Recyclable biocatalytic composites of lipase-linked magnetic macro-/nano-particles for glycerol carbonate synthesis

Article abstract of DOI:10.1016/j.apcata.2012.06.016

Lipase immobilized biocatalysts were prepared via enzyme binding onto functionalized surface of magnetic micro-/nano-particles. In order to achieve an efficient biocatalytic composite the immobilization parameters (e.g. lipase concentration, pH of the imm

Full text of DOI:10.1016/j.apcata.2012.06.016

Applied Catalysis A: General 437–438 (2012) 90–95
Contents lists available at SciVerse ScienceDirect
Applied Catalysis A: General
journal homepage: www.elsevier.com/locate/apcata
Recyclable biocatalytic composites of lipase-linked magnetic
macro-/nano-particles for glycerol carbonate synthesis
∗
Madalina Tudorache, Loredana Protesescu, Alina Negoi, Vasile I. Parvulescu
University of Bucharest, Department of Organic Chemistry, Biochemistry and Catalysis, Bd. Regina Elisabeta 4-12, Bucharest 030016, Romania
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 April 2012
Received in revised form 11 June 2012
Accepted 12 June 2012
Available online 19 June 2012
Lipase immobilized biocatalysts were prepared via enzyme binding onto functionalized surface of mag-
netic micro-/nano-particles. In order to achieve an efficient biocatalytic composite the immobilization
parameters (e.g. lipase concentration, pH of the immobilization phase, activation reagent) were correlated
with support morphology and type of the functional group on the support surface. The characterization of
the lipase-particle composites was made using FTIR and UV–Vis techniques. The biocatalyst activity was
evaluated in the transesterification reaction of glycerol with DMC (dimethyl carbonate). Under solvent-
free conditions the conversion of glycerol was of 48.6% with the selectivity in glycerol carbonate (GlyC)
of 85%. The biocatalyst composites were easily recycled using the magnetic properties of the support.
Compared to free enzyme, recycling experiments demonstrated that the operational stability of the het-
erogeneous biocatalyst was improved (fifteen cycles for bio-composites vs. four cycles for free enzyme).
Keywords:
Biocatalyst
Magnetic nano-/micro-particles
Lipase
Glycerol carbonate
©
2012 Elsevier B.V. All rights reserved.
1
. Introduction
facilitating the synthesis under mild reaction conditions. So far,
there are only few reports on this topic. The lipase from Candida
antarctica source immobilized on resins (Novozyme 435) catalyzed
the transesterification of glycerol with DMC in THF [9] or solvent-
free [10] conditions. Aspergillus niger lipase is also able to catalyze
this reaction under solvent-free conditions [11]. In fact it is already
obvious that a biocatalytic synthesis occurs under green conditions.
The immobilization of the enzyme onto a solid support makes
even greener the biocatalytic synthesis of GlyC. Immobilization
provides a physical support for enzymes conferring additional sta-
bility against their denaturation and sometimes modulating the
catalytic activity [12–14]. However, in most of the cases the design
of such biocatalysts is not trivial. Generally, it involves binding
the enzyme via simple adsorption or covalent bonds [15] to a
carrier (support) with different composition and geometry (e.g.
ion exchange resin, mesoporous silica, polypropylene hollow fiber,
glass balls, cellulose membrane and magnetic beads) [16]. The main
advantages of immobilization are the easy separation of the enzyme
from the reaction mixture and the reusability for tens of time. These
lead to a reduction of the amount of used enzyme and, indirectly,
of the process cost [17]. In the particular case of lipase, the immo-
bilization can lead to changes of the enzyme activity and stability
depending on the source of the enzyme, the type of the support
and the immobilization method [13,14,18,19]. The selection of the
proper support and of the immobilization method is thus very
important.
Glycerol carbonate (GlyC) is a rather new compound with
promising high potential in the chemical industry [1]. It is included
in the category of so called “green solvents” due to the low toxicity,
good biodegradability and high boiling point. Up to date it has been
investigated as an additive of coatings, paints and detergents as well
as component of gas separation membranes [2]. Also, it is part of
the technological processes of polycarbonates and polyurethanes
production as monomer [3].
