Spatial co-localization of multi-enzymes by inorganic nanocrystal-protein complexes

Article abstract of DOI:10.1039/c4cc05478d

We report a simple precipitation method for the construction of spatially co-localized multi-enzyme systems based on inorganic nanocrystal-protein complexes. A spatially controlled multi-enzyme system exhibits enhanced overall catalytic performance, allowing for sensitive detection of glucose in solution. This journal is

Full text of DOI:10.1039/c4cc05478d

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Spatial co-localization of multi-enzymes by
inorganic nanocrystal–protein complexes†
Cite this: Chem. Commun., 2014,
0, 12465
5
a
a
a
b
a
a
Zhixian Li, Yifei Zhang, Yechao Su, Pingkai Ouyang, Jun Ge* and Zheng Liu*
Received 16th July 2014,
Accepted 22nd August 2014
DOI: 10.1039/c4cc05478d
www.rsc.org/chemcomm
We report a simple precipitation method for the construction of
spatially co-localized multi-enzyme systems based on inorganic
nanocrystal–protein complexes. A spatially controlled multi-enzyme
system exhibits enhanced overall catalytic performance, allowing for
sensitive detection of glucose in solution.
Compartmentalization is the key to achieve high efficiency of
biological reactions in living cells, where precisely co-localized
multiple enzymes catalyze various multi-step cascade reactions.
This compartmentalization in vivo inspires the construction
of artificial multi-enzyme systems enabling many promising
Scheme 1 Schematic illustration of the preparation of multi-enzyme incor-
porated complexes. (a) Randomly distributed multi-enzyme complexes as a
control; (b) spatially co-localized multi-enzyme complexes.
1
–3
applications.
Co-immobilization of enzymes on polymeric
4
,5
6–10
11,12
capsules, DNA scaffolds,
protein scaffolds,
and other
1
3–17
systems
has been proven to be effective for constructing
complex multi-enzyme systems. However, a robust and easy-to-use simple method, the spatial co-localization of enzymes can be
method for fabricating spatially co-localized multi-enzyme systems conveniently achieved.
with high catalytic efficiency has remained a challenge. In this study,
We chose horseradish peroxidase (HRP) and glucose oxidase
we present a simple method that produces multi-enzyme–inorganic (GOx) as the model enzymes for a cascade reaction. In the
nanocrystal complexes with pre-defined spatial co-localization of synthesis of spatially co-localized multi-enzyme complexes, as
enzymes, mimicking in vivo compartmentalization. We demonstrate shown in Scheme 1b, 50 mL of phosphate buffer saline solution
À1
that the spatially controlled co-localization of enzymes plays a crucial (PBS, 10 mM, pH 7.4) containing 0.25 mg mL of HRP was first
role in the overall enzyme activity enhancement of the artificial mixed with 1.5 mL of CuSO in water solution (200 mM) at 4 1C
4
multi-enzyme system.
for 24 h to form the HRP-incorporated complexes. Then, the
1
8
19
Recently, Ge et al. and Zeng and co-workers reported a HRP-incorporated complexes were incubated in another PBS
method of preparing enzyme–inorganic nanocrystal complexes solution containing 0.25 mg mL of GOx for another 24 h at
À1
with flower-like shapes by simply adding metal ions to the 4 1C to adsorb the second enzyme on the surface of the complexes
2+
protein solution in phosphate buffer. Protein molecules were by the coordination interaction between Cu and amino acids
complexed with copper phosphate nanocrystals and served as of proteins. Then the GOx@HRP complexes (GOx@HRP) were
‘
glue’ to bind the nanocrystals together to form the protein– obtained after centrifugation and washed with deionized water to
1
8
inorganic hybrid nanoflowers. In the present study, we remove the loosely adsorbed proteins. To examine the compart-
extended the above mentioned co-precipitation method to mentalization effect, the above mentioned method was also applied
construct multi-enzyme complexes. As shown in Scheme 1, by this to synthesize HRP@GOx in which GOx was used in the first step,
HRP in the second. Randomly distributed multi-enzyme complexes
a
Key Lab for Industrial Biocatalysis, Ministry of Education, Department of
(
5
GOx–HRP) were prepared as the control (Scheme 1a), in which
Chemical Engineering, Tsinghua University, Beijing 100084, China.
E-mail: junge@mail.tsinghua.edu.cn
0 mL of phosphate buffer saline (PBS, 10 mM, pH 7.4) solution
À1
À1
b
containing 0.25 mg mL of GOx and 0.25 mg mL of HRP was
mixed with 1.5 mL of CuSO in water solution (200 mM), followed
College of Biotechnology and Pharmaceutical Engineering,
Nanjing University of Technology, Nanjing 211816, China
4
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/c4cc05478d by a stationary incubation for 24 h at 4 1C.
