Entrapment of Enzymes within Sol-Gel-Derived Magnetite

Article abstract of DOI:10.1021/acs.chemmater.6b00193

Magnetically controlled enzymatic composites have received much attention for both therapeutic and industrial applications. Until now, such materials have been composed of at least four components: the enzyme, magnetic nanoparticles, their stabilizing com

Full text of DOI:10.1021/acs.chemmater.6b00193

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Article
Entrapment of enzymes within sol-gel derived magnetite
Andrey S. Drozdov, Olga E. Shapovalova, Vladimir Ivanovski, David Avnir, and Vladimir V. Vinogradov
Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.6b00193 • Publication Date (Web): 16 Mar 2016
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Chemistry of Materials
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Andrey S. Drozdova, Olga E. Shapovalovaa, Vladimir Ivanovskib, David Avnirc and Vladimir V. Vinogradova*
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aLaboratory of Solution Chemistry of Advanced Materials and Technologies, ITMO University, St. Petersburg, 197101, Russian Fed-
eration
bFaculty of Natural Sciences and Mathematics, Institute of Chemistry, Ss. Cyril and Methodius University in Skopje, Skopje, 1000,
Republic of Macedonia.
cInstitute of Chemistry and the Centre for Nanoscience and Nanotechnology, the Hebrew University of Jerusalem, Jerusalem, 91904,
Israel
ABSTRACT: Magnetically controlled enzymatic composites have received much attention for both therapeutic and industrial applications.
Until now, such materials have been composed of at least four components: the enzyme, magnetic nanoparticles, their stabilizing compo-
nents, and an organic or inorganic (or hybrid) matrix as a carrier. However, such compositions, affect the magnetic response and the enzymat-
ic activity, and also pose obstacles for intravenous administration due to regulatory restrictions. Here we present a methodology for the crea-
tion of magnetic bioactive nanocomposites composed of only two biocompatible components: an enzyme and magnetite nanoparticles. A
series of magnetic biocomposites with a full set of therapeutical and industrial proteins (carbonic anhydrase, ovalbumin, horseradish peroxi-
dase, acid phosphatase, proteinase and xylanase) were successfully created by the direct entrapment of proteins within a sol-gel magnetite
matrix specially developed for these aims. The activity of entrapped enzymes was studied at different temperatures and concentrations, and it
was found that the entrapped enzymes showed remarkable thermal stabilization induced by the ferria matrix. For instance, entrapped carbon-
o
ic anhydrase catalyzed the decomposition of p-nitrophenylacetate at a temperature of 90 C, while free enzyme completely loses activity and
denaturates already at 70 oC. Magnetic characterization of the obtained biomaterials is provided.
INTRODUCTION
oping an ultrasonic approach for the preparation of a variety of
stable oxide hydrosols, without the need for any stabilizing agents
and at neutral pH, that is, highly pure oxide hydrosols composed
only of the nanometric oxide particles and water.11-13 Alumina was
studied intensively by this new methodology, resulting in a series of
active biomaterials with unprecedented stabilities and performanc-
es, including therapeutic14 and biotechnological enzymes,15 syner-
gistically-active wound-healing materials,16 and materials with effi-
cient thrombolytic properties.17, 18
Our current knowledge of sol-gel derived biomaterials is largely
based on intensive studies of silica: With this oxide methodologies
of entrapment were developed, stability studies were carried out,
and enzymatic activities were analyzed.1 On the advantages side,
silica sol-gel materials not only preserve the activity of the bioactive
dopants, but also provide exceptional stability to a variety of pro-
tein and bioactive molecules against various deterioration process-
es.2-5. And yet, an elementary key-limitation of silica that has been a
major obstacle towards development of medical applications is the
fact that this oxide is not approved by medical regulators for injec-
tion. However, this limitation on sol-gel materials can be addressed
if the scope is broadened beyond silica. Among the common ox-
ides, ferria (iron oxides in general) and alumina are approved for
parenteral administration into the human body.6 Magnetite is ap-
proved as a component for magnetic resonance imaging (Refidex
and Resovist),7 and for treatment of iron deficiency (anaemia) in
people with chronic kidney failure (Ferumoxytol, Feraheme®),8
while alumina is the most widely used vaccine adjuvant.9 These two
oxides are therefore perfect candidates for development of sol-gel
derived carriers of proteins, antibodies,10 drugs, and other bioactive
components, aimed at their stabilization, delivery and release.
