Immobilizing and de-immobilizing enzymes on mesoporous silica

Article abstract of DOI:10.1039/c5ra19568c

Beta glucosidase was immobilised as a model enzyme within mesoporous silica (MCF) at a high loading (80 mg g^-1). The enzyme was further entrapped within the material by precipitating additional silica within the channels. This entrapment was pe

Full text of DOI:10.1039/c5ra19568c

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Immobilizing and de-immobilizing enzymes on mesoporous silica
Vladimir Zlateski, Tobias C. Keller, Javier Pérez-Ramírez and Robert N. Grass*
Received 00th January 20xx,
Accepted 00th January 20xx
Beta glucosidase was immobilised as a model enzyme within mesoporous silica (MCF) at a high loading (80 mg/g). The
enzyme was further entrapped within the material by precipitating additional silica within the channels. This entrapment
was performed by the polycondensation of tetraethoxysilane under very mild conditions (pure water). Although
unreactive while entrapped, in this state the enzyme was highly stable towards heat treatments of 60–70°C. Upon release
from the matrix by a mild silica dissolution step involving a fluoride comprising buffer, the enzyme regained most of its
original activity. With this we developed a novel protein entrapment/release scheme, which is designed along the
principles of orthogonal protection group chemistry as the protection/deprotection steps do not affect the integrity of the
(bio)molecule. The principle can be adopted to many previously developed mesoporous silica/enzyme biocomposites and
will allow the application of enzyme dependent diagnostic devices in applications involving demanding environmental
storage requirements.
DOI: 10.1039/x0xx00000x
www.rsc.org/
simplicity, support stability and large surface area.6, 8-11 Despite
its high loading, intrinsic problems remain: immobilized
enzymes are less active than free enzymes; activity may be
Introduction
Mild and environmentally friendly reaction conditions in
combination with high chemo-, regio- and stereoselectivity as
well as high turnover rates compared to synthetic catalysts,
contributed to the increased use of enzymes in the last
decades, thus fostering the idea of using sustainable
methodologies for chemical reactions.1, 2 Even though enzymes
are extremely efficient as biocatalysts for many chemical
reactions, their application is often hampered by the lack of
long-term storage stability, considering the fact that many
temperature sensitive enzymes need to be continuously stored
at -20°C. Operational stability, namely inactivation caused by
mechanical treatment or heat denaturation, and difficulties in
recovery and recycling are other every-day problems.3 The
effort invested to circumvent these issues led to the
development of enzyme immobilization techniques on solid
supports (physical adsorption or covalent binding),
entrapment and cross-linking, which have proven to enhance
enzyme stability and enable re-use.1, 4-6
further lost12 due to enzymes leaching out from the support
and due to spatial constraints, the reaction of immobilized
enzymes
with
large
substrates
(proteins/DNA/polysaccharaides) is very limited.
Previous studies have demonstrated attempts to tackle the
leaching and stability problems by fine-tuning the channel to
the enzyme size (snug fit).13,
14
However, this approach is
enzyme specific and results in low protein loadings and
substrate diffusion problems.15 Another strategy is based on
selective silylation thus reducing the size of the pore openings
of the mesoporous supports. In their work, He and co-workers
managed to slightly reduce the pore opening diameter of the
lipase immobilized MPS by employing 3-(trimethoxysilyl)propyl
methacrylate (PMA) and polymerization of the anchored vinyl
groups with free PMA.16 Similar work was done by Ma and co-
workers, where they employed the same enzyme but a
different grafting strategy.17 However, the conditions used in
both cases are quite harsh (toluene, 70 °C or 35 °C in the latter
case) for most enzymes to survive and be active again. Besides,
mechanical and thermal stability of the proteins was not
investigated. Both strategies however, have not solved the
large substrate limitations.
