Biocompatible magnetic cellulose-chitosan hybrid gel microspheres reconstituted from ionic liquids for enzyme immobilization

Article abstract of DOI:10.1039/c2jm33033d

Magnetic Fe₃O₄-cellulose-chitosan hybrid gel microspheres were prepared by sol-gel transition technology using ionic liquids as solvent for cellulose and chitosan dissolution and regeneration. The synthesized microspheres were characterized by XRD, FTIR, SEM, and vibrating sample magnetometer (VSM). The XRD and FTIR results showed that chitosan and cellulose had been successfully coated onto the surface of Fe₃O ₄ after the preparation. SEM presented that the synthesized microspheres were regular sphere with a mean diameter of about 10 μm. Furthermore, it was found that glucose oxidase (GOx) could be successfully immobilized on the hybrid gel microspheres via glutaraldehyde technique. The immobilized enzyme presented higher thermostability, wider range of pH optima and improved storage stability in comparison with free GOx. The glucose conversion could reach 91.5% within 4 h and retained 84.2% of the initial activity after 15 cycles of use. These results demonstrate their potential applications in the field of biocatalysis. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2jm33033d

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PAPER
Biocompatible magnetic cellulose–chitosan hybrid gel microspheres
reconstituted from ionic liquids for enzyme immobilization
*
Zhen Liu, Haisong Wang, Bin Li, Chao Liu, Yijun Jiang, Guang Yu and Xindong Mu
*
Received 7th March 2012, Accepted 30th May 2012
DOI: 10.1039/c2jm33033d
Magnetic Fe3O4–cellulose–chitosan hybrid gel microspheres were prepared by sol–gel transition
technology using ionic liquids as solvent for cellulose and chitosan dissolution and regeneration. The
synthesized microspheres were characterized by XRD, FTIR, SEM, and vibrating sample
magnetometer (VSM). The XRD and FTIR results showed that chitosan and cellulose had been
successfully coated onto the surface of Fe3O4 after the preparation. SEM presented that the synthesized
microspheres were regular sphere with a mean diameter of about 10 mm. Furthermore, it was found that
glucose oxidase (GOx) could be successfully immobilized on the hybrid gel microspheres via
glutaraldehyde technique. The immobilized enzyme presented higher thermostability, wider range of
pH optima and improved storage stability in comparison with free GOx. The glucose conversion could
reach 91.5% within 4 h and retained 84.2% of the initial activity after 15 cycles of use. These results
demonstrate their potential applications in the field of biocatalysis.
liquids (ILs) to dissolve biomass has been found to be a very
interesting method in order to create possibilities for the
production of new types of materials, such as cellulose fibers,
beads, and film.9 Therefore, cellulose was widely used as a solid
support for enzyme immobilization. For example, Luo et al.10
synthesized magnetic cellulose microspheres for protein delivery.
Bagheri et al.11 prepared the cellulose–dendrimer composites for
immobilization of laccase. Turner et al.12 reported the prepara-
tion of cellulose–polyamine composite film and beads for bio-
catalyst. Most of the enzyme was immobilized to the carriers via
glutaraldehyde by covalent attachment, which was one of the
simplest and gentlest coupling methods in enzyme technology.
Glutaraldehyde can react with several functional groups of
proteins, such as amine, thiol, phenol, and imidazole.13 Some
authors investigated the ability of aldehydes to react with amino
acids, and they ranked the reactive moieties of the amino acids in
decreasing order of reactivity as follows: 3-amino, a-amino,
guanidinyl, secondary amino, and hydroxyl groups.14,15 The
presence of amino groups in chitosan turns it into a good option
for the enzyme immobilization through glutaraldehyde
technique.15
Introduction
During the past decades, enzymology and enzyme technology
have been widely used in the fields of food industry, textiles,
biotechnology, analysis and diagnostics, fine chemicals and
pharmaceuticals.1 The use of immobilized enzymes normally
offers several advantages over free enzymes, such as higher
stability, being more convenient to handle, easier separation
from the product, as well as more efficient recovery and reuse of
costly enzymes.2 A broad variety of immobilization techniques
have been applied to immobilize enzymes, including adsorption
to solid supports, covalent attachment, and polymer entrapment.
The formation of covalent bonds provides powerful links
between the enzymes and carrier supports. Therefore, it has been
the most studied method.
