Encapsulation of enzyme in large mesoporous material with small mesoporous windows

Article abstract of DOI:10.1039/c2cc33592a

Trypsin has been encapsulated in the mesopores of a hierarchical mesoporous silica material synthesized via Cu(i) catalyzed azide-alkyne click reaction between azide functionalized large spherical SBA-15 particles and alkyne functionalized mesoporous sili

Full text of DOI:10.1039/c2cc33592a

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COMMUNICATION
Encapsulation of enzyme in large mesoporous material with small
mesoporous windowsw
Bharmana Malvi and Sayam Sen Gupta*
Received 18th May 2012, Accepted 11th June 2012
DOI: 10.1039/c2cc33592a
Trypsin has been encapsulated in the mesopores of a hierarchical
mesoporous silica material synthesized via Cu(I) catalyzed
azide-alkyne click reaction between azide functionalized large
spherical SBA-15 particles and alkyne functionalized mesoporous
silica nanoparticles (MSNs). Encapsulated trypsin functions as an
efficient biocatalyst and can be recycled several times.
Recently, Bein et al. have attached alkyne labeled trypsin
covalently on the surface of azide functionalized SBA-15
by Cu(I) catalyzed azide alkyne click chemistry (CuAAC)
approach.5b However, after covalent immobilization there is
limited control of the spatial orientation of the immobilized
enzyme within the pores which in turn affects the reactivity.
Another approach to prevent enzyme leaching is via a
‘‘ship-in-a-bottle’’ approach where the pores of the mesopore
are partially closed after adsorption of the enzyme; thereby
preventing the enzyme from leaching out while the diffusion of
the substrate and product remains unaffected.5c,d For example,
Wang and Caruso have encapsulated enzymes in bimodal
mesoporous silica (BMS) by enzyme adsorption followed by
assembly of a multilayer shell on the particle surface by
layer–by-layer electrostatic assembly of oppositely charged
species to prevent the enzyme from leaching.5e However with
changes in pH or salt concentration, these multilayer shell may
disassemble or cause changes in their permeability properties
thereby limiting possible applications in catalysis.5f Hence,
there is a need to generate new porous materials that can
efficiently immobilize native enzymes that have activity similar
to the native enzyme and can also be recycled several times
without loss of activity.
Enzymes are versatile biocatalysts that perform chemo-, regio-
and stereo-selective reactions in aqueous media under mild
conditions.1 The specificities of enzymes carry tremendous
promise as catalysts in industrial applications, but the short
lifetime of enzymes presently limit their usefulness. Therefore,
improvements in enzyme stability and reusability is required
to increase their lifetime so as to further enable practical
applications.1 One of the approaches to improve enzyme
reusability is via enzyme immobilization which represents the
attachment or incorporation of enzyme molecules into large
structures that render them insoluble in water. This allows
efficient recovery of the enzyme after the reaction and hence
can be reused several times. Various inorganic solid supports
have been used for enzyme immobilization including alumina,2a
silica,2b zeolites2c,3aand mesoporous silica materials.1c,3b,3c
In particular mesoporous silica materials are very good
candidates for the immobilization of enzymes due to their
ease of synthesis, high surface area (up to 1000 m2 gÀ1) and
tuneable pore size (2–50 nm) which enables researchers to
tailor these materials suitably so that the encapsulation of
a variety of proteins and enzymes is feasible.3d Thus, the
development of mesoporous material based biocatalysts has
attracted a lot of attention. Most of these materials have been
prepared by immobilization of the enzymes into the pores of
the mesoporous channels by physical adsorption.3e,f Although
simple and efficient, this process is severely limited by the fact
that with time these adsorbed enzymes leach out into the solution.
To overcome this, enzymes have been covalently immobilized
by chemically attaching them to an organo-functionalized
mesoporous material via amino acid side-chains like lysine.4,5a
Herein, we report the synthesis of a hierarchical mesoporous
silica based hybrid material and its application as a support
for the immobilization of trypsin via a ‘‘ship-in-a-bottle’’
approach. Enzyme was adsorbed onto an azide functionalized
mesoporous spherical SBA-15 (diameter 5–8 mm) with pores
larger than the diameter of the enzyme. This was then capped
with alkyne functionalized mesoporous silica nanoparticles
(MSNs; diameter B100 nm) by covalent linkage using
CuAAC (Scheme 1). The mesoporous silica nanoparticles
(MSNs) have pores that are large enough to allow small
molecules such as reactants and products to diffuse in and
out of SBA-15 but do not allow enzyme to leach out of
SBA-15. Hence, in this hierarchical silica mesoporous hybrid
material, the pores of the MSNs on the surface of SBA-15 act
as windows for the enzyme and effectively stop the enzyme
from leaching. Trypsin, an industrially important enzyme for
the hydrolysis of peptides in food processing, was chosen as a
model enzyme for this study.5g,6a Developed hierarchical
mesoporous solid material has several attributes that make it
very attractive: (i) spherical SBA-15 particles with high surface
area can be synthesized in various pore sizes (3 nm–13 nm)6b
CReST Unit, Chemical Engineering Division, National Chemical
laboratory, Dr Homi Bhabha Road, Pune, India 411008.
E-mail: ss.sengupta@ncl.res.in; Fax: +91 20 25902621;
Tel: +91 20 25902747
w Electronic supplementary information (ESI) available: Experimental
details and characterization. See DOI: 10.1039/c2cc33592a
c
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Chem. Commun., 2012, 48, 7853–7855 7853

