Amine-functionalized magnetic nanocomposite particles for efficient immobilization of lipase: effects of functional molecule size on properties of the immobilized lipase

Article abstract of DOI:10.1039/c5ra02471d

A cost-effective design of reusable enzyme-functionalized particles with better catalytic activity is of great scientific interest due to their applications in a wide range of catalytic reactions in several industrial processes. In this work, a systematic

Full text of DOI:10.1039/c5ra02471d

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Amine-functionalized magnetic nanocomposite
particles for efficient immobilization of lipase:
effects of functional molecule size on properties of
the immobilized lipase†
Cite this: RSC Adv., 2015, 5, 33313
Parvaneh Esmaeilnejad-Ahranjani,ab Mohammad Kazemeini,b Gurvinder Singhc
a
*
and Ayyoob Arpanaei
A cost-effective design of reusable enzyme-functionalized particles with better catalytic activity is of great
scientific interest due to their applications in a wide range of catalytic reactions in several industrial
processes. In this work,
a systematic approach for preparing amine-functionalized magnetic
nanocomposite particles through the surface modification of core/shell type Fe3O4 cluster@SiO2
particles by the small molecules of 3-(2-aminoethyl)aminopropyltrimethoxysilane (AAS) or the large
molecules of polyethyleneimine (PEI) with two different molecular weights, as the support materials for
enzyme immobilization, has been demonstrated. The functional nanocomposite particles were
characterized by STEM, XRD, EDX, VSM, TGA, FTIR, oxygen elemental analysis and zeta potential
measurement techniques. Lipase from Pseudomonas cepacia, chosen as
a model enzyme, was
covalently immobilized on glutaraldehyde-activated particles. The free and immobilized lipases were
characterized by UV-vis, FTIR and CD spectroscopic methods. It has been shown that the size of the
functional molecule has a significant effect on the concentration of binding sites on the particles and
consequently on the lipase immobilization efficiency and loading capacity as well as the conformation
and activity of the immobilized lipase. Resulting from the increased binding site concentration on the
low and high molecular weight PEI-functionalized particles' surface, high lipase immobilization
efficiencies (87 and 97%, respectively) and loading capacities (803 and 817 mg gꢀ1, respectively) were
obtained. Upon the immobilization of lipase, the activity, thermal and storage stability as well as
reusability were improved under harsh reaction conditions in the order of the lipase immobilized on the
low molecular weight PEI > high molecular weight PEI > AAS functionalized particles. This study offers
insight into the design of functionalized magnetic particles for efficient immobilization of enzymes as
well as improvement of the immobilized enzymes properties.
Received 8th February 2015
Accepted 1st April 2015
DOI: 10.1039/c5ra02471d
www.rsc.org/advances
difficulties associated with their recovery and reusability
making the processes less economically feasible.2,3 Immobili-
Introduction
zation of enzymes on various supports has been considered to
overcome these problems by providing a more stable, recover-
able and reusable biocatalyst from the reaction media.4,5
Nanoscale materials can potentially be used as supports for
enzyme immobilization due to their size-dependent unique
physical properties and high specic surface area.6 Among
various materials, iron oxide nanoparticles have been greatly
favored because of their easy and inexpensive synthesis, low
toxicity and rapid magnetic separation from a reaction media
averting the use of expensive liquid chromatography, lters,
centrifuges and other instruments for the separation.7,8 In
particular, magnetite nanoparticles with a size below 20 nm
have attracted more attention due to their superparamagnetic
behavior avoiding magnetically induced self-aggregation of
particles.9,10 However, the iron oxide nanoparticles are
Enzymes are widely employed to catalyze a broad range of
reactions in industry.1 However, their application in native form
is oen limited, mainly because of their short lifetime, and
aDepartment of Industrial and Environmental Biotechnology, National Institute of
Genetic Engineering and Biotechnology, PO Box: 14965/161, Tehran, Iran. E-mail:
arpanaei@yahoo.com; Fax: +98-21-4458039; Tel: +98-21-44580463
bDepartment of Chemical and Petroleum Engineering, Sharif University of Technology,
Tehran, Iran
cDepartment of Chemical Engineering, Norwegian University of Science and
Technology, Trondheim, Norway
† Electronic supplementary information (ESI) available: Zeta potential values of
the particles vs. pH; effect of the pH value of the buffer solution on removing
the unattached lipase molecules; CD data of the free and immobilized lipases
pre-treated according to the thermal and storage assays; hydrolysis reactions'
kinetic curves; thermal stability of the lipases at 40 ꢁC. See DOI:
10.1039/c5ra02471d
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chemically unstable under acidic and oxidative industrial
reaction environments.9,11 Therefore, coating of a protective
layer on the magnetic particles is necessary to maintain their
stability.12,13 Design of composite materials such as a core/shell
morphology encapsulating magnetic nanoparticles inside a
shell of silica can be effective for extension of their applications
due to the unique characteristics of silica shells including high
stability under extreme reaction condition, good dispersion in
aqueous solution, biocompatibility and ability to provide a
platform for further surface modication due to abundant
hydroxyl groups on the silica surface.7,14,15
Moreover, different factors can inuence the conformation
and activity of the immobilized enzyme on nanoparticles, such
as immobilization techniques, concentration of functional
groups on the particles surface and reaction physico-chemical
parameters.16,17 Among the different enzyme immobilization
methods, covalent immobilization has been reported as a better
approach to bind enzyme molecules onto the solid support
providing no or negligible enzyme leakage.18,19 The number of
functional groups on the surface of solid support can be an
important parameter in terms of achieving the desired enzyme
stability, conformation and activity under relevant physico-
chemical reaction condition as well as the appropriate enzyme
loading capacity and immobilization efficiency.17,20 Various
methods have been explored for the chemical binding of
enzymes to magnetic particles functionalized with small func-
tional molecules such as aminopropyltriethoxy silane (APTES),21
3-glycidoxypropyltrimethoxysilane (GLYMO),22 3-glycidoxy-
propyltrimethoxysilane (GPTMS) and 3-mercaptopropyl-
trimethoxysilane (MPTMS).23 However, these methods usually
result in low loading capacities of the particles for enzyme
immobilization and poor conformational and catalytically
stabilities of the immobilized enzymes.
An alternative solution can be functionalization of particles
surface by polymer molecules which enhance enzyme immo-
bilization efficiency and loading capacity of the particles
through providing a high concentration of functional groups on
the particles surface.18,24,25 It is also expected that the high
number of covalent bonds between the polymer-functionalized
particles surface and the enzyme molecules result in the
immobilized enzymes with stable conformations and activities
under harsh reaction conditions.26–29 Adsorption of the polymer
molecules on the surface of magnetic particles via electrostatic
interactions has been reported as a simple approach for surface
modication;30 however, electrostatic interactions are not
strong enough and vary by changes of temperature or pH
resulting in desorption of the polymer molecules from the
particles surface.31,32 Plasma polymerization has also been used
as an alternative strategy for modifying the particle surface with
polymer molecules.7,27,33 However, this process is a very complex
technique and less efficient for industrial scale modication of
submicron particles. Therefore, despite numerous previous
studies for modication of the particle surface with polymers,
there is still demand for a simple and cost-effective approach to
obtain a core/shell type polymer-functionalized nanocomposite
Scheme 1 Schematic illustration of (a) the synthesis of amine-func-
tionalized magnetic silica nanocomposite particles and lipase immo-
bilization; (b) the correlation between particles functional parameters
and properties of the immobilized enzyme. AAS; 3-(2-amino-
ethylamino)propyltrimethoxysilane,
polyethyleneimine.
GA;
glutaraldehyde,
PEI;
concentration of binding sites on the enzyme loading capacity
of the polymer-functionalized particles as well as on the
immobilized enzyme's conformation and activity-related prop-
erties are still needed to be elucidated.
