Lipase-embedded silica nanoparticles with oil-filled core-shell structure: Stable and recyclable platforms for biocatalysts

Article abstract of DOI:10.1039/c2cc17896f

Lipase enzyme was embedded within silica nanoparticles with oil-filled core-shell structure. The enzyme embedded within such architecture retained all of its activity and showed high catalytic performance both in water and in organic media with optimal stability and recyclability. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc17896f

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COMMUNICATION
Lipase-embedded silica nanoparticles with oil-filled core–shell structure:
stable and recyclable platforms for biocatalystsw
Yasutaka Kuwahara,a Takato Yamanishi,a Takashi Kamegawa,a Kohsuke Mori,a Michel Cheb
and Hiromi Yamashita*a
Received 18th December 2011, Accepted 27th January 2012
DOI: 10.1039/c2cc17896f
Lipase enzyme was embedded within silica nanoparticles with
oil-filled core–shell structure. The enzyme embedded within such
architecture retained all of its activity and showed high catalytic
performance both in water and in organic media with optimal
stability and recyclability.
increasing the stability and recyclability: enzymes are directly
entrapped within spherical silica nanoparticles having oil-filled
core and oil-induced mesoporous silica shell. The advantages
of this synthetic route are the ease of fabrication, ambient
synthetic conditions and unique architecture including oil in its
core. We selected Candida Antarctica Lipase A (CALA) for this
work because it has been widely investigated as a promising
biocatalyst for transesterification reaction involved in biodiesel
production from vegetable oils.14 The CALA-embedded oil-filled
silica nanoparticles (CALA@OSN) were synthesized by an
oil-in-water emulsion templating method,15 which was carried
out by adding enzymes (30 mg per g SiO2) into the emulsion
consisting of oleic acid (OA) and water (pH 5.3). After
addition of silicon sources (TEOS and APTES), the resulting
solution was aged for 24 h at 50 1C, during which the pH value
was 9.6 at the maximum and finally approached the neutral
value of 7.3 (Fig. S1 in ESIw).
Biocatalysts are highly selective entities, but by nature show
limited stability and recyclability. Robust immobilization that
preserves the activity of biocatalysts has been a technique of
great importance for a range of applications in biotechnology
including food processing, enzymatic catalysis, drug-delivery
systems and biosensors.1 A common strategy for immobilizing
enzymes is their encapsulation inside sol–gel derived silica
matrices, which function to efficiently protect the encapsulated
enzymes from the surrounding environment.2–6 However, due
to nonporous structure, most studies showed lower specific
activity than that of the free enzymes. Silica hollow spheres have
been also envisaged as promising carriers for biomolecules because
the void volume can be used for encapsulation of various guests
and the silica shell acts as a physical barrier to protect them from
the surrounding environment.7 However, the synthetic methodo-
logy requires intricate multiple preparation steps and careful
control of the synthetic conditions to circumvent the denaturation
of enzymes. Although chemical/physical fixation within the pore
channels of functionalized mesoporous silica8–12 or onto the void
cavities of silica-based macrocellular foam13 has been widely used
because of their rigid and uniform open-pore structure, the
complicated synthetic procedures and significant leaching during
their use limit their applicability. A worthwhile challenge to this
issue is to develop a facile and scalable immobilization approach
while preserving catalytic performance of enzymes.
Fig. 1 summarizes the results of structural analyses of
CALA@OSN. The SEM image of CALA@OSN shows
monodispersed spherical silica nanoparticles with an average
particle size of 246 nm (Fig. 1(b)). XRD and N2 adsorption
measurements identified nonporous structure of as-synthesized
CALA@OSN, but confirmed OA-induced mesoporous structure
of the calcined sample. The calcined sample exhibits a broad
In this communication, we present a novel protocol for
immobilizing enzymes retaining all of their activity and
a Division of Materials and Manufacturing Science, Graduate School
of Engineering, Osaka University, 2-1 Yamada-oka, Suita,
Osaka, Japan. E-mail: yamashita@mat.eng.osaka-u.ac.jp;
Fax: +81 6-6879-7457; Tel: +81 6-6879-7457
b Institut Universitaire de France and Laboratoire de Re´activite´ de
´
Surface, Universite Pierre et Marie Curie-Paris 6, France.
