Transesterifications and peracid-assisted oxidations in aqueous media catalyzed by Mycobacterium smegmatis acyl transferase

Article abstract of DOI:10.1002/cctc.201300683

Hydrolases catalyze synthetic reactions in nonaqueous media, whereas they perform hydrolysis under aqueous solutions. An acyl transferase from Mycobacterium smegmatis (MsAcT) is able to catalyze synthetic reactions in buffer because of its highly hydrophobic active site, which enables efficient transesterification reactions even at 99.9 % v/v buffer solution. This unique feature of MsAcT among hydrolases may open new opportunities to conduct synthetic (bio)catalysis in aqueous media. With these goals in mind, this paper explores some evidence of such potential: MsAcT can perform enantioselective transesterifications (e.g., (S)-2-octanol), which could be combined with other aqueous multistep (asymmetric) reactions; 5-hydroxymethylfurfural (HMF) can be esterified to produce more hydrophobic and easily extractable HMF esters (e.g., for downstream processing or wastewater treatment); and upon addition of dilute H₂O₂, MsAcT works efficiently as a perhydrolase to form in situ peracids - in bulk water - that can be used for oxidations (e.g., furfural to furoic acid oxidation). Overall, these and many other new applications can be envisaged by using MsAcT in aqueous solutions. Challenging thermodynamics? As a result of its highly hydrophobic active site, an acyl transferase from Mycobacterium smegmatis catalyzes synthetic reactions in buffer in which other hydrolases could only conduct hydrolysis. Among several options, opportunities for downstream processing, enantioselective (multistep) aqueous syntheses, and peracid-mediated epoxidations and oxidations are envisaged. Copyright

Full text of DOI:10.1002/cctc.201300683

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DOI: 10.1002/cctc.201300683
Transesterifications and Peracid-Assisted Oxidations in
Aqueous Media Catalyzed by Mycobacterium smegmatis
Acyl Transferase
[
a]
[b]
[a]
[a]
Lotte Wiermans, Sebastian Hofzumahaus, Christiane Schotten, Lisa Weigand,
[c]
[b]
[a]
Marcus Schallmey, Anett Schallmey, and Pablo Domꢀnguez de Marꢀa*
Hydrolases catalyze synthetic reactions in nonaqueous media,
whereas they perform hydrolysis under aqueous solutions. An
acyl transferase from Mycobacterium smegmatis (MsAcT) is able
to catalyze synthetic reactions in buffer because of its highly
hydrophobic active site, which enables efficient transesterifica-
tion reactions even at 99.9% v/v buffer solution. This unique
feature of MsAcT among hydrolases may open new opportuni-
ties to conduct synthetic (bio)catalysis in aqueous media. With
these goals in mind, this paper explores some evidence of
such potential: MsAcT can perform enantioselective transesteri-
fications (e.g., (S)-2-octanol), which could be combined with
other aqueous multistep (asymmetric) reactions; 5-hydroxyme-
thylfurfural (HMF) can be esterified to produce more hydro-
phobic and easily extractable HMF esters (e.g., for downstream
processing or wastewater treatment); and upon addition of
dilute H O , MsAcT works efficiently as a perhydrolase to form
2
2
in situ peracids—in bulk water—that can be used for oxida-
tions (e.g., furfural to furoic acid oxidation). Overall, these and
many other new applications can be envisaged by using
MsAcT in aqueous solutions.
Introduction
The use of enzymes as biocatalysts for synthetic purposes has
grown significantly in recent decades—stimulated by develop-
ments in biology—with many examples both at academic and
tions in different (non)conventional media. Following these
considerations, recently an acyl transferase from Mycobacteri-
[4]
um smegmatis (MsAcT) was biochemically characterized. In-
terestingly, MsAcT is able to catalyze acyl transfer reactions,
namely, (trans)esterification and perhydrolysis, in bulk aqueous
[
1]
industrial level. In this area, hydrolases play a central role be-
cause of their robustness and their capability to catalyze many
[4]
(
promiscuous) diverse reactions without the need of cofac-
solutions under process conditions in which other hydrolases
could only perform hydrolytic conversions as the excess of
water as nucleophile would hamper any synthetic reverse at-
tempt. The reason for such a peculiar feature is that MsAcT
bears an octameric structure (PDB: 2Q0Q) that creates a huge
restriction in the morphology of the active site morphology,
which limits the access of solvent molecules and creates
[
1,2]
tors.
