Self-assembled lipase nanosphere templated one-pot biogenic synthesis of silica hollow spheres in ionic liquid [Bmim][PF6]

Article abstract of DOI:10.1039/c5ra22543d

The spontaneous self-assembly of hydrophobic enzymatic protein triacylglycerol acylhydrolase (commonly known as lipase and a member of the serine hydrolase family) in hydrophobic 1-butyl-3-methylimidazolium hexafluorophosphate [Bmim][PF₆] and in hydrophilic 1-butyl-3-methylimidazolium tetrafluoroborate [Bmim][BF₄] ionic liquids resulted in the formation of lipase enzyme nanocapsules of different morphology. The lipase enzyme capsules were found to retain varying enzyme activity in both cases with both kinds of lipase capsules acting as self-catalyzing functional templates for the hydrolysis of silica precursors into silica. The presence of silica and its interaction with biomolecules was proved by X-ray Photoemission Spectroscopy (XPS). Interestingly, hollow silica spheres were obtained in the case of [Bmim][PF₆] ionic liquid, while solid silica spheres were obtained in the case of [Bmim][BF₄] ionic liquid for the same enzyme. The structural orientation of the enzyme within the capsules, their functional templating to obtain silica particles of varying morphology and finally their combined catalytic activity depend on the initial lipase-ionic liquid interaction. The enzyme activity of all these materials was evaluated against the esterification reaction between oleic acid (fatty acid) and butanol, i.e. biodiesel production. The relative enzyme activity was found to be 93.30% higher in the case of lipase nanocapsules synthesized in [Bmim][PF₆] and its in situ templating action to make hollow silica spheres further enhanced the residual activity. Furthermore time dependent kinetics of esterification by hollow silica spheres has also been shown here. Hollow silica spheres can also be used as a reusable catalyst for up to 6 cycles. This work demonstrates that the choice of ionic liquid is critical in controlling the self-assembly of enzymes as the ionic liquid-enzyme interaction plays a major role in retaining capsule activity and enzyme function.

Full text of DOI:10.1039/c5ra22543d

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PAPER
Self-assembled lipase nanosphere templated one-
pot biogenic synthesis of silica hollow spheres in
ionic liquid [Bmim][PF6]†
Cite this: RSC Adv., 2015, 5, 105800
c
a
Sampa Sarkar,a Kshudiram Mantri,ab Dinesh Kumar, Suresh K. Bhargava
*
a
*
and Sarvesh K. Soni
The spontaneous self-assembly of hydrophobic enzymatic protein triacylglycerol acylhydrolase (commonly
known as lipase and a member of the serine hydrolase family) in hydrophobic 1-butyl-3-methylimidazolium
hexafluorophosphate [Bmim][PF6] and in hydrophilic 1-butyl-3-methylimidazolium tetrafluoroborate
[Bmim][BF4] ionic liquids resulted in the formation of lipase enzyme nanocapsules of different
morphology. The lipase enzyme capsules were found to retain varying enzyme activity in both cases with
both kinds of lipase capsules acting as self-catalyzing functional templates for the hydrolysis of silica
precursors into silica. The presence of silica and its interaction with biomolecules was proved by X-ray
Photoemission Spectroscopy (XPS). Interestingly, hollow silica spheres were obtained in the case of
[Bmim][PF6] ionic liquid, while solid silica spheres were obtained in the case of [Bmim][BF4] ionic liquid
for the same enzyme. The structural orientation of the enzyme within the capsules, their functional
templating to obtain silica particles of varying morphology and finally their combined catalytic activity
depend on the initial lipase-ionic liquid interaction. The enzyme activity of all these materials was
evaluated against the esterification reaction between oleic acid (fatty acid) and butanol, i.e. biodiesel
production. The relative enzyme activity was found to be 93.30% higher in the case of lipase
nanocapsules synthesized in [Bmim][PF6] and its in situ templating action to make hollow silica spheres
further enhanced the residual activity. Furthermore time dependent kinetics of esterification by hollow
silica spheres has also been shown here. Hollow silica spheres can also be used as a reusable catalyst for
up to 6 cycles. This work demonstrates that the choice of ionic liquid is critical in controlling the self-
assembly of enzymes as the ionic liquid–enzyme interaction plays a major role in retaining capsule
activity and enzyme function.