Typical solid supports used for immobilization of enzymes
include glass,3 silica,4 polymer,5 and electrode materials.6 Among
the various types, natural polymer is of great interest for its
biocompatible, nontoxic, and chemical stabilization. Cellulose
and chitosan are the two most abundant biorenewable natural
materials with many significant properties, such as biodegrad-
able, biocompatible, and bioactive, and they are very promising
raw materials in chemical and biological industries.7 However,
the potential materials have not been fully exploited because they
cannot be dissolved in conventional solvents due to the strong
inter- and intra-molecular hydrogen bonding.8 The use of ionic
Glucose oxidase (GOx, EC 1.1.3.4) has been found various
industrial applications, such as removal of glucose or oxygen
from food products and monitoring of the glucose levels in
miscellaneous samples using biosensors based on immobilized
enzyme.16 Therefore, it was chosen as a model oxidizing enzyme
to demonstrate the suitability of the cellulose based supports for
enzyme immobilization.
Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess
Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao
266101, People’s Republic of China. E-mail: wanghs@qibebt.ac.cn;
muxd@qibebt.ac.cn; Fax: +86 532 80662724; Tel: +86 532 80662723
Herein, we reported a simple method for preparing magnetic
cellulose–chitosan gel microspheres and investigated their
potential application in enzyme immobilization. The magnetite
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(Fe3O4) coated with biocompatible chitosan and cellulose can
enhance the stability of the synthesized microspheres.17 The
presence of amino groups in chitosan serves as stabilizer to
increase the stability of magnetic composites, which tend to
aggregate due to the strong magnetic dipole–dipole attractions
between particles. Moreover, the microspheres are easier to be
recovered and reused for the development of economically
feasible process by simple magnetic separation.
surface-bound enzyme. Finally, the products were stored in a DI
ꢀ
H2O bath at 2 C to be ready for all the tests.
Measurement of the yield of enzyme immobilized on the
composite microspheres
The amount of immobilized protein was calculated as
Q ¼ (Cpi ꢁ Cpf)V/W1
where, Q was the amount of immobilized protein on composite
microspheres (mg mgꢁ1), Cpi and Cpf were the protein concen-
tration in the enzyme solution before and after immobilization
(mg mLꢁ1), V was the volume of the enzyme solution (mL), and
W was the weight of the dried composite microspheres (mg).
Each sample was measured spectrophotometrically from 200 to
600 nm and protein content was estimated using the 215/225 nm
difference measurements.19 The GOx concentration (mg mLꢁ1) in
each solution was calculated from a calibration curve con-
structed from spectrophotometric measurements of known
concentrations (mg mLꢁ1) of the enzyme.
Experimental section
Chemicals
Glucose oxidase (GOx) from Aspergillus niger was purchased
from Sigma. Dissolving pulp (a-cellulose content was higher
than 92.5 wt%) was supplied by ChenMing Co. Ltd. (Shandong,
China). Chitosan (with a deacetylation degree of 90%) was
purchased from Sinopharm Chemical Reagent. The IL used in
this study was 1-butyl-3-methylimidazolium chloride (bmimCl),
which was obtained from Lihua Co. Ltd. (Henan, China). All
other reagents were of analytical grade and used without further
purification.
Activity of immobilized enzymes
The activity of immobilized enzymes was calculated by
measuring the amount of hydrogen peroxide (H2O2) formed
using a spectrophotometric method provided by Megazyme.20
GOx catalyzes b-D-glucose to D-glucono-d-lactone and releases
H2O2, which participate in a second reaction with the presence of
peroxidase (POD) involving p-hydroxybenzoic acid and 4-ami-
noantipyrine. The released quinoneimine dye was measured
spectrophotometrically at 510 nm.
Preparation of cellulose, cellulose–chitosan and magnetic
cellulose–chitosan composite microspheres18
The microspheres were prepared with water-in-oil suspension
and regeneration. The cellulose, chitosan and bmimCl were dried
at 80 ꢀC for 24 h in a vacuum oven before use. Cellulose, or
cellulose and chitosan were dissolved in bmimCl at 100 ꢀC for 30
min to obtain a 7 wt% (0.7 g biomass in 10 g bmimCl) clear and
viscous solution. For the preparation for magnetic cellulose–
chitosan composite microspheres, a certain amount of Fe3O4
(30 wt%, according to the weight of cellulose and chitosan) was
added to the solution and homogenized by continuous agitation
for 20 min. After that, the mixture was dispersed in 100 mL
vacuum pump oil with 5 wt% Span 80 under the agitation of 1000
rpm for 4 h at 100 ꢀC and cooled down at the rate of 5 ꢀC per 10
min to room temperature. During stirring, ethanol was added to
the suspension to further regenerate the microspheres from ILs.