Herein, hydrolytic strategies are feasible if hydrolases
are used in aqueous solutions and water acts as the nucleo-
phile, whereas a broad array of synthetic options (e.g., (trans)-
esterifications, amidations, epoxidations, CÀC bond forming re-
actions, etc.) can be reached if nonaqueous, nonconventional
media are used, such as organic solvents, ionic liquids, solvent-
[
1–3]
[4]
free processes, etc.
a highly hydrophobic microenvironment. This allows for
Given their importance and practical interest at (industrial)
synthetic level, there is a constant quest for new enzymes in
general, and for hydrolases in particular, that might catalyze
other relevant reactions or might enable new innovative op-
a water-free active site in which “nonaqueous” synthetic reac-
[4]
tions may proceed even in buffer solutions. Moreover, for the
same reason, MsAcT also displays a high perhydrolase activity
in aqueous solutions and is able to form organic peracids if
dilute H O2 is added to the aqueous solution. In this case,
2
dilute H O competes efficiently with bulk water as a nucleo-
2
2
[
a] L. Wiermans, C. Schotten, L. Weigand, Dr. P. Domꢀnguez de Marꢀa
Institut fꢁr Technische und Makromolekulare Chemie (ITMC)
RWTH Aachen University
Worringer Weg 1, 52074 Aachen (Germany)
Fax: (+49)241-8022177
phile. Based on this, free or immobilized MsAcT has been as-
sessed in different oxidative processes by forming in situ per-
acetic acid from ethyl acetate. Several examples of the discol-
oration of dyes, decontamination, tooth whitening, bleaching,
or surface disinfection have been reported at the patent
E-mail: dominguez@itmc.rwth-aachen.de
[
b] S. Hofzumahaus, Dr. A. Schallmey
Junior Professorship for Biocatalysis
Institute of Biotechnology
[5,6]
level.
However, despite the foreseeable interest in MsAcT for prac-
tical synthetic biocatalysis in aqueous media, which provides
virtually unique and new properties among hydrolases, its syn-
thetic potential has surprisingly not been explored so far. The
possibility of the addition of a new hydrolase to the portfolio
that could catalyze synthetic reactions in aqueous solutions
RWTH Aachen University
Helmertweg 3, 52074 Aachen (Germany)
[
c] M. Schallmey
Forschungszentrum Jꢁlich GmbH
Institute of Bio- and Geosciences, IBG-1: Biotechnology
5
2425 Jꢁlich (Germany)
ꢁ
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might open applications from asymmetric synthesis and/or
coupling to multistep reactions in aqueous media to further
opportunities in downstream processing, by making products
more hydrophobic through transesterifications and thus more
easily extractable, or for wastewater treatment upon the oxida-
tion of chemicals. Based on all these promising and largely un-
explored options, in this paper the first proofs-of-concept for
MsAcT-catalyzed aqueous-based synthesis are reported, in
which we focus on several selected examples as benchmark
cases: the enantioselective transesterification of (S)-2-octanol,
the transesterification of 5-hydroxymethylfurfural (HMF) for re-
moval from water effluents in biorefineries, and the peracid-
mediated oxidation of furfural to afford furoic acid.
some unreacted substrate, presumably because of equilibrium
reactions. In any case, these results may be expected, as many
other hydrolases can perform efficient (trans)esterifications in
[7]
biphasic media. Therefore, to assess the new biocatalytic per-
formance that MsAcT might bring, the enzyme was assessed
in-depth towards the transesterification of neopentylglycol in
monophasic systems that used buffer solution (95% v/v) and
ethyl acetate as the acyl donor (5% v/v) at low enzyme load-
À1
ings (0.0025 mgmL ). Under these more challenging reaction
conditions, other lipases (e.g., CAL-B) did not display any ob-
servable synthetic activity (data not shown) as hydrolysis
would be the dominating direction. The results for MsAcT are
depicted in Figure 2.