Received 28th October 2015
Accepted 9th December 2015
DOI: 10.1039/c5ra22543d
www.rsc.org/advances
various supports and nanomaterials. There are various enzyme
immobilization techniques and these include adsorption,2,3
Introduction
cross linked enzyme aggregates, entrapment and encapsula-
tion,4 covalent attachment and addition of lipase during silica
condensation.5
Enzymes can be considered biocatalysts and have various
advantages in environmentally friendly processes due to their
ability to perform their action at near ambient temperature,
with high specicity and enabling otherwise non-attainable
reactions.1 There are various factors that restrict the routine
use of enzymes, such as instability under high temperatures,
extreme pH conditions, interaction with organic media and
their specic solubility.1 To overcome these problems, consid-
erable research has been carried out to immobilize enzymes on
Bio-molecular self-assembly is omnipresent in biological
systems and motivates the development of a range of complex
biological architectures which give rise to different metabolic
functions in biology. These self-assembled structures can be
reproduced under laboratory conditions and are of interest for
various applications in emerging areas of biomaterial sciences.6
Various stimuli such as changes in ionic strength, temperature,
pH and the addition of certain chemical entities have been used
to trigger self-assembly process of molecular building blocks
and especially protein7 and peptide amphiphile structures8
based on self-assembled structures are well-known exam-
ples.9–11 These studies prompted the use of peptide-based
surfactants and peptides with programmed sequences leading
to induce b-sheet or coil–coil interactions,12–14 which were
shown to undergo self-assembly in aqueous solutions.15 Instead
aCentre for Advanced Materials and Industrial Chemistry, RMIT University, 3001,
Australia. E-mail: sarveshkumar.soni@rmit.edu.au; sarvesh.soni@monash.edu
bReliance Industries Limited, Vadodara, India
cBanasthali University, Rajasthan, India
† Electronic supplementary information (ESI) available: TEM images of protease
template self-assembled hollow silica spheres, self-assembled lipase capsule at
lower concentration, illustration for esterication reaction and tables and gure
for time dependent kinetics of esterication reaction by hollow silica spheres.
See DOI: 10.1039/c5ra22543d
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of using peptides, protein design could be used to manipulate production.33,34 Lipases liberate glycerol and fatty acids aer
devices through a ‘bottom up’ approach whereas proteins/ acting on carboxyl ester bonds present in triacylglycerol-
biomacromolecules are used as monomeric building blocks typically under aqueous conditions. Long-chain triacylglycer-
for the fabrication of structures of higher order via self- ols are the natural substrates of lipases, which are much less
assembly.16
soluble in polar solvents like water; the catalysis generally
Most of the studies mentioned above have hitherto used occurs at the lipid–water interface. Lipases have the exclusive
aqueous solvents as the media to explore the self-assembly of ability to perform the reverse reaction, (under micro-aqueous
organic moieties. It is well known that bio-macromolecular self- conditions) leading to esterication and alcoholysis. There are
assembly processes are known to be affected by the surrounding a number of lipase immobilization methods that have been
continuous phase. In fact, there is a very high possibility that if reported, namely immobilized onto a macroporous acrylic
regularly used aqueous solvent phases are replaced with resin, covalent binding of lipase onto commercial Eupergit (R)
solvents of unique properties (such as ionic liquids-ILs) addi- supports and glyoxyl agarose beads.2,35 Other examples of lipase
tional advantages to control the self-assembly processes may be immobilization include adsorption onto silica nanoparticles36,37
attained rather than using the arduous synthetic method for cross-linking using glutaraldehyde38,39 and sol–gel entrap-
self-assembly process via programmed amino acid sequences.17 ment.40 Many of these methods, however, suffer from tedious
Surrounding media can denitely provide a simple approach for and expensive preparation methods, poor stability, or the use of
the self-assembly of bio-macromolecules without much change toxic or hazardous chemicals.
in their functionality.
In this article, we have reported the controlled self-assembly
Ionic liquids (ILs-commonly known as room temperature of a relatively hydrophobic enzymatic protein lipase (tri-
liquid salts) recently emerged as green solvents for the synthesis acylglycerol acylhydrolase) (E.C. 3.1.1.3) from Pseudomonas u-
of organic compounds and nano-biomaterials due to their orescens (a member of serine hydrolase family) in the ionic
unique physico-chemical properties.18 Recently, applications of liquid 1-butyl-3-methylimidazolium hexauorophosphate
these ionic liquids have started to be explored in the eld of ([Bmim][PF6]). Self-assembly of lipase enzyme in environmen-
bionanosciences, especially in areas such as enzyme stabiliza- tally friendly green solvent [Bmim][PF6]-‘hydrophobic in nature’
tion,19 protein crystallization,20 pGFP gene transformation,21 leads to the formation of lipase enzyme capsules further utilized
anti-cancer drug delivery,22 biocatalysis,23 enzyme-templated as functionally active templates for the in situ growth of silica
metal nanospheres24 and bio-fuel cells.25
resulting in hollow silica spheres. These synthesized silica
However, the stability of these structures remains a chal- hollow nanospheres were found to function as nanocontainers
lenge. Self-assembled nanostructures need to be supported by by immobilizing lipase in situ and retaining its enzymatic
inorganic materials that can provide the required stability activity for possible industrial applications. Most of the earlier
without compromising their activity. The rapid development in studies have shown the immobilisation of lipase on the already
the area of new advanced functional nano materials and synthesized silica or that the enzyme was added during the
organic–inorganic hybrid nano-scaffolds, plays an increasingly process of silica synthesis, however in our study we employed
signicant role in the self-assembly of biomacromolecular the principles of biomolecular (lipase) self-assembly in ionic
building blocks.26,27 Silica has been a technologically important liquid for its immobilisation in a single step (and one-pot)
inorganic material for wide range of commercial applications resulting into biogenic silica.
