Enzyme-Catalyzed Synthesis of Esters in Water

Article abstract of DOI:10.1002/cctc.201801991

MsAcT catalyzes the esterification of primary alcohols in water. When utilizing acid and alcohol as starting materials low yields dictated by thermodynamics were observed. However, with activated esters such as ethyl acetate and vinyl acetate very high yields of the desired ester can be achieved in combination with the appropriate alcohol. This study investigated both the intrinsic kinetic properties of MsAcT for the hydrolysis and transesterification of esters in water as well as the thermodynamics of the reaction. In comparison to the chemical or enzymatic ester synthesis using either toxic reagent, and harsh organic solvents, the MsAcT-catalyzed synthesis of esters of primary alcohols can be achieved efficiently in water without neutralization steps.

Full text of DOI:10.1002/cctc.201801991

Accepted Article
Title: Enzyme Catalyzed Synthesis of Esters in Water
Authors: Luuk Mestrom, Jord Claessen, and Ulf Hanefeld
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10.1002/cctc.201801991
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Enzyme Catalyzed Synthesis of Esters in Water
Luuk Mestrom[a], Jord G.R. Claessen[a], and Ulf Hanefeld*[a]
Dedication ((optional))
Abstract: MsAcT catalyzes the esterification of primary alcohols in
water. When utilizing acid and alcohol as starting materials low
yields dictated by thermodynamics were observed. However, with
activated esters such as ethyl acetate and vinyl acetate very high
yields of the desired ester can be achieved in combination with the
appropriate alcohol. This study investigated both the intrinsic kinetic
properties of MsAcT for the hydrolysis and transesterification of
esters in water as well as the thermodynamics of the reaction. In
comparison to the chemical or enzymatic ester synthesis using
either toxic reagent, and harsh organic solvents, the MsAcT-
catalyzed synthesis of esters of primary alcohols can be achieved
efficiently in water without neutralization steps.
catalyzed by MsAcT is strictly speaking a transesterification
since the reactions all utilize ethyl acetate or more reactive acid
derivatives.[3] While this is similar to the classical ester synthesis
which utilizes acid chlorides or anhydrides it raises the question
whether MsAcT catalyzes the direct synthesis of esters, too.
This has been described for lipases and other catalysts;[4] but in
all those examples the yield was determined by the
thermodynamic stability of the product under reaction conditions.
Therefore, the direct esterification and transesterification of
alcohols catalyzed by MsAcT in water was studied in parallel. As
a catalyst MsAcT should accelerate the reaction but not alter its
overall equilibrium.[5] High transesterification yields with MsAcT
can only be obtained under kinetic control using activated acids.
MsAcT is known to be an excellent catalyst for the
transesterification of primary alcohols and amines with a variety
of acyl donors[3d, 6]. Benzyl alcohol and isobutyl alcohol were
chosen for this study in combination with acetic acid in the form
of potassium acetate buffer or ethyl-, vinyl- and phenyl-acetate
as activated acids. The kinetic properties of MsAcT were
investigated for both the hydrolysis and transesterification rate of
vinyl acetate. This acyl donor irreversibly tautomerizes upon
hydrolysis, simplifying the reaction kinetics of the
transesterification.
Introduction
The synthesis of esters is textbook-knowledge and is performed
according to standard protocols that are taught unaltered
already for many decades.[1] An activated acid, i.e. an acid
chloride or anhydride, reacts with an alcohol in the presence of a
base, typically pyridine, often catalyzed by DMAP, in an organic
solvent. Alternatively, the acid is activated in situ with reagents
such as DCC. In all cases considerable amounts of waste are
generated: the solvent and stoichiometric amount of salt from
neutralization steps. Furthermore the activation, in situ or not,
also generates considerable amounts of waste. The direct
esterification catalyzed by an acid requires azeotropic removal
of water to shift the equilibrium towards ester formation and the
reaction conditions are so drastic that it is limited to stable
starting materials and products. Moreover it still leaves the
problem of the organic solvent and the neutralization steps
generating salt waste.[1a-c] Similarly the enzyme catalyzed
synthesis of esters is performed in dry organic solvents with
activated acids (acyl donors), often generating much waste
which needs to be removed in additional work up steps (Scheme
1).[2] Since the activated acids in the enzyme catalyzed ester
synthesis are esters themselves, these reactions are
transesterifications.
