Thermal responsive microgels as recyclable carriers to immobilize active proteins with enhanced nonaqueous biocatalytic performance

Article abstract of DOI:10.1039/c3cc46161k

We describe the preparation of a thermoresponsive microgel, which can non-covalently immobilize active proteins with enhanced biocatalytic performance in organic solvents and easy reusability due to the porous microstructure and temperature responsive property.

Full text of DOI:10.1039/c3cc46161k

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Thermal responsive microgels as recyclable carriers to
immobilize active proteins with enhanced nonaqueous
biocatalytic performance†
Cite this: Chem. Commun., 2013,
49, 11299
Received 12th August 2013,
Accepted 10th October 2013
Qing Wu, Teng Su, Yanjie Mao and Qigang Wang*
DOI: 10.1039/c3cc46161k
www.rsc.org/chemcomm
We describe the preparation of a thermoresponsive microgel, which in responsive microgels incorporating these properties would
can non-covalently immobilize active proteins with enhanced bio- exhibit particularly fascinating performance in biological catalysis.
catalytic performance in organic solvents and easy reusability due to
the porous microstructure and temperature responsive property.
Here, a typical thermoresponsive microgel was prepared by
copolymerization of N-isopropylacrylamide (NIPAM) with acryloylated
bovine serum albumin (BSA), non-covalently immobilized bioactive
The immobilization of enzymes aims at solving the problems of cata- hemoglobin (Hb) or horseradish peroxidase (HRP) via a swelling
lytic efficiency, stability and reusability of enzymes to some extent,1 effect at low temperature. This microgel serves as an efficient matrix
which is exceedingly important for the application of enzymes in for heterogeneous biocatalysis in various solvents. As a cyto-
various fields, including tissue engineering, fine chemical industry, compatible component, BSA is the most ample protein in plasma
bioremediation, and so on.2 One important approach to improve the and acts as a carrier for various compounds in the blood stream. As a
activity and stability of the immobilized enzymes is to explore thermo-sensitive component, poly(N-isopropylacrylamide) (PNIPAM)
suitable carrier materials with low diffusional limitation, large has been attracting increasing attention due to its uniqueness of
specific surface area, and high enzyme loading. The entrapped undergoing a volume phase transition around 32 1C; moreover, this
proteins can interact with solid supports by covalent coupling and transition is perfectly reversible.10 Therefore, the loading of Hb can
non-covalent forces. The robust chemical crosslinking method be mildly realized by lowering the temperature to approx. 4 1C. After
includes glutaraldehyde molecules3 and Lentikatss polymers,4 raising the temperature to room temperature (approx. 25 1C), the
which exhibit higher stability but heavy activity loss due to the immobilized active proteins (Hb and HRP) within the microgel will
change in enzyme structure. Recently, nano-structured materials5 not be expelled from the network due to the non-covalent interaction
have been applied to mildly immobilize proteins by non-covalent with the microgel. Sequentially, the enzymes entrapped in the
forces. Hamachi et al. have reported that supramolecular hydrogels microgel would be removed out of the network because of the
can preserve the active sites of enzymes and serve as a transportable weakening of hydrogen bonds by raising the temperature above
scaffold for biological activity.6 Therefore, non-covalent incorpora- the lower critical solution temperature (LCST). At last, the repro-
tion of enzymes into hydrogels is an alternative method.7 Moreover, ducible shrinking and re-swelling is beneficial to the recyclability
the enzymes immobilized within peptide-based supramolecular of both enzymes and the matrix in biocatalysis.