and various inorganic silica structures have been reported uti-
A plausible mechanism based on function activity of lipase
lising enzyme and self-assembled peptide bres14,28 as self- for esterication of oleic acid and butanol (as a model bio-
assembling template. The ability to tune hydrophilic enzyme catalytic reaction of commercial importance) and solid nano-
phytase29,30 to synthesize enzymatically functional (hollow structure obtained in [Bmim][BF4], a hydrophilic IL via self-
silica) and non-functional silica spheres (solid silica) by using assembly process has been proposed and hollow silica nano-
hydrophilic [Bmim][BF4] and hydrophobic [Bmim][PF6] ionic containers further tested for enzyme reusability for the afore-
liquid, respectively has been shown.19 It would be intriguing to mentioned reaction.
look at the self-assembling properties of a relatively hydro-
phobic enzyme in hydrophilic and hydrophobic media. Control
over nanoparticle shape can be achieved by tuning the template
property (in the earlier study the authors tuned the solvent)
Results and discussion
Lipase self-assembly in ionic liquid [Bmim][PF6]
while bio-molecular self-assembly in ionic liquid has not yet Lipase enzyme of bacteria Pseudomonas uorescens was extrac-
been shown elsewhere.
We have used lipase enzyme in this study as lipases are using deionized water as extraction media.
considered to be of relatively hydrophobic nature.31 Lipases
Lipase (20 mg mLꢀ1
was mixed with [Bmim][PF6]-
ted from the commercial lyophilized matrix by shaking for 2 h
)
have also been used as key enzymes in rapidly expanding hydrophobic IL, and kept for 24 h shaking. Transmission elec-
biotechnology/bioprocess industry because of their multifac- tron micrograph has been shown in Fig. 1A, interconnected
eted characteristics, which are used in various applications, lipase capsules were obtained aer controlled self-assembly of
such as pharmaceutical industry, bio-based surfactants in lipase enzyme in IL [Bmim][PF6].
detergents, food technology, biomedical sciences,32 and more
recently in the renewable energy industry in biodiesel lipase capsules and these retained their morphology. This
Sample preparation for TEM did not lead to disintegration of
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Fig. 1 Lower (A) and higher magnification (B) TEM images of self-
assembled lipase (20 mg mLꢀ1) capsules synthesized in ionic liquid
[Bmim][PF6].
might be interesting in the case of biomacromolecular self-
assembled so matters. The lipase enzyme capsules are
morphologically spherical, interconnected and apparently have
a smooth surface with an average diameter ranging from 100–
150 nm. Notably, due to the organic nature of enzyme, under
the high energy electron shower of transmission electron
microscope, these nanocapsules began to burst and/or burn
during focusing, which is shown as a TEM micrograph of higher
magnication in Fig. 1B, wherein self-assembled lipase nano-
capsules start fusing with each other and showing relatively big
interfused lipase hollow spheres in the centre, thus showing its
interior. The self-assembly of lipase enzyme during a control
experiment in water was not observed and gave a clear indica-
tion that ionic liquid [Bmim][PF6] promoted the self-assembly
process. A TEM image of a lipase capsules synthesized by
using 10 mg mLꢀ1 enzyme concentration has also been shown in
Fig. S1.† These capsules were of similar size (ꢁ100–150 nm) and
not much change in morphology was observed.
Fig. 2 TEM images of hollow silica nanoparticles synthesized in ionic
liquid [Bmim][PF6] using self-assembled lipase capsules as catalytic
templates. Lipase template capsules were self-assembled using 20 mg
mLꢀ1 lipase enzyme and inset shows the thickness of hollow silica
nanoparticles (ꢁ40 nm) at a higher magnification image of one of the
particles from the same sample.
Apparently, it is remarkable that the self-assembly of a rela-
tively hydrophobic enzyme lipase in a hydrophobic IL [Bmim]
[PF6] resulted in the synthesis of enzyme nanocapsules and the
aforementioned were utilized for the in situ synthesis of hollow
silica nanospheres with variable wall thickness.
Formation of hollow silica spheres without any external
hydrolyzing agent is quite notable and suggests the role of self-
assembled lipase capsule as a functional template for the
synthesis of hollow silica nanocapsules and simultaneously
enzymatically hydrolyzing the TEOS.
XPS analysis of lipase and silica hollow spheres
Synthesis of hollow silica spheres in ionic liquid [Bmim][PF6]
XPS analysis of self-assembled lipase in IL ([Bmim][PF6]) and
Lipase capsules obtained in [Bmim][PF6] were further used for lipase templated hollow silica nanostructures was further
synthesizing hollow silica nanoparticles and acted as templat- carried out to investigate the nature of interaction between the
ing nanoreactors. Hollow silica nanospheres were obtained by lipase-IL nanostructures and the hollow silica nanostructures.
adding tetraethyl orthosilicate (TEOS), a silica precursor to pre- Fig. 3 shows the F 1s, N 1s, C 1s, O 1s and Si 2p core-level spectra
assembled lipase capsules (using 20 mg mLꢀ1 lipase) in [Bmim] collected from the lipase-IL nanostructures and the lipase-IL
[PF6]. The TEM micrograph of hollow silica nanospheres nanostructures templated silica hollow nanospheres. The
synthesized in situ with [Bmim][PF6]-hydrophobic IL has been presence of F 1s component (BE 686.0 eV) observed both in the
illustrated in Fig. 2.