To address the problem the acyltransferase from
Mycobacterium smegmatis (MsAcT) was employed as catalyst.[3]
This enzyme was described to enable the synthesis the esters in
water. With MsAcT it should thus be possible to avoid the use of
organic solvents and to eradicate the need for neutralization
steps commonly employed to remove the base utilized in ester
syntheses. In all cases described to date, the ester synthesis
Scheme 1. Textbook chemical and enzymatic synthesis strategies for esters.
Results and Discussion
To establish the thermodynamic equilibrium of the reaction
and to probe whether MsAcT had the potential to overcome the
macroscopic thermodynamic forces of the reaction[3b] the direct
esterification of benzyl alcohol with acetic acid at pH 7.5 in
potassium phosphate buffer (KPi) was studied. Investigation of
both the synthesis (10 mM benzyl alcohol) and hydrolysis (10
mM benzyl acetate) reaction catalyzed by MsAcT revealed a
final concentration of benzyl acetate of 0.12 mM. A Keq of
0.00012 could thus be established. The uncatalyzed control
[a]
L. Mestrom, J.G.R. Claessen, Prof. Dr. U. Hanefeld
Biokatalyse, Afdeling Biotechnologie
Technische Universiteit Delft
van der Maasweg 9, 2629HZ Delft (The Netherlands)
E-mail: u.hanefeld@tudelft.nl
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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reactions yielded essentially no product. MsAcT is thus a
classical catalyst, catalysing a reaction but not altering it.
with 212 mM vinyl acetate with less than a µg mL-1 of MsAcT
was studied. Full conversion of isobutanol toward isobutyl
acetate was observed[3d] and no ester of the gem-diol was
observed (Fig. S6-S9). Next, the substrate concentration was
increased to 97 mM isobutanol and 20-fold more MsAcT was
added. Under these conditions the starting materials are all
soluble but the product will be insoluble in water. During the
course of the reaction a second organic phase appeared due to
the insolubility of isobutyl acetate (Fig. 2, Fig. S10, and video
SV). No hemiacetal ester was observed. After prolonged
reaction time the reaction mixture became clear again
demonstrating the hydrolysis of isobutyl acetate. This occurred
after all acyl donor had been consumed, indeed no isobutyl
acetate was detectable after 18 hours of reaction time at this
stage of the reaction. MsAcT catalyzed only the hydrolysis of the
product ester, leading to the final reaction equilibrium; that of
acid, alcohol, ester and water.
To prepare the desired ester it is therefore necessary to
employ activated acids. As was demonstrated earlier this can be
simple ethyl acetate, a benign solvent that can be used
monophasic, i.e. dissolved in water, or as a separate layer.[3]
Here, all studies were performed under monophasic conditions
with the acyl donor dissolved in the pH 7.5 KPi buffer. In order to
characterize the affinity and activity of activated acyl donor ethyl,
vinyl and phenyl acetate to MsAcT, the optimal enzyme
concentration was investigated. At less than 50 ng mL-1 MsAcT
the conversion of benzyl alcohol to benzyl acetate was not
complete. In line with earlier observations,[3d] a significant
amount of benzyl acetate was hydrolyzed again at high enzyme
concentrations (Fig. 1). The trend in conversion of vinyl acetate
> phenyl > ethyl acetate after one hour of reaction time was in
line with the reactivity of the acyl donor.[2a,b] Vinyl acetate clearly
gave the highest conversion for ester synthesis. Moreover it is
synthesized via an atom efficient catalytic process from acetic
acid and ethylene making its production environmentally
benign.[7] Additionally it is very readily available as it is a
monomer for polymer synthesis. Also the side product
acetaldehyde does on laboratory scale not require special work
up steps. On industrial scale measures to handle it are well
established.[8] Therefore, this activated acyl donor is relatively
environmentally benign. It does not require neutralization steps
in down-stream processing nor are toxic compounds produced
at a large scale. The characterization of this acyl donor was
pursued in more detail in water to also avoid organic solvents
necessary when used in combination with lipases.
