Rapid protein immobilization for thin film continuous flow biocatalysis

Article abstract of DOI:10.1039/c6cc04210d

A versatile enzyme immobilization strategy for thin film continuous flow processing is reported. Here, non-covalent and glutaraldehyde bioconjugation are used to immobilize enzymes on the surfaces of borosilicate reactors. This approach requires only ng o

Full text of DOI:10.1039/c6cc04210d

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Rapid protein immobilization for thin film continuous flow
biocatalysis
Joshua Britton [a,b], Colin L. Raston [b]* and Gregory A. Weiss [a]
*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A
versatile enzyme immobilization strategy for thin film improve sustainability metrics by avoiding hazardous solvents
continuous flow processing is reported. Here, non-covalent and and toxic metals. Combining the benefits of continuous flow
glutaraldehyde bioconjugation are used to immobilize enzymes on and biocatalysis offers numerous advantages such as
the surfaces of borosilicate reactors. This approach requires only processing with immobilized enzymes and rapid scale-up.
ng of protein per reactor tube, with the stock protein solution Continuous flow biocatalysis has thus increasingly become a
readily recycled to sequentially coat >10 reactors. Confining focus of many laboratories, as shown in a few examples.21-24
reagents to thin films during immobilization reduced the amount
Immobilizing enzymes can increase their industrial viability
of protein, piranha-cleaning solution, and other reagents by ~96%. by creating reusable biocatalysts with potentially improved
Through this technique, there was no loss of catalytic activity over reactivity, purity, specificity, selectivity, thermal stability and
10 h processing. The results reported here combines the benefits pH tolerance.25-30 Given this importance, many immobilization
of thin film flow processing with the mild conditions of strategies have been described, including attachment to
biocatalysis.
magnetic nanoparticles and nanomaterials,31,32 supports
through antibody-specific epitopes and crosslinking,33 and also
entrapment within
a
polymer network.34 Glutaraldehyde
Nature builds diverse and complex natural products through
assembly line biosynthesis. Polyketide synthases for example,
are multi-domain proteins that perform iterative processes to
crosslinking was chosen here due to its simplicity, commercial
availability, and success in previous immobilization studies.35,36
Recently, our laboratories have focused on utilizing thin
films to mediate protein folding,37 biocatalysis38 and molecular
assembly line processes.39 This involves processing in a vortex
fluidic device (VFD) which confines reagents to a ≈250 µM thin
film. Here, micromixing, shear stress and mechanical
vibrations40,41 can operate upon reagents to increase reaction
synthesize
a
large range of secondary metabolites.1,2
Continuous flow has emerged as an analogous, in vitro
process, for synthesizing compounds through multistep
processes.
In the laboratory, enzymes can perform a wide range of
transformations
including
reductions,3,4
oxidations,5,6
cyclization,7,8 aziridinations9 and nitration reactions.10
Improving the performance of these enzymes typically relies
on directed evolution11,12 and computational design.13,14 These
widely used techniques can improve reaction rates and
enzyme promiscuity to accept non-natural substrates.
Although such approaches increase the utility and adoption of
biocatalyzed transformations, scaling up enzyme-catalyzed
reactions can be challenging.
