Peroxidase Encapsulated in Peroxidase Mimics via in situ Assembly with Enhanced Catalytic Performance

Article abstract of DOI:10.1002/cctc.201902055

Enzymatic catalysis is of great importance due to its high catalytic activity and high selectivity. However, intrinsic fragile property of enzyme limits its application in a wide range, where inactive inorganic materials have proven promising in enhancing the stability of the enzyme in industry. Here, Prussian blue nanoparticles (PB) as peroxidase mimics were adopted for encapsulation of peroxidase, Cytochrome c (Cyt c). Prussian blue/Cytochrome c (PB?Cyt c) composite developed via in-situ assembly exhibited a 2.6-fold higher apparent enzymatic activity than the sum activity of equivalent Cyt c and PB due to synergistic effect. Mechanism investigation showed that interaction between irons ions from PB and carbonyl group and nitrogen from enzyme led to a favorable catalytic conformation of Cyt c. Moreover, PB shielded the encapsulated Cyt c against harsh conditions (e. g. high temperature and organic solvents). This new efficient hybrid catalyst would open opportunities for wide applications in biosensors and biocatalysis.

Full text of DOI:10.1002/cctc.201902055

Accepted Article
Title: Peroxidase encapsulated in peroxidase mimics via in-situ
assembly with enhanced catalytic performance
Authors: Xiaoling Wu, Jun Xiong, Shuli Liu, Bin Chen, Shan Liang,
Wenyong Lou, and Min-hua Zong
This manuscript has been accepted after peer review and appears as an
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To be cited as: ChemCatChem 10.1002/cctc.201902055
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COMMUNICATION
Peroxidase encapsulated in peroxidase mimics via in-situ
assembly with enhanced catalytic performance
Xiaoling Wu*[a], Jun Xiong[a], Shuli Liu[a], Bin Chen[a], Shan Liang[a], Wenyong Lou*[a,b] and Minhua
Zong[a]
Abstract: Enzymatic catalysis is of great importance due to its high
catalytic activity and high selectivity. However, intrinsic fragile
property of enzyme limits its application in a wide range, where
inactive inorganic materials have proven promising in enhancing the
stability of the enzyme in industry. Here, Prussian blue nanoparticles
(PB) as peroxidase mimics were adopted for encapsulation of
peroxidase, Cytochrome c (Cyt c). Prussian blue/Cytochrome c
(PB@Cyt c) composite developed via in-situ assembly exhibited a
2.6-fold higher apparent enzymatic activity than the sum activity of
equivalent Cyt c and PB due to synergistic effect. Mechanism
investigation showed that interaction between irons ions from PB and
carbonyl group and nitrogen from enzyme led to a favourable catalytic
conformation of Cyt c. Moreover, PB shielded the encapsulated Cyt c
against harsh conditions (e.g. high temperature and organic solvents).
This new efficient hybrid catalyst would open opportunities for wide
applications in biosensors and biocatalysis.
Moreover, nanostructured shells were not limited to metal
phosphate crystals[10] and metal organic frameworks (MOFs).[11]
The inactive carrier outside the enzymes has been regarded as
one of the most effective strategies for protecting enzymes.
Another attempt to eliminate the environmental attack for
enzymatic catalysis was via the de-novo design of inorganic
nanostructured materials to mimic the complex structure and
active site of the natural enzyme. Due to the relative inert property
of the inorganic materials, inorganic enzyme mimics can be
adopted in a wide operating range in harsh environments. To date,
developed enzyme mimics can be categorized into three classes:
carbon-,[12] metal-,[13] and metal-oxide-based[14] nanomaterials,
according to their structure[15], which has found their potential
applications in the biosensor development, diagnosis, cancer
therapy, environmental protection and so on. For example,
enzyme mimics serving as anticarcinogen has been rapidly
developed in recent decades. In 2018, Qu and etc[16] fabricated
homogeneous cerium oxide nanoparticles (CeO2 NPs) with
oxidase-like activity and ATP deprivation capacity, which
exhibiting high efficiency in cancer therapy. Biomimetic
MnO2@PtCo nanoflowers[17] which can accelerate cascade
intracellular chemical reaction to produce ROS were reported to
induce cell apoptosis and thus significantly inhibit tumor growth.
