Ester synthesis using Candida rugosa lipase immobilized on magnetic nanoparticles

Article abstract of DOI:10.3109/10242422.2010.550044

Magnetic nanoparticles were synthesized by co-precipitation under hydrothermal conditions. The average diameter of the magnetic nanoparticles was found to be in the range of 15 ± 5 nm with an average surface area of 112.15 m² g^-1. Immobilization of lipase on magnetite nanoparticles was confirmed by FTIR, differential scanning calorimetry and thermal gravimetric analysis. The activation energy of the free enzyme was 1.9-fold higher than that of the immobilized lipase for hydrolytic reactions. Additionally, the lower KM and higher Vmax values of the immobilized enzyme for hydrolysis of 4-nitrophenyl palmitate indicated an increased efficiency of the immobilized lipase. The immobilized lipase exhibited higher esterification efficiency compared with free lipase for synthesis of ethyl isovalerate. It also exhibited fairly good reusability, with about 8.5% reduction in esterification efficiency for ethyl isovalerate synthesis over ten cycles of reuse.

Full text of DOI:10.3109/10242422.2010.550044

Biocatalysis and Biotransformation, January–February 2011; 29(1): 37–45
ORIGINAL ARTICLE
Ester synthesis using Candida rugosa lipase immobilized
on magnetic nanoparticles
VRUSHALI DANDAVATE, HARESH KEHARIA & DATTA MADAMWAR
BRD School of Biosciences, Sardar Patel University,VallabhVidyanagar, Gujarat, India
Abstract
Magnetic nanoparticles were synthesized by co-precipitation under hydrothermal conditions.The average diameter of the
magnetic nanoparticles was found to be in the range of 15 Ϯ 5 nm with an average surface area of 112.15 m2 g–1. Immo-
bilization of lipase on magnetite nanoparticles was confirmed by FTIR, differential scanning calorimetry and thermal
gravimetric analysis. The activation energy of the free enzyme was 1.9-fold higher than that of the immobilized lipase for
hydrolytic reactions. Additionally, the lower KM and higher Vmax values of the immobilized enzyme for hydrolysis of
4-nitrophenyl palmitate indicated an increased efficiency of the immobilized lipase.The immobilized lipase exhibited higher
esterification efficiency compared with free lipase for synthesis of ethyl isovalerate. It also exhibited fairly good reusability,
with about 8.5% reduction in esterification efficiency for ethyl isovalerate synthesis over ten cycles of reuse.
Keywords: Magnetite nanoparticles, immobilization, esterification, lipase
Introduction
successfully designed a number of immobilization
Lipases (acylglycerol acyl hydrolases, EC 3.1.1.3)
catalyze the hydrolysis of esters in aqueous media,
but they can also catalyze esterification and trans-
esterification in organic media (Inada et al. 1984;
Inada 1986; Wu et al. 1996). Lipases are active
against a broad range of substrates and are highly
stable at extremes of temperature and pH (Deleuze
et al. 1987). Thus lipases are among the most
important enzymes in biotechnology today with
wide industrial applications in detergents, oils
and fats, baking, organic synthesis, hard surface
cleaning, and the leather and paper industries
(Anderson et al. 1998; Schmid & Verger 1998;
Jaeger et al. 1999).
In order to enhance lipase performance, several
strategies such as solvent and protein engineering
(Svendsen 2000), chemical and surface modifica-
tion of lipase (Klibanov & Dabulis 1993; Basri et
al. 1995; Okahata et al. 1995; Noda et al. 1997),
use of biphasic and reversed micellar systems (Wu
et al. 2001), enzyme immobilization and enzyme
bioimprinting (Mingarro et al. 1995; Palomo et al.