After stewing for 12 h, the upper oil phase was poured out, and
the microspheres were washed five times with ethanol followed
by five washings with deionized (DI) water to remove the residual
ILs. Finally, the reconsꢀtituted hybrid microspheres were stored
in DI water at about 2 C.
The reactions involved are:
GOx
ƒƒƒƒƒ!
b-D-glucose þ O þ H O
D-glucono-d-lactone þ H O
2
2
2
2
(1)
2H2O2 þ p-hydroxybenzoic acid
POD
ƒƒƒƒƒƒƒ!
þ 4-aminoantipyrine
quinoneimine dye þ 4H O
2
(2)
Stability of the gel microspheres and the enzyme
For the stability testing of the synthesized gel microspheres, 50
mg gel microspheres were dispersed in HCl (pH ¼ 1) solution for
24 h, and the iron ion of the solution was analyzed by the
inductively coupled plasma optical emission spectroscopy (ICP-
OES, Optima 7000, PerkinElmer, USA). The long term storage
stability of the microspheres was studied by putting the micro-
Immobilization of enzymes on the reconstituted hybrid
microspheres
The hybrid microspheres were removed from DI water and
immersed in a mixed aqueous solution of 25 mL of 25 wt%
glutaraldehyde and 25 mL of 0.1 M phosphate buffer (pH 7.0)
with the agitation of 50 rpm for 18 h at room temperature. The
glutaraldehyde-bound microspheres were then mixed with 50 mL
of 0.1 M phosphate buffer solution (pH 7.0) containing 10 mg of
glucose oxidase. Enzyme immobilization was carried out at room
temperature for 4 h, followed by the continuous reaction at 2 ꢀC
for 16 h. After that, the products were washed with phosphate
buffer and followed by DI H2O to remove electrostatically
ꢀ
spheres into a sealed glass bottle for 2 months at 2 C, and fol-
lowed by ICP-OES tests.
The stability of the enzyme was studied in the presence of
different pH value, higher temperature, long term storage and
organic solvents. The pH of the solution (phosphate buffers)
ranged from 2 to 10. For the thermal stability test, the enzyme
solution was incubated at different temperature (up to 50 ꢀC) for
28 h and residual activity of the enzyme was determined as
described above. The storage stability of the enzyme was studied
15086 | J. Mater. Chem., 2012, 22, 15085–15091
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by assaying the residual activity at different time intervals. In
addition, organic solvents (methanol, ethanol and acetone)–
buffer mixtures with 20 wt% concentration of organic solvent
were prepared and the residual activity was assayed. All the
measurements were performed in duplicates.
The FTIR spectra of chitosan, cellulose, regenerated cellulose–
chitosan, and Fe3O4–cellulose–chitosan microspheres are dis-
played in Fig. 1. The absorption bands at 1655 and 1590 cmꢁ1 in
the chitosan spectra (Fig. 1b) were due to the absorption of
amide I and II groups (H–N–C]O), respectively.23 In the spectra
of the cellulose–chitosan (Fig. 1c) and Fe3O4–cellulose–chitosan
(Fig. 1d), the band at 1590 cmꢁ1 became weaker, which may be
due to the strong hydrogen bonding among Fe3O4, chitosan, and
cellulose.10,24 The band at about 1425 cmꢁ1 in the spectra of the
cellulose (Fig. 1a) and chitosan (Fig. 1b), assigned to the
symmetric CH2 bending vibration, shifted to a higher wave
number (1462 cmꢁ1) in the spectra of the cellulose–chitosan
(Fig. 1c) and Fe3O4–cellulose–chitosan (Fig. 1d).25 That was
because a change of the conformation of CH2OH at C-6.26 The
peak at 1154 cmꢁ1 in the spectra of the cellulose–chitosan
(Fig. 1c) and Fe3O4–cellulose–chitosan (Fig. 1d) was attributed
to the C–O–C stretch vibration, which was the contribution of
both the bands at 1161 cmꢁ1 in the spectra of the cellulose
(Fig. 1a) and 1148 cmꢁ1 in the spectra of the chitosan (Fig. 1b).