However, the catalytic activity of artificial enzymes still can not
compete with natural enzymes.
In this study, the idea of integrating inorganic enzyme
mimicry and natural enzyme was realized by utilizing Prussian
blue nanoparticles (PB) with mimic peroxidase activity as
protecting shell to encapsulate the native peroxidase Cytochrome
c (Cyt c) (Scheme 1). Our research showed that Cyt c embedded
into PB via in-situ assembly process exhibited “ultra-activity” due
to the synergistic effect. The as-synthesized Prussian
blue/Cytochrome c (PB@Cyt c) composite showed a 2.6-fold
higher activity than the sum activity of equivalent Cyt c and PB.
Moreover, owing to the shielding effect from PB, the encapsulated
Cyt c showed enhanced stability when exposed to harsh reaction
conditions (such as high temperature and organic solvents). The
strategy proposed here is demonstrated to integrate enzyme with
enzyme mimics to generate enhanced catalytic performance, thus,
may provide a facile and versatile approach for fabrication of
efficient hybrid biocatalysts in biosensors and biocatalysis.
Bioactive molecules, with remarkable capability of manipulating
plenty of substrates, tend to deactivate and lost their biological
function when exposed to industrial conditions including high
temperature and organic solvents.[1] The past decades have
witnessed worldwide interest in the development of immobilized
enzyme which intends to protect the enzyme from attack of
extreme artificial environments.[2] In nature, one of the self-
protection mechanism was biomineralization, where an inorganic
shell formed around biomolecules during a biologically induced
in-situ self-assembly process.[3] The inorganic shell endowed
biomolecules with enhanced mechanical properties and protected
them against harsh environments.[4] Learning from nature,
researchers have developed a general method for encapsulating
biomolecules into the inorganic nanostructure via in-situ assembly.
Ge[5] and co-authors reported the first enzyme encapsulated in
inorganic copper phosphate hybrid catalyst, where biomolecules
formed a complex with copper ions and served as nucleation sites
for further growth of copper phosphate shell. The resultant
enzyme hybrid nanoflower exhibited remarkably enhanced
thermal stability. The versatility of this process has driven the
construction of bioactive molecules with protective inorganic
shells including enzymes,[6] antibodies,[7] vaccine[8] and cells.[9]
[a]
Dr. X. Wu, J. Xiong, S. Liu, B. Chen, S. Liang, Prof. W. Lou, Prof. M.
Zong
Lab of Applied Biocatalysis
School of Food Science and Engineering
South China University of Technology
Guangzhou 510640 (P. R. China)
E-mail: wuxl18@scut.edu.cn
[b]
Prof. W. Lou
Innovation Center of Bioactive Molecule Development and
Application
South China Institute of Collaborative Innovation
Dongguan 221116 (P.R. China)
E-mail: wylou@scut.edu.cn
Scheme 1. Schematic showing preparation of PB@Cyt c composite via
biomimetic mineralization.
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The ultraviolet-visible (UV-Vis) spectrum (Figure 1a) of free
Cyt c showed a peak at around 410 nm from the typical soret band
of heme group of Cyt c, which can also be found in the PB@Cyt
c composite, demonstrating the successful incorporation of Cyt c
into the PB. Typical absorption at 700 nm from PB was observed
in the freshly synthesized PB and showed a slightly shift to ~ 710
cm in the PB@Cyt c composite, indicating the possible interaction
between Cyt c and PB. XRD patterns of PB@Cyt c composite
showed diffraction peaks in line with PB (Figure 1b), implying the
presence of Cyt c in the composite did not alter the cyanometalate
structure of PB nanoparticles. The Cyt c loading amount in the
PB@Cyt c composite was investigated by thermal gravity analysis
(TGA) in the air (Figure 1c). As can be seen,in the range of 240
to 400°C, PB only exhibited 17 wt% of decomposition. While in
the PB@Cyt c composite, an obvious decline of 47 wt% was
observed. The apparently higher decomposition percentage can
be attributed to the decomposition of Cyt c within this range,
calculated to be 30 wt%. To further evaluate the encapsulation
efficiency of this biomineralization, protein concentration in the
supernatant immediately after the synthesis was measured by
Coomassie brilliant blue assay. Almost no protein was detected
in the supernatant, implying 100% encapsulation efficiency of this
process, which was also consistent with the result of TGA. The
dynamic light scattering (DLS) analysis showed that PB
nanoparticles can be evenly dispersed in the aqueous solution
(Figure 1d). Scanning electron microscopy (SEM) images of PB
itself exhibited as nanocubes with a size of ~ 50 nm (Figure 1e),
interfered with the biomineralization.