2002) have been investigated. Researchers have
techniques such as adsorption or deposition of
enzyme on a porous support, entrapment in a gel
matrix, or covalent attachment of enzyme to an
immobilization carrier (Reetz et al. 1996; Gitlesen
et al. 1997; Persson et al. 2000; Dyal et al. 2003),
with the objectives of enhancing enzyme reusability
and providing better control of enzyme-catalyzed
reactions. It is believed that immobilization leads to
dispersion of the enzyme over a large surface area,
thus preventing its aggregation and improving mass
transfer (Nagayama et al. 2002). Immobilization
of lipase has been investigated extensively for
improvement in stability, separation and reuse as
well as for the development of continuous pro-
cesses. Currently the focus in enzyme immobiliza-
tion technology is shifting towards use of nanosize
materials as supports, in particular owing to their
higher specific surface area. A variety of nanosup-
ports have been used for enzyme immobilization,
such as phospholipid bilayers (Xiao et al. 2006),
Langmuir–Blodgett films (Guiomar et al. 1997),
self-assembled monolayers (Sun et al. 1998),
silica nanotubes (Nakaminami et al. 1999), and
Correspondence: D. Madamwar, BRD School of Biosciences, Sardar Patel Maidan, Vadtal Road, PO Box 39, Satellite Campus, Sardar Patel University,
Vallabh Vidyanagar – 388 120, Gujarat, India. Tel: ϩ 91-2692-229380. Fax: ϩ 91-2692-231042/236475. E-mail: datta_madamwar@yahoo.com
(Received 2 September 2010; revised 11 December 2010; accepted 19 December 2010)
ISSN 1024-2422 print/ISSN 1029-2446 online © 2011 Informa UK, Ltd.
DOI: 10.3109/10242422.2010.550044

38 V. Dandavate et al.
magnetite (Kouassi et al. 2005), silver (Ren et al.
2005) and gold nanoparticles (Phadtare et al. 2002).
Magnetic nanoparticles have received considerable
attention for enzyme immobilization due to their higher
surface area, lower mass transfer resistance, reduced
fouling and ease of separation of immobilized enzyme
from the reaction mixture by application of a magnetic
field (Halling & Dunnill 1980). Magnetite (Fe3O4) is
one of the most widely employed magnetic materials
owing to its biocompatibility, low toxicity and super-
paramagnetic properties (Dresco et al. 1999). Magne-
tite nanoparticles have been used as a support for
covalently attaching several enzymes including yeast
alcohol dehydrogenase, lipase, glucose oxidase, choles-
terol oxidase and bacterial esterase (Liao & Chen 2001;
Dyal et al. 2003; Guo et al. 2003).
We have been working on the development of
an efficient process for ethyl isovalerate synthesis
using Candida rugosa lipase (CRL) and encouraging
results were obtained using surfactant-coated
CRL immobilized in Sodium bis-(2-ethylhexyl)
(AOT)-based organogels (Dandavate & Madamwar
2007). A major limitation in the development of a
lipase-mediated esterification process has been
recovery of enzyme for its efficient reuse. This, in
principle, can be overcome by immobilization of
lipase; however, since the esterification reaction
needs to be carried out under non-aqueous condi-
tions, most conventional immobilization carriers
designed for aqueous systems are incompatible.
Furthermore, water is produced as a by-product
during the esterification reaction,and if not removed
will accumulate and shift the equilibrium of the
reaction towards hydrolysis. In the present study,
we have immobilized CRL by covalent attachment
to the surface of magnetite nanoparticles prepared
in our laboratory.This paper deals with the charac-
terization of CRL immobilized on nanoparticles
and their use in the synthesis of esters.
Methods
Preparation of magnetic nanoparticles
Magnetic nanoparticles (Fe3O4) were prepared by
chemical co-precipitation of Fe2ϩ and Fe3ϩ ions in
a solution of ammonium hydroxide under hydro-
thermal conditions (Halling & Dunnill 1980; Huang
et al. 2003). Fe2ϩ and Fe3ϩ (molar ratio 1:2) were
dissolved in nanopure water (to a final concentration
of 0.25 M) and chemically precipitated at room tem-
perature (25°C) by adding NH4OH solution (30%
v/v) to give pH 10. The precipitates were heated at
80°C for 35 min under continuous mixing and
washed four times in MilliQ water followed by sev-
eral washes in ethanol. During washing, the mag-
netic nanoparticles were separated from the
supernatant using a magnetic separator of strength
greater than 20 MOe.The particles were finally dried
in a vacuum oven at 70°C.The dried particles exhib-
ited strong magnetic attraction.