Physical adsorption is based purely on hydrogen bonds,
electrostatic and hydrophobic interactions between the
support surface and the protein of interest. Compared to the
other immobilization methods it is the simplest, with which
denaturation/deactivation of the enzymes can be avoided and
good enzymatic activities can be maintained.7 Adsorption of
enzymes on pre-fabricated porous inorganic supports, such as
mesoporous silicates (MPS), is currently one of the most
attractive enzyme immobilization methods due to the offered
Following on previous unsuccessful attempts to directly
encapsulate proteins in silica in analogy to encapsulating
DNA/RNA (see supporting info),18-21 we took advantage of
literature knowledge on enzyme immobilization on
mesoporous materials. To further overcome the disadvantages
that immobilized enzymes have during application, we herein
report a novel approach to stably entrap enzymes and release
them on demand (Scheme 1).
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Scheme 1. Silica entrapment and fluoride buffer triggered release of enzymes.
was used to determine the external surface area (Smeso). Pore
size distributions were determined by applying the BJH model
to the adsorption branch of the isotherm (Table 1).
Experimental
MCF mesoporous silica synthesis
Mesocellular foam (MCF) was prepared according to a method
reported previously with some modifications.22 The
mesoporous silica was synthesized from TEOS (tetraethyl
orthosilicate) (Sigma-Aldrich), pluronic P123 (poly(ethylene
Small angle X-ray scattering (SAXS)
The small-angle X-ray scattering (SAXS) curve was recorded on
an Empyrean powder diffractometer (PANalytical B.V., The
Netherlands), operating in transmission mode with Cu Kα
radiation (45 kV, 40 mA). The interlayer spacing was calculated
by the Bragg`s law (n x λ = 2 x d x sin(θ), where λ = 0.154 nm.
glycol)-block-poly(propylene
glycol)-block-poly(ethylene
glycol)) (Sigma-Aldrich) and mesitylene (Sigma-Aldrich). First,
pluronic P123 (4 g) was added to water (120 mL), followed by
KCl (6.1 g). The mixture was stirred on a magnetic stirring plate
(Heidolph) at 500 rpm at room temperature until it became
translucent. Next, mesitylene (3 g) and 12 N HCl (23.6 g, Sigma-
Aldrich) were added and the mixture was stirred for 2 h at
room temperature. TEOS (8.5 g) was then added to the
mixture and all together stirred vigorously for 30 min at room
temperature. The solution was transferred to a Teflon-lining
steel autoclave and was aged for 24 h at 35 °C in an incubator
(Binder GmbH). Later, the solution was subsequently aged for
an additional 24 h at 130 °C in a drying oven (T 6030, Heraeus
Instruments), filtered and washed with water and ethanol.
After all the ethanol was evaporated, the produced powder
was calcined in a furnace (Nabertherm) at a heating rate of 60
°C/h and held at 500 °C for 6 h.
TEM and SEM analysis
For transmission electron microscopy (TEM), the samples were
dispersed in ethanol and some droplets of the suspension
were deposited on a lacey carbon foil supported on a Cu grid.
TEM was performed on a Tecnai F30 (FEI, field emission gun
(FEG), SuperTwin lens (point resolution ca. 0.2 nm), operated
at 300 kV). For scanning electron microscopy (SEM), the
samples were resuspended in i-PrOH, and loaded onto
copper/carbon grids. The microscope (FEI Nova NanoSEM) was
operated at 30 kV.
β-glucosidase immobilization
MCF (20 mg) was suspended in MQH2O (0.5 mL) by 30 s
ultrasonicating and 15 s vortexing. Separately, β-glucosidase
from almonds (20 mg, Sigma-Aldrich) was dissolved in MQH2O
(1 mL) and split into two eppendorf tubes (0.5 mL each). In
one, the MCF suspension was added, whereas in the second
MQH2O (0.5 mL) was added. Both tubes were shaken on an
orbital shaker (VXR basic, IKA) for 3 h at room temperature
followed by centrifugation at maximum speed for 4 min
(CT15E, Hitachi Koki Co., Ltd). The pellet (mesoporous silica
plus enzyme) was washed with MQH2O (1 mL) and finally
suspended in the same volume of MQH2O.