Herein, we report on preparation and characterization of
amine-functionalized magnetic nanocomposite particles with a
core/shell structure for enzyme immobilization. In the rst step
of synthesis, oleic acid-coated magnetite (Fe3O4) nanoparticles
were synthesized. Magnetite particles were then coated and
stabilized with a nonporous silica layer. PEI molecules, with two
different molecular weights, were anchored to the as-prepared
magnetic silica composite (MS) particles via a simple covalent
attachment approach. The particles modied by a small mole-
cule of AAS were also prepared. To demonstrate the utility of
these functionalized composite particles as magnetically sepa-
rable supports for immobilization of enzymes, lipase, which has
a wide range of industrial applications,1,34 was chosen as a
model enzyme and covalently immobilized on the functional-
ized particles surface using glutaraldehyde (GA) as the cross-
linker agent. The sequential steps involved in the preparation
of nanocomposite particles and lipase immobilization are
illustrated in Scheme 1a. The correlation between the particles
functional parameters and properties of the immobilized
enzyme is illustrated in Scheme 1b. We studied the effects of
functional molecule size on the binding sites concentration of
particles and consequently on the enzyme loading capacity and
immobilization efficiency, as well as conformation, activity,
stability, reusability and catalytic reaction kinetics of the
immobilized lipase.
Experimental
Materials
particles with a stable polymer layer. Moreover, the effects of Ferrous chloride tetrahydrate (FeCl2$4H2O), ferric chloride
parameters such as polymer molecular weight and the hexahydrate (FeCl3$6H2O), oleic acid (99 vol%), ammonia
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(25 vol%), ethanol (99.9 vol%), toluene (99.9 vol%), tetraethyl the mixture under continuous stirring.37 The reaction was
orthosilicate (TEOS, 99 vol%), 3-(2-aminoethyl amino)propyl- allowed to proceed for 1 h and then the product was collected
trimethoxysilane (AAS, 97 vol%), glutaraldehyde (GA, 25 vol%) with a magnet, washed excessively with ethanol and water and
were purchased Merck. Polyethyleneimine (PEI1, average nally dispersed in an aqueous sodium phosphate buffer
molecular weight 10 000, 99 wt%) was received from Alfa-Aesar. solution (2.5 mL). Unless it is otherwise specied, 0.1 mol Lꢀ1
4-Nitrophenyl palmitate (pNPP), polyethyleneimine (PEI2, sodium phosphate buffer solution (pH 7.4) was used
average molecular weight 750 000, 50 wt% in H2O) and lipase throughout in the present study.
from Pseudomonas cepacia were purchased from Sigma-Aldrich.
To prepare PEI-functionalized particles through covalent
All reagents were used as received without further purication. attachment method, the AAS-MS particles were activated with
Milli-Q water (18.2 MU cm) was used throughout the work.
GA through the following procedure. 100 mL of GA was added to
2.5 mL of 2 mg mLꢀ1 solution of AAS-MS particles and the
resulting solution was continuously shaken at 60 oscillations
minꢀ1 and 4 ꢁC for 60 min. Aer the reaction, the GA-activated
AAS-MS particles (hereaer shown as GA-AAS-MS) were washed
three times with the buffer solution, and then dispersed in 2.5
mL of buffer solution. GA is known as the cross-linker agent
which forms an imine bond in the reaction with primary amine
groups and leaves the other aldehyde group free for further
conjugation of molecules containing primary amine groups.23,38
Then, 2.5 mL of either 0.2 mmol Lꢀ1 solution of PEI1 or 2.7 ꢂ
10ꢀ3 mmol Lꢀ1 solution of PEI2 in the buffer solution were
added to the GA-AAS-MS particles solution and allowed to shake
at 60 oscillations minꢀ1 for 2 h. The nal mass concentration of
both polymer solutions were 2 g Lꢀ1. The resulting PEI-
functionalized particles, hereaer referred as PEI1-MS and
PEI2-MS, were washed three times with a buffer solution and
nally dispersed in a buffer solution (2.5 mL).
Preparation of Fe3O4 nanoparticles
Oleic acid-coated magnetite nanoparticles were prepared by the
co-precipitation method as described previously.4,35 Briey,
540.0 mg of FeCl3$6H2O together with 199.0 mg of FeCl2$4H2O
was added to a 150 mL three-necked ask and mixed with 60 mL
of water under a nitrogen atmosphere with mechanical stirrer at
1000 rpm. Oleic acid (0.1 mL) and ammonia (7.5 mL) were
added to the solution and the resulting mixture was stirred and
ꢁ
heated to 80 C. Then, 0.4 mL of oleic acid was continuously
added to the solution in four steps every 5 min. The reaction was
then allowed to proceed at 80 ꢁC and 1000 rpm for 30 min. The
resulting dark brown suspension was cooled down to room
temperature and the nanoparticles were collected with a
magnet and washed with water several times to remove
nonmagnetic by-products.
Preparation of Fe3O4 cluster@SiO2 (MS) nanocomposite
particles
Lipase immobilization on functionalized MS particles
The synthesis of magnetite/silica core/shell nanocomposite
For immobilization of lipase molecules on the functionalized
particles, the primary amine functional groups on the AAS-MS,
PEI1-MS and PEI2-MS particles surface were rst activated with
GA. 100 mL of GA was added to 2.5 mL of the as-prepared
functionalized particles solution and the resulting mixture
was shaken at 60 oscillations minꢀ1 and 4 ꢁC for 60 min. Once
amine groups were activated, the samples were washed three
times with a buffer solution and dispersed in 2.5 mL of the
buffer solution. The GA-activated particles hereaer are referred
as GA-AAS-MS, GA-PEI1-MS and GA-PEI2-MS. Then, 2.5 mL of
lipase solution with various concentrations (2, 4, 6, and 8 mg
mLꢀ1) was rapidly added to all the GA-activated particles solu-
tions. According to the literature,4 lipase from Pseudomonas
cepacia preserves its maximum activity at pH 7.4, hence the
immobilization procedure was ꢁcarried out at this pH value.
Aer overnight incubation at 4 C, the lipase-immobilized GA-
AAS-MS (L-AAS-MS), GA-PEI1-MS (L-PEI1-MS) and GA-PEI2-MS
(L-PEI2-MS) samples were rst washed three times with a
sodium phosphate buffer solution (pH 7.4). Since the surface
charges of the particles and the enzyme are opposite at pH 7.4,
the enzyme molecules can physically adsorb onto the particles
¨
(MS) was performed by the Stober method via the hydrolysis-
condensation of TEOS in the presence of Fe3O4 nano-
particles,36 with slight modications. Briey, the as-prepared
magnetite nanoparticles (75 mg) were dispersed in toluene (5
mL) and then re-dispersed in a mixed solution of ethanol (80
mL), deionized water (20 mL) and ammonia (2.5 mL). Emulsion
drops were formed by ethanol as the emulsier once toluene
suspended particles were added to the ethanol. The mixed
solution was homogenized at room temperature and 400 rpm
for 20 min to form a uniform dispersion of nanoparticles. Then,
1 mL of TEOS was added to the solution by dropwise addition in
30 min under stirring of 400 rpm at room temperature. The
resulting mixture was stirred at room temperature for 4 h to
complete the synthesis of MS nanoparticles. The product was
separated using a magnet and washed several times with
ethanol and water to remove nonmagnetic by-products. The
ꢁ
collected nanoparticles were dried at 50 C.
Preparation of ASS- and PEI-functionalized Fe3O4
cluster@SiO2 nanocomposite particles
The surface of MS particles was modied by AAS molecules to surface via various interactions such as electrostatic attractive
render a platform for covalently attachment of either enzyme forces. Therefore, the samples were washed with acetate buffer
molecules or PEI molecules. For synthesis of the AAS- solutions of two pH values (0.1 mol Lꢀ1, pH 3 and 5, twice with
functionalized MS (AAS-MS) particles, 5 mg of the as-prepared each solution) and then with sodium phosphate buffer solu-
MS particles were dispersed in 4.6 mL of ethanol. Then, water tions of three pH values (0.1 mol Lꢀ1, pH 6, 8 and 9, twice with
(0.2 mL), acetic acid (0.1 mL) and AAS (0.1 mL) were added to each solution) to ensure the removal of all the noncovalently-
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attached or loosely-bounded lipases. The washed samples were
Characterization techniques
nally dispersed in a phosphate buffer solution (pH 7.4) and
stored at 4 C.
Scanning transmission electron microscopy (STEM) coupled
with energy dispersive X-ray (EDX) spectroscopy was performed
using a Hitachi S-5500 operating at 30 kV on the particles.
