CL-140228
Received: March 15, 2014 | Accepted: April 8, 2014 | Web Released: April 19, 2014
Immobilization of a Lipase on Mesocellular Foam of Silica for Biocatalysis
in Low-water-containing Organic Solvents
Kayambu Kannan,1 Joyeeta Mukherjee,2 and Munishwar N. Gupta*1
1Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi,
Hauz Khas, New Delhi, India 110016
2Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, India 110016
(E-mail: munishwar@chemistry.iitd.ac.in)
Unfunctionalized mesocellular foam of silica was used for
well-known applications in low water media. Lipases are known
to catalyze transesterification reactions in anhydrous organic
solvents.3 The synthesis of a flavor compound called hexyl
butyrate12,13 by a transesterification reaction was studied. Also
the synthesis of biodiesel from soybean oil and ethanol by
transesterification in a solvent-free medium was investigated.5
The superparamagnetic Fe3O4 nanoparticles were prepared
and coated with PEI by a method previously described by
Solanki and Gupta (2011).10 Siliceous MCF was synthesized
using the procedure reported by Kannan and Jasra (2011).7
The lipase from Thermomyces lanuginosus (TLL) was
immobilized on mesocellular foam (MCF) with polyethylene-
imine (PEI)-Fe3O4 nanocomposites by two methods. In one
method PEI-Fe3O4 nanoparticles (4 mg) were added to 50 mM
Tris-HCl (pH 7, 1 mL) buffer and dispersed by sonication (Elma
transonic digital ultrasonic unit, model T 490 DH, Germany) at a
fixed frequency of 40 kHz with 110 W power rating for 20 min.
To the above, 3.5 U TLL (50 ¯L) was added and kept overnight
at 4 °C with constant shaking at 200 rpm. After incubation, the
enzyme-adsorbed nanoparticles were separated by magnetic
separator. The excess of enzyme was removed by washing with
50 mM Tris-HCl (pH 7) buffer. The enzyme activity bound
to nanoparticles was calculated by determining the unbound
activity in the supernatant. To the above TLL-bound PEI-
Fe3O4 nanoparticles [called (PEI-Fe3O4)-TLL], 10 mg of MCF
suspended in 1 mL of buffer was added and kept at 4 °C with
constant shaking at 200 rpm for 2 h. Finally they were washed
with ethanol twice and have been designated as [(PEI-Fe3O4)-
TLL]-MCF throughout the work.
In another method, PEI-Fe3O4 nanoparticles (4 mg) were
added to 50 mM Tris-HCl (pH 7, 1 mL) buffer and dispersed
by sonication as mentioned above. To the above, MCF (10 mg
in 1 mL of the same buffer) was added and kept at 4 °C with
constant shaking at 200 rpm for 2 h [called MCF-(PEI-Fe3O4)].
The excess of PEI-Fe3O4 nanoparticles was removed by
washing with buffer. To this 3.5 U TLL (50 ¯L) in 1 mL of
buffer was added and kept overnight at 4 °C with constant
shaking at 200 rpm. This preparation has been called [MCF-
(PEI-Fe3O4)]-TLL throughout this work.
The hydrolytic activity of the lipase was monitored by
following the rate of hydrolysis of p-nitrophenyl palmitate at
410 nm.14 The transesterification reaction and biodiesel synthesis
catalysed by the lipase preparation was carried out as described
in the Supporting Information.15
immobilization of the lipase from Thermomyces lanuginosus. In
the first approach, lipase was adsorbed onto polyethyleneimine-
coated Fe3O4 nanoparticles, and these particles in turn were
bound to the foam interior to obtain the immobilized preparation
[(PEI-Fe3O4)-TLL]-MCF. In the second method, PEI-coated
Fe3O4 nanoparticles were bound to the foam, and the lipase was
bound to the composite to obtain [MCF-(PEI-Fe3O4)]-TLL as
the immobilized preparation. The second preparation was found
to be better in terms of surface area (397 m2 g¹1), pore diameter
(112 ¡), and total pore volume (2.5 cm3 g¹1). This preparation
also performed better for synthesis of hexyl butyrate and
biodiesel.
Enzyme catalysis in low-water-containing organic solvents
is used for many applications in organic synthesis, biotransfor-
mations, and kinetic resolution of racemic compounds.1-3
Lipases constitute the largest subclass of enzymes which have
been used for various applications in such media.3-5 Immobi-
lization on various supports has been extensively studied in this
regard.3 Mesoporous silica with its large surface, nonvulner-
ability to microbial degradation, robustness and environmental
acceptability has been especially considered an attractive choice
for use as a support for immobilization.6,7 Mesocellular foams
of silica constitute one such material in this category and have
several features useful for enzyme immobilization. The large
pores allow high enzyme loadings and larger accessibility to the
substrate. These foams have interconnected cages which allow
“ship-in-a-bottle” type entrapment of the enzyme.6 Both ad-
sorption and covalent attachment have been used for enzyme
immobilization in such foams. Generally, functionalization of
the foam has been required before significant immobilization
could be achieved.8,9
In the present work, we describe our efforts in immobiliza-
tion of a lipase on a mesocellular foam of silica in two novel
ways. In the first approach, the lipase was coated on magnetic
nanoparticles of Fe3O4 by a method described earlier with a
different lipase.10 This nanoconjugate was then in turn adsorbed
into the pores of the foam. The second approach involved first
adsorbing the Fe3O4 nanoparticles into the pores of the silica
foam. Subsequently, the lipase was adsorbed on these nano-
particles which were inside the silica foam pores. Both immobi-
lized preparations were found to be superparamagnetic. The use
of superparamagnetic support allows the recovery (and subse-
quent reuse) of the biocatalyst even from viscous reaction media.
A commercially available and relatively inexpensive prep-
aration of lipase from Thermomyces lanuginosus11 was used in
the present work. The immobilized lipase was evaluated for two
The two different approaches for the immobilization of
lipase on silica foam were evaluated in terms of the immobi-
lization efficiency as measured by the hydrolysis of p-nitro-
phenyl palmitate14 (Table 1). The lipase in the first approach was
bound to the PEI-coated Fe3O4 nanoparticles.10 The PEI-coated
© 2014 The Chemical Society of Japan
Kannan, Kayambu
