Polyelectrolytes Tailored Enzyme Cascades with Enhanced Stability and Activity for One-pot Synthesis

Article abstract of DOI:10.1002/cctc.201801532

Cascade enzymes from the same origin or different resources often differ in their pH windows of stability and activity, thus impeding one-pot enzyme cascade reactions. Here we present a method to assemble enzyme cascades using enzyme-polyelectrolyte as the building block, in which the polyelectrolytes not only assemble enzymatic cascades via electrostatic interactions but also create favorable microenvironments for enzymes to expand their pH windows of stability and activity. We first demonstrated the effectiveness of this method for the oxidization of p-xylene to p-toluic acid using chloroperoxidase (CPO) and xanthine oxidase (XO) cascade, in which CPO from Caldariomyces fumago and XO from bovine milk were covalently linked to polymethacrylic acid (PMAA) and chitosan quaternary ammonium salt, respectively. Conjugation of the enzymes with PMAA and chitosan expanded the stability and activity pH windows for CPO and XO and increased the yield of p-toluic acid production from p-xylene by 7.6-fold in comparison to that obtained from their native counterparts in soluble form. We then applied this method to assemble glucose oxidase (GOx) and horseradish peroxidase (HRP) cascade. The GOx-PMAA@HRP-chitosan displayed a lower K_m and higher apparent activity at low glucose concentration, as compared to GOx/HRP cascade in soluble form, suggesting that the assembly method boosted the outcome of the one-pot enzyme cascade reaction.

Full text of DOI:10.1002/cctc.201801532

Accepted Article
Title: Polyelectrolytes tailored enzyme cascades with enhanced
stability and activity for one-pot synthesis
Authors: Zheyu Wang, Junqian Wang, Gong Chen, Weina Xu,
Zhongwang Fu, Guoqiang Jiang, Jianzhong Wu, and Zheng
Liu
This manuscript has been accepted after peer review and appears as an
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To be cited as: ChemCatChem 10.1002/cctc.201801532
Link to VoR: http://dx.doi.org/10.1002/cctc.201801532
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Polyelectrolytes tailored enzyme cascades with enhanced
stability and activity for one-pot synthesis
Zheyu Wang,[a] Junqian Wang,[a] Gong Chen,[a] Weina Xu,[a] Zhongwang Fu,[a] Guoqiang Jiang,[a]
Jianzhong Wu,[b] and Zheng Liu*[a]
Abstract: Cascade enzymes from the same origin or different
resources often differ in their pH windows of stability and activity,
thus impeding one-pot enzyme cascade reactions. Here we present
constructing enzyme cascades can be divided into two
categories. One is protein engineering approach[3] such as
protein scaffold[4] and site-specific attachment,[5] which is
effective in tuning the content, position and orientation of
cascaded enzymes. The other group hinges on synthetic
chemistry and material engineering[6] such as crosslinking,[7]
encapsulation,[8] self-assembly,[9] co-electrospinning,[10] co-
precipitation,[11] and layer-by-layer deposition.[12] In this case, the
distance between enzymes can be shortened to several
nanometers. Idan and Hess[13] proved that the increase in direct
substrate transfer due to the proximity provides an initial but
temporary boost to the throughput of the cascade and loses its
impact as intermediates accumulate in the surrounding container.
Zhang et al.[14] confirmed that an enhanced activity of GOx/HRP
on DNA scaffold is contributed by a favorable local pH by DNA
nanostructures. It is often difficult to establish a tailored local pH
suitable for multiple enzymes that differ in their pH windows of
stability and activity. A compromised pH condition may not
conducive to the maximum performance of the enzymatic
cascade.
Complementary to protein engineering approach such as
site mutation[15] and directed evolution,[16] enzyme immobilization
on suitable matrices such as mesoporous silica,[17] chitosan,[18]
and polyethylenimine-agarose[19] has been proven to be effective
in enhancing enzyme stability at unfavorable pH. Vonklitzing et
al.[20] have demonstrated the procedure by tuning the local pH
inside a polyelectrolyte thin film via the outer polyelectrolyte
layer and the ionic milieu of the adjacent solution. Goldstein et
al.[21] have found that the pH-activity profile of trypsin can be
tuned toward more alkaline pH by immobilization on insoluble
polyanionic carriers of high charge densities. Hess et al.[22] have
extended the optimal pH window of cytochrome c to more
alkaline conditions by conjugation with polymethacrylic acid.
