Enhanced thermal and ultrasonic stability of a fungal protease encapsulated within biomimetically generated silicate nanospheres

Article abstract of DOI:10.1016/j.bbagen.2010.01.004

Background: Dendrimers are highly branched synthetic macromolecules with a globular shape. They have been successfully used for generation of nanospheres at mild conditions via biomimetic silicification. Encapsulation of enzyme molecules within these nano

Full text of DOI:10.1016/j.bbagen.2010.01.004

Biochimica et Biophysica Acta 1800 (2010) 459–465
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbagen
Enhanced thermal and ultrasonic stability of a fungal protease encapsulated within
biomimetically generated silicate nanospheres
b
c
a
a
Ashkan Madadlou a, , Daniela Iacopino , David Sheehan , Zahra Emam-Djomeh , Mohammad E. Mousavi
⁎
a
Department of Food Science & Engineering, Faculty of Technology, Campus of Agriculture & Natural Resources, University of Tehran, Karadj, Iran
Nanotechnology Group, Tyndall National Institute, Lee Maltings, Cork, Ireland
Laboratory of Protein & Proteomics, Department of Biochemistry, Faculty of Science, University College Cork, Lee Maltings, Cork, Ireland
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Background: Dendrimers are highly branched synthetic macromolecules with a globular shape. They have
been successfully used for generation of nanospheres at mild conditions via biomimetic silicification.
Encapsulation of enzyme molecules within these nanospheres during their synthesis is a promising method
for rapid and efficient entrapment of several enzymes. However, encapsulation of proteolytic enzymes has
been rarely done via biomimetic silicification. As well, the operational stability of encapsulated enzyme has
not been systematically reported.
Received 30 September 2009
Received in revised form 15 December 2009
Accepted 15 January 2010
Available online 25 January 2010
Keywords:
Methods: A proteolytic enzyme, either α-Chymotrypsin or a fungal protease from Aspergilus Oryzea was
encapsulated along with iron oxide nanoparticles within particles yielded via biomimetic silicification of
different generations of polyamidoamine (PAMAM) dendrimers. Stability of encapsulated enzyme was
compared to that of free enzyme during storage at room temperature. As well, their thermal and ultrasonic
stabilities were measured. Scanning electron microscopy, transmission electron microscopy and optical
microscopy were used to investigate the morphology of nanospheres.
Enzyme
Dendrimer
Immobilization
Microscopy
Biomimetic silicification
Ultrasound
Results: Determination of encapsulation efficiency revealed that ∼85% of fungal protease with concentration
1.4 mg mL−1 stock solution was immobilized within particles yielded by generation 0. Based on microscopic
images the generated particles interconnected with each other and had spherical morphologies independent
of generation. Kinetic analysis of encapsulated fungal protease demonstrated that Mechaelis-Menten
constant (Km) slightly increased.
Conclusion: PAMAM dendrimer generation 0 could be effectively used for rapid encapsulation of a fungal
protease from Aspegilus Oryzae.
General significance: Encapsulation significantly enhances the thermal and ultrasonic stabilities of enzymes,
suggesting a range of diverse applications for them.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
strates toward their active sites [3]. Smaller porous particles owing to
their shortened diffusion path of substrates can generally reduce
Amongst the several approaches to improve the operational
stability of enzymes, enzyme immobilization can overcome some of
major problems inherent with the use of free enzymes. It prevents
from product contamination with enzyme molecules and simplifies
their separation from the reaction mixture which also provides the
possibility to reuse those [1]. The immobilized enzyme molecules do
not contact with the external hydrophobic interfaces such as gas
bubbles originated for example by harsh mixing, sonication or
supplying the required gases. The hydrophobic interfaces otherwise
will inactivate the soluble enzymes [2]. The immobilized enzymes
however, commonly suffer from low specific activities compared to
their free counterparts due to the mass transfer limitations for sub-
the mass transfer resistance [4]. In theory, there is no attrition prob-
lem for a nanometric carrier. It is however almost impossible to
separate them in a bioreactor by conventional methods like filtration
[5]. Paramagnetic nanoparticles being an ideal alternative can be
easily separated from the medium by using a magnetic field [4]. These
particles retain no magnetism after removal of the magnetic field [6].