Immobilization of CRL on magnetic
nanoparticles
Magnetic nanoparticles (50–250 mg) were sus-
pended in 1 mL of phosphate buffer (0.05 M, pH
7.2). To this, 0.5 mL of carbodiimide solution
(0.025 g/mL in 0.05 M phosphate buffer, pH 7.2)
was added and the mixture kept in a sonicator bath
for 10 min. Then 2.5 mL of lipase (5 mg mL–1)
was added and the reaction mixture was sonicated
for a further 30 min at 4°C. Magnetic nanoparti-
cles with bound lipase were recovered from the
reaction mixture using a strong magnetic separa-
tor. The precipitates were washed with 0.05 M
phosphate buffer (pH 7.2), then with 0.1 M Tris–
HCl (pH 8), and used for activity measurements
and characterization. In order to determine the
binding efficiency, the residual unbound protein in
the supernatant was estimated and the percentage
binding was determined by considering the initial
protein as 100%.
Materials and methods
Materials
Characterization
CRL (1140 units mg–1) and ethyl carbodiimide hydro-
chloride N-(3-dimethylaminopropyl)-N’-ethyl carbo-
diimide hydrochloride were purchased from
Sigma-Aldrich, Steinheim, Germany. Ferrous chloride
(FeCl2) was obtained from Loba Chemie, Mumbai,
India. Ferric chloride (FeCl3) and ammonium hydrox-
ide were obtained from Sisco Laboratories, Mumbai,
India. n-Hexanol was purchased from Fluka, Germany
and p-nitrophenol and Triton X-100 from Hi-Media,
Mumbai, India. 4-Nitrophenyl palmitate (pNPP) was
obtained fromAlfaAesar, Lancaster, UK.All substrates
and standards of esters for esterification were obtained
from Merck, Sigma-Aldrich or Fluka.
The size and morphology of the magnetic nano-
particles were determined by transmission electron
microscopy (TEM) (Tecnai 20 electron micro-
scope; Philips, Hillsboro, Oregon, USA.). The
sample for analysis was prepared by placing a drop
of a solution of magnetic nanoparticles dispersed
in absolute ethanol on a Formvar-covered copper
grid and evaporating off the ethanol in air at room
temperature.
X-ray diffraction (XRD) was used to determine
the crystal structure of the magnetic nanoparticles
before and after lipase immobilization using a
Philips X’Pert X-ray diffractometer with Cu-Kα

Ester synthesis using lipase immobilized on magnetic nanoparticles 39
radiation (λ ϭ 0.154 nm). The functionalized
nanoparticles, free lipase and magnetic nanopar-
ticles with bound lipase were examined using
FTIR. The surface area of the nanoparticles was
determined from the nitrogen adsorption–desorp-
tion isotherm using the Brunauer–Emmett–Teller
(BET) method (Brunauer et al. 1938). Thermal
gravimetric analysis (TGA) and differential scan-
ning calorimetry (DSC) of magnetite nanoparticles
with and without bound enzyme were conducted
using a Perkin–Elmer Pyris 1 thermogravimetric
analyzer and a Perkin–Elmer Pyris 1 differential
scanning calorimeter (Melville, New York, USA),
respectively.
Figure 1. TEM image of magnetic nanoparticles.
Kinetic parameters
The kinetic parameters for both free lipase and lipase
immobilized on magnetic nanoparticles (maximum
specific activity Vmax and Michaelis constant KM)
were determined by measuring the initial hydrolysis
rate of pNPP (100–900 mM) using lipase concentra-
tion of 5 mg mL–1. The influence of immobilization
of lipase on the activation energy for lipase-catalyzed
hydrolytic reaction of pNPP ester was determined
from Arrhenius plots obtained for the enzyme within
the temperature range of 25–40°C.
Analytical procedures
Detection and quantification of esters.