Figure 2. Transesterification of isobutyl alcohol with MsAcT resulting in the
reversible formation of an emulsion. a) desired reaction and potential side
reaction; b) initial reaction mixture at t=0; c) reaction mixture at t=5 min; d)
reaction mixture at t=60min.
Having ruled out hemiacetate esters as side product and
established the transient formation of the desired ester, optimal
reaction conditions were established. The concentration of vinyl
acetate was varied at constant benzyl alcohol and MsAcT
concentration (Scheme 2 and Fig. 3c). At a 1:1 ratio of benzyl
alcohol:vinyl acetate the benzyl ester is quickly formed and
hydrolyzed, full conversion is never reached (Fig. 3a). However,
at 1:10 benzyl alcohol:vinyl acetate excellent conversions are
obtained with a prolonged stability of the synthesized ester (Fig.
3b). Indeed, by increasing the acyl donor concentration the yield
is increased and hydrolysis is outcompeted until all acyl donor is
consumed.
Figure 1. Transesterification of benzyl alcohol with the acyl donor vinyl acetate
(red), phenyl acetate (black), and ethyl acetate (blue) using different MsAcT
concentrations. Reaction conditions: KPi buffer (200 mM, pH 7.5), 10 mM
benzyl alcohol, 100 mM acyl donor, 5 – 500 ng MsAcT, 1 mL, 1000 rpm, 1
hour, 21°C. The experiment was performed in triplicates and the error bars
show the standard deviation.
To firmly establish all possible products and side products,
such as hemiacetal esters, in-situ 1H-NMR analysis was
performed. In a previous study, we have shown that MsAcT
catalyzes the transesterification of isobutanol in water.[3d] Based
on those results and since transesterification of benzyl alcohol
1
and vinyl acetate could not be followed via in-situ H-NMR due
to overlapping NMR signals, the acylation of 11 mM isobutanol
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Scheme 2: MsAcT catalyzed transesterification of benzyl alcohol with vinyl
acetate.
The transesterification of alcohols with activated acids
catalyzed by MsAcT follows overall a synthesis/hydrolysis
pattern as is well established for the amide bond synthesis.[9]
Initially the alcohol is the preferred substrate of MsAcT and the
desired ester, the kinetic product, is formed. In parallel both the
activated acid and the ester are also subject to hydrolysis. Once
all acyl donor is consumed the hydrolysis reaction leads to the
final reaction equilibrium and thermodynamics dictate the ester
yield (Scheme 2 and Fig. 3a).
After the synthesis of esters in water with different acyl
donors or enzyme concentrations the kinetic parameters of
MsAcT catalyzed transesterification in water was investigated.
To firmly establish the transesterification of benzyl alcohol with
vinyl acetate the ester formation was monitored using GC
analysis for the determination of the Michaelis-Menten constants
of two-substrate reactions[10]
.
The MsAcT-catalyzed
transesterification of benzyl alcohol with vinyl acetate gave for
benzyl alcohol a Km of 9.1 mM ± 2.8 mM and kcat of 3.16 x 103 s-
1
,
demonstrating high transesterification efficiencies. More
interestingly, the transesterification was revealing a KM and kcat
of 30.1 mM ± 6.3 mM and 2.49 x 103 s-1 respectively.
The formation of acetaldehyde via the hydrolysis of vinyl
acetate was monitored using a coupled spectrophotometric
activity assay, as is shown in table 1 (fig. S11-S13). The
hydrolysis of vinyl acetate with MsAcT showed high affinity to
the acyl donor vinyl acetate, with moderate catalytic efficiency.