Given these efforts, a method that displays simultaneously
scaffold and tailors local pH for individual enzymes is needed for
one-pot enzyme cascade reactions.
a
method to assemble enzyme cascades using enzyme-
polyelectrolyte as the building block, in which the polyelectrolytes not
only assemble enzymatic cascades via electrostatic interactions but
also create favorable microenvironments for enzymes to expand
their pH windows of stability and activity. We first demonstrated the
effectiveness of this method for the oxidization of p-xylene to p-toluic
acid using chloroperoxidase (CPO) and xanthine oxidase (XO)
cascade, in which CPO from Caldariomyces fumago and XO from
bovine milk were covalently linked to polymethacrylic acid (PMAA)
and chitosan quaternary ammonium salt, respectively. Conjugation
of the enzymes with PMAA and chitosan expanded the stability and
activity pH windows for CPO and XO and increased the yield of p-
toluic acid production from p-xylene by 7.6-fold in comparison to that
obtained from their native counterparts in soluble form. We then
applied this method to assemble glucose oxidase (GOx) and
horseradish peroxidase (HRP) cascade. The GOx-PMAA@HRP-
chitosan displayed a lower Km and higher apparent activity at low
glucose concentration, as compared to GOx/HRP cascade in soluble
form, suggesting that the assembly method boosted the outcome of
the one-pot enzyme cascade reaction.
Enzymes are powerful biocatalysts enabling all living systems on
our planet, for example, nitrogen fixation and conversion,
photosynthesis, and metabolic activities of microorganisms.
Enzyme cascades also hold great promise for green and
efficient synthesis of fine chemicals with complex structures.[1]
The proximity and compartmentation are essential features of
high performance enzyme cascade in vivo, in which the
proximity of consequential enzymes enables substrate
channeling and direct transport of intermediates thus prevents
either their degradation and/or their adverse effects on enzyme
catalysis.[2] Compartmentation provides
a
tailored local
environment ensuring high activity and stability of each enzyme
in cascade reactions. To reproduce above mentioned
mechanisms for chemical synthesis, recent efforts in
Here we present a novel method to assemble enzyme
cascades using enzyme-polyelectrolytes as building blocks.
Scheme 1 outlines the key steps for preparation of CPO-
PMAA@XO-chitosan conjugates. The first step involves the
formation of enzyme-polyelectrolyte conjugates. In connecting
chloroperoxidase (CPO) with polymethacrylic acid (PMAA), the
carboxyl groups of PMAA were firstly activated with N-(3-
dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC)/
N-hydroxysulfosuccinimide sodium salt (sulfo-NHS) and then
grafted with the amine groups of CPO. The SDS-PAGE result
confirms disappearance of free CPO band and appearance of a
new band with larger molecular weight (Figure 1a), which also
[a]
Z. Wang, J. Wang, Dr. G. Chen, W. Xu, Z. Fu, Prof. G. Jiang, Prof.
Z. Liu
Department of Chemical Engineering
Tsinghua University
Beijing 100084 (P.R. China)
E-mail: liuzheng@mail.tsinghua.edu.cn
Prof. J. Wu
[b]
Department of Chemical and Environmental Engineering
University of California, Riverside
Riverside CA 92521 (USA)
indicates
a high conjugation yield. The result of 2,4,6-
trinitrobenzenesulfonic acid (TNBS) assay shows that around 3
lysines per CPO are conjugated with PMAA (Figure 1b), and that
Supporting information for this article is given via a link at the end of
the document.