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components (600 1C). During this stage, the total weight loss is
around 10%, confirming that the total weight percentage of proteins
loaded in complexes is approximately 10 wt%, agreeing well with
the BCA protein concentration measurements. However, the indi-
vidual weight percentage of GOx and HRP loaded onto the three
complexes were varied. For GOx–HRP, the content of HRP was
calculated by UV absorption at 403 nm, while the total protein
content was calculated by the BCA assay. For GOx@HRP and
HRP@GOx, the content of GOx and HRP was determined by the
BCA assay in each step of the preparation. The loading amounts of
GOx and HRP in GOx@HRP, HRP@GOx and GOx–HRP were
(2 wt%, 8 wt%), (8 wt%, 2 wt%) and (5 wt%, 5 wt%), respectively.
The overall enzymatic activities of GOx@HRP, HRP@GOx,
and GOx–HRP at the same total protein content were measured
using 20 mM glucose and 1 mM ABTS as the substrates in PBS
solution (Fig. 2a). The free GOx and HRP systems in solution
with the same protein amount and the corresponding GOx and
HRP ratios were utilized as controls. As shown in Fig. 2b, compared
to the free enzyme systems, all the three types of multi-enzyme
complexes showed increased overall enzymatic activity (140–310%).
Fig. 1 Structural characterizations of multi-enzyme incorporated complexes.
(
a) A typical transmission electron microscopy (TEM) image of GOx@HRP
inset: high-resolution TEM of one petal); (b) a typical scanning electron
microscopy (SEM) image of GOx@HRP (inset: high-resolution SEM of petals);
(
(
(
c)–(e) confocal microscopy images of (c) HRP@GOx, (d) GOx@HRP, and
e) GOx–HRP (HRP and GOx were labeled with RhB (red) and FITC (green), Obvious activity loss often occurs during enzyme immobiliza-
respectively).
tion or encapsulation, which is caused by either denaturation
of the enzymes or hindered mass transfer within the solid
Fig. 1a and b show the transmission electron microscopy carriers. A nanoscale matrix offers a way to reduce the mass
TEM) and scanning electron microscopy (SEM) images of the
(
complexes containing HRP and GOx. The high-resolution TEM
image of one petal of the complex shows the crystal structure of
the complex (Fig. 1a inset). The complexes have an average size
of B10 mm with highly porous structures (Fig. 1b). The high-
resolution SEM image of the petals shows that the complex is
formed by the stacked nanocrystals (Fig. 1b inset). The powder
X-ray diffraction pattern of the multi-enzyme incorporated
complexes fits that of Cu
agrees well with the results of our previous study. As we
discussed in our previous study, proteins induce the crystal-
3
(PO
4
)
2
Á3H
2
O (Fig. S1a, ESI†), which
1
8
1
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lization of Cu (PO ) Á3H O and bind the nanocrystals together to
3
4 2
2
assemble into flower-like structures. To investigate the spatial
distribution of enzymes, GOx and HRP were labeled with fluores-
cent probes fluorescein isothiocyanate (FITC) and rhodamine B
(RhB), respectively. The multi-enzyme complexes were observed
under a confocal microscope. Core–shell structures are clearly
observed for both HRP@GOx (Fig. 1c) and GOx@HRP (Fig. 1d).
And the Z-stack confocal images of GOx@HRP further confirmed
the structures (Fig. S2 and Movie S1, ESI†). In contrast, GOx–HRP
(
Fig. 1e) appears to have a random distribution of HRP and GOx
molecules.
The total enzyme loadings for all the three types of multi-
enzyme incorporated complexes were around 10 wt%, calculated by
measuring the protein concentration in solution using a standard
BCA method. Furthermore, we confirmed the incorporation of
Fig. 2 Overall activities of the multi-enzyme incorporated complexes.
a) A schematic representation of the cascade reaction catalyzed by a
(
enzymes by the thermal gravimetric analysis (TGA). For GOx@HRP GOx–HRP system; (b) comparison of overall activities of three types of
multi-enzyme incorporated complexes (absorbance was measured after
(
Fig. S1e, ESI†), the first-stage decomposition of the hybrid com-
incubation for 10 min). The GOx and HRP contents in GOx@HRP, HRP@GOx
and GOx–HRP were (2 wt%, 8 wt%), (8 wt%, 2 wt%) and (5 wt%, 5 wt%),
respectively. The overall activities of multi-enzyme incorporated com-
plexes were compared with the activities of free enzyme systems with the
plexes in air starts from 25 1C and finishes around 200 1C, caused by
the removal of physically adsorbed and bound water molecules. The
decomposition of enzymes starts at around 200–300 1C, which is
much lower than the decomposition temperature of the inorganic corresponding GOx and HRP contents.
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transfer distance, and thus allows enzymes to exhibit a similar retained B90% initial activity after incubating in water solution at
20–30
activity as their native counterparts.
