Self-Assembling All-Enzyme Hydrogels for Flow Biocatalysis

Article abstract of DOI:10.1002/anie.201810331

Continuous flow biocatalysis is an emerging field of industrial biotechnology that uses enzymes immobilized in flow channels for the production of value-added chemicals. We describe the construction of self-assembling all-enzyme hydrogels that are compris

Full text of DOI:10.1002/anie.201810331

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Accepted Article
Title: Self-assembling all-enzyme hydrogels for flow biocatalysis
Authors: Theo Peschke, Patrick Bitterwolf, Sabrina Gallus, Yong Hu,
Claude Oelschlaeger, Norbert Willenbacher, Kersten S. Rabe,
and Christof M. Niemeyer
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201810331
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Self-assembling all-enzyme hydrogels for flow biocatalysis
Theo Peschke, Patrick Bitterwolf, Sabrina Gallus, Yong Hu, Claude Oelschlaeger, Norbert
Willenbacher, Kersten S. Rabe and Christof M. Niemeyer[*]
Abstract: Continuous flow biocatalysis is an emerging field of
industrial biotechnology that uses enzymes immobilized in flow
channels for the production of value-added chemicals. We describe
the construction of self-assembling all-enzyme hydrogels that are
comprised of two tetrameric enzymes. The stereoselective
dehydrogenase LbADH and the cofactor-regenerating glucose 1-
dehydrogenase GDH were genetically fused with a SpyTag or
SpyCatcher domain, respectively, to generate two complementary
homo-tetrameric building blocks that polymerize under physiological
conditions into porous hydrogels. Mounted in microfluidic reactors,
the gels show excellent stereoselectivity with near quantitative
conversion in the reduction of prochiral ketones along with high
robustness under process and storage conditions. The gels function
as compartment that retains intermediates thus enabling
extraordinary high total turnover numbers of the expensive cofactor
NADP(H).
enzyme hydrogels would provide an ideal solution for the
loading with maximum possible quantities of active biocatalyst.
Hydrogels are porous polymers that can be constructed from
natural or synthetic structural proteins.[9] A recently established
protein gelation strategy utilizes a pair of genetically encoded
reactive partners, SpyTag and SpyCatcher, that spontaneously
form
a
covalent isopeptide linkage under physiological
conditions.[10] While these protein hydrogels are being explored
for applications in biomedical sciences, such as cell
encapsulation and tissue engineering, strategies for their
exploitation in biocatalysis remain underdeveloped.
We here present a self-assembling all-enzyme hydrogel
that displays extraordinary high space-time yields in biocatalytic
flow processes. We choose two widely used homotetrameric
enzymes, the highly (R)-selective alcohol dehydrogenase (EC
1.1.1.2) from Lactobacillus brevis (LbADH) and the nicotinamide
adenine dinucleotide phosphate (NADPH)-regenerating glucose
1-dehydrogenase GDH (EC 1.1.1.47) from Bacillus subtilis. Both
enzymes were genetically fused with either the SpyTag (ST) or
the SpyCatcher (SC) in addition to a hexahistidin (His) tag
tethered to the same terminus of the protein (Figure 1a). Given
the molecular weights of LbADH, GDH, His-SC and ST-His (27,
28, 13 and 3kDa, respectively), this leads to a hydrogel whose
mass consists of 77% enzymes. The ST/SC system enables the
rapid cross-linking of the two tetravalent protein building blocks
through the formation of covalent isopeptide bonds under
physiological conditions.[11] The proteins were overexpressed in
E. coli and purified to homogeneity by Ni-NTA affinity
chromatography (Figure S1). Initial electrophoretic analysis of
enzyme gelation confirmed that polymerization only occurs when
both enzymes bear the complementary binding sites (Figure S2).
A more detailed investigation of the polymerization reaction by
dynamic light scattering (DLS) analysis (Figure 1b, c) revealed
that the time-dependent formation of protein clusters occurred
on time scales of minutes to hours, depending on the
concentration of the two enzyme building blocks (Figure 1b). In
homogeneous solution particles with average size of up to 65
nm were formed that further fused to a viscous liquid and even
free-standing hydrogel pieces upon further desiccation of the
solvent (Figure 1a, b). Variation of the stoichiometric ratio of the
two enzyme building blocks showed the fastest increase of the
hydrodynamic diameter at equimolar ratio (Figure 1c). Analysis
of the gel’s morphology by scanning electron microscopy (SEM)
and atomic force microscopy (AFM) revealed no clearly
distinctive ultrastructure, however, particle-like features were
evident in both SEM and AFM images (Figure 1a, S3).
