In recent years, magnetic nanoparticles (MNPs) have been applied to numerous biological systems. The nanoparticles are particularly useful in separating biological molecules due to its low price, scalable ability and very little interference. Here, MNPs, which can efficiently separate biocatalysts from reaction media by external magnet, was used to immobilize an alkaline protease (Alcalase®). Covalent attachment of the enzyme to MNPs began with the functionalization of the MNPs' surface with amines (APTES). Then, glutaraldehyde was introduced to link the MNP surface amines with enzyme surface amine residues, typically lysine. Successful covalent bonds were checked by FT-IR. Our results showed the attached enzyme did not affect superparamagnetic property of MNPs, therefore the MNPs-attached enzyme was easily recovered after the reaction. The immobilized enzyme maintained its activities after 10 times of recycle uses

Huu, Cao Xuan

Linh, Nguyen Bao

Long, Dang Duc

Yen, Nguyen Thi Hoang

Vietnam Academy of Science and Technology: Journals Online

Communications in Physics, Vol. 24, No. 3S1 (2014), pp. 121-126
DOI:10.15625/0868-3166/24/3S1/5464
IMMOBILIZING ALCALASE R© ENZYME ONTO MAGNETIC
NANOPARTICLES
CAO XUAN HUU
Department of Electronics & Telecommunication Engineering,
Da Nang University of Technology
NGUYEN BAO LINH, NGUYEN THI HOANG YEN, DANG DUC LONG
Department of Chemistry Engineering, Da Nang University of Technology
E-mail: huucao78@gmail.com
Received 04 April 2014
Accepted for publication 24 May 2014
Abstract. In recent years, magnetic nanoparticles (MNPs) have been applied to numerous biological systems. The
nanoparticles are particularly useful in separating biological molecules due to its low price, scalable ability and very
little interference. Here, MNPs, which can efficiently separate biocatalysts from reaction media by external magnet,
was used to immobilize an alkaline protease (Alcalase R©). Covalent attachment of the enzyme to MNPs began with
the functionalization of the MNPs’ surface with amines (APTES). Then, glutaraldehyde was introduced to link the
MNP surface amines with enzyme surface amine residues, typically lysine. Successful covalent bonds were checked
by FT-IR. Our results showed the attached enzyme did not affect superparamagnetic property of MNPs, therefore the
MNPs-attached enzyme was easily recovered after the reaction. The immobilized enzyme maintained its activities after
10 times of recycle uses.
Keywords: magnetic nanoparticles, alkaline protease, enzyme immobilization, glutaraldehyde.
I. INTRODUCTION
In recent years, magnetic nanoparticles (MNPs) have been applied to numerous biological
systems due to their high saturation magnetization, their biocompatibility, their stability under
hard condition, and the simplicity of their preparation process [1,2]. MNPs have been used in
drug delivery system, protein separation, enzyme and protein immobilization [3] and diagnostics
[4]. In those applications, the surface of MNPs needs to be functionalized and attach to various
molecules such as enzyme, antibodies, drug moieties, biopolymers.
Enzymes immobilized on superparamagnetic nanomaterials can be easily separated from
the reaction media due to a strong interaction between external magnet and nanoparticles in so-
lution. Furthermore, those immobilized enzymes with very small sizes can penetrate into solid
substances; thus eliminating one of main limitations of commercial immobilized enzymes. Bare
MNPs are hydrophobic, so they tend to aggregate in aqueous solutions and are difficult to link to
other molecules. To improve the hydrophilicity of MNPs, a layer of silica derivatives is often made
onto the surface of MNPs. 3-amino propyltrethoxysilane (APTES) containing an amino group is
preferred to use to activate this silica layer to react with various compounds. With this functional
c©2014 Vietnam Academy of Science and Technology