These results indicated the strong interactions among chitosan,
Fe3O4 and cellulose.
Characterization
The microspheres were in the form of hydrogels (water content
was about 92 wt%). Before the XRD, FTIR, SEM and VSM
characterization, the hydrogels were dried for 24 h to remove the
water. XRD patterns were recorded on the X-ray diffractometer
(D8-Advance, Bruker, Germany) with Cu Ka radiation (l ¼
ꢀ
ꢀ
1.5406 A) at 40 kv and 30 mA in the range of 10–80 at room
temperature. The morphology of the microspheres was deter-
mined with scanning electron microscopy (SEM, S-4800, Hita-
chi, Japan) with an accelerating voltage of 10 kV. Prior to image,
the samples were sputter-coated with gold thereby making the
samples conductive. Magnetization measurement of the micro-
spheres was performed with a vibrating sample magnetometer
(VSM, Lake Shore, 7410, USA) at room temperature under
ambient atmosphere. The Fourier transform infrared (FTIR)
spectra of all the samples were measured on a Nicolet iN 10 IR
spectrometer (Thermo Fisher Scientific Inc., USA) over the
frequency range of 4000–400 cmꢁ1 at a resolution of 4 cmꢁ1. The
KBr disk method was adopted to conduct the FTIR experiment.
The crystalline structure of the composite microspheres is
shown in Fig. 2. The six characteristic peaks ((220), (311), (400),
(422), (511), and (440)) for Fe3O4 (Fig. 2e) were consistent with
the JCPDS file (PDF No. 65-3107), which was the standard
pattern for crystalline magnetite with spinel structure.27
The original cellulose diffraction pattern (Fig. 2a) showed the
typical cellulose I structure, with a sharp peak at 22.5ꢀ and a wide
peak between 12ꢀ and 18ꢀ.28 The crystalline structure of chitosan
(Fig. 2b) was almost the same as that of cellulose, with a sharp
peak at 20ꢀ and a wide peak between 12ꢀ and 16ꢀ. The regen-
erated cellulose from ILs had the typical cellulose II structure
with a wide peak at about 12ꢀ, 20ꢀ and 22ꢀ (Fig. 2c) as reported in
literature.29 However, the combination of Fe3O4 with cellulose–
chitosan resulted in obvious crystal change. The peak at 20ꢀ ꢂ22ꢀ
(Fig. 2d) disappeared, indicating that chitosan and cellulose had
been successfully coated onto the surface of Fe3O4.
Results and discussion
The proposed scheme of the preparation for the magnetic Fe3O4–
cellulose–chitosan microspheres, the immobilization of GOx on
the microspheres, and the catalytic oxidation of glucose using the
immobilized microspheres were shown in Scheme 1. In the
process of preparing magnetic Fe3O4–cellulose–chitosan gel
microspheres, Fe3O4 particles could be easily coated by the
interactions with –NH2–OH groups of chitosan or cellulose.21
The cellulose also served as a support linked through electro-
static adsorption and hydrogen bonding.22 The immobilization
was achieved by using glutaraldehyde through the crosslink
between the surface of amine-functionalized microspheres and
the enzymes.
Scheme 1 Schematic illustration of the preparation for the magnetic
Fe3O4–cellulose–chitosan microspheres, the enzyme immobilization onto
the magnetic cellulose–chitosan microspheres, and their use for catalytic
oxidation of glucose.
Fig. 1 FTIR spectra for cellulose (a), chitosan (b), regenerated chito-
san–cellulose (c), and Fe3O4–chitosan–cellulose microspheres (d).
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Fig. 4 SEM images of the microspheres with different composition
ratios. (a) Cellulose; (b) cellulose : chitosan : Fe3O4 ¼ 2 : 1 : 1; (c) cellu-
Fig. 2 XRD of the cellulose (a), chitosan (b), regenerated chitosan–
cellulose microspheres (c), regenerated Fe3O4–chitosan–cellulose micro-
spheres (d), and the naked Fe3O4 (e).
lose : chitosan : Fe3O4
1 : 2 : 1.