Samples for STEM analysis were prepared by the evaporating of
a dilute solution of particles onto 300 mesh carbon-coated
copper grid. Scanning electron microscopy (SEM) images were
obtained on a scanning electron Seron Technology AIS-2100
microscope. The average values of core size, shell thickness
and overall diameter of the particles, and the standard devia-
tion were calculated by counting over 100 particles using the
ImageJ soware.
ꢁ
Immobilization efficiency (i.e., mass ratio of the immobi-
lized lipase to the initial lipase, wt%) and loading capacity (i.e.,
mass ratio of the immobilized lipase to the functionalized
particle, mg gꢀ1) of all prepared samples were estimated by
Bradford assay using bovine serum albumin as a standard
protein by measuring the content of lipase in the original lipase
solution, supernatant and collected washing solutions.
Catalytic activity assays
Bruker AXS D8 advanced diffractometer with Cu Ka radiation
Lipase activity study was performed according to the procedure
reported in the literature,24 with a slight modication. Briey,
450 mL of 1 mmol Lꢀ1 4-nitrophenyl palmitate (pNPP) solution
was added to 50 mL of 2 mg mLꢀ1 solution of either free lipase or
immobilized lipase in a buffer solution. All the reaction systems
contained a similar amount of lipase in either free or immobi-
lized state. The resulting mixture was shaken at 110 rpm for an
equilibrium reaction time of 15 min. Control experiments were
also performed on the buffer solutions and particles without the
immobilized lipase. As accurate measurement of enzyme
activity requires complete conversion of 4-nitrophenol product
into 4-nitrophenoxide; hence, NaOH was added to the reaction
mixture, aer the completion of reactions and recovering the
particles using a magnet, and the pH value of the solution was
adjusted to 10 to ensure more than 99% ionization of the pNP
product. The molar concentration of the hydrolysis product, i.e.,
p-nitrophenol (pNP), which is equal to the molar concentration
of 4-nitrophenoxide, was measured using the spectrophotom-
etry method (T80 + UV/VIS Spectrophotometer, PG Company,
Germany) at 405 nm.39 One unit of lipase activity was dened
as the amount of lipase liberating 1 mmol of pNP per minute
under the assay conditions and referred to as specic activity
(U mgꢀ1).
˚
(l ¼ 1.5418 A) was used for collecting X-ray diffraction (XRD)
patterns. Vibrating sample magnetometer (VSM, Meghnatis
Daghigh Kavir Co., Iran) operating at room temperature was
employed to measure the magnetic properties of the particles.
The temperature dependence of the magnetization of nano-
particles under zero-eld-cooled (ZFC) and eld-cooled (FC)
(applied eld of 100 Oe) conditions was studied using a
superconducting quantum interference device (SQUID)
magnetometer (MPMS, Quantum Design). Zeta (z) potential
values and hydrodynamic diameters of the particles were
measured using a Malvern Zetasizer Nano instrument (S90, UK)
equipped with the dynamic light scattering (DLS) system.
Fourier-transform infrared (FTIR) spectra were recorded from a
Bruker Tensor 27 FTIR spectrophotometer with a resolution of 4
cmꢀ1 using pellets made by 1 mg samples in 100 mg KBr.
Thermogravimetric analysis (TGA) and differential thermal
analysis (DTA) using TG/DTA6300 instrument was performed
on the dried particles at a heating rate of 10 ꢁC minꢀ1 under air
atmosphere. Sample studied by TGA were also analysed by
CHNS–O Elemental Analyzer instrument (Costech ECS 4010,
USA) for quantitative determination of oxygen content of the
particles.
The secondary conformational characteristics of the free and
immobilized lipase molecules were studied by circular
dichroism (CD) method using a J-715 spectropolarimeter (Jasco,
Japan) over the wavelength rage of 190–260 nm on 0.2 mg mLꢀ1
solution of either free lipase or immobilized lipase in a sodium
phosphate buffer solution (0.1 mol Lꢀ1, pH 7.4) at a desired
temperature. Blank spectra of the buffer solution with and
without particles were obtained at identical conditions for
background correction.
The kinetic parameters of Michaelis–Menten equation for
the free and immobilized lipases were determined by
measuring the initial rates of the hydrolysis reaction through
varying the substrate (pNPP) concentration,40 in a range of
0.05–5 mmol Lꢀ1 at 40 C and 50 C, at a constant pH 7.4.
To investigate the effect of temperature on hydrolytic activity
of the free and immobilized lipases, the hydrolysis reactions
was carried out at different temperatures, ranging from 30 ꢁC to
ꢁ
ꢁ
ꢁ
60 C, at a constant pH 7.4.
Thermal stability of the free and immobilized lipases was
determined by evaluating the residual enzymatic activities aer
incubation in a phosphate buffer solution (pH 7.4) at two
Data analysis
different temperatures of 40 ꢁC and 50 ꢁC for 1–10 h. The Analysis of variance (ANOVA) was used to determine whether
storage stability of free and immobilized lipases was deter- the effects of the examined parameters are statistically signi-
mined by measuring the ꢁcatalytic activities at 40 ꢁC aer storing cant (p < 0.05). The signicance of differences was analyzed by
in a buffer solution at 4 C for a certain period of time.
the Duncan test as a post-hoc test. All samples were analyzed in
Reusability of the immobilized lipases was measured triplicate and mean ꢃ standard deviation values were calculated
ꢁ
through repeated uses in the hydrolysis reaction at 50 C and and reported. The TGA, XRD, EDX, VSM, FTIR, oxygen
pH 7.4. The immobilized lipases were magnetically recovered elemental analysis and hydrodynamic diameter and zeta
and washed excessively with a buffer solution prior to be used in potential measurements were assessed in duplicate. The CD
the next reaction batch.
spectroscopy analysis was performed in triplicate.
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Results and discussion
Characterization of nanocomposite particles
Fig. 1a–e presents the electron microscopy images of the
synthesized particles. The Fe3O4 nanoparticles have a uniform
diameter of 7.8 ꢃ 0.9 nm. The MS particles, which were
obtained via encapsulating of magnetite nanoparticles in a shell
of silica, are near-spherical (210 ꢃ 50 nm) and possess a core/
shell structure with a 130 ꢃ 30 nm core and a 40 ꢃ 10 nm
silica shell. The AAS-MS, PEI1-MS and PEI2-MS nanocomposite
particles exhibit the same morphology as that of the MS parti-
cles with a mean diameter of 210 ꢃ 50 nm.
The existence of 33 ꢃ 4% Fe content in all the composite
particles was veried from EDX spectra. Taking into account the
mass (estimated from data obtained using TGA study, Fig. 2a
and Table 1) and density of the organic/polymer layer, and the
size of the MS particles, the thickness of organic layers on the
dried PEI1-MS and PEI2-MS particles surface are calculated to
be about 1.4 ꢃ 0.2 nm and 2.3 ꢃ 0.2 nm, respectively. The
almost similar morphologies observed for all the samples
indicates that the very thin organic/polymer layers were not
detectable through imaging the particles using the electron
microscopy. However, the size measurement through DLS
revealed the larger diameters for PEI1-MS (310 ꢃ 30 nm) and
PEI2-MS (370 ꢃ 30 nm) compared to the values determined
from STEM images, most likely due to the swelling of the
hydrophilic polymer layers in the aqueous solution. The
hydrodynamic diameters of the MS (210 ꢃ 30 nm) and AAS-MS
(215 ꢃ 30 nm) particles were almost similar to those determined
from the electron microscopy images.
Fig. 1f shows the XRD patterns of the as-prepared particles.
The presence of six diffraction peaks in diffractogram of
magnetite nanoparticles conrms the inverse spinel structure
of Fe3O4 nanoparticles (JCPDS card no. 85-1436). These
magnetite nanoparticles retain their crystal structure upon the
encapsulation in the silica shell and the subsequent function-
alization with organic/polymeric molecules. A small reduction
in the intensity of the corresponding XRD peaks for the MS,
AAS-MS, PEI1-MS and PEI2-MS particles can be attributed to the
presence of an amorphous structure of silica shell, where it is
seen that the very thin organic/polymeric layers do not more
affect the corresponding peaks' intensity.