Results and Discussion
A codon-optimized synthetic gene of MsAcT was ordered and
cloned into vector pET28a(+) for overexpression of the
enzyme in E. coli BL21 (DE3) at 308C for 24 h. After cell lysis
and the removal of cell debris by centrifugation, the resulting
cell-free extract was lyophilized and used as a crude enzyme
preparation in all reactions. When we looked at the previous
literature, the only example of MsAcT-catalyzed transesterifica-
tion was the formation of monoesters of neopentylglycol in
different buffer/ethyl acetate aqueous solutions of up to 95%
[
4]
v/v buffer content. To start the biocatalytic characterization
of the overexpressed MsAcT, the same reaction was assessed
in a biphasic system (buffer/ethyl acetate 50:50 v/v) with differ-
ent enzyme loadings (Figure 1).
Figure 2. Kinetic profile of the MsAcT-catalyzed transesterification of neo-
pentylglycol in a monophasic system of ethyl acetate/buffer. Conditions: Po-
tassium phosphate buffer (50 mm, pH 8, 95% v/v), ethyl acetate 5% v/v, ne-
À1
opentylglycol 100 mm, enzyme loading 0.0025 mgmL , room temperature.
[
4]
1
In agreement with the literature, MsAcT was able to cata-
lyze transesterification reactions in such biphasic conditions
Conversions were determined by H NMR spectroscopy.
(
buffer/ethyl acetate 50:50% v/v) and showed remarkable ac-
MsAcT was able to perform this synthetic reaction efficiently.
The monoester was rapidly formed at the beginning of the re-
action, whereas diester started to appear at later stages, until
equilibria were reached between the different species. To
obtain further data on these equilibria and the enzyme perfor-
mance, in a subsequent set of experiments smaller amounts of
ethyl acetate were assessed for the same model reaction and
conversions were measured at longer reaction times (65 h).
The results are shown in Figure 3.
tivities in short reaction times (30 min, Figure 1). At higher
enzyme loadings, a mixture of mono- and diesters of neopen-
[
4]
tylglycol was found (not reported previously ), together with
Remarkably, MsAcT was able to perform synthetic reactions
even in virtually pure buffer (99.9% v/v with 0.1% ethyl ace-
tate). Albeit the kinetics was slower at these low concentra-
tions of ethyl acetate, in all cases an equilibrium profile of sub-
strate–monoester–diester was reached after long reaction
times. Subsequently and envisaging the potential of this
enzyme as an option for synthetic biocatalysis, other substrates
such as alcohols were evaluated as substrates in transesterifica-
tion reactions. Remarkably, glycerol, 2-butanol, 1-octanol, ben-
zylalcohol, and 1-phenylethanol were accepted by MsAcT as
substrates, which showed kinetically influenced conversions in
transesterifications in the range of 10–25% (data not shown).
For other more sterically hindered substrates, such as 3-octa-
nol, traces of the ester were found. Overall, these first results
provide promising prognoses on the use of MsAcT as a unique
biocatalyst with a broad substrate range to perform synthetic
Figure 1. MsAcT-catalyzed transesterification of neopentylglycol in a biphasic
system of ethyl acetate/buffer at different enzyme loadings. Conditions: Po-
tassium phosphate buffer (50 mm, pH 8, 50% v/v), ethyl acetate 50% v/v,
neopentylglycol 100 mm, different enzyme loadings, 30 min reaction time at
1
room temperature. Conversions were determined by H NMR spectroscopy.
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ment of the activity and stability of MsAcT to match actual
process conditions.
