Comparative study of ASNase immobilization on tannic acid-modified magnetic Fe3O4/SBA-15 nanoparticles to enhance stability and reusability

Article abstract of DOI:10.1039/d0nj00127a

In this work, l-asparaginase was immobilized on tannic acid-modified magnetic mesoporous particles. In brief, Fe₃O₄/SBA-15/tannic acid magnetic particles were synthesized, and their structures and morphologies were fully characterized using various methods. The properties of the free and immobilized enzyme were examined in terms of pH, temperature, thermal stability, storage stability, and reusability. Moreover, the effects of metal ions, inhibitors and organic solvents on the activity of the immobilized enzyme were investigated. Compared to the free enzyme, the immobilized enzyme possessed better tolerance to changes in ambient temperature and pH. Additionally, thermal incubation results showed that the free enzyme lost its activity, while the immobilized enzyme exhibited the opposite behavior. Most strikingly, the immobilized l-asparaginase exhibited a high degree of activity (70%) after being reused 16 times while also demonstrating 71% and 63% storage stability of the initial activity even after 28 days at 4 °C and room temperature, respectively. Together with these results, l-asparaginase was successfully immobilized upon Fe₃O₄/SBA-15/tannic acid magnetic nanoparticles with improved stability properties. This support holds great potential and opens up a novel perspective for growing applications.

Full text of DOI:10.1039/d0nj00127a

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Page 1 of 33
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DOI: 10.1039/D0NJ00127A
Comparative study of ASNase Immobilization on Tannic Acid-Modified
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Magnetic Fe3O4/SBA-15 Nanoparticles to Enhance Stability and Reusability
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Samir Abbas Ali Noma1, Ahmet Ulu1, Ömür Acet2, Raúl Sanz3, Eloy S. Sanz-Pérez4, Mehmet
Odabaşı2, Burhan Ateş1*
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1Department of Chemistry, Science and Literature Faculty, İnönü University, 44280, Malatya, Turkey
2Aksaray University, Faculty of Arts and Science, Chemistry Department, Aksaray, Turkey
3Department of Chemical and Environmental Technology, ESCET, Universidad Rey Juan Carlos, C/ Tulipán s/n,
28933 Móstoles, Madrid, Spain
3Department of Chemical, Energy, and Mechanical Technology, ESCET, Universidad Rey Juan Carlos, C/
Tulipán s/n, 28933 Móstoles, Madrid, Spain
*Corresponding Author: Burhan ATEŞ
E-mail: burhan.ates@inonu.edu.tr
Tel: (+) 90-422-3773888
Fax: (+) 90-422-3410037
Address: Department of Chemistry, Faculty of Arts and Science,
İnönü University, 44280, Malatya, Turkey
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Abstract: In this work, L-asparaginase was immobilized on tannic acid-modified magnetic
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mesoporous particles. In brief, Fe3O4/SBA-15/tannic acid magnetic particles were synthesized
and fully characterized by using various methods as structural and morphological. The
properties of free and immobilized enzyme were examined in terms of pH, temperature, thermal
stability, storage stability, and reusability. Moreover, the effects of metal ions, inhibitors and
organic solvents were investigated on activity of the immobilized enzyme. As compared to free
enzyme, the immobilized enzyme possessed better tolerance to changes in ambient temperature
and pH. Additionally, thermal incubation results showed that free enzyme lost its activity while
the immobilized enzyme exhibited the opposite behavior. Most strikingly, the immobilized L-
asparaginase exhibited a high degree of activity (70%) after being reused 16 times while also
demonstrating 71% and 63% storage stability of the initial activity even after 28 days, at 4 oC
and room temperature, respectively. Together with these results, L-asparaginase was
successfully immobilized upon Fe3O4/SBA-15/tannic acid magnetic nanoparticles with
improved stability properties. This support holds great potential and opens up a novel
perspective for its growing applications.
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Keywords: Enzyme immobilization; Magnetic nanoparticles; Surface functionalization;
Asparaginase; Tannic acid
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1.