case of lipase-IL and lipase-IL–SiO2 nanostructures, clearly
The silica particles obtained in this process were found to reveals that these nanostructures consist of IL units and an
have a hollow interior. The diameter of these hollow silica integral part of the nal structures. The intensity of P 2p level
nanoparticles ranges from 150 to 200 nm with a spherical spectral component was too weak to be detected. Since the
morphology and rough surface. Thick-walled interconnected source of uorine comes only from IL, therefore the absence of
hollow silica nanospheres (wall thickness of ꢁ40 nm) without P 2p signal still indicates the incorporation of IL units within
much signicant difference in overall nanosphere size were the nanostructures. Another major evidence of the presence of
observed (Fig. 2 inset). When we checked the effect of higher IL units comes from the N 1s spectral data of lipase-IL and
lipase concentration up to 100 mg mLꢀ1, the hollow interior lipase-IL–SiO2 nanostructures. Lipase-IL nanostructures clearly
started to reduce in size and TEM imaging of these materials showed two distinct N 1s components and their binding ener-
appeared to be of solid spheres or hollow spheres with a very gies observed at 399.3 eV and 401.5 eV. The higher binding
small narrow interior having the diameter varying between 1–5 energy N 1s component (401.5 eV) corresponds to the imida-
nm (image not shown for brevity).
zolium part of IL and the lower binding energy N 1s component
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led to the major changes in N1s core-level spectra and their
binding energies. This was also supported from the C 1s core
level spectra, collected from lipase-IL nanostructures and
lipase-IL–SiO2 nanostructures. C 1s level that corresponds to the
carboxylic acid carbon (BE 287.8 eV) and the carbon attached
with the nitrogen (BE 286.2 eV) present in the lipase-IL nano-
structures showed change in their binding energy aer the
silica nanoparticles formation around the nanostructures. This
was probably due to the silica nanoparticles binding with the
imidazolium group of IL moieties and amine groups of lipase
enzyme, which resulted in liing the weak electrostatic inter-
action between the carboxylic acid groups of the enzyme with
the amine and imidazolium moieties. However, signicant
changes in the O 1s core-level spectra observed aer lipase-IL
nanostructures mediated the hydrolysis of TEOS to form silica
nanostructures, as shown in the O 1s core-level spectra are
shown in Fig. 3c. Since IL [BMIM][PF6] does not contain any
oxygen, the changes observed in the case of the O 1s core-level
spectra were a direct consequence of any modication that
occurred in the lipase-IL nanospheres and the presence of
silica. In the case of lipase-IL, O 1s component exhibit its BE at
531.9 eV, which was assigned to the carboxylic oxygen atoms
present in the amino acid residues of lipase enzyme. In lipase-
IL–SiO2, three chemically distinct components of O 1s were
observed and the binding energy of the lipase carboxylic oxygen
was shied to 532.3 eV. The additional two O 1s components
observed at BEs 530.9 and 533.1 eV, were attributed to the SiO2
and Si–OH components. Fig. 3e shows the Si 2p core level
spectra recorded from lipase-IL–silica nanostructures and the
prominent peak observed at BE 103.3 eV that corresponds to
silicon present in the SiO2 nanostructures. This is a clear indi-
cation of the presence of silica structures (as a result of TEOS
hydrolysis) around the lipase-IL nanostructures.
Plausible mechanism of synthesis of hollow silica spheres
Lipases are considered to be hydrophobic (due to their affinity
to hydrophobic surfaces: oil microcapsules41) in nature and
soluble in both hydrophilic and hydrophobic solvents while
also having large hydrophobic amino acids domains around the
active site.31 However lipases are rather diverse from other
hydrolytic enzymes because of their catalytic activity in non-
aqueous systems, relatively hydrophobic nature and interfa-
cial activation phenomenon.31 Lipase enzyme of bacteria Pseu-
domonas uorescens used in this study was obtained
commercially and this enzyme belongs to the serine hydrolases
family which is typically known to acts at the interface of lipid–
water or hydrophobic–hydrophilic boundary. The catalytic
active site domain comprises Ser-Asp/Glu-His and usually the
serine of the active site domain is also found to be surrounded
by a conserved sequence (Glyx-Ser-x-Gly). The a/b-hydrolase
fold, a typical characteristic of mostly lipases, is also revealed in
the three-dimensional structural maps.42 The role of catalyti-
cally active histidine in this sequence can be proposed as it is
known to hydrolyse the substrate in a two-step reaction.31 The
hydrolysis of TEOS, leading to hollow silica nanospheres can be
associated with such catalytically active domains. Lipase has
Fig. 3 XPS core-level spectra recorded from lipase and lipase–silica
after interaction with ionic liquid [Bmim][PF6]. (a) F 1s core-level
spectra. (b) N 1s core-level spectra. (c) C 1s core-level spectra. (d) O 1s
core-level spectra. (e) Si 2p from lipase-IL and lipase-IL–silica nano-
structures. The solid lines correspond to nonlinear least-squares fits to
the experimental data as shown by symbols.
