Biocatalytic synthesis of the Green Note trans-2-hexenal in a continuous-flow microreactor

Article abstract of DOI:10.3762/bjoc.14.58

The biocatalytic preparation of trans-hex-2-enal from trans-hex-2-enol using a novel aryl alcohol oxidase from Pleurotus eryngii (PeAAOx) is reported. As O₂-dependent enzyme PeAAOx-dependent reactions are generally plagued by the poor solubility of O₂ in aqueous media and mass transfer limitations resulting in poor reaction rates. These limitations were efficiently overcome by conducting the reaction in a flow-reactor setup reaching unpreceded catalytic activities for the enzyme in terms of turnover frequency (up to 38 s^-1) and turnover numbers (more than 300000) pointing towards preparative usefulness of the proposed reaction scheme.

Full text of DOI:10.3762/bjoc.14.58

Biocatalytic synthesis of the Green Note trans-2-hexenal in a
continuous-flow microreactor
Morten M. C. H. van Schie1, Tiago Pedroso de Almeida1, Gabriele Laudadio2,
Florian Tieves1, Elena Fernández-Fueyo3, Timothy Noël*2, Isabel W. C. E. Arends1
and Frank Hollmann*1
Letter
Open Access
Address:
Beilstein J. Org. Chem. 2018, 14, 697–703.
1Department of Biotechnology, Delft University of Technology, Van
doi:10.3762/bjoc.14.58
der Maasweg 9, 2629 HZ Delft, The Netherlands, 2Department of
Chemical Engineering and Chemistry, Micro Flow Chemistry &
Process Technology, Eindhoven University of Technology, Den
Dolech 2, 5612 AZ Eindhoven, The Netherlands and 3Centro de
Investigaciones Biológicas, CSIC, Madrid, Spain
Received: 20 October 2017
Accepted: 15 March 2018
Published: 26 March 2018
This article is part of the Thematic Series "Integrated multistep flow
synthesis".
Email:
Timothy Noël* - t.noel@tue.nl; Frank Hollmann* -
f.hollmann@tudelft.nl
Guest Editor: V. Hessel
*
Corresponding author
© 2018 van Schie et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
alcohol oxidase; alcohol oxidation; aldehyde; flow chemistry
Abstract
The biocatalytic preparation of trans-hex-2-enal from trans-hex-2-enol using a novel aryl alcohol oxidase from Pleurotus eryngii
(
PeAAOx) is reported. As O2-dependent enzyme PeAAOx-dependent reactions are generally plagued by the poor solubility of O2
in aqueous media and mass transfer limitations resulting in poor reaction rates. These limitations were efficiently overcome by con-
ducting the reaction in a flow-reactor setup reaching unpreceded catalytic activities for the enzyme in terms of turnover frequency
(
up to 38 s−1) and turnover numbers (more than 300000) pointing towards preparative usefulness of the proposed reaction scheme.
Introduction
trans-2-Hexenal is well-known as a major component of the first sight an oxidation of primary alcohols to the correspond-
Green Notes of fruits and vegetables such as apples, straw- ing aldehydes does not appear to be a major challenge, the
berries, cherries and more. It is widely used in the flavour and methods of the state-of-the-art are mostly plagued by undesired
fragrance industry as fresh flavour ingredient in foods and side reactions [1]. Also some of the stoichiometric oxidants
beverages.
used are questionable from an environmental and/or toxicolog-
ical point of view and therefore are not compatible with con-
One attractive access to trans-2-hex-2-enal is the oxidation of sumer products such as Green Notes. Therefore, we turned our
the corresponding allylic alcohol to the aldehyde. Though at attention to biocatalytic oxidation methods. For clean conver-
697