The room-temperature magnetization curves reveal the
superparamagnetic behavior of all the as-prepared particles
(Fig. 1g). The MS particles present a relatively high saturation
magnetization (Ms) value (24.4 emu gꢀ1), which supports the
use of simple separation of the particles by an external magnetic
eld. The organic/polymeric layer on the MS particles have a
negligible effect on the reduction of the Ms values for the AAS-
MS, PEI1-MS and PEI2-MS because the corresponding layers
thickness is negligible as compared to the silica shell thickness.
The lower Ms value of the composite particles compared to the
Fe3O4 nanoparticles, i.e., 60 emu gꢀ1 reported in our previous
work for the same magnetite particles,4 is due to the contribu-
tion of the nonmagnetic silica shell around the Fe3O4 particles.
It should be mentioned that all the nanocomposite particles
Fig. 1 Electron microscopy images of magnetite (a), MS (b), AAS-MS
(c), PEI1-MS (d) and PEI2-MS (e). XRD patterns (f), room temperature
magnetization curves (g) and FC-ZFC curves (h) of bare and func-
tionalized MS particles.
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The superparamagnetic behavior of the as-prepared particles
under the experimental conditions is also identied from FC-
ZFC curves as the FC and ZFC curves coincide at high temper-
atures and exhibit a paramagnetic-like decay with the increase
of the temperature (Fig. 1h). The FC and ZFC curves split with
the decrease of the temperature; the ZFC curves reach maxima
at 80 K and 65 K corresponding to the blocking temperatures of
the magnetite and composite nanoparticles, respectively, and
then start to decrease, while the FC curves keep increasing. All
the bare and functionalized MS particles exhibit the similar FC
and ZFC curves; hence, the shi in the blocking temperature for
the composite particles to a lower value in comparison to that of
the magnetite particles can be due to the rather thick
nonmagnetic silica shell around the magnetite clusters. It has
been reported that the nonmagnetic shell reduces the interac-
tions established between the magnetic nanoparticles, altering
the superparamagnetic anisotropy energy barrier, and causes a
shi in the blocking temperature to lower values.41 The
decreased magnetic interactions between the nanoparticles
upon coating with silica is also evident in the slopes of ZFC
curves below blocking temperature since the steeper slopes are
observed for the composite particles as compared to the
magnetite nanoparticles.
In order to estimate the amount of functional molecules on
the surface of particles aer each functionalization step, we
applied TGA technique (Fig. 2a). DTA proles are presented
along with the TGA proles to establish where the mass loss is
occurring (Fig. 2b). For all the samples, the mass loss below
150 ꢁC is mostly related to desorption of the water physically
adsorbed to the surface as presented as the endothermic peak
in DTA proles. For the bare MS particles, no further mass loss
or endothermic/exothermic peaks are observed, in agreement
with the literature,42 revealing that the decomposition of silica
ꢁ
does not occur at temperatures below 600 C. For the AAS-MS
and GA-AAS-MS particles, the mass losses occur between 150
ꢁ
ꢁC and 300 C, due to the thermal decomposition of the corre-
sponding organic components, which are also evident as the
exothermic peaks in the DTA proles. TGA proles of the as-
prepared PEI-functionalized particles show mass losses
between 200 ꢁC and 500 ꢁC, due to the thermal decomposition
of PEI molecules and the organic component (i.e., AAS and GA)
under the attached PEI molecules. The DTA proles of the PEI-
functionalized particles present wide exothermic peaks and do
not show individual exothermic peaks for the combustion of
polymeric and organic components separately. In agreement
with results reported previously,13,31 the exothermic peak of PEI
molecules is very wide. Therefore, these results imply that most
likely the combustion of all organic layer components happens
in a similar temperature range, hence, the small exothermic
peaks related to the decomposition of AAS and GA molecules
are hidden within the exothermic peak produced through the
decomposition of PEI molecules. Aer activation of the PEI-
functionalized particles with GA (GA-PEI1-MS and GA-PEI2-
MS), the mass loss increases again due to the decomposition
Fig. 2 TGA (a) and DTA (b) profiles of bare and functionalized MS
particles. FTIR spectra (c) of pure PEI molecules, bare and function-
alized MS particles.
could be easily separated from the suspension of 5 mg mLꢀ1 of the attached GA molecules. The DTA proles of the GA-
particles in water with a magnet within 30 s at room tempera- activated particles exhibit a sharp peak cantered at 200 ꢁC
ture (Fig. 1g inset) and then re-dispersed by shaking.
associated with the decomposition of GA molecules and a wide
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Table 1 Surface characteristics of nanocomposite particles
Samples
AAS-MS
GA-AAS-MS
PEI1-MS
87.12
8.71
GA-PEI1-MS
PEI2-MS
188.12
0.25
GA-PEI2-MS
Mass ratio of functional layer to MS
31
—
—
—
particles (mF, mg gꢀ1
)
Concentration of functional molecules
on particles (CF, mmol gꢀ1
Mass ratio of GA to MS particles
(mGA, mg gꢀ1
239
—
—
—
)
—
15.98a (17.68)b
—
40.37 (46.03)
—
88.32 (99.10)
)
Concentration of amine functional
239
—
543
—
1366
—
groups on particles (CNH , mmol gꢀ1
)
2
Concentration of binding sites on
—
190
—
—
480
—
—
1050
—
particles (CCHO, mmol gꢀ1
)
Zeta (z) potential at pH 7.4 (mV)
ꢀ8.2 ꢃ 1.3
15.7 ꢃ 1.8
19.7 ꢃ 2.0
a
Mass ratio of GA to MS particles calculated from oxygen elemental analysis results. b Mass ratio of GA to MS particles calculated from TGA analysis
results.
shoulder corresponding to the thermal decomposition of other the surface of the GA-activated particles, the oxygen elemental
organic components.
analysis were performed on the GA-AAS-MS, GA-PEI1-MS and GA-
The mass of the organic/polymeric layer on the MS particles PEI2-MS. The oxygen content related to the free aldehyde groups
aer each functionalization step are summarized in Table 1. on the aforementioned GA-activated particles was estimated aer
The obtained results reveal that the mass of the functional subtracting the oxygen content of the AAS-MS particles, taken as
molecule on the MS particles surface (mF) increases as the size the reference material, (mO,ref) from that of the GA-activated
of the applied molecules enhances, i.e., PEI2-MS > PEI1-MS > particles (mO,GA). It is also mentioned that the oxygen elemental
AAS-MS.
analysis results for the PEI1-MS and PEI2-MS particles represents
However, the number of the attached functional molecules no oxygen content once subtracting the oxygen content of the
on the particles surface (CF, mmol gꢀ1), calculated using eqn (1), reference material (AAS-MS).
changes in the converse order. This can be due to this fact that
The free aldehyde groups concentration (CCHO, mmol CHO
the steric/electrostatic repulsive interactions between organic gꢀ1 MS particles), which is equal to the oxygen concentration,
molecules of higher molecular weights, i.e., PEI, are severe and was estimated using eqn (2) and reported in Table 1.
prohibit these molecules to attach closely on the surface of
mO;GA ꢀ mO;ref
CCHO
¼
(2)
particles, which leads to the lower concentration of attached
molecules of high molecular weights (PEI) compared to the
small molecules of AAS.
Mw;O
where Mw,O is the atomic weight of oxygen element (i.e., 16).
According to Table 1, the CCHO value increases as the size of
the functional molecule enhances as the ratio of CCHO for GA-
AAS-MS : GA-PEI1-MS : GA-PEI2-MS can be estimated to be
mF
CF ¼
(1)
Mw;F
where Mw,F is the molecular weight of the corresponding 1 : 6.68 : 14.26. Although, as mentioned previously, CF decrease
attached functional molecule (i.e., AAS, PEI1 or PEI2). We as the functional molecule size increase, the larger functional
further performed TGA analysis on both PEI1-MS and PEI2-MS molecule possessing the more numbers of primary amine
particles once incubated in sodium phosphate buffer solution functional groups in one molecule, i.e., the ratio of primary
(0.1 mol Lꢀ1, pH 5, 7.4 and 9) at room temperature for 60 days to amine groups on AAS : PEI1 : PEI2 is 1 : 82 : 6198, provides a
evaluate the stability of polymer layers. No noticeable difference platform for binding of the more numbers of GA molecules and
was observed, suggesting the stable coating of PEI molecules on consequently leads to the higher value of CCHO
the MS particles (data not shown). To understand the possible crosslinking of the primary
.