originates from the amine group of amino acid residues from
the lipase enzyme. However, aer the silica structures forma-
tion, N 1s core level spectra was found to be different from the
lipase-IL nanostructures. The two distinct N 1s components
were merged into single component and the binding energy was
observed at 399.6 eV. There are many reports in the literature
that demonstrated that hydrolysis of TEOS to form silica can be
catalysed by amine groups of amino acids and imidazolium
groups of IL. Their role in TEOS hydrolysis and their electro-
static interaction with negatively charged silica nanostructures
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more hydrophobic domains even though it is amphiphilic in added, its hydrophobic nature tends to stay on the hydrophobic
nature.31 Therefore, dispersing them in a non-polar ionic liquid domains of the enzyme capsules, which also comprises the
like [Bmim][PF6] tends to drive the self-assembly of many active site domains for hydrolysis of TEOS.43
individual enzyme units by orienting their hydrophobic
For the enzyme encapsulation and formation of hollow silica
domains towards the [Bmim][PF6] continuous phase (Scheme nanospheres with a thin silica layer, the TEOS hydrolysis was
1). Since spherical structures are known to have lower surface initiated on the surface of a nanocapsule template made of self-
energy, self-assembly of lipase enzyme molecule in [Bmim][PF6] assembled lipase units that grew from the surface to inwards.
resulted in the formation of spherical structures as revealed Its TEM image has been illustrated in Fig. 2 and thick walled
from the TEM images (Fig. 1).
(ꢁ40 nm) hollow silica spheres (Fig. 2 inset) were obtained
In addition, it is also proposed that due to inter-molecular primarily due to the availability of active sites on the surface of
hydrogen bonding the amino acids of respective domains of the template; also more lipase lamellar layers formed in the
enzyme may also come in proximity with each other along with capsule so that TEOS hydrolysis was facilitated on the surface of
the hydrophobic domains of lipase, with hydrophobic tail the lipase nanocapsule. This also interestingly proposes that the
packing between them, nally leading to the formation of self-assembled lipase nanocapsules obtained in [Bmim][PF6]
a structure that looks more like a nanocapsule of lipase.
acted as a template for silica growth while concurrently enzy-
In hydrophobic IL [Bmim][PF6], this hierarchal self-assembly matically hydrolyzing the silica precursor.
of lipase should result in a hydrophilic core moving away from
[Bmim][PF6], and its hydrophobic groups aligning to [Bmim]
[PF6].
Catalytically active sites and surrounding amino acid
Synthesis of silica solid spheres in hydrophilic ionic liquid
[Bmim][BF4] and plausible mechanism
As per the above-mentioned mechanism, when lipase enzyme
was added to a IL [Bmim][BF4], hydrophilic in nature using
similar reaction conditions, it was anticipated that the hydro-
phobic lipase enzyme molecules would undergo a different kind
domains of lipase31 are considered hydrophobic in nature and
these domains of active site are most probably expected to be in
close vicinity with [Bmim][PF6]. When silica precursor (TEOS) is
Scheme 1 Schematic illustration for the structure–function relationship of self-assembled lipase enzyme in hydrophobic [Bmim][PF6] and
hydrophilic ionic liquids [Bmim][BF4]. Lipase enzyme structure from Pseudomonas (http://www.ebi.ac.uk/pdbe/entry/pdb/3a70) taken from
a protein data bank in Europe (with due permission) is shown here to represent enzyme molecules. Self-assembly using hydrophobic IL [Bmim]
[PF6] leads to lipase nanospheres with-active hydrophobic domains facing ionic liquid; while in hydrophilic [Bmim][BF4] catalytically-active
hydrophobic domains are sequestered towards the center. Addition of TEOS to the self-assembled lipase leads to the formation of a hollow silica
nanosphere in [Bmim][PF6], but results in a solid silica sphere in [Bmim][BF4]. An interesting correlation between the synthesized nanostructures
and relative enzyme activity was also observed.
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of structural packing during the self-assembly process within
the lipase capsules, which differs from the structural packing
that was observed in the case of lipase capsules formed in
hydrophobic IL.
Therefore, in the lipase nanocapsules self-assembled in IL
[Bmim][BF4], the hydrophilic regions of lipase enzyme should
position it orienting outwards (towards the hydrophilic
continuous phase of IL [Bmim][BF4]), while the lipase enzyme's
hydrophobic domains should be packed inside away from
[Bmim][BF4]. Since catalytically active sites of lipase are present
in the hydrophobic region of lipase (which are sequestered
towards the centre in a hydrophilic ionic liquid), as shown in
Scheme 1, hydrolysis of silica on self-assembled lipase
templates in IL [Bmim][BF4] should happen in an inward to
outward manner. Since TEOS is also hydrophobic in nature,
TEOS tends to infuse within the hydrophobic domains of the
lipase capsules, thus staying on the surface.