Mercury intrusion experiment
Hg intrusion in the pressure range of 0.01-400 MPa was
carried out in a Micromeritics Autopore IV 9510 instrument
assuming a contact angle = 140° with a pressure equilibration
time of 10 s. Pore size distributions were smoothed using a
2nd order Savitzky-Golay filter over a window of 10 points to
eliminate noise from the differentiation.
Nitrogen sorption experiment
Nitrogen sorption at −196°C was carried out in a Micromeriꢀcs
TriStar II instrument. The MCF was evacuated for 3 h at 300°C,
whereas the enzyme-loaded analogues were outgassed at
room temperature. The total surface area (SBET) of the samples
was determined by the BET method, and the t-plot method
β-glucosidase entrapment in silica
MCF-β-glucosidase (1 mg, corresponding to 50 μL suspension
after immobilization) was suspended in MQH2O (0.45 mL) by
30 s ultrasonicating and 15 s vortexing, followed by TEOS
addition (4 μL). The final mixture was left shaking for 5 days at
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500 rpm on an orbital shaker, with subsequent additions of was transferred to a plastic disposable cuvette and absorbance
TEOS (4 μL) after 24, 48 and 72 h. Afterwards, the sample was was monitored each minute for up to 30 minutes.
washed twice with MQH2O and stored in the fridge until the
Enzymatic activity calculation
next activity measurement. In parallel, the two control
samples (1) β-glucosidase in water and 2) MCF-β-glucosidase) The specific enzymatic activities (U/mg) of both free and
were treated the same way but without any TEOS addition.
immobilized
calculated with the given formula: Specific Activity = (∆A x Vt x
Df)/( x l x Vs x C) where ∆A = (∆A Test - ∆A Blank)/min. at the
β
-glucosidase and
α-chymotrypsin were
Enzyme release
ε
The release of the enzyme was triggered by the fluoride buffer, desired wavelength (390 or 405 nm); Vt = total volume of the
which was added to the entrapped enzymes in order to reaction mixture; Df = dilution factor; ε = extinction
dissolve the silica support. The buffer was prepared in coefficient; l = path length; Vs = volume of the sample; C =
polyethylene, polypropylene or Teflon containers according to protein concentration. The extinction coefficient in the case of
the following protocol: for 10 mL fluoride buffer we dissolved the p-nitrophenol (β-glucosidase assay) was calculated under
0.23 g of NH4FHF in 5 ml of H2O and 0.19 g of NH4F in 5 ml of
H2O eventually pooling the two solutions together (pH
our assay conditions; ε
4; of the p-nitroaniline (α-chymotrypsin assay)
= 13394.43 (M-1 x cm-1) and in the case
= 12500 (M-1
x
∼
ε
measure pH with pH paper and not with a pH electrode).This cm-1). The path length is l=1 cm.
solution is stable at room temperature for at least 2 months. In
Protein concentration measurement
order to release the enzyme, enough fluoride buffer was
added in order to obtain a clear solution. In the case of the The amount of protein bound on the mesoporous material was
fluoride buffer at pH 5, the pH was adjusted by carefully estimated from the C, H, N percentage mass increase after
adding NaOH (1 M, Merck). Fluoride comprising waste as immobilization obtained by elemental microanalysis
collected in a saturated calcium carbonate solution.18
measurement (Vario Micro Cube, Elementar) and from
knowing the elemental content of the enzymes (% N and % C).
Enzymatic activity assays
β-glucosidase assay
Thermal stability test
Two samples, β-glucosidase free in water and β-glucosidase
entrapped in the silica material, were submitted to thermal
stress by incubating them for 1 h at 4 different temperatures:
room temperature, 50°C, 60°C and 70°C in a thermomixer
(compact, Eppendorf). The samples were let to cool down to
and enzymatic activity was measured where the enzyme
entrapped in silica was first dissolved with a proper amount of
fluoride buffer (as described in the “β-glucosidase assay“
enzymatic activity section).