Triggered by the promising results obtained with different
substrates in transesterification reactions in aqueous solutions,
in the next step preliminary studies on the potential enantiose-
lectivity of the enzyme were conducted. To this end, the trans-
esterification of (R)-2-octanol or (S)-2-octanol in buffer were as-
sessed, and the results are depicted in Figure 5.
Figure 3. MsAcT-catalyzed transesterification of neopentylglycol with ethyl
acetate at different acyl donor loadings (5–0.1% v/v). Conditions: Potassium
phosphate buffer (50 mm, pH 8, 95 to 99.9% v/v), ethyl acetate 5 to 0.1% v/
À1
v, neopentylglycol 100 mm, enzyme loading 0.0025 mgmL , 65 h reaction
1
time at room temperature. Conversions were determined by H NMR
spectroscopy.
reactions in conventional media and/or to combine such a syn-
thetic aqueous-based transesterification reaction with other
[8,9]
(bio)catalysts in the same buffer in a multistep approach.
Subsequently, the pH activity profile of the enzyme was as-
sessed. Ideally, a broad pH range of activity might allow the
combination of MsAcT with other (bio)catalysts with different
pH requirements. The results of the tests with the benchmark
reaction of neopentylglycol transesterification with ethyl ace-
tate are depicted in Figure 4.
Figure 5. MsAcT-catalyzed transesterification of (R)-2-octanol or (S)-2-octanol
with ethyl acetate in buffer. Conditions: Potassium phosphate buffer
(
50 mm, pH 8, 97.5% v/v), ethyl acetate 2.5% v/v, (R)-2-octanol or (S)-2-octa-
À1
nol 100 mm, enzyme loading 0.0025 mgmL , room temperature. Conver-
sions were determined by H NMR spectroscopy.
1
As observed, MsAcT displayed around eightfold kinetic selec-
tivity for the S enantiomer over the R one, which shows that it
is possible to perform asymmetric synthesis with this enzyme
in aqueous solutions (97.5% v/v buffer and 2.5% v/v ethyl ace-
tate). To the best of our knowledge, this is the first example of
an asymmetric synthetic reaction performed by enzymes in
monophasic aqueous solution. Although the stereobias was
not as satisfactory as that of wild-type MsAcT as the R enantio-
mer is still accepted by the enzyme, the conceptual result
opens the possibility to apply protein engineering tools to
design improved biocatalyst variants with higher enantioselec-
tivities under aqueous conditions.
Figure 4. MsAcT-catalyzed transesterification of neopentylglycol with ethyl
acetate at different pH values. The buffers used were (50 mm): pyridine
buffer, pH 4; potassium phosphate buffer, pH 6 and 8; tris buffer, pH 9–11.
Conditions: Buffer 95% v/v, ethyl acetate 5% v/v, neopentylglycol 100 mm,
À1
enzyme loading 0.0025 mgmL , 3 h reaction time at room temperature.
Once the transesterification-based synthetic performance of
MsAcT was fully demonstrated in aqueous solution, which
paves the way for aqueous-based asymmetric biocatalysis, em-
phasis was put on the possibility to use this enzyme for appli-
cations in biomass processing. Herein, we envisaged that
MsAcT could be used to enhance the hydrophobicity of some
compounds in aqueous solutions—through transesterifica-
tion—to subsequently extract them in a more straightforward
manner with the aid of a (bio-based) solvent. To validate the
idea, the MsAcT-catalyzed transesterification of HMF in aque-
1
Conversions were determined by H NMR spectroscopy.
Gratifyingly, MsAcT displayed aqueous-based synthetic activ-
ity in a broad range of pH values, with significant enzymatic
activities still observed at pH 11 and 4. The latter might
become relevant, as potential (trans)esterifications of bio-
based carboxylic acids obtained by fermentation (e.g., succinic
[
7]
acid) could be envisaged with this enzyme. Needless to men-
tion, protein engineering approaches may enable the improve-
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[
4–6]
ous solution was assessed. HMF is produced by the acid-cata-
lyzed triple dehydration of glucose and it is considered as a val-
peracids in aqueous solutions as previously reported.