The enzymatic assay
was performed by using hydrogen peroxide and ABTS as
substrate. PB, as a peroxidase mimicry has been demonstrated
[19]
to show comparable activity with free Cyt c. When physically
mixing free Cyt c with obtained PB (denoted as PB/Cyt c mixture),
the activity of PB/Cyt c mixture was just sum up of activity of
separate Cyt c and PB. While PB@Cyt c composite exhibited a
2.6-fold higher activity than PB/Cyt c mixture (Figure 2a). This
enhanced activity was possibly attributed to the synergistic effects
generated during the biomimetic mineralization.[20]
To demonstrate the above hypothesis, we investigated in-
depth the interaction between Cyt c and PB by using Fourier
transform
infrared
spectroscopy
(FTIR),
fluorescence
spectroscopy and X-ray photospectroscopy (XPS) analysis
together. As shown in Figure 2b, a strong absorption band around
2090 cm-1 was ascribed to the characteristic stretching mode of
C≡N from PB. The band at around 1650 cm-1 which was ascribed
to the amide I vibration can be found in the spectra of Cyt c.
However, this peak showed a slight shift to higher wavenumber in
the PB@Cyt c composite, confirming the successful incorporation
of Cyt c during the biomineralization process. This shift also
indicated that metal ions interact with the carbonyl groups, thus,
regulating the activity of the encapsulated Cyt c. To further
confirm the conformational change of PB@Cyt
c
after
biomineralization, bands of amide I of enzyme in FTIR spectrum
were analyzed. The contents of β-pleated sheet (1610-1640 cm-
1), random coil (1640-1650 cm-1), α-helix (1650-1658 cm-1) and β-
turn (1660-1700 cm-1) were calculated by using PeakFit 4.2
software.[21] As shown in Figure S1 and Table S1, random coil
constituted 29.34% of the secondary structure in PB@Cyt c
composite, which showed a 2.52% increase compared with that
of free Cyt c. While β-pleated sheet of PB@Cyt c composite
accounted for 14.42%, lower than that of free Cyt c. The increase
of random coil and decrease of β-pleated sheet in PB@Cyt c may
lead to a favorable secondary structure, which can partly explain
a 2.0
b
Cyt c
PB
PB@Cyt c
PB
PB@Cyt c
1.6
1.2
0.8
0.4
0.0
10 20 30 40 50 60 70 80
2 theta (degree)
300
400
500
600
700
800
hyperactivation of PB@Cyt
c
composite.[22] Fluorescence
Wavelength (nm)
c 100
90
100
PB
spectroscopy can reveal the tertiary structure change of protein.
It is known that the active site of Cyt c lies in the heme group.[23]
Moreover, four tyrosine residues and a tryptophan residue located
near the heme group and thus affected the microenvironment of
the active site.[24] The conformational change of these arranged
amino acids around Cyt c can be reflected in the fluorescence
spectra of Cyt c and affected the catalytic performance of the Cyt
c.[25] As shown in Figure 2c, a 20 nm shift was observed in the
fluorescence spectrum of PB@Cyt c composite with emission
peak of 358 nm, demonstrating that the chemical interaction
between free Cyt c and PB during the biomimetic mineralization
process. The microenvironment change possibly resulted from
the interaction of iron ions from PB with amide bond of Tyr67
residue of Cyt c, benefiting the consequent displacement of Met80
near the heme group and further exposure of heme group of Cyt
c, which would promote catalysis,[26] and contribute to enhanced
enzymatic activities.
d
PB
PB@Cyt c
75
50
25
0
80
70
60
50
40
30
1
10
100 1000
Size (d. nm)
10000
100 200 300 400 500 600
Temperature (oC)
e
f
Figure 1. (a) UV-Vis spectra of Cyt c, PB and PB@Cyt c composite. (b) XRD
patterns of PB and PB@Cyt c composite. (c) TGA analysis of PB and PB@Cyt
c composite. (d) DLS measurement of PB in aqueous solution. (e-f) SEM
images of PB (e) and PB@Cyt c composite (f).