122 IMMOBILIZING ALCALASE R© ENZYME ONTO MAGNETIC NANOPARTICLES
group, MNPs are able to bind to biological molecules, such as enzymes. However, to increase the
efficiency of binding, glutaraldehyde (GA) with two aldehyde groups in their molecules is used
as a bridge between amino group on MNPs surface and amino group from residues of enzyme,
typically lysine [5].
Alcalase R© is an alkali protease used widely in many applications, for example in detergent
formulations. The immobilized Alcalase R© can offer several advantages of reusability and high
hydrolytic activity [6]. However, obstacles still exist to realize those advantages. Therefore, the
aim of the present study was to immobilize the enzyme onto MNPs in order to achieve complete
enzyme reusability through the unique features of the MNPs located in a magnetic field. The
structure and characterization of the MNPs used were examined while the stability and activity of
the bound Alcalase R© were assessed.
II. MATERIALS AND METHODS
II.1. Materials
Alcalase R© 2.4L FG was purchased from Novozyme, Danmark. MNPs coated with an ionic
coating (Fe3O4.SiO2) were kindly provided by a laboratory of Hanoi University of Natural Sci-
ences, Hanoi National University. The protein assay standard was bovine serum albumin (BSA)
obtained from Bio-Rad, USA; 3-aminopropyl triethoxysilane and glutaraldehyde (GA) were pur-
chased from Sigma-Aldrich, USA. Other chemicals were purchased from Sinopharm Chemical
Reagent Co. Ltd. (Shanghai, China).
II.2. Methods
Preparation of immobilized enzyme
The purchased MNPs were washed in distilled water and ethanol 50%. Amine-derived
SiO2-MNP was prepared by modifying the MNP (coated with SiO2) with APTES to introduce
amine groups on the particles’ surface. Ten milligrams of SiO2-MNP was treated in 940µl ethanol
96% and 940µl distilled water with an ultrasonic processor at 37Hz, 30 minutes. And then 20µl 3-
APTES and 100µl glycerol was added. The solution was shaken at 0oC, 150 rpm in an incubator
for 5 hours, continued ultrasound in 10 minutes. The modified MNPs were recovered with a
magnet after washing five times with ethanol 50%. After washing with ethanol, they were washed
three more times with phosphate buffer of 1M. The washed solid was mixed well in 0.5ml aqueous
GA wt. 10% solution. The mixture was incubated in an hour with a light shaking and washed five
times with the above phosphate buffer to remove the redundant GA. At the final washing step, the
mixture was centrifuged at 4000xg for 10 minutes and then the pellet was rewashed with a 0.1 M
borate buffer of pH 7.4. The obtained solid was mixed with 64 µl enzyme and 436 µl of the borate
buffer and incubated overnight with a light shaking. Then the mixture was added with 500 µl of
NaCNBH3 1% (wt.) in 0.2M Tris buffer and incubated for an hour. The particles were washed
four times, each time used 500 µl of the borate buffer plus Tween 20 0.05% in a cool centrifuge
(4oC) at 4000xg for 10 minutes. The resulted immobilized enzymes on MNPs were stored in the
borate buffer solution containing 0.1% Tween 20 and 0.05% wt. NaN3.
Determination of enzyme concentration
The concentration of enzyme was measured by A280 assay, using SmartSpecTMPlus Spec-
trophotometer BioRad [7].
CAO XUAN HUU, NGUYEN BAO LINH, NGUYEN THI HOANG YEN, AND DANG DUC LONG 123
Determination of enzyme activity
The activity of Alcalase R© was determined by Anson assay with a small modification [8].
Briefly, the reaction mixture was prepared by following method: 1ml casein 2% and enzyme at
55oC was mixed in 15 minutes, 55oC, adding 0.5ml TCA to 0.5ml above solution, centrifuged
to remove the solid. Adding 0.8ml Na2CO3 6% and 0.2ml folin 5X onto 0.2ml supernatant in 15
minutes, measured absorbance of sample at 750nm. The same process also was performed without
reaction between enzyme and casein. This sample is control sample.
III. RESULTS AND DISCUSSION
III.1. Characterization of the support
and immobilized protease
 
