Improving the properties of β-galactosidase from Aspergillus oryzae via encapsulation in aggregated silica nanoparticles

Article abstract of DOI:10.1039/c3nj00685a

In this study, a new immobilization method was exploited to encapsulate β-galactosidase (β-gal) from Aspergillus oryzae using aggregated core-shell silica nanoparticles as a matrix. Transmission electron microscopy (TEM) and Fourier transform infrared (FTIR) spectroscopy were used to characterize the material encapsulated β-gal. Compared to the free β-gal, the encapsulated β-gal shows a broader pH tolerance and thermal stability. Furthermore, the encapsulated β-gal shows better storage stability over 30 days. After nine cycles of hydrolytic reaction, the encapsulated β-gal still maintains 94.2% of its initial activity, which indicates that the β-gal exhibits excellent reusability after encapsulation.

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Improving the properties of b-galactosidase from
Aspergillus oryzae via encapsulation in aggregated
Cite this: DOI: 10.1039/c3nj00685a
silica nanoparticles
Zhuofu Wu,ab Zhi Wang,b Buyuan Guan,a Xue Wang,a Ye Zhang,a Yu Xiao,a
Bo Zhi,a Yunling Liu,a Zhengqiang Li*b and Qisheng Huo*a
In this study, a new immobilization method was exploited to encapsulate b-galactosidase (b-gal) from
Aspergillus oryzae using aggregated core–shell silica nanoparticles as a matrix. Transmission electron
microscopy (TEM) and Fourier transform infrared (FTIR) spectroscopy were used to characterize the
material encapsulated b-gal. Compared to the free b-gal, the encapsulated b-gal shows a broader
pH tolerance and thermal stability. Furthermore, the encapsulated b-gal shows better storage stability
over 30 days. After nine cycles of hydrolytic reaction, the encapsulated b-gal still maintains 94.2% of its
initial activity, which indicates that the b-gal exhibits excellent reusability after encapsulation.
Received (in Montpellier, France)
25th June 2013,
Accepted 29th August 2013
DOI: 10.1039/c3nj00685a
www.rsc.org/njc
In this study, we used silica cross-linked micellar nano-
particles as the building units to create the host material for
1. Introduction
enzyme immobilization.20,21 The silica nanoparticles consist of
hydrophobic cores containing poly propylene oxide segments
and hydrophilic shells formed by poly propylene oxide segments
surrounded by a thin silica layer. These high-quality silica nano-
particles can be easily synthesized while possessing good dispersion
in water and biocompatibility. Due to the existence of the silica
shell, the nanoparticles easily assemble together due to a state of
lower surface energy.22 As a simple and irreversible process, lyophi-
lization can promote the aggregation of the silica nanoparticles
under the stress of freezing and dehydration. The interstices formed
by the stacked nanoparticles provide sufficient space to encapsulate
enzymes. Furthermore, the stacked nanoparticles possess abundant
three dimensional mass transfer voids which will be beneficial for
the diffusion of substrates. The silica encapsulated b-gal was
prepared by lyophilization of a mixture of the silica nanoparticles
and b-gal. The encapsulated b-gal shows good pH stability, thermal
stability, storage stability and reusability.
Immobilization is an important technique in the enzyme
engineering field, which has been widely used to improve the
enzyme performance (enzyme activity, stability, etc.).1–6 Many
types of immobilization technology have been developed and
the sol–gel method is one of the most widely used immobilization
techniques.7–10 Especially, enzyme immobilization in silica gels
has been well studied during the past few decades.11–14 For
example, Ellerby et al. encapsulated metalloprotein and heme
proteins in porous silica glass matrices under mild conditions
using a standard sol–gel process and found that the encapsulated
enzymes maintained their enzymatic activity and spectroscopic
properties.15 Gill and Ballesteros carried out the entrapment of
proteins in poly(glyceryl silicate) sol–gel and verified that the
entrapped enzyme had high stability.16 Ferrer et al. encapsulated
horseradish peroxidase by an alcohol-free sol–gel route and found
that the embedded enzyme retained a more native conformation
than that produced by a regular sol–gel process.17 Although great
efforts have been made, some disadvantages of the sol–gel
method, such as the brittleness of the sol–gel matrix, the complex
pH control when preparing a biocompatible sol–gel matrix and
the pore collapse during xerogel formation, have yet to be over-
come.18,19 Therefore, a simple and practical method is required to
overcome these problems.