Beilstein J. Org. Chem. 2018, 14, 697–703.
sion of primary alcohols to aldehydes principally two biocat- correlates with the interfacial area between aqueous medium
alytic approaches are available (Scheme 1) [2-5]. Alcohol dehy- and the gas phase. Large interfacial surface areas can be
drogenases catalyse the reversible oxidation of alcohols in a achieved via heterogeneous intake, by bubbling, stirring, etc.
Meerwein–Ponndorf–Verley-type of reaction (Scheme 1A). The Soluble enzymes, however, are often rather unstable under these
poor thermodynamic driving force of this reaction, however, conditions, possibly owing to the mechanical stress leading to
necessitates significant molar surpluses of the stoichiometric irreversible inactivation of the biocatalyst [7,8]. Methods of
oxidant (such as acetone). This not only negatively influences bubble-free aeration have been described in the literature to
the environmental impact of the reaction [6] but also compli- alleviate the inactivation issue described above [9-12].
cates downstream processing. Furthermore, the nicotinamide
cofactor (even if used in catalytic amounts only) causes addi- The continuous-flow microreactor technology has emerged as a
tional costs.
safe and scalable way to approach oxidation reactions [13,14].
Due to its small dimensions, hazardous reactions can be easily
controlled, owing to the large surface-to-volume ratio which
can minimise hot-spot formation and allows for control over
mixing and heating phenomena [15,16]. Furthermore, a well-
defined gas–liquid regime can be easily maintained [17,18].
High mass-transfer coefficients are generally the consequence
of small vortices induced by the segmented flow regime. This
flow pattern guarantees an enhanced contact between the two
phases and provides a uniform gas concentration in the liquid
segment.
Therefore, it is not very astonishing that also the biocatalysis
community is showing interest in flow chemistry. Several
biocatalytic processes have been reported in flow reactors [19],
mostly advocating easier process intensification in combination
with enzyme immobilization [20-23]. Also the higher oxygen-
transfer rates in flow reactions compared to batch reactions
have been emphasised by several groups. Here, reactor designs
ranging from simple flow reactors, tube-in-tube reactors [24],
agitated tube reactors [25,26] and continuous agitated cell reac-
tors [27] have been reported.
Scheme 1: Enzymatic reaction schemes for the selective oxidation of
trans-hex-2-enol. A: Alcohol dehydrogenase (ADH)-catalysed oxida-
tion producing stoichiometric amounts of NAD(P)H, which needs to be
recycled in situ; the overall reaction is reversible requiring surpluses of
the cosubstrate (e.g., acetone) to shift the overall equilibrium to the
side of trans-hex-2-enal. B: Envisioned aerobic oxidation using alcohol
oxidases (AOx). H2O2 is formed as byproduct and dismutated by cata-
lase into H2O and O2.
Encouraged by these contributions, we asked ourselves whether
a slug-flow approach may combine mechanically less
demanding conditions with high O2-transfer rates thereby
enabling efficient and robust oxidase-catalysed oxidation reac-
Therefore, we concentrated on alcohol oxidase-catalysed reac- tions.
tion schemes (Scheme 1B. Oxidases utilise O2 as terminal elec-
Results and Discussion
Selection and characterisation of the
tron acceptor for the oxidation reaction yielding H2O2 as sole
byproduct. The latter can be disproportionated easily by using
catalase (Scheme 1B). Furthermore, O2 reduction adds suffi- biocatalyst
cient thermodynamic driving force to the reaction to make it As biocatalyst for this study we focussed on the recombinant
essentially irreversible.
aryl alcohol oxidase from Pleurotus eryngii (PeAAOx) [28-31].
Especially the availability as recombinant enzyme (enabling
The benefits of using O2, however, also come with the disad- future at-scale production and protein engineering) and its
vantage of its very poor solubility in aqueous media (ca. promising activity on allylic alcohols make PeAAOx a promis-
0
.25 mM at room temperature). Hence, in the course of an oxi- ing starting point. Commercially available alcohol oxidases
dation reaction dissolved O2 is consumed rapidly and diffusion from Pichia pastoris and Candida boidinii showed no signifi-
of O2 into the reaction medium can easily become overall rate- cant activity for the substrate under the same conditions. As
limiting. The O2 diffusion rate into the reaction medium directly trans-2-hex-enol had not been reported as substrate for
698