E-mail: michel.che@upmc.fr
w Electronic supplementary information (ESI) available: Detailed
information on the material synthesis, XRD and sorption data for
CALA@OSN and CALA@MCM-41 and activity characterization
results. See DOI: 10.1039/c2cc17896f
Fig. 1 (a) Illustration of structure, (b) SEM micrograph (inset shows
a particle size distribution diagram) and (c) TEM micrograph of
CALA@OSN.
c
2882 Chem. Commun., 2012, 48, 2882–2884
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diffraction peak associated with 1-D wormhole-like mesopores
at 1.231 and a distinct pore distribution with an average pore
diameter of 4.2 nm (Fig. S2 in ESIw). The specific surface area
and total pore volume were determined to be 26.2 m2 gÀ1 and
0.284 cm3 gÀ1 for an as-synthesized sample and 325 m2 gÀ1 and
0.472 cm3 gÀ1 for a calcined sample. While such meso-porosity in
the silica shell is not observed in the TEM image because of
disordered arrangement of the pores (Fig. 1(c)), these observations
evidence that the CALA@OSN consists of the core of OA and the
mesoporous silica shell filled with OA as illustrated in Fig. 1(a).
In order to disclose the whereabouts of the incorporated
enzymes, bovine serum albumin–fluorescein isothiocyanate conju-
gate (BSAf), whose molecular dimensions (7.0 Â 7.0 Â 5.0 nm)8a–c
are close to those of CALA (6.3 Â 5.6 Â 4.2 nm),16 was used as a
guest molecule instead of CALA. The suspension of BSAf-
embedded silica nanoparticles is separated into a yellow precipitate
and a clear supernatant by centrifugation (Fig. 2(a)). The remain-
ing enzyme amount in the supernatant and wash fractions was
negligible, suggesting that most of the soluble biomolecules were
incorporated within the composite materials. Furthermore,
the fluorescence micrograph unambiguously confirms that
the biomolecules are mostly sequestered within the silica shell,
not in the oil phase (Fig. 2(b)). These results indicate that our
enzyme immobilization protocol spontaneously leads to the
incorporation of the enzymes within the silica matrix of the
shell during silica network formation.
on MCM-41 (CALA@MCM-41: for N2 adsorption data, see
Fig. S3 and Table S1 in ESIw). As shown in Fig. 3(A),
CALA@OSN exhibits as high a reaction rate as that of free
enzyme, indicating negligible loss of enzymatic activity upon
immobilization. Furthermore, the reaction was quenched after
recovery of the silica particles from the reaction media by
simple filtration, whereas free enzyme is unrecoverable due to
its homogeneity. More significantly, the catalytic efficiency is
at least up to 10 times reproduced for CALA@OSN while
retaining more than 97% of the initial activity in each cycle
(Fig. 3(B)), demonstrating its excellent recyclability. The retention
of enzymatic activity and fine reproducibility can be attributed to
the mild synthetic conditions (ambient temperature and narrow
pH range) and the robust immobilization ability of the silica
matrix without affecting the active sites and disruption of subunits,
respectively. In comparison, CALA@MCM-41 obtained by
conventional wet-impregnation showed a quite poor reaction
rate (16.4% conversion after 6 h) and significant decrease in
enzymatic activity during repeated use due to enzyme denatura-
tion and substantial leaching to the reaction medium. One of
the critical disadvantages of the conventional immobilization
on silicate materials is the low catalytic efficiency of enzymes
mainly derived from nonporosity of the silica-gel or close
packing within the mesopore channels, thereby resulting in a
lower activity than that of free enzymes. Contrarily to these
approaches, this synthetic route enables us to achieve as high a
catalytic efficiency as that of free enzyme. This is presumably
because of high accessibility between enzymes and reactants; the
thickness of the silica shell determined from the TEM image is
ca. 10 nm (Fig. 1(c)), which is slightly larger than the molecular
dimensions of CALA, indicating that CALA molecules are
being embedded in the vicinity of the outer surface. It is
deduced that such a configuration allows reactants to efficiently
access to the active sites of enzymes.
We examined the activity of CALA@OSN by hydrolysis of
n-butylacetate in phosphorous buffer (pH 7.0) as a test reaction
and compared with that of free enzyme and CALA immobilized
It is worth mentioning that CALA@OSN can be used as an
efficient heterogeneous biocatalyst in the hydrolysis and trans-
esterification reactions of triglycerides using n-heptane as an
organic solvent owing to its amphiphilicity. As shown in Fig. 4,
CALA@OSN retains most of its activity in multiple catalytic
cycles in both catalytic reactions, whereas CALA@MCM-41
loses its whole activity after a few cycles (for detailed catalytic
results, see Fig. S4 and S5 and Table S1 in ESIw), suggesting
that triglycerides in the organic medium have easy access to the
active sites of the embedded CALA. It is believed that the
OA-induced organophilicity of OSN increases the affinity with
the organic media and leads to a prominent catalytic activity,17
because enzyme itself is insoluble in the organic media. The
attainment of high catalytic performance in organic media
using enzyme-based heterogeneous biocatalysts is currently
the research area of great industrial importance, because most
of pharmaceutical or biofuel end products are insoluble in
water.13 The above results, therefore, suggest the availability
of this material to be used as an industrially practicable
heterogeneous biocatalyst.