2. Experimental
2.1 Materials
b-Galactosidase (b-gal) obtained from Aspergillus oryzae was
purchased from Amano Enzymes Co. (Nagaya, Japan). o-Nitro-
phenol-b-D-galactoside (ONPG) was purchased from BBI Co.
Ltd. (Boston, MA, USA). Tetraethyl orthosilicate (TEOS), Pluronic
F127 and diethoxydimethylsilane (Me2Si(OEt)2, DEDMS) were
commercially available from Sigma-Aldrich (St. Louis, Missouri,
USA). Octanoic acid was purchased from Tianjin Guangfu
Fine Chemical Research Institute of China. All other chemicals
a State Key Laboratory of Inorganic Synthesis and Preparative Chemistry,
College of Chemistry, Jilin University, Changchun 130012, China.
E-mail: huoqisheng@jlu.edu.cn; Fax: +86-431-85168602; Tel: +86-431-85168602
b Key Laboratory for Molecular Enzymology and Engineering of the Ministry of
Education, College of Life Sciences, Jilin University, Changchun 130012, China.
E-mail: lzq@jlu.edu.cn; Fax: +86-431-85155201; Tel: +86-431-85155201
c
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and reagents were of analytical grade. All aqueous solutions system contained 100 ml of ONPG (50 mM) and 800 ml of HAc–
were prepared with Milli-Q water.
NaAc buffer (50 mM, pH 4.5). The hydrolytic reaction was triggered
by adding the enzyme. And then, the reaction mixture was
incubated in a water bath at 37 1C for 10 min. Thereafter, the
2.2 Synthesis of silica cross-linked micellar nanoparticles
In a typical preparation, 0.54 g of Pluronic F127 was dissolved reaction was stopped by adding 2 ml of Na2CO3 solution (0.5 M)
in 10 g of HCl solution (0.57 M) at room temperature. Octanoic followed by centrifugation at 3500g for 3 min. The amount of
acid (0.8 g) was added to the mixture followed by ultrasound produced ortho-nitro-phenol (ONP) was determined at 410 nm.
treatment for 1.5 h and then was stirred for 2 days. 1 g of TEOS The enzyme activity (1 U) was defined as the amount of b-gal that
was added to the above solution. The mixture was stirred at liberates 1 mmol ONP per min under the above experimental
room temperature for 0.5 h, and 0.08 g of DEDMS was added. conditions.
Stirring was continued at room temperature for 0.5 h followed
by filtration using a Teflon filter (0.22 mm).
2.6 Stability of encapsulated b-gal
HCl and EtOH (resulting from the hydrolysis of TEOS and
pH stability. The pH stability of the free b-gal and the
DEDMS) were removed by dialysis (Millipore, MW cutoff 8000– encapsulated b-gal was determined by incubating the enzyme
12 000) against pure water with stirring at room temperature. in different buffers (pH 2.5–10.8) for 1 h at 25 1C. The buffers
The water was refreshed every 2 hours. The dialysis process was used were citric acid–sodium citrate (pH 2.5–4.6), K2HPO4–
monitored by measuring pH of the solution and terminated at KH2PO4 (pH 5.8–8.0) and Gly–NaOH (pH 9.3–10.8). The encap-
pH 7.0. Then, the silica cross-linked micellar nanoparticles sulated b-gal was collected by centrifugation (3500g for 3 min)
solution (40 mg silica nanoparticle per ml) was obtained.
and air dried. And then the residual b-gal activity was deter-
mined at its optimal pH according to the assay in Section 2.5.
Temperature stability. Free or the encapsulated b-gal was
2.3 Encapsulation of b-gal
Crude b-gal was purified as follows: 2 g of crude b-gal was added to HAC–NaAC buffer (50 mM, pH 4.5), followed by
dissolved in 10 ml of HAC–NaAC buffer (50 mM, pH 4.5) in an incubation at different temperatures (40–70 1C) for 120 min.
ice bath. The b-gal solution was centrifuged at 3500g for 3 hours The specific activity of free or the encapsulated b-gal was
using an ultrafiltration device (Millipore, MW cutoff 10 000). assayed according to the description in Section 2.5.