Nowadays, the common routes to GlyC production involve
glycerol carboxylation with various compounds delivering acyl
groups (e.g. ethylene carbonate [4], urea [1] and carbon dioxide
[
5]). Dimethyl carbonate (DMC) is another acyl donor for glyc-
erol carboxylation. Scheme 1 presents the synthesis route of GlyC
4). Glycerol dicarbonate (5) and diglycerol tricarbonate (6) are
secondary products. Typically, this requires large quantities of
Brønsted base as catalyst (e.g. K CO ), n-Bu Sn(OCH ) , macrop-
(
2
3
2
3 2
orous resins or alkaline X/Y zeolite [6–8] and results in moderate
yields of targeted product. In addition, the synthesis is carried out
under harsh reaction conditions.
Recently, biocatalytic preparation of GlyC has been accom-
plished based on lipase-catalyzed glycerol and DMC reaction, thus
∗ Corresponding author. Tel.: +40 21 4100241; fax: +40 21 4100241.
E-mail addresses: vasile.parvulescu@g.unibuc.ro, v parvulescu@yahoo.com
In recent years, magnetic supports (i.e. Fe O4 micro-/nano-
3
(
V.I. Parvulescu).
spheres coated with polymeric layer(s)) have showed attractive
0
926-860X/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.apcata.2012.06.016

M. Tudorache et al. / Applied Catalysis A: General 437–438 (2012) 90–95
91
free enzyme. Another aim of this study was to establish a detailed
evaluation of the relationship between the characteristics of the
solid surface and the catalytic behavior of lipase. FTIR and UV–Vis
investigations were carried out in this scope.
2. Experimental
2.1. Chemicals and solutions
The lipase from Aspergillus niger was purchased from
Sigma–Aldrich (USA). The enzyme was immobilized on the surface
of magnetic particles (Chemicell, Rostock, Germany) with differ-
ent size and structure (Fig. 1). As immobilization reagents were
used 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-
hydroxysuccinimide (NHS) and glutaraldehyde (GA). A solution
of 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES), pH 4.7/7.4
was used as a coupling buffer for the enzyme attachment. Phos-
phate buffer saline (PBS) of 10 mM concentration (8 g NaCl, 0.2 g
KCl, 1.43 g Na HPO × 2H O and 0.34 g KH PO in 0.1 l distilled
2
4
2
2
4
water) at pH 7.4 containing 0.1% BSA and 0.05% sodium azide was
used for blocking active sites of the solid surface after enzyme
immobilization. All the synthesis reagents were purchased from
Sigma–Aldrich (USA). Derivatization reagents (BSTFA:TMCS = 99:1
and pyridine) were purchased from Macherey-Nagel Corp. (Duren,
Germany) and Fluka (Switzerland). The organic solvents used in all
the experiments were of the analytic purity.
2.2. Biocatalyst preparation
The biocatalyst design assumed the immobilization of the
enzyme onto the magnetic particles surface via a covalent binding.
The magnetic particles used for the preparation of biocatalysts were
spheres of maghemite ꢀ-Fe O3 (micro-particles of 500 nm exter-
2
nal diameter) (M1 and M ) and magnetite Fe O (nano-particles of
2
3
4
5
0 nm external diameter) (M and M ). Their surface was function-
3 4
alized with a coating layer (Fig. 1) consisting of a polymeric layer of
silicone derivatives with COOH (MP ) and NH2 (MP2 and MP3)
1
terminal groups. Ethylene diamine derivatives were also used to
generate NH2 terminal groups (MP ).
4
Lipase enzyme from Aspergillus niger source was attached on
the magnetic micro-/nano-spheres via covalent immobilization
using EDC or GA reagents. They were further denominated as EDC
and GA routes. In this scope 0.1 mL suspension of magnetic beads
were placed in an Eppendorf vial where the beads were separated
from the solution using a permanent magnet (Chemicell, Rostock,
Germany). The particles were washed with 0.1 M MES by separating
and re-suspending them in the liquid phase. Then, the immobiliza-
tion conditions depended on the reagent has been used. For EDC
route, a solution containing 10 mg/mL EDC and a given amount of
lipase (21, 43, 86 ␮g/mL) in 0.1 M MES (pH 4.7) were added in the
Eppendorf vial containing the magnetic support pre-treated with
MES. The suspension was stirred gently for 2 h. Then, the lipase-
beads were washed with a PBS solution (0.1 M, pH 7.4) for 3 times
and re-suspended in the blocking solution (0.1% BSA and 0.05%
sodium azide in 0.1 M PBS) to remove the enzyme excess and to
avoid the unspecific binding on the particle surface.
