Penicillium expansum lipase-coated magnetic Fe3O 4-polymer hybrid hollow nanoparticles: A highly recoverable and magnetically separable catalyst for the synthesis of 1,3-dibutylurea

Article abstract of DOI:10.1039/c4ra04156a

Herein, amino-epoxy supports were innovatively imported onto magnetic nanoparticles (Fe₃O₄-polymer hybrid nanospheres) for immobilizing enzymes. This new support has a coating layer with dual functional groups (epoxy and amino-epoxy). Consequently, this support has great anionic exchange power and a high number of epoxy groups. The acquired immobilized Penicillium expansum lipase in combination with this heterofunctional support represents a novel class of heterogeneous catalyst towards the synthesis of 1,3-dibutylurea from ethylene carbonate and butylamine, which has not been very commonly catalyzed by enzymes. After optimization of the reaction conditions, the yield of 1,3-dibutylurea was 77% under solvent free conditions at 60 °C. Moreover, after completion of reaction, the catalyst was simply recovered by an external conventional magnet and recycled without significant loss in the catalytic activity (up to ten cycles). the Partner Organisations 2014.

Full text of DOI:10.1039/c4ra04156a

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Penicillium expansum lipase-coated magnetic
Fe3O4–polymer hybrid hollow nanoparticles: a
highly recoverable and magnetically separable
catalyst for the synthesis of 1,3-dibutylurea
Cite this: RSC Adv., 2014, 4, 25983
*
Jun Liu, Wenjing Wang, Huiwen Liu, Yaoliang Zhou, Haibo Zhang and Xiaohai Zhou
Herein, amino-epoxy supports were innovatively imported onto magnetic nanoparticles (Fe3O4–polymer
hybrid nanospheres) for immobilizing enzymes. This new support has a coating layer with dual functional
groups (epoxy and amino-epoxy). Consequently, this support has great anionic exchange power and a
high number of epoxy groups. The acquired immobilized Penicillium expansum lipase in combination
with this heterofunctional support represents a novel class of heterogeneous catalyst towards the
synthesis of 1,3-dibutylurea from ethylene carbonate and butylamine, which has not been very
commonly catalyzed by enzymes. After optimization of the reaction conditions, the yield of 1,3-
dibutylurea was 77% under solvent free conditions at 60 ꢀC. Moreover, after completion of reaction, the
catalyst was simply recovered by an external conventional magnet and recycled without significant loss
in the catalytic activity (up to ten cycles).
Received 5th May 2014
Accepted 22nd May 2014
DOI: 10.1039/c4ra04156a
www.rsc.org/advances
higher catalytic activity and stability despite their cumbersome
preparation, painful separation and poor efficiency. Currently,
Introduction
using magnetic properties is a pinpoint protocol to solve these
problems. With the rapid development of nanotechnology,
magnetic nanoparticles for enzyme immobilization are now
being studied extensively all over the world.22–29 However,
immobilization on nanoparticles has also some drawbacks:
enzymes immobilized on nanoparticles can easily lose their
activity due to the interaction with external interfaces or even
with the enzyme molecules immobilized on other particles.
This may be solved by covering the immobilized enzymes with
polymers, which could provide a porous structure to stabilize
the enzyme.30
Nowadays, with the increasing use of green chemistry,
magnetic chitosan (CS)–Fe3O4 nanoparticles with their poten-
tial application for enzyme immobilization have attracted
extensive interest because of chitosan's excellent properties
such as non-toxicity, biocompatibility, mucus-adhesion and
biodegradation.31–41 Zhi et al.42 prepared magnetic CS–Fe3O4
nanoparticles in situ with small pools of a water-in-oil micro-
emulsion, which contains CS and the ferrous salt as micro-
reactors, by adding a basic precipitant such as NaOH into the
micro-emulsion. The defect of this method is poor productivity;
in addition, it uses a lot of surfactant and co-surfactant
compounds that are pernicious to the environment. Wu et al.43
prepared magnetic CS–Fe3O4 nanoparticles by the covalent
binding of CS and tripolyphosphate onto Fe3O4 nanoparticles
by a one-step method. The problem of this method is that the
magnetisation process is complicated and hard to control.
