Tuneable 3D printed bioreactors for transaminations under continuous-flow

Article abstract of DOI:10.1039/c7gc02421e

A method to efficiently immobilize enzymes on 3D printed continuous-flow devices is presented. Application of these chemically modified devices enables rapid screening of immobilization mechanisms and reaction conditions, simple transfer of optimised conditions into tailored printed microfluidic reactors and development of continuous-flow biocatalytic processes. The bioreactors showed good activity (8-20.5 μmol h^-1 mg_enz^-1) in the kinetic resolution of 1-methylbenzylamine, and very good stability (ca. 100 h under flow).

Full text of DOI:10.1039/c7gc02421e

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Tuneable 3D printed bioreactors for transaminations under
continuous-flow
Received 00th January 20xx,
Accepted 00th January 20xx
Edgar Peris,a Obinna Okafor,b,d Evelina Kulcinskaja,c Ruth Goodridge,b Santiago V. Luis,a Eduardo
Garcia-Verdugo,a Elaine O’Reilly,c Victor Sans,b,d
*
DOI: 10.1039/x0xx00000x
www.rsc.org/
A method to efficiently immobilize enzymes on 3D printed attention.14 The development of reliable methods for chemical
continuous-flow devices is presented. Application of these modification has enormous potential to reduce cost and
chemically modified devices enables rapid screening of deliver functional devices for rapid screening of immobilized
immobilization mechanisms and reaction conditions, simple enzymes. Furthermore, the freedom of design inherent to 3D
transfer of optimised conditions into tailored printed microfluidic printing allows parallelized fast screening platforms to be
reactors and development of continuous-flow biocatalytic combined with intensified continuous-flow reactors featuring
processes. The bioreactors showed good activity (8-20.5 ꢀmol h-1 advanced geometries and optimized mixing properties.
mgenz-1) in the kinetic resolution of 1-methylbenzylamine, and very Here, we report the first example of modified 3D printed
good stability (ca. 100 h under flow).
devices (Figure 1A) for the immobilization and application of
-transaminase (ω-TA) enzymes in continuous-flow. The
ω
The development of biocatalytic processes is emerging as a
new paradigm in the sustainable manufacturing of high value
chemicals for the pharmaceutical industry.1, 2 The application
of biocatalysts can provide alternative synthetic approaches to
those relying on more traditional chemistry, reducing the need
for toxic reagents, transition-metal catalysts and avoiding
costly purification steps and protecting group manipulations.3
The immobilization of enzymes onto solid supports has
facilitated the development of biocatalytic industrial
processes, by simplifying the recovery and reuse of the
enzymes and enabling their transition into continuous flow
reactors.4-6 However, the number of commercially available
systems is currently limited, mainly due to the absence robust
immobilization strategies.
sequential chemical functionalization of the printed devices
(Figure 1B) enables a fine control of the physico-chemical
properties of the reactor surface, thus enabling diverse types
of interaction with target enzymes and allowing rapid
screening of experimental conditions. The strategy combines
effective enzyme immobilization and reutilization with the
efficiency of continuous-flow and represents a significantly
more sustainable approach to the development of biocatalytic
processes compared to those in traditional batch reactors.5b
Additive manufacturing, commonly known as 3D printing, has
the potential to enable the rapid assembly of complex
reaction system geometries, which is often challenging using
traditional manufacturing techniques.7 The development of 3D
printed devices for chemical synthesis, coined “reactionware”,
has gained a great deal of attention in recent years,8 and
continuous-flow reactors with varying degrees of
sophistication have been described in the literature for a
variety of applications.9-13 However, the chemical post-
modification of printed devices has received much less
Figure 1. Schematic representation of the preparation of 3D printed bioreactors. A)
Printing process of Nylon employing an FDM printer, including a CAD file of a simple
fluidic device (left), the printing process (centre) and the device (right). B) Schematic
representation of the post-functionalisation of the printed reactor.
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increase in the intensity of the C=O band associated with this
functionality (Figures 2B-C, spectra P4).
The application of transaminases for the synthesis of optically
pure chiral amines from readily available ketones has received
considerable attention from both the academic and industrial
communities, due to the challenging nature of this
transformation.19-24 Additionally, there are only a handful of
reports describing their successful application in continuous
flow.25-28 We selected the commercially available (R)-ω-TA,
ATA117, to demonstrate our flow methodology. The enzyme
was supported following a literature procedure (see ESI) and
the immobilization confirmed by ATR-FTIR (Figure 2, polymer
P5). An assay was used to confirm that the enzyme had
retained activity (see ESI).