In our study, the higher 25 1C for 7 days. In contrast, the free enzyme system in solution
enzymatic activity compared to the native counterparts is due to lost nearly 80% of the overall activity within 1 day. The enhanced
the interaction between copper ions in nanocrystals and the enzyme stability is due to the confined configuration of the
2
0–23
incorporated metalloenzymes, mechanism for which has been incorporated proteins, which prevents protein denaturation.
18
well elaborated in our previous study. More interesting, besides The almost unchanged activity of GOx@HRP also confirms that
the effect of copper phosphate nanocrystals, the spatial distribu- the enzyme-nanocrystal complex formed by co-precipitation and
tion of the two enzymes has an obvious influence on the overall adsorption is very stable in water solution, without obvious the
activity. GOx@HRP which has GOx on the outer shell and HRP in leakage of enzyme.
the core of the complex gave the highest overall activity, about
10% of activity compared to the free enzyme system.
We propose that the highest performance of GOx@HRP is
attributed to the ordered substrate transport on GOx@HRP in
3
As a demonstration, we applied GOx@HRP, which has the coherence with enzymatic cascade reactions. In the case of
highest activity, for the convenient, sensitive detection of glucose GOx@HRP, glucose and oxygen molecules first migrate to GOx
in solution. The cascade reaction catalyzed by GOx@HRP produces on the shell while the product of the GOx-catalyzed reaction,
ꢀ
+
ABTS which is UV-Vis detectable. In a typical experiment, the
2 2
H O , is subsequently directed to HRP in the core of the
absorbance at 405 nm was measured after 5 min of incubation of complexes. Substrate and intermediate concentration gradients
GOx@HRP in PBS solution containing glucose (0–100 mM) and help to drive and facilitate the GOx–HRP cascade, efficiently
ABTS (1 mM) at room temperature (Fig. S3a, ESI†). As shown in yielding a colored product. Especially, oxygen molecules that
Fig. 3a, a good linearity between the absorbance and the concen- are needed for the first reaction are more accessible when GOx
tration of glucose in the range of 0–20 mM was obtained (R = 0.995). is located on the surface of the complexes. To confirm this, we
The limit of detection (LOD) is 0.3 mM, lower than most of the studied the transport of the substrate and product molecules in
31–34
reported colorimetric glucose sensors.
visible detection of glucose can be achieved by using GOx@HRP scopy. By using the fluorescent glucose analogue 6-NBDG as an
Fig. S3b, ESI†). In comparison, free enzymes, HRP@GOx and indicator, the uptake of the substrate by GOx@HRP was visua-
In addition, a direct the GOx@HRP catalyzed reactions (Fig. 4a) by confocal micro-
(
HRP–GOx produced less sensitive signals (Fig. S3c, ESI†). The long- lized as shown in Fig. 4b. The fluorescent glucose analogue was
term storage stability of enzymes was improved upon incor- first captured by the complexes from solution due to the strong
poration into the complexes. As shown in Fig. 3b the GOx@HRP adsorption capability of the highly porous microspheres. Then
the substrate molecules gradually diffused into the inside of
microspheres (Fig. 4b and Movie S2, the video record can be
found in the ESI†). By utilizing glucose as the substrate for
GOx and Amplex Red as the substrate for HRP which can be
converted to the highly fluorescent resorufin, we observed the
generation of resorufin in the core of the complex where
HRP is located, immediately after subjecting the substrates
to GOx@HRP. As the enzymatic reaction proceeded, more
resorufin molecules were generated and gradually diffused
from the core to the bulk solution (Fig. 4c and Movie S3, the
video record can be found in the ESI†).
Fig. 4 Transport of substrates and products on GOx@HRP. (a) the cas-
Fig. 3 Detection of glucose in solution. (a) Using GOx@HRP with the
concentration of glucose in solution ranging from 0–100 mM and 0–20 mM
cade reaction catalysed by GOx and HRP using fluorescent substrates;
(b) confocal images showing the transport of fluorescent glucose analogue
6-NBDG on GOx@HRP; (c) confocal images showing the transport of
resorufin on GOx@HRP.
(inset) (absorbance was measured after incubation for 5 min); (b) stability of
GOx@HRP in aqueous solution at room temperature.
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In summary, we presented a facile method that yields multi-
enzyme–inorganic nanocrystal complexes with defined spatial
localization of enzymes. Using GOx and HRP as model enzymes,
we demonstrated that the high catalytic efficiency of the compart-
mentalized multi-enzyme systems is attributed to the directed
substrate transport in coherence with enzymatic reactions. The
ease of operation makes this method available for fabricating
multi-enzyme systems with defined spatial distribution of enzymes
so as to enable promising applications in biotechnology, diagnosis,
biosensing and biomedical devices.
This work was supported by the National High Technology
Research and Development Program (‘‘863’’ Program) of China
under the grant number of 2014AA020507, the National Natural
Science Foundation of China under the grant numbers 21036003
and 21206082, and the Tsinghua University Initiative Scientific
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