The time has come now to study intensively the second regulatory
approved oxide, ferria (iron oxide), and in particular magnetite
because of its added value of magnetic maneuverability. Here we
report the first successful entrapment of enzymes within magnetite
ferria, and the finding that thermal protectability, typical of sol-gel
materials, is obtained with this class as well. The ability to move-on
to ferria became possible following a recent solution13 of the well-
known problem of ferria hydrosols: These are so unstable (because
of low surface charge of the particles), that massive use of additives
or use of extreme pH values are needed in order to achieve sol sta-
bility. However, ferria hydrosols so obtained are not friendly for
protein entrapment. To overcome this problem and produce mag-
netic responsive biomaterials, magnetite nanoparticles were co-
entrapped with enzymes within oxides such as silica19 or titania,20
or, alternatively, proteins were covalently linked to magnetite na-
noparticles.21 We have recently solved the problem of producing
pure ferria hydrosols (magnetite nanoparticle and water only)
avoiding stabilizing agents and retaining neutral pH. The approach,
The use of the sol-gel approach for these purposes is primarily de-
pendent on the development of bio-friendly synthetic procedures
for the preparation of the oxide hydrosols – alumina and ferria in
our context - from which the doped sol-gel materials are obtained.
In a series of recent papers we have addressed that need by devel-
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described in detail in ref. 13, is a carefully tailored process which
hours. The CAB@ferria composite was rinsed with Tris-buffer (pH
combines an unconventional non-stoichiometric Fe2+/Fe3+ ratio,
ultra-sonic treatment and the Schikorr reaction;13 all of these give
rise to a high-enough positive surface charge of the magnetite parti-
cles, thus forming a stable hydrosol at neutral pH.
7.4) solution to ensure removal of any adsorbed protein. The en-
trapment of CAB was full, as indicated by the lack of activity of the
washings; this is true for all other enzymes in this study.
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OVA@ferria: 10 mL of the freshly prepared ferria sol was mixed
with 1 ml of the protein water solution, prepared separately using
10 mg of the protein. The resulting protein-containing sol was
dried in a vacuum desiccator for 24 hours at room temperature.
The weight content of protein in the final composites was 5%.
Comparative adsorption experiments were carried out by exposing
10 ml of the same OVA solution to ferria xerogel obtained from 10
ml of 30 min ultrasonically treated ferria sol (see Fig 2S, Supple-
mentary Material for IR characterization and comparison to ad-
sorption).
In this report we focus on bovine carbonic anhydrase (CAB), one
of the most efficient and well-studied enzymes22 and a member of
an important class of enzymes used in Alzheimer disease treat-
ment.23 Recently, CAB has also been the topic of a report on the
ability of the sol-gel process to renature thermally denatured en-
zyme into enzyme with higher activity compared to the free one
(the “Phoenix effect”).24 Following the detailed study of
CAB@ferria described below, which has revealed enhanced ther-
mal stability, we screened over a number of proteins to test both
the generality of the entrapment procedure in magnetite and the
gained stability. These proteins are ovalbumin (OVA), horseradish
peroxidase (HRP), a member of the peroxidase enzymes family,
which is used for conversion of pro-drugs to active ones; acid phos-
phatase (AcP), important for prostate cancer treatment;25 xylanase
(XY), a key biotechnological enzyme widely used in the paper in-
dustry and in the production of biodiesel;26 and proteinase (PR),
protease-class enzyme used in the food and leather industries.27 We
believe that our report provides conclusive evidence that sol-gel-
derived ferria is a matrix worth attention as a bio-carrier of en-
zymes, being at the same time stabilizing, magnetic and injectable.