Introduction
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Enzyme immobilization is defined as the chemical and/or physical interaction of different types
of carriers to reduce the mobility of free or dissolved enzyme. The immobilized enzyme is more
stable than free enzyme against factors such as pH, high temperature, various denaturing
organic solvents.1 In recent years, immobilize enzyme technology has been commonly used in
multidisciplinary studies for the analysis of medicine, antibiotic production, drug metabolism,
food industry, biodiesel production, biosensor, bioremediation and clinical.2,3 The most
important problem encountered in soluble enzymes applications is rapid activity loss and high
cost of some enzymes. Most importantly, the control of enzymes is very difficult and is
impossible to remove the enzymes from the reaction medium. Therefore, various
immobilization techniques such as adsorption, covalent binding, entrapment and crosslinking
have been developed in order to improve enzyme activity.2,4
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On the other hand, the choice of suitable carrier support material for enzyme
immobilization has a critical role to ensure the performance and stabilization of the enzyme. In
recent years, the trend supports commonly used for enzyme immobilization are nanoparticles
and their modified forms. In this respect, SBA-15 (Santa Barbara Amorphous) produced in
1998 by Zhao et al. 5 has attracted high interest because of some properties such as high surface
area, large pore volume, complement channels 6 and the combination of micro and mesoporous
constructions.7,8 These excellent structural and textural features, which make them very desired
matrices for some implementations such as adsorption, catalysis and drug delivery.9–11 And
also, when compared to other particles, silica-based structures are non-toxic, biodegradable and
relatively cheap. The main reasons for choosing SBA-15 in adsorption process are primarily to
combine the immobilization and purification processes in one step and to save time and cost.4
Magnetic nanoparticles (MNPs) have also been frequently preferred as a support
material for the immobilization of enzymes because of their high surface area, leading to
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improved thermal stability and a higher residual activity of the immobilized enDzOyIm: 10e.10u39n/dD0eNrJ00127A
harsh solvent conditions, as compared to the free enzyme. Nevertheless, the interaction between
mesoporous silica and enzyme is too weak for immobilization, so the surface functionalization
must carry out with appropriate organic groups to improve stability of the immobilized
enzyme.12 In our previous works, we discovered that functionalized magnetic nanoparticles
exhibited excellent results with a high immobilization yield in immobilization of enzyme.13–16
The surface functionalization of MNPs has been carried out by binding different compounds
containing functional groups such as amino (NH2), hydroxyl (OH), carboxylic acid (COOH),
or thiol (SH).17 However, many chemical reagents used in the modification of MNPs are not
only expensive but also harmful to human health and the environment.18 Therefore, to avoid
the use of these chemicals, they should be replaced with new, inexpensive and non-toxic
reagents.
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Tannic acid (TA) is a biocompatible water-soluble polyphenol used in applications such
as different metal ions adsorption, as a polymeric coagulant and flocculants for water treatment.
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Besides, it is used in the construction of multifunctional coatings in surface modification.
Recently, TA modified carrier supports were widely used for immobilization of various
enzymes such as trypsin 17,20, glucose oxidase 19, catalase 21, nitrile hydratase 22 and β-agarase.18
L-asparagine is a nutritional requirement for the growth of both normal and leukemia
cells.23 L-asparaginase (ASNase, EC 3.5.1.1) is important enzyme catalysis the hydrolysis of
L-asparagine into aspartic acid and ammonia.24 In presence of ASNase, the leukemia cells
cannot continue to proliferate and consequently dies. The chemotherapeutic effect of the
ASNase enzyme is based on this principle. However, there are two important factors limit the
medical use of ASNase: First, it is a microbial product derived from Escherichia coli or Erwinia
chrysanthemi.25 Therefore, it shows a very short half-life due to rapid clearance from the body.
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Secondly, it may cause serious side effects after therapy.26 The effective and simple method
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used to avoid the two disadvantages is enzyme immobilization.
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Till now, many studies about the different materials as the support have been reported
for ASNase immobilization. In current work, for the first time, we present in detail how the
design of hybrid TA-modified magnetic particles affects the effectiveness of immobilization,
activity, and performance of ASNase after immobilization. Moreover, it is also known that TA
has anticancer and antioxidant properties. Therefore, the use of support containing TA for
ASNase immobilization may lead to a synergistic effect. The ASNase immobilization processes
on Fe3O4/SBA-15/TA are shown in Fig. 1. The samples were characterized by various
techniques such as ATR-FTIR, XRD, VSM, SEM, EDX and zeta potential measurements.
Subsequently, ASNase was successfully immobilized on Fe3O4/SBA-15/TA, and the
biochemical properties of immobilized ASNase were investigated in detailed. As we predicted,
the immobilized ASNase displayed high tolerance against extreme conditions, excellent
reusability, long-term incubation stability compared to free enzyme. We hope that this detailed
work will contribute to improving the immobilized ASNase in broad practical applications such
as biotechnology, industrial catalysis, and biomedical engineering.
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Fig. 1. The schematic diagram of the preparation of magnetic Fe3O4/SBA-15/TA nanoparticles
(a), the immobilization of ASNase on Fe3O4/SBA-15/TA (b), and the catalytic activity of
immobilized enzyme (c). The picture of ASNase was taken from following article.27
2.
Materials and method
2.1. Materials
All the reagents were used without further purification. All enzyme and substrate solutions were
freshly prepared before being used. The lyophilized ASNase powder obtained from Escherichia
coli was purchased from ProSpec. The L-asparagine, trichloroacetic acid (TCA),
tris(hydroxymethyl)aminomethane, HCl, and TA all purchased from Sigma–Aldrich.
FeCl2·4H2O, FeCl3·6H2O, and NH3 were obtained from Merck. The other chemicals used were
of analytical purity. Ultra-pure water was used to prepare all the aqueous solutions required
throughout the study.
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2.2. Synthesis of magnetic Fe3O4
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Fe3O4 was synthesized according to method reported previously.28 FeCl2·4H2O (4.23 g) and
FeCl3·6H2O (11.48 g) were dissolved in 500 mL of distilled water, then the reaction mixture
was heated to 90 °C and refluxed under Argon gas for 6 h. After that, an aqueous solution of
NH3 (25% w/v) (10 mL) was added drop by drop to the mixture over 30 min, and the reaction
allowed to proceed for a further 2 h at 90 °C. The black precipitate was separated via an external
magnet and washed several times with distilled water/ethanol. Fe3O4 particles were dried at 100
°C overnight.