A key advantage of the use of 3D printing is the ability to
rapidly generate multiplexed reactors that can increase the
experimental throughput, thus speeding up the discovery and
optimization of new compounds or processes.29 This can be
invaluable for the rapid screening of immobilization
conditions, which usually relies on trial and error approach,
being time and resource intensive. Indeed, the use of
microplate based immobilization techniques has been
described to rapidly establish the best immobilization
techniques for esterases.30 Exploiting the same materials for
enzyme immobilization and the development of 3D printed
flow reactors has the potential to significantly speed up time
and reduce labour intensive assays due to the rapid translation
of immobilization conditions.
Figure 2. Chemical modification of 3D printed material for enzyme immobilization.
A) A 3D printed nylon part (P1) was treated with HCl 5M (P2) and then the
superficial amine groups were reacted with glutaraldehyde (P3) to generate
covalent link points for the enzyme. The reaction with polyethyleneimine (PEI)
(polymer P3’) and glutaraldehyde led to a polymer with increased linkage points
(P4). B) Detail of the aldehyde region in the ATR-IR spectra from polymers P1-P4. C)
Disappearance of the aldehyde band (blue region) of polymer P4 in contact with a
ω
-TA enzyme and amide-II bands observed at 1550 cm-1 (brown region).
An extrusion based 3D printing platform, Lulzbot Taz 5, with
commercially available nylon 6 filaments was employed to
fabricate bespoke devices. The surface was sequentially
modified to enable the adhesion of enzymes, allowing the
design of biocatalytic systems for screening in multiple well-
plate and continuous-flow biocatalytic reactors. Nylon is a
particularly convenient immobilization matrix for enzymes.15, 16
It is inexpensive, chemically inert, non-toxic, has good
mechanical properties and is commercially available in
multiple formats. Indeed, several studies report the
immobilization of various enzymes like proteases,
glucosidases, endocellulases and laccases onto different nylon
matrices.15, 17 Various types of nylon are widely employed for
additive manufacturing. Powder based nylon 11/12 is
commonly employed material of choice for selective laser
sintering (SLS) methods.18 These platforms are capable of
producing robust parts with highly intricate geometries and
fine layer resolution of 100 ꢀm. Therefore, there is an
untapped potential to develop 3D printed supported
Figure 3. Evaluation of the effect of immobilisation on the enzymatic activity of an ω-TA
ATA117. A) reaction scheme for the benchmark reaction. B) Experimental results
obtained for the different immobilisation conditions. C) Image and scheme of the
chemically modified 3D printed wells (left) and the experimental design (right),
bioreactors fabricated in nylon.
The initial goal was to functionalise 3D printed nylon. To this
end, small pieces of commercially available Nylon Taulman 645
(1x0.6x0.2 cm) were printed and chemically modified following
the scheme shown in Figure 2A. After brief treatment with HCl
(5M), a functional surface was generated with glutaraldehyde,
as evidenced by the presence of a characteristic aldehyde C=O
band at 1719 cm-1 in the ATR-IR spectra (Figure 2B, P3). This
modification was accompanied with a change of colour from
white to light orange. In order to increase the functionality of
the surface, polymer P3 was treated with polyethylenimine
(PEI, 60k Mn) to form the corresponding imine leading to
multiple free amines at the nylon surface (Polymer P3’). The
consisting of
a combination of blank reactors without chemical modification nor
enzyme (green box), control reactions in chemically modified wells without enzyme
(blue box) and chemically modified wells containing enzyme (purple). Reaction
conditions: Each well was filled with a 785 µL solution of methylbenzylamine (5mM)
((R)-MBA in wells 1 & 2 and (Rac)-MBA in well 3), pyruvate (5mM) and PLP (0.1mM) in
potassium phosphate buffer, pH=8.0. Reactions run at 30 oC for 17 hours.
A multi-well plate (dimensions: 1 cm i.d. × 1 cm height, Figure
3C) was manufactured in Nylon (Taulman 645). Four different
immobilization strategies were simultaneously screened (see
ESI, Figure S2 for the proposed immobilization mechanisms).