Scheme 1. GlyC synthesis using the glycerol carboxylation route.
support for lipase immobilization with the final goal to prepare
biocatalysts [16]. Magnetic particles were chosen as supports for
enzyme immobilization due to their fast and facile separation from
the reaction medium that can be effectively controlled by applying
a magnetic field [20,21]. Beside the easy way of manipulation, the
catalytic properties of the enzyme-magnetic particle composites
were often improved compared with the free enzyme [22–24]. Min-
imized diffusion limitation, increase of the reaction kinetic, high
exposed area for immobilization, avoiding the nonchemical sepa-
ration of biocatalyst from the reaction mixture are only few of the
specific advantages of the magnetic supports [25]. Consequently,
the lipase immobilization strategy on these materials appears as a
promising alternative to prepare performing and sustainable bio-
catalysts [16,24,26–28].
For GA route, the enzyme was immobilized via the interaction of
the NH2 terminal groups with glutaraldehyde. For this purpose,
the magnetic support pre-treated with MES was suspended in a
MES solution containing 0.1% glutaraldehyde and lipase (21, 43,
86 ␮g/mL). Then, the procedure followed a similar route as for EDC.
The present study proposes the immobilization of a lipase
enzyme from Aspergillus niger source onto a functionalized mag-
netic micro-/nano-particle surface. The catalytic behavior of the
resulted bio-composites was tested in a solvent-free system
designed for GlyC synthesis and compared to the behavior of the
2.3. Biocatalyst characterization
The evidences of the enzyme immobilization on the function-
alized magnetic micro-/nano-spheres were obtained from FTIR

9
2
M. Tudorache et al. / Applied Catalysis A: General 437–438 (2012) 90–95
Fig. 1. Magnetic particles structure used for lipase biocatalyst preparation.
spectra by comparing the data collected for free enzyme, support
and lipase-magnetic beads composites. The loading of immobilized
lipase was calculated based on a UV–Vis procedure using bovine
serum albumin as a standard [29]. The absorbance of the enzyme
in the immobilization solution was measured at 280 nm before and
after immobilization. The loading of the immobilized enzyme was
calculated from the difference between the initial concentration of
enzyme and the remanent concentration after the enzyme immo-
bilization.
3. Results and discussion
3.1. Lipase immobilization on magnetic particles
Lipase enzyme was covalently attached on the magnetic parti-
cles surface. Evidences of the enzyme immobilization are presented
in Fig. 2, which compares the FTIR spectrum of enzyme-magnetic
beads composite with those of the magnetic support and free
enzyme. The spectrum of the biocatalyst presents an absorption
−
1
band at 1063 cm that is characteristic for lipase [21]. The pres-
ence of this band in all the investigated catalysts represents an
important proof for the immobilization of enzyme. However, the
shift of this band and also of the other bands in the spectrum of
lipase account also for the immobilization of the enzyme. Notewor-
thy, this spectrum has been not altered even after the 15th recycle
(Fig. 2).