Transmission electron micrograph of lipase enzyme and
silica nanostructures obtained in IL [Bmim][BF4]-hydrophilic
aer lipase self-assembly and hydrolysis of TEOS is shown in
Fig. 4 and 5, respectively. Solid silica nanoparticles of broad size
distribution (diameter 100–250 nm) and quasi-spherical in
shape were obtained. These are quite different in comparison
with the above-mentioned hollow silica particles obtained in
[Bmim][PF6]. Fig. 5B inset shows the TEM image for silica solid
spheres in higher magnication, showing a dense core while
the intensity towards the periphery fades away. This TEM
micrograph suggested that both the TEOS hydrolysis and
nucleation of silica start from the hydrophobic protein domain
that has been sequestered towards the centre as a dense core
was observed in solid silica nanoparticles while using [Bmin]
[BF4]. Interestingly, the same enzymatic protein lipase results in
hollow silica nanospheres when in hydrophobic IL [Bmim][PF6]
and in solid silica particles when in hydrophilic IL [Bmim][BF4].
We envisage that the self-assembly of enzymes may have led
to the formation of superstructures involving structural changes
on each enzyme unit in the superstructure as directed by the
polarity of the ionic liquid. The relatively hydrophobic lipase
undergoes self-assembly to form capsules in two ionic liquids,
which differ only in their polarity as a result of their counter
anions. However, the function of the enzyme capsules was
found to be different because of the structural reorganization of
Fig. 5 Lower (A) and higher magnification (B) TEM images of solid
silica nanoparticles synthesized in ionic liquid [Bmim][BF4] using 20 mg
mLꢀ1 lipase enzyme.
each enzyme unit within the capsule. As a result, hydrolysis of
TEOS was found to occur at catalytic sites in different regions of
the enzyme capsules. There are no reports in the literature that
show the synthesis of silica hollow and solid nanoparticles by
using a hydrophobic enzyme. In summary, a commonly known
hydrophobic enzyme lipase in a hydrophobic ionic liquid
[Bmim][PF6] results in a hollow self-assembled functional
template that hydrolyses the silica precursor and results in
functional hollow silica nanoparticles. The same hydrophobic
enzyme in hydrophilic ionic liquid [Bmim][BF4] results in solid
silica nanospheres. This strongly suggests a complementary
advantage over earlier approaches which require the modica-
tion of bio-macromolecules before their self-assembly can be
performed.44 As a result, the nal structures of silica enzyme
capsules were found to be different. Eventually, the structure
and function of these capsules with and without silica particles
was reected in the natural enzymatic activity of the lipase.
Self-assembly of protease enzyme and synthesis of hollow
silica spheres in [Bmim][BF4]
Self-assembly of hydrophilic protease (papain) enzyme in
hydrophilic ionic liquid [Bmim][BF4] leads to the synthesis of
hollow silica spheres containing enzymes (ESI Fig. S2†). This
can be a versatile approach tosynthesize and immobilize
enzyme into biogenic silica nanocapsules utilizing ionic
liquids, in a single step.
Structure–activity relationship of lipase encapsulated hollow
and solid silica spheres for esterication reaction for biodiesel
production
In this work we report the results on the esterication of oleic
acid with n-butanol (Fig. S3†) using immobilized lipase as bio-
catalyst (prepared by single step self-assembly process) which
leads to enantiomeric esters (biodiesel)34 with >98% of
selectivity.
Gas Chromatography (GC) was used as the method to
quantify esters of fatty acids produced and residual fatty acid i.e.
oleic acid. Fig. 6 represents the comparative activities of the
lipase enzyme in its different environments for the esterica-
tion of oleic acid with n-butanol. Lipase in butanol is much
active than in water due to its better solubility in butanol. A
Fig. 4 Lower (A) and higher magnification (B) TEM images of self-
assembled lipase (20 mg mLꢀ1) structures obtained in ionic liquid
[Bmim][BF4].
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compounds and potential drug intermediates.31 As there was in
situ growth of silica nanospheres in ILs, in which self-
assembled lipase capsules were acting as templates, there is
a very high possibility of nding embedded enzyme molecules
in both hollow and solid silica nanostructures during their
synthesis mediated by lipase enzyme (in [Bmim][PF6] and
[Bmim][BF4], respectively). Hence, enzyme reusability studies
were performed with hollow and solid silica nanocontainers.
Hollow silica nanospheres synthesized in [Bmim][PF6] signi-
cantly showed bio-catalytic conversion of oleic acid and 1-
butanol by a well-known esterication reaction and retained its
88% activity at-least up to six cycles of enzyme reusability
(Fig. 7).
Aer the rst cycle solid silica spheres retained only 45% of
their initial activity (Fig. 7) and hence the enzymatic activity of
these particles was not tested further. It is particularly inter-
esting to know the retention of lipase enzyme activity onto
hollow silica spheres as the activity of the native enzyme
generally highly sensitive to environmental factors such as
solvent system, temperature and pH, variation in any one of
these factors can lead to changes to the enzyme's native struc-
ture leading to loss of enzymatic activity. Time dependent
kinetic studies have also been performed (ESI Fig. S4†) with
hollow silica spheres. The conversion of oleic acid was checked
every 15 minutes and tested for 2 hours. The maximum
conversion in 2 h was 85% (ESI Table S2†), that normalised to
100% and the subsequent conversions were plotted for the
different time intervals. It has been demonstrated that the
choice of ionic liquids is critical in controlling the self-assembly
of enzymes due to their specic interaction with the enzymes,
its major role in retaining the function of the enzyme and the
activity of the capsules.