Fig. 1. The schemes of the immobilization process
 Fig. 2. FTIR spectra of (a) SiO2-coated Fe3O4 nanopar-
ticle, (b) APTES-linked nanoparticle
In this work, APTES was used as
a reactive agent to attach –NH2 on the
surface of MNPs. This process improved
the MNPs’ capacity to immobilize enzymes
owing to the increase of the density of
-NH2 in these beads. These amine groups
were linked with other amino groups on
the enzyme surface via GA bridge. The
whole immobilization process were illus-
trated in Fig. 1.
Reacting with APTES is an impor-
tant step to activate MNPs. FT-IR spec-
troscopy was used to confirm the establish-
ment of silanizated layer onto MNPs sur-
face. Fig. 2 shows the FT-IR spectra of
the SiO2-coated Fe3O4 nanoparticles and
APTES-nanoparticles. For the SiO2-coated
Fe3O4 nanoparticles, the characteristic ab-
sorption peaks at 464.76 cm−1 are attrib-
uted to the Fe-O structure [9]. APTES is
resided on the magnetite nanoparticles sur-
faces by Fe-O-Si bands, because of the ab-
sorption band corresponding to this band
appears at around 500 cm−1 and therefore
overlaps with the Fe-O band, the latest band
cannot be seen in the FT-IR spectrum. The
band at 1096.33 cm−1, which dues to the stretching vibration of Si-O linkage, can be seen on
both of the FT-IR spectra. However, after the silanization of APTES on the MNPs’ surface, the
transmission at this band increase a lot in the case of the APTES-linked MNPs. This indicates
the success of silanization reaction. The two bands at 3305.6 and 1639.1 cm−1 due to the N-H
stretching vibration and -NH2 bending mode of free -NH2 groups again confirm the existence of
APTES.
124 IMMOBILIZING ALCALASE R© ENZYME ONTO MAGNETIC NANOPARTICLES
 
 
Fig. 3 
 
Fig. 3. TEM images in Fe3O4.SiO2-Alcalase (A) and Fe3O4.SiO2 (B)
-20 -10 0 10 20
-40
-30
-20
-10
0
10
20
30
40
M
ag
n
e
tiz
at
io
n
 
(em
u
/g
)
Applied Field (kOe)
 MNP+Enzyme
 MNP
 
Fig. 4. Superparamagnetic property of samples
MNP and MNP-binding APTES examined by
vibrating sample magnetometer
Overlap bands at 3305.6 cm−1 for N-H
stretching is a crucial reason for not to use
FT-IR spectra for confirmation of covalent binding
between GA and APTES, or between GA and en-
zyme. Transmission electronic microscopy (TEM)
images of the particles were taken (Fig. 3). The
diameter of the MNPs coated with enzyme (about
23.0 nm in Fig. 3A) increased compared to that
of SiO2-Fe3O4 nanoparticles (about 17.5 nm in
Fig. 3B). The enzyme modification looks do not
change the surface morphology of the MNPs. When
the samples were examined by magnetic measure-
ments, the superparamagnetic property of MNPs
was conserved after APTES binding (Fig. 4). The
reduction in magnetization of MNPs coated with
enzyme is acceptably predicted by higher dissolu-
bility of MNPs-enzyme ones in solution.
III.2. The activity of the immobilized Alcalase R©
From the FT-IR spectra (Fig. 2) and TEM images (Fig. 3), a covalent binding of enzyme
on activated MNPs is suggested but cannot be firmly concluded. In a further experiment, a certain
amount of enzyme Alcalase R© was incubated with different kinds of MNPs, including of Fe-SiO2,
Fe-SiO2-APTES, Fe-SiO2-GA, Fe-SiO2-APTES-GA, and a bead of Fe-SiO2-APTES-GA which
not incubated with enzyme as a control at the same condition, and then the beads were separated
by external field and washed several times.
After that, the proteolytic activities of these nanoparticles were assessed. The results are
shown in the Table 1. The proteolytic activities of absorbed enzyme on Fe3O4.SiO2 with GA
(0.0062 units/mg enzyme) or APTES (0.0208 units/mg enzyme) was low. This is because the
CAO XUAN HUU, NGUYEN BAO LINH, NGUYEN THI HOANG YEN, AND DANG DUC LONG 125
Table 1. The proteolytic activity of the immobilized Alcalase of five different conditions.
Samples Fe3O4.SiO2- Fe3O4.SiO2- Fe3O4.SiO2-E Fe3O4.SiO2- Fe3O4.SiO2-
GA-E APTES-E APTES-GA APTES-GA-E
Proteolytic
activity 0.0062 0.0208 0.0102 0 0.3686
(units/mg enzyme)
physically absorbing processes allow very limited amounts of protease stay on the MNPs after
washing. The same result happened for Fe3O4.SiO2 without covered by APTES and GA (0.0102
units/mg enzyme). The other experiments proved that the Fe3O4.SiO2 nanoparticles, which only
were covered by APTES and GA, had no proteolytic activity, either. Meanwhile, under the same
condition, the proteolytic activity of the enzyme on Fe3O4.SiO2 nanoparticles, which underwent
the reactions with APTES and GA, was 0.3686 units/mg enzyme. It can be seen clearly from
these results that Alcalase R© on Fe3O4.SiO2-APTES-GA nanoparticles has been immobilized on
the MNPs more stably than in the normal absorption. This only can be the result of the covalent
attachment of the enzyme on the MNPs.
III.3. Effect of pH and temperature to activity of immobilized Alcalase R©
  