In general, enzymes are covalently attached on magnetic nanoparti-
cles using several ligands [7]. It is worthy to consider that enzymes
immobilized on nanoparticles are able to interact with the hydro-
phobic interfaces and even with enzyme molecules immobilized on
other particles [2]. The enzyme molecules immobilized on magnetic
nanoparticles have been therefore, coated with aldehyde dextran thus
avoiding the enzyme inactivation by gas bubbles in a stirred system
[1].
Encapsulation of enzyme molecules within nanospheres produced
via bio-(mimetic) silicification is a promising method for rapid and
⁎
Corresponding author. Tel.: +98 860380258, +98 9128614065.
E-mail address: Ashkan.madadlou@gmail.com (A. Madadlou).
0304-4165/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagen.2010.01.004

460
A. Madadlou et al. / Biochimica et Biophysica Acta 1800 (2010) 459–465
efficient entrapment of several enzymes. A set of posttranslationally
modified polycationic peptides called silaffins isolated from the walls
of diatoms, single-cell algae; generate nanospheres within seconds
when added to a silicic acid solution. There are seven highly homol-
ogous repeating units (R1 to R7) in the structure of silaffins [8]. In
addition to biosilicification, the synthesized unmodified parent
sequence of R5 unit (H2N-SSKKSGSYSGSKGSKPRIL-CO2H) is also
capable to condense the silicic acid through self-assembling to larger
structures [9]. This process, being appealing is called biomimetic
silicification. Luckarift et al. [10] physically entrapped the 90% of
butyrylcholinesterase within the nanospheres produced via biomi-
metic silicifiction of R5 peptide. The biomimetic silicification process
is a one-pot procedure in which synthesis of silica and entrapment of
enzyme molecules occur simultaneously at room temperature [11]
and the peptides are captured within the precipitated nanospheres
[12]. Other biological and biomimetic amine containing polymers
have been also investigated to produce silica nanospheres. Dendri-
mers are highly branched synthetic macromolecules with a globular
shape. They are structurally homogenous and biocompatible [13]
polymers whose functionality can be tuned through the delicate
choice of branching elements and terminal groups. Amine-terminated
dendrimers, specifically polyamidoamine (PAMAM) and polypropy-
lenimine (PPI) dendrimers have been successfully used for biomi-
metic silica synthesis. PPI dendrimers are the synthetic analogous to
one of the major posttranslational modifications in the lysines of
silaffins, while PAMAM dendrimers are reminders of the unmodified
lysines within the R5 unit [12]. Based on the number of amino groups
PAMAM and PPI dendrimers of different generations are possible,
with each generation having double the number of groups as the
former one [13].
The encapsulation of proteolytic enzymes has been rarely done via
biomimetic silicification. As well, the operational stability of encap-
sulated enzyme has not been systematically reported. In the present
communication, the encapsulation efficiency of two proteolytic
enzymes within the nanospheres originated by three different
generations (0, 2 and 4) of PAMAM dendrimers is compared. Then,
the enzyme with the higher encapsulation efficiency is further studied
for characteristics of nanospheres and its activity and stability is
compared with those of free enzyme.