Beilstein J. Org. Chem. 2018, 14, 697–703.
PeAAOx we evaluated its catalytic properties, particularly the reaction mixture in the flow reactor (and thereby the reaction
substrate concentration-dependency of the enzymatic oxidation. time, Figure 2).
Initial rate measurements (performed in 1 mL cuvettes) revealed
a Michaelis–Menten dependency of the enzyme activity
(
Figure 1). Apparent KM and kcat values of approximately 1 mM
and 22 s−1 were estimated, respectively. These values are in the
same order of magnitude as those for benzyl alcohol substrates
reported previously [29]. The slightly decreasing enzyme activi-
ty at elevated substrate concentrations may be an indication for
a slight substrate inhibition. Performing these initial rate mea-
surements in the presence of varying product concentrations
showed a pronounced product inhibition (Supporting Informa-
tion File 1, Figure S2, vide infra).
Figure 2: The influence of the residence time on the conversion of
trans-hex-2-enol (red squares) to trans-2-hexenal (black diamonds) in
a flow reactor. Conditions: 3 mL flow reactor, 50 mM KPi buffer (pH 7,
3
6
0 °C), [trans-hex-2-enol]0 = 10 mM, [PeAAOx] = 0.25 µM, [catalase] =
00 U mL−1.
A full conversion of the starting material into the desired trans-
hex-2-enal was observed at residence (reaction) times of
approximately 40 min corresponding to a turnover number (TN)
for the biocatalysts of 32400 and an average turnover frequen-
cy (TF) of 13.5 s−1. Even more interestingly, at higher flow
rates apparent TF of up to 38 s−1 (RT = 5 min) were observed.
This value exceeds the previously determined kcat(PeAAOx)
Figure 1: Michaelis–Menten kinetics of the PeAAOx-catalysed oxida-
tion of trans-hex-2-enol. Conditions: 50 mM KPi buffer (pH 7, 30 °C),
[
trans-hex-2-enol]0 = 3 mM, [PeAAOx] = 0.044 µM, [horseradish perox-
(
Figure 1) significantly. We attribute this observation to an in-
idase] = 500 U mL−1, [ABTS] = 2 mM.
creased oxygen-transfer rate at high flow rates. In the case of
the 5 minutes residence time this corresponds to an O2-transfer
rate of roughly 0.25 mM min−1. Similarly high values could be
Continuous-flow reactor enzymatic oxidation
Next, we performed the PeAAOx-catalysed oxidation of trans- obtained previously only under mechanically demanding reac-
hex-2-enol in a slug-flow reactor setup (Supporting Informa- tion conditions or using surfactant-stabilised emulsions [7].
tion File 1, Figure S1 and Figures S9–S11). In a first set of ex- Varying the ratio of gas to liquid had no significant effect on the
periments we systematically varied the residence time of the overall rate of the reaction (Table 1).
Table 1: Effect of variation of the gas-to-liquid ratio on the rate of the PeAAOx-catalysed aerobic oxidation of trans-hex-2-enol.
ratio
liquid:gas)
liquid flow
[mL min−1]
gas flow
[mL min−1]
residence time [min]
[product]
[mM]
(
1:1
1:3
1:5
0.20
0.10
0.20
0.30
15
15
16
5.48 (± 0.01)
5.18 (± 0.32)
4.99 (± 0.49)
0.067
0.333
Conditions: 3 mL flow reactor, 50 mM KPi buffer (pH 7, 30 °C), [trans-2-hexen-1-ol]0 = 10 mM, [PeAAOx] = 0.25 µM, [catalase] = 600 U mL−1.
699