Fig. 2 (a-1) An aqueous solution of bovine serum albumin–fluorescein
isothiocyanate conjugate (BSAf) and suspensions of BSAf@OSN (a-2)
before and (a-3) after centrifugation. (b-1) Fluorescence microscopy
image and (b-2) optical microscopy image of BSAf@OSN with huge
particle size taken using an Olympus confocal system.
Fig. 3 (A) Hydrolysis of n-butylacetate in Na-phosphate buffer (pH 7.0)
catalyzed by (a) free CALA, (b) CALA@OSN, (c) after removal of
CALA@OSN by filtration of the reaction mixture (the reaction had
previously been allowed to proceed for 1 h) and (d) CALA@MCM-41.
(B) Reproducibility test. Reaction conditions: catalyst including 1.0 mg of
CALA, n-butylacetate aqueous solution (50 mM, 1 mL), Na-phosphate
buffer (50 mM, 9 mL), 40 1C.
To evaluate the suitability of OSN as stable platforms for
enzymes, we also examined the enzymatic activity as a function of
solution pH and temperature. As seen in Fig. 5, the CALA@OSN
exhibits a higher enzymatic activity than that of free enzyme over a
wide pH range. The free enzyme in solution is readily denatured
c
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demonstrated that the enzyme embedded within such architecture
retained all of its activity and showed high catalytic performance
both in water and in organic media with increased stability and
recyclability. The enzyme immobilization protocol presented in
this study provides several advantages: mild synthetic conditions
that preserve the integrity of enzymes, low cost and convenience
in preparation, efficient recovery and recyclability of the
catalysts, amphiphilicity induced by oil-filled core–shell structure
and versatility in choice of enzymes. With those favourable
characteristics, this method will be a promising approach for
synthesizing highly stable and readily recoverable biocatalysts.
This work was financially supported by the Grant-in-Aid
for Scientific Research (KAKENHI) from the Ministry of
Education, Culture, Sports, Science and Technology of Japan
(No. A233603560). The authors are grateful to Dr Eiji Taguchi
and Prof. Hirotaro Mori at the Re-search Center for Ultra-High
Voltage Electron Microscopy, Osaka University, for assistance
with TEM measurements. Y. K. thanks the JSPS research
Fellowships for Young Scientists.
Notes and references
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Fig. 4 Comparison of recyclability of (K) CALA@OSN and (’)
CALA@MCM-41 in (A) hydrolysis of tricaprylin (C27H50O6) in
water-saturated n-heptane and (B) transesterification of tricaprylin
with ethanol in n-heptane. Reaction conditions: catalyst containing
4.0 mg of CALA, tricaprylin (10 mmol), either water-saturated
n-heptane or 120 mmol of ethanol-containing n-heptane (10 mL), 40 1C.
during the reaction at 60–80 1C (51–83% decrease in relative
activity). In contrast, CALA@OSN retains most of its activity
under the same conditions, proving enhanced thermostability
for this composite material. This is attributed to the stabilizing
effect of the silica matrix, which prevents extensive conformational
changes typical of thermal denaturation.18 The ability to retain
enzyme activity in wide pH ranges and at high temperatures
provides a number of processing advantages such as improved
reaction rate and substrate solubility, thereby expanding the range
of applications of enzymes.
In summary, we fabricated a new type of heterogeneous
biocatalyst by integrating multi-components, i.e., enzymes, silica
shell and oil core, into isolated spherical silica nanoparticles, and
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Fig. 5 Enzymatic activity of (K) CALA@OSN and (m) native CALA
at different (A) pH and (B) temperature. Relative activity was assessed
by hydrolysis of n-butylacetate, which was performed in the mixture of
1 mL of n-butylacetate aqueous solution (50 mM), 9 mL of 50 mM
buffer solution and the catalyst including 1.0 mg of CALA.
The activities were normalized by that of free CALA at pH 7.0 and
at 40 1C.
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77–90% of relative activity was observed at 80 1C.
c
2884 Chem. Commun., 2012, 48, 2882–2884
This journal is The Royal Society of Chemistry 2012