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2.3. Synthesis of SBA-15
SBA-15 mesostructured silica was prepared following the original synthesis procedure by
Stucky and co-workers 29 with a modification in order to get a large-scale batch.30,31 Pluronic
P123 (PEO20-PPO70-PEO20, M ≈5,800, Sigma-Aldrich) was used as a structure directing agent
and tetraethyl orthosilicate (TEOS, 98 %, Sigma-Aldrich) as a silica source. Briefly, 1.9 M HCl
was used to dissolve Pluronic P123. TEOS was added to this mixture and kept at 40 ºC for 20
h to perform a hydrolysis step. This was followed by an ageing step at 110 ºC for 24 h without
stirring. The obtained SBA-15 material was filtered and calcined at 550 ºC for 5 h to remove
the surfactant from the porous structure.
2.4. Synthesis of Fe3O4/SBA-15 particles
0.5 g of SBA-15 was dissolved in (25 mL) of dry toluene. A solution of Fe3O4 (0.25 g, 5 mL)
in dry toluene was added dropwise to the dispersed SBA-15 solution under continuous stirring.
The reaction mixture was kept under refluxing conditions for about 24 h under inert atmosphere
by using triethylamine as initiator. Then, the product was allowed to cool at room temperature.
After cooling, an external magnet was used to collect the product. After that, the product was
washed several times with distilled water/ethanol and dried at 70 °C for 24 h.
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2.5. Surface modification
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Fe3O4/SBA-15 (0.5 g) was dissolved in a flask containing dried acetonitrile. Afterward, TA (0.6
g) and triethylamine (0.15 ml) were added into the reaction flask. The mixture was refluxed at
80 °C for 24 h. Then, the obtained solid was separated by using an external magnet and was
washed with water/ethanol for several times to remove unreacted TA. The obtained
Fe3O4/SBA-15/TA was filtrated before drying at 60 °C overnight.32
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2.6. Immobilization of ASNase on Fe3O4/SBA-15/TA
The obtained magnetic Fe3O4/SBA-15/TA particles (100 mg) were dispersed in 1 mL Tris-HCl
buffer solution (50 mM, pH 8.6) by sonication. After separation, the mixture involving free
enzyme (100 U) and Fe3O4/SBA-15/TA was directly transferred into plastic flasks. The mixed
solution was incubated at 4 °C in a shaker with a continuous shaking rate (120 rpm) for 6 h
until the enzyme was completely immobilized. Then, Fe3O4/SBA-15/TA/ASNase was
separated by magnetic separation with help of an external magnet and was washed with
deionized water and Tris-HCl buffer (pH 8.6) three times to remove the unbound enzymes from
the supports. Finally, the Fe3O4/SBA-15/TA/ASNase was stored at 4 °C for activity tests.
Immobilization yield (IY) and immobilization efficiency (IE) were calculated by using Eq. (1)
and Eq. (2), respectively.
C1 - C2
IY (%) =
x 100 (1)
C1
Where C1 is the concentration of protein for total enzyme and C2 is concentration of protein
present in the supernatant and washing solutions after immobilization.
The activity of Fe3O4/SBA - 15/TA/ASNase
IE (%) =
x 100 (2)
The activity of free ASNase
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2.7. Enzymatic activity assay
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ASNase activity was measured spectrophotometrically by using L-asparagine as the substrate.
Briefly, L-asparagine solution (10 mM) was prepared, first with Tris-HCl buffer (pH:8.6). A
certain amount of free ASNase and Fe3O4/SBA-15/TA/ASNase were placed into a plastic tube,
and the reaction was initiated by addition of substrate solution. The incubation time was 15 min
at 37 oC. At the end of the time, TCA solution (1.5 M) was added to stop the reaction and 100
μl of product mixture was afterward added to 100 μl Nessler’s reagent. The resulting mixture
was incubated in dark for 10 min and then absorbance was measured using a microplate reader
at 480 nm. One unit (U) of enzyme activity is defined as the amount of ASNase consumed to
produce 1 µmol ammonia per min.
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2.8. Determination of optimal temperature and pH
The reaction temperature and pH are important critical factors influencing the catalytic activity
of enzyme. Therefore, the optimum temperature was analyzed by assaying enzyme activity after
incubating the enzyme samples in the 25-70 °C range. To determine the optimum pH of free
and immobilized enzyme, the activities were measured under standard assay conditions by
using sodium-acetate (50 mM, pH:4.0–6.0), sodium-phosphate (50 mM, pH:7.0–8.0), and Tris-
HCl (50 mM, pH:8.5-10.0) buffers.
2.9. Thermal and storage stability
In fact, thermal inactivation is a major challenge for industrial enzymes. Exposure to high
temperatures causes changes in the 3D structure of the enzyme. Considering it’s significant, the
stability of free and immobilized enzyme was examined after preincubation at 50 °C for 180
min. Enzyme aliquots were removed after 30 min and the percentage of residual activities
calculated.