2. Materials and methods
2.1. Materials
PAMAM dendrimers with ethylendiamine cores of generations 0, 2
and 4 (20 wt% in methanol solution for generations 0 and 2 and 10 wt%
for generation 4) were purchased from Aldrich and used without further
purification. Fig. 1 presents thechemical structureof PAMAM dendrimer
with ethylendiamine cores of generations 2. α-Chymotrypsin from
bovine pancreas (a serine protease that hydrolyzes peptide bonds
with aromatic or large hydrophobic side chains (Tyr, Trp, Phe, Met) on
the carboxyl end of the bond) with molecular weight of 25 kDa and
Flavourzyme (a protease from Aspegilus Oryzae that contains both
endoprotease and exopeptidase activities) were purchased from Fluka
and Sigma, respectively. N-Succinyl-L-alanyl-L-alanyl-L-prolyl-L-
phenylalanine p-nitroanilide and L-Leucine-p-nitroanilide were
purchased from Sigma and Fluka, respectively. Iron oxide nanopar-
ticles solution (5 mg mL−1 in toluene) with an average particle size
of 5 nm was purchased from Aldich. It contained <0.1% oleic acid as
stabilizing ligands.
Fig. 1. Chemical structure of PAMAM dendrimer of generation 2 with ethylendiamine core.

A. Madadlou et al. / Biochimica et Biophysica Acta 1800 (2010) 459–465
461
2.2. Purification of iron oxide magnetic nanoparticles
2.6. Microscopic analyses
1 mL of magnetic nanoparticles solution was washed three times
with 20 mL of distilled water through shaking at 50 rpm for 30 min
[14]. The particles were then accumulated with a magnet and sus-
pended twice in absolute ethanol each of which for 15 min while
shaking at 50 rpm to remove the stabilizing ligands [7]. The nano-
particles were subsequently resuspended in 3 mL deionized water
and stored.
Scanning electron microscopy (SEM) images were acquired using
a field emission scanning electron microscope (JSM-6700F, JEOL
UK Ltd.) operating at beam voltage 7 kV. Solution of SiO2/enzyme
(diluted 1:20 with aqueous solution of sodium dodecyl sulfate
(SDS)) was deposited on acid cleaned highly doped Si wafer. After
solvent evaporation, a thin layer of gold was evaporated with a
vacuum coating (Edwards RV3 scancoat 6, Edwards Laboratories,
Milpitas, CA) in order to avoid sample charging during SEM imaging.
For optical microscopy images, drop deposited from SDS aqueous
suspension onto acid cleaned glass microscope slides, were acquired
using a calibrated epi-fluorescence microscope (Zeiss Axioskop II
Plus, Carl Zeiss U.K.) equipped with a 100 W halogen lamp and a CCD
camera (Optronics DEI-750). Images were analysed using Image Pro
Express software (Media Cybernetics Inc., USA). Dark-field images
were acquired with the same microscope equipped with a dark-field
condenser with numerical aperture (NA) 0.9 and working distance
1.5 – 2 mm. A Jeol 2100 Transmission Electron Microscope was used
at an accelerating voltage of 200 kV to image the samples. A drop of
dendrimer generation 0 sample diluted (1:20) with SDS was depos-
ited for 5 min on a formvar/carbon coated copper grid (400 mesh).
Excess solution was removed by wicking and the grid was dried in
air.
2.3. Enzyme encapsulation procedure
Dendrimer solutions of different generations were diluted with
phosphate buffer 10 mM to a dendritic amine concentration of
80 mM. α-Chymotrypsin was dissolved in 10 mM phosphate buffer
whilst, fungal protease being liquid was diluted with phosphate buffer
10 mM to destined protein concentrations. 265 µL enzyme aliquot
was charged with 125 µL dendrimer and 60 µL iron oxide solutions.
This was followed by adding 50 µL silicic acid resulting in rapid
formation of silica precipitate. The generated silica particles were
removed either by a magnet or through centrifugation (5 s at
20500 g), washed twice with deionized water and stored in phos-
phate buffer 10 mM. Silicic acid was obtained from a 60 min hydro-
lysis of 75 µL tetraethylorthosilicate (TEOS) in 425 µL 1 mM HCl
[15]. For free enzyme treatments, TEOS was substituted with
deionized water to prevent from the silicification of dendrimer
templates. The encapsulation efficiency was determined by compar-
ing the enzymatic activity of free enzyme and that of supernatant
following the reaction [16]. For α-Chymotrypsin, N-Succinyl-L-alanyl-
L-alanyl-L-prolyl-L-phenylalanine p-nitroanilide 5 mM and for fungal
protease L-Leucine-p-nitroanilide 40 mM were used as substrates.