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HRP@ferria: 200 µL of freshly prepared ferria sol was transferred
to a cuvette, then 25 µL of HRP (20 U mL-1) solution in glycine-
HCl buffer (pH 4.7) was added and the resulting sol was mixed.
The sol was dried under vacuum for 24 hours. The HRP@ferria
composite was rinsed with glycine-HCl buffer (pH 4.7) solution.
AcP@ferria: 200 µL of freshly prepared ferria sol was transferred to
a cuvette, then 5 µL of AcP (200 U mL-1) solution in glycine-HCl
buffer (pH 5.0) was added and the resulting sol was mixed. The sol
was dried under vacuum for 24 hours. The composite was rinsed
with glycine-HCl buffer (pH 5.0) solution.
Proteins@ferria for DSC analyses: 12 mg of the corresponding
protein (CAB, OVA, HRP, AcP, XY, PR) was added to 6 mL of the
sol at room temperature so that final fraction was 10 % wt. The
suspension was aged for 3 h and dried at 20°C for 2 days in a vacu-
um desiccator.
EXPERIMENTAL DETAILS
Chemicals
Carbonic anhydrase from bovine erythrocytes (CAB, cat.#
C-2624), ovalbumin (OVA), horseradish peroxidase (HRP,
cat.#P-8375), acid phosphatase from potato (AcP, cat.# P-
0157), β-xylanase from Trichoderma longibrachiatum (XY),
proteinase from Aspergillus melleus (PR), p-nitrophenylacetate
(pNPA), 2,2’-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid)
(ABTS), hydrogen peroxide 30%, p-nitrophenylphosphate
(pNPP), were all obtained from Sigma-Aldrich. Glycine buffers
were made from glycine solution (0.05 M, from Sigma-Aldrich)
with a desired volume of 1 M HCl. Tris-buffer was made from 0.1
M Trizma-hydrochloride solution with a desired volume of 1 M
NaOH. The hydrosol was prepared from iron (II) chloride tetra-
hydrate, iron (III) chloride hexahydrate and ammonia, all from
Sigma-Aldrich.
Thermal stability of CAB@ferria
After rinsing the bioactive hybrid (10 % wt. enzyme loading) with 1
mL of Tris-buffer (pH 7.4) for 10 minutes, 3 mL of Tris-buffer (pH
7.4) solution was added to it and the cuvette was incubated at tem-
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perature of 65 C for periods ranging from 4 to 60 min. After incu-
bation, 125 µL of pNPA solution (0.8 M in acetone) was added,
and enzymatic activity was spectrophotometrically measured at 40
oC by following the formation of p-nitrophenolate (pNP, Fig. 1.1)
through the absorption at 405 nm. Testing for possible activity of
the rinsing solutions was carried out by adding 1 mL of Tris-buffer
and 60 µL of pNPA solution to it. For comparative analysis of the
free enzyme, 10 µL of the enzyme solution (17500 U mL-1) and 3
mL of Tris-buffer were mixed and treated similarly to the bioactive
composite.
Preparation of the protein@ferria biocomposites
Ferria pure hydrosol: This was prepared ultrasonically as described
is ref. 13. Briefly, 2.5 g FeCl2·4H2O and 5 g FeCl3·6H2O were dis-
solved in 100 mL of deionized water under constant stirring (500
rpm). Then, 12 mL of aqueous ammonia solution was added under
constant stirring (500 rpm) at room temperature. Using a magnet,
the formed magnetite precipitate was collected and washed with
deionized water until neutral pH. The washed black precipitate was
mixed with 100 mL of deionized water and subjected to ultrasonic
treatment (37 kHz, 110 W) under constant stirring (300 rpm) for
120 minutes. The resulting magnetite sol was then cooled to room
temperature. The mass fraction of the magnetite nanoparticles in
the resulting sol 2.2%, with an average particle size 10 nm, as de-
termined from HR-SEM imaging, Fig. 1S, Supplementary Materi-
al).