Enzymes are versatile biocatalysts with high specicity, and
their applications have been found in areas of biomaterials and
organic synthesis.1,2 However, the major drawbacks for their
industrial applications are their low thermal and solvent
stability as well as difficulty in recycling. Applications of lipase
can be achieved more economically and efficiently by immobi-
lization to enhance its activity, selectivity, and operational
stability.3–8 For example, the activity of lipase could be improved
by adsorption of lipase on hydrophobic supports through the
hydrophobic areas surrounding the active centre and thus
stabilizing the open form of the lipase.9 Enantiomeric ratio may
be changed from 1 to almost 100 simply by using different
immobilized preparations in some cases.10,11 Moreover, the
stability of lipase could be enhanced through rigidication of
the enzyme structure by multipoint covalent immobiliza-
tion.12,13 Therefore, a lot of fruitful efforts have been made on
the preparation of lipases in immobilized forms, which involve
a variety of both support materials and immobilization
methods.14–19
Nanophase materials have many advantages in immobiliza-
tion due to their unique size and physical properties.9,20,21
Lipases, immobilized onto nanoparticles, which have a high
specic surface area and low diffusion resistance, possess much
College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072,
China. E-mail: haibozhang1980@gmail.com; Fax: +86-027-87218534; Tel: +86-027-
87218534
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44
´
Siodmiak et al. prepared CS–poly[N-benzyl-2-(methacryloxy)-
N,N-dimethylethanaminium bromide]-coated magnetic nano-
particles by co-precipitation method via the epichlorohydrin CS
cross-linking reaction. The disadvantage of this method is that
it uses some poisonous compounds, such as benzyl bromide,
which is nocuous and not environmentally benign. Ding et al.45
illustrated a successful and green synthetic route of magnetic
hollow Fe3O4–polymer hybrid nanospheres (MHHNs) by the
Scheme
carbonate and amine.
1
Synthesis of 1,3-disubstituted urea from ethylene
addition of Fe3O4 nanoparticles to an aqueous solution of strategy to synthesize 1,3-disubstituted urea is the composite of
polymer–monomer pairs composed of the cationic polymer, CS, an amine and ethylene carbonate (EC) (Scheme 1). Although
and the anionic monomer, acrylic acid (AA), which is followed ethylene carbonate is a green reagent and solvent that is
by polymerization of AA and selective crosslinking of CS at the industrially obtained from an insertion reaction of carbon
end of the polymerization process. This innovative method with dioxide into epoxides,60,61 some base catalysts such as NaOMe,62
the highlights of simple operation, high performance, cost- CaO,63 1,5,7-triazabicyclo[4.4.0]dec-5-ene and thioureas64 or
effectiveness, and environmental friendliness, will usher in rosy Cs2CO3,65 were used to catalyze this reaction. However, all of
ꢀ
prospects for magnetic nanospheres in the application of these catalysts operate under extreme conditions (above 90 C)
immobilized enzymes.
and are complicated to recycle. Moreover, most of these
In this paper, we introduce this kind of magnetic CS–Fe3O4 conventional catalysts contain metals, which are considered to
nanoparticles into the immobilization of lipases. MHHNs were be environmentally hazardous. Nowadays, with a universal
activated by epoxy chloropropane. Aer that, Penicillium awareness of environmental protection, eliminating the metal
expansum lipase (PEL) was immobilized on this support by the component involvement in catalyst design and avoiding volatile
covalent linkage between the epoxy groups on the support organic solvent usage in the process of chemical synthesis is
surface and the amino group on the enzymes surface to form more important and necessary for our future.
the immobilized PEL magnetic Fe3O4–polymer hybrid nano-
In this study, we extended the scope of application of these
spheres (MHHNs-lip). Recently, the production, purication, nanoparticles in the synthesis of 1,3-disubstituted ureas. We
and application of PEL have been discussed in various report the immobilization of PEL-coated magnetic hollow
reviews.46,47 Immobilization on this kind of support follows a Fe3O4–polymer hybrid nanospheres, which represents a novel
two-step mechanism:48 (1) an ionic exchange with the amino class of heterogeneous catalyst for the synthesis of 1,3-dibuty-
groups in the support or hydrophobic adsorption is the rst lurea from EC and butylamine. The main advantages behind
step in the immobilization process. (2) The protein, which has using the immobilized PEL magnetic Fe3O4–polymer hybrid
been previously adsorbed, is covalently attached to the epoxy nanospheres (MHHNs-lip) as the catalyst are as follows: (1)
groups present on the surface of the support. Finally, to compared to chemical catalysts, lipase is regarded as an opti-
complete the protein-support reaction, the epoxy groups can be mized potential candidate for environmentally friendly reac-
easily blocked by reaction with very different thiol or amine tions. (2) the synthesis of MHHNs-lip is simple and allows the
compounds under mild conditions,49 preventing further large-scale preparation of nanospheres, which also follows an
uncontrolled reactions between the support and the enzyme, eco-friendly and non-toxic route. (3) The catalyst can be easily
which could decrease its stability. Obviously, the protocol is separated by an external magnet without using extra organic
simple and allows the large-scale preparation of other enzymes, solvents and additional ltration steps. (4) The chemical reac-
like Aspergillus niger lipase and Rhizopus chinensis satio lipase, tion with the catalyst of MHHNs-lip can be carried out under
ꢀ
which are still under investigation in our laboratory.
mild conditions (60 C and atmospheric pressure). All of these
At the same time, we have investigated the application of advantages are in agreement with the principles of green
MHHNs-lip for the synthesis of 1,3-disubstituted ureas, which chemistry and sustainable development.