¼
1 : 1 : 1; (d) cellulose : chitosan : Fe3O4
¼
Naked magnetic Fe3O4 particles are very susceptible to air
oxidation and aggregation in aqueous systems,30 resulting in
reduced saturation magnetization. This research indicated that
chitosan had high affinity to Fe3O4 particles, which enhanced the
stability of Fe3O4 by preventing their aggregation. The hysteresis
loop of naked Fe3O4 particles and Fe3O4–cellulose–chitosan
(1 : 1 : 2) microspheres were shown in Fig. 3. As can be seen, the
saturation magnetization (Ms) of the naked Fe3O4 and Fe3O4–
The rough materials (Fig. 4c and d) with higher chitosan content
could immobilize more proteins and could be correlated to
enzymatic activity (Table 1).
The magnetic Fe3O4–cellulose–chitosan microspheres were
used as supports for GOx immobilization as a model system to
demonstrate the applications in the field of biocatalysis. The
protein content loaded on the microspheres and specific activities
of the free and immobilized GOx are presented in Table 1, which
show that the amount of bound protein increased from 36 mg gꢁ1
to 108 mg gꢁ1, with the increase of the concentration of chitosan.
That should attribute to the more protein attachment sites of
primary amine groups (–NH2) introduced at the higher chitosan
percentage. However, the immobilized enzymes had lower
activity (13–36 U mgꢁ1) compared with free enzymes (50 U
mgꢁ1). The loss is most probably due to the orientation of the
enzyme or reacting with lysine resides within the protein and
making them non functional.15
cellulose–chitosan particles was 82.5 emu gꢁ1 and 14.7 emu gꢁ1
,
respectively, while the remanent magnetization (Mr) of the naked
Fe3O4 and Fe3O4–chitosan–cellulose microspheres was 21.3 emu
gꢁ1 and 2.6 emu gꢁ1, respectively. The results indicate that the
Fe3O4–cellulose–chitosan microspheres possess good magnetic
properties and will have a promising application in magnetic-
field assisted separation.
SEM images of cellulose–chitosan composite microspheres
with different composition ratios were shown in Fig. 4. The
shape of the cellulose microspheres was regular sphere and the
mean diameter was about 10 mm. The shape was distorted and
the surface became rough as the content of chitosan increased.
The immobilized enzyme was used for catalytic oxidation of
glucose and the experiment was performed with immobilized
GOx gel microspheres (0.3 g) in 50 mL of 0.1 M phosphate buffer
solution at 35 ꢀC with an initial glucose concentration of 580 mg
Lꢁ1. The dependence of glucose conversion on reaction time with
different catalysts was shown in Fig. 5. As can be seen, for the
catalyst (a), the enzymes were simply adsorbed at the surface of
cellulose without chemical bonding, where the interaction was
much weaker compared with the covalent bond interactions
between chitosan and enzymes through glutaraldehyde linkage
(b to d).
Therefore, the protein bound to the pure cellulose surface was
much less than that on cellulose–chitosan composite materials
and the conversion of glucose was thus much lower. With the
increase of the chitosan content ratio, the catalytic activities also
increased markedly. The best results were obtained on catalyst
(d), and the conversion of glucose quickly increased to 75.8%
within 2 h and finally reached 91.5% in 4 h.
The stability of the magnetic hybrid gel microspheres was
studied, as that was of critical importance for its successful
Fig. 3 Hysteresis loop of magnetic Fe3O4 particles and Fe3O4–cellulose–
chitosan microspheres.
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Table 1 The amounts and activities of enzymes immobilized on the magnetic cellulose–chitosan microspheres
Specific activity
(U mgꢁ1
)
Samplea
Bound proteinb mg gꢁ1
IEc %
Free
Cellulose
—
—
50 ꢃ 1
36 ꢃ 2
67 ꢃ 3
85 ꢃ 4.2
108 ꢃ 5
21.3 ꢃ 1.7
42.6 ꢃ 2.6
53.7 ꢃ 2.7
67.8 ꢃ 3.2
13 ꢃ 1.2
24 ꢃ 1.6
28 ꢃ 1.4
36 ꢃ 2
Cellulose/chitosan ¼ 2 : 1
Cellulose/chitosan ¼ 1 : 1
Cellulose/chitosan ¼ 1 : 2
a
The concentration of cellulose/chitosan was 7 wt% according to the weight of ILs and the weight of Fe3O4 particles accounted for 30 wt% of the weight
of cellulose/chitosan. b The bound protein was calculated according to the weight of the protein (mg) in the per g of the dried magnetic cellulose–chitosan
composite microspheres. IE is immobilization efficiency, which was defined as protein concentration after immobilization subtracted from initial
c
protein concentration in the percentage of initial protein concentration.
pH stability of the magnetic hybrid microspheres. On the other
hand, the good long term storage stability of the synthesized
microspheres was verified by the fact that there was no detectable
decomposition of the gel microspheres after storage in water for
2 months.