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each PMAA chain contains about 220 carboxyl groups. From the
transmission electron microscopy (TEM) images, we estimate
that the average size of the CPO-PMAA conjugate is 24 nm
(Figure 1c), whereas the size of a free CPO is 10 nm (Figure S1
and S2). The sizes of CPO-PMAA and free CPO are consistent
with those from the dynamic light scattering (DLS) analysis,
which are 29 and 10 nm, respectively (Figure S3).
FITC-labeled XO-chitosan, in which a reduction of color intensity
of the upper solution is observed after 10 min incubation,
indicating adsorption of CPO-PMAA on the surface of XO-
chitosan by electrostatic interactions. The confocal laser
scanning microscopy (CLSM) further confirms co-localization of
RhoB-labeled CPO-PMAA (red) and FITC-labeled XO-chitosan
(green) (Figure 2c). We carried out the FRET measurement to
determine the distance between CPO and XO in the conjugate.
The efficiency of FRET is determined as 40.6% according to the
acceptor photobleaching method (Figure 2d). Using the method
described by Xia[23] (See Supporting Information for details), we
estimate the average distance between the donors and the
acceptors to be 6.2 nm.
Scheme 1. Preparation of CPO-PMAA and XO-chitosan conjugates and self-
assembly of CPO-PMAA@XO-chitosan bienzyme based on electrostatic
interactions.
Using glutaraldehyde as the cross linker, xanthine oxidase
(XO) was covalently linked with chitosan quaternary ammonium
salt. We estimated from the scanning transmission electron
microscopy (STEM) images the average size of XO before and
after chitosan-conjugation, 10 nm and 200 nm, respectively
(Figure 1d, 1e and Figure S4). The particle diameters agree well
with the corresponding results determined from DLS, 12 and 460
nm (Figure S5). The reason for the large particle size of the XO-
chitosan conjugate may be attributed to the self-assembly of
chitosan quaternary ammonium salt. Because of the drastic
difference in size, we applied size exclusion chromatography
(SEC) to determine the yield of XO-chitosan conjugate. As
shown in Figure 1f, XO-chitosan has a retention time of 11.9 min
whereas that corresponding to free XO is 15.6 min. The overall
yield of XO-chitosan conjugate at above condition is over 90%,
which was calculated from the XO concentration in the
supernatant as determined by SEC. BCA protein assay with
bovine serum albumin (BSA) as standard protein was applied to
determine the concentrations of free enzymes and enzyme
conjugates.
Figure 1. (a) SDS-PAGE of the free CPO (F), protein marker (M) and CPO-
PMAA conjugate (C). (b) TNBS assay for free amino groups of free CPO and
CPO-PMAA conjugate. (c) TEM image of the CPO-PMAA conjugate (scale bar
is 100 nm). (d) BF-STEM image of the XO-chitosan conjugate (scale bar is
100 nm). (e) SEM image of the XO-chitosan conjugate (scale bar is 100 nm).
(f) Size exclusion chromatograms of the free XO (0.5 mg ml-1) and XO-
chitosan conjugate (0.5 mg ml-1) solution.
In order to determine an optimal pH for the self-assembly of
CPO-PMAA@XO-chitosan conjugates, which is driven by
electrostatic interactions between oppositely charged enzyme
conjugates, we measured their zeta-potentials from pH 4.0 to
9.0. Attachment of polyelectrolytes alters the local solution
properties and thus the magnitude of the zeta-potential of the
enzyme conjugate. The change in the electrostatic potential
facilitates the assembly of enzyme cascade based on
electrostatic interaction. As shown in Figure 2a, the zeta-
potential of CPO-PMAA falls from -12.1 to -22.5 mV in response
to the pH increase from 4.0 to 9.0. By contrast, the zeta-potential
of XO-chitosan remains positive but its magnitude decreases
from 15.4 to 7.0 mV. It can be concluded from Figure 2a that a
broad pH range is available for forming CPO-PMAA@XO-
chitosan assembly.