are very essential intermediates in the preparation of pharma-
ceuticals and biochemicals. 1,3-Disubstituted ureas have
extensive applications such as dyes, antioxidants, corrosion
inhibitors, and intermediates for the preparation of pharma-
Results and discussion
Characterization of immobilized lipase
ceuticals and agricultural chemicals.50–52 Recently, these Typical TEM micrographs of pure Fe3O4 and the MHHNs-lip
compounds have attracted extensive interest because nanoparticles are shown in Fig. 1. A salient feature of Fig. 1b–d
substituted ureas, which contain amino acid groups, have been is that these nanospheres have an intense dark circle within the
used for brain cancer treatment and have been proven to have a shells of the spheres and dark spots on the surface of some
marked inhibitory effect on the HIV protease enzyme.52 The spheres, which suggests that the Fe3O4 particles were coated by
currently studied alternative routes for the synthesis of ureas polymer acrylic acid (PAA), CS and PEL. The mean size of Fe3O4
through phosgene substitutes are as follows: (a) the oxidative particles was 30 ꢁ 5 nm.
carbonylation of amines with carbon monoxide under various
The thermal stability of the samples was investigated by
metal catalysts,53–56 (b) the direct reaction of carbon dioxide with thermogravimetric analysis (TGA). The TGA curves for the
amines,57,58 and (c) the use of some phosgene substitutes such particles of bare Fe3O4, PEL, MHHNs, and MHHNs-lip are
as carbonates, which are less toxic and more stable.59 Another shown in Fig. 2. As shown in Fig. 2, in all the samples, the
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Fig. 3 FT-IR spectra of: (a) bare Fe3O4, (b) Penicillium expansum, and
(c) MHHNs-lip.
Fig. 1 TEM images of the (a) bare Fe3O4 nanoparticles and (b–d)
MHHNs-lip.
illustrates a characteristic amide II band with a maximum at
1414 cmꢂ1 due to N–H bending with contribution from C–N
stretching vibrations. The presence of the amide III band with a
maximum at 1323 cmꢂ1 is due to N–H bending with C–Ca and
C–N stretching vibrations. The absorption band at 2938 cmꢂ1
was due to the stretching vibrations of C–H. These signicant
bands were observed in the free lipase as well as MHHNs-lip
(Fig. 3c), indicating that the lipase was successfully coated onto
the surface of the Fe3O4 magnetic nanoparticles. The FT-IR
spectrum of MHHNs-lip (Fig. 3c) shows that the amide I and II
bands of the puried acidic lipase overlapped at 1647 cmꢂ1 with
the absorption bands of the C]O groups in PAA and the
ammonium groups in CS. This band became weaker, which
may be due to the strong hydrogen bonding between Fe3O4 and
lipase.69 The characteristic peak for the Fe–O group of magnetite
at 598 cmꢂ1 can be observed in Fig. 3c, which indicates the
successful generation of MHHNs-lip particles.
Fig. 2 TGA of (a) bare Fe3O4, (b) MHHNs, (c) MHHNs-lip, and (d)
Penicillium expansum.
weight loss within 200 ꢀC was attributed completely to the loss
of adsorbed water molecules.66 MHHNs showed a higher weight
loss than bare Fe3O4 because of the loss of the PAA and CS
components (Fig. 2b). Aer immobilization of PEL, the weight
loss shown in Fig. 2c increased again, which was due to the
increased organic components on the MHHNs-lip. From this
gure, the protein loading content was determined to be 56 mg
gꢂ1, which was very close to the calculated value (the total
amount of PEL protein assayed according to the Coomassie
Brilliant Blue G-250 method was 58 mg gꢂ1).
The magnetic properties of the magnetic nanoparticles were
analyzed by VSM at room temperature. Fig. 4 shows the
hysteresis loops of the samples. The saturation magnetization
was found to be 38.79 emu gꢂ1 for MHHNs, which is less than
that for the pure Fe3O4 nanoparticles (67.08 emu gꢂ1). This
difference suggests that a large amount of chitosan and polymer
Fig. 3 shows the FT-IR spectra of (a) Fe3O4 nanoparticles, (b)
Penicillium expansum, and (c) MHHNs-lip. There was a strong
peak observed at 598 cmꢂ1 in Fig. 3a, assigned to the charac-
teristic absorbance of Fe3O4.62 In addition, the peaks at 1622
and 1362 cmꢂ1 were assigned to the absorbance of the asym-
metric and symmetric stretching vibration of COOꢂ of the citric
acid modier, respectively, indicating the presence of Fe3O4
nanoparticles coated by the COOꢂ group.67 The FT-IR spectra of
free lipase contained a broad envelope at 3421 cmꢂ1 due to the
overlap of the –NH stretching of the amide group in the proteins
(Fig. 3b). The FT-IR absorption spectrum of lipase generally
shows three major bands caused by peptide group vibrations ꢂin1
the range of 1800–1300 cmꢂ1 68 The amide I band at 1605 cm
.