The increase in absorbance (production of p-nitrophenol) over
time at a wavelength of 405 nm was measured on a micro-titer
plate reader (Infinite f200 Tecan) in a transparent flat bottom
96-well plate (TPP) at 25 °C. The assay mixture of both free and
immobilized
enzyme
contained
4-nitrophenyl
β-D-
glucopyranoside (11 mg, 0.037 mmol, Sigma-Aldrich) dissolved
in sodium phosphate buffer (1.404 mL, 0.1 M, pH 6.5) to which
enzyme solution was added (48 µL). Samples (242 µL) were
added to a stopping sodium carbonate buffer solution (62 µL,
0.5 M, pH 10.8) and transferred to the 96-well microplate for
measurement. Reaction took place over time (final time of 4
min) with samples being taken after every minute.
Fluoride buffer influence on enzymatic activity
Both β-glucosidase and α-chymotrypsin free in solution were
used to check the influence of the fluoride buffer on the
enzymatic activity. The same concentrations of free enzyme in
solution as used for the immobilization process were used and
enzymatic assays were performed by using 10 μL of the
enzyme solutions and 25 μL of the following fluoride buffer
solutions: 1) fluoride buffer pH 4 (preparation steps shown in
the “enzyme release” section of this materials and methods
chapter), 2) 1:10 diluted fluoride buffer pH 4 and 3) fluoride
buffer pH 5 (pH adjusted to 5 with NaOH). The enzyme
solutions were incubated in the presence of the respective
fluoride buffer for 2-3 minutes and then transferred to a glass
vial for neutralization of the excess F- ions. The enzyme assay
solutions were added into the vials and the desired
absorbance was subsequently measured.
In the case of the silica-entrapped enzyme we took 10 µL of
the reaction mix (as described in the “β-glucosidase
entrapment in silica” section) and diluted up to 100 µL with
water (1:10). Then from the dilution we used 48 µL in the
assay (as mentioned above). In the cases where fluoride buffer
was utilized for either enzyme release or fluoride buffer
resistance tests, 25 µL of the fluoride buffer were added to the
10 µL of the enzyme sample, the volume was brought up to
100 µL with water and again 48 µL were taken for the assay.
α-chymotrypsin assay
The increase in absorbance (production of p-nitroaniline) over
time at a wavelength of 390 nm was measured on a
spectrophotometer (Nanodrop 2000c, Thermo scientific) at
room temperature. The assay mixture contained: tris/HCl
buffer pH 7.8 (1.42 mL, 80 mM, Fluka), substrate solution (1.4
mL, 1.18 mM N-benzoyl-L-tyrosine-p-nitroanilide, Sigma-
Aldrich, dissolved in 1:1 water/DMSO mixture), CaCl2 (80 µL, 2
M, Fluka) and 100 µL of the sample to make up a final volume
of 3 mL. Immediately after the enzyme addition, the mixture
Results and Discussion
For entrapment, we combined the well-known advantages of
the mesoporous silicas with the simplicity of the adsorption
process in order to obtain high loadings of highly active
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Table 1. Structural properties of the mesoporous silica after synthesis (MCF), after β-
glucosidase addition (MCF-β-Glu) and following additional silica growth (MCF-β-Glu-
SiO2).
Sample
dpore
(nm)
Vpore
Smeso
SBET
(cm3/g) (m²/g) (m²/g)
MCF
23
21
13
1.95
1.70
0.48
410
323
234
434
368
341
MCF-β-Glu
MCF-β-Glu-SiO2
enzymes immobilized on the MCF carrier. The ultralarge cage-
like mesopores of this support are ideal for entrapping
enzymes of different dimensions in high loadings.23
Fig. 2 TEM and SEM images of the MCF (a) and c)) and MCF-β-Glu-SiO2 (b) and d)). The
texture of the material after silica deposition drastically changed and the obvious
mesopore openings in the original material were no longer visible.