The suspension was lyophilized. The b-gal solution (8 mg mlÀ1
)
Storage stability. The encapsulated b-gal was kept at room
was prepared by re-dissolving the lyophilized b-gal in the buffer temperature under dry conditions (air-dried) for 30 days. The
(pH 4.5) at 4 1C. residual activity was checked from time to time according to the
Purified b-gal solution (5 ml, 8 mg mlÀ1) was added to 10 ml description in Section 2.5.
of the silica cross-linked micellar nanoparticle solution with
Reusability. The encapsulated b-gal was used in successive
slow stirring at 4 1C. The resulting solution was frozen at batches. The relative activity of the encapsulated b-gal was
À20 1C for 30 min and then lyophilized. The lyophilized b-gal measured according to the assay in Section 2.5. After each
was repeatedly rinsed with Milli-Q water at room temperature, cycle, the reaction mixture was centrifuged at 3500g for 3 min.
until no absorbance of the protein in the supernatant could be The obtained precipitate was washed with HAC–NaAC buffer
detected. The wet sample was lyophilized again. Finally, the (50 mM, pH 4.5) to remove any residual substrate or product
lyophilized powder was stored at 4 1C.
and then kept overnight in a vacuum oven at room temperature
The protein content of the enzyme solution was determined for complete drying. After drying, the powder was used in the
using the Bradford method.23 Bovine serum albumin was used next cycle under otherwise equivalent conditions.
as standard. The encapsulation yield of b-gal is 89.2% in this
experiment.
3. Results and discussion
2.4 Characterization of silica cross-linked micellar
nanoparticles
3.1 Synthesis and assembly of silica nanoparticles
We designed a novel method for encapsulating b-galactosidase
Transmission electron microscopy was performed using an FEI (b-gal) from Aspergillus oryzae through assembly of the silica
Tecnai G2 F20 s-twin (FEI, American) at 200 keV. The particle nanoparticles (Fig. 1).
size of the silica nanoparticles was measured by a Malvern
Uniform spherical particles (90 nm) can be observed in
Zetasizer Nano-S instrument at room temperature using the Fig. 2a, which is consistent with the results of light scattering
Dynamic Light Scattering (DLS) principle with a HeNe laser measurements (Fig. 2c). The inset of Fig. 2a demonstrates that
(633 nm). The FTIR spectrum was recorded on a Nicolet 5700 an individual silica nanoparticle is a solid sphere and its
FTIR spectrometer with a resolution of 4 cmÀ1 through the KBr surface does not possess any large porous structure. From
method.
Fig. 2b, the aggregation of the silica nanoparticles can be found
after lyophilization and the shape of the silica nanoparticles
during the aggregation does not change. Furthermore, the
2.5 Enzyme activity assay (ONPG assay)
The enzyme activity of b-gal was measured using ONPG aggregated silica nanoparticles can form many interstices
(O-nitrophenyl-b-D-galactopyranoside) as a substrate according to (about 20 nm  20 nm  20 nm, calculated by the formulation
our previous method with a slight modification.24 The reaction of the interstice (hole) in closest-packing of spheres25), which
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Fig. 1 The new immobilization method for encapsulating b-gal in the aggre-
gated silica nanoparticles. The gray sphere and the black ellipsoid represent the
silica nanoparticles and b-gal, respectively.
Fig. 3 FTIR spectrum of: (a) the aggregated silica nanoparticles, (b) the aggre-
gated silica nanoparticles loaded with b-gal, and (c) pure b-gal.
Fig. 2 (a) TEM images of the silica nanoparticles. Inset: individual silica nano-
particle. (b) The matrix loaded with b-gal. (c) Light scattering measurement of the
silica nanoparticles.
might provide enough room to accommodate b-gal (16 nm Â
11 nm  11 nm).26
It’s known that the lyophilisation process can induce the
aggregation of nanoparticles.27 In this study, lyophilisation
accelerates water removal so as to promote the formation of
the silica nanoparticle matrices. Furthermore, freezing and
dehydration stresses generated by lyophilisation also help to
form the silica nanoparticles matrices. Moreover, no obvious
difference between the aggregated silica nanoparticles with
b-gal and the counterpart without b-gal was found in TEM
images.
Fig. 4 pH stability of free b-gal (’) and encapsulated b-gal (J) at different pH
media.
