Accelerating Enzymatic Catalysis Using Vortex Fluidics

Article abstract of DOI:10.1002/anie.201604014

Enzymes catalyze chemical transformations with outstanding stereo- and regio-specificities, but many enzymes are limited by their long reaction times. A general method to accelerate enzymes using pressure waves contained within thin films is described. Ea

Full text of DOI:10.1002/anie.201604014

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International Edition: DOI: 10.1002/anie.201604014
German Edition: DOI: 10.1002/ange.201604014
Biocatalysis
Accelerating Enzymatic Catalysis Using Vortex Fluidics
Joshua Britton, Luz M. Meneghini, Colin L. Raston,* and Gregory A. Weiss*
Abstract: Enzymes catalyze chemical transformations with
outstanding stereo- and regio-specificities, but many enzymes
are limited by their long reaction times. A general method to
accelerate enzymes using pressure waves contained within thin
films is described. Each enzyme responds best to specific
frequencies of pressure waves, and an acceleration landscape
for each protein is reported. A vortex fluidic device introduces
pressure waves that drive increased rate constants (kcat) and
enzymatic efficiency (kcat/Km). Four enzymes displayed an
average seven-fold acceleration, with deoxyribose-5-phosphate
aldolase (DERA) achieving an average 15-fold enhancement
using this approach. In solving a common problem in enzyme
catalysis, a powerful, generalizable tool for enzyme acceler-
ation has been uncovered. This research provides new insights
into previously uncontrolled factors affecting enzyme function.
E
nzymes make life possible by catalyzing diverse and
challenging chemical transformations with exquisite specific-
ity. Applications in both industry[1] and academia[2] rely on the
selectivity and power of enzymes to catalyze otherwise
challenging transformations. Biocatalysts offer remarkable
rate accelerations compared to the uncatalyzed reactions,
with typical rate accelerations (kcat/kuncat) of 105- to 1015-fold
faster.[3] Though some enzymes are diffusion-limited,[4] the
catalytic rates of enzymes are more typically limited by their
catalytic efficiency (kcat/Km); additionally, molecular crowd-
ing, along with product and substrate inhibition, can reduce
enzyme efficiency.[5] Though some enzymes catalyse trans-
formations with rapid rates (for example, laccases, fumarases,
and alcohol dehydrogenases),[6] other enzymes operate at
only modest reaction rates, requiring long reaction times and
carefully optimized conditions; for example, DERA requires
long processing times (hours to days), and is substrate-
inhibited.[7] We report a process that accelerates four different
enzymes at standard temperature and pressure, but many
other water-soluble enzymes could be accelerated as well.
Recently, vortex fluidic devices (VFDs) have been used to
accelerate covalent and noncovalent bond formation. VFDs
Figure 1. The VFD and its Faraday waves. A) A representation of the
VFD. B) A VFD sample tube, containing 5 mL of solution rotating at
3500 rpm at a tilt angle q of 458, shows Faraday waves formed within
the thin film. Such pressure waves, shown as an overlaid representa-
tion, can affect the enzyme–substrate complex. C) Enlargement of the
VFD-generated Faraday waves. Additional images of the Faraday waves
can be found in the Supporting Information, Figure S14.
process solutions in thin films by the rapid rotation of
a sample tube (Figure 1).[8] Within the thin film, species are
subjected to high levels of shear stress, mass transfer, and
vibrational energy input at specific rotational speeds. For
example, the VFD demonstrated the effective folding of four
different proteins within minutes at standard temperature and
pressure.[9] The VFD has also been used to improve the
synthesis of lidocaine[10] and several other organic trans-
formations.[11] In a continuous flow regime, flow rates of up to
20 mLminÀ1 can be achieved to process up to 30 L per day in
the current benchtop configuration. Since VFD processing
increased the rates of organic reactions and protein folding,
we hypothesized that biocatalysis, which requires both
reactivity and the correct protein fold, could benefit as well.
Control reactions with alkaline phosphatase demon-
strated the requirements for high, specific rotational speeds
of the VFD to generate a thin film containing the enzyme for
accelerated catalysis (Supporting Information, Figures S2–4).
VFD-mediated acceleration of four biocatalysts was com-
[*] J. Britton, Prof. Dr. C. L. Raston
Chemical and Physical Sciences, Flinders University
Bedford Park, Adelaide, 5001 (Australia)
E-mail: Colin.Raston@flinders.edu.au
J. Britton, Prof. Dr. G. A. Weiss
Department of Chemistry, University of California, Irvine
Irvine, CA 92697-2025 (USA)
E-mail: Gweiss@uci.edu
L. M. Meneghini, Prof. Dr. G. A. Weiss
Department of Molecular Biology and Biochemistry
University of California, Irvine
Irvine, CA 92697-2025 (USA)
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201604014.