Fig. 4 Magnetic hysteresis curves of (a) bare Fe3O4, (b) MHHNs, and
is mainly due to C–O stretching vibrations, whereas free lipase
(c) MHHNs-lip.
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of acrylic acid was coated onto the surface of the Fe3O4 nano-
particles. The lower saturation magnetization of MHHNs-lip
(25.99 emu gꢂ1) compared with MHHNs suggests that PEL was
immobilized successfully in the MHHNs. With large saturation
magnetization, MHHNs-lip could be separated from the reac-
tion medium rapidly and easily in a magnetic eld. In addition,
there is no hysteresis in the magnetization with both the
remanence and coercivity being zero, suggesting that these
magnetic nanoparticles are superparamagnetic. Moreover,
when the external magnetic eld is removed, the magnetic
nanoparticles could be dispersed by gentle shaking. These
magnetic properties are critical in the applications within the
biomedical and bioengineering elds.
Scheme 2 Illustration of the preparation, activation of the epoxy
groups and immobilization of the lipase.
Fig. 5 shows the XRD patterns of pure Fe3O4, MHHNs and
MHHNs-lip. It is apparent that the diffraction pattern of our
Fe3O4 nanoparticles is similar to the standard pattern for crys-
talline magnetite (Fig. 5a). The characteristic diffraction peaks
marked by their indices (220), (311), (400), (422), (511), and
(440) could be indexed to the inverse cubic spinel structure of
Fe3O4 (JCPDS card no. 85-1436), and they were also observed for
MHHNs (Fig. 5b) and MHHNs-lip (Fig. 5c). This reveals that the
graed and polymerized process, on the surface of Fe3O4
nanoparticles did not lead to their crystal phase change. The
average crystallite size D was about 30.3 nm, which is in good
agreement with that measured by TEM. The average crystallite
size was obtained from the Sherrer equation D ¼ Kl/(b cos q),
where K is constant, l is the X-ray wavelength, and b is the peak
width of half-maximum. However, due the existence of the
organic layer (CS and PAA) on the surface of the Fe3O4 nano-
particles in MHHNs (Fig. 5b), it may lead to a decrease in the
intensity for the Fe3O4 specic peaks (Fig. 5a), which could also
explain the decrease in intensity in Fig. 5c compared to Fig. 5b,
because of the extra lipase layer on the surface of MHHNs.
The preparation procedure is illustrated schematically in
Scheme 2. We selected CS, which bears amino groups, as the
cationic polymer and AA, which bears acid groups, as the
anionic monomer. The CS and AA polymer–monomer pair and
Fe3O4 nanoparticles stabilized by poly(vinyl alcohol) (PVA) were
mixed and formed micelles loaded with Fe3O4 nanoparticles;
the cores consist of the polyionic complexes of CS and AA (i.e.
positively charged protonated CS chains and negatively charged
dissociated AA) and the shells consist of protonated CS chains.
Initiation of the polymerization of AA with potassium persulfate
(K2S2O8) followed by cross-linking of the shells with glutaral-
dehyde (GA) at the end of the polymerization process led to the
formation of magnetic hollow Fe3O4–polymer hybrid
nanospheres.45
Following the procedures described in the literature, it is
hard to give an exact structure of the groups formed by the GA.
However, whatever the exact structure of the GA on the
support,70,71 the nal result is a support having a fairly hydro-
phobic moiety formed by the GA chains and free amino groups72
(the ratio73 of aldehyde groups from GA to the amino groups
from CS was 1 : 2). In the next activation procedure with epoxy
chloropropane, the epoxy activation will proceed via the amino,
hydroxyl67 and carboxyl groups.74 Therefore, the nal support
will be an amino-epoxy heterofunctional one (Scheme 3), which
contributes a lot to promote the absorption via ionic exchange
at low ionic strength.75 However, due to the tendency of the
open form of the lipases to become adsorbed versus the
hydrophobic interface, even though immobilization is per-
formed at low ionic strength, the enzyme will still be immobi-
lized by both immobilization mechanisms: interfacial
activation and ionic exchange.76–78 Aer enzyme hydrophobic
adsorption, the reactive groups of the enzyme near the support
surface may produce some covalent bonding reactions, which
may be produced aer enzyme immobilization, and there is no
guarantee that the enzyme will nally have any covalent
attachment with the support.75 Therefore, aer completion of
the immobilization, in order to protein-support reaction,
aspartic acid was added to block the epoxy groups to prevent
further uncontrolled reactions between the support and the
enzyme that could decrease its stability.48,49
On the basis of the two-step mechanism of enzyme immo-
bilization,67,79 the adsorption of the enzymes on the support
Fig. 5 XRD pattern of (a) bare Fe3O4, (b) MHHNs, and (c) MHHNs-lip. Scheme 3 Illustration of the activation of the epoxy groups.