General protein content of enzyme solutions was quantified using the
Bradford assay method.
3. Results and discussion
3.1. Encapsulation of enzymes
The encapsulation efficiency was compared for two types of
proteases using different generations of PAMAM dendrimer. It is
clear from Fig. 2 that α-Chymotrypsin had significantly lower
encapsulation efficiency at any given generation of dendrimer. An
increase in the concentration of -Chymotrypsin from 80 µg mL−1 to
4.5 mg mL−1 stock totally failed to encapsulate the enzyme. Both
the PAMAM dendrimers and α-Chymotrypsin are positively
charged at physiological pH as the dendrimers have a pKa ∼ 9.5
and α-Chymotrypsin has a pI ∼ 9.1. The positively charged enzyme
molecules repel the protonated surface of dendrimers, leading to
minor electrostatic interactions between them. Under this condi-
tion, the enzyme is solely entrapped non-specifically in the aggre-
gating silica matrix during precipitation of particles [16]. Of
note, the fungal protease had an acidic pI and therefore, interacted
extensively with the cationic surface of dendrimer causing a
noticeable degree of encapsulation. A decrease in the concentra-
tion of fungal protease in stock from 6.9 to 1.4 mg mL−1 increased
2.4. Enzyme stability experiments
The residual activity of encapsulated and free fungal protease was
measured during storage at room temperature for 10 days. For
determination of thermal stability, free and encapsulated protease
solutions were incubated at 30, 40, 50, 60 and 70 °C for 5 min and
their residual activity was measured. For determination of ultrasonic
stability, free and encapsulated protease solutions were sonicated at a
frequency of 23 kHz using a MSE Soniprep 150 bench mounted
ultrasonic disintegrator (Wolf Laboratories Ltd., York, England). The
amplitude of titanium probe was adjusted either on ∼4 or 22 µm for
15 min and 60 seconds, respectively. These resulted in actual power
dissipated to solution of ∼4.5 and 27 W, respectively. The power was
measured as described previously [17]. Temperature of solutions was
controlled over the sonoprocessing by an ice bath except for power
measurements.
2.5. Kinetic of fungal protease
The Mechaelis-Menten constant (Km) of fungal protease was
determined by the hydrolysis of L-Leucine-p-nitroanilide (0.1-100 µM)
at room temperature. The absorbance increase at 410 nm due to
release of p-nitroaniline was monitored using an infinite M200
microplate reader (Tecan Trading AG, Switzerland). For encapsulat-
ed enzyme, silica particles were removed by centrifugation before
determination of absorbance [10]. No enzyme activity was observed
at supernatant after removal of particles. Kinetic constant Km was
obtained through the non-linear regression analysis at 95% confi-
dence level by using HYPER32.EXE software Version 1.0.0 (Liverpool
University, Liverpool, UK).
Fig. 2. Encapsulation efficiency of enzyme within silica spheres yielded via biomimetic
silicification of different generations of PAMAM dendrimer.

462
A. Madadlou et al. / Biochimica et Biophysica Acta 1800 (2010) 459–465
Table 1
the encapsulation efficiency suggesting a limited capability for silica
particles to entrap the enzyme molecules within them. For both
types of proteolytic enzymes, PAMAM template of generation 0
provided the highest encapsulation efficiency. An efficiency of ∼85%
was achieved for fungal protease with 1.4 mg mL−1 stock encapsu-
lated via biomimetic silicification of PAMAM generation 0.
Diameter of spheres yielded via silicification of different generations of PAMAM
dendrimer.