It is well known that if a suitable concentration of GA is amine groups on the surface of the functionalized-MS particles
employed in the reaction of GA with surface primary amine through the activation with GA molecules, the concentration of
groups, one of the aldehyde groups of GA forms an imine (C]N) free primary amine groups (CNH , mmol NH2 gꢀ1 MS particles)
2
bond and the other aldehyde group is le free, which can be on the AAS-MS, PEI1-MS and PEI2-MS particles were estimated
employed in the formation of another imine bond with an amine using eqn (3), as summarized in Table 1, and the obtained
group existing in protein or other types of molecules,23,38 as results were compared with the CCHO value for the corre-
illustrated in Scheme 1a. However, due to homofunctional nature sponding GA-activated particles.
of the bis-aldehyde crosslinker (i.e., GA), the formation of bonds
between both aldehyde groups with adjacent amine groups
existing on the surface of particles is inevitable.23 Therefore, in
order to estimate the exact concentration of the free aldehyde
groups, which are called binding sites in this paper, present on
mF
CNH
¼
ꢂ nNH ;F ꢀ CCHO
(3)
2
2
Mw;F
where nNH ,F is the number of primary amine groups in one
2
molecule of the corresponding functional molecule, i.e., AAS,
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PEI1 or PEI2. It is noted that the CCHO value for AAS-MS in eqn covalent attachment of PEI molecules on the AAS-MS particles
(3) is zero. For PEI1-MS and PEI2-MS, as the GA-AAS-MS parti- using GA as the cross-linker agent.43,44
cles were used for attachment of the PEI molecules and the
The z potential values of the functionalized MS particles
oxygen elemental analysis of PEI1-MS and PEI2-MS represents recorded at pH 7.4 are presented in Table 1. Alteration of
no oxygen content once subtracting the oxygen content of the surface charges of all the particles by changes in the pH of the
reference material (AAS-MS), it was indicated that all the free solution is shown in Fig. S1.† The MS particles show a negative z
aldehyde groups on the GA-AAS-MS were bonded to some of the potential value (ꢀ34.8 ꢃ 2.3) due to the presence of a high
primary amine groups in the PEI molecules. Therefore, the number of hydroxyl groups on the surface in an aqueous
concentration of the GA-bonded primary amine groups of PEI medium.37 A considerable increase in z potential is noticed aer
molecules, which is equal to the concentration of the aldehyde modifying the particle surface with organic materials contain-
groups (CCHO) on the GA-AAS-MS, is subtracted from the total ing amine groups. The extent of increase in the z potential
PEI amine groups estimated from data obtained using TGA (the depends on the concentration of amine groups (CNH , Table 1)
2
rst term of eqn (3)).
introduced on the particles surface. The recorded z potential
Comparing the results obtained from eqn (2) and (3) indi- value for the AAS-MS particles is fairly in agreement with
cates that the CCHO values for GA-AAS-MS, GA-PEI1-MS and GA- previous studies showing an almost similar change in z
PEI2-MS are about 11–23% lower than the CNH values for AAS- potential value of magnetic silica particles through functional-
2
MS, PEI1-MS and PEI2-MS (Table 1). These results can conrm ization with AAS, where the remained hydroxyl groups on the
the above hypothesis on the formation of some imine bonds silica affects the surface charge of the amine-functionalized
between both aldehyde groups of GA molecules with primary particles.7,37 The higher positive surface charge of the PEI2-MS
amine groups present on the surface of AAS-MS, PEI1-MS or particles as compared to the PEI1-MS ones can be attributed
PEI2-MS particles.
to high concentration of amine functional groups on the
To further verify this hypothesis, the mass of the attached GA former.
molecules on the GA-activated particles (mGA (mg GA gꢀ1 MS
particles)) was calculated using the data obtained from the
Characterization of immobilized and free lipases
oxygen elemental analysis (eqn (4)), and then compared with
those obtained from the TGA analysis.
The conjugation of lipase onto the particles occurred via the
formation of imine bonds between the aldehyde groups on the
GA-activated particles and the amine groups in the lipase
molecules as illustrated in Scheme 1a. Aer immobilization
procedure, the samples were washed excessively with buffer
solutions of various pH values to ensure the removal of the
physically or electrostatically adsorbed lipase molecules. The
total amount of unattached enzyme molecules collected aer
each washing step (Fig. S2†) was used to determine the lipase
immobilization efficiency and loading capacity of the particles.
The inuence of lipase initial concentration on the immo-
bilization efficiency and loading capacity are displayed in Fig. 3.
A continuous decrease in the immobilization efficiency is found
with the increase of lipase initial concentration, while the
enzyme loading capacity initially increases with the increase of
lipase concentration up to 2 mg mLꢀ1 and then starts to
decrease with further increase in the lipase initial concentra-
tion. As veried in the literature,14,45 strong protein–protein
interactions lead to the formation of protein aggregates in the
solution, limit the immobilization of lipases and consequently
result in the reduction of the lipase loading capacity at the
lipase concentrations above 2 mg mLꢀ1, in this study. The
results of lipase immobilization efficiency and loading capacity
obtained at this lipase concentration (2 mg mLꢀ1), which is
used as the optimum value through this study, are summarized
in Table 2. Obviously, there is a direct relation between the
lipase immobilization efficiency or loading capacity and the
binding site concentration on the particles at all the lipase
initial concentrations. For instance, at 2 mg mLꢀ1 enzyme
initial concentration, the lipase immobilization efficiency and
loading capacity ratios of L-AAS-MS : L-PEI1-MS : L-PEI2-MS
particles are 1 : 1.77 : 1.97 and 1 : 1.68 : 1.72, respectively.
mO;GA ꢀ mO;ref
mGA
¼
ꢂ Mw;GA
(4)
Mw;O
where Mw,GA is the molecular weight of the attached GA
molecule.
The results indicated that the mass of the attached GA
molecules estimated from oxygen elemental analysis data (eqn
(3)) is lower than that obtained using TGA analysis (Table 1),
further conrming the above hypothesis. Because in the
calculated mass of the GA on the particles from TGA analysis
result, all GA molecules are taken into account regardless of
having one free aldehyde group or both aldehyde groups taking
part in the formation of imine bonds with surface amine
groups. But, only the mass of the GA molecules with one free
aldehyde group is measurable using the data obtained from
oxygen elemental analysis method.
The functional groups of the modied and unmodied MS
particles were further characterized by FTIR spectroscopy, as
illustrated in Fig. 2c. For all the samples, the absorption band at
600 cmꢀ1 is assigned to the vibration of Fe–O bonds.24 The
stretching vibration of Si–O–Si, Si–OH and bending vibration of
Si–O bonds make absorption peaks at 810, 973 and 1099 cmꢀ1
,
respectively.26 The AAS-MS, PEI1-MS and PEI2-MS particles as
well as the pure PEI1 and PEI2 molecules spectra exhibit two
low-intensity absorption bands at 1617 and 1701 cmꢀ1 associ-
ated with the bending vibration of N–H bonds and a peak at
3245 cmꢀ1 corresponding to stretching vibration of N–H bonds
in amine groups.8,43 The absorption peak at 1394 cmꢀ1 is
assigned to stretching vibration of C–N bonds.8 For the PEI1-MS
and PEI2-MS, the stretching vibration of C]N bonds make
absorption peaks at 1488, 1570 and 1630 cmꢀ1, verifying the
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of the binding site concentration and lipase loading capacity of
the particles, the number of linking points for each lipase
molecule conjugated onto the particles, represented as the
molar ratio of the binding sites to the immobilized lipase
molecules (Table 2), is estimated to be in the increasing order of
L-PEI2-MS > L-PEI1-MS > L-AAS-MS.
FTIR spectra of the free and immobilized lipases are illus-
trated in Fig. 4a. The free lipase spectrum exhibits two
absorption peaks at 1605 and 1707 cmꢀ1 corresponding to C–O
stretching vibration mode in amide I bonds and an absorption
band at 1414 cmꢀ1 associated with N–H bending mode and C–N
stretching vibration mode in an amide II bonds. The bending of
N–H bonds and stretching vibration of C–N bonds make
absorption peak at 1323 cmꢀ1 verifying the amide III bonds. The
absorption peak at 2938 cmꢀ1 is assigned to stretching vibra-
tion of C–H bonds.4,24 These bands are observed in the immo-
bilized lipases spectra as well, indicating that the lipase was
successfully attached on the surface of functionalized particles.