Enzyme stabilization was also taken into consideration in
order to realize their full potential usage as catalysts. The
stability of the catalyst was studied in the presence of pH,
organic solvents, thermal stability and considerable periods of
time to demonstrate the viability of this methodology for bio-
catalysis. Immobilization of enzyme to solid carriers is thought
to be the most useful strategy to improve operational stability
of biocatalysts.31 The immobilized enzyme had wider pH range,
higher thermal stability and improved storage stability
compared with the free one. Free GOx showed maximum
activity at pH 6, while pH optimum of the immobilized GOx
was much wider, in the pH range of 4–7. Also, the immobilized
Fig. 5 The dependence of glucose conversion on reaction time with
enzyme still had 63% of the initial activities after heating at 50
ꢀC for 28 h with pH 7, while the free enzyme only had 22% of
different catalysts. (a) Cellulose microspheres (b) magnetic cellulose–
chitosan microspheres with the ratio of cellulose to chitosan was 2 : 1, (c)
its initial activities under the same conditions. The results were
in accordance with those reported in literature.16,32 After
1 : 1, and (d) 1 : 2.
ꢀ
storage at 2 C for 2 months, the free GOx retained 80% of the
initial activity, while the immobilized GOx retained 92% of the
applications. The pictures of the microspheres dispersed in
water (a) and after crosslinked with glutaraldehyde (b) were
shown in Fig. 6. The well dispersed solution indicated that
glutaraldehyde was most likely to conjugate enzyme GOx onto
the surface of chitosan–cellulose–magnetic microspheres rather
than to crosslink the magnetic microspheres to form larger
aggregates.
ICP-OES results showed that no Fe2+or Fe3+ was detected in
the microsphere solution (pH ¼ 1) after 24 h, indicating the good
Fig. 7 Reuse of magnetic cellulose–chitosan microspheres (the ratio of
cellulose to chitosan was 1 : 2) in the enzymatic catalytic oxidation of
glucose.
Fig. 6 The pictures of the microspheres dispersed in water (a) and after
crosslinked with glutaraldehyde (b).
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6 M. Hiller, C. Kranz, J. Huber, P. Bauerle and W. Schuhmann, Adv.
Mater., 1996, 8, 219.
initial activity. The activity of immobilization of enzymes on
cellulose based supports in the presence of organic media was
studied as well, and the results showed that in all organic
solvent–buffer mixtures, the activity was decreased with the
increase of solvent concentration. However, the immobilized
enzyme showed higher activity than the free enzymes. The
activities of the immobilized enzyme in methanol–buffer,
acetone–buffer and ethanol–buffer mixtures were 87%, 92%
and 85%, respectively. The immobilization on cellulose based
supports could prevent unfolding of the enzyme by the organic
solvents.33
7 W. K. Czaja, D. J. Young, M. Kawecki and R. M. Brown Jr,
Biomacromolecules, 2007, 8, 1–12; D. Klemm, B. Heublein,
H. P. Fink and A. Bohn, Angew. Chem., Int. Ed., 2005, 44, 3358–
3393; J. K. F. Suh and H. W. T. Matthew, Biomaterials, 2000, 21,
2589–2598; M. Kumar, R. A. A. Muzzarelli, C. Muzzarelli,
H. Sashiwa and A. J. Domb, Chem. Rev., 2004, 104, 6017–
6084.
8 N. Sun, R. P. Swatloski, M. L. Maxim, M. Rahman, A. G. Harland,
A. Haque, S. K. Spear, D. T. Daly and R. D. Rogers, J. Mater.
Chem., 2008, 18, 283–290; R. Rinaldi, R. Palkovits and F. Schueth,
Angew. Chem., Int. Ed., 2008, 47, 8047–8050.