Enzymatic activity of the protein@ferria biocomposites
CAB@ferria:
After rinsing of the bioactive hybrid (4 mg, 12 % wt. enzyme load-
ing) with 1 mL of Tris-buffer (pH 7.4) for 10 minutes, 3 mL of
Tris-buffer (pH 7.4) solution was added and the cuvette was incu-
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bated at temperatures ranging from 20 C to 90 C for 5 minutes.
After incubation, 125 µL of pNPA solution (0.8 M, in acetone) was
added, and the enzymatic activity was measured at the same tem-
perature following the formation of p-nitrophenolate (pNP, Fig. 1-
1) spectrophotometrically through the absorption at 405 nm. A
small hydrolytic effect of the ferria matrix without enzyme was
determined similarly and the enzymatic activity results were cor-
rected accordingly (5-10 % correction, depending on the tempera-
ture of the experiment). Testing for possible activity of the rinsing
solutions was carried out by adding 1 mL of Tris-buffer and 60 µL
of pNPA solution; no activity was found. For comparative analysis
CAB@ferria: Different quantities (from 0 to 112 µL) of CAB solu-
tion (17500 U mL-1) in Tris-buffer (pH 7.4) were mixed with 200
µL of freshly prepared ferria sol and dried under vacuum for 24
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Chemistry of Materials
of the free enzyme, 10 µL of the enzyme solution (17500 U mL-1)
and 3 mL of Tris-buffer were mixed and treated similarly to the
bioactive composite.
RESULTS AND DISSCUSSION
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CAB@ferria
HRP@ferria: The enzymatic activity was measured using ATBS as
a substrate (Fig. 1-2). The substrate solution is composed of 0.1
mL of 30 % H2O2 and 1 mL of 0.5 mM ABTS in 2 mL of 50 mM
glycine-HCl buffer (pH 4.7). After rinsing with 1 mL of glycine-
HCl buffer (pH 4.7), 4 mg of the bioactive composite containing
HRP@ferria (0.3 % wt. enzyme loading) was transferred into a
cuvette, where 300 µL of glycine-HCl buffer (pH 4.7) was added
As mentioned in the Introduction, the entrapment of proteins with-
in sol-gel derived magnetite (or, for that matter, within any form of
ferria) has not been described before. The entrapment procedure
described in the Experimental details represents a carefully opti-
mized method, which, as also described in the Introduction, is
based on the newly developed bio-friendly pure magnetite hydro-
sol.12 One of the most important optimized parameters was the
enzyme loading. Fig. 2a shows the conversion rate and specific
activity of CAB@ferria as a function of the amount of entrapped
enzyme. It is seen that while the absolute activity increases with
loading, the specific activity (defined as absolute activity divided by
the mass fraction of enzyme in the composite) levels off at about
12% mass fraction. Such threshold is usually indicative of the onset
of formation of aggregates of the dopant,13 that is, a larger portion
of enzyme with loading beyond this threshold is buried inside the
aggregates and becomes inaccessible. An approximate model esti-
mate shows that about 20 magnetite nanoparticles (~10 nm in
diameter, shown in Fig. 2b) are needed to coat and entrap one
protein molecule (6 6  24 nm,21 Fig. 2c), that is, at higher loads
there is a deficiency in nanomagnetite particles to coat each enzyme
molecule individually. 12% loading was therefore used in the fol-
lowing measurements reported. The explanation of why activity
with entrapped enzyme is possible at all, lies in the porosity of the
matrix. Typical surface areas, obtained from the BET analysis of the
nitrogen adsorption isotherms are 120 m2/g for both the pure
magnetite and for CAB@ferria, with BJH average pore diameters of
8.5 and 8.2 nm respectively (Fig. 3S and Table 1S, Supplementary
Material).
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and comparative incubation was carried out at 25 C and 65 C for
1 h. Then, 3 ml of the substrate solution was added and the enzy-
matic activity was followed spectrophotometrically at 420 nm and a
o
temperature of 30 C. Testing of possible enzymatic activity of the
rinsing solutions was carried out under similar conditions – none
was found. For the free enzyme, 1 µL of HRP (20 U mL-1) was add-
ed to 300 µL of glycine-HCl buffer and was treated similarly to the
entrapped enzyme.