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Table 1 Screening the lipase sources for 1,3-dibutylurea synthesis.
Conditions: catalyst: 0.7 g; EC: 40 mmol; butylamine: 80 mmol;
temperature: 60 ꢀC; time: 24 h
surface was the rst step. In the second step, with enzymes
absorbed onto the surface of the support successfully, the high
concentration of reactive groups in the PEL and the epoxy
groups in the support could make a rapid “intramolecular”
covalent reaction (Scheme 4). It has been reported that the
probability of the formation of multiple covalent linkages
between PEL and epoxy could be enhanced, leading to an
increase in the stability of the PEL immobilized micro-
spheres.67,80 The immobilization of the enzymes inside the
hollow MHHNs may increase the stability by preventing any
intermolecular process and by preventing the enzyme from
interacting with external interfaces.81 Therefore, the cavities in
the cellulose microspheres could provide not only a space for
storage of the enzyme but also a wall shell to protect its struc-
ture and nature.
Entry
Catalyst
Yield (%)
1
2
3
4
5
6
7
8
9
10
Penicillium expansum
Penicillium neutral expannsum
Aspergillus niger
Aspergillus oryzae
Rhizopus chinensis satio
Porcine pancreatic
69
37
28
15
9
6
13
5
Novozym 435
Neural proteolytic enzyme
Acidic proteolytic enzyme
Alkaline proteolytic enzyme
3
4
Table 2 Synthesis of 1,3-dibutylurea. Conditions: catalyst: 0.7 g; EC:
40 mmol; butylamine: 80 mmol; temperature: 60 ꢀC; time: 24 h
Screening of the biocatalyst for 1,3-dibutylurea synthesis
Entry
Catalyst
Yield (%)
The catalytic activity of lipase from various natural sources has
been directly evaluated in the transesterication of EC with
butylamine. All the available lipases were screened based on
their activities for the production of 1,3-dibutylurea. As shown
in Table 1, PEL showed high catalytic performance for the
production of 1,3-dibutylurea, while other lipases revealed little
activity. Based on these results, we selected PEL as the lipase for
immobilization.
1
2
3
4
5
PEL
Fe3O4
MHHNs
MHHNs-lip
Epoxy sepabeads-lip
69
6
4
77
70
supports (epoxy Sepabeads). This different effect on the enzyme
activity of the immobilization when using the different supports
(although both of them have epoxy groups as the reactive group)
may be related to different enzyme orientations of the enzyme
on the support. This indicates that the enzyme likely reacts with
the support via different areas (the areas of the protein surface
that have more hydrophobic residues or areas of the protein
that have more negative charge) with concomitant different
effects on enzyme activity.48
The temperature inuence on the transesterication process
has also been investigated. As seen from Table 3, the tempera-
ture increase is favourable for the production of 1,3-dibutylurea,
since the enzyme activity increases with an increase in temper-
ature. The yield of 1,3-dibutylurea increased gradually with the
temperature till a maximum at 60 ꢀC. However, a further
increase in temperature (up to 70 ꢀC) led to a slight decrease in
the yield of 1,3-dibutylurea. Nevertheless, we adopted 60 ꢀC for
As seen in Table 2, when bare Fe3O4 was used in the reaction
medium, the yield of reaction was 6% in 24 h. Using bare
MHHNs in the reaction at room temperature gave a 4% yield. In
the experiment using MHHNs-lip gave 77% yield. This value is
higher than that using the same lipase in its free form, where
the yields ranged from 69% to 77%. It seems that aer immo-
bilization, there was an enhancement of the enzymes activity, as
shown by Dalla-Vecchia in esterication reactions of carboxylic
acids with n-pentanol in organic media by Rhyzopus oryzae
lipase.82 However, the activity of the immobilized lipase
decreased when PEL was immobilized onto conventional epoxy
Table 3 Influence of reaction temperature on the synthesis of 1,3-
dibutylurea. Conditions: MHHNs-lip catalyst: 0.7 g; EC: 40 mmol;
butylamine: 80 mmol; time: 24 h
Temperature
(ꢀC)
Yield (%)
20
30
40
50
60
70
9
23
35
52
77
76
Scheme 4 Illustration of the immobilization of lipase.
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Table 4 Influence of incubation time on the 1,3-dibutylurea synthesis.