Biocatalysis is a green and sustainable technology that is
widely considered as a key domain of industrial (‘white’)
biotechnology, which is expected to have an enormous impact
on the emergence of biobased economy.[1] Towards this goal,
bioinspired, multistep enzymatic cascade reactions are currently
attracting much attention.[2] Their exploitation for technical
processes requires compartmentalized flow systems to prevent
multiple reactions from spreading and unproductive crosstalk.
While this approach is well implemented in ‘continuous flow
chemistry’ that has yielded impressive synthesis campaigns for
small molecules,[3] the implementation of enzyme-based
production processes, i.e., continuous flow biocatalysis, is far
less developed.[2g, 4]
Biocatalytic flow processes are difficult to realize because
the heterogeneous catalysis regime calls for effective surface
immobilization techniques that are more demanding for enzymes
than for conventional organo(metallic) catalysts.[5] Common
methods for enzyme immobilization inside microstructured flow
channels, such as physisorption, chemical cross-linking, or
genetically encoded immobilization tags,[6] have proven their
applicability,[2g, 4c-g] however, there remains the problem that the
amount of immobilized biocatalyst is limited by the effective
surface area. To overcome this limitation, pseudo-3D interfacial
layers comprised of synthetic polymers or micro-/nanoparticles[7]
can be used to increase the loading capacity for enzymes.[8]
These approaches also waste the limited reactor space and also
often require additional coupling steps with potential drawbacks
for biocatalytic activity. Therefore, in situ generation of pure
To further elucidate the material properties, the enzyme
hydrogels were analyzed by optical microrheology based on
multiple particle tracking (MPT) analysis[12] (Figure S4). The
[*]
Dr. T. Peschke, M. Sc. P. Bitterwolf, M. Sc. S. Gallus, M. Sc. Y. Hu,
Dr. K. S. Rabe, Prof. Dr. C. M. Niemeyer
Karlsruhe Institute of Technology (KIT)
Institute for Biological Interfaces (IBG 1)
Hermann-von-Helmholtz-Platz 1
method revealed that the hydrogel has
a homogeneous
structure on the micrometer length scale with a G0= 20±7 Pa, an
average mesh size = 60±7 nm and a pore size <200 nm. Since
the pore size is in the range of typical microfiltration
membranes,[13] the gels should be well suited for flow reactions.
D-76344 Eggenstein-Leopoldshafen, Germany
E-mail: niemeyer@kit.edu
Dr. C. Oelschlaeger, Prof. Dr. N. Willenbacher
Karlsruhe Institute of Technology (KIT)
Institute for Mechanical Process Engineering and Mechanics
Gotthard-Franz-Straße 3, D-76131 Karlsruhe, Germany
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The kinetics of NDK reduction was then compared
between the GDH-ST/SC-LbADH hydrogel and a control of the
unassembled mixed proteins. To this end, hydrogels were
prepared into which NADP+ was included during polymerization
and solvent evaporation. After swelling of the hydrogel, reaction
buffer, containing NDK and glucose, was added and product
formation was monitored by chiral HPLC analysis. We found that
the hydrogel was rapidly forming the (R,R)-diol 3d, whereas the
unassembled enzymes produced almost exclusively the
hydroxyketones 2c/d (Figures 2b, S7). Moreover, the total
activity of the unassembled enzyme system was higher than that
of the hydrogel (Figure S7). We attribute both observations to
mass transport limitations due to restricted diffusion in between
the hydrogel and the surrounding medium. To confirm this
hypothesis, the hydrogel matrix was broken up by sonication
and, indeed, NDK conversion was almost equal to unassembled
enzymes (Fig 2c).
Figure 1. Design, formation and morphological characterization of the self-
assembled all-enzyme hydrogels. a) Schematic illustration of the two
homotetrameric enzyme building blocks, GDH-ST and SC-LbADH, that can
self-assemble to
a hydrogel via formation of covalent isopeptide bonds.
Photograph and representative SEM image of the hydrogel; scalebar 300 nm;
for additional morphological characterization see Figure S3. b) Time- and
concentration-dependent increase in hydrodynamic diameter (Z-average),
determined at 25°C by DLS. The control contained equimolar amounts of
GDH-ST and LbADH lacking the SC domain. c) Stoichiometry-dependent
increase in particle diameter observed in the initial 30 min after mixing of the
two enzyme building blocks (100 µM total subunit concentration, 25 °C).