The transesterification of benzyl alcohol with vinyl acetate was
repeated using the spectrophotometric assay to ensure
saturation conditions for two-substrate reactions of vinyl acetate
with initial rate conditions[11], were a >100-fold higher kcat was
observed for the transesterification over hydrolysis. The ratio
between the catalytic rate constants of the hydrolysis and
synthesis has been utilized to classify acyltransferase
efficiencies,[12] indicating that MsAcT classifies as an efficient
acyltransferase in water. The affinity for vinyl acetate is high
during the hydrolysis towards acetaldehyde, but during
transesterification in the presence of benzyl alcohol the apparent
affinity is significantly reduced indicating that competitive
inhibition
occurs.
Taken
together,
MsAcT
favors
transesterification over hydrolysis towards benzyl acetate under
kinetic control while hydrolyzing benzyl acetate to its alcohol
under thermodynamic control.
Figure 3. The synthesis of an ester in water. In a) the synthesis of an ester
under kinetic control followed by reverse hydrolysis into the thermodynamic
equilibrium; b) solely kinetic control is demonstrated by a 10-fold excess of
vinyl acetate; c) one can observe the decrease in reverse hydrolysis by 1, 2.5-,
5-, and 10-fold excess of acyl donor. Reaction conditions: KPi buffer (200 mM,
pH 7.5), 10 mM benzyl alcohol, 0 - 100 mM acyl donor, 50 ng MsAcT, 1 mL,
1000 rpm, 21°C. The error bars show the standard deviation of triplicates.
Table 1. Kinetic parameters of MsAcT catalyzed hydrolysis and
esterification in water
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Conclusions
Compound[a]
Ki (mM)
KM (mM)
kcat (s-1)†
182 ± 2.5
52.7 ± 3.5
2586 ± 2.4
Vinyl acetate[a]
Benzyl acetate[b]
Benzyl alcohol[c]
n.a.*
2.28 ± 0.42
n.a.*
0.0123 ± 0.0012
0.0308 ± 0.0087
13.6 ± 1.7
The synthesis of esters in high yields can be achieved in water
with activated acids using MsAcT. The transesterification in
water proceeds under kinetic control and careful monitoring of
the reaction is necessary to avoid reverse hydrolysis. The kinetic
parameters for MsAcT demonstrated a high synthesis to
hydrolysis ratio. If the reaction is performed at high
concentrations of benzyl alcohol, the product will form a
separate layer leading to in-situ product removal essential for
reducing enzyme inhibition under synthesis conditions. From a
reaction engineering perspective, since the side product
acetaldehyde is volatile, the laboratory scale reaction is
straightforward to perform. On a larger scale standard measures
to prevent acetaldehyde from escaping into the environment
need to be taken.[8] These are well-established for industrial
scale. Overall, organic solvents and wasteful neutralization steps
can be avoided.
†kcat is based on the molecular weight of MsAcT (23.3 kDa).
**n.a.: not applicable
[a] Assay conditions: Vinyl acetate (0 – 10 mM), NADH (0.3 mM), KPi (200 mM, pH 7.5),
MsAcT (24 ng mL-1), ScADH (50 U mL-1); [b] Assay conditions: Benzyl alcohol (0 – 100
mM), vinyl acetate (10 mM), NADH (0.3 mM), KPi (200 mM, pH 7.5), MsAcT (24 ng mL-
1), ScADH (50 U mL-1); [c] Assay conditions: Benzyl acetate (0.00 – 9.45 mM), NAD (5
mM), KPi (200 mM, pH 7.5), MsAcT (24 ng mL-1), HLADH-E (5 U mL-1).
More interestingly, using a coupled spectrophotometric
assay the hydrolysis of benzyl acetate was measured
demonstrating high affinity and uncompetitive inhibition for
MsAcT. When benzyl acetate was added to the hydrolysis of
vinyl acetate the apparent affinity towards vinyl acetate was
significantly lower representing competitive inhibition (Fig. 4).
Recently, a computational study suggested that benzyl acetate
would have high affinity towards the aromatic active site.[6b] In
this study, we demonstrated that the kinetics of the enzymatic
reaction are severely inhibited by the addition of benzyl acetate
indicating that ligand exchange severely lowers the reaction
rates.