After initiation of esterification, 500 μL samples
from the reaction mixture were withdrawn periodi-
cally and analyzed immediately by gas chromatogra-
phy (model Clarus 500 instrument; Perkin–Elmer)
using flame ionization detection and a 30 m Rtx-R-
20 (crossbond 80% dimethyl–20% diphenyl polysi-
loxane) capillary column. Nitrogen served as carrier
gas at a split flow rate of 90 mL min–1; the column
temperature was programmed to increase from 40
to 210°C at the rate of 6°C min–1 and from 210 to
280°C at the rate of 15°C min–1. The injector and
detector temperatures were 250°C and 280°C,
respectively. Ester identification and quantification
were done by comparing the retention time and the
peak area of the sample with a standard.
Synthesis of esters using lipase immobilized
on magnetic nanoparticles
The reaction mixture, comprising 20 mL hexane
containing equimolar concentrations (100 mM) of
isovaleric acid and ethanol, was placed in a 250-mL
glass-stoppered flask on an orbital shaker and the
reaction was initiated by adding magnetite-bound
lipase (40 mg). The mixture was kept at 37°C
and shaken at 150 rpm for ethyl isovalerate
production. Samples were withdrawn at 24 h
intervals and analyzed for accumulation of ethyl
isovalerate. In order to determine the effect of
fatty acid chain length, esterification of ethanol
with butyric acid, caprylic acid and acetic acid was
also investigated using immobilized and free
enzyme. The esterification reaction for different
esters was carried out as described above for ethyl
isovalerate, except that different fatty acids were
used in place of isovaleric acid in the reaction
mixture.
25
20
15
10
5
Reusability of immobilized lipase
For reusability studies, the esterification reaction was
carried out as described above for ethyl isovalerate
synthesis. Upon completion of each cycle the immo-
bilized enzyme was recovered magnetically, then
washed once with anhydrous hexane and used for
the next cycle of esterification. This procedure was
repeated for several cycles.
0
8
10
12
14
Size (nm)
16
18
20
22
Figure 2. Size distribution of magnetite particles obtained from
electron micrographs.

40 V. Dandavate et al.
Results and discussion
Determination of lipase activity
The activity of free and bound lipase was determined
spectrophotometrically using pNPP as substrate at
37°C. The substrate solution consisted of 37.7 mg
pNPP dissolved in 10 mL propan-2-ol, 0.207 g bile
salts and 0.1 g gum arabic in 90 mL of 0.05 M phos-
phate buffer (pH 7.2). Nanoparticles with bound
lipase (100 μL) were added to a test tube containing
3.5 mL of substrate solution. The increase in p-nitro-
phenol (pNP) concentration was measured at 410 nm.
One unit of lipase activity was defined as the amount
of enzyme required to release 1 μmole of pNP per
milliliter of solution per minute at 37°C and pH 7.2.
Properties of magnetite nanoparticles
Fine magnetic nanoparticles with a mean diameter
of 10–20 nm were synthesized (Figure 1). The par-
ticle size was evaluated using five different TEM
images and varied from 9 to 22 nm, with most par-
ticles having a size of about 11 nm (Figure 2). A
similar size range of magnetite nanoparticles with a
mean diameter of 12.7 Ϯ 3.1 nm was obtained by
Huang et al. (2003) using the same method. The
XRD patterns of magnetite particles with and with-
out bound lipase exhibited six characteristic peaks
for Fe3O4 (2θ ϭ 30.1°, 35.6°, 43.3°, 53.6°, 57.2° and
Figure 3. XRD patterns of magnetite nanoparticles without (A) and with (B) bound lipase.

Ester synthesis using lipase immobilized on magnetic nanoparticles 41
62.5°) as shown in Figure 3. The XRD patterns of
to the bonding of enzyme to the nanoparticles. The
presenceofacharacteristicbandforproteins(1631.15
cm–1) in the spectrum of lipase to bound magnetite
nanoparticles confirmed the binding of enzyme to the
nanoparticles. Furthermore, the characteristic –NH2
group band at 1593.92 cm–1 observed for the magne-
tite nanoparticles (spectrum A) disappeared after the
lipase was bound (spectrum C), suggesting that
binding occurred between the amine group on
the nanoparticles and the carboxyl group of lipase
after being activated by carbodiimide (Huang et al.