Figure 2. (a) Zeta-potentials of free CPO, CPO-PMAA, free XO, and XO-
chitosan. (b) Addition of RhoB-labeled CPO-PMAA to FITC-labeled XO-
chitosan after 0 min (left) and 10 min (right) incubation. (c) Confocal laser
Figure 2b illustrates the formation of the CPO-PMAA@XO-
chitosan conjugate by adding RhoB-labeled CPO-PMAA to
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scanning microscope images of the CPO-PMAA@XO-chitosan conjugate,
CPO and XO were labeled with rhodamine-B (RhoB, red) and fluorescein
(FITC, green), respectively (scale bars are 2 μm). (d) Confocal laser scanning
microscope images of the CPO-PMAA@XO-chitosan conjugate after acceptor
photobleaching (scale bars are 10 μm).
We explored the polyelectrolyte effects on the activity and
stability of conjugated enzymes. CPO is
a monomeric
hemoprotein.[24] The pH-activity profile of free CPO and CPO-
PMAA conjugate was determined by a colorimetric method using
H2O2 and guaiacol as the substrates[25] at 470 nm (Figure S6).
Here the extinction coefficients of product tetraguaiacol at
different pHs were calibrated because its absorption is pH-
dependent (Table S1). Figure 3a shows that both free CPO and
CPO-PMAA conjugate appear the highest activity at pH 5.5,
which is consistent with a previous report.[26] In the case of CPO,
the activity of free CPO at pH 5.5 is set as 100%. In the case of
XO, the activity of free XO at pH 8.0 is set as 100%. The relative
activity of CPO-PMAA is 18% higher than that of free CPO at pH
5.5. The activity of free CPO is highly dependent on pH and
decreases dramatically as pH increases, which can be seen
from its relative activity of 46% and 7% at pH 7.0 and 8.0,
respectively. By contrast, the CPO-PMAA conjugate exhibits
high activity in a wide pH range from 4.0 to 8.0, and 52% of its
maximum activity is retained at pH 8.0. Previous studies suggest
that the decrease of apparent activity is caused mainly by
unfavorable conformational transition of the enzyme.[27] However,
circular dichroism spectroscopy (CD) measurement shows no
significant change in the CPO conformation (Figure 3b). The
number of carboxyl groups and the dissociation behavior of
CPO-PMAA were determined by titration (Figure 3c). The results
indicate that a highly charged polyelectrolyte can attract protons
to their vicinity and form a partial acidic microenvironment. We
calculated the pH profile near the surface of CPO-PMAA using
the Poisson-Boltzmann model[14, 20] and found that the local pH
depends on the bulk pH and the surface potential. The higher
bulk pH, the larger pH difference between the local and bulk
solution (see Figure 3d and Supporting Information for details).
We also compared the p-tolualdehyde throughput catalyzed by
free CPO or CPO-PMAA at different pHs. The enhancement
effect magnifies as bulk pH increases (Figure 3e and 3f),
suggesting that PMAA may be utilized to maintain a more acidic
microenvironment in the vicinity of enzyme surface. The
polyelectrolyte conjugate enables enzymes to display high
activity within a pH range wider than that of the bulk solution.
Figure 3. (a) Relative pH-activity profile of free CPO, CPO-PMAA conjugate,
free PMAA, free XO, XO-chitosan conjugate, and free chitosan. (b) CD
spectrum of native CPO at pH 5.5, 6.0, and 7.0 (0.1 mg ml-1). (c) Titration
curves of free CPO and CPO-PMAA conjugate (0.8 mg enzyme). (d) pH profile
near the CPO-PMAA conjugate at different pHs. (e-f) The time dependence of
p-tolualdehyde production of free CPO (e) and CPO-PMAA conjugate (f) at
different pH values.
We then determined the pH-activity profiles of free XO and
the XO-chitosan conjugate using a colorimetric method[28] at 510
nm (Figure S7). XO is a homodimer, in which each of the
monomers acting independently in catalysis.[29] The dissociation
behavior of XO-chitosan was determined by titration (Figure S8).