Experimental Section
Materials and methods
Ampicillin (Sigma-Aldrich), benzyl acetate (Sigma-Aldrich), benzyl alcohol
(Acros Organics), bovine serum albumin (ThermoFischer), diethyl ether
(VWR), DNAse I (bovine pancreas, Sigma-Aldrich), dodecane (Janssen),
ethyl
acetate
(VWR),
imidazole
(VWR),
isopropyl
β-D-1-
thiogalactopyranoside (ThermoFischer), lysozyme (chicken egg white,
Sigma-Aldrich), magnesium sulfate (VWR), neopentyl glycol (Sigma-
Aldrich), phenyl acetate (sigma-aldrich), potassium acetate (Sigma-
Aldrich), potassium phosphate dibasic (Honeywell), potassium phosphate
monobasic (Merck), sodium sulfate (Merck), vinyl acetate (Sigma-
Aldrich). LB-medium consists of 1.0% (w/w) tryptone, 0.5% (w/w) yeast
extract, 1.0% NaCl, and autoclaved at 121°C for 20 minutes. All media
was supplemented with 100 μg mL-1 ampicillin before use. A multimode
spectrophotometer plate reader (Synergy 2, BioTek) was used for
spectrophotometric measurements. The pH electrode (Metrohm) was
calibrated before use with standards of pH 4.00 and pH 7.00 with R2
>
0.98. 1H and 13C NMR spectra were recorded on a Varian 400 (400 MHz)
spectrometer in CDCl3 (buffer for the NMR-monitored reaction). The
chemical shifts are given in ppm relative to the solvent signal (1H: δ
(CHCl3) = 7.26 ppm) and (13C: δ (CDCl3) = 77.16 ppm, for centerline of
CDCl3 triplet).
Expression and purification of MsAcT
A previous expression protocol was followed with slight modifications[3d]
.
A synthetic gene of MsAcT (GenBank accession: ABK70783) from
Mycobacterium smegmatis MC2 155 was cloned in pET16b-MsAcT
containing a C-terminal HisTag and expressed in E. coli BL21(DE3). A
baffled 5L shake flask containing 1 liter of TB-media was inoculated with
10 mL preculture and shaken until the OD600 reached 0.5 (25 °C, 180
rpm). Expression was induced with 0.1 mM isopropyl β-D-1-
thiogalactopyranoside (IPTG) and the culture was shaken overnight
(25 °C, 180 rpm). The cells were harvested with centrifugation (10 min,
10 000 rpm, 4 °C, Sorvall RC 6+) and the supernatant was discarded.
The cell pellet was washed with KPi buffer (20 mM, pH 7.5) and the
supernatant was discarded again. The cell pellet was suspended in KPi
buffer (20 mM, pH 7.5) containing 20 mM imidazole, lysozyme (0.5 mg
mL-1, chicken egg white, Sigma-Aldrich), DNase I (0.1 mg mL-1, bovine
Figure 4. Product inhibition of the synthesis of benzyl acetate shown in figure
a), while the competitive inhibition of the hydrolysis of vinyl acetate is shown in
figure b). Reaction conditions: (i) product inhibition: benzyl acetate (0.0 – 12.0
mM), vinyl acetate (5.4 mM), benzyl alcohol (80 mM), NADH (0.25 mM), KPi
(200 mM, pH 7.5), MsAcT (24 ng mL-1), ScADH (50 U mL-1); (ii) competitive
hydrolysis: Benzyl acetate (0.00 – 5.6 mM), vinyl acetate (5.4 mM), NADH
(0.25 mM), KPi (200 mM, pH 7.5), MsAcT (24 ng mL-1), ScADH (50 U mL-1).
The error bars show the standard deviation of triplicates.