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Figure 2. Parameters for accelerated biocatalysis of the four enzymes. Fold acceleration was determined as the ratio of the VFD-mediated
substrate conversion to that of an identical enzyme–substrate solution not treated with the VFD. A) A time dependent study at a fixed rotational
speed (8000 rpm) reveals processing times for further optimization. As indicated with a ?, DERA required longer reaction times of 60, 80, 100,
120, 140, 160, and 180 min. B) Simultaneous changes to the substrate and enzyme concentrations at a 8000 rpm rotational speed mapped the
reaction landscape (Supporting Information, Table S2A–D). C) Rotational speed scans in 50 rpm increments identify harmonic oscillations
associated with Faraday wave-promoted biocatalysis. Error bars are larger for DERA than for any other enzyme due to the non-linear fluorescence
calibration curve of the product. D) Varying the tilt angle of the sample tube when processing alkaline phosphatase and its substrate identified
458 as the optimal angle. Thus, a 458 tilt angle was used herein. E) The addition of PEG dramatically affects alkaline phosphatase catalysis in the
non-VFD control. However, the VFD-processed solution demonstrated significant catalytic activity. Error bars indicate the standard deviation
around the mean (n=3 with three independent measurements on three different VFDs). With the exception of a single data point requiring 90%
confidence limits, all data reported have no overlapping errors within 95% confidence limits. The concentrations of the enzymes and substrates
are as follows: fast alkaline phosphatase (6.77 nm) and its substrate p-nitrophenol phosphate (0.17 mm), b-glucosidase (19.3 nm) and its
substrate 4-nitrophenyl b-d-glucopyranoside (7.5 mm), esterase (0.12 nm) and its substrate p-nitrophenol acetate (44 mm), and DERA (7.69 mm)
and its fluorogenic substrate (0.52 mm) unless otherwise indicated, and as described in the Supporting Information, Table S2A–D.
pared to identical unprocessed enzyme-substrate solutions for
efficient reaction optimization (Figure 2). The VFD process-
ing times were varied to identify short time periods (2 min to
3 h) suitable for further optimization (Figure 2A). Esterase
produced a lower VFD-based enhancement (two-fold) com-
pared to the other three enzymes; esterase also needed longer
reactions times due to the enzymeꢀs requirements for low
substrate concentrations.[12] In general, after long time
periods, the substrate is expended and the unprocessed
solution can reach similar levels of substrate conversion.
Furthermore, the VFD-processed solutions have parallel
activities to their non-VFD counterparts for the first few
minutes before the rapid acceleration of the former (for
example, alkaline phosphatase in the Supporting Information,
2
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Figure S9). A similar lag period before rate acceleration has
trations of substrate and steric crowding suggests that the
been described previously for ultrasound-accelerated enzyme
VFD could be applied to processes requiring complex
mixtures and minimal amounts of solvent.
acceleration.[13]
The substrate and enzyme concentrations were simulta-
neously varied for the rapid scanning of reaction space to find
effective reaction conditions (Figure 2B). This optimization
unexpectedly revealed that VFD-mediated enzyme reactions
are less susceptible to substrate inhibition than the conven-
tional conditions. For example, b-glucosidase without VFD
processing encounters substrate inhibition at around 3.1 mm
4-nitrophenyl b-d-glucopyranoside; VFD processing prevents
the onset of substrate inhibition up to an almost three-fold
higher concentration (Supporting Information, Figure S10B).
With the exception of DERA, the three other enzymes
tolerated higher concentrations of substrate without losing
VFD-mediated acceleration. This decrease in substrate
inhibition suggests that the VFD increases the enzymatic
kcat, as further demonstrated below. DERA catalyzed the
retro-aldol reaction of a pro-fluorophore at 144 mmolhÀ1 LÀ1
when processed in the VFD (7900 rpm rotational speed),
compared to 10.7 mmolhÀ1 LÀ1 under non-VFD conditions.
DERA has previously been employed to synthesize high-
value, complex, polyoxygenated compounds.[7b] The VFD-
mediated DERA reaction achieved an average 15-fold
enhancement. Conventional approaches to improving
DERA have applied extensive screening[7b] and multiple
rounds of error-prone PCR. For example, screening
20000 colonies yielded a 10-fold increase in DERA activity.[14]
The efforts required to achieve a greater than 10-fold
acceleration by the VFD in several days compared with
conventional protein engineering, highlight the power of the
approach reported here.
The dependence on rotational speeds was also specific to
each enzyme (Figure 2C and the Supporting Information,
Figure S11). Such requirements likely reflect differences in
enzyme size, structure, and dynamics. Esterase, for example,
was highly dependent on a single rotational speed for
enhanced activity. When processing esterase under VFD-
mediated conditions, the only rotational speed to generate an
enhancement was 8000 rpm; at all other rotational speeds, the
enzyme behaved similarly to the non-VFD-mediated con-
ditions. To map out the fine details of such resonances, a high-
resolution scan of rotational speeds examined the acceler-
ation of alkaline phosphatase and b-glucosidase (Figure 3).
The rotational landscapes are intricate with little overlap of
the optimal rotational speeds for each enzyme. Device-
specific variations in rotational landscapes were also
observed, likely due to differences between device bearings
and components (for example, the Teflon collar, which wears
out due to friction from the sample tube); thus, Figure 3
depicts two enzymes processed by a single VFD. In addressing
this issue of wear, and avoiding variable vibrations, we turned
to 3D printing. Fabricating the collar out of high-density ABS
plastic allowed an interchangeable sleeve to be incorporated.