2.4. Biocatalytic synthesis of GlyC
Enzymatic synthesis of GlyC has been performed in solvent-
free system. Given amounts of Gly and DMC (Gly:DMC = 1:10 molar
ratio) were mixed together with the lipase-magnetic particle bio-
catalyst (2–8%, w/w) in a 1.5 mL reaction vial (Eppendorf tube). The
In order to perform an efficient coupling enzyme-support, the
lipase molecules must be at the neutral form (i.e. negative and
positive enzyme charge must be equal) which means that the pH
of the immobilization solution has to be close to the isoelectic
point (IP) of the enzyme (IP 4.0–4.5 [30]). Indeed, Table 1 shows
◦
mixtures were incubated for 6 h under stirring at 60 C using a ther-
mostated shaker. After the reaction was completed, the biocatalyst
was recovered from the reaction mixture using a magnetic field. A
permanent magnet was placed closed to the reaction vessel wall (on
the external part) gathering the biocatalyst. The liquid phase was
◦
evaporated at 50 C under vacuum in order to eliminate the DMC
Table 1
excess and the produced methanol. Dried reaction products (GlyC
and secondary products) were analyzed using gas chromatography
Influence of the immobilization approach on the biocatalyst activity (expressed by
glycerol conversion, %). Conditions for GlyC synthesis: 50 mg glycerol, 1 mL DMC,
◦
60
C temperature and 6 h incubation time.
(
GC) coupled to flam ionization (FID) detector (Schimadzu GC-2014,
Thermo Electron Scientific Corporation, USA), after a pre-silylation
step (100 ␮L silylation agent BSTFA:TMCS = 99:1 and 100 ␮L pyri-
dine). The efficiency of the lipase-biocatalyst was evaluated based
on the glycerol conversion calculated as the ratio of the moles of
transformed glycerol to the moles of glycerol introduced in the
reaction. The catalytic efficiency of the lipase-composites was also
Catalyst
Immobilization approach
EDC pH value
GA
4.7
7.4
MP1
MP2
MP3
MP4
31.7
29.1
48.6
29.5
1.4
11.6
15.5
18.4
–
10.5
15.7
29.7
−
1
evaluated by calculating the turnover frequency (TOF, h ), i.e. the
glycerol moles converted by one lipase mol per time unit.

M. Tudorache et al. / Applied Catalysis A: General 437–438 (2012) 90–95
93
Fig. 2. FTIR spectra of (a) free lipase, (b) magnetic particles support, (c) biocatalytic composite before reaction and (d) after 15th reaction cycle.
very high differences between the catalytic efficiency of enzyme
immobilized at pH 4.7 and that at 7.4, for EDC route (Scheme 2).
isoeletric point (pH 7.4), the biomolecules were positively charged
affecting thus the enzyme immobilization via NH2 function.
Consequently, the catalytic efficiency of the bio-composites dimin-
ished due to the electrostatic intermolecular interactions of lipase
enzyme.
The largest difference was found for MP , when the catalytic effi-
1
ciency was even 22 times more for the enzyme immobilized at
pH 4.7. When the lipase was exposed to a pH value above the
Scheme 2. Lipase immobilization on the magnetic particles surface using (a) EDC and (b) GA immobilization method.

9
4
M. Tudorache et al. / Applied Catalysis A: General 437–438 (2012) 90–95
Fig. 4. Effect of the support composition on the biocatalyst activity. Biocatalyst
preparation based on the EDC immobilization approach. Conditions for GlyC syn-
◦
Fig. 3. Optimization of the enzyme concentration involved in the immobilization
approach (21, 43 and 86 ␮g/mL lipase). The biocatalyst was prepared based on EDC
thesis: 50 mg glycerol, 1 mL DMC, 60 C temperature and 6 h incubation time.
immobilization approach. Conditions for GlyC synthesis: 50 mg glycerol, 1 mL DMC,
◦
6
0
C temperature and 6 h incubation time.
5
−1
2
.11–2.30 × 10 h ). The selectivity of the biocatalytic process was
Using GA route, the binding of the enzyme to the magnetic beads
similar (in the range of 85–90% selectivity in GlyC) for all of the
bio-composites (lipase – MP , MP , MP and MP ). The domi-
occurs through the GA linker (Scheme 2). In this case, the NH2
terminal groups of both the support and enzyme were coupled via
the GA linker. The prepared biocatalyst exhibited poorer perfor-
mances compared to the EDC case (Table 1). An explanation of this
could be done based on the activity loss of lipase biocomposites
caused by the GA tendency to form large-molecular-weight com-
posites generated by an uncontrollable polymerization during the
cross-linking process [31].
1
2
3
4
nant by-product (5–15% selectivity) resulted after reaction was
diglycerol tricarbonate (6) (Scheme 1). The other secondary prod-
uct detected was glycerol dicarbonate (5) (Scheme 1). Insignificant
traces of glycerol dicarbonate were found in the reaction mixture
for MP biocomposite (5% selectivity in glycerol dicarbonate).