To further reveal the local chemical environment of Fe ions,
X-ray photoelectron spectroscopy (XPS) was carried out. The full
spectra were shown in Figure 2d, showing the existence of Fe, C,
N and H. The binding energy of Fe before and after the
corporation of Cyt c was compared (Figure 2e). Peaks at 721.6
eV (Fe 2p1/2) and 713.1eV (Fe 2p1/2) were attributed to the FeNx
which is consistent with the previous report.[18] After incorporation
of Cyt c into the PB nanoparticles, a plate like structure was
observed (Figure 1f), indicating that the introduction of protein
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10.1002/cctc.201902055
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unit. Peaks at 708.4 eV (Fe 2p3/2) were ascribed to [FeII(CN)6]
group of PB. While in the high resolution XPS spectra of Fe in the
PB@Cyt c composite, higher binding energy (708.6 eV) was
observed, indicating the lower electron density of Fe. The N 1s
high resolution spectra showed a positive shift from 397.6 eV in
the PB to 400.2 eV in the PB@Cyt c composite (Figure 2f). The
shift of binding energy of Fe and N together provided evidence
that the self-assembly of Cyt c into PB was driven by the
coordination between Fe ions and nitrogen from the amide bond
of enzyme. Thus, results of XPS further corroborated that Cyt c
interacted with PB via coordination during the biomineralization
process thus altered the microenvironment of heme, which
facilitated the displacement of Met80 near the heme group of Cyt
c and benefited Cyt c-catalyzed peroxidase reaction.[26] Moreover,
nitrogen from Cyt c may also interact with iron ions from PB and
generate more catalytic active sites with structure similar to heme
groups and catalyze the reaction. Thus, based on the above
investigation, the enhanced apparent activity can be attributed to
synergistic effect resulting from the change of microenvironment
of Cyt c and the generated catalytic active sites between Cyt c
and PB during the biomineralization process.
showed a slightly higher Km of 7.1 mM. As expected, PB@Cyt c
composite showed kcat of 75.9 s-1, which is 2.1-fold higher than
the sum kcat of free PB and Cyt c, consistent with the enhanced
enzymatic activity after biomineralization. Moreover, PB@Cyt c
composite exhibited remarkably enhanced catalytic efficiency
compared with PB/Cyt c mixture, with kcat/Km of 10.7 s-1·mM-1 for
PB@Cyt c composite and 1.5 s-1·mM-1 for PB/Cyt c mixture,
respectively. These results further demonstrate that PB@Cyt c
composite prepared via biomineralization exhibited improved
catalytic capability.
Furthermore, the coating of PB around Cyt c provided
shielding effect for encapsulated Cyt c, which endowed Cyt c
enhanced stability when exposed to harsh conditions. The
stability of enzyme under harsh conditions was vital for
biochemical analysis. As can be seen in Figure 3b, when Cyt c
was incubated in aqueous solutions under 80 oC for 3 h, it lost its
original conformation, leading to exposure of heme group and
increased activity of 3-fold was observed.[20] While the PB@Cyt c
composite retained almost 100% of its activity, implying that the
conformation of Cyt c encapsulated inside PB was retained.