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pancreas, Sigma-Aldrich) were incubated at 0 °C for 30 minutes. The
cells were lysed by cell disruption (Constant Cell disruption systems) for
three consecutive rounds (1.35 kbar). The cell-free extract was obtained
after centrifugation (10 min, 10 000 rpm, 4 °C, Sorvall RC 6+). The cell
free extract was filtered using a syringe filter (0.45 µm) before sample
application on Ni Sepharose 6Fast Flow resin (12 mL, GE Healthcare)
equipped in a XK 16/20 adapter (GE Healthcare) connected to a NGC
system (Bio-Rad). Before sample application the system and resin were
flushed with 20% ethanol, deionized water, and equilibration buffer
containing KPi (20 mM, pH 7.5) with 20 mM imidazole. The protein was
flushed through the column until all MsAcT protein was bound and eluted
with a gradient of elution buffer KPi (20 mM, pH 7.5) and 500 mM
imidazole. The eluted fractions containing MsAcT were desalted with a
PD-10 desalting column and flash frozen with liquid nitrogen prior to
storage at -80 °C.
Esterification of benzyl alcohol with potassium acetate
under monophasic reaction conditions
For the synthesis of benzyl acetate from benzyl alcohol and potassium
acetate the following reaction conditions were applied. MsAcT (12000 ng)
was added to KPi buffer (200 mM, pH 7.5) containing 10 mM benzyl
alcohol and 100 mM potassium acetate to yield a monophasic reaction
mixture of 1 mL. For the hydrolysis of benzyl acetate to benzyl alcohol
and potassium acetate the following reaction conditions were applied.
MsAcT (12000 ng) was added to KPi buffer (200 mM, pH 7.5) containing
10 mM benzyl acetate and 100 mM potassium acetate to yield a
monophasic reaction mixture of 1 mL. The reaction mixture was shaken
at 1000 rpm at 21 °C. Samples were taken after 1, 2, 3, 4, 5 and 24
hours. Samples were quenched by addition of 500 µL diethyl ether
containing dodecane (10 mM) as external standard. The mixture was
rapidly vortexed for 30 seconds and centrifuged at 13 000 rpm to remove
enzyme precipitates. The organic layer was collected, dried with an
excess of dry MgSO4, and analyzed by GC. The amount of benzyl
alcohol and benzyl acetate was quantified using an external calibration
curve, as is shown in figure S4.
SDS-PAGE analysis
The purity of MsAcT (24 kDa) was analyzed with SDS-PAGE analysis.
The protein samples were denatured using a XT Sample Buffer (Bio-
Rad) and XT Reducing Agent (Bio-Rad) at 95 °C for 5 to 10 minutes. A
Criterion XT Bis-Tris MOPS 4-12% precast gel (Bio-Rad) equipped with a
Precision Plus Protein Unstained Standard (Bio-Rad) and denatured
protein samples was run at 180 V in MES buffer (Bio-Rad) for 40
minutes. The gel was stained with SimplyBlue SafeStain
(ThermoFischer).
Reversible hydrolysis and synthesis of isobutyl acetate
by MsAcT
The reaction was initiated by the addition of MsAcT to a reaction solution
resulting in the final concentration of 11 mM isobutanol, 212 mM vinyl
acetate, KPi (200 mM, pH = 7.5), and 20 μg mL-1 MsAcT. The solution
was resuspended vigorously during addition for 1 - 3 seconds and
measured immediately with 1H-NMR (Agilent, 400 MHz). Also, the
reaction was repeated and recorded with a dual pixel 12MP OIS (F1.7)
camera to show the reversible synthesis and hydrolysis.
For the synthesis of isobutyl acetate from isobutanol and vinyl acetate
the following reaction conditions were applied. The reaction was initiated
by the addition of MsAcT to a reaction solution resulting in the final
concentration of 11 mM isobutanol, 212 mM vinyl acetate, 200 mM KPi
(200 mM, pH = 7.5), and 0.5 μg mL-1 MsAcT. The solution was
resuspended vigorously during addition for 1-3 seconds and measured
immediately with 1H-NMR (Agilent, 400 MHz).