1
The catalytic behavior of immobilized enzyme is strongly influ-
enced by the support properties [16]. In our case they refer to both
the support and polymeric inter-layer (Fig. 1). Among the prop-
erties reported to influence the catalytic behavior are: the nature
of the support surface, the composition and conformation of the
support. In the specific case of lipase the catalytic behavior is dic-
tated by two conformations in equilibrium, i.e. the open-active and
closed-inactive ones. Modulation of the support properties leads to
shift the equilibrium to one of these enzyme forms improving or
even diminishing the enzyme catalytic capacity [18].
The lipase concentration of the immobilization solution was set
up at an optimum value for each type of support (MP , MP , MP
1
2
3
and MP ) in order to generate a catalytic active enzyme layer totally
4
covering the support. The results are shown in Fig. 3. A concen-
tration of 21 ␮g/mL lipase allowed the preparation of biocatalyst
composites characterized by a maximum glycerol conversion for
both micro-structure supports (32.6% and 34.9% glycerol conver-
sion for MP1 and MP ). The maximum loading of the immobilized
2
enzyme was of 0.10 and 0.15 ␮g enzyme/mg support for MP1
Based on crystallographic investigations, Brzozowski et al.
showed that the catalytic site of lipase is covered by a helical
part called “lid”, which has two clearly defined regions: i.e. one on
each side of the lid, e.g. hydrophobic and hydrophilic region [32].
The hydrophobic (non-polar) medium (e.g. hydrophobic support
surface) around the enzyme molecule may interact with hydropho-
bic part of the enzyme lid pushing out site the lid and exposing
the catalytic site. Also, a hydrophilic medium (as example water
molecules) in the enzyme micro-environment may bury the lid in
the open position by interactions with its hydrophilic side [32].
The catalytic behavior of our biocomposites (MP , MP , MP and
and MP , respectively. Further increase of the enzyme loading
2
corresponded in a decrease of the catalytic efficiency (Fig. 3). Over-
agglomeration of the lipase on the support surface led to a sterical
inhibition, blocking of the catalytic sites of the enzyme. Mass trans-
fer limitations can also contribute to this behavior.
Nano-beads (MP3 and MP ) offered larger available areas for
4
the enzyme immobilization compared to micro-supports (MP1
and MP ). Indeed, the optimum concentration of the immobilized
2
enzyme was of 43 ␮g/mL lipase (48.6% glycerol conversion) (Fig. 3)
with an enzyme loading of 3.1 ␮g enzyme/mg support for MP sup-
3
1
2
3
port. The MP support exhibited higher capacity of immobilization.
In this case larger amounts than 86 ␮g/mL lipase were necessary to
saturate the support surface.
Coating matrixes of magnetic particles exhibit different
hydrophilicity/hydrophobicity that may also influence the enzyme
catalytic efficiency. Fig. 1 depicts the morphology of the inves-
tigated bio-composites. The functionalized cover of magnetic
micro-particles (500 nm external diameter) corresponds to a com-
MP ) can be explained based on this model. Lipase-MP biocompos-
4
4
3
5
−1
ite showed higher catalytic capacity (TOF = 3.52 × 10 h ) based
on MP3 properties [33] that can be associate to the hydrophobic
properties of the external layer of the support allowing the access
of substrates to the catalytic site. In addition, the hydrophobic
interactions between the support and enzyme may help a bet-
ter docking of the lipase on the magnetic particle surface [34].
Beside this such a structure may provide an easy exchange of water
molecules between beads coating matrix and external medium (i.e.
the hydrophobic area around the lipase lid) (Fig. 1).
pact non-porous hydrophobic silica layer leaving COOH (MP ) and
1
NH2 (MP ) terminal groups (Fig. 1) for the covalent attachment
2
of the enzyme. Nano-beads (50 nm external diameter) cover exhib-
ited combined hydrophobic (associated to aminosilane for MP4)
and hydrophilic (associated to water molecules which filled the
holes of the polymeric matrix) properties. The catalytic efficiency
of these biocatalysts is presented in Fig. 4. The best efficiency was
Among the investigated catalysts MP , MP2 and MP4 supports
1
are characterized by dominant hydrophobic surfaces (Fig. 1) [33].
