead of interpreting this as a disadvantage,
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As shown in Figure 3, robust oxyfunctionalisation of all three
model starting materials over at least 5 days could be achieved
using methanol as sacrificial electron donor. Very promising
turnover numbers for rAaeUPO and AoFOx of up to 49000 were
observed (equimolar concentrations of biocatalysts used). The
differences in the overall rates of cyclohexan hydroxylation, ethyl
benzene hydroxylation and cis--methyl styrene epoxidation
(0.05 mMh-1, 0.23 mMh-1 and 0.32 mMh-1, respectively) can be
attributed to the relative activity of the peroxygenases for these
substrates.[12]
Nevertheless, already at present stage, the catalytic
performance of the biocatalysts with turnover numbers as high as
49000 for rAaeUPO and AoFOx, respectively represent a good
starting point for further characterisation and optimisation of the
reaction system.
Experimental Section
Enzyme preparation. Recombinant expression and purification
of the evolved unspecific peroxygenase mutant from A. aegerita
in P. pastoris was performed following a previously described
procedure.[10a] Formate oxidase from Aspergillus oryzae RIB40
(AoFOx) was produced recombinantly in E. coli BL21(DE3) as
reported before with slight modifications.[9] During desalting step
with HiTrap, an additional buffer exchange was applied by using
a phosphate potassium buffer (25 mM, pH 6.0) for elution of the
target enzyme of the column. A final protein concentration of
2.16 ± 0.06 mg mL-1 was measured by BCA assay. AoFOx purity
of approximately 60% was determined by SDS-PAGE. With
consideration of the extinction absorption at 472 nm,[16] a molar
protein concentration of 21 µM was calculated.
Enzyme activity assay. AoFOx kinetics were determined
indirectly by (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid ) ABTS assay. ABTS assay was performed with 50 mM of
acetate buffer at pH 4.5 or phosphate buffer at pH 6.0,
respectively. A final concentration of 1 mM of ABTS, 10 U mL-1 of
HRP (horseradish peroxidase) and different concentration of
methanol or formaldehyde were used. ABTS was either dissolved
in 50 mM acetate buffer (pH 4.5) or 50 mM phosphate buffer (pH
6.0). HRP was stored in 25 mM phosphate buffer (pH 7.5). The
reactions were measured with an UV-Vis spectrometer at 420 nm
for 1 min at a controlled temperature of 25 °C. For calculation, an
extinction coefficient of 36 000 M-1 cm-1 was used.
Enzymatic reaction. Reactions of 200 µL volume were
performed in GC glass vials of 1.5 mL. Thermoshakers were used
for temperature and stirring control. All components were mixed
and the reactions started by the addition of either methanol,
formaldehyde or formate. For each time point, a full reaction is
extracted with ethyl acetate (equivolume) containing 5 mM n-
octanol as internal standard. The extraction is then dried over
MgSO4 and analysed by gas chromatography (temperature
profiles in supporting information). Further information for the
negative control can be found in supporting information.
Figure 3. Time-courses of the selective oxyfunctionalisation of (●) ethyl
benzene, (▲) cis-β-methylstyrene and (■) cyclohexane using the
MeOH/AoFOx/rAaeUPO cascade. Conditions: 10 %(v/v) methanol, c(substrate)
= 100 mM, c(rAaeUPO) = 1 µM, c(AoFOx) = 1 µM, 25 °C, 100 mM KPi (pH 6.0),
600 rpm.
Overall, with the current contribution we have demonstrated
that methanol-driven peroxygenase reactions are principally
possible using just one biocatalyst (AoFOx), in addition to
rAaeUPO. At present, the major limitation of this approach is the
comparably high KM value of the wild-type AoFOx for MeOH,
which may be addressable through protein engineering.
Envisioning preparative scale applications, further issues are
likely to occur such as the poor solubility of O2 in aqueous media,
which we will address by either using non-aqueous reaction
media[13] and/or innovative reactor concepts such as the bubble
column[14] or flow-chemistry[15] to attain higher kL values for O2.
Acknowledgements
We thank The Netherlands Organisation for Scientific Research
for financial support through a VICI grant (No. 724.014.003).
Keywords: Biocatalysis, Formate oxidase, Methanol,
Oxyfunctionalisation, Peroxygenases
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Entry for the Table of Contents
COMMUNICATION
Sébastien J.-P. Willot, Manh Dat Hoang,
Caroline E. Paul, Miguel Alcalde, Isabel
W.C.E. Arends, Andreas S. Bommarius,
Bettina Bommarius Frank Hollmann*
Page No. – Page No.
Formate oxidase from Aspergillus oryzae (AoFOx) also oxidises methanol and
formaldehyde producing one equivalent of H2O2 in each oxidation step. Thus,
AoFOx is a promising catalyst for the in situ generation of H2O2 to drive
peroxygenase-catalysed oxyfunctionalisation reactions. The preparative usefulness
of AoFOx is demonstrated in combination with the recombinant peroxygenase from
Agrocybe aergerita (rAaeUPO) resulting in robust oxyfunctionalisation reactions.
FOx News: Towards methanol-driven
biocatalytic oxyfunctionalisation
reactions
This article is protected by copyright. All rights reserved.