With
these considerations in mind, the oxidation of furfural to furoic
acid in aqueous solution was assessed for the first time.
Herein, two different acyl donors, ethyl acetate and dimethyl
carbonate (DMC), were used successfully. Both of these sub-
strates have also been reported as substrates for CAL-B-medi-
[10]
uable biomass-derived platform chemical for the future. Re-
cently, our group showed that HMF esters can be accessed
straightforwardly by lipase-catalyzed (trans)esterifications in
nonaqueous media to deliver new bio-based compounds with
promising envisaged applications in many chemical seg-
[6,12]
ated oxidations in nonaqueous solutions.
Moreover, these
[
11]
ments. However, HMF is also formed in aqueous solutions
during (ligno)cellulose acid-based pretreatments because of
the often harsh chemical conditions applied, which can cause
growth inhibition of micro-organisms if such (crude) fermenta-
ble sugars are used as a carbon source in fermentations.
Herein, we envisaged that the mild MsAcT-catalyzed esterifica-
tion of HMF in aqueous solutions would enhance its hydropho-
bicity by producing HMF esters, which thus allows a more
straightforward recovery—and subsequent valorization of such
esters—before aqueous crude sugar effluents are delivered to
fermenters. The results are depicted in Figure 6.
results would explore the MsAcT acceptance of different acyl
donors, which would certainly enhance possibilities for differ-
ent applications. Very recently we showed how peracids can
perform this reaction for furans (furfural and HMF) efficiently
[13]
by using lipases and nonaqueous solutions. Herein, the pro-
cess was conducted in buffer, and the results are shown in
Figure 7.
Figure 7. MsAcT-catalyzed oxidation of furfural to furoic acid by using ethyl
Figure 6. MsAcT-catalyzed transesterification of HMF with ethyl acetate (2%
v/v) in buffer (98% v/v). Conditions: Potassium phosphate buffer (50 mm,
pH 8, 98% v/v), ethyl acetate 2% v/v, HMF 100 mm, enzyme loading
2 2
acetate and H O in buffer. Conditions: Potassium phosphate buffer
(200 mm, pH 8), acyl donor. Variable amounts of buffer and acyl donor
(EtOAc or DMC) depending on the equivalents added; furfural 100 mm,
À1
1
À1
2
mgmL , room temperature. Conversions were determined by H NMR
enzyme loading 2.5 mgmL , 3 h reaction time at 408C. H
2
O
2
(30% v/v) was
1
spectroscopy.
added stepwise (6ꢂ) every 30 min. Conversions were determined by H NMR
spectroscopy. a) 15 equiv. EtOAc, 8 equiv. H ; b) 12 equiv. EtOAc, 4 equiv.
; c) 8 equiv. EtOAc, 4 equiv. H ; d) 8 equiv. DMC, 8 equiv. H
2
O
2
H
2
O
2
2
O
2
2 2
O .
MsAcT was able to accept HMF as a substrate to yield HMF
acetate esters in virtually pure aqueous conditions (98% v/v
buffer). Equilibrium under these conditions was reached at
À1
around 20–25% conversion (corresponding to 5 gL ester,
Gratifyingly, MsAcT was able to mediate the oxidation of fur-
formed in 10–15 h). The set-up of a continuous process or the
use of higher ethyl acetate loadings may certainly improve the
reaction outcome reported here (see Figure 2). The fact that
fural under these reaction conditions, which opens new op-
tions for wastewater treatment, oxidation, and/or the removal
of furans from aqueous environments before fermentative
strategies are applied. After the judicious choice of loadings of
H O and ethyl acetate, almost full conversions of furfural were
[
5a,b]
MsAcT can be successfully immobilized and reused
paves
the way to envisage highly integrated continuous systems to
remove HMF from sugar-containing effluents by converting it
2
2
achieved to form furoic acid in high purities (Figure 7). Herein,
[
11]
to an ester with promising properties.