Conditions: MHHNs-lip catalyst: 0.7 g; EC: 40 mmol; butylamine: 80
mmol; temperature: 60 ꢀ
C
Incubation time
(h)
Yield (%)
1
3
6
12
24
36
22
34
46
58
77
77
Fig. 7 Reusability of catalyst.
this work, which is in agreement with the our earlier reports for
the appropriate reaction temperature of this lipase.83
As shown in Table 4, the inuence of incubation time on the
transesterication process was also investigated. It was
apparent that increasing the reaction time from 0 to 24 h is
more advantageous for the reaction process, giving rise to
improved yields. When increasing the reaction time further
from 24 h to 36 h, no apparent change on the yield of 1,3-
dibutylurea yield can be seen, indicating the appropriate reac-
tion time was 24 h.
may display a higher stability than the original lipase. The
activity of the rst batch was taken to be 100%. The MHHNs-lip
exhibited a better reusability, and it retained over 94.8% of its
residual activity aer being used 10 times. Moreover, there was
no further loss in activity aer these cycles. Fig. 8 shows the FT-
IR spectra of the MHHNs-lip before and aer the catalyst was
used 10 times in the reaction medium. As seen in Fig. 8, no
change in the FT-IR spectra of the catalyst was observed aer 10
cycles. Moreover, according to elemental analysis, the organic
content in the catalyst aer 10 cycles was found to be C ¼
35.5%, Fe ¼ 14.6%, and N ¼ 39.9%, and no considerable
change was seen in the CHN data aer recycling.
The storage stability study of free lipase and MHHNs-lip was
investigated and the immobilized lipase, which retains its
original catalytic activity, was found to be appreciably stable for
a period of 60 days (Fig. 9). The immobilized enzyme provided
excellent yield up to 77% of desired product, whereas
commercially available lipase provided 69% yield aer a period
of 60 days.
The recyclability and storage stability of lipase
To make the process more economical, it is necessary to study
the recyclability of the immobilized lipase, which is regarded as
the most important advantage of immobilized enzymes when
compared to the free enzyme. Aer completion of the reaction,
the catalyst in the free PEL catalyst reaction was recovered by
centrifugation of the resulting suspension and washed using
acetone. The recovery free PEL obtained was dried at 45 ꢀC
under reduced pressure overnight (at 1 Torr for 24 h), and then
used for the next reaction. At the same time, the catalyst in the
MHHNs-lip catalysed reaction was magnetically separated from
the reaction medium directly (Fig. 6) and run in another reac-
tion vessel under the same conditions. The residual activity of
both catalysts during their reuse is presented in Fig. 7, which
shows that MHHNs-lip has a better recyclability compared to
free PEL. This suggests that the lipase immobilized on MHHNs
Thermal stability of the immobilized PEL
The thermal stability of the immobilized PEL is one of the most
important criteria when dealing with its possible applications.
Fig. 10 shows the relative activity of the free and the
Fig. 8 FT-IR spectra of the MHHNs-lip before and after the catalyst
was used 10 times.
Fig. 6 Magnetic separation of catalyst by an external magnet.
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simple, easily prepared, and environmental friendly process will
have wide applications in the eld of biocatalysis.
Experimental
Chemical reagents
Crude lipase from Penicillium expansum (PEL; 5000 U mgꢂ1
solid), Penicillium neutral expannsum (PNEL; 10 000 U mgꢂ1
solid), Aspergillus niger (ANL; 120 000 U mgꢂ1 solid), Rhizopus
chinensis satio (RCSL; 10 000 U mgꢂ1 solid), Aspergillus oryzae
(AOL; 30 000 U mgꢂ1 solid), Porcine pancreatic (PPL; 135 U mgꢂ1
solid), neural proteolytic enzyme (50 000 U mgꢂ1 solid), acidic
proteolytic enzyme (60 000 U mgꢂ1 solid), alkaline proteolytic
enzyme (40 000 U mgꢂ1 solid) were kindly donated by Shenzhen
Leveking Bio-engineering Co. Ltd., China. These enzymes were
produced by spraying the concentrated supernatant from
fermentation with the addition of a certain amount of starch as
a thickening agent. Immobilized C. antarctica lipase B (Novo-
zyme 435; EC 3.1.1.3) was donated by Novozymes Investment
Co. Ltd (China). Epoxy Sepabeads, EC, butylamine, Fe3O4
nanoparticles and all other chemicals were purchased from the
Aladdin reagent company, China and were used without further
purication. The water used in the study was prepared using a
Milli-Q Water Purication System (Millipore, Bedford, MA,
USA).
Fig. 9 The storage stability of the immobilized lipase.