Additionally,
MCFs
possess
a
three-dimensional,
interconnected pore structure which would facilitate substrate
diffusion.24, 25 Having that as a starting point, we developed a
procedure to further entrap enzymes within the porous
material, by growing additional silica inside the MCF cells in a
protein-friendly environment (water).
Fig S2). Analysis of the scattering data shows that no indexing
of higher order peaks to any plane or space group (ex. p6mm)
is possible and no SBA-15-like structure could be found (Fig. 3).
β-glucosidase from almonds was chosen as a model
enzyme to be immobilized and further entrapped in the silica
matrix. Typically, proteins enable binding interactions for
adsorption to take place mainly due to the amino and
carboxylic acid groups present on their surface. Knowing that
the surface of the silicas carries a negative charge at pH values
above 2,29 which is much lower than the isoelectric point (pH
at which no overall electric charge is carried) values of most
proteins, we are able to tune the charge of the protein surface
by changing the pH of the solution. However, if the proteins
MCF material with a mesopore size of 23 nm was
synthesized as the immobilization support of choice. The
mesopores of the calcined MCF were confirmed by measuring
nitrogen adsorption–desorption isotherms of the dried sample
and plotting the corresponding pore-size distribution curve.
(Fig. 1, black squares). N2 sorption analysis evidences the
typical type IV isotherm (well defined hysteresis loop) obtained
for uniform-size mesoporous materials26 and gives an external
surface of 410 m2 g-1 with a total pore volume of 1.95 cm3 g-1
(Fig. 1, Table 1). The large uniform mesopores (dpore = 23 nm),
which are in good agreement with the microscopy analysis
get too much positively charged strong self-repulsion may
30
Taking advantage of improved adsorption in
occur.8,
(Fig. 2, a) and c) and the X-ray data (Fig. 3), can be attributed
27, 28
Hg
to the organic cosolvent mesitylene addition.22,
solutions which have a pH near the pI value of the enzyme, we
immobilized β-glucosidase (pI = 7.3) in water at pH 7. We
managed to retain activity up to 95 % after immobilization
with an enzyme loading of ~80 mg/g. The pore network is well-
intrusion experiment resulted in a pore window size of ~ 11
nm and 75 % of the mesopore volume determined by nitrogen
sorption was proven to be accessible for Hg intrusion (Fig. S1,
Fig. 1 N2 sorption isotherms and pore-size distribution curves (inset) obtained from the
adsorption branch by the Barret-Joyner-Halenda (BJH) method of: MCF (black squares),
MCF-β-Glu (blue circles), and MCF-β-Glu-SiO2 (red triangles).
Fig. 3 Small-angle X-ray scattering (SAXS) from a MCF with an interlayer spacing of 25.6
nm. No ordered structure is evident.
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preserved after the loading of the enzyme, although the size of
the mesopores, and consequently their volumes are reduced
(Fig. 1 (blue circles), Table 1).