hydrogels can even exhibit apparent ‘‘superactivity’’ due to their
hydrophilic nature in the network.7a,8
Preparation of this microgel is very simple. Fig. 1 illustrates the
detailed procedure to prepare the microgel via an inverse emulsion
Relative to bulk hydrogels, microgels have several important polymerization method.11 The W/O inverse emulsion was created by
advantages, namely, good control over the particle size, fast dropwise addition of aqueous solution to the continuous oil phase
response to external stimuli and low diffusional limitation. and then sonication. Span80 (1.25%, w/w) dissolved in mineral oil
In addition, due to the uniqueness of exhibiting stimulus- constituted the continuous oil phase, while the aqueous phase was
responsive behaviour to external stimuli like temperature, pH formed by acryloylated BSA and NIPAM. The emulsion was homo-
and ionic strength, the responsive microgels are particularly attrac- genized by sonication and then initiated by g-ray irradiation for poly-
tive carriers for encapsulating drugs and proteins for drug delivery merization. Incorporation of enzymes into the microgel was carried
and biosensor applications.9 Therefore, the enzymes immobilized out by alternating changes in the external temperature. A constant
amount of microgel was mixed with a certain concentration of enzyme
solution and stored at 4 1C for 24 h to achieve equilibration. Then, the
whole solution containing the microgel was transferred to room tem-
Department of Chemistry, and Advanced Research Institute, Tongji University,
Shanghai 200092, PR China. E-mail: wangqg66@tongji.edu.cn
perature (approx. 25 1C). At last, the Hb containing microgel was
separated from the solution via centrifugation. The amount of the
† Electronic supplementary information (ESI) available: Experimental details and
supplementary figures. See DOI: 10.1039/c3cc46161k
c
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Fig. 1 The scheme for the preparation of microgels and their application as
active protein carriers.
bound active protein was calculated by subtracting the residue
from the total amount in solution analyzed by UV-Vis spectro-
scopy. The bound active protein has less than 5% loss in the
24 h incubation period.
Fig. 3 (a) Comparison of activities of Hb(U) and Hb(I) in various media. (b) The
initial reaction course of o-phenylenediamine (OPD) (10.0 mM) and H2O2 (40.0 mM)
in four kinds of organic solvents catalyzed by various Hbs (0.1 g LÀ1). (c) The
reusability of Hb(I) in toluene. (d) The relative catalytic activity of Hb(U) and Hb(I) at
temperatures from 25 1C to 37 1C in water.
We investigated the temperature-dependent sizes of the selected
microgels using SEM and DLS measurements. Fig. 2a and b show
the SEM morphology of the freeze-dried microgel particles, indicat-
ing preferably spherical shapes and monodispersity. Fig. 2c and d
display the temperature-dependent variation in the average dia-
meter of the particles from 10 1C to 55 1C. DLS measurements were
executed to determine the hydrodynamic diameter (DH) of the
microgels and their size distribution at different temperatures
varying from 4 1C to 55 1C. The measurements were executed after
allowing the sample to equilibrate for 5 min at each temperature.
The change in hydrodynamic diameter (DH) as a function of tem-
perature is displayed in Fig. 2c. It reveals that DH of the microgel
becomes smaller with the increase of temperature, illustrating that
the microgel exhibits pronounced thermosensibility. The value of
the swelling ratio (a) was found to be 53.0 by calculating the volume
ratio before and after phase transition according to the literature.12
The high value of a indicates the excellent swelling behavior of the
microgel, which makes the microgel suitable to encapsulate the
enzymes by lowering the temperature to approx. 4 1C.
unconfined Hb (Hb(U)) and immobilized Hb (Hb(I)). As shown in
Fig. 3a and b, the OPD oxidation reaction was demonstrated in
different solvents, e.g., including toluene, ethylacetate, and dioxane,
respectively. And the kinetic constants of Hb(U) and Hb(I) in various
environments are listed in Table 1. From Fig. 3a, it can be observed
that the activity of Hb(I) is almost the same as that of Hb(U) in water.
The microgel without Hb has no activity under the same conditions.
Hb(I) exhibits higher activity than Hb(U) in toluene, ethylacetate, and
dioxane. The results indicate that the microgel can provide the
aqueous microenvironment within organic solvents. The activities of
both Hb(U) and Hb(I) increase with the decrease of the polarity of the
organic solvent. As shown in Fig. 3b, Hb(I) exhibits the maximum
initial rate in hydrophobic toluene. Relative to Hb(U) in other organic
solvents, Hb(I) also shows observable enhancement in the initial
reaction rate. Even in the most hydrophilic dioxane, Hb(I) still has a
certain activity, while Hb(U) powder shows almost no catalytic ability.