AcP@ferria: After rinsing the bioactive composite with 1 mL of
glycine-HCl buffer (pH 5.0), 4 mg of it was transferred into a cu-
vette and 300 µL of glycine-HCl buffer (pH 5.0) was added. Com-
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parative incubation was carried out at 25 C and 65 C for 30 min.
After that, 3 mL of 10.8 mM pNPP solution in glycine-HCl buffer
(pH 5.0) was added, and the enzymatic activity (Fig. 1-3) was
measured spectrophotometrically by following the absorbance at
o
405 nm and a temperature of 30 C. Rinsing solutions were tested
as described above. For the free enzyme, 1 µL of AcP(200 U mL-
1)was added to 300 µL of glycine-HCl buffer (pH 5.0) and was
treated similarly to the entrapped enzyme.
Characterization methods: Specific surface area pore volume and
pore size distribution were investigated using Quantachrome Nova
1200e by nitrogen adsorption at 77 K, analyzed by the BET and
BJH equations. Prior to analysis, all samples were degassed at room
temperature for 48 hours. For scanning electron microscopy (SEM,
ultra-high resolution Magellan 400L electron microscope), the final
suspension of the entrapped enzyme was coated on silicon wafer
and fully dried under vacuum. The samples for TEM were obtained
by dispersing a suspension drop on copper mesh covered with car-
bon (FEI TECNAI G2 F20, at an operating voltage of 200 kV).
ATR spectra were recorded on a Perkin-Elmer System 2000 FT-IR
Differential scanning calorimetry (DSC) measurements of the free
and entrapped enzyme provided strong indication of a thermal
stability effect of the matrix. We recall that DSC has proved in a
series of recent studies11,14,15 as a perfect analytical tool for detection
of thermal stabilization of entrapped enzymes; the idea has been to
follow the denaturing temperature, which behaves in this analysis as
any other phase transition. The results of the measurements (Fig.
o
3a) show that the denaturation temperature of CAB - 68 C – is
pushed up to 98 oC,
spectrometer, with
a Golden Gate reflectance accessory
(SPECAC) equipped with diamond ATR crystal and ZnSe lenses.
DSC curves were obtained with a 204 F1 Phoenix NETZSCH ap-
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paratus and a heating rate of 10 C min-1 was used from 30 C to
o
150 C under nitrogen. The spectrophotometrical measurement of
enzymatic activity was carried out using an Agilent Cary HP 8454
Diode Array spectrophotometer with TEC.
O
O
O
CAB
H2O
N+
O
N+
N+
O
1
2
3
OH
O
O
O
O
N
O
S
O
S
N+
OH
HRP
H2O
N
OH
S
S
N
O
O
N
S
N
S
O
N+
O
S
S
N
HO
HO
O
O
O
O
O
O
AcP
H2O
Figure 2. (a) The conversion rate (left axis) and the specific enzy-
matic activity (right axis) of the CAB@ferria composite. (b) HR-
SEM of the CAB@ferria colloid particles. (c) A model of an enzyme
molecule entrapped within a matrix aggregated cage.
N+
N+
O
N+
OH
O
O
O
O
P
HO
OH
Figure 1. Enzyme-catalyzed reactions used in this study
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Figure 3. (a) DSC curves of free CAB and of the CAB@ferria composite. (b) Comparison of the relative enzymatic activities of free CAB and CAB@ferria
composite. (c) Comparison of the relative enzymatic activity of free CAB and CAB@ferria composite after exposure to 65 oC for an hour.
apparent activation energy, Ea’ and an apparent Arrhenius pre-
factor, A’, can be estimated (T is the temperature, R is the gas con-
stant). Fig.’s 4a,b show this analysis both for the free CAB (Fig. 4a)
and for the entrapped CAB (Fig. 4b). Two different slopes are evi-
dent for the free and the entrapped enzyme – up to the temperature
where denaturation commences, and beyond it. The right-hand
slope relates to the range where temperature is accelerating the
reaction (positive activation energy), while the left slope is the
higher temperatures range where the enzyme is destroyed, giving
rise to negative slopes; we focus only the former.
that is, about 30 °C higher (!). Following this encour-
aging observation, we performed a comparative study of
the relative enzymatic activity of CAB and CAB@ferria at dif-
ferent temperatures. The results are shown in Fig. 3b. It can be
seen that the relative activity of the free enzyme reaches its
maximum at a temperature of 40 C, which corresponds to
o
absolute activity of 8 µM min-1, and that above 60 C there is a
rapid loss of activity down to a full stop at 70 oC. This denatur-
ation temperature is in good agreement with the literature22.