Fig. 5. Effect of environmental conditions to activity of immobilized alcalase: a) effect
of temperature; b) effect of pH.
The activity of the immobilized enzyme is affected by environmental conditions, such as
pH, temperature. When the Alcalase R© was immobilized on Fe3O4.SiO2 nanoparticles by bonds
between groups amino of enzymes and groups cacboxyl of GA, the bonds can change ionic of
protein. Therefore, that can influent the pH of the proteolytic reaction. To determine that impact,
we performed experiments to investigate the proteolytic activity of immobilized enzyme at five
different pH: 5.5, 7.4, 8.5, 9.4, 10.5 at the same temperature of 55oC. The activity of immobilized
Alcalase R© was increasing as pH increased, until it peaked at pH 9.4 (Fig. 5a). This is consistent
with enzyme’s characteristics, which is alkaline protease. Therefore, the enzymatic activity in
acidic reaction pH was low and a steady rise to neutral pH. However, when pH increased to 10.5,
the activity of enzyme dropped suddenly. Then, the optimum reaction temperature of the immo-
bilized enzyme was tested. When the reaction temperature went up, kinetic energy of molecules
also rises, thereby increasing the possibilities of collisions between the protease and casein and
126 IMMOBILIZING ALCALASE R© ENZYME ONTO MAGNETIC NANOPARTICLES
the casein fitting into the enzyme. However, the temperature exceeding the optimum limit, the
Alcalase R© starts to degrade dues to the breaking of chemical bonds, that loss active sites.
To see this change, the hydrolysis reactions were performed in the range of 37oC-90oC
and at pH 9.4. It can be seen clearly from the Fig. 5b, the activity of immobilized Alcalase R©
increased with greater temperature was 55oC and then it slipped back. This may be due to high
heat resistance of Alcalase R© , which is limited at 55oC.
III.4. Reusability assay
 
Fig. 6. Proteolytic activity of immobilized en-
zyme after number of recycles
The reusability of immobilized enzyme is
important advantages as it finds wide applica-
tions in the economical point of view. In this
study, number of recycles was performed with ca-
sein in pH 9.4 and 55oC. After that, immobilized
Alcalase R© was separated from the reaction solu-
tion by magnetic field and reached next reactions
after it was washed with buffer clearly. For each
cycle, the corresponding activity was determined.
Fig 6 shows that there was a steady decreasing
from the first to thirteenth times. This loss of the
proteolytic activity may be caused due to many
reasons such as protein denaturation, lost enzyme
during washing.
IV. CONCLUSIONS
An easily recoverable and reusable support for the immobilization of Alcalase R©, a broadly
used protease, is synthesized by a covalent attachment of the enzyme to magnetite nanoparticles.
The attachment reaction’s conditions were optimized. The obtained particles are thoroughly char-
acterized. They possess a higher enzyme immobilization efficiency and high superparamagnetism,
which enable a fast enzyme recovery. The immobilized enzyme was more thermally stable than
the free enzyme. The immobilized enzyme has also been efficiently recycled after more than ten
cycles.
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[4] Elaı¨ssari, A. and V. Bourrel, Journal of Magnetism and Magnetic Materials 225 (2001) 151-155.
[5] Voliker Herzog and H. Dariush Fahimi, Journal of Cell Biology 60 (1974) 303-311
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