Various kinetic constants of various Hbs were employed to
demonstrate the effect of the carrier and the solvent. The kinetic
constant values of Hb were calculated from the Lineweaver–Burk
plots established by the initial reaction rates at different o-phenylene-
diamine (OPD) concentrations (Fig. S1, ESI†). As shown in Table 1,
the activity of Hb(I) (Vmax = 0.25 mM sÀ1) is 0.76 times that of Hb(U)
(0.33 mM sÀ1) in bulk water, which does not display the positive effect
of immobilization. However, in the organic solvents, Hb(I) exhibits
enhanced activity relative to Hb(U). The Kcat values of Hb(I) in toluene
The H2O2 oxidation of o-phenylenediamine (OPD) to phenazine
was selected as a model reaction to explore the catalytic activity of
Table 1 Reaction kinetic constants of various Hbs in organic solvents and water
Vmax
Km
(mM)
Kcat
Kcat/Km
Solvent
(mM sÀ1
)
(sÀ1
)
(sÀ1 mMÀ1
)
Hb(I)
Toluene
Ethylacetate
Dioxane
Water
0.18
0.02
0.01
0.25
16.17
2.85
1.73
0.78
0.12
0.007
0.005
0.004
0.22
0.014
0.007
0.17
Hb(U)
Toluene
Ethylacetate
Dioxane
Water
0.007
0.001
0.0003
0.33
3.57
—
—
0.005
0.0007
0.0002
0.23
0.002
—
—
Fig. 2 SEM images of the microgels (a and b), hydrodynamic diameter of the
microgels as a function of temperature (c), and particle size distribution of the
microgels from 10 1C to 55 1C (d).
1.05
0.22
c
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(log P = 2.5), ethylacetate (log P = 0.73), and dioxane (log P = À1.1) were investigation of the immobilization of enzymes, which differ in their
25.7, 20, and 33 times higher than those of Hb(U), respectively. This sensitivity to hydrophobic environments.15 The Hb(I) in the microgel
trend indicates that the activity of Hb(I) appears to be affiliated with shows a slowing down of the catalytic rate at temperatures above
the solvent polarity. The enhancement of the solvent hydrophobicity 32 1C. The suitable operative temperature should be from 25 to 32 1C.
(log P) prompts the higher catalytic activity. Based on the Vmax values In contrast, Hb(U) always shows a slow increase of the catalytic rate
presented in Table 1, Hb(U) follows evidently the same tendency as along with the increasing temperature. The shrinkage of the microgel
Hb(I) in biological catalytic activity. This correlation is consistent with network above the transition temperature can lead to the inconve-
the theoretical explanation, which illustrates the enzyme catalytic nient diffusion of the product and slow activity.
ability in organic solvents.13 The low intrinsic activity of Hb(U) initiated
In summary, we demonstrate a simple and useful approach to
by the polar solvent may be due to their high hydrophilicity to remove immobilize enzymes in the thermo-sensitive microgel by changing
the essential water layer around the Hb surface. Thus, the enhanced the temperature from 4 1C to 25 1C. The desirable characteristics of
activity of the Hb(I) in the microgel could be attributed to the aqueous microgel-immobilized Hb with enhanced catalytic performance
environment which prevents the water extraction from the Hb surface have also been shown in aqueous and organic solvents. The catalytic
by organic solvents. Another active protein, HRP, displays much activity of the enzymes can also be modulated by temperature over a
higher catalytic ability and similar trends in organic solvents relative certain range. Our microgel is emerging to be a recyclable enzyme-
to Hb for the same biocatalytic reaction (Fig. S2 and Table S1, ESI†). immobilizing carrier for a large variety of applications.
The Vmax of immobilized HRP can reach about 74% of that in water.
In toluene, the immobilized HRP can achieve 10-fold activity com- Foundation of China (21274111), the Program for New Century
pared to the unconfined one. Excellent Talents in University of Ministry of Education of
This work was supported by the National Natural Science
In the three selected organic solvents, the Km (Michaelis con- China (NECT-11-0386), the Fundamental Research Funds for
stant) value for the encapsulated enzyme was found to increase with Central Universities, the Pujiang talents program (11PJ1409500),
the enhancement of hydrophobicity of the solvent (from dioxane to and the Recruitment Program of Global Experts.
toluene). Because of the low catalytic activity, the kinetic parameter
Km for Hb(U) can only be measured in toluene, among the three
kinds of organic solvents used for investigation. It is apparent from
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other bulk carriers, which are usually subject to remarkable mass
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c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 11299--11301 11301