3.6–9.3 (Fig. 4). As for the encapsulated b-gal, the pH stability
curve exhibits a plateau over a broad pH range of 2.5–10.6
(Fig. 4). The experimental data demonstrate that the pH stability
has been improved by encapsulation of b-gal in aggregated silica
nanoparticles.
To confirm the encapsulation of b-gal in the matrix, the
FT-IR spectra of the silica nanoparticles, b-gal and the matrix
loaded with b-gal were studied. The characteristic bands of
Si–O–Si could be observed at 808 and 1100 cmÀ1 in Fig. 3a28,29
and the characteristic bands of the b-gal can be observed at
1644 and 1552 cmÀ1 in Fig. 3c.30 After immobilization, all these
characteristic bands for both b-gal and Si–O–Si can be found in
Fig. 3b, which verifies that b-gal is successfully entrapped in the
aggregated silica nanoparticles.
Wang et al. found catalase encapsulated in nanoporous
silica spheres to have improved stability against pH change
compared with the free enzyme by the physical adsorption
method, which was consistent with our results.31
The thermal inactivation of b-gal often limits its application.
In this study, the thermal-stability of free and the encapsulated
b-gal in the range of 40–70 1C has been investigated to assess
the potential advantage of the encapsulated b-gal and the
results are shown in Fig. 5. After 120 min incubation, the free
and the encapsulated b-gal were stable at lower temperature
(40–50 1C) and became vulnerable to inactivation at higher
3.2 Stability
pH stability is an important parameter for an immobilized temperature (50–70 1C) at which the specific activity of encap-
enzyme. We investigated the pH stability of the encapsulated sulated b-gal decreased at a slower rate than that of the free
b-gal by first incubating the encapsulated b-gal at different one. These results suggested that the encapsulated b-gal could
pH values (2.5–10.6) for 30 min and then measuring the activity exhibit a better thermo-stability at higher temperatures. The
at its optimal pH. The free b-gal is stable between pH values encapsulation of b-gal in aggregated silica nanoparticles might
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of inactivation of b-gal as a result of encapsulation in aggre-
gated silica nanoparticles.
It’s known that the immobilized enzyme can be reused to
decrease the cost of enzymes for an enzymatic reaction. We
investigated the reusability of the encapsulated b-gal and found
that the enzyme activity remained at almost 94.2% of its initial
activity even after nine reaction cycles (Table 2).
4. Conclusions
In this study, a novel encapsulation method was exploited to
immobilize b-galactosidase (b-gal) in aggregated silica nano-
particles through a simple lyophilization step. Once the assembly
of the silica nanoparticles occurred, the matrix formed by the
building units exhibited perfect stability in aqueous solution. The
interstice formed by the aggregated silica nanoparticles provided
a comfortable microenvironment for b-gal. The encapsulated b-gal
exhibited good pH stability, temperature stability, storage stability
and reusability. In the future, core–shell nanoparticles modified
by organically modified silicates or loaded with metal ions could
be employed to entrap enzyme molecules, providing multi-
functional materials.
Fig. 5 Thermal stability of free b-gal (’) and encapsulated b-gal (J).
Table
1
Storage stability of the encapsulated b-gal for hydrolytic activity
towards ONPG
Storage time (day)
Activity (U mgÀ1
)
Residual activity (%)
0
5
10
15
20
25
30
653.0
649.7
633.5
626.7
602.0
591.0
571.0
100.0
99.5
97.1
96.0
92.2
90.5
87.4
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (grant no. 21171064 and 21071059) and
the National High Technology Research and Development
Program of China (2006AA02Z232).
preserve the tertiary structure of the protein and protect b-gal
from disassembling of the active center caused by the diminu-
tion of non-covalent forces at higher temperatures.32,33 The
encapsulated b-gal with higher thermal stability is beneficial to
the hydrolysis of the lactose in milk at higher temperature,
reducing bacterium contamination and accelerating reaction
rates in a continuous-flow system.
The practical application of enzymes requires that they
could be maintained for long periods. In this study, the storage
stability of the encapsulated b-gal has been investigated and
the results are shown in Table 1. We found that the enzyme
activity of the encapsulated b-gal gradually decreases with the
increasing storage time and the encapsulated b-gal could
maintain a higher enzyme activity (87.4% of its initial activity)
even after 30 days of storage. The high storage stability of the
encapsulated b-gal may be attributed to a reduction in the rate
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