Owing to its relevance for stereochemistry and natural
product synthesis,[14] we chose the prochiral CS-symmetrical 5-
nitrononane-2,8-dione (NDK) 1 (Figure 2a) as the substrate for
benchmarking the biocatalytic activity of the all-enzyme
hydrogels. Depending on the stereoselectivity of
a given
ketoreductase, either one or both carbonyl groups of NDK are
reduced to form diastereomeric hydroxyketones 2 or diols 3,
respectively (see also Figure S5), and all products can be
readily quantified by chiral HPLC analysis.[14a] We had previously
established that particle-immobilized LbADH converts NDK with
very high stereoselectivity into (R)-syn/anti-hydroxyketones 2c/d
(e.r.>99:1; d.r. ~60:40), which are further reduced to form the
(R,R)-configured pseudo C2-diol 3d.[4d] We now used the NDK
reaction to initially profile the SC-LbADH and GDH-ST building
Figure 2. NDK 1 reduction employing the GDH-ST/SC-LbADH hydrogel or
unassembled enzymes (control). (a) Reaction scheme of the (R)-selective
conversion of NDK 1 to (R)-syn/anti 2c/d hydroxyketone which is further
reduced to (R,R)-configured pseudo C2 diol 3d. (b) Distribution of all reaction
products after 2 hours. Note that the hydrogel rapidily reduces NDK 1 to diol
3d, whereas the unassembled control enzymes predominantly form the
hydroxyketones 2c/d. A corresponding time dependent kinetic analysis is
shown in Figure S8. (c) NDK conversion of an assembled GDH-ST/SC-LbADH
hydrogel (black) or the unassembled enzyme control (grey), respectively. Error
bars indicate the standard deviation, obtained in at least two independent
experiments.
blocks, which revealed
a slightly decreased (30%) and
increased (22%) specific activity, respectively, as compared to
the untagged enzymes (Figure S6,Table S1).
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Figure 3. Stereoselective continuous flow biocatalysis with GDH-ST/SC-LbADH hydrogel-loaded microfluidic reactors. a,b) Relative Activity of the hydrogel and
unassembled enzymes (control) in the presence of variable concentrations tetrahydrofuran (THF), a) and acetonitrile (ACN, b). c) Time dependent NDK-
conversion, determined from the outflow of the enzyme-loaded reactors perfused with continuous 5mM substrate and continuous cofactor supply (1 mM NADP+)
at a flowrate of 10 µl/min. d) Storage stability of gel-loaded reactor chips after storage for one month at 30°C. The bars show NDK-conversion obtained with a
fresh and stored reactor after continuous operation for one day. e) Flow rate dependent productivity (black bars) and corresponding space-time yields (STY, grey
line), obtained with continuous cofactor supply. f) Time dependent NDK conversion of a reactor loaded with a gel containing 1 mM entrapped NADP+, in the
absence of continuous cofactor supply. g) Total Turnover Numbers of NADPH (TTNNADPH) obtained with reactors bearing polymerized hydrogels (black) or
unassembled enzymes (grey). For detailed time dependent data, see Figure S11. h) Continuous flow conversion of NDK 1, acetophenone 4, 4′-
chloroacetophenone 6 and trans-4-phenyl-3-buten-2-one 8 to their corresponding (R)-configured alcohols 5a, 7a and 9a with a hydrogel-loaded micro reactor.
Note that conversion of 8 required addition of 1 % (v/v) ACN due to its low solubility in aqueous buffers. All error bars indicate the standard deviation, obtained
from at least two independent analyses.
To overcome the limited mass transport and to take
advantage of the high enzyme concentrations, we evaluated the
use of the gels in biocatalytic flow processes. Initial
assessments showed that the gels and the unassembled
enzymes have a similar stability against elevated temperature
(Figure S8) and pH-shifts (Figure S9). However, the gels reveal
a higher tolerance against commonly used organic solvents,
such as tetrahydrofuran (THF) and acetonitrile (ACN) (Figure
3a,b). We then set up a microfluidic reactor system, wherein a
PDMS chip with a flow channel of 150 µl volume was connected
to syringe pumps for substrate delivery and a fraction collector
for automated sampling of the outflow. The flow channel was
filled with swollen hydrogel and covered with a glass slide
(Figure S10).