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The long-term storage stability was investigated by incubating free and immobiliDzOeId: 10e.1n0z39y/mD0eNJ00127A
under dry conditions for 28 days. The samples were withdrawn to measure the residual activity
at different intervals, and the first activity was considered as 100%.
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2.10. Operational stability
To perform the operational stability of the immobilized enzyme, a known amount of
Fe3O4/SBA-15/TA/ASNase was transferred into a reaction tube containing substrate solution,
and the reaction was performed under optimal conditions. The Fe3O4/SBA-15/TA/ASNase was
separated from the reaction mixture via an external magnet and washed three times with Tris-
HCl (pH: 8.5). After washing, the Fe3O4/SBA-15/TA/ASNase was subjected to repeated use by
adding fresh substrate. In the first use, the relative activity was considered to be 100%.
2.11. Determination of kinetic parameters
Kinetic parameters (Km and Vmax) for free ASNase and Fe3O4/SBA-15/TA/ASNase were
calculated using the Lineweaver-Burk plot at different concentrations (0.25, 0.5, 0.75, 1.0, 2.0,
4.0, 6.0, 8.0, and 10.0 mM) of L-asparagine at optimal assay conditions.
2.12. The effects of different metal ions, inhibitors and solvents on ASNase activity
The both free and immobilized ASNase was measured by using the assay procedure after 24 h
incubation with the different metal ions (10 mM, Ag+, Mg2+, Ba2+, Zn2+, Hg2+, Sn2+, Pb2+, and
Al3+), potential inhibitors (5%, EDTA, SDS, Tween 80 and Triton X-100) and 5% of organic
solvents (acetone, acetonitrile, chloroform, tetrahydrofuran (THF), dimethylformamide (DMF)
ethanol, methanol, and n-hexane).
2.13. Instruments for characterizations
N2 adsorption-desorption isotherms were used to investigate the Brunauer, Emmett, and Teller
(BET) surface area and Barrett-Joyner-Halenda (BJH) pore size distribution at 77 K in a
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Micromeritics Tristar 3000 apparatus for SBA-15. The surface area was obtained DbOyI:u10s.i1n03g9/tDh0eNJ00127A
BET equation in the P/P0 range between 0.05 and 0.20. Pore size distribution was calculated
from the adsorption branch of the isotherms through the BJH model.33 An 8 h degasification
step in nitrogen flux was performed at a temperature of 200 ºC. To detect different functional
groups in samples, FTIR spectra (Perkin Elmer) were obtained in ATR mode within the
wavelength range 400-4000 cm-l. The XRD (Rigaku) analysis was used to analyze the structure
of crystalline materials with Cu Kα radiation and the 2θ angle range of 10-80°. The saturation
magnetization values of prepared particles were measured by using a VSM (PPMS) at room
temperature. Knowledge about surface morphology and elemental composition of samples was
taken from energy dispersive x-ray spectroscopy (EDS) in scanning electron microscopy (SEM)
(LEO, 440 SEM-EDX system, Leica-Zeiss, DSM-960). The zeta potential of the samples was
measured by using Zeta potential analyzer with at least 3 replicates for each sample. The
enzyme activity measurements were conducted on Microplate Reader (BioTek).
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2.14. Statistical analysis
Data were statistically analyzed using GraphPad Prism 5.0. P<0.05 was considered to be
statistically significant.
3.
Results and discussion
3.1. Materials characterizations
N2 adsorption-desorption isotherm and pore size distribution of SBA-15 is shown in Fig. 2. As
seen, the isotherm fits within the type IV group of isotherms corresponding to mesostructured
solids, as described by the IUPAC.33 Siliceous SBA-15 presented a wide and open porous
structure. Its main textural properties are specific surface of 533 m2/g, pore volume of
0.87 cm3/g and a mean pore diameter of 8.5 nm. Thus, these solids present an open pore
structure interesting for its further application.
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Relative Pressure (P/Po)
Pore Size (nm)
Fig. 2. a) N2 adsorption-desorption isotherm SBA-15 silica, and b) pore size distribution of
SBA-15 silica.
The FTIR spectra of Fe3O4, SBA-15, pure TA, and Fe3O4/SBA-15/TA samples are shown in
Fig. 3. The band at 540 cm-1 are attributed to Fe–O vibration of Fe3O4.34 For SBA-15,
characteristic peaks strong broadband at 1000-1250 cm−1 and a weak band at 812 cm−1 are
assigned to the Si-O-Si symmetric and asymmetric stretching vibration, respectively.8 The
essential characteristic bands of the pure TA compound are at 3100-3600 cm-1 (O-H stretching
of the phenolic and methylol group of TA), 1700 cm-1 (C=O stretching), 1610 cm-1 (aromatic
C=C stretching), 1191 cm-1 (phenolic C-O stretching), 870 cm-1 (the deformation out of the
plane (C-H) for benzene ring band), 758 cm-1 (C-H vibration of benzene ring).35 The spectrum
of Fe3O4/SBA-15/TA contains not only the peaks of Fe3O4/SBA-15 but also the peaks of TA.