The first row was left unmodified and without enzyme to
function as a blank for the reaction. The wells of row 2 were
maintained unmodified (i.e. with polymer P1) to study the
further reaction with glutaraldehyde led to
a highly-
functionalised polymer P4, characterized by the significant
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possible non-covalent immobilization directly on the surface
by weak interactions (hydrogen bonds, hydrophobic effects,
etc.). Polymer P2 (Figure 3C, row 3) provides a surface capable
to immobilize the enzyme through ionic pairing and hydrogen
bonds. Non-covalent immobilization is sometimes preferred to
covalent immobilization as a minimal distortion of enzyme
structures is expected. The covalent immobilization of the
enzyme by polymer P3 in row 4 and multi-punctual covalent
immobilization row 5. After the modifications, the wells of
rows 2-5 and columns 1 and 2 were filled with a solution of a
(R)-ω-TA (ATA117, 1 mg/ml solution of the enzyme in 0.1M K
phosphate buffer pH=8.0, with 0.1 mM PLP). Column 3 was
employed as a control, containing only buffer and PLP. The
plate was left overnight at 4 oC. After this period, the wells
were emptied and washed with buffer. Wells in rows 4 and 5
were then additionally washed with a solution of NaCl (0.5 M)
for
2 hours to remove any non-covalently immobilized
enzyme.
The conversion of (R)-methyl-benzylamine into acetophenone
was selected as a model transamination reaction to evaluate
the performance of the ω-TA immobilized in the nylon wells
(Figure 3A). The wells were filled with 785 µL of a solution
containing the reagents required for the biotransformations
(see figure 3 legend for details). After 17 hours, the solution
was removed from the wells, extracted with ethyl acetate and
analysed by GC/MS. The results obtained are summarized in
the Figure 3B. The control experiments (row 1 and column 3)
showed only residual activity (< 2%) indicating no catalytic
activity from the different polymers without enzyme. Only
polymer P4 (row 4, columns 1 and 2) showed some level of
activity (ca. 25%). The ω-TA immobilized on the modified
surface with higher number of superficial aldehydes, which can
potentially bind to the enzyme in multiple points (row-5) did
not exhibited any level of activity. This was rather surprising,
and it might be due to the presence of multiple covalent bonds
between the enzyme and the support inducing changes in the
structure of the enzyme or due to the adsorption of the
substrates. In light of these results, polymer P4
(glutaraldehyde functionalized nylon) appeared to be the more
efficient way to immobilize the ω-TA tested. The diffusion of
substrate outside the wells was visually observed, indicating an
imperfect inter-bonding layer (see ESI TableS1).
ATA117 as the most suitable ω-TA enzyme for immobilization
by covalent attachment to 3D printing well-plate device
(polymer P4’).
Figure 4. A) 3D-printed Nylon wells used to perform the screening of the different
enzymes. B) Representation of the enzyme distribution in the wells. C) Average
conversion and standard deviation (error bars) observed for the three experiments of
each of the enzymes tested. Conditions: 5mM (R)-MBA (in the case of ATA117) or S-
MBA (in the case of ATA256 and 3HMU), 5mM pyruvic acid, 0.1mM PLP, r.t., 30 °C.
0.785 ml of a solution of the reagents in 0.1 M K phosphate buffer pH 8.0 was
introduced in each well.
Crucially, the results of the screening can be readily
transferred into continuous-flow micro-structured devices. The
bioreactor devices were built in Nylon Taulman 618, Figure 4A,
using the same platform described for the fabrication of the
test wells. An iterative approached described in the ESI was
employed to optimize the reactor manufacturing conditions.
The nozzle temperature, build chamber temperature and
model design were adjusted in other to produce robust leak-
free devices. A ‘track and hole’ support structure (ESI, Table
S1) was incorporated into the design of the reactor to break up
the large surface area of the part printed per layer, aiming to
minimize warping. This design also reduces temperature build
up at the mid regions of the part as allows air flow through
these areas. The reactor device was connected with PTFE
tubing using rheodyne metal connectors.
A new type of Nylon 6 (Taulman 618), which showed better
inter-layer adhesion, was employed to manufacture a new set
of well plates with the same dimensions and to evaluate the
effect on a small panel of ω-TAs. A plate containing 6 wells was
printed and modified to generate the equivalent of polymer
P4, called P4’ for simplicity. Three different ω-TAs, two of them
(S) selective (256 and 3HMU) and one (R) selective (ATA117)
were immobilized in triplicate under same conditions
previously employed. The benchmark reaction, the conversion
of (R)- or (S)-methyl-benzylamine into acetophenone, was
employed in order to compare the activity of the enzymes. The
results demonstrated satisfactory reproducibility for the
triplicate assays. The (S)-selective ω-TAs displayed a lower
activity than the (R)-selective enzyme (ATA117 > 256 > 3HMU)
(Figure 4). In summary, this simple screening highlighted
The reactor modification was performed under stop-flow
conditions for the HCl and glutaraldehyde solutions (see ESI for
details). The immobilization of the enzyme was achieved via
recirculation at 25 µL·min-1 a 15 mL of solution of ATA117 (5
mg/mL in 0.1 M of K phosphate buffer pH=8.0 and 0.1mM PLP)
at r.t. for 24h. and washed with buffer solution afterwards.