Beilstein J. Org. Chem. 2018, 14, 697–703.
Within the experimental error, the conversion in all experi- Pleasingly, already in these first experiments a TN for the en-
ments was identical indicating that even at a comparably low zyme of more than 300000 was observed at long residence
volumetric ratio of 1:1 the O2 availability was already suffi- times. This also underlines the robustness of the enzyme under
cient not to be overall rate-limiting.
the flow conditions. Compared to Figure 2 somewhat lower TFs
for PeAAOx were observed here, which again can be attributed
It is worth mentioning here that under batch reaction conditions, to a lower O2-transfer rate at lower flow rates. The quasi-linear
similar progression curves were only attainable under mechani- relationship shown in Figure 3 also suggests that even higher
cally very demanding conditions (i.e., very vigorous stirring and TN may be attainable – however at the expense of longer reac-
bubbling of O2 directly into the reaction mixture, Supporting tion times. Therefore, further investigations will focus on identi-
Information File 1, Figure S3). These conditions also caused a fying conditions satisfying the demand for high TNs and short
significant evaporation of the substrate at higher substrate con- reaction times. Encouraged by these results, we also tried a
centration (Supporting Information File 1, Figure S4), which semi-preparative scale reaction using 5 g L−1 (50 mM) sub-
was much less the case in the flow-reaction setup.
strate loading in a total of 50 mL with 0.75 μM PeAAOx. As a
result, 90% conversion was achieved after 18 h of total reaction
From an economical point-of-view the catalyst performance in time (roughly 80 minutes of residence time in the 6 mL reactor).
terms of turnover number (TN) is of utmost importance as it The product was purified chromatographically resulting in 200
directly correlates with the cost-contribution of the catalyst to mg of pure trans-hex-2-enal (as determined by NMR) in 81%
the production costs [32-34]. Therefore we evaluated the TN isolated yield thereby demonstrating the preparative potential of
attainable for PeAAOx in the flow setup (Figure 3). For this the proposed reaction setup.
lower PeAAOx concentrations as well as significantly in-
creased residence times were applied. The increased residence Conclusion
times were achieved by decreasing the flow rates and using a Alcohol oxidase-catalysed oxidation of alcohols to aldehydes
longer flow reactor (6 mL volume instead of 3 mL).
bears a significant potential for preparative biocatalysis. The
reaction is independent from expensive and instable nicotin-
amide cofactors (and the corresponding cosubstrates/coprod-
ucts as well as possible regeneration enzymes) and produces
only water as byproduct. These advantages, however, are coun-
teracted by the generally low reaction rates caused by the poor
O2 availability. Flow chemistry is a promising technique to
provide the aqueous reaction mixture with O2 needed for the
oxidation. It enables high O2 transfer rates while avoiding en-
zyme robustness issues frequently observed with ‘traditional’
aeration methods.
Future developments in our laboratories will concentrate on the
characterisation, extension and preparative demonstration of
this powerful combination of oxidase catalysis and flow chem-
istry.
Experimental
General
Turnover numbers (TN) and turnover frequencies (TF) reported
in this manuscript were calculated based on Equation 1 and
Equation 2.
Figure 3: Increasing the PeAAOx turnover numbers (TN) by increas-
ing the residence time. Conditions: 6 mL flow reactor, 50 mM KPi
buffer (pH 7, 30 °C), [trans-hex-2-enol]0 = 40 mM, [PeAAOx] = 0.02
µM, [catalase] = 600 U mL−1. The TN value was calculated based on
the GC yield of every run. The TN was obtained by dividing the prod-
uct concentration (as determined chromatographically) by the biocata-
lyst concentration.
(
1)
2)
(
700