2003). DSC analysis of magnetite nanoparticles with
bound enzyme revealed an exothermic peak at 155°C
(ΔH ϭ –88.003 J g–1) which also supports cross-
linking of the enzyme on carbodiimide-activated
magnetite nanoparticles (Figure 5).TGA showed that
magnetite nanoparticles with bound lipase suffered
20% weight loss in the temperature range 100–450°C,
which can be attributed to decomposition of the
enzyme; a further increase in temperature up to
900°C did not cause any additional significant weight
loss (Figure 6, trace B). In contrast, unbound magne-
tite nanoparticles did not exhibit any significant change
up to 900°C (Figure 6, trace A).
magnetite nanoparticles before and after lipase bind-
ing matched the standard XRD data file of magnetite
(JCPDS card no. 019–0629). Further, the XRD data
did not reveal any significant difference between free
magnetite nanoparticles and those with bound lipase,
suggesting that covalent attachment of lipase to the
surface of the particle did not cause any alteration
in its crystal phase. The size of magnetite nanopar-
ticles with and without bound lipase was calculated
using the Debye–Scherrer equation, and agreed with
the sizes observed by TEM. The surface area of
magnetite nanoparticles was determined by the
BET method to be 112.15 m2 g–1.
Immobilization of lipase on nanoparticles
In our studies we preferred to immobilize lipase on
nanoparticles by covalent attachment since sorption-
based immobilization of enzymes is often associated
with problems of enzyme leaching from the surface
during the reaction (Palomo et al. 2002).The lipase
upon carbodiimide activation was covalently attached
to –NH2 groups on magnetite nanoparticles (Huang
et al. 2003; Kouassi et al. 2005) and immobilization
was monitored by FTIR analysis. Figure 4 shows the
FTIR spectra of magnetite nanoparticles, lipase bound
to carbodiimide-activated magnetite and free lipase.
The band at 592 cm–1 is due to Fe–O of magnetite.
Weak bands at 2924 and near 2900 cm–1, which are
not present in magnetite, are due to symmetric and
asymmetric C–H stretching vibrations of the enzyme.
Shifting of the characteristic band at 1122.18 cm–1
in the spectrum of the free enzyme to 1062 cm–1 with
reduced intensity in the spectrum of the enzyme
bound to magnetite nanoparticles can be attributed
Binding efficiency of enzyme on nanoparticles
The binding efficiency of the enzyme was investigated
for increasing amounts of magnetic nanoparticles at
a fixed concentration of lipase (3.125 mg protein mL–1).
Figure 7 shows that the percentage of bound lipase
increased with increasing amount of nanoparticles
and, at a concentration of magnetite particles above
30 mg mL–1, the efficiency of binding was found to
be 100%. Thus a weight ratio of lipase to magnetite
of 0.104 was required for complete binding of lipase
Figure 4. FTIR spectra of magnetic nanoparticles without and with bound lipase: (A) magnetite nanoparticles; (B) free lipase;
(C) magnetite ϩ carbodiimide ϩ lipase.

42 V. Dandavate et al.
120
100
80
60
40
20
0
0
10
20
30
40
50
60
Fe3O4 (mg mL–1
)
Figure 5. DSC analysis of magnetite nanoparticles with bound
lipase.