Fig. 6 Comparative lipase enzyme activity for the esterification of
oleic acid with n-butanol (in terms of conversion of oleic acid). 1.
Lipase dissolved in de-ionized water, (2) lipase dissolved in butanol, (3)
self-assembled lipase in [Bmim][BF4], (4) self-assembled lipase
capsules synthesized in [Bmim][PF6], (5) lipase encapsulated solid silica
nano spheres synthesized in [Bmim][BF4], (6) lipase encapsulated in
hollow silica nanocapsules synthesized in [Bmim][PF6].
cartoon illustrating the structure and activity relationship of
native enzyme, self-assembled lipase enzyme capsules, silica
nanospheres (hollow-synthesized in IL [Bmim][PF6] and solid-
synthesized in IL [Bmim][BF4]) and residual enzymatic activity
(tested for esterication reaction) for all the samples is eluci-
dated in Scheme 1. Lipase-encapsulated solid silica nano-
spheres synthesized in hydrophobic IL were found relatively
55% less active than hollow silica nanospheres formed in
hydrophilic IL (conversion of oleic acid to ester by hollow silica
spheres was considered 100%).
The highly porous walls of hollow silica nanospheres facili-
tate the dispersion of lipase on support and substrate and
therefore products are diffused during enzymatic reaction via
these pores. This might lead to the possibility of in situ TEOS
hydrolysis by the lipase capsules formed in hydrophobic
[Bmim][PF6], once enhanced enzymatic activity was expressed
in hollow silica capsules (enzyme–silica nanobio-hybrid)
formed in [Bmim][PF6] in comparison to solid silica nano-
structures synthesized in [Bmim][BF4] (Fig. 6 and Table S1†).
Scheme 1 represents these results in an illustrative way for
better clarity of the structure–function relationship of lipase
hollow and silica nanospheres' enzymatic activity.
Experimental
Materials
Commercial Amano Lipase AK of Pseudomonas uorescens and
tetraethyl orthosilicate (TEOS) were procured from Sigma-
Reusable biocatalysts and peptide loaded nano-vehicles45
have attracted a lot of attention due to their widespread appli-
cations in therapeutics with emphasis on the immobilization of
commercially and technologically signicant biomolecules onto
robust supports as an area for wide-ranging research and
development. However, the main challenge linked with enzyme/
protein immobilization strategies is to preserve the native
enzyme activity onto a reusable substrate without any leakage or
denaturation of the enzyme. One way to address these issues
and to preserve enzyme activity is to use ILs with unique solvent
properties.46 The lipase enzyme employed in this particular
study are known for their signicant industrial and biomed-
ical31 value in the synthesis of enantiomeric therapeutic
Fig. 7 Reusability study of lipase enzyme encapsulated within hollow
silica nanospheres during their synthesis in ionic liquid [Bmim][PF6] for
esterification of oleic acid with 1-butanol.
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Aldrich. Ionic liquids (ILs) 1-butyl-3-methylimidazolium tetra- with 20 mg mLꢀ1 concentration). The 1 mL reaction contents
uoroborate ([Bmim][BF4]) and 1-butyl-3-methylimidazolium were incubated at 37 ꢄ 0.1 ꢃC for 24 h under stirring conditions,
hexauorophosphate ([Bmim][PF6]) were purchased from during which all the reactions involving lipase became turbid,
Ionic Liquid Technologies (IoLiTec). All chemicals were used as indicating TEOS hydrolysis. Aer 24 h of reaction, samples were
received, unless specied.
centrifuged at 8000 rpm followed by washing with deionized
water and acetonitrile to remove the viscous ionic liquid. Silica
nanostructures thus obtained were further analyzed by TEM
and used for enzyme reusability studies. In a control experi-
ment, 10 mL of deionized water were added to 500 mL of TEOS
stock solution and 490 mL IL, and the reaction was pursued
along with the main experiment. However, no turbidity was
observed in the control reaction containing water, thereby
negating the possibility of water-mediated hydrolysis of TEOS in
IL [Bmim][PF6] and [Bmim][BF4] and suggesting the role of
lipase towards TEOS hydrolysis in ILs. Washing the reaction
products with deionized water at 37 ꢃC (instead of washing with
acetonitrile) did not show any signicant difference in the silica
nanoparticles formed, however samples not washed with
acetonitrile were difficult to image in TEM due to the residual
presence of ILs in those samples. The TEM images shown in
this article correspond to nanoparticles that were washed with
acetonitrile.
Lipase extraction
Lipase was extracted from the dried commercial powder. 10 mg
of lyophilized lipase on inert matrices was suspended in 1 mL of
deionized water and kept shaking at room temperature for 4 h.
The dissolved lipase was recovered by centrifugation at 14 000
rpm for 10 minutes in supernatant. The extracted protein
concentration was checked by Bradford method47 and diluted
accordingly with deionized water to make nal a concentration
of 10 mg mLꢀ1
.