9 R. P. Swatloski, S. K. Spear, J. D. Holbrey and R. D. Rogers, J.
Am. Chem. Soc., 2002, 124, 4974–4975; H. Xie, S. Zhang and
S. Li, Green Chem., 2006, 8, 630–633; D. A. Fort, R. C. Remsing,
R. P. Swatloski, P. Moyna, G. Moyna and R. D. Rogers, Green
Chem., 2007, 9, 63–69; I. Kilpelainen, H. Xie, A. King,
M. Granstrom, S. Heikkinen and D. S. Argyropoulos, J. Agric.
Food Chem., 2007, 55, 9142–9148; J. Cai and L. Zhang,
Macromol. Biosci., 2005, 5, 539–548; J. Cai, L. Zhang, J. Zhou,
H. Qi, H. Chen, T. Kondo, X. Chen and B. Chu, Adv. Mater.,
2007, 19, 821.
In addition, the reusability of the immobilized enzyme was
investigated and the results are exhibited in Fig. 7, which shows
that the immobilized enzyme retained 84.2% of its initial activity
after 15 uses. The higher stability of the enzyme immobilized on
magnetic hybrid microspheres could be ascribed to the covalent
binding of enzymes, which prevented the enzymes detaching
from the carrier.
10 X. Luo, S. Liu, J. Zhou and L. Zhang, J. Mater. Chem., 2009, 19,
3538–3545.
11 M. Bagheri, H. Rodriguez, R. P. Swatloski, S. K. Spear, D. T. Daly
and R. D. Rogers, Biomacromolecules, 2008, 9, 381–
387.
12 M. B. Turner, S. K. Spear, J. D. Holbrey and R. D. Rogers,
Biomacromolecules, 2004, 5, 1379–1384; M. B. Turner, S. K. Spear,
J. D. Holbrey, D. T. Daly and R. D. Rogers, Biomacromolecules,
2005, 6, 2497–2502.
13 A. F. S. Habeeb and R. Hiramoto, Arch. Biochem. Biophys., 1968,
126, 16.
14 D. Hopwood, C. R. Callen and M. McCabe, Histochem. J., 1970, 2,
137–150; M. E. Ortiz-Soto, E. Rudino-Pinera, M. E. Rodriguez-
Alegria and A. Lopez Munguia, BMC Biotechnol., 2009, 9, DOI:
10.1186/1472-6750-9-68.
15 I. Migneault, C. Dartiguenave, M. J. Bertrand and K. C. Waldron,
BioTechniques, 2004, 37, 790.
16 A. Vikartovska, M. Bucko, D. Mislovicova, V. Paetoprsty, I. Lacik
and P. Gemeiner, Enzyme Microb. Technol., 2007, 41, 748–
755.
17 Y. Wang, A. S. Angelatos and F. Caruso, Chem. Mater., 2008, 20,
848–858; J. M. R. Grech, J. F. Mano and R. L. Reis, J. Mater.
Sci.: Mater. Med., 2010, 21, 1855–1865.
Conclusion
In this research, magnetic Fe3O4–cellulose–chitosan micro-
spheres were prepared by a simple process utilizing ionic liquids
as solvent and these microspheres were proved to be excellent
carriers for enzyme immobilization. GOx (glucose oxidase) was
immobilized on the prepared microspheres and characterized as
a model protein. The GOx-immobilized microspheres exhibited
good catalytic activity for glucose oxidation and retained
significant activity (84.2% of the initial activity) after 15 cycles of
use through simple magnetic separation. Further studies on the
immobilization of different biocatalysts are under way, and this
type of biocompatible material is proposed to have promising
potential in biomedical, magnetic-field assisted drug delivery
systems, cell/enzyme immobilization, and many other related
fields.
18 R. P. Swatloski, J. D. Holbrey, J. L. Weston and R. D. Rogers, Chim.
Oggi, 2006, 24, 31.
19 P. Wolf, Anal. Biochem., 1983, 129, 145–155.
20 A. Aitken and M. Learmonth, Protein Determination by UV
Absorption, 1996.
21 S. Hong, Y. Chang and I. Rhee, J. Korean Phys. Soc., 2010, 56, 868–
873.
22 S. Alila, S. Boufi, M. N. Belgacem and D. Beneventi, Langmuir, 2005,
21, 8106–8113.