Moreover, enzyme molecules tend to go through
a
microenvironment change of active sites when exposed to
organic solvents, which usually cause the loss of essential layer
of water around and activity fluctuation thus, hindering its
application in industry.[27] Here, free Cyt c and PB@Cyt c
composite were incubated in water, dimethylsulfoxide (DMSO),
500
a
c
e
b
400
300
200
100
0
o
acetonitrile (MeCN), and acetone (CP) at 30 C for 30 min and
samples were taken out for enzymatic assay. As shown in Figure
3c, free Cyt c exhibited 1.8-fold, 2-fold, 3-fold higher activity in
DMSO, MeCN and CP than its reference in aqueous buffer,
respectively. The hyperactivation of Cyt c after exposure to
organic solvents would lead to poor reproducibility during
industrial conditions with organic solvents. While almost no
fluctuation of activity was observed in the sample of PB@Cyt c
composite. The long-term storage stability of Cyt c after
encapsulation was also measured (Figure 3d). Free Cyt c and
PB@Cyt c composite retained almost 100% of activity even after
PB
PB/Cyt c
PB@Cyt c
Cyt c
PB PB@Cyt c PB/Cyt c
4000
3000
2000
1000
Wavenumber (cm-1)
d
C
PB@Cyt c
PB
O
Fe
N
PB@Cyt c
Cyt c
320
340
360
380
400
1000 800 600 400 200
Binding energy (eV)
0
Wavelength (nm)
incubated in aqueous buffer for
8
days under 37 oC,
f
Fe 2p
N 1s
708.2 eV
397.6 ev
demonstrating its stability in aqueous solution. These results
together suggested that PB shell around Cyt c during the
biomimetic mineralization endowed the encapsulated Cyt c with
enhanced stability against organic solvents and high temperature.
Furthermore, the recycle performance of PB@Cyt c composite
was investigated (Figure S3). PB@Cyt c composite retained 87.5%
of its initial activity after 5 batches. After 10 cycles, PB@Cyt c
composite loss 37.5% of activity, which was ascribed to the loss
of nanoparticles and partly enzymatic activity during
centrifugation process for next cycle. Thus, PB@Cyt c composite
exhibited good reuse performance.
PB
PB
708.6 eV
400.2 eV
PB@Cyt c
730
PB@Cyt c
740
720
710
410
405
400
395
390
Binding energy (eV)
Binding energy (eV)
Figure 2. (a) Relative activity of PB, Cyt c, PB/Cyt c mixture and PB@Cyt
c composite. (b) FTIR spectra of PB, PB/Cyt c mixture and PB@Cyt c
composite. (c) Fluorescence spectra of Cyt c and PB@Cyt c composite.
(d-f) XPS measurement of PB and PB@Cyt c composite. Full spectra (d),
high resolution spectra of Fe (e) and N (f).
Kinetic parameters to evaluate the catalytic capability of PB,
Cyt c, PB@Cyt c composite and PB/Cyt c mixture were obtained
by fitting experimental data with Michaelis-Menten equation
(Figure 3a, Figure S2 and Table S2). Free PB and Cyt c exhibited
Km of 1.3 mM and 1.2 mM, respectively. After integrating Cyt c
with PB via in-situ assembly, the obtained PB@Cyt c composite
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Cyt c
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In summary, peroxidase was in-situ encapsulated into
peroxidase mimics via a biomimetic mineralization process. The
as-synthesized PB@Cyt c composite exhibited a 2.6-fold higher
apparent activity than that of simple mixture of PB and Cyt c. The
enhanced apparent activity was attributed to the favorable
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We acknowledge the financial support from the National Natural
Science Foundation of China (21908070, 21676104, 21878105),
the National Key Research and Development Program of China
(2018YFC1603400, 2018YFC1602100), China Postdoctoral
Science Foundation (BX20180102, 2019M652902) and the
Fundamental Research Funds for the Central Universities
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Keywords: Enzymes • Immobilization • in-situ assembly •
synergistic effect
[1]
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Huisman, R. J. Kazlauskas, S. Lutz, J. C. Moore, K. Robins, Nature 2012,
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Entry for the Table of Contents (Please choose one layout)
Layout 2:
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Xiaoling Wu*[a], Jun Xiong[a], Shuli Liu[a],
Bin Chen[a], Shan Liang[a], Wenyong
Lou*[a,b] and Minhua Zong[a]
Page No. – Page No.
Peroxidase encapsulated in
peroxidase mimics via in-situ
assembly with enhanced catalytic
performance
Peroxidase encapsulated in peroxidase mimics via in-situ assembly exhibited a 2.6-
fold higher apparent catalytic activity and enhanced stability under harsh conditions.
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