Template
generation weight
Molecular Template
Number of
Enzyme Nanosphere
diameter (Å) surface amino loaded
groups
diameter SD1
(nm)
0
517
15
4
No
198 41(25%)
405 54 (75%)
306 79
The particles originated by different generations of PAMAM had
similar microstructural features. Optical microscopy images of
particles yielded by PAMAM template of generation 0 are demon-
strated in Fig. 3a and b. It is observed that particles were extensively
interconnected with each other forming a widespread network.
Higher magnifications by scanning electron microscopy (Fig. 3c-e)
revealed that particles had spherical morphology. It was also observed
by SEM and TEM that a proportion of silica particles were partially
coalesced whilst retaining their spherical shape (Fig. 3f and g). The
average diameter of silica nanospheres yielded by different genera-
tions was measured by SEM and is reported in Table 1. Since the
concentration of phosphate buffer has been recognized as a critical
factor that influences the size of spheres [18], enzyme-free spheres
were generated with the same concentration of phosphate buffer in
final solution, i.e. deionized water was diluted with phosphate buffer
10 mM instead of liquid enzyme during sample preparation. The
enzyme-free nanospheres yielded by generation 0 presented a
bimodal distribution with smaller particle diameters averaging
198 nm and larger particle diameters averaging 405 nm. The former
particles consisted ∼25% in population while the latter ones did ∼75%.
0
2
4
517
3256
14215
15
29
45
4
16
64
Yes
Yes
Yes
328 83
275 69
1. Standard deviation.
The bimodal distribution of spheres yielded by PAMAM generations 0
and 1 has been previously reported and is not observed for higher
generations [12]. This kind of distribution is probably due to the
agglomeration of smaller particles leading to the rise of larger ones
[16]. The enzyme containing spheres yielded by generation 0
presented a unimodal size distribution averaging 306 nm. This
suggests that the presence of enzyme molecules within spheres
prevented from their agglomeration. A number of uncharged surface
amino groups of dendrimers [1], regarded as a generalized acid-base
catalyst, accept a proton from monosilicic acid. The positively charged
patches on the surface of dendrimers likely interact with negatively
charged silica species, causing the condensation of these species on
Fig. 3. Bright field (a) and dark field (b) optical microscopy images of spheres containing protease from A. Oryzae with 1.4 mg mL−1 stock yielded via biomimetic silisification of
PAMAM dendrimer of generation 0; c-f) SEM images of spheres; g) TEM image of spheres.

A. Madadlou et al. / Biochimica et Biophysica Acta 1800 (2010) 459–465
463
the surface of dendrimers [12]. Neutralization of growing negatively
charged silicate surface is required for further growth and agglom-
eration of particles. This neutralized surface is formed through
interaction of cations with silicate surface which decreases the elec-
trostatic repulsions thus permitting the particles to grow and agglom-
erate to larger sizes [18]. The negatively charged molecules of fungal
protease likely interacted with cationic surface of dendrimer tem-
plates, followed by their physical entrapment in the growing silica
spheres. The enzyme molecules not only made the silica matrix
discontinuous but also repelled the silica species and prevented from
the extensive agglomeration of silica particles.
3.2. Kinetics and stability of encapsulated protease
Fungal protease with concentration of 1.4 mg mL−1 stock solution
encapsulated via silicification of PAMAM generation 0 was further
studied for enzymatic function. Fig. 4 shows the representative
hyperbola obtained for free and encapsulated fungal proteases. The
entrapment of protease within nanospheres increased the Km value
from 6.515 1.257 to 9.606 2.341 mM, indicating a decreased
affinity of enzyme for its substrate [19] because of an increase in
mass transfer limitation for substrate through silica matrix [4]. A
minor change in the structure of protease due to encapsulation within
spheres could also play a role in the increased Km. The increase in Km
was however small whilst, that of sol-gel entrapped enzymes undergo
a significant increase from 250% [20,21] up to 600% [16]. This suggests
that silica matrix did not significantly hinder the access of substrate to
Fig. 5. Stability of free (
temperature (a) and incubated at different temperatures for 5 min (b).