stable conversion of the NDK-substrate for more than 6 days,
whereas unassembled enzyme mixtures were rapidly washed
out of the reactor (Figure 3c). Notably, hydrogel-loaded chips
stored for 30 days at 30°C showed a similar activity in flow
experiments than freshly prepared chips (Figure 3d). This
suggests that ready-to-use chips can be stored for prolonged
times and shipped at typical conditions of 4°C. NDK-conversion
and corresponding space-time yields (STY, Figure 3e) revealed
that the reactor could be operated at 200-fold higher flowrates
(200 µl/min; corresponding with a reactor residence time of 45 s)
and performed with a >68-fold higher time yield and a more than
4.5-fold higher STY than previously reported packed-bed micro
reactors that contained bead-immobilized GDH and LbADH.[4d]
The fact that the hydrogel rapidly forms the (R,R)-diol 3d
while the unassembled enzymes produced almost exclusively
the hydroxyketones 2c/d (Figure 2b, S8) suggested that
intermediate species cannot easily escape from the gels due to
the spatially and kinetically coupled GDH-ST and SC-LbADH
enzymes. We reasoned that this phenomenon could also be
To investigate the process stability of the GDH-ST/SC-
LbADH hydrogels under continuous flow conditions, the channel
was perfused with reaction buffer containing NADP+, glucose
and NDK at a flowrate of 10 µl/min. As expected, the hydrogel
effectively retained the immobilized enzymes and showed a
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harnessed to prevent escape of the redox cofactor species
NADP(H) from the gel under flow conditions. To investigate this
hypothesis, microreactors were loaded with hydrogels bearing 1
mM co-entrapped NADP+ and perfused with reaction buffer
containing only glucose and NDK. Indeed, the NDK was
continuously converted for more than 30 h, indicating the
effective retainment of the entrapped cofactor inside the
hydrogel over ≥124 reactor column volumes (Figure 3f). By
decreasing the NADP-concentration to 1 µM the total turnover
number of NADPH (TTNNADPH) was greater than 14000 (Figure
3g, S11). This is more than 13-fold higher than that of a recently
reported self-sufficient heterogeneous biocatalyst, based on
bead-bound ketoreductases with electrostatically co-immobilized
NADP(H)[15]. To the best of our knowledge, the TTNNADPH
observed here is the highest value ever reported for flow
processes in devices lacking supportive membranes, thereby
clearly meeting the demands determined for economically
feasible processes.[16]
monooxygenases[21], imine reductases[22] or transaminases.[23]
Conventional approaches for cofactor retainment in flow
systems use ultra- and nanofiltration membranes or specifically
modified surfaces that retain cofactors through electrostatic
attraction or even covalent immobilization. While these
approaches have led to increased TTNNADPH values, they can
increase the complexity of production processes and costs,
24]
thereby leading to limited economic viability.[15,
Our self-
assembly approach, in contrast, is straightforward, scalable[25]
and, owing to the gel’s intrinsic material properties, can be
readily implemented in arbitrary reactor geometries. We
therefore believe that this work paves the way for the
development of novel catalytic biomaterials for applications in
continuous flow biocatalysis.
Acknowledgements
As a further demonstration of the hydrogels’ utility for
applications in continuous flow biocatalysis, hydrogel-loaded
micro reactors were used for continuous production of chiral (R)-
configured alcohols (Figure 3h). To this end, the chip was
sequentially perfused with solutions of four different methyl
ketone substrates that were converted to their respective (R)-
alcohols (5a, 7a and 9a, in Figure 3h). Each substrate
administration was conducted for 10 h and reaction products in
the outflow were analyzed by chiral HPLC. We found that all
substrates were reduced to the corresponding (R)-configured
alcohols with near quantitative conversion and stereoselectivities
of >99%. These results clearly show that the novel biocatalytic
This work was supported by the Helmholtz programme
“BioInterfaces in Technology and Medicine” and DFG project
Ni399/15-1. We thank Ishtiaq Ahmed, Jens Bauer, Anke Dech,
Leonie Hacker, Silla Hansen, Arnold Leidner, Esther Mittmann,
Hatice Mutlu and Volker Zibat for experimental help.
Keywords: enzymes • flow biocatalysis • immobilization •
microreactors • stereoselective reactions
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COMMUNICATION
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chiral alcohol about 100 chips would have to be operated for six days.
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Hydrogels comprised entirely of
enzymes, self-assemble under
physiological conditions by covalent
SpyTag/SpyCatcher interaction to
porous hydrogels. Mounted in
microfluidic reactors, the gels exhibit
excellent stability, stereoselectivity
and cofactor retention without the
need for additional carriers or
membranes.
Theo Peschke, Patrick Bitterwolf,
Sabrina Gallus, Yong Hu, Claude
Oelschlaeger, Norbert Willenbacher,
Kersten S. Rabe and Christof M.
Niemeyer*
Page No. – Page No.
Self-assembling all-enzyme
hydrogels for flow biocatalysis
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