ꢀ
ꢀ
ꢀ
Sample
tube
ꢀ
ꢀ
+
NH3
ꢀ
ꢀ
b
c
Vortex Fluidic
Device
O
O
O
OH
OH
OH
Si
Si
Si
NH2
NH2
C3H6
C3H6
C3H6
O
O
OH
OH
a
e
O
O
OH
OH
NH2
O
O
OH
OH
Translating reactions into continuous flow can increase
safety,15,16
d
reaction
yields
and
aid
multistep
f
transformations,17,18 and decrease human effort and
waste.19,20 Furthermore, enzymes in synthetic pathways can
Fig. 1 Enzyme immobilization onto the surface of the VFD reactor. (a) First, the
surface of the sample tube is coated with APTES (3-aminopropyl triethoxysilane)
to generate a high concentration of surface-bound amines; a simplified depiction
of this surface coating is shown here. (b) β-glucosidase is added directly to the
APTES-coated sample tube for non-covalent immobilization. (c) After
derivatization of the APTES layer with glutaraldehyde, -glucosidase is attached
in this simplified structure of the linker and cross-link. (d) The imine-
glutaraldehyde is reduced with NaBH3CN. (e) The immobilization efficiency was
tested by VFD processing in the presence of the -glucosidase substrate, 4-
β
β
nitrophenyl β-D-glucopyranoside. (f) The sample tube can be regenerated
through rapid treatment with a thin film of piranha solution. Some of the
reactions in this manuscript were performed in continuous flow, further
information on the reactor setup has been previously reported.39
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150
150
100
80
60
40
20
0
a)
b)
100
Rate/ rate loss
Rate: rate loss
Rate
Rate (ꢁM/min)
(ꢁM minꢀ1
)
120 ꢀ 135
120.00ꢀ135.00
80
60
40
20
0
60 ꢀ 70
60.00ꢀ70.00
105 ꢀ 120
105.00ꢀ120.00
50 ꢀ 60
50.00ꢀ60.00
90 ꢀ 105
90.00ꢀ105.00
40 ꢀ 50
40.00ꢀ50.00
75 ꢀ 90
75.00ꢀ90.00
30 ꢀ 40
30.00ꢀ40.00
60 ꢀ 75
60.00ꢀ75.00
20 ꢀ 30
20.00ꢀ30.00
45. 0ꢀ .00
0 60
45 ꢀ 60
10 ꢀ 20
10.00ꢀ20.00
0 ꢀ 10
30 ꢀ 45
30.00ꢀ45.00
0.00ꢀ10.00
15 ꢀ 30
15.00ꢀ30.00
0 ꢀ 15
0.00ꢀ15.00
0
0.1
0.2
0.3
0.5
1
2
0
0.1
0.2
0.3
0.5
1
2
Enzyme concentration (mg mLꢀ1
)
Enzyme concentration (mg mLꢀ1
)
d)
c)80
OH
OH
OH
O
60
40
20
0
NO2
OH OH
OH
O
OH
OH
O
OH OH
NO2
βꢀGlucosidase
λmax – 405 nm
1
2
3
4
5
6
7
8
9
10
pH of attachment buffer
Fig. 2 Non-covalent immobilization using β-glucosidase and 4-nitrophenyl β-D-glucopyranoside for optimization. (a) The enzyme and buffer salt concentration were
varied during the immobilization step. The contour plot reveals that a 0.3 mg mL-1 enzyme concentration in a 60 mM NaCl PBS buffer is optimal for high substrate
conversion. (b) Decreased catalytic activity due to enzyme leaching is depicted in this contour plot. Thus, higher salt concentrations are revealed as beneficial for
immobilization longevity. (c) Varying the pH of the attachment buffer established optimal immobilization in PBS at pH 8.0. The deviation from the trend at pH 5.0 is
due to the isoelectric point of
used.
each reactor was assayed six times. Two separate reactors were used per data point, and the error is a standard deviation around the mean (n=12).
β-glucosidase. (d) For all optimization experiments, a β-glucosidase - 4-nitrophenyl β-D-glucopyranoside (10 mM, 1.50 mL) system was
β
-glucosidase hydrolyses the substrate, releasing p-nitrophenol (λmax 405 nm) and β-D-glucopyranoside. Each assay was performed in the VFD for five min, and
yields and efficiencies. Processing in a single VFD with a 20 mm cleaning efficiency, whilst reducing the volume of this highly
external diameter reactor can achieve flow rates up to 20 mL hazardous fluid by 94%. After washing and drying, the reactor
min-1. Larger scale processing is possible by applying multiple surface is then derivatized with a dilute APTES solution (79.5
VFDs. In pursuing new multistep transformations, we have mM, 60 µL in 3 mL MeOH). Again, confining reagents to a thin
recently embarked on exploring thin film continuous flow film reduced the quantities of MeOH and APTES required by
biocatalysis. In future experiments, using enzymes alone, or in 94%. Lastly, the APTES-modified surface is heated to 160 °C to
conjunction with organic reagents will require immobilization drive the condensation reaction to completion (Fig. 1a).
of minute quantities of protein for efficient continuous flow
reactors.