) and encapsulated (-■-) protease from A. Oryzae at room
-•-
active site of enzyme or release of product by creating a diffusion
barrier in the system [16].
The encapsulated enzyme retained more than 90% of its initial
activity when stored at room temperature for 10 days. The free
enzyme however, progressively lost its activity (Fig. 5a). The thermo-
stability of free and encapsulated enzymes was investigated to find
out if the silica nanospheres could protect the encapsulated enzyme
molecules from thermal denaturation or not. The activity of free
enzyme was progressively decreased with the increasing temperature
of incubation. Free enzyme lost ∼70% of its initial activity when
incubated at 70 °C, indicating a profound sensitivity to heat. The
encapsulated enzyme however, retained 100% of its initial activity at
any incubation temperature (Fig. 5b). Immobilization of enzyme
molecules onto beads [10] and nanoparticles [14] has a quite poor
effect on thermal stability of enzymes and the immobilized enzymes
exhibit very similar stability to their free counterparts. In agreement
to the results obtained at the present study Luckarift et al. [10]
reported that encapsulation of butyrylcholinesterase within nano-
spheres generated via biomimetic silicification of R5 peptide caused a
100% retention of its activity upon incubation at 65 °C for 1 h. The
significantly enhanced thermostability of encapsulated protease is
due most probably to the fixation of enzyme molecules in silica matrix
of nanospheres that hinders the thermal fluctuations [22] and
Fig. 4. Hyperbolic plots for free (a) and encapsulated fungal protease (b).

464
A. Madadlou et al. / Biochimica et Biophysica Acta 1800 (2010) 459–465
prevents from conformational changes upon heating. The significant
thermostability of encapsulated protease provides a number of
processing advantages such as reduced microbial risk, lower viscosity,
improved mass transfer rates and substrate solubility [10]. Stability of
free and encapsulated enzymes to sonication was investigated to find
out if silica nanospheres could protect the encapsulated enzyme
molecules against ultrasonic treatment or not. Free enzyme lost ∼25%
and 55% of its initial activity due to sonication at 4.5 W and 27 W for
15 and 1 min, respectively (Fig. 6). This is attributed to a series of
phenomena originated from cavitation. Bubbles generated by
ultrasound undergo a number of oscillations during their existence.
Diffusion of gases into and out of bubbles during oscillations can
create microcurrents around them [23] which may lead in conjunc-
tion with microstreaming to turbulence and eventually to intermo-
lecular collisions. The capability of microstreaming to disrupt DNA
or disaggregate bacteria has been reported [24]. Inactivation of
enzymes at interfaces is also of note. It proceeds via destabilization of
electrostatic, hydrophobic and hydrogen bonds of proteins, leading
to the irreversible denaturation of enzyme molecules [14]. Besides,
the oscillating bubbles generated by low frequency ultrasound have
significant pounding force because of their large resonance and
maximum expansion radii [25] during their long life [26]. As well, the
shearing force of imploding bubbles can extensively disintegrate the
enzyme molecules in a solution. Finally, highly reactive chemical
species such as hydroxyl, hydrogen and organic radicals generated
by thermolysis of water vapor molecules and volatiles inside the
cavities [25,27] can extensively degrade the proteins [28]. It is
worthy to note however, that at low frequencies as 23 kHz applied in
the present study, number of generated radicals is low and therefore
played a minor role in the inactivation of enzyme molecules. The
encapsulated fungal protease not only retained 100% of its initial
activity upon sonication at 4.5 W for 15 min but interestingly its
relative activity increased up to ∼112% due to ultrasonic treatment at
27 W (Fig. 6). It is clear that encapsulation within silica matrix
protected the enzyme molecules from inactivation at the air-liquid
interface and preserved them against pounding and shearing forces
of microbubbles. An enzyme activity corresponding to the increased
relative activity of encapsulated enzyme due to sonication at 27 W
was detected in supernatant. This indicates that a proportion of silica
nanospheres were disrupted near to the end of harsh sonoproces-
sing, leading to release of some enzyme molecules into the sur-
rounding medium. A prolonged sonication of encapsulated enzyme
solution at such a high acoustic power could logically disrupt a
higher number of silica spheres and subsequently inactivate the
released enzyme molecules.