The analysis of the enzyme solution after the immobilization
process indicated that 170 µg of ω-TA were attached to the
surface of the reactor channel.
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Figure 5. Continuous-flow biocatalytic reactors for the kinetic resolution of rac-methylbenzylamine. A) CAD of the bioreactor and main reactor parameters. B) Kinetic resolution of
rac-methylbenzylamine employing ATA 117 supported on the 3D printed bioreactor. C) Experimental set-up employed for the continuous-flow biocatalytic transformation. D)
Productivity and enantiomeric excess observed as a function of time. The first two data points correspond to experiments assayed at 25 µL·min-1, while the rest were assayed at 10
µL·min-1. The plot represents two separate runs under continuous-flow, the first of them for 24 h, followed by a 48 h storage in fresh buffer and a subsequent run for further 78 h.
The results indicate the activity and stability of the enzyme, by keeping its activity over extended periods of time (ca. 100 h) under continuous-flow streams.
The results obtained for the kinetic resolution of rac- productivity of the system is greatly enhanced by the flow
methylbenzylamine are summarized in Figure 5D. At the initial conditions. Indeed, a total of 105 catalytic cycles were
flow rate assayed 25 µL·min-1 of a solution of 5mM rac- performed with the same enzyme. This work has
methylbenzylamine, 5mM pyruvic acid and 0.1mM PLP and demonstrated the first example of enzyme immobilization on
after 5 h of time on stream, a conversion of the 48% of the (R) modified 3D printed continuous flow biocatalytic reactors and
enantiomer on the amine into ketone was observed. Thus, (S)- paves the way for numerous applications in industrial
methylbenzylamine was obtained with an enantiomeric excess biotechnology. The possibility of rapidly testing different
of 93±2% e.e., as determined by HPLC. This corresponds to a immobilization mechanisms and conditions, that are easily
remarkably low residence time of 20 min and a productivity of transferred to continuous-flow bioreactors with tailored
20.5
ꢀ
experiment performed under similar conditions, thus applications. Significantly, the
mol h-1 mgenz
.
This is comparable to the batch geometries will allow for the development of numerous
-TA enzymes tested showed a
ω
indicating that the enzyme activity is retained. Then, the flow comparable activity to the free enzyme and were successfully
rate was reduced to 10 µL·min-1 in order to test the temporal recycled. Next steps will include demonstrating the approach
stability of the bioreactor and the reaction was carried out for on a diverse set of enzymes, employing different printing
an additional 20 hours. At this flow rate, corresponding to a techniques and assessing alternative immobilisation methods
residence time of 50 min, almost full conversion of the (R)- by modifying the surface linking agents.
enantiomer was observed (>49 %) yielding the (S)-amine with
an enantiomeric excess >99 % e.e. In order to evaluate the
Acknowledgements
stability of the biocatalytic system, the reactor was flushed
with buffer and stored at 4 oC for 48 hours. Afterwards, a
second cycle was performed under the same experimental
conditions (10 µL·min-1, 5mM rac-methylbenzylamine, 5mM
The authors acknowledge the Royal Society (RG150021) for
funding and the Research Priority Area (RPA) of UoN in Industrial
pyruvic acid, 0.1mM PLP) and the flow system was maintained
for a further 78 hours. During these period, the activity and
enantioselectivity of the bioreactor was maintained with e.e.
Biotechnology for funding. Research leading to these results has
also received funding from the BBSRC (BB/M021947/1). This
work was partially supported by G. V. (PROMETEO 2016-071)
>94%. These results demonstrate that the enzyme can be and MINECO (CTQ2015-68429R). E. Peris thanks MICINN for
personal financial support (FPU13/00685)
effectively immobilized on printed flow bioreactors, and the
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29 P. J. Kitson, R. J. Marshall, D. L. Long, R. S. Forgan and L.
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