Procedure for the preparation of magnetic hollow Fe3O4–
Fig. 10 Relative activity of the free and the immobilized PEL incubated
in 10 mM phosphate buffer (pH 7.0) at 80 ꢀC for 12 h.
polymer hybrid nanospheres
The procedure for the preparation of magnetic hollow Fe3O4–
polymer hybrid nanospheres was carried out as previously
reported.45 10 mL of a PVA-stabilized magnetite nanoparticle
suspension (z1 ꢃ 1018 Fe3O4 particles mLꢂ1) was added to 50
mL of an aqueous solution of CS–AA, which consisted of 0.5 g of
puried CS (Mw ¼ 200 kDa, degree of deacetylation ¼ 90%) and
0.22 g of AA with a stoichiometric molar ratio of 1 : 1.1
(glucosamine unit–acid). Polymerization was then initiated by
K2S2O8 at 70 ꢀC under a stream of nitrogen. As the reaction
system appeared opalescent, the reaction was allowed to
ꢀ
immobilized PEL incubated in a phosphate buffer at 80 C for
12 h. As shown in Fig. 10, the relative activity of the immobilized
PEL was much higher than that of free PEL. Clearly, the
immobilized PEL was more stable than free PEL at relatively
high temperatures, indicating a better thermal stability. The
increase in the thermal stability of immobilized PEL was caused
by its stabilization through the weak intermolecular forces and
the prevention of autolysis of the PEL.67 Therefore, the thermal
stability of the PEL was signicantly improved through its
immobilization on the MHHNs. In addition, no protein could
be observed from the immobilization of PEL (MHHNs-lip) even
aer a period of 48 h under magnetic stirring, which indicates
that the mechanical stability of MHHNs-lip is also high.
ꢀ
proceed for another 100 minutes at 50–60 C. Aerwards, 0.5
mL of GA, a bifunctional cross-linker, was added to the reaction
system at 40 ꢀC to crosslink the chitosan selectively. The
resultant suspension was separated by an external magnet and
washed using acetone. The amino bands of the shells were
cross-linked with glutaraldehyde, in a ratio of glucosamine unit
in CS to aldehyde unit in GA of 2. The amine moieties were
quantied by measuring the color obtained by the reaction of
the primary amino groups on the support and an excess of
picrylsulphonic acid solution.84,85 However, in order to immo-
bilize the lipase, we needed to activate the MHHNs with epoxy
chloropropane.
Conclusion
We have developed a novel class of heterogeneous biocatalyst,
which is particularly attractive in the environmentally friendly
synthesis of 1,3-dibutylurea. PEL was immobilized successfully
in the hollow structure of activated MHHNs. The immobilized
PEL exhibited high effective activity and thermal stability as well
as good reusability. Moreover, the cellulose magnetic micro-
spheres loaded with PEL could be conveniently and easily
Activation of the magnetic hollow Fe3O4–polymer hybrid
nanospheres
magnetically separated from reaction solution, leading to MHHNs (4 g) was added to a solution of 12 mL of epoxy
recovery of the catalyst. The immobilization carriers prepared chloropropane in 40 mL of DMSO; then, 40 mL of 2 N NaOH was
from CS and acrylic acid solution in aqueous solvent via a slowly dripped into the reaction mixture system. The mixture
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was then allowed to stand for 6 h at room temperature; subse- section, except that the reaction mixture was continuously
quently, the resulting solution was removed by magnetic sepa- stirred during the reaction. The activity yield remaining aer
ration. The activated MHHNs were obtained aer washing with immobilization was dened as follow:
acetone and distilled water until the eluate was neutral.67 The
C
Activity yield ð%Þ ¼ ꢃ 100
(1)
epoxy groups in MHHNs were determined by a reaction between
the oxirane ring and sodium thiosulfate, followed by titration
with 0.01 mol Lꢂ1 hydrochloride acid to neutralize any OHꢂ
released in the reaction.86 The amount of epoxy groups in the
activated MHHNs was estimated to be 1.68 mmol gꢂ1 (dry). The
epoxide group readily reacts with the enzyme. Under relatively
mild conditions, these groups in the cellulose support may react
with both amino groups and carboxyl group in the enzyme.
A
and the immobilization yield was calculated by
A ꢂ B
Immobilization yield ð%Þ ¼
ꢃ 100
(2)
A
where A is the total activity of enzyme added in the initial
immobilization solution, B is the activity of the residual enzyme
in the immobilization and washing solution aer the immobi-
lization procedure, and C is the activity of the immobilized PEL.
The maximum activity was dened as 100%, and the relative
activity refers to the percentage that an enzyme activity accounts
for the highest one. The concluded activity yield and immobi-
lization yield were 55.6% and 44.8%, respectively.
Preparation of the immobilized PEL
The immobilization was carried out as follows. About 1.0 g of
the activated magnetic carriers was mixed with 1.0 g of lipase in
25 mL of 10 mM sodium phosphate buffer at pH 7.0 to avoid
diffusional problems that could alter the apparent properties of
the immobilized enzymes under continuous and gentle stirring.