However, the peak related to the stretching mode of the Fe-O bond shifted to shorter
wavenumbers (540 and 443 cm-1) after the addition of TA.36 These results indicated that
Fe3O4/SBA-15 was successfully coated with TA compound.
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Fig. 3. FTIR spectra of Fe3O4 (a), SBA-15 (b), TA (c), and Fe3O4/SBA-15/TA (d).
The magnetic hysteresis loops for Fe3O4, Fe3O4/SBA-15, and Fe3O4/SBA-15/TA were shown
in Fig. 4. According to the curves, the magnetization values of Fe3O4, Fe3O4/SBA-15, and
Fe3O4/SBA-15/TA were 28.14, 8.14, and 7.94 emu g-1, respectively. The saturation
magnetization of the prepared Fe3O4 NPs was lower than the previously reported Fe3O4 due to
size-dependent magnetization and the presence of the residues on the surface of the Fe3O4
NPs.37,38 When the SBA-15 was immobilized on Fe3O4, the saturation magnetization was
remarkably decreased. In addition, the small decrease was observed in magnetization value
after covering with TA. The decreases in overall magnetization values showed that the Fe3O4
surface was successfully covered with nonmagnetic materials such as SBA-15 and TA.39,40
Consequently, the Fe3O4/SBA-15/TA can be dispersed in aqueous solution and easily collected
from aqueous solution by using an external magnet.
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Fig. 4. Magnetic hysteresis loops of Fe3O4, Fe3O4/SBA-15, and Fe3O4/SBA-15/TA.
Fig. 5 showed the XRD patterns of Fe3O4, SBA-15, Fe3O4/SBA-15, and Fe3O4/SBA-15/TA.
The six diffraction peaks observed at 30.43°, 35.56°, 43.18°, 53.35°, 57.19°, and 62.67° are
well accordance with the inverse cubic spinel phase of Fe3O4 (Fig. 5a).41 The strong and sharp
peaks indicated well crystallization of synthesized Fe3O4. For SBA-15, the broad diffraction
peak between 15° and 30° is ascribed to the silicon material (Fig. 5b).40 From Fig. 5c and 5d, it
can be observed that both of Fe3O4/SBA-15 and Fe3O4/SBA-15/TA showed the same pattern.
However, the strong peaks for both samples compared with Fe3O4 were decreased due to the
presence of amorphous SBA-15.42 The XRD results verified the production of Fe3O4/SBA-
15/TA nanocomposite.
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Fig. 5. XRD patterns (a) Fe3O4, (b) SBA-15, (c) Fe3O4/SBA-15 and (d) Fe3O4/SBA-15/TA.
3.2. Morphological assessment
The SEM micrographs of Fe3O4, SBA-15, Fe3O4/SBA-15, Fe3O4/SBA-15/TA and Fe3O4/SBA-
15/TA/ASNase at different magnifications were shown in Fig. 6. As seen in Fig. 6a-b, the Fe3O4
nanoparticles displayed homogeneous size distribution and uniform morphology. However,
some clusters of particles may be observed because of the magnetic interaction between the
particles. Fig. 6c-d showed the typical morphology of SBA-15, and it consists of vermicular-
shaped particles as described by Souza et al.43 Although Fe3O4/SBA-15 particles were non-
homogeneous in size, and increased surface roughness was observed due to the presence of
Fe3O4 particles (Fig. 6e-f). As demonstrated in Fig. 6 g-h, the Fe3O4/SBA-15/TA displayed
heterogeneous morphology, but it had still vermicular-shaped. Finally, Fig. 6i-j revealed that
no significant morphological changes were observed on the surface of the material after enzyme
immobilization. The obtained SEM images were similar to those previously reported in the
literature.17,44
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Fig. 6. SEM micrographs of (a, b) Fe3O4, (c, d) SBA-15, (e, f) Fe3O4/SBA-15, (g, h)
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Fe3O4/SBA-15/TA and (i, j) Fe3O4/SBA-15/TA/ASNase at different magnifications.
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3.3. EDX analysis
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The surface composition of Fe3O4, Fe3O4/SBA-15, Fe3O4/SBA-15/TA, and Fe3O4/SBA-
15/TA/ASNase was determined through EDX as shown in Fig. 7. The peaks of iron (Fe) and
oxygen (O) appeared in all of the samples. Fig. 7b confirmed that Fe3O4 was coated with SBA-
15 because of in the presence of silica (Si). TA presence on the surface of Fe3O4/SBA-15/TA
was verified by appearing of carbon (C) peak (Fig. 7c). Also, the presence of characteristic
peaks of sulphur (S) and nitrogen (N) in Fig. 7d proved that ASNase was successfully
immobilized onto Fe3O4/SBA-15/TA.
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Fig. 7. EDX patterns of (a) Fe3O4, (b) Fe3O4/SBA-15, (c) Fe3O4/SBA-15/TA and (d)
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Fe3O4/SBA-15/TA/ASNase.
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3.4. Zeta potential
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The zeta potential values of Fe3O4, SBA-15, Fe3O4/SBA-15, and Fe3O4/SBA-15/TA were
measured at pH:7, and each measurement was repeated three times (Fig. 8A). Fe3O4, SBA-15,
and Fe3O4/SBA-15 showed a zeta potential of −32.7 mV, −30.0 mV, and -24.4, respectively.