4. Conclusion
In the current communication, capability of PAMAM dendrimer
generation 0 for effective and rapid encapsulation of a fungal protease
from Aspegilus Oryzae was represented. We are optimistic that other
enzymes preferentially with acidic pI may also be immobilized
effectively within nanospheres originated via biomimetic silicification
of dendrimers. The encapsulated protease could be easily separated
from the reaction solution by a magnet or via centrifugation,
terminating the proteolysis without need to thermal or acidic
denaturation of enzyme molecules. This also provides the possibility
to purify the nutraceuticals produced through enzymatic hydrolysis of
proteins such as bioactive peptides. The encapsulated protease
possessed a significant stability during storage at room temperature
and against heat treatment making it an ideal choice for proteolysis at
conditions when high temperatures are required. As well, the
significantly improved ultrasonic stability of encapsulated protease
expands the range of its applications for example to ultrasound-assisted
proteolysis in proteomics.
References
[1] L. Betancor, H.R. Luckarift, Bioinspired enzyme encapsulation for biocatalysis,
Trends Biotechnol. 26 (2008) 566–572.
[2] C. Mateo, J.M. Palomo, G. Fernandez-Lorente, J.M. Guisan, R. Fernandez-Lafunete,
Improvement of enzyme activity, stability and selectivity via immobilization
techniques, Enzyme Microb. Technol. 40 (2007) 1451–1463.
[3] I. Magario, X. Ma, A. Neumann, C. Syldatk, R. Hausmann, Non-porous magnetic
micro-particles: comparison to porous enzyme carriers for a diffusion rate-
controlled enzymatic conversion, J. Biotechnol. 134 (2008) 72–78.
[4] J. Kim, H. Jia, C. Lee, S. Chung, J.H. Kwak, Y. Shin, A. Dohnalkova, B. Kim, P. Wang, J.W.
Grate, Single enzyme nanoparticles in nonporous silica: a hierarchical approach to
enzyme stabilization and immobilization, Enzyme Microb. Technol. 39 (2006)
474–480.
[5] X. Gao, K.M. Kerry, K.Y. Tam, S.C. Tsang, Colloidal stable silica encapsulated nano-
magnetic composite as a novel bio-catalyst carrier, Chem. Commun. (2003)
2998–2999.
[6] Z. Yang, S. Si, C. Zhang, Magnetic single-enzyme nanoparticles with high activity
and stability, Biochem. Biophys. Res. Commun. 367 (2008) 169–175.
[7] A.K. Johnson, A.M. Zawadzka, L.A. Deobald, R.L. Crawford, A.J. Paszczynski, Novel
method for immobilization of enzymes to magnetic nanoparticles, J. Nanopart.
Res. 10 (2008) 1009–1025.
[8] N. Kröger, R. Deutzmann, M. Sumper, Polycationic peptides from diatom biosilica
that direct silica nanosphere formation, Science 286 (1999) 1129–1132.
[9] M.R. Knecht, D.W. Wright, Functional analysis of the biomimetic silica
precipitating activity of the R5 peptide from Cylindrotheca fusiformis, Chem.
Commun. 9 (2003) 3038–3039.
[10] H.R. Luckarift, J.C. Spain, R.R. Naik, M.O. Stone, Enzyme immobilization in a
biomimetic silica support, Nat. Biotechnol. 22 (2004) 211–213.
[11] R.R. Naik, M.M. Tomczak, H.R. Luckarift, J.C. Spain, M.O. Stone, Entrapment of
enzymes and nanoparticles using biomimetically synthesized silica, Chem.
Commun. (2004) 1684–1685.