See Fig. 4S, Supplementary Material for specific plots of tem-
perature effects on the kinetics on both free and entrapped
CAB). CAB@ferria on the other hand shows a distinctly dif-
ferent behaviour: The relative activity of the entrapped en-
The apparent activation energy thus calculated for the free enzyme
is 14 kJ mol-1 with an Arrhenius prefactor of 0.02 s-1. For the en-
trapped enzyme the apparent activation energy is higher, 38 kJ mol-
1, and particularly interesting is the three orders of magnitude in-
crease in the prefactor, to 45 s-1. The higher apparent activation
energy is a reflection of the well-known diffusional limitations into
the pore-system of the sol-gel matrix. It is for this reason - and also
in order to interpret the increase in the pre-factor - that we also
took a more advanced model developed for diffusional restricted
heterogeneous catalytic process29. The general picture remains the
same, but with the following corrected values (see Supplementary
Material for details): the corrected activation energy is approxi-
mately twice the apparent one, namely 76 kJ mol-1, and the correct-
ed pre-factor is smaller, 13 s-1, but still 2.5 orders of magnitude
higher compared to solution. A similar effect of pre-factor increase
upon entrapment was also reported for the entrapment of CAB in
alumina based composites14,24 and was interpreted as reflecting the
directing effect that pores leading to the enzyme active site, have.
This phenomenon is possible if active site of the enzyme is average-
ly oriented away from the surface and into the pore space. With
CAB and ferria, this situation is indeed favorable, due to the follow-
ing charge considerations: On one hand, at the experimental pH
7.4 the surface of our ferria is positively charged13 (Fig. 7S) and
therefore repels the neighborhood of the active site of CAB which
has large electropositive zones (Fig. 6S)30, but attracts the other
portions of the protein which are negatively charged at pH 7.4 (be-
cause its isoelectric point of the CAB is 5.9), thus positioning the
enzyme in a more substrate-orientation.
o
zyme grows continuously up to 60 C, after which the activity
o
gradually decreases, maintaining even at 90 C, namely, very
close to the denaturation point (Fig. 3a). The exceptional
thermal stability of CAB when entrapped in magnetite
(CAB@ferria) is further demonstrated in Fig. 3c, where the
composite was incubated for a period of 60 minutes at a tem-
perature of 65 C, (close to the maximum for free enzyme -
Fig. 3a). It can be seen that while the free enzyme lost 45 % of
its initial activity, the entrapped enzyme retained 95 % of its
efficiency, representing a destruction rate of 0.9% of total en-
zyme-molecules/minute for the free CAB, compared to 0.08%
of total enzyme-molecules/minute for CAB@ferria, that is an
increase in thermal stability by a full order of magnitude.
The experimental data of Fig. 3b allows one to estimate apparent
kinetic parameters of the enzymatic reaction, namely a pseudo-first
order pNPA-hydrolysis. A preliminary estimation was first made by
taking the experimental rate constant k’ and analyzing them using
the classical Arrehnius equation, k’=A’exp(-Ea’/RT). Doing so, an
Figure 4. – Arrhenius plots of the hydrolysis enzymatic reaction of pNPA
by free (a) CAB, and (b) by CAB@ferria.
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Figure 7. (a) Magnetic manipulation with CAB@ferria. (b) The magneti-
zation curves of OVA@ferria at different protein loadings. The absence of
hysteresis loop is showed in the zoomed area
amples are shown in Fig. 6, for the two therapeutical enzymes,
HRP and AcP. Specifically, for HRP, after 1 hour of incubation
at 65 oC, the relative activity of the free enzyme dropped to 5%
of the initial activity, while the composite material still showed
75% of the enzymatic activity measured prior to heating; and
for AcP, after 1 hour of heating the activity of free enzyme was
10 times lower (remaining activity was 10 % of the initial one),
while entrapped enzyme was at 33% of its initial activity.