However the covering layer of the supports has compact structure
avoiding the hosting of the water molecules closed to the lipase.
This allowed the enzyme binding to the support but was not able
to provide a proper environment for a high catalytic efficiency of
the enzyme in the transesterification process. Consequently, the
5
−1
obtained for the MP3 supported biocatalyst (TOF = 3.52 × 10 h ).
The other bio-composites (lipase – MP , MP and MP ) were
1
2
4
characterized by lower catalytic efficiencies (TOF in the range of
biocomposites activity for MP , MP2 and MP4 was lower than MP3.
1

M. Tudorache et al. / Applied Catalysis A: General 437–438 (2012) 90–95
95
particles were successfully used for fifteen reaction cycles (90 h
of reaction) without any significant loss of the catalytic efficiency,
while the free enzyme allowed only four cycles (16 h of reaction).
Acknowledgments
This work was financially supported by PN II HR YT-96 program,
contract no. 91/2010 from CNCSIS.
References
[
[
[
[
[
[
1] M. Aresta, A. Dibenedetto, F. Nocito, C. Ferragina, J. Catal. 268 (2009) 106–114.
2] D. Randall, R. de Vos, Patent EP419114 (1991).
3] V. Plasman, T. Caulier, N. Boulos, Plast. Addit. Compd. 7 (2005) 30–33.
4] H.-J. Cho, H.-M. Kwon, J. Tharun, D.-W. Prk, J. Ind. Eng. Chem. 16 (2010) 679–683.
5] J. George, Y. Patel, M. Pillai, P. Munshi, J. Mol. Catal. A: Chem. 304 (2009) 1–7.
6] M. Aresta, A. Dibennedetto, F. Nocito, C. Pastore, J. Mol. Catal. A: Chem. 257
(
2006) 149–153.
[
7] G. Rokicki, P. Rakoczy, P. Parzuchowski, M. Sobiecki, Green Chem. 7 (2005)
529–539.
Fig. 5. Operational stability of lipase-beads biocatalyst in GlyC synthesis system.
Conditions for GlyC synthesis: 50 mg glycerol, 1 mL DMC, 60 C temperature and 6 h
incubation time.
◦
[8] Z. Mouloungui, J.W. Yoo, C.A. Gachen, A. Gaset, G. Vermeersch, EP0739888
1996).
9] S.C. Kim, Y.H. Kim, H. Lee, D.Y. Yoon, B.K. Song, J. Mol. Catal. B: Enzym. 49 (2007)
5–78.
[10] E.Y. Lee, K.H. Lee, C.-H. Park, Bioprocess Biosyst. Eng. 33 (2010) 1059–1065.
[11] M. Tudorache, L. Protesescu, S. Coman, V.I. Parvulescu, Green Chem. 14 (2012)
(
[
7
It must be mentioned that the core (ꢀ-Fe O ) of the magnetic
2
3
particles (support composition) does not catalyze the reaction of
GlyC synthesis. Also, glycerol does not react with dimethylcarbon-
ate in the absence of the biocatalyst.
478–482.
[
[
[
12] C. Mateo, J.M. Palomo, G. Fernandez-Lorente, J.M.D. Guisan, R. Fernandez-
Lafuente, Enzyme Microb. Technol. 40 (2007) 1451–1463.
13] J.M. Palomo, G. Mu n˜ oz, G. Fernández-Lorente, C. Mateo, M. Fuentes, J.M. Guisan,
R. Fernández-Lafuente, J. Mol. Catal. B: Enzym. 21 (2003) 201–210.
14] J.M. Palomo, C. Ortiz, G. Fernández-Lorente, M. Fuentes, J.M. Guisán, R.
Fernández-Lafuente, Enzyme Microb. Technol. 36 (2005) 447–454.
3.2. Recyclability and stability
The biocomposite exhibiting the best catalytic efficiency was
[15] R.A. Sheldon, Appl. Microbiol. Biotechnol. 92 (2011) 467–477.