The mixture was shaken at room temperature for 24 h at 250
rpm. Then, the remaining epoxy groups were blocked by incu-
bation with 3 M aspartic acid at pH 8.5 for 24 h.87 The super-
natant was removed by magnetic separation, and the MHHNs-
lip was washed with deionized water, respectively, to remove the
noncovalently coupled enzymes on the support. The MHHNs-
lip was stored at 4 ꢀC. The total amount of PEL protein was
assayed according to the Coomassie Brilliant Blue G-250
method with bovine serum albumin (BSA) used as the
standard.88
Characterization of free and immobilized lipase
Transmission electron microscopy (TEM) was performed using
a JEM-2010FEF eld-emission electron microscope operating at
200 kV equipped with an EDAX Phoenix EDS analyzer. The
thermogravimetric analysis (TGA) was carried out using a Q-
series 600 analyzer. About 8–10 mg of samples were placed in a
ceramic crucible and the analysis programmed from 30 to 800
ꢀC with a 10 ꢀC minꢂ1 rise in temperature, under a 99.99% pure
nitrogen atmosphere with ow rate of 100 mL minꢂ1. The
reference run was carried out with an empty sample crucible
pan and the results were recorded accordingly. The immobi-
lized biocatalysts and free lipase were investigated for their
native conrmation using FT-IR analysis (Bruker. Vertex 70).
The magnetic properties of Fe3O4, MHHNs and MHHNs-lip
were determined using a vibrating sample magnetometer (Lake
Shore 7410 VSM). X-ray diffraction of the magnetic micro-
spheres was carried out on the X-ray diffractometer (Rigaku D/
MAX-2400 XRD with Ni-ltered Cu Ka radiation). The XRD
patterns were recorded in the region of 2q from 10 to 80 ꢀC.
Samples were ground into powders and dried in a vacuum oven
Measurement of enzyme activity
Soluble PEL Activity. The hydrolytic activity of the PEL in an
aqueous solution was determined according to the method
described in the literature89 with a slight modication. The PEL
solution (ꢄ0.23 U mlꢂ1, based on the activity in catalyzing the
hydrolysis of p-nitrophenyl palmitate (pNPP) at 37 ꢀC in 50 mM
phosphate buffer, pH 6.0) was obtained as the supernatant aer
dissolution of 1.5 g of the enzyme powders in 10.0 ml phosphate
buffer (50 mM, pH 8.0) followed by centrifugation. A substrate
mixture was prepared by mixing 1 ml of 15.0 mM pNPP in iso-
propanol and 9 ml of a 50 mM buffer solution (phosphate
buffer, pH 5.0–8.0; Tris–HCl buffer, pH 7.0–9.0; barbiturate–HCl
ꢀ
at 60 C for 48 h before characterization.
Procedure for the synthesis of 1,3-dibutylurea from EC and
butylamine
buffer, pH 7.5–10.0) containing 0.1% (w/v) arabic gum and 0.4% The reaction was performed in a 25 mL conical ask. Aer 40
(w/v) Triton X-100. A cuvette containing 2.4 ml of this substrate mmol of EC, 80 mmol of butylamine and the lipase catalyst was
mixture was placed in a Pharmacia Biotech Ultraspec 2000UV/V charged into the reactor, the mixtures were incubated for 24 h
spectrophotometer equipped with a thermostatic cell, for pre- under stirring at temperatures in the range of 60 ꢀC with an
equilibration at 37 ꢀC, and the reaction started by the addition agitation speed of 180 rpm. At the end of the reaction, the
of 0.1 ml of the PEL solution. The variation in the absorbance at catalyst was separated by an external magnet and washed using
410 nm of the assay against a blank without enzyme was acetone. The recyclable immobilized lipase obtained was dried
ꢀ
monitored for 2–5 min. The reaction rate was calculated from at 45 C under reduced pressure overnight (at 1 Torr for 24 h),
the slope of absorbance versus the time curve using the molar and it was then used for the next reaction. Aer separating the
extinction coefficient for p-nitrophenol (pNP), obtained as catalyst, the reactor was cooled to room temperature and the
measured above.
reaction mixture dispersed in 50 mL of water. Then, the
Immobilized PEL Activity. The enzyme activity of immobi- resulting mixture was stirred for 1 h at room temperature. The
lized PEL was measured in the same manner as in the deter- solid formed was ltered off and washed with water. The
mination of the soluble PEL activity described in the previous product yield was determined from the weight of the solid. The
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product was characterized by gas chromatography, gas chro- 23 X. Wang, C. Zhao, P. Zhao, P. P. Dou, Y. Ding and P. Xu,
matography-mass spectrometry and 1H NMR spectroscopy and
Bioresour. Technol., 2009, 100, 2301–2304.
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25 H. D. Li, Z. Li, T. Liu, X. Xiao, Z. H. Peng and L. Deng,
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26 Y. Jiang, C. Guo, H. Xia, I. Mahmood, C. Liu and H. Liu,
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Acknowledgements
We gratefully acknowledge the nancial support of the National
Natural Science Foundation of China (no. 20972120, 21202037). 27 Y. Zhang and C. Xia, Appl. Catal., A, 2009, 366, 141–147.
28 Y. Zhang, Y. Zhao and C. Xia, J. Mol. Catal. A: Chem., 2009,
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