Material characterisation
TEM studies were made on a JEOL JEM-2100F instrument
equipped with a slow-scan CCD camera and an accelerating
voltage of the electron beam (200 kV). The preparation of TEM
samples involved sonication in isopropanol for 2–5 min, fol-
lowed by deposition of a drop on a copper grid supporting
a perforated carbon lm and allowed to dry. The nature of the
material synthesized (lipase capsules and hollow silica nano-
spheres) in ionic liquid [Bmim][PF6] characterized by XPS and
measurements was carried out on a Thermo K Alpha XPS
instrument at a pressure greater than 1 ꢂ 10ꢀ9 Torr (1 Torr ¼
1.333 ꢂ 102 Pa). The general scan and N 1s, P 2p, O 1s, and C 1s
core-level spectra of hollow silica spheres, as well as core-level
spectra from the respective samples were recorded with mon-
ochromated aluminum Ka radiation (photon energy ¼ 1486.6
eV) at a pass energy of 20 eV and an electron take off angle
(angle between the electron emission direction and surface
plane) of 90ꢃ. The core-level binding energies (BEs) were aligned
with the adventitious carbon binding energy of 285 eV.
Esterication of oleic acid with 1-butanol and reusability of
bio-catalyst. The preparation contained only one kind of lipase
and no other enzymes which had esterifying activity. To deter-
mine the enzymatic stability of lipase in hollow silica nano-
spheres aer encapsulation, hollow silica nanoparticles
synthesized using 80 mg mLꢀ1 lipase in IL [Bmim][PF6] were
dispersed in n-butanol.
The reaction mixture (3.6 mM oleic acid with 36 mmol n-
ꢃ
butanol) was then incubated at 50 C for 2 h at 300 rpm, fol-
lowed by centrifugation to obtain a clear supernatant and
nanoparticles pellet. The conversion of oleic acid in to ester due
to enzyme activity was determined in supernatant by gas chro-
matography as mentioned.48 The products of the reaction were
analysed directly by injection of a 0.2 mL sample into the GC.
The products were analyzed by Shimadzu Gas Chromatograph
14A, Ultra-1 (25 m ꢂ 0.3 mm capillary column) equipped with
Ionic liquid-mediated synthesis
Lipase nanocapsules. A typical synthesis reaction of lipase FID and helium as a carrier gas. The major product was n-butyl
nanocapsules in ILs was performed in 0.5 mL volume contain- oleate which was compared with the standard. Conversion and
ing 490 mL of the respective IL [Bmim][PF6] and 10 mL of puried selectivity were calculated using following formula.
lipase enzyme (1 mg mLꢀ1 dissolved in water), thus achieving
Conversion of oleic acid ð%Þ ¼
a nal enzyme concentration of 20 mg mLꢀ1 in the reaction. The
IL-lipase mixture was incubated at 37 ꢄ 0.1 ꢃC for 24 h with
gentle reciprocal shaking, aer which samples were centrifuged
at 14, 000 rpm, followed by washing with deionized water and
acetonitrile to remove the viscous IL. Lipase nanocapsules thus
obtained were further analysed by TEM and XPS.
Hollow and solid silica nanospheres. To obtain silica nano-
spheres, 20 mM tetraethyl orthosilicate (TEOS) stock solution
was prepared in the respective ILs viscous [Bmim][PF6] and
[Bmim][BF4]. 500 mL of TEOS stock solution (20 mM) in ILs was
added to 500 mL of the aforementioned reaction mixture con-
taining IL and lipase capsules. The nal concentration of lipase
became 10 mg mLꢀ1 and TEM image of lipase capsules (10 mg
mLꢀ1) has been shown in Fig. S1 under ESI.† These capsules
looked similar to the lipase capsules shown in Fig. 1 (obtained
moles of oleic acid consumed
moles of oleic acid initially added
ꢂ 100
Selectivity of n-butyl oleate ð%Þ ¼
n-butyl oleate produced
ꢂ 100
moles of all products
A time-dependent kinetics study was also performed with the
interval of 15 minutes. To check the recyclability of the immo-
bilized enzyme, the nanoparticle pellet obtained aer centri-
fugation (containing hollow silica nanocontainers with
encapsulated enzyme) was washed twice with 1-butanol and
fresh reactants were added for the next reaction cycle, as in the
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rst cycle. The experiment was continued for 6 cycles and the
enzymatic activity obtained in rst cycle was considered as
100% for comparison with subsequent cycles. Experiments were
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In summary, we have shown that ionic liquids can be utilized as
non-aqueous self-assembling media for hydrophilic as well as
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S. K. S. and SS thanks Australian Government, Department of
Education for an Endeavour Research Award and School of
Applied Sciences, RMIT University for an ECR-start up grant to
SKS. The authors duly acknowledge the RMIT Microscopy and
Microanalysis Facility (RMMF) for providing access to their
instruments used in this study. The authors would also like to
thank the protein data bank in Europe49 for granting permis-
sion to use the Pseudomonas sp. MIS38 lipase50 enzyme that was
used to represent lipase enzyme molecules in Scheme 1.
Authors are also very grateful to Ms. Ana Maria Martins for
proofreading and correcting grammatical errors in this article.
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