In such a case, both free XO and the XO-chitosan conjugate
exhibit similar activities in a wide pH range from 5.0 to 7.0 and
the activity of XO-chitosan is only 88% of that for free XO at pH
8.0 (Figure 3a). Control experiments indicate that both PMAA
and chitosan have no catalytic activity at all pH. To understand
this activity enhancement in XO-chitosan conjugate, we checked
and compared the stability of free CPO, CPO-PMAA, free XO
and XO-chitosan at pH 5.5, 6.0, and 7.0. Figure 4a shows both
free and immobilized CPO are stable at pH 5.5 and 6.0, whereas
the immobilized CPO appears a significantly improved stability
at pH 7.0. Figure 4b shows that XO exhibits more significant
reduction of the activity in response to the decrease in the bulk
pH. For example, free XO loses 88% of its original activity after 9
h incubation at pH 5.5, whereas XO-chitosan retains over 82%
of its original activity. The drastic improvement of activity
indicates the stabilization effect of the conjugated chitosan. CD
measurement shows changes in the XO conformation (Figure
S9). We also compared the p-toluic acid throughputs catalyzed
by free XO or XO-chitosan under different pH conditions. As
shown in Figure 4c and 4d, the yield decreases for free XO after
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9 h as the bulk pH decreases. However, the yield for XO-
chitosan is almost consistent, irrespective to the bulk pH. The
enhanced stability may be attributed to the strengthened pH
tolerance of XO-chitosan compared to native XO, which is
deactivated at acidic pHs.
hours of reaction. As shown in Figure 5b, 5c and 5d, CPO-
PMAA@XO-chitosan conjugate gives the highest yield at pH 5.5,
6.0 and 7.0, compared with the free enzyme solution or any
combination of free enzyme and the conjugated cascade
enzyme. Moreover, pH 6.0 is optimal for the CPO-PMAA@XO-
chitosan conjugate, at which the yield is 7.6-fold of that obtained
by the free enzyme cascade after 9 hours of reaction (Figure
S13). It is also noteworthy that, the yield by CPO-PMAA@XO-
chitosan conjugate at both pH 5.5 and 7.0, though less below
the peak value at pH 6.0, are significantly higher than those
corresponding to their free native counterparts. In particular, the
free CPO and XO cascade has no conversion at pH 7.0. Above
results indicate that CPO-PMAA@XO-chitosan conjugate has a
wider operational pH range and this is advantageous for process
practice of enzymatic catalysis. The superior catalytic
performance of the CPO-PMAA@XO-chitosan might be
attributed to two important factors: the proximity enhancing
intermediate transport and the conjugated polyelectrolytes
enabling tailored local microenvironment in terms of pH for
individual enzymes, respectively.
Figure 4. (a) Stability of free CPO and CPO-PMAA conjugate at pH 5.5, 6.0,
and 7.0. (b) Stability of free XO and XO-chitosan conjugate at pH 5.5, 6.0, and
7.0. (c-d) The time dependence of p-toluic acid production of free XO (c) and
XO-chitosan conjugate (d) at different pH values.
To demonstrate its workability and advantages over the
native counterpart in free solution, the CPO-PMAA@XO-
chitosan conjugate was used to catalyze the oxidation of p-
xylene to p-toluic acid. As shown in Figure 5a, CPO converts p-
xylene into p-tolualdehyde by consuming H2O2, p-tolualdehyde
is further oxidized into p-toluic acid catalyzed by XO. In situ H2O2
formation is one advantage of the CPO/XO cascade because it
enables oxidation with less addition of H2O2 feedstock, making it
convenient to handle in particular at large scale. During a typical
run, 1.0 ml mixture of free CPO (0.5 mg ml-1) and XO (1.0 mg ml-
1) or equal amounts of CPO-PMAA@XO-chitosan conjugate was
added into a 10.0 ml solution containing 5%(v/v) acetone, 6.0
mM p-xylene, and dissolved oxygen gas at saturation. The
reaction was initiated by addition of 30 μl H2O2 (30 wt. % in
water). The concentrations of the enzymes in the following
experiments are equal. GC-MS was employed in order to
determine the individual components, as detailed in Figure S10
and S11. The concentrations of p-xylene, p-tolualdehyde, and p-
toluic acid were analyzed with GC (Figure S12).