For all the L-AAS-MS, L-PEI1-MS and L-PEI2-MS samples, the
absorption bands at 1488 and 1570 cmꢀ1 corresponding to
C]N vibration mode43,44 indicate the covalent attachment of
lipase molecules to the particles using GA as the cross-linker
agent.
The CD spectroscopy was also performed on the free and
immobilized lipases to provide information on the changes in
the secondary conformation of the immobilized lipases as
compared to the free lipase as a function of the available
binding sites concentration on the functionalized particles
surface. The CD spectra of the fresh free and immobilized
lipases, suspended in buffer and stored at 4 ꢁC for 15 min,
exhibit a broad negative band from 208 to 222 nm (Fig. 4b), as
the characteristic of enzyme molecules such as lipase, which
contain both a-helices, making two intensive peaks at 208 and
222 nm, and b-sheets, making a peak at 217 nm.46,47 The
proportional contents of secondary structural elements, i.e.,
a-helices, b-sheets, b-turns and random coils in the free and
immobilized lipases are summarized in Table 3. Upon the
immobilization of lipase, the a-helical and b-sheet contents
partially decrease and the content of unordered elements
marginally increases in the order of L-PEI2-MS > L-PEI1-MS >
L-AAS-MS as compared to the free lipase. By considering the
molar ratio of concentration of binding sites to the concentra-
tion of immobilized lipases (Table 2), it is revealed that there is
a direct relation between the conformational changes of lipase
and the number of covalent bonds between the lipase and
particles. The alteration of enzyme conformation upon the
covalent linkage onto the particles surface is well known, where
the hydrogen bonds between the functional groups of an
enzyme molecule, which give a specic structure for the enzyme
molecule, are reduced as they are involved in the covalent
linkage to the particles surface.20 Therefore, according to the
literature,9 as the concentration of binding sites on the particles
increases, the interactions between the lipase molecules and
the particles increases and consequently the lipases suffers
more conformational changes.
Fig. 3 Effect of lipase initial concentration on (a) lipase immobilization
efficiency and (b) loading capacity of the amine-functionalized
magnetic nanocomposite particles.
The signicantly higher values of lipase immobilization
efficiency and loading capacity of the PEI1-MS and PEI-MS
particles as compared to the AAS-MS particles can be attrib-
uted to higher binding sites concentration on the formers
which create a microenvironment favoring more interactions
and bond formations with the higher numbers of lipase mole-
cules. The above mentioned ratios are not exactly according to
the ratio of the binding sites concentration on the particles, i.e.,
1 : 2.52 : 5.53 (Table 1), which could be due to steric barriers
created by the large branched PEI molecules toward diffusion of
the higher number of the lipase molecules within the polymer
matrix. By calculating the number of lipase molecules on the
surface of the AAS-MS particles (0.033 immobilized lipase
molecules per nm2 AAS-MS) and taking into account the size of
the lipase molecule (average diameter of 5 nm), it is revealed
that the lipase molecules are packed closely together in one
layer on the AAS-MS particles surface. In contrast, the high
concentration of the lipase molecules on the PEI-functionalized
particles should be dispersed within the binding sites of the
large molecules of PEI. Moreover, taking into account the values
The catalytic performance of the free and immobilized
lipases was characterized at the optimum reaction condition
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Table 2 Evaluation of enzyme immobilization efficiency and loading capacity of various amine-functionalized magnetic nanocomposite
particles and activity-related properties of free and immobilized lipases
Samples
Free lipase
L-AAS-MS
L-PEI1-MS
L-PEI2-MS
Enzyme immobilization efficiencya (wt%)
—
—
49.2 ꢃ 0.4
477 ꢃ 4
11
87.3 ꢃ 0.4
803 ꢃ 3
21
97.1 ꢃ 0.3
817 ꢃ 2
36
Enzyme loading capacityb (mg gꢀ1
)
Molar ratio of binding sites on particles (CCHO
to immobilized lipase molecules (mol molꢀ1
Specic activity per unit weight of lipasec (U mgꢀ1
)
)
)
2.32 ꢃ 0.05
2.11 ꢃ 0.03
91.3 ꢃ 0.3
1.98 ꢃ 0.04
84.3 ꢃ 0.2
1.89 ꢃ 0.03
78.8 ꢃ 0.2
Specic activity ratio of immobilized lipase to free
—
lipase (activity retention, %)
Activity per unit weight of particled (U mgꢀ1
)
—
1.0 ꢃ 0.01
0.62 ꢃ 0.05
3.29 ꢃ 0.02
1.58 ꢃ 0.03
1.54 ꢃ 0.01
Km (mmol Lꢀ1
)
0.53 ꢃ 0.0e
0.83 ꢃ 0.03 (0.44 ꢃ 0.05)f
1.07 ꢃ 0.02 (0.78 ꢃ 0.04)
2.83 ꢃ 0.03 (3.66 ꢃ 0.04)
Vmax (U mgꢀ1
)
3.51 ꢃ 0.0e
3.14 ꢃ 0.05 (4.11 ꢃ 0.03)f
a
Mass percentage of the immobilized lipase to the initial lipase used for immobilization; the amount of immobilized lipase was calculated by
b
subtracting the amount of un-immobilized lipase from the total amount of the lipase used for the immobilization. Mass ratio of the
c
immobilized lipase to the particles. One unit of lipase activity dened as the amount of lipase liberating 1 mmol of pNP per minute under
d
hydrolysis reaction of pNPP. Specic activity per unit weight of lipase ꢂ enzyme loading capacity. e Kinetic parameters at reaction temperature
of 40 ꢁC. f Kinetic parameters at reaction temperature of 50 ꢁC is shown in parentheses.
ꢁ
(40 C, pH 7.4, in this work) required for maximum activity of
the free lipase from Pseudomonas cepacia, as common reaction
conditions used to study the effect of immobilization on the
catalytic properties of enzymes.27,48 The sodium phosphate
buffer solution and the particles without immobilized lipase
taken as controls showed no decomposition of substrate. The
results summarized in Table 2 indicate that the specic activity
per unit weight of lipase and specic activity retention follow
the order of L-AAS-MS > L-PEI1-MS > L-PEI2-MS as compared to
the free lipase. The immobilized lipases activities per unit
weight of particles display a converse trend, indicating that as
the lipase loading capacity of the particle increases, the lower
amount of the corresponding particle is utilized to achieve a
constant amount of lipase in the reaction media.
The variation of specic activity of lipase upon immobiliza-
tion can be understood by looking into the reaction kinetic
parameters. The kinetic characteristics of the free and immo-
bilized lipases were also determined at this reaction condition
ꢁ
(40 C, pH 7.4), as illustrated in Fig. S3.†
The calculated Michaelis–Menten constant, i.e., Km, and the
initial maximum reaction velocity, i.e., Vmax, values indicate that
the affinity of enzymes for the substrate and the rate of reaction
proceeding, decrease in the order of free lipase > L-AAS-MS >
L-PEI1-MS > L-PEI2-MS (p < 0.05) (Table 2). Considering the CD
analysis data recorded for the free and immobilized lipase at
40 ꢁC which are not considerably different from the CD data
recorded at 4 ꢁC (Table 3), the lower affinity of immobilized
lipases for substrate as compared to the free lipase is likely due
to the conformational changes in the enzyme molecules upon
immobilization onto the particles, which are in the order of
L-PEI2-MS > L-PEI1-MS > L-AAS-MS. Conformational changes of
enzyme molecules usually leads to the change in the active site
and consequently the lower affinity of active site to the substrate
molecules.20,27 Immobilization also led to the lower Vmax values
for the immobilized lipases as compared to the free lipase. This
phenomenon is commonplace in the enzyme immobilization
process and is due to mass transfer limitation for substrate
Fig. 4 FTIR spectra (a) and CD spectra (b) of free and immobilized
lipases. CD analysis condition: 2 mg mLꢀ1 solution of fresh free or
immobilized lipase in a sodium phosphate buffer solution (pH 7.4)
stored at 4 ꢁC.