BCA assay
Protein content was determined with the bicinchoninic acid (BCA) protein
quantitation kit (Thermo Scientific, Carlsbad, USA). Standard curves
were prepared with bovine serum albumin (BSA) in the range of 0.003 to
1.38 mg mL–1 in a (poly)styrene 96-well plate. Samples were measured
in triplicate and monitored at 562 nm utilizing
spectrophotometer (Synergy 2, BioTek).
a microtiter plate
MsAcT activity assay with neopentyl glycol (NPG)
Optimizing MsAcT concentration for the acylation of
benzyl alcohol with vinyl-, phenyl-, and ethyl acetate
The activity assay was started with the addition of 25 µL from a solution
of MsAcT (0.02 mg mL-1) dissolved in KPi buffer (200 mM, pH 8) to 975
µL ethyl acetate containing the external standard dodecane (10 mM) and
NPG (100 mM). The biphasic reaction mixtures were shaken for 30
seconds at 2500 rpm at room temperature. Reactions were quenched
after 0, 2, 4, 6 and 8 minutes by addition of excess sodium sulfate. 50 µL
of each reaction mixture was pipetted into a GC vial and 950 µL ethyl
acetate was added prior to GC analysis. The amount of NPG
monoacetate was quantified using external calibration curve, as is shown
in figure S5. Activity is reported as micromoles of NPG monoacetate
produced per minute per milligram of purified MsAcT.
MsAcT (50 - 12000 ng) was added to KPi buffer (200 mM, pH 7.5)
containing 10 mM benzyl alcohol, and 100 mM acyl donor being either
vinyl-, phenyl-, and ethyl acetate to yield a monophasic reaction mixture
of 1 mL. The mixture was shaken at 1000 rpm at 21 °C for 60 minutes.
Product and substrate were extracted twice by addition of 500 µL diethyl
ether containing dodecane (10 mM) as external standard. The mixture
was rapidly vortexed for 30 seconds and centrifuged at 13 000 rpm to
remove enzyme precipitates. The organic layer was collected, dried with
an excess of dry MgSO4, and analyzed by GC. The amount of benzyl
alcohol and benzyl acetate was quantified using external calibration
curve (fig. S4). Initial rates were calculated from the linear slope of benzyl
acetate concentration over time.
MsAcT activity assay with p-nitrophenylbutyrate (pNPB)
The activity assay was started by adding 20 µL of MsAcT to 180 µL
buffer containing KPi buffer (50 mM, pH 7.5) in transparent 96-well
polystyrene plates. The rate of hydrolysis was monitored at 37 °C at 405
nm using a multimode spectrophotometer plate reader (Synergy 2,
BioTek). Buffer and 96-well plate were pre-heated to 37 °C before the
start of the reaction. An external calibration curve of p-nitrophenol was
constructed between 0.00 – 1.00 mM.
Determination kinetic parameters for vinyl acetate and
benzyl alcohol using GC analysis
For the kinetic parameters for benzyl alcohol the following reaction
conditions were applied. MsAcT (50 ng) was added to KPi buffer (200
mM, pH 7.5) containing either 0, 1, 2.5, 5, 10, 15, and 20 mM benzyl
alcohol, and 100 mM vinyl acetate to yield a monophasic reaction mixture
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of 1 mL. For the kinetic parameters for vinyl acetate the following reaction
conditions were applied. MsAcT (50 ng) was added to KPi buffer (200
mM, pH 7.5), 10 mM benzyl alcohol, and 0, 10, 25, 50, 100, and 200 mM
vinyl acetate to yield a monophasic reaction mixture of 1 mL. The mixture
was shaken at 1000 rpm at 21 °C for 0, 2, 5, 10, 20, and 30 minutes.
Product and substrate were extracted twice by addition of 500 µL diethyl
ether containing dodecane (10 mM) as external standard. The mixture
was rapidly vortexed for 30 seconds and centrifuged at 13 000 rpm to
remove enzyme precipitates. The organic layer was collected, dried with
an excess of dry MgSO4, and analyzed by GC. The amount of benzyl
alcohol and benzyl acetate was quantified using external calibration
curves (figure S4) and the data is shown in figure S14. Initial rates were
calculated from the linear slope of benzyl acetate concentration over
time. The curves were fitted to the Michaelis-Menten equation using the
fit function of Gnuplot 5.2[13], as is shown in table S1.