Beilstein J. Org. Chem. 2018, 14, 697–703.
Production of PeAAOx
PeAAOx were pooled, concentrated and desalted using HiTrap
E. coli cultivation
desalting columns (GE Healthcare) and 10 mM sodium phos-
For the production, activation and purification of PeAAOx, a phate buffer, pH 5.5. The PeAAOx concentration was calcu-
slightly modified literature protocol was used [28]. Pre-cultures lated based on the absorbance using the molar extinction coeffi-
of LB media containing 100 μg mL−1 of ampicillin were cient of ε463 11,050 M−1 cm−1.
inoculated with E. coli W3110 containing pFLAG1-AAO and
incubated overnight at 37 °C and 180 rpm. Overexpression Activity assay
was carried out in 5 L flasks with 1 L of TB medium supple- The activity of PeAAOx was determined by UV–vis spectros-
mented with 100 μg mL−1 of ampicillin. The medium was copy, using an Agilent Cary 60 UV–vis spectrophotometer,
inoculated with the pre-culture to an OD of 0.05 and grown following the oxidation of ABTS (ε405 = 36,800 M−1 cm−1) by
at 37 °C and 180 rpm. At an OD600 of 0.8, 1 mM isopropyl horseradish peroxidase (POD) at the expense of hydrogen
β-D-thiogalactopyranoside (IPTG) was added and the cultures peroxide. In general, 0.044 µM PeAAOx was used to convert
were incubated for additional 4 h at 37 °C and 180 rpm. The 3 mM of trans-2-hex-2-enol. The hydrogen peroxide formed in
bacterial pellets, obtained after harvesting the cells, were this reaction was subsequently used to convert 2 mM of ABTS
re-suspended in a total volume of 40 mL 50 mM Tris/HCl to ABTS·+ by an excess of POD (500 U mL−1). The reactions
buffer, pH 8.0, containing 10 mM EDTA and 5 mM dithio- were performed at 30 °C in oxygen-saturated 50 mM KPi buffer
threitol (DTT).
at pH 7.0.
Refolding
Flow reactor experiments
The re-suspended cells were disrupted by incubation with PFA microreactor coils (750 μm ID) with a volume of 3 and
2
mg mL−1 lysozyme for 1 h at 4 °C. Afterwards, 0.1 mg mL−1 6 mL were constructed. The reaction mixture was introduced
DNase, 1 mM MgCl2 and 0.1 mM PMSF were added followed via a syringe pump (Fusion 200, Chemyx), while the pure
by sonication. The insoluble fraction was collected by centrifu- oxygen flow was controlled by a mass flow controller (EL-
gation (30 min at 15,000 rpm and 4 °C), re-suspended and FLOW, Bronkhorst), resulting in a segmented flow (Supporting
washed three times with 20 mL 20 mM Tris/HCl buffer, pH 8.0, Information File 1, Figure S8). Residence times were taken as
containing 10 mM EDTA and 5 mM DTT using a potter the time between the solution entering and exiting the coil and
homogenizing device. The pellets obtained after centrifugation were varied by altering the flow, keeping the ratio of oxygen to
(
15 min at 15,000 rpm and 4 °C) were solubilized in a total liquid at three to one. Samples were collected on ice and as
volume of 30 mL 20 mM Tris/HCl buffer, pH 8.0, containing soon as enough volume was collected, extracted with ethyl
mM EDTA, 50 mM DTT and 8 M urea. After incubation on acetate and analysed by GC (vide infra).
ice for 30 min, the solution was cleared by centrifugation
15 min at 15,000 rpm and 4 °C). The obtained supernatant was GC analysis
2
(
used as stock solution for the in vitro refolding.
The collected reaction mixtures were extracted into an equal
volume of ethyl acetate, dried with magnesium sulphate and
The PeAAOx was solubilized using 150 µg mL−1 protein in analysed on a CP-wax 52 CB GC column (50 m × 0.53 m ×
2
1
4
0 mM Tris/HCl buffer, pH 9.0, containing 2.5 mM GSSG, 2 µm) (GC method: 60 °C for 3 min; 30 °C/min to 105 °C;
mM DTT, 0.02 mM FAD, 34% glycerol and 0.6 M urea at 105 °C for 7 min; 30 °C/min to 250 °C; 250 °C for 1 minute).
°C for 80 h. After the incubation for PeAAOx activation/ Dodecane (5 mM) was added as standard.
refolding, the refolding mixture was concentrated to 100 mL
and the buffer exchanged against 10 mM sodium phosphate Work-up semi-preparative scale
buffer, pH 5.5 by diafiltration (DV 20) and subsequently The reaction mixture was directly collected in deuterated
concentrated using an Amicon Ultra 15 mL centrifugal filter chloroform at the end of the flow reactor followed by recording
(
MWCO 10 kDa). After centrifugation (overnight at 15,000 rpm the NMR spectrum in order to evaluate the conversion (see Sup-
and 4 °C), the soluble fraction was further purified using anion- porting Information File 1). The organic mixture was diluted
exchange chromatography.
and introduced into a separation funnel and washed with brine.
The aqueous phase was backwashed once with DCM. The
collected organic phase was dried over MgSO4, filtered and
Purification
The concentrated PeAAOx solution was purified using a 58 mL concentrated under reduced pressure. Purification of the isolat-
Q Sepharose column (GE Healthcare). PeAAOx was eluted ed mixture was performed by flash chromatography on silica
with a linear NaCl gradient (0–0.6 M over 6 CV) using 10 mM (pure DCM). The final product was obtained as colourless oil
sodium phosphate buffer, pH 5.5. Fractions containing (200 mg).
701

Beilstein J. Org. Chem. 2018, 14, 697–703.
1
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Supporting Information File 1
General information and supporting figures.
[https://www.beilstein-journals.org/bjoc/content/
doi:10.1007/3418_2015_152
supplementary/1860-5397-14-58-S1.pdf]
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ORCID® iDs
Gabriele Laudadio - https://orcid.org/0000-0002-2749-8393
Timothy Noël - https://orcid.org/0000-0002-3107-6927
Frank Hollmann - https://orcid.org/0000-0003-4821-756X
2
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