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diffusion and accessibility to the active site of the immobilized
enzymes.48,49 L-PEI1 and PEI2-MS exhibit the lower Vmax values
as compared to L-AAS-MS because of the higher hampered
accessibility of substrate molecules to the active sites of
immobilized lipases within the large molecules of PEI. On the
other hand, the lipases immobilized on the AAS-MS particles,
which are scattered in one layer on the particles, are directly
exposed to the substrate molecules. Such changes in the kinetic
parameters of the lipase upon immobilization lead to the
reduction of the specic activities of the immobilized lipases.
However, the Vmax values for the immobilized lipases are about
80–93% of that for the free lipase, fairly implying that the
resulting immobilized lipases can provide efficient catalytic
ability similar to that of the free lipase.
Effect of temperature on activity of the free and immobilized
lipases
Fig.
5
Effect of temperature on hydrolytic activity of free and
Fig. 5 shows the effect of temperature on the hydrolytic activity
of the free and immobilized lipases. The maximum activity
value of the free and immobilized lipases at optimal conditions
was dened as 100%, and the ratio of the specic activity to the
maximum specic activity was referred as the relative activity.
As seen in Fig. 5, the optimum temperature (i.e., the temperature
at which the activity is maximum) of the tested samples varies
from 40 ꢁC to 55 ꢁC in the order of L-PEI1-MS > L-PEI2-MS >
L-AAS-MS > free lipase. From Fig. 5 inset, it is also observed that
L-PEI1-MS and L-PEI2-MS exhibit 23 and 9% more activity at their
own optimum temperatures (55 and 50 ꢁC, respectively) than the
native lipase at its optimum temperature (40 ꢁC), while L-AAS-MS
immobilized lipases at pH 7.4. Inset: maximum activity of free and
immobilized lipases at their own optimum temperatures; free lipase
(40 ꢁC), L-AAS-MS (45 ꢁC), L-PEI1-MS (55 ꢁC) and L-PEI2-MS (50 ꢁC).
The differences in the optimum temperatures and the effect
of temperature on the lipases activities can be explained by
considering the CD data of the free and immobilized lipases
recorded aer incubation in a buffer solution at 50 ꢁC for 15
min (Table 3). The free lipase undergoes decreases of 8.4% and
4.3% in its a-helices and b-sheets, respectively, and equally an
increase in the content of unordered structures at 50 ꢁC as
compared to those of the free lipase at 40 ꢁC, suggesting the fast
denaturation of the free lipase at temperatures above 40 ꢁC as a
main reason of its reduced activity.27,43
For L-AAS-MS, decreases of 6.4% and 3.6% in its a-helical
and b-sheet contents are observed at 50 ꢁC, respectively, as
compared to those of L-AAS-MS at 40 ꢁC, fairly implying the low
conformational stability of L-AAS-MS at elevated temperatures.
L-PEI1-MS does not show major changes in its unordered
elemental contents and exhibits a decrease of 6% in its a-helical
contents and equally an increase in the b-sheet contents at 50 ꢁC
as compared to those obtained at 40 ꢁC. L-PEI2-MS shows a
decrease of 5% in its a-helices and an increase of 5.3% in its b-
sheets at 50 ꢁC as compared to those obtained at 40 ꢁC.
ꢁ
sample retains 95% of the native lipase activity at 45 C as its
optimum temperature.
The inuence of temperature on the activity of enzymes is
possibly raised from two distinct opposite phenomena. First,
the hydrolytic reaction rate increases thermodynamically
through increasing the temperature. Second, an increase in the
temperature beyond the so-called optimum temperature leads
to the changes in the enzyme conformation and reduces the
enzyme activity negating the positive effect of temperature upon
the reaction rate.8,27 The obtained results reveal that the
conformational changes and the denaturation of the free lipase
may occur at lower temperatures compared to the immobilized
lipases.
Table 3 Secondary structural content (%) of lipase in free and different immobilized states
a-Helix (%)
b-Sheet (%)
40 ꢁ
b-Turn (%)
4 ꢁ 40 ꢁ
Random coil (%)
4 ꢁ 40 ꢁC 50 ꢁ
Sample
4a ꢁC 40b ꢁC 50c ꢁC R-50d ꢁC 4 ꢁ
C
C
50 ꢁC R-50 ꢁ
16
C
C
C
50 ꢁ
25
24.5
20.5
25.9
C
R-50 ꢁ
C
C
C
R-50 ꢁ
C
Free lipase 31.6
L-AAS-MS 30.2
L-PEI1-MS 28.1
L-PEI2-MS 26.3
31.4
29.4
27.9
24.5
22.7
23
21.5
17
20.4 20.3
19.9 19.1
18.7 19
18.1 18.3
18.9 20.5
20.5 21.6
23.4 24.8
29.9 30
31 31
33.3
35
18
21
15
15.5
25
10.6
23.3
19.1
30.9
22.6
29.2
40.5
33.1
36.7
32.7 32.5
33.2 35
32.6
34
17.1 17.7
23.1
a
Fresh lipases dispersed in a buffer solution at 4 ꢁC and CD recorded at 4 ꢁC. b Lipases pretreated in a buffer solution at 40 ꢁC for 15 min and CD
recorded at 40 ꢁC. c Lipases pretreated in a buffer solution at 50 ꢁC for 15 min and CD recorded at 50 ꢁC. d Lipases recovered at the end of the 5th
cycle of reusability assay performed at 50 ꢁC and then dispersed in a buffer solution at 50 ꢁC, and CD recorded at 50 ꢁC.
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ꢁ
Enhancement of the conformational stability of the covalently was also evaluated at 40 C (Fig. S4†). All the free and immo-
attached enzymes at elevated temperatures has also been bilized lipases represent the rather more retained activities at 40
documented in the literature.28,48,50 The more stable conforma- ꢁC compared to those at 50 ꢁC, i.e., 40, 76, 94 and 89% for free
tions of L-PEI1-MS and L-PEI2-MS compared to L-AAS-MS can lipase, L-AAS-MS, L-PEI1-MS and L-PEI2-MS, respectively.
also be related to rigidication of lipase molecules through However, as seen in Fig. 6a and S4,† the deactivation rate
multi-point covalent attachments with the large numbers of follows the same order of free lipase > L-AAS-MS > L-PEI2-MS >
binding sites on the PEI1-MS and PEI2-MS particles (Table 1). L-PEI1-MS at both temperatures. According to the literature,43
The secondary structure of the lipases immobilized on PEI- the higher thermal stability of the immobilized lipases
ꢁ
functionalized particles was also studied at 55 C. The L-PEI1- compared to the free lipase can be due to the higher resistivity
MS sample did not show signicant changes in its conforma- of the lipases against the denaturation through the covalent
tion, but L-PEI2-MS represented decreases of 3% and 2.1% in immobilization. The results of secondary structure of the free
the a-helical and b-sheet contents, respectively, as compared to and immobilized lipases aer incubation in a phosphate buffer
ꢁ
those obtained at 50 ꢁC. Owning to the conformational exi- solution at 50 C for 10 h can conrm this hypothesis (Table
bility characteristic of the polymer chains,51 the more severe S1†). For L-AAS-MS, L-PEI1-MS and L-PEI2-MS, decreases of
changes in the conformation of L-PEI2-MS compared to L-PEI1- 5.3%, 2.1% and 5% in the a-helical contents, and decreases of
MS may be due to the larger conformational changes of high 8.1%, 2% and 4.2% in the b-sheet contents are observed aer 10
molecular weight PEI molecules chains (PEI2) upon increasing h-heat treatment, respectively, as compared to those obtained
the temperature compared to the low molecular weight PEI
molecules (PEI1). The conformational changes of the PEI2
molecules may impose more striking conformational changes
in the immobilized enzyme molecules and consequently lead to
the lower activity of L-PEI2-MS at the reaction temperature of
55 ꢁC as compared to L-PEI1-MS. However, further investigation
is required to identify mechanisms governing the conformation
changes in immobilized protein molecules imposed by confor-
mational changes of polymer chains used as the immobilization
matrix, upon increasing the temperature. These results also imply
the importance of the b-sheet content and its crucial role in the
retaining of enzyme activity upon immobilization at elevated
temperatures. The L-PEI1-MS and L-PEI2-MS represent about
4.7% and 2.8% more b-sheets at 50 ꢁC, respectively, as compared
to the free lipase at 40 ꢁC (Table 3). It can be concluded that the
ꢁ
ꢁ
higher activities of L-PEI1-MS at 55 C and L-PEI2-MS at 50 C,
compared to the free lipase at 40 ꢁC, are resulted from not only
the thermodynamically enhancement of the reaction rates at
elevated temperatures, but also an increase in their b-sheet
contents. According to the literature,46,52 the enhancement of b-
sheet content in some enzymes leads to opening of the lipase's
active site and increasing of the lipase affinity for substrate
molecules. The kinetic parameters (Km and Vmax) of the lipases
immobilized on PEI-functionalized particles also conrm the
enhancement of these immobilized lipases affinity for substrate
molecules and reaction rates at 50 ꢁC (Table 2).