Figure 7. Lipase-binding capacity of carbodiimide-treated
magnetite nanoparticles. The error bars in all figures represent
standard error from mean.
to magnetite nanoparticles, which gave a specific
activity of immobilized lipase of 43.24 units per
milligram of magnetite-bound lipase.
as a catalyst for pNPP hydrolysis compared with free
lipase, as inferred from the ratio of Vmax to KM
values. These changes in the kinetic parameters
suggest that covalent binding of lipase to magnetite
nanoparticles probably stabilized a favored confor-
mation of the enzyme on the nanoparticle surface,
resulting in improved accessibility to the active site
(Huang et al. 2003). The effect of temperature on
the activities of free and magnetite-bound lipase can
be seen in Arrhenius plots (Figure 9). The free and
immobilized lipase exhibited linear relationships in
the temperature range 25–40°C and the correspond-
ing activation energies were found to be approxi-
mately 7.8 and 4.1 kJ mol–1, respectively.The lower
activation energy of immobilized lipase in compari-
son to free lipase supports the proposed change in
conformation of the enzyme on the surface of nano-
particles (Kouassi et al. 2005).
Kinetic parameters
Vmax and KM for free and magnetite-bound lipase
were determined using Lineweaver–Burk plots for
lipase-catalyzed hydrolysis of pNPP (Figure 8). KM
of the immobilized enzyme (0.48 mM) was 2.97-fold
lower than that of free lipase (1.427 mM), while Vmax
of immobilized lipase (0.357 mmol mg–1 min–1) was
13.7-fold higher than that of free lipase (0.026 mmol
mg–1 min–1). The lower KM value indicates a higher
affinity of the enzyme for the substrate and the higher
Vmax value indicates a higher activity of the enzyme
at saturating levels of substrate. Thus, the immobi-
lized lipase was found to be 40 times more efficient
Ester synthesis
The lipase immobilized on magnetite nanoparticles
was used for the synthesis of ethyl isovalerate. Table
1 shows that immobilized enzyme exhibited higher
esterification efficiency than free lipase in the syn-
thesis of ethyl isovalerate. However, no significant
difference in esterification efficiency was observed
for synthesis of ethyl acetate or ethyl butyrate, while
free enzyme was found to favor synthesis of ethyl
caprylate in comparison to immobilized lipase.These
results suggest that the conformational change result-
ing from covalent binding of lipase on the surface
of magnetite nanoparticles influenced the substrate
specificity of the enzyme. In our previous studies,
we demonstrated that unmodified CRL catalyzed
Figure 6.TGA scans of magnetite nanoparticles without (A) and
with (B) bound lipase.

Ester synthesis using lipase immobilized on magnetic nanoparticles 43
A
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
2.1
2.6
3.1
1/T×103 (1/K)
3.6
4.1
0
0.002 0.004 0.006 0.008 0.01
1/[pNPP] (mmol–1
0.012
)
Figure 9. Arrhenius plots for lipase-catalyzed hydrolysis of pNPP:
(♦) free lipase; ( ) magnetite-bound lipase.
0.018
0.016
0.014
0.012
0.01
B
recovery from the reaction system and ease of con-
trolling water activity during its reuse. In the case
of lipase immobilized in microemulsion-based
organogels when employed for esterification reac-
tion, water accumulates in the gel during the reac-
tion which can alter the equilibrium of the reaction
towards hydrolysis, thus lowering the esterification
efficiency during reuse. This accumulated water
from the gel then has to be removed intermittently
using a liquid dessicant in order to restore the ester-
ification activity (Jenta et al. 1997; Dandavate &
Madamwar 2007, 2008), which is time-consuming
and cumbersome. Figure 11 shows that the lipase
attached to magnetite nanoparticles could be reused
for ten cycles of ethyl isovalerate synthesis with only
about 8.5% decrease in esterification efficiency.
Thus, this process is more efficient in terms of reuse
than lipase immobilized in microemulsion-based
organogels,wheretheesterificationactivitydecreases
drastically within five cycles of reuse owing to accu-
mulation of water produced during ester synthesis
within the gel matrix (Dandavate & Madamwar
2007, 2008).