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Scheme 1 Encapsulation of trypsin in hierarchical mesoporous
material. The particle in blue represents azide functionalized SBA-15
(diameter 5–8 mm) and the particle in grey represents alkyne functio-
nalized MSNs (diameter B100 nm).
Fig. 1 (a) TEM of spherical SBA-15 (AZP-SBA) (Inset) TEM of an
individual AZP-SBA, scale bar represents 2 mm; (b) TEM of the outer
surface of Trypsin encapsulated hierarchical mesoporous material
(Trypsin-SBA-MSN) showing MSN particles attached onto the surface
of SBA-15 (Inset) TEM of an individual Trypsin-SBA-MSN, scale bar
represents 2 mm (c) Fluorescein labelled trysin absorbed onto AZP-SBA;
(d) AZP-SBA clicked with rhodamine B labelled MSNs showing that the
MSN particles are present on the outer surface of SBA-15
depending upon the size of the enzyme to be encapsulated;
(ii) native enzymes can be used for immobilization; (iii) the
bio-orthogonal6d nature of CuAAC ensures that the efficient
capping of organoazide groups on spherical SBA-15 particles
can be carried out with alkyne functionalized MSNs without
denaturing the enzyme6c and (iv) the inner pore surface of
MSNs can be further functionalized to tune the hydrophobic/
hydrophilic property of the surface for selective diffusion of
either hydrophobic or hydrophilic substrates for chemo-
selective reaction. These materials have been characterized
by various analytical techniques such as powder XRD,
SEM, TEM, FT-IR, confocal laser scanning microscopy
(CLSM) and nitrogen adsorption-desorption experiments
and their catalytic activity for the hydrolysis of BAPNA has
been shown over 10 catalytic cycles.
After the click reaction the enzyme encapsulated material
(Trypsin-SBA-MSN) was washed with 10% PEG solution in
50 mM pH 8 tris buffer to leach out any weakly adsorbed
trypsin on the surface of the material.10a The amount of
trypsin adsorbed was estimated to be 31 Æ 1 mg (B1.3 mmol)
of trypsin per gram of Trypsin-SBA-MSN from TGA of
Trypsin-SBA-MSN (Fig. S8, ESIw) and UV-Vis analysis of
the residual trypsin that remained unadsorbed (ESIw). TEM
image of Trypsin-SBA-MSN shows trypsin adsorbed spherical
SBA-15 particles were very well covered by MSNs also
demonstrating the formation of the hierarchical mesoporous
structure (Fig. 1a and 1b). For complete characterization of
the hierarchical mesoporous hybrid material formed using
CuAAC, we synthesized another material by clicking AZP-
SBA (without trypsin adsorbed) with ALK-MSN to yield
CLICK-SBA-MSN and it was extensively characterized by
FT-IR, nitrogen adsorption-desorption and 13C CP-MAS
NMR spectroscopy experiments. FT-IR spectra show about
25% decrease in the integrated intensity of nas(N3) at
2100 cmÀ1 (Fig. S5, ESIw).7 Nitrogen adsorption-desorption
experiments gave a multipoint BET surface area of 542 m2 gÀ1
and total pore volume 0.96 cc gÀ1. BJH pore size distribution
shows presence of hierarchical pore structure having pore sizes
8.18 nm and 2.2 nm (Fig. S6(c), Table S1, ESIw). Formation of
hierarchical mesopore structure was further confirmed by
13C CP-MAS NMR spectroscopy, the peaks at 124 and
147 ppm observed are due to formation of triazole by the
click reaction between AZP-SBA and ALK-MSN (Fig. S7(c),
ESIw).7 To investigate the distribution of trypsin in the pores
of AZP-SBA, fluorescein labeled trypsin was immobilized on
AZP-SBA (Trypsin-AZP-SBA) and observed under CLSM. In
another experiment AZP-SBA was clicked with rhodamine B
functionalized MSN (ALK-RH-MSN, ESIw) and the resultant
material was imaged by CLSM. This hierarchical material
shows, a uniform coverage of rhodamine B functionalized
MSNs (red) on the outer surface of AZP-SBA (Fig. 1d).10b
The azide functionalized mesoporous spherical SBA-15
(AZP-SBA) was prepared by post synthetic grafting of
AzPTES (3-azido-propyltriethoxysilane) on calcined spherical
SBA-15 (CAL-SBA) using reported procedures (ESIw).6b,7 The
average pore size and surface area was estimated to be 8.2 nm
and 666 m2 gÀ1 respectively; while the amount of azide groups
grafted on the surface of AZP-SBA was determined to be
0.6 mmol gÀ1 (0.09 nmol cmÀ2) by elemental analysis. MSNs
having alkyne groups present on the outer surface (ALK-MSN)
were synthesized in a two-step procedure (ESIw). In the first
step, as-synthesized MSNs8,9 were subjected to outer surface
amine grafting using 3-amino-propyltriethoxysilane (APTES)
followed by removal of template to yield NH2-MSN. The
amount of amine groups grafted on the surface of NH2-MSN
was determined to be 0.78 mmol gÀ1 by elemental analysis. In the
second step, NH2-MSN was coupled with N-(4-Pentynoyloxy)-
succinimide to yield alkyne grafted MSNs (ALK-MSN). The
average pore size and surface area of ALK-MSN was esti-
mated to be 2.2 nm and 587 m2 gÀ1 respectively. ALK-MSN
was characterized by a variety of analytical techniques such as
powder XRD, TEM and 13C CP-MAS NMR spectroscopy
(ESIw).
For the encapsulation of trypsin, the azidopropyl labeled
SBA-15, AZP-SBA was first subjected to trypsin adsorption in
phosphate buffer (100 mM, pH 7) at 4 1C and then subjected
to CuAAC with ALK-MSN using CuSO4 and sodium
ascorbate at 4 1C as described in the literature (ESIw).6c,7,9b
c
7854 Chem. Commun., 2012, 48, 7853–7855
This journal is The Royal Society of Chemistry 2012