[
[
16] M.M.M. Elnashar, J. Biomater. Nanobiotechnol. 1 (2010) 61–77.
17] M. Tudorache, D. Mahalu, C. Teodorescu, R. Stan, C. Bala, V.I. Parvulescu, J. Mol.
Catal. B: Enzym. 69 (2011) 133–139.
recycled for twenty times in order to evaluate the catalyst sta-
bility. The experimental data showed not significant differences
on the biocatalyst efficiency for the first fifteen cycles (e.g. the
glycerol conversion was close to the initial values 48.6%) (Fig. 5).
Also, the selectivity of enzyme was conserved during the reaction
cycles (around 85% in GlyC). The stability of the biocomposite in
operating conditions was confirmed by both the results in con-
version and similarity of the IR spectra of biocatalyst (Fig. 2).
These results demonstrated that the enzyme immobilization
improved significantly the operational stability of the lipase-beads
composites compared to free enzyme. In terms of time, this cor-
responded to an operational stability of 90 h for bio-composites,
while the operational stability of the free enzyme was only
[18] R. Fernandez-Lafuente, J. Mol. Catal. B: Enzym. 62 (2010) 197–212.
[19] G. Bayramoglu, B. Kaya, M.Y. Arica, Food Chem. 92 (2005) 261–268.
[20] S. Huang, M. Liao, D. Chen, Biotechnol. Progr. 19 (2003) 1095–1100.
[21] W. Xie, J. Wang, Biomass Bioenergy 36 (2011) 373–380.
[22] D.S. Jiang, S.Y. Long, J. Huang, H.Y. Xiao, J.Y. Zhou, Biochem. Eng. J. 25 (2005)
5–23.
1
[
23] M.M. Zheng, L. Dong, Y. Lu, P.M. Guo, Q.C. Deng, W.L. Li, Y.Q. Feng, F.H. Huang,
J. Mol. Catal. B: Enzym. 74 (2012) 16–23.
[24] C.H. Kuo, Y.C. Liu, C.M.J. Chang, J.H. Chen, C. Chang, C.J. Shieh, Carbohydr. Polym.
87 (2012) 2706–2711.
[
[
[
[
25] I. Magario, X. Ma, A. Neumann, C. Syldatk, R. Hausmann, J. Biotechnol. 134
(
2008) 72–78.
26] Y. Yong, Y.-X. Bai, Y.-F. Li, L. Lin, Y.-J. Cui, C.-G. Xia, Process Biochem. 43 (2008)
1179–1185.
27] Y. Cui, X. Chen, Y. Li, X. Liu, L. Lei, S. Xuan, Appl. Microbiol. Biotechnol. (2011)
1
6 h [11].
1–10.
28] X. Liu, L. Lei, Y. Li, H. Zhu, Y. Cui, H. Hu, Biochem. Eng. J. 56 (2011) 142–149.
4
. Conclusions
[29] E.F. Hartree, Anal. Biochem. 42 (1972) 422–427.
[
[
[
[
30] R. Sharmaa, Y. Chistib, U. Chand Banerjee, Biotechnol. Adv. 19 (2001)
27–662.
31] G.T. Hermanson, Bioconjugate Techniques, Academic Press, Elsevier, New York,
1996.
32] A.M. Brzozowski, U. Derewenda, G.G. Dodson, D.M. Lawson, J.P. Turkenburg, F.
Bjorkling, B. Huge-Jensen, S.A. Patkar, L. Thim, Nature 351 (1991) 491–494.
33] www.chemicell.com.
6
A biocatalyst design was developed for GlyC synthesis follow-
ing the covalently immobilization of lipase enzyme on magnetic
particles leading to a biocatalyst with a three times higher cat-
5
−1
alytic efficiency (3.52 × 10 h TOF) comparing with free lipase (i.e.
5
−1
1
.16 × 10 h TOF). Also, the immobilized lipase was more stable
[34] D.H. Zhang, L.X. Yuwen, Y.L. Xie, W. Li, X.B. Li, Colloids Surf. B: Biointerfaces 89
2012) 73–78.
(
and even recyclable compared with free lipase. Lipase-composite