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Figure 5.The DSC shifts upon thermal denaturation of free com-
pared to the entrapped protein. (a) Ovalbumin (adsorbed OVA is
shown as well). (b) Horseradish peroxidase. (c) Acid phosphatase.
(d) Proteinase. (e) Xylanase.
Magnetic properties
Obviously, a key property of these entrapped enzymes is that they
are magnetically-responsive (Fig. 7a). Due to the simplicity of the
composition and the absence of coupling agents and stabilizing
compounds, the magnetic moments of the obtained materials are
very high. As described above, in our case the average particle size
of the magnetite is around 10 nm (Fig.’s 2b and 1S) and the result-
ing superparamagnetic behavior is clearly evident (Fig. 7b) by the
complete absence of hysteresis. The magnetic moment obtained at
6000 Oe amounts to 88 emu/g for the pure magnetite matrix. En-
trapment of OVA as model protein only slightly decreased magnet-
ic moment to 80 emu/g at 10% wt. loading (Fig. 7b).
The generality of the entrapment and of the stabilization
Following the development of the procedure of entrapment of
CAB in magnetite, and the observation of enhanced thermal
stability of that entrapped enzyme, we have screened over
several other enzymes in order to evaluate the generality of
these observations. These proteins are ovalbumin (OVA),
horseradish peroxidase (HRP), phosphatase (AcP), xylanase
(XY) and proteinase (PR), all (as mentioned in the Introduc-
tion) having specific applications in medicine and in biotech-
nology. Fig. 5 shows that the stabilization effect is general. For
OVA@ferria (Fig. 5a) the denaturation temperature is higher
CONCLUSIONS
compared to the free protein by 10 C. Also shown in this
figure is the known de-stabilization of proteins upon adsorp-
tion on oxides.28 The difference in the denaturation tempera-
tures between destabilized and stabilized enzyme upon ad-
sorption and entrapment, respectively, is an important mani-
festation of the inherent differences between these two het-
erogenization methods. For AcP@ferria this difference is 22
In this study we developed and demonstrated, for the first
time, an entrapment methodology of proteins within magnet-
ite sol-gel matrixes. The key has been the use of the newly de-
veloped magnetite hydrosol synthesis which does not employ
stabilizers and is carried out at a neutral pH. Taking CAB as a
model system, we observed enhanced thermal stability of the
entrapped protein and a shift of the denaturation temperature
to a higher value. This effect was also shown for five additional
enzymes of therapeutical and industrial importance. Entrap-
ment of the enzymes not only maintained the biocatalytic ac-
tivity but allowed performing the enzymatic reactions at tem-
peratures higher than the denaturation temperatures of the
free enzymes. These findings open up a new methodology for
the preparation of magnetic-responsive enzymatic catalysis.
Such materials provide new options and possibilities for ther-
apeutic agents or heterogeneous enzymatic reaction catalysts.
The capability of moving and positioning the material by im-
posing a magnetic field, also facilitates applications of enzymes
in these two fields. From a medical point of view, thermal sta-
bility of proteins is of significant importance, and the biocom-
patibility and injectability make these composites even more
interesting. The approach we developed is general: With min-
imum adaptation it is possible to create bioactive magnetic
o
oC, leading to a 82 C denaturation temperature of the stabi-
lized enzyme (Fig. 5b); for HRP@ferria the stabilization leads
o
to 25 C higher temperature (Fig. 5b); for PR@ferria it is ele-
vated by 27 °C (Fig. 5d); and for XY@ferria by 22 °C (Fig.
5e). Kinetics analyses likewise confirm this generality, and two
typical ex-
Figure 6. Comparison of the relative of the enzymatic activity of (a) free
HRP and HRP@ferria composite; (b) free AcP and AcP@ferria
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