23 J. Brugnerotto, J. Lizardi, F. M. Goycoolea, W. Arguelles-Monal,
J. Desbrieres and M. Rinaudo, Polymer, 2001, 42, 3569–
3580.
24 P. R. Chang, J. Yu, X. Ma and D. P. Anderson, Carbohydr. Polym.,
2011, 83, 640–644.
Acknowledgements
This work was supported by the Natural Science Foundation of
China and Shandong Province (no. 21003146, no. 20803038,
ZR2010BQ014), the Key Science and Technology Program of
Shandong Province (no. 2008GG20002038), the Qingdao Key
Technology Program (no. 09-1-4-1-nsh), the Key Science and
Technology
Program
of
Shandong
Province
(no.
2007GG2QT07006), and the Knowledge Innovation Program of
the Chinese Academy of Sciences (no. KSCX2-EW-J-10).
25 T. Kondo and C. Sawatari, Polymer, 1996, 37, 393–399; K. Kamide,
K. Okajima and K. Kowsaka, Polym. J., 1992, 24, 71–
86.
26 S. Y. Oh, D. I. Yoo, Y. Shin, H. C. Kim, H. Y. Kim, Y. S. Chung,
W. H. Park and J. H. Youk, Carbohydr. Res., 2005, 340, 2376–
2391; D. Ruan, L. N. Zhang, Y. Mao, M. Zeng and X. B. Li, J.
Membr. Sci., 2004, 241, 265–274.
References
1 S. A. Cetinus and H. N. Oztop, Enzyme Microb. Technol., 2003, 32,
889–894; P. Bajpai, Biotechnol. Prog., 1999, 15, 147–157.
2 R. A. Sheldon, Adv. Synth. Catal., 2007, 349, 1289–1307; C. Mateo,
J. M. Palomo, G. Fernandez-Lorente, J. M. Guisan and
R. Fernandez-Lafuente, Enzyme Microb. Technol., 2007, 40, 1451–
1463.
3 T. Laurell, J. Drott and L. Rosengren, Biosens. Bioelectron., 1995, 10,
289–299.
4 S. E. Letant, B. R. Hart, S. R. Kane, M. Z. Hadi, S. J. Shields and
J. G. Reynolds, Adv. Mater., 2004, 16, 689.
27 Z. Y. Ma, Y. P. Guan and H. Z. Liu, J. Polym. Sci., Part A: Polym.
Chem., 2005, 43, 3433–3439.
28 A. P. Mathew, K. Oksman and M. Sain, J. Appl. Polym. Sci., 2005,
97, 2014–2025; G. Cheng, P. Varanasi, C. Li, H. Liu,
Y. B. Menichenko, B. A. Simmons, M. S. Kent and S. Singh,
Biomacromolecules, 2011, 12, 933–941; I. P. Samayam,
B. L. Hanson, P. Langan and C. A. Schall, Biomacromolecules,
2011, 12, 3091–3098.
5 A. Vilkanauskyte, T. Erichsen, L. Marcinkeviciene, V. Laurinavicius
and W. Schuhmann, Biosens. Bioelectron., 2002, 17, 1025–
1031.
15090 | J. Mater. Chem., 2012, 22, 15085–15091
This journal is ª The Royal Society of Chemistry 2012

View Article Online
29 P. Mansikkamaki, M. Lahtinen and K. Rissanen, Cellulose, 2005, 12,
233–242; Z. Liu, H. Wang, Z. Li, X. Lu, X. Zhang, S. Zhang and
31 P. V. IyerandL. Ananthanarayan, Process Biochem., 2008, 43, 1019–1032.
32 L. Betancor, F. Lopez-Gallego, A. Hidalgo, N. Alonso-Morales,
G. Dellamora-Ortiz, J. M. Guisan and R. Fernandez-Lafuente, J.
Biotechnol., 2006, 121, 284–289.
K.
227.
Zhou,
Mater.
Chem.
Phys.,
2011,
128,
220–
30 J.-F. Liu, Z.-S. Zhao and G.-B. Jiang, Environ. Sci. Technol., 2008, 42,
6949–6954.
33 A. Fishman, I. Levy, U. Cogan and O. Shoseyov, J. Mol. Catal. B:
Enzym., 2002, 18, 121–131.
This journal is ª The Royal Society of Chemistry 2012
J. Mater. Chem., 2012, 22, 15085–15091 | 15091