Thermal stability and storage ability of the free and
immobilized lipases
Thermal stability of enzymes is one of the most important
criteria for different applications.2 The inactivation courses of
the free and immobilized lipases incubated in a buffer solution
at 50 ꢁC are shown in Fig. 6a. The relative activity is dened as
the ratio of the measured specic activity to the initial specic
activity. The free lipase is inactivated at a faster rate compared
to the immobilized lipases as it preserves about 18% of its
initial activity aer 10 h heat treatment. L-AAS-MS, L-PEI1-MS
and L-PEI2-MS retain 41, 81 and 72% of their initial activities,
Fig. 6 Thermal stability at 50 ꢁC and pH 7.4 (a), and storage stability at
respectively. Thermal stability of free and immobilized lipases pH 7.4 and 4 ꢁC (b) for free and immobilized lipases.
33324 | RSC Adv., 2015, 5, 33313–33327
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aer pre-treatment at 50 ꢁC for 15 min. In contrast, the free
lipase suffers more conformational changes and exhibits
decreases of 15% and 9.5% in its a-helices and b-sheets,
respectively, over the heat treatment as compared to those
ꢁ
obtained aer pre-treatment at 50 C for 15 min.
As seen in Fig. 6a and Table S1,† the higher thermal stabil-
ities of the lipases immobilized on PEI-functionalized particles
compared to L-AAS-MS can be due to the rigidication of their
conformations through several covalent attachments on these
particles (see molar ratio of binding sites to immobilized lipase
molecules in Table 2). Enhanced thermal stability of enzymes
through multi-point covalent bindings on particles has also
been documented in the literature.23,53 The less thermal and
conformational stability of L-PEI2-MS compared to L-PEI1-MS,
is probably due to the changes in the conformation of the
high molecular weight PEI molecules (PEI2) at elevated
temperatures, as described in the previous section, which lead
to the higher extent of conformational changes in the lipase
molecules immobilized on the PEI2-MS particles.
Fig. 7 Reusability of the immobilized lipases at 50 ꢁC and pH 7.4.
Storage stability of the free and immobilized lipases was also
examined through the measurement of the remaining lipase
To further investigate how the loss of activity can be related to
the enzyme conformational changes, the secondary structure of
the samples aer 5th cycle was studied by CD analysis (Table 3).
The changes in the conformation of the immobilized lipases, as
compared to those pre-treated at 50 ꢁC for 15 min (Table 3), are in
the increasing order of L-AAS-MS > L-PEI2-MS > L-PEI1-MS. For
L-AAS-MS, decreases of 5% and 4.9% in the a-helices and
b-sheets can be observed, respectively, as compared to those
ꢁ
ꢁ
activity at 50 C aer storing in a buffer solution at 4 C for 20
days (Fig. 6b). The free lipase and L-AAS-MS lose 60% and 47%
of their initial activities within 20 days, respectively, while
L-PEI1-MS and L-PEI2-MS retain almost 86 and 73% of their
initial activities, respectively. The secondary structures of the
free and immobilized lipase were also studied aer storage in a
ꢁ
phosphate buffer solution at 4 C for 20 days (Table S1†). The
obtained results indicate that the decreases in the a-helical and
b-sheet contents are 1.4–9.3% and 1.6–6.8%, respectively, as
compared to those estimated for the fresh ones, and follow the
order of L-PEI1-MS < L-PEI2-MS < L-AAS-MS < free lipase.
Compared to the free lipase and L-AAS-MS, the higher storage
stabilities of the lipases immobilized onto the PEI-
functionalized samples can be due to the multi-point covalent
binding of the lipases on these particles, which retains the
lipases in stable conformations.53
It should be mentioned that no detectable lipase molecules
were observed in the immobilized lipases stocks prepared for
the thermal and storage stability studies, as the Bradford assay
was employed, verifying that the activity loss through these
above-mentioned assays was not due to the possible leaching of
the lipase molecules from the nanocomposite particles surface.
ꢁ
obtained aer pre-treatment at 50 C for 15 min, revealing the
unfolding of this lipase as a main reason of its activity loss over
repeated uses. L-PEI1-MS suffers only decreases of 1% and 1.7%
in its a-helices and b-sheets, respectively, while L-PEI2-MS
undergoes decreases of 2% and 4% in its a-helices and
b-sheets, respectively, aer ve times reusing as compared to
those obtained aer pre-treatment at 50 ꢁC for 15 min. The
formation of the larger number of multipoint covalent linkages
between the PEI-functionalized particles surface and lipases
leads to an increase in the conformational stability of lipases
over a multi-cycle reuse as compared to the L-AAS-MS sample.
As explained in the previous section, the lower stability of
L-PEI2-MS compared to L-PEI1-MS over repeated uses may be
related to the more severe conformational changes of the high
molecular weight PEI molecules (PEI2) imposing higher extents
of conformational changes in the enzyme molecules at a high
temperature (50 ꢁC, in this test).
Reusability of the immobilized lipases
For development of a cost-effective enzymatic process, the
reusability of immobilized lipases is an important issue.2 The
reusability of the immobilized lipases through the hydrolysis
Conclusions
reaction was easily performed by rapid separation and recov- In the present work, an easy, practical and low cost approach
ering the biocatalysts with a magnet. Fig. 7 demonstrates that was employed to functionalize magnetic silica nanocomposite
L-AAS-MS, L-PEI1-MS and L-PEI2-MS have good reusability and particles with the large molecules of PEI and the small mole-
retain nearly 57, 88 and 74% of their initial activities in the 5th cules of AAS for lipase immobilization. We found that the size of
cycle, respectively. In general, enzymes are very sensitive to the applied functional molecule is a crucial parameter strikingly
environmental changes, and frequent treatment, i.e., recovering inuencing the properties of the functionalized particles and
and washing with buffer and then using in a new reaction batch, immobilized lipases. The binding site concentration, lipase
would unavoidably make them denatured.13,27
immobilization efficiency, loading capacity and conformational
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change upon immobilization were directly correlated to each 11 Y. Saylan, L. Uzun and A. Denizli, Ind. Eng. Chem. Res., 2015,
other and they varied in the order of L-PEI2-MS > L-PEI1-MS > 54, 454.
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and L-PEI2-MS at the elevated temperatures fairly suggested the 15 S. Abramson, C. Meiller, P. Beaunier, V. Dupuis,
direct correlation of the lipase activity and the b-sheet content,
which increased in the order of L-PEI1-MS > L-PEI2-MS > L-AAS-
MS as compared to the free lipase.
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among the three samples presented in this work, to improve
R.
C.
Rodrigues
and
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Fernandez-Lafuente,
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study opens up new perspectives for the design of polymer- 19 G. Zhang, J. Ma, J. Wang, Y. Li, G. Zhang, F. Zhang and
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Acknowledgements
This work is nancially supported by the National Institute of
Genetic Engineering and Biotechnology in Iran. The authors
thankfully acknowledge the NTNU NanoLab for providing
STEM characterization facility. The Research Council of Norway 22 S. Lin, Z. X. Lin, G. P. Yao, C. H. Deng, P. Y. Yang and
is acknowledged for the support to NTNU NanoLab through the X. M. Zhang, Rapid Commun. Mass Spectrom., 2007, 21, 3910.
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