As shown in Figure 3a, CPO and XO have vastly different
optimal pH conditions, the maximum activities for CPO and XO
are observed at pH 5.5 and 8.0, respectively. We compared the
catalytic performance of CPO-PMAA@XO-chitosan conjugate
with that corresponding to their native counterparts in free form
or that for a combination of one free enzyme with a conjugated
cascade enzyme. The latter is chosen to illustrate the effect of
mass transport for both p-tolualdehyde and H2O2 on the overall
catalytic performance. These measurements were taken after 9
Figure 5. (a) Schematic of one-pot synthesis of p-toluic acid from p-xylene by
CPO/XO bienzyme cascade reaction. (b-d) Relative activity and p-toluic acid
throughput of the free CPO with free XO, CPO-PMAA with free XO, XO-
chitosan with free CPO and CPO-PMAA@XO-chitosan conjugate at pH 5.5 (b),
6.0 (c), and 7.0 (d).
To demonstrate the effectiveness of this electrostatic-
interaction enabled assembly method, we also prepared GOx-
PMAA and HRP-chitosan conjugates and confirmed the
assembly of FITC-labeled GOx-PMAA and RhoB-labeled HRP-
chitosan using CLSM (Figure S14). Moreover, we used
GOx/HRP cascade to examine the effects of the polyelectrolyte
on the reaction kinetics of enzyme cascade, in which H2O2
generated by GOx served as substrate for co-localized HRP in
the assembly, similar to the CPO/XO cascade reaction (Figure
6a). The specific activity unit of the GOx/HRP bienzyme system
is defined as 1.0 mg of total enzymes that catalyze 1.0 μmole of
o
glucose per minute at pH 7.0 and 25 C. At all conditions, the
GOx-PMAA@HRP-chitosan conjugate exhibits activities higher
than those corresponding GOx-PMAA with the same amount of
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free HRP (Figure 6b). For example, when the glucose
concentration is 1.0 mM, the specific activity of the GOx-
PMAA@HRP-chitosan conjugate is 41.6% higher than its free
counterparts. We determined the Michaelis-Menten parameters
of GOx in GOx-PMAA@HRP-chitosan conjugate (Figure 6c and
Table 1). The Km value for GOx-PMAA@HRP-chitosan is the
lowest, meaning the highest substrate affinity. The higher
specificity constant (kcat/Km) and higher overall activity at low
glucose concentration confirm that this assembly method boosts
the outcome of the one-pot enzyme cascade reaction.
cascades, and on the other hand, creates a favourable
microenvironment in terms of pH in the vicinity of enzyme
surface leading to enhanced activity and stability of the
conjugated enzymes. The expanded stability- and activity-
windows of CPO-PMAA and XO-chitosan lead to a 7.6-fold
increase in the conversion of p-xylene to p-toluic acid, as
compared to their native counterparts in free solution. The
increased overall activity and enhanced substrate affinity is
observed in GOx-PMAA and HRP-chitosan cascade. The
advantages of this method include ease of construction,
facilitated intermediate transport, and the expanded pH window
of stability and activity. The diverse availably of polyelectrolytes
enables virtually unlimited construction of multi-enzymes for
one-pot cascade reactions and other applications of enzymatic
catalysis.
Experimental Section
Synthesis of CPO-PMAA conjugate
During a run, 6.0 mg N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (EDC) and 26.6 mg polymethacrylic acid (PMAA) were
added into 1.6 ml MES buffer (10 mM, pH 5.5) and stirred for 5 min. Then
8.0 mg N-hydroxysulfosuccinimide sodium salt (sulfo-NHS) was added
into the mixture to activate PMAA and stirred for 15 min. Then 1.6 ml of
1.25 mg ml-1 CPO was mixed with the activated PMAA and stirred 12
hours at 25 oC. After stopping the reaction, the excess small molecule
agents were removed by ultrafiltration using 30 kDa MWCO centrifugal
filter units.