excess ethyl acetate (15 equiv.) and H O2 (8 equiv.) were
2
Apart from these transesterification concepts for different
chemical purposes, asymmetric synthesis, multistep aqueous-
based processes, downstream unit operations, etc, MsAcT also
displays perhydrolase activity and is thus able to form organic
needed to achieve this. A possible explanation for the large
amount of H O needed could be the presence of catalases in
2
2
the crude extract used, which would decompose some of the
H O . Under these best process conditions, approximately
2
2
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À1
1
0 gL of furfural was oxidized in 3 h, which provides a prom-
Protocol for furfural oxidations
Furfural (1 mmol) was added to a solution of enzyme (2.5 mgmL
ising prognosis for use in wastewater oxidation.
À1
)
in different mixtures of potassium phosphate buffer (200 mm,
pH 8) and acyl donor (ethyl acetate or DMC; 10 mL). The reactions
Conclusions
were started by the stepwise addition (every 30 min) of H
O (from
2 2
3
0% v/v H O ) until final equivalents (according to Figure 7). After
2
2
We have successfully explored the possibility to conduct syn-
thetic reactions—transesterifications and peracid-mediated oxi-
dations—in aqueous solutions by using an acyl transferase
from Mycobacterium smegmatis (MsAcT) as the catalyst. As it
has an octameric structure with a highly buried hydrophobic
active site, this enzyme can prioritize synthesis over hydrolysis
in aqueous media. This performance may open new options in
biocatalysis, which range from asymmetric synthesis or multi-
step processes in buffer solutions to strategies for downstream
processing, wastewater treatment, effluent purification over
oxidation, etc. We hope that our work will stimulate other re-
search groups to focus on the new possibilities that MsAcT can
bring to the (bio)catalytic arena.
stirring at RT for 3 h, the products were extracted with ethyl ace-
tate (3ꢂ10 mL), and the solvent was evaporated under reduced
pressure. The conversion of the reactions was determined by
1
H NMR spectroscopy.
Acknowledgements
This work was performed as part of the Cluster of Excellence
“Tailor-Made Fuels from Biomass”, which is funded by the Excel-
lence Initiative of the German Research Foundation to promote
science and research at German universities. Furthermore, finan-
cial support from DFG training group 1166 “BioNoCo” (“Biocataly-
sis in Non-conventional Media”) is gratefully acknowledged as
well.
Experimental Section
Keywords: biocatalysis · enantioselectivity · enzyme catalysis ·
hydrolases · transferases
Chemicals
All chemicals were purchased from Sigma–Aldrich and were used
without further purification. Restriction enzymes and T4 ligase
were obtained from New England Biolabs.
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A codon-optimized synthetic gene that encoded MsAcT (GenBank
accession: ABK70783) from Mycobacterium smegmatis str. MC2 155
was ordered from Life Technologies. The gene was cloned into
vector pET28a(+) (Life Technologies) by using restriction enzymes
NdeI and BamHI and T4 ligase for ligation. The resulting vector
pET28-MsAcT was afterwards transformed into electrocompetent
E. coli BL21 (DE3) for heterologous expression. E. coli BL21 (DE3)
pET28-MsAcT was grown at 378C in terrific broth (TB) medium that
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were resuspended in 50 mm potassium phosphate buffer pH 7.4
and disrupted in an EmulsiFlex-C3 French press (Avestin Europe
GmbH) with five cycles at 1500 bar and 48C. The cell debris was re-
moved by centrifugation for 30 min at 47808 g and 48C. The re-
sulting cell-free extract was lyophilized overnight, and the crude
enzyme powder stored at 48C.
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[
Alcohol (2 mmol) was added to different mixtures of potassium
phosphate buffer (50 mm, pH 8) and ethyl acetate (20 mL). After
the addition of the enzyme, the reaction mixtures were stirred at
RT. The product was obtained by extraction with ethyl acetate (3ꢂ
1
0 mL) and evaporation of the solvent under reduced pressure.
1
The conversion of the reactions was assessed by H NMR
spectroscopy.
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Received: August 15, 2013
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Published online on November 6, 2013
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