Non-covalent immobilization is sometimes preferred to
covalent immobilization as introducing random covalent bonds
Unlike other continuous flow systems, the VFD reactor is can distort enzymes’ structures.28 For testing a large number
made from borosilicate glass. This material can simplify of non-covalent immobilization variables,
a colorimetric
bioconjugation, as explored systematically here. APTES (3- enzyme-substrate assay was used, β-glucosidase and 4-
aminopropyl triethoxysilane) was coupled to the reactor nitrophenyl β-D-glucopyranoside, respectively. This assay
surface to create a layer of nucleophilic amines (Fig. 1a). This offers high throughput conditions (5 min per reaction), an
APTES modified reactor was then used for rapid covalent and effective quench solution, and stability to vortexing conditions
non-covalent immobilization. Non-covalent immobilization can (Fig. 2d).38
be achieved though surface-exposed functionalities on the β-Glucosidase and buffer salt concentrations play an
protein interacting with the APTES layer through salt bridges integral role in non-covalent immobilization efficiency and
and hydrogen bonds (Fig. 1b). In contrast, covalent activity. Varying both of these variables simultaneously
immobilizations used surface-exposed lysine sidechains (also generated
a
contour plot (Fig. 2a).
A
β-glucosidase
thiols, phenols and imidazoles36) on the protein to form imine concentration of 0.3 mg mL-1 was optimal, with variation either
and amine bonds with a glutaraldehyde-modified APTES linker side of this concentration decreasing immobilization efficiency.
(Fig. 1c and d). The structure of the glutaraldehyde linker and Furthermore, confining the protein solution to a thin film for
resultant cross-link has been simplified in Fig. 1; in aqueous immobilization reduced the volume of protein solution used
solution, for example, many different forms of glutaraldehyde from 50 to 3 mL, ie. a reduction of 94% (15 mg to 0.9 mg).
can exist.35,36
Additionally, 60 mM NaCl in PBS was found to be optimal, but
Coating the reactor with APTES required optimizing a taking into account rate loss over time revealed that 150 mM
three-step process. First, treatment with piranha solution NaCl in PBS is superior, with no decrease in substrate
exposes high concentrations of silinols on the reactor surface. transformation rate over 30 min (Fig. 2b). The higher salt
Although the reactor can be filled with piranha solution (50 concentration during adsorption could increase the strength of
mL), confining 3 mL to a thin film for one min offers the same the enzyme-APTES interaction.42
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a)
b) 1.25
200
1
150
100
50
0.75
0.5
0.25
0
Nonꢀcovalent
Covalent (imine)
Covalent (amine)
Alkaline phosphatase
βꢀglucosidase
Phosphodiesterase
0
0
0
2
4
6
8
10
2
4
6
8
10
Processing time (h)
Processing time (h)
c)
d)
1
0.75
0.5
300
PBS alone
No buffer
PBS and protease inhibitor
200
100
0.25
0
0
1
2
3
4
5
6
7
8
9
10 11 12
0
1
2
3
4
Storage time (weeks)
Sample tube number
Fig. 3 The conditions optimized here are general for a range of proteins. The protein solutions used in the immobilization step can be recycled to coat more than
ten sample tubes, with the coated sample tubes still maintaining catalytic activity for weeks. (a) Switching from non-covalent to covalent attachments increased
substrate conversion levels dramatically. (b) Applying the symmetrical amine-glutaraldehyde cross linker optimized for -glucosidase to alkaline phosphatase and
-glucosidase solution (3 mL,
β
phosphodiesterase establishes the generality of the method, with all three proteins having good stability over 10 h of processing. (c) The
β
0.3 mg mL-1) used in the immobilization step can be recycled to coat more than ten sample tubes, with the first sample tube having the same substrate
transformation rate as the last. We anticipate that this solution could coat tens of sample tubes given the small amount of protein used in each immobilization. (d)
Storing the enzyme-immobilized tubes devoid of buffer allowed >20% catalytic activity after one month. The rates displayed above are the average rates as described
in Fig. 2 (n=12). The data in (a) and (b) were from continuous flow experiments with a flow rate of 1.0 mL min-1.