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of amount of trypsin leached out shows that 89% of trypsin
leached out during PEG solution treatment (ESIw).10a These
results conclusively show that encapsulated trypsin in the
hierarchically porous silica material functions as an efficient
bio-catalyst and can be reused several times without loss of
activity.
In summary, we have reported a facile and efficient route for
the development of hierarchical mesoporous materials and its
application in the encapsulation of enzymes. The encapsulated
enzyme functions as an efficient biocatalyst. In general, the
method can be used for the encapsulation of other enzymes
of interest. Such encapsulated enzymes can be used in the
development of continuous flow bio-reactors. Further, chemo-
selective reactions can be achieved by the modification of inner
pores surface of MSNs which can act as a gate-keeper for the
substrates. Such efforts are under way in our laboratory.
S. S. G. acknowledges Sushma Kumari for confocal images,
and DST, New Delhi (Grant No: SR/S1/PC-56/2008) for
funding. B. M. acknowledge CSIR, New Delhi for fellowship.
Fig. 2 Activity of trypsin encapsulated or immobilized on different
silica materials using 2 mM BAPNA in 50 mM, pH 8 tris buffer.
The spectroscopy data of various hierarchical mesoporous
materials discussed above indicates successful formation of
hierarchical mesoporous materials and encapsulation of enzyme
in the large pores of this material via CuAAC approach.
To investigate the activity of the encapsulated trypsin,
Trypsin-SBA-MSN was treated with 2 mM BAPNA in tris
buffer (50 mM, pH 8).10c The activity of the encapsulated
trypsin was found to be 44 mmol gÀ1 minÀ1 and remained
almost constant even after 10 cycles. (Fig. 2) This shows that
the enzyme is active and remains encapsulated efficiently. This
encapsulated enzyme has comparable activity with the activity
of the physically adsorbed trypsin on post synthetic functio-
nalized SBA-15 with thiols, amines and carboxylic acids.10c
For further comparison of the activity, two control experi-
ments were carried out. In one case, trypsin was physically
adsorbed on AZP-SBA and then ALK-MSN was added in the
same ratio as was used for encapsulation of trypsin. This
material is referred as PHY-SBA-MSN (ESIw). In another
control, the PHY-SBA-MSN hybrid material described above
was subjected to the same workup protocol that was carried
out during the encapsulation reaction (washing with 10%
PEG solution in 50 mM, pH 8 tris buffer). This is referred
as PEG-SBA-MSN (ESIw). Both the materials were subjected
to activity study with BAPNA solution. The results (Fig. 2)
show that even though the activity of PHY-SBA-MSN is
about two-fold higher than that of the activity of the encap-
sulated enzyme (Trypsin-SBA-MSN), the activity of the PHY-
SBA-MSN decreases significantly with each cycle due to
leaching of the adsorbed enzyme in the solution.10c After
10 cycles activity of the adsorbed enzyme was decreased by
B75% where as encapsulated enzyme shows almost no loss in
activity. Two-fold decrease in activity of the trypsin upon
encapsulation in comparison to activity of adsorbed enzyme
observed may be due to increased mass transport limitations;
as BAPNA molecules have to diffuse through the pore channels
of MSNs and SBA-15 to reach to the encapsulated trypsin.
It is likely that the BAPNA molecules being positively charged
were strongly adsorbed inside pore channels of MSNs which
resulted in slow diffusion of BAPNA molecules onto the
immobilized trypsin inside the pores of SBA-15.3f On the other
hand, PEG-SBA-MSN showed 87% less activity as compared
to activity by PHY-SBA-MSN. UV-Vis studies on estimation
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c
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Chem. Commun., 2012, 48, 7853–7855 7855