0.008
0.006
0.004
0.002
0
0
0.002 0.004 0.006 0.008 0.01
1/[pNPP] (mmol–1
0.012
)
Figure 8. Lineweaver–Burk plots for hydrolysis of pNPP by free
(A) and immobilized (B) lipase.
synthesis of ethyl isovalerate with a lower efficiency
than ethyl butyrate, whereas modifying CRL with
cationic surfactant treatment resulted in altered cat-
alytic efficiency with higher yields for ethyl isovaler-
ate than for ethyl butyrate (Thakar & Madamwar
2005; Dandavate & Madamwar 2007). In the pres-
ent study, the lipase immobilized on magnetite nano-
particles catalyzed ethyl isovalerate synthesis with a
maximum esterification efficiency of 55% after 196
h of incubation, which is five times higher than that
obtained with free lipase (Figure 10). Although the
esterification efficiency of lipase immobilized on
magnetite nanoparticles was lower than that observed
for surfactant-coated lipase immobilized in micro-
emulsion-based organogels reported by us earlier
(Dandavate & Madamwar 2007), the lipase immo-
bilized on magnetite nanoparticles would be more
suitable for commercial applications due to its easy
Table I. Esterification efficiency of free and immobilized lipase.
% Esterification
Ester
Free lipase
Immobilized lipase
Ethyl acetate
43.6 Ϯ 2.6
60.0 Ϯ 4.3
10.3 Ϯ 0.9
65.6 Ϯ 5.7
53.0 Ϯ 3.5
63.7 Ϯ 4.8
55.0 Ϯ 4.5
46.7 Ϯ 4.3
Ethyl butyrate
Ethyl isovalerate
Ethyl caprylate
The reactions were carried out under agitated conditions (150 rpm)
at 37°C in glass stoppered flasks.
Values after ‘Ϯ’ represent standard error from mean for %
esterification values calculated from results of three independent
sets of experiments.

44 V. Dandavate et al.
60
ferrous ions under hydrothermal conditions. C.
rugosa lipase was immobilized on the magnetite
nanoparticles by covalent attachment using carbodi-
imide activation, which was demonstrated by FTIR
and DSC. The kinetics of free and immobilized
enzyme suggests that the enzyme undergoes a con-
formational change during immobilization that
results in lower activation energy for hydrolysis of
pNPP.This was also reflected in an altered substrate
specificity of the immobilized lipase which exhibited
fivefold higher esterification activity for ethyl isoval-
erate synthesis in comparison to the free enzyme,
although there was no significant difference observed
for synthesis of ethyl butyrate and ethyl acetate.The
immobilized lipase exhibited fairly good reusability
property with a decrease in esterification efficiency
of about 8.5% over ten cycles of reuse. This study
demonstrates that lipase immobilized on magnetite
nanoparticles can be used for synthesis of esters.
50
40
30
20
10
0
0
50
100
Time (h)
150
200
250
Figure 10. Ethyl isovalerate synthesis catalyzed by free lipase (♦)
and magnetite-bound lipase ( ).
With lipase immobilized on magnetite nano-
particles the water produced during the esterification
reaction diffuses away from the enzyme into the bulk
solvent phase, from which the enzyme can be sepa-
rated using a magnetic field. Thus, the question of
accumulation of water in the vicinity of the immobi-
lized lipase does not arise, making it a more efficient
system for reuse. This also creates the possibility of
developing a continuous enzyme bioreactor for ester
synthesis, wherein lipase attached to magnetite par-
ticles is held on the surface of magnetic strips in a
reactor having inlet and outlet at opposite ends.The
solvent containing substrates can be made to flow at
a controlled rate from one end of the reactor, while
the solvent containing ester (product) and residual
reactants can be collected from the other end. How-
ever, before such a bioreactor can be designed, more
work is needed to search for a better lipase with
higher catalytic efficiency and stability.
Acknowledgements
The authors thank the Sophisticated Instrument
Centre for Applied Research and Testing, Vallabh
Vidyanagar, India, for material analysis.
Declaration of interest: The authors are grateful
to the University Grants Commission (UGC) for
providing financial support. The authors report no
conflicts of interest. The authors alone are respon-
sible for the content and writing of the paper.
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Conclusions
Magnetite nanoparticles of uniform size were syn-
thesized by chemical co-precipitation of ferric and
70
60
50
40
30
20
10
0
0
1
2
3
4
5
6
7
Number of cycles
8
9
10 11
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Supplementary material available online
SupplementaryTable 1.