Next we next examined the conditions required for this immobilization strategy (0.9 mg), we were able to explore
covalent immobilization. Covalent immobilization can increase phosphodiesterase,
a poorly overexpressing recombinant
enzymes’ stability greatly through the addition of short spacers protein. Immobilizing phosphodiesterase and a commercially
off the reactor surface.27 Reacting glutaraldehyde with the available alkaline phosphatase via amine-glutaraldehyde
APTES-coated reactor, followed by the sequential addition of immobilization (Fig. 1c) resulted in stable levels of substrate
β
-glucosidase solution afforded an imine linker for conversion for 10 h in continuous flow (1 mL min-1, Fig. 3b).
immobilization (Fig. 1c). Furthermore, this imine can be The final criterion for this immobilization method was to
reduced to the amine with NaBH3CN solution (Fig. 1d).43,44 increase immobilization efficiency. This process already uses a
Notably, lysine residues in the active site are typically low quantity of protein, but, to address efficiency further, it
uninvolved in catalysis, and this immobilization strategy is would be useful to know how much of protein is on the
therefore unlikely to perturb enzyme function.36 Once again, surface of the reactor. Two complimentary experiments
these steps were performed in the thin film, resulting in a 96% revealed that 15.4 to 69.8 ng of β-glucosidase are present on
reduction in quantity of buffer and reagents required. the surface of the reactor after covalent immobilization (Fig.
Switching to covalent immobilization increased the yields S1-S2). This surprising result opened up the possibility to
of conjugated enzyme with a concomitant increase in the rates recycle the protein stock solution (0.3 mg mL-1). Indeed,
of substrate conversion. Although a slight increase in enzyme recycling the stock solution of
β-glucosidase allowed the
immobilization efficiency and reaction rate results from coating of 12 reactor tubes with no observable decrease in
switching to covalent immobilization, the reduction of imine to substrate conversion between the first tube and the last (Fig.
amine provides a dramatic improvement. This reduction 3c). We were unable to identify why recycling the enzyme
prevents hydrolysis of the imine, thus increasing the solution increased substrate conversion levels for sample
concentration of protein on the surface of the reactor tube tubes 2-8. This trend subsides with additional sample tubes,
(Fig. 3a). To test the stability of these immobilization and we believe that it is an experimental artifact.
strategies, each immobilized enzyme was subjected to a
Lastly, sample tube storage was investigated, which was
continuous flow reaction at 1.0 mL min-1; all immobilizations deemed important given that sample tubes are often
demonstrated excellent stability, with no loss of activity after transported to other laboratories. Surprisingly, a dry sample
10 h of processing recorded with the amine linker (Fig. 3a).
tube bearing surface bound β-glucosidase provided reasonable
Our second requirement for this immobilization strategy substrate conversion after one month of storage (4°C storage,
was to make it general. Given that proteins have a hydrophilic 19 µM min-1 conversion, Fig. 3d). Presumably the decrease in
surface, most enzymes have a surface-exposed lysine residue substrate conversion results from a combination of protein
for immobilization. As small quantities of protein are used in leaching and unfolding.
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In conclusion, a rapid and general technique for protein
immobilization onto a thin film continuous flow reactor has
been developed. Importantly, using thin films for reagent
confinement reduced the volume of protein solution, piranha
solution, APTES, MeOH, glutaraldehyde, NaBH3CN and a range
of buffers by an average of 95%. The ability to use a small
amount of protein (900 µg) to coat >10 sample tubes provides
a general strategy to increase the efficiency of enzyme-
mediated transformations in continuous flow. Incorporating
biocatalysts into multistep processes offers the potential to
create complex molecules using nature’s machinery. The
findings reported here will facilitate biocatalysts by allowing
low expressing proteins to be used in complex substrate
transformations such as natural products and pharmaceutical
ingredients.
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Acknowledgments
JB was supported in part by a Taihi Hong Memorial award. GW 26. U. Guzik, K. Hupert-Kocurek and D. Wojcieszyńska,
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gratefully acknowledges the National Institute of General
Medical Sciences of the NIH (1RO1-GM100700-01). CR
acknowledges the Australian Research Council and the
Government of South Australia for their financial support
during this project.
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