[12] M.R. Knecht, D.W. Wright, Amine-terminated dendrimers as biomimetic
templates for silica nanosphere formation, Langmuir 20 (2004) 4728–4732.
[13] M. Bagheri, H. Rodríguez, R.P. Swatloski, S.K. Spear, D.T. Daly, R.D. Rogers, Ionic
liquid-based preparation of cellulose-dendrimer films as solid supports for
enzyme immobilization, Biomacromolecules 9 (2008) 381–387.
[14] L. Betancor, M. Fuentes, G. Dellamora-Ortiz, F. López-Gallego, A. Hidalgo, N.
Alonso-Morales, C. Mateo, J.M. Guisán, R. Fernández-Lafuente, Dextran aldehyde
coating of glucose oxidase immobilized on magnetic nanoparticles prevents its
inactivation by gas bubbles, J. Mol. Catal. B-Enzym. 32 (2005) 97–101.
[15] N.A. Pchelinstev, F. Neville, P.A. Millner, Biomimetic silication of surfaces and its
application to preventing leaching of electrostatically immobilized enzymes, Sens.
Actuators B 135 (2008) 21–26.
[16] S.A. Miller, E.D. Hong, D. Wright, Rapid and efficient enzyme encapsulation in a
dendrimer silica nanocomposite, Macromol. Biosci. 6 (2006) 839–845.
[17] A. Madadlou, M.E. Mousavi, Z. Emam-Djomeh, M. Ehsani, D. Sheehan, Sonodis-
ruption of re-assembled casein micelles at different pH values, Ultrason.
Sonochem. 16 (2009) 644–648.
Fig. 6. Stability of free and encapsulated A. Oryzae protease sonicated at different
conditions.
[18] M.R. Knecht, S.L. Sewell, D.W. Wright, Size control of dendrimer-templated silica,
Langmuir 21 (2005) 2058–2061.

A. Madadlou et al. / Biochimica et Biophysica Acta 1800 (2010) 459–465
465
[19] B.D. Hames, N.M. Hooper, Biochemistry, BIOS Scientific Publishers Ltd, New York,
2001, p. 82.
[20] D.L. Jürgen-Lohmann, R.L. Legge, Immobilization of bovine catalse in sol-gels,
Enzyme Microb. Technol. 39 (2006) 626–633.
[21] Y. Yi, S. Kermasha, R. Neufeld, Characterization of sol-gel entrapped chlorophyl-
lase, Biotechnol. Bioeng. 95 (2006) 840–849.
[25] N.H. Ince, G. Tezcanli, R.K. Belen, Î.G. Apikyan, Ultrasound as a catalyzer of aqueous
reaction systems: the state of the art and environmental applications, Appl. Catal.
B-Environ. 29 (2001) 167–176.
[26] P.R. Gogate, I.Z. Shirgaonkar, M. Sivakumar, P. Senthilkumar, N.P. Vichare, A.B.
Pandit, Cavitation reactors: efficiency assessment using a model reaction, AIChE J.
47 (2001) 2526–2538.
[22] M. Yan, J. Ge, Z. Liu, P. Ouyang, Encapsulation of single enzyme in nanogel with
enhanced biocatalytic activity and stability, J. Am. Chem. Soc. 128 (2006) 11008–11009.
[23] L. Zheng, D.-W. Sun, Innovative applications of power ultrasound during food
freezing process-a review, Trends Food. Sci. Technol. 17 (2006) 16–23.
[24] T.G. Leighton, What is ultrasound? Prog. Biophys. Mol. Biol. 93 (2007) 3–83.
[27] S.-K. Han, S.-N. Nam, J.-W. Kang, OH radical monitoring technologies for AOP
advanced oxidation process, Water Sci. Technol. 46 (2002) 7–12.
[28] M. Barteri, M. Diociaiuti, A. Pala, S. Rotella, Low frequency ultrasound induces
aggregation of porcine fumarase by free radicals production, Biophys. Chem. 111
(2004) 35–42.