The negative charge of Fe3O4, SBA-15, and Fe3O4/SBA-15 was due to the presence of surface
hydroxyl groups and silanol groups.45,46 The zeta potential of Fe3O4/SBA-15/TA was -24.5 mV.
The zeta potential value was not significantly changed after modification by TA at pH:7.0. In
this study, Fe3O4/SBA-15/TA was used as support for ASNase immobilization. Therefore, we
measured zeta potential values of Fe3O4/SBA-15/TA from pH:4.0 to 10.0 and the estimated
zeta potential values were presented in Fig. 8B. As expected, the zeta potential values of
Fe3O4/SBA-15/TA were negative in the analyzed pH range.
Fig. 8. Average zeta potential of Fe3O4, SBA-15, Fe3O4/SBA-15, and Fe3O4/SBA-15/TA at
pH:7.0 and the zeta potential values of Fe3O4/SBA-15/TA at different pH conditions (from 4 to
10).
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3.5. Immobilization yield and immobilization efficiency
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The Fe3O4/SBA-15/TA showed an immobilization yield of 92% and an immobilization
efficiency of 96% for ASNase. The high immobilization efficiency and efficiency can be
attributed not only to the high surface area of the nanoparticles but also to the modification of
the nanoparticles with TA. Similar results were observed in studies using TA modified
nanoparticles as immobilization support.17
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3.6. Determination of optimal pH and temperature
The optimum temperatures of free ASNase and Fe3O4/SBA-15/TA/ASNase were determined,
as shown in Fig. 9A. The free ASNase possessed maximum activity at 50 °C and maintained
80% of its activity at temperatures of 45 to 55 °C. At temperatures above 55 °C, the catalytic
activity of free ASNase rapidly declined due to denaturation of the peptide structure and lost
all activity at 65 °C. On the other hand, the activity of Fe3O4/SBA-15/TA/ASNase significantly
improved with increasing temperature, and it exhibited its highest activity at 65 °C. In addition,
the relative activity of it is more than 40% at even 70 °C. Free ASNase undergoes
conformational changes with high temperature, resulting in activity decreasing at a faster rate
than that of the Fe3O4/SBA-15/TA/ASNase.1 In contrast, the Fe3O4/SBA-15/TA
microenvironment protected the enzyme from denaturation under harsh conditions, and the
immobilization process preserved more the enzyme stability and activity. Similar results were
also obtained for the optimum pH of free ASNase and Fe3O4/SBA-15/TA/ASNase. As shown
in Fig. 9B, free ASNase displayed maximum activity at pH 8.5, while the optimum pH of
Fe3O4/SBA-15/TA/ASNase, as expected, shifted especially to alkaline pH range (7.5-10.0). It
is well known that cationic or anionic support matrix influences the catalytic properties of
enzyme after the immobilization.47 The isoelectric point of ASNase is 8.74 and the optimum
pH value is 8.50. At this pH, Fe3O4/SBA-15/TA possessed highly surface negative charges
because of hydroxyl group on TA (zeta potential is −37.4 mV). Therefore, this shift in optimum
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pH might be attributed to the microenvironment due to the weak acidity of Fe3O4/SBA-15/TA.
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The results obtained in terms of temperature and pH coincide with the data reported for both
enzymes in the literature.48,49
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3.7. Thermal and storage stability
To investigate thermal stability, free ASNase and its counterpart were incubated for 3 h at 50
°C (Fig. 9C). After 3 h incubation, it was observed that free ASNase lost almost half activity,
while Fe3O4/SBA-15/TA/ASNase retained 73% of the initial activity. Thanks to Fe3O4/SBA-
15/TA, stability and instinct structure of the enzyme were retained due to not being direct
exposure to heat. It was evident from the results that hybrid composites provide a stuck
protection effect and prevent denaturation under harsh conditions.50 Similar studies reported
that the thermal stability of free ASNase enhanced after immobilization. For instance, Uygun
et al. reported that free ASNase maintained only 5% of its initial activity after 5 h incubation at
65 °C, whereas immobilized ASNase retained 30% of its initial activity under the same
conditions.51 In another study, trypsin was immobilized on TA modified Fe3O4.17 The
immobilized trypsin was found to be more thermostable than the free trypsin after 2 h
incubation at 45 °C. It is indicated that the novel hybrid composite played a key role in thermal
stability because of its stabilizing effect.
In terms of industrial scale, storage stability is one of the important parameters. To test the long-
term storage stability of the enzymes, free and immobilized enzyme were stored at 4 °C and
room temperature (nearly 25 °C). However, a slight difference between the relative activity of
free ASNase and Fe3O4/SBA-15/TA/ASNase was observed. As shown in Fig. 9D, the
Fe3O4/SBA-15/TA/ASNase retained about 71% of its initial activity at 4 °C and 63% at room
temperature for 28 days, respectively, while free ASNase possessed 66% and 59% of the overall
activity, respectively, under similar conditions. These results confirmed that the storage
stability of Fe3O4/SBA-15/TA/ASNase improved slightly in comparison to free one after
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immobilization. This might be explained by the low protective effect of the support material
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Fig. 9. (A) optimum temperature; (B) optimum pH; (C) thermal stabilities at 50 oC; (D) storage
stabilities of free ASNase and Fe3O4/SBA-15/TA/ASNase. All measurements were run in
triplicate (n=3).