Changing the insert upon wear insures reproducibility of the
reported experiments (Supporting Information, Figure S19).
Michaelis–Menten-based experiments were performed
with b-glucosidase, and the kinetic constants derived for
both the VFD- and non-VFD processed solutions (Table 1).
The kcat in the VFD-mediated reaction was around 2.5-fold
faster than the non-VFD reaction (Figure 3 and Table 1). A
lower Michaelis–Menten constant (Km) was also obtained for
the VFD-processed enzyme–substrate solution; 2.50 mm
compared to 3.76 mm for a non-VFD-mediated reaction.
The decrease in Km demonstrates the higher affinity for the b-
Enzyme acceleration by the VFD is sensitive to the tilt
angle of the sample tube and the viscosity of the solution
(Figure 2D,E). A tilt angle of 458 produced the strongest
response, as has been previously observed in other VFD
experiments.[8b]
more, high concentrations of
viscous, steric-crowding
Further-
reagents that decrease or
terminate enzymatic cataly-
sis in the non VFD-mediated
control conditions were over-
come in the VFD. Biocata-
lytic acceleration was ach-
ieved, for example, in high
concentrations of PEG 8000
(6.00 mgmLÀ1, 0.75m), a con-
dition that suppresses enzy-
matic catalysis in non-VFD-
mediated reactions. Through
rapid micro mixing or other
associated
VFD-processed
phosphatase tolerated high
concentration of PEG 8000,
resulting in a circa 9-fold
enhancement. The relative
phenomena,
alkaline
Figure 3. The rotational landscape of b-glucosidase and alkaline phosphatase. Though the two enzymes have
similar levels of response at some rotational speeds, distinctly different rotational landscapes are revealed.
The results demonstrate the enzyme specificity of VFD-mediated acceleration. Each data point represents the
mean (n=2) for a 10 min reaction at the indicated rotational speed. The alkaline phosphatase enzyme–
substrate solution used fast alkaline phosphatase (6.77 nm) and p-nitrophenol phosphate solution (0.17 mm)
whilst the b-glucosidase enzyme–substrate system used b-glucosidase (19.3 nm) and 4-nitrophenyl b-d-
indifference to high concen- glucopyranoside (7.5 mm).
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Table 1: Michaelis–Menten parameters for the VFD versus non-VFD-
mediated processing of b-glucosidase.[a]
Acknowledgements
Parameter
Non VFD-mediated
reaction
VFD-mediated rate acceler-
ation
J.B. thanks the Taihi Hong Memorial Foundation and Kritika
Mohan for photography. L.M.M. acknowledges the Miguel
Velez Scholarship at UCI. G.A.W. gratefully acknowledges
the National Institute of General Medical Sciences of the
NIH (RO1-GM100700-01). C.L.R. acknowledges the Aus-
tralian Research Council and the Government of South
Australia for their financial support during this project.
Vmax [nmsÀ1
Km [mm]
]
128Æ5.71
3.76Æ0.15
13.4Æ0.59
3.55Æ0.19
309Æ52.4
2.50Æ0.44
32.1Æ5.45
13.32Æ4.03
k
cat [sÀ1
]
kcat/Km
[mmÀ1 sÀ1
]
[a] For the VFD process with b-glucosidase (9.26 nm), a rotational speed
of 7600 rpm was used, as this provided the most consistent enhance-
ment over sustained time periods. Errors indicate the standard deviation
around the mean (n=3). There was no overlapping error at 95%
confidence limits. The raw data was fitted to the Michaelis–Menten
equation using a least squares fitting (LSF) approach (Supporting
Information, Figures S17and S18 and Table S3).
Keywords: aldolases · biocatalysis · enzyme acceleration ·
hydrolases · vortex fluidics
glucosidase–substrate interaction under VFD-mediated con-
ditions. The increase of kcat and the decrease in Km leads to an
approximately 3.5-fold increase in enzyme efficiency (kcat/Km)
for the VFD-mediated reaction.
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We hypothesize that enzymes are accelerated in the VFD
by the instantaneous pressure changes generated by Faraday
waves. Three possible mechanisms could harness such pres-
sures. First, transient pressurization of the active site around
the substrate could occur. In this situation, a decrease in the
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the turnover number of the enzyme; such enhancement
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enzyme speeds uncovered through such experiments. Thus,
the VFD-driven rate acceleration could simply shift the
distribution of enzyme conformational states to favor cata-
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In conclusion, we have demonstrated that the VFD
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Received: April 25, 2016
Published online: && &&, &&&&
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Communications
Biocatalysis
Enzymes, they’re picking up good vibra-
tions: A simple, generalizable approach
to accelerate enzymes through the use of
pressure waves in thin films has been
developed. Each enzyme responds best
to specific vibrations uncovering a previ-
ously unappreciated aspect of biocataly-
sis.
J. Britton, L. M. Meneghini, C. L. Raston,*
G. A. Weiss*
&&&& — &&&&
Accelerating Enzymatic Catalysis Using
Vortex Fluidics
6
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