The assay was performed in polyacrylate 1 cm cuvettes by monitoring
the conversion of 0.25 mM NADH at 340 nm and 20 °C by using an
extinction coefficient of 6.221 mM–1 cm–1. Before the addition of MsAcT,
the reaction was monitored until no chemical background hydrolysis
occurred. Enzymatic reactions were started by the addition of MsAcT
with a final concentration of benzyl acetate (0.00 – 5.6 mM), vinyl acetate
(5.4 mM), NADH (0.25 mM), KPi (200 mM, pH 7.5), MsAcT (24 ng mL-1),
ScADH (50 U mL-1). The error bars show the standard deviation of
triplicates. All measurements were performed in triplicates. The addition
of additional ScADH did not result in a higher response for indirect
acetaldehyde detection.
Coupled spectrophotometric activity assay with ScADH
of benzyl acetate synthesis with MsAcT in the presence
of varying amounts of product
Coupled spectrophotometric activity assay with ScADH
of vinyl acetate hydrolysis with MsAcT
The assay was performed in polyacrylate 1 cm cuvettes by monitoring
the conversion of 0.25 mM NADH at 340 nm and 20 °C by using an
extinction coefficient of 6.221 mM–1 cm–1. Before the addition of MsAcT,
the reaction was monitored until no chemical background hydrolysis
occurred. Enzymatic reactions were started by the addition of MsAcT
with a final concentration of benzyl acetate (0.0 – 12.0 mM), vinyl acetate
(5.4 mM), benzyl alcohol (80 mM), NADH (0.25 mM), KPi (200 mM, pH 7.5),
The assay was performed in polyacrylate 1 cm cuvettes by monitoring
the conversion of 0.25 mM NADH at 340 nm and 20 °C with an extinction
coefficient of 6.221 mM–1 cm–1. Before the addition of MsAcT, the
reaction was monitored until stable. Enzymatic reactions were started by
the addition of MsAcT with a final concentration of vinyl acetate (0 – 10
mM) in KPi (200 mM, pH 7.5), ScADH (50 U mL-1), MsAcT (24 ng uL-1).
All measurements were performed in triplicates. The addition of
additional ScADH did not result in a higher response for indirect
acetaldehyde detection. The Michaelis Menten curve is shown in figure
S11. The curves were fitted to the Michaelis-Menten equation using the
fit function of Gnuplot 5.2,[13] as is shown in table S1.
MsAcT (24 ng mL-1), ScADH (50
U
mL-1). All measurements were
performed in triplicates. The addition of additional ScADH did not result in
a higher response for indirect acetaldehyde detection.
Acknowledgements ((optional))
We thank Carolin Bisterfeld, Nicolas de Leeuw, and Laura van
der Weel for fruitful discussions and support.
Coupled spectrophotometric activity assay with ScADH
of benzyl acetate synthesis with MsAcT
The assay was performed in polyacrylate 1 cm cuvettes by monitoring
the conversion of 0.25 mM NADH at 340 nm and 20 °C by using an
extinction coefficient of 6.221 mM–1 cm–1. Before the addition of MsAcT,
the reaction was monitored until stable. Enzymatic reactions were started
by the addition of MsAcT with a final concentration of benzyl alcohol (1 –
100 mM), vinyl acetate (10 mM) in KPi (200 mM, pH 7.5), ScADH (50 U
mL-1), MsAcT (0.24 ng uL-1). All measurements were performed in
triplicates. The addition of additional ScADH did not result in a higher
response for indirect acetaldehyde detection. The Michaelis Menten
curve is shown in figure S12. The curves were fitted to the Michaelis-
Menten equation using the fit function of Gnuplot 5.2[13], as is shown in
table S1.
Keywords: acyl transferase • transesterification • water •
Myobacterium smegmatis • thermodynamic equilibrium • kinetic
control
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MsAcT catalyzes the synthesis of
primary alcohols in water. Starting
from ethyl acetate or vinyl acetate and
an alcohol of choice essentially
quantitative yields are achieved with
very little waste generation. This
opens up a path to clean and green
ester syntheses.
Luuk Mestrom, Jord G.R. Claessen, and
Ulf Hanefeld*
Page No. – Page No.
Enzyme Catalyzed Synthesis of
Esters in Water
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