3.8. Operational stability
Fig. 10 presents the percentages of the residual activity of the Fe3O4/SBA-15/TA/ASNase after
multiple uses. After first 5 and 10 recycles, it maintained about 92% and 87% of its original
activity, respectively. The Fe3O4/SBA-15/TA/ASNase exhibited better operational stability that
was able to maintain 70% of the initial activity after 16 consecutive cycles. Many studies
reported the reusability of immobilized ASNase on different supports. For instance, the two
different immobilized ASNase maintained 42% and 29% of the initial activity after 8 cycles in
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study of Monajati et al.52 In another study, Agrawal and Kango evaluated the reusability of
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immobilized ASNase on the functionalized aluminum oxide nanoparticles and titanium oxide
nanoparticles, and the residual activities were reported as 91.8% and 95.1%, respectively, after
9 cycles.53 Such amazing recycling efficiency demonstrated that suggested carrier can be used
in various industrial bioprocesses.
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Fig. 10. The operational stability of Fe3O4/SBA-15/TA/ASNase. All measurements were run in
triplicate (n=3).
3.9. Determination of kinetic parameters
The Michaelis constant (Km) and maximum reaction velocity (Vmax) was estimated by using
the Lineweaver–Burk plot method at different concentrations of L-asparagine as substrate (Fig.
11). Additionally, the kinetic values were summarized in Table 1. The Km values of free
ASNase and Fe3O4/SBA-15/TA/ASNase were calculated to be 0.34±0.03 and 0.67±0.02 mM,
respectively. After ASNase immobilization, an obvious increase in Km (~1.97-fold) indicated
that the Fe3O4/SBA-15/TA/ASNase had a significantly lower affinity for its substrate than free
ASNase due to steric inhibition of the solid support. The Km was increased approximately 1.3
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times for immobilized ASNase compared to free ASNase.54 They attributed this increase to the
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structural changes in the enzyme or due to the lower accessibility of the substrate. On the other
hand, Vmax values for free ASNase and Fe3O4/SBA-15/TA/ASNase were determined and this
value decreased from 47.52±1.13 to 42.00±1.30 μmol min-1 after immobilization. The decrease
in Vmax value could be due to the conformational changes in the enzyme after immobilization.
Monajati prepared aspartic acid functionalized graphene oxide nanosheet for ASNase
immobilization.52 They reported that Vmax value for immobilized enzyme decreased about 1.3
times after immobilization. In another study, Uygun et al. reported a similar trend.51 This
increase in Km and decrease in Vmax are consistent with our results.
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Fig. 11. The Lineweaver-Burke plots of free ASNase and Fe3O4/SBA-15/TA/ASNase. All
measurements were run in triplicate (n=3).
Based on the Student’s t-test results, Table 1 also demonstrated that the increase in Km value
and the decrease in Vmax value were statistically significant at P<0.05.
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Table 1. Km and Vmax values of free ASNase and Fe3O4/SBA-15/TA/ASNase.
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Free ASNase
0.34±0.03
Fe3O4/SBA-15/TA/ASNase Sig. (2-tailed)
Km (mM)
0.67±0.02
.000a
.000b
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Vmax (μmolmin-1)
a, b P<0.05
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42.00±1.30
3.10. Effect of metal ions, inhibitors, and organic solvents
The effects of different metal ions were studied on free ASNase and Fe3O4/SBA-
15/TA/ASNase. The results were summarized in Table 2. The free ASNase lost 61% of its
original activity in the presence of Ag+, while silver ions showed no action on the Fe3O4/SBA-
15/TA/ASNase (109%). On the other hand, the divalent metals tested had inhibitory effect on
both free ASNase and Fe3O4/SBA-15/TA/ASNase except for Mg2+. Zuo et al. 55 and Agrawal
& Kango 53 reported that ASNase activity may be slightly stimulated in the presence of Mg2+.
According to our results, the relative activity of free ASNase and Fe3O4/SBA-15/TA/ASNase
enhanced approximately 64.8% and 69.0%. Besides, this phenomenon was observed by
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Mahajan et al. and Hassan et al. Ba2+ (54%), Hg2+ (85%), Sn2+ (79%), Pb2+ (82%), Zn2+
(84%) and Al3+ (63%) showed the high inhibitory effect on free ASNase activity. Similarly,
Fe3O4/SBA-15/TA/ASNase was also inhibited, however, it exhibited more activity in
comparison to free ASNase. For instance, Fe3O4/SBA-15/TA/ASNase maintained high activity
in presence of the strong inhibitors such as Pb2+ andZn2+. The highest inhibition effect was also
observed with Hg2+ for both enzyme samples. A similar trend was reported for previous
immobilized L-ASNase.54 In another work, the ASNase was immobilized on aluminum oxide
pellets. Immobilized enzyme retained 78% of its initial activity in presence 5 mM Hg2+, whereas
the free ASNase maintained 41% of its initial activity 58. These inhibitions may be due to
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charged ions interact with the side chain groups of amino acids in the enzyme whicDhOaI:f1f0e.1c03ts9/tDh0eNJ00127A
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conformation and stability of the enzyme.59
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Table 2. The effects of different metal ions on free ASNase and Fe3O4/SBA-15/TA/ASNase
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activity. All measurements were run in triplicate (n=3).