Figure 6. (a) Schematic of GOx/HRP cascade reaction. (b) Specific activities
of free GOx with free HRP, GOx-PMAA with free HRP, HRP-chitosan with free
GOx and GOx-PMAA@HRP-chitosan conjugate at different glucose
concentrations. (c) The Michaelis-Menten plots of GOx in the mixture of the
free GOx and free HRP, GOx-PMAA@HRP-chitosan conjugate, GOx-PMAA
and free HRP, and free GOx and HRP-chitosan and the concentration of GOx
and HRP were 0.2 μg ml-1 and 0.8 μg ml-1, respectively.
Synthesis of XO-chitosan conjugate
During a run, 10.0 mg chitosan quaternary ammonium salt (chitosan) and
2.0 μl glutaraldehyde (50 wt. % in water) were added into 1.0 ml
phosphate buffer (50 mM, pH 7.0) and stirred for 40 min. Then 5.0 mg
XO was added into the mixture and stirred for 1 hour at 25 oC. After
stopping the reaction, the excess small molecule agents were removed
by ultrafiltration using 30 kDa MWCO centrifugal filter units.
Table 1. Michaelis-Menten parameters for the enzymatic kinetics of GOx-
PMAA@HRP-chitosan, GOx-PMAA and free HRP, free GOx and HRP-
chitosan, and free GOx and HRP mixture.
Km
[mM]
vmax
[μM s-1]
kcat
[s-1]
kcat/Km
[s-1 mM-1]
Synthesis of p-toluic acid by CPO/XO cascade reaction
During a typical run, 1.0 ml mixture of free CPO (0.5 mg ml-1) and XO
(1.0 mg ml-1) or 1.0 ml solution of CPO-PMAA@XO-chitosan conjugate
was added into a 10.0 ml solution containing 5%(v/v) acetone, 6.0 mM p-
xylene, and dissolved oxygen gas at saturation. The reaction was
initiated by addition of 30 μl H2O2 (30 wt. % in water). The reaction
mixture was magnetically stirred under room temperature. The p-xylene,
p-tolualdehyde and p-toluic acid concentrations were analyzed by GC. At
set intervals (0, 1, 2, 4, 6, 9 hours), 1.0 ml solution was added into a
mixture of 1.5 ml diethyl ether and 50 μl HCl (37 wt. % in water) to extract
the products. The diethyl ether was centrifuged at 10000 rpm for 10 min
and filtered through a 0.22 μm membrane before the GC analysis (See
Supporting Information for details).
GOx-PMAA @HRP-
chitosan
4.31±0.47
6.48±0.67
7.08±0.42
6.66±0.66
1.70±0.05
1.64±0.04
1.31±0.02
1.92±0.05
1360±36
1310±36
1050±17
1540±41
316±26
203±15
148±6
GOx-PMAA and free
HRP
free GOx and HRP-
chitosan
free GOx and free
HRP
231±17
In summary, we have established a novel method for
assembling an enzyme cascade using enzyme-polyelectrolyte
as the building block and demonstrated its efficiency using
CPO/XO and GOx/HRP as cascade examples. Conjugation of
enzymes with polyelectrolytes, on one hand, provides
electrostatic driving forces for the assembly of enzymatic
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (21676157 and 21520102008) and
Tsinghua University (2014Z22077). J.W. acknowledges partial
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financial support from the US National Science Foundation
(Grant No. NSF-CBET-8200852353).
Keywords: enzyme cascades • multiple enzymes • pH matching
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10.1002/cctc.201801532
ChemCatChem
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Zheyu Wang, Junqian Wang, Gong
Chen, Weina Xu, Zhongwang Fu,
Guoqiang Jiang, Jianzhong Wu and
Zheng Liu*
Page No. – Page No.
Polyelectrolytes tailored enzyme
cascades with enhanced stability and
activity for one-pot synthesis
A new method to assemble enzyme cascades using enzyme-polyelectrolyte as the
building block is proposed, in which the polyelectrolytes not only assemble
enzymatic cascades via electrostatic interactions but also create favorable
microenvironments in the vicinity of enzymes to expand their pH windows of
stability and activity. It enables chloroperoxidase (CPO) and xanthine oxidase (XO)
to produce p-toluic acid with high catalytic activity.
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