NJC
PAPER
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
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
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013
New J. Chem.