Metal ions
Concentration
Relative activity (%)
Free ASNase
Fe3O4/SBA-15/TA/ASNase
100±12.1
Control
Ag+
-
100±4.1
39.2±6.6
164.8±8.9
46.0±0.7
15.4±2.4
21.3±0.3
18.4±1.5
16.0±1.1
37.2±2.4
10 mM
10 mM
10 mM
10 mM
10 mM
10 mM
10 mM
10 mM
109.9±5.9
169±16.5
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54.7±2.8
79.0±3.9
Pb2+
59.6±2.7
Zn2+
Al3+
61.6±5.1
68.5±1.1
As shown in Fig. 12A, the stability of free ASNase and Fe3O4/SBA-15/TA/ASNase were
investigated in the presence of various inhibitors. The results revealed that the relative activity
of free ASNase significantly decreased in EDTA, SDS, Tween 80 and Triton X-100 by about
83%, 63%, 71% and 66%, respectively. Although the relative activity of the Fe3O4/SBA-
15/TA/ASNase decreased by about 31% in Triton X-100, its relative activity almost stable in
the presence of SDS and Tween 80. El-Naggar et al. investigated the effect of several inhibitors
on the purified L-asparaginase from Streptomyces brollosae NEAE-115 60. They reported that
Tween 80 acted as activators for ASNase activity. This increase was also in agreement with our
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result. Surprisingly, its relative activity significantly increased by about 1.5-fold DaOftI:e1r0.1E03D9/TD0ANJ00127A
incubation. A similar enhancement was reported for ASNase immobilization on titanium oxide
nanoparticles. The relative activity was increased approximately 40% compared to control
(100%), which is similar with our results.53
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The effects of different organic solvents on the activity of free ASNase and Fe3O4/SBA-
15/TA/ASNase were also investigated, and the results are shown in Fig. 12B. After 24 h of
incubation, the residual activity of free ASNase was 80.8, 87.3, 53.0, 71.7 and 93.6%, whereas
Fe3O4/SBA-15/TA/ASNase maintained 95.5, 111.5, 113.1, 104.4, 107.8% of its initial activity
in chloroform, DMF, THF, ethanol and n-hexane, respectively. However, interestingly, a
noticeable increase in Fe3O4/SBA-15/TA/ASNase activity was observed approximately 23.8,
28.1, and 57.6% in the presence of methanol, acetone, and acetonitrile, respectively. Similar
findings related to organic solvents stability for immobilized enzyme was also reported by
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Agrawal and Kango and Agrawal et al. . The modified ASNase showed relative activity
168, 138, 114, 112 and 107% in presence of ethyl acetate, acetone, acetonitrile, ethanol and
methanol by respectively. These results are in good agreement with our outcome in terms of
organic solvents stability tests.
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Page 27 of 33
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DOI: 10.1039/D0NJ00127A
Fig. 12. The effects of different inhibitors (A) and organic solvents (B) on the activity of free
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ASNase and Fe3O4/SBA-15/TA/ASNase. All measurements were run in triplicate (n=3).
4. Conclusions
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In summary, the present work demonstrated the successful immobilization of ASNase on the
Fe3O4/SBA-15 support functionalized with TA to improve stability and biochemical properties
of ASNase. The use of the Fe3O4/SBA-15/TA for ASNase immobilization is important in two
aspects. On the one hand, thanks to the functional groups on surface, ASNase was immobilized
quickly and easily on these surfaces as a result of interactions. On the other hand, Fe3O4/SBA-
15/TA can be rapidly separated and propelled from the reaction system to provide practical and
low-cost applications. In this respect, the carrier matrix was verified via ATR-FTIR, SEM,
EDX, VSM, and XRD analysis. A significant improvement in pH and thermal stability of
Fe3O4/SBA-15/TA/ASNase was observed when compared to its free counterpart. Additionally,
the Fe3O4/SBA-15/TA/ASNase exhibited high thermal stability, repeated usage as well as
extended storage. However, the drawbacks of this support can be summarized as follows: (1)
As expected, slight loss of activity was observed due to mass transfer limitations after
immobilization. (2) It is very well known that adsorption has disadvantages such as enzyme
leakage from the support material, activity loss and contamination of the product. Despite these
disadvantages, the results show that the Fe3O4/SBA-15/TA material can be satisfactorily
applied to biomedical, drug delivery systems, cell/enzyme immobilization, and many other
related areas. Also, further studies on the immobilization of different biocatalysts are under
way.
Disclosure statement
No potential conflict of interest was reported by the authors.
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New Journal of Chemistry
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DOI: 10.1039/D0NJ00127A
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