With the aim to increase the enzyme stability and prevent
enzyme leaching, we further entrapped the enzymes within
the matrix. For this we utilized silica sol-gel synthesis, a well-
known and fundamental reaction that brings about the
conversion of silicate precursors (i.e. TEOS) to silica gels.31 In
order to minimize any perturbation of the enzyme integrity,
we performed the polycondensation reaction by simply mixing
the immobilized enzyme with highly diluted tetraethyl
orthosilicate (TEOS) in water (opposed to the traditional
procedure in alcohol and base catalysis). The entrapment
process was monitored by evaluating the activity of the
enzyme after various timepoints. As shown on Fig. 4, this
entrapment process was relatively slow and proceeded over
several days, which is due to the slow polycondensation
reaction of TEOS in the absence of a suitable catalyst.32 After a
silica growth process of 5 days, the enzyme activity had
dropped by > 80% indicating that new silica material had
formed within the MCF cells hindering the substrate diffusion
to the enzyme. This observation was confirmed by nitrogen
sorption and electron microscopy, evidencing that the silica
deposition led to a pronounced textural modification (Fig. 2 &
Table 1). After the reaction the material evidences both micro-
and shallow mesopores. The mesopore volume is reduced by
75%, and the formerly uniform pore-size is transformed into a
broader distribution centred at around dpore = 13 nm (Fig. 1,
red triangles), indicating that the cells are gradually filled with
amorphous silica. Furthermore, the broad hysteresis loop
points towards the presence of ink-bottle-like pores, i.e. pores
Fig. 5 Highly active β-Glu (white columns) was released after MCF-β-Glu-SiO2 (black
columns) was treated with fluoride-containing buffer. The activities of the MCF-β-Glu
(sparse columns) and β-Glu free in solution (dense diagonal lines) are given for
comparison. The storage stability was assessed after 15 days and further shows the
advantage of the silica entrapment/release scheme for enzyme storage.
of the isotherm at p/p0 = 0.45.33, 34
While the above shows that the entrapment of the enzyme
within the support was successful, it also displays that as long
as the enzyme is entrapped within the inorganic material, it is
not very active. As both the support structure (MCF) and the
additional material grown within the pores consists entirely of
silica, we investigated on a de-encapsulation scheme by
dissolving the silica, again without perturbing the enzyme
structure. It is well known that silica dissolves rapidly in
fluoride comprising buffers (e.g. buffered oxide etch, a pH
stabilized ammonium fluoride solution). However, these
reagents are very rarely used in biochemistry as they are
feared due to their toxicological potential und unclear
implications with proteins. Still the application of fluoride
solutions is a commonplace in classical protection group
chemistry (e.g. potassium fluoride or tetra butylammonium
fluoride) and even dentistry. So in order to avoid the handling
of dangerous HF, we prepared small volumes of a 4 wt% F-
buffered oxide etch solution from ammonium fluoride and
ammonium hydrogen fluoride. For reference, dental care
products (fluoride gels) contain up to 1.23 wt% F- and 5 mg F-
/kg bodyweight is considered the probable toxic dose for
humans.18
accessible only through
a
narrow opening, further
corroborated by the increased intensity of the forced closure
In order to de-encapsulate the entrapped enzymes, 20 μg
of the material was mixed with a small volume of buffered
oxide etch (50 µl), resulting in a clear solution. To evaluate the
intactness of the active site of the released enzyme the β-
glucosidase activity of the resulting solution was measured in
an appropriate buffer. As shown on Fig. 5 this procedure
resulted in a great boost in the catalytic activity, indicating that
the silica support had been dissolved and that the enzyme was
released unharmed. The activity of the released enzyme went
up to ~250 % of the entrapped one and was comparable to the
activity of β-glucosidase free in solution after 5 days storage in
the fridge (Fig. 5, dense columns), attributing almost no
activity loss to the entrapment/release process itself. It is
Fig. 4 Enzymatic activities during the sol-gel synthesis of MCF-β-Glu-SiO2 (stars) and β-
Glu free in solution (squares). In order to rule out that the loss of activity is due to
enzyme degradation during the 5 day reaction, free enzyme was subjected to the same
conditions (mechanical stress and temperature / but no TEOS addition) and showed
only a minor loss of activity.
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worth mentioning is that no increase in activity is evident connection with proteins, DNA and polysaccharides is
when the immobilized (not entrapped) enzyme is incubated in currently ongoing.
fluoride buffer (Fig, S3). As an additional control to this we
Since there is already extensive work and knowledge on the
measured the compatibility of β-glucosidase with fluoride loading of enzymes to nanoporous silica supports we anticipate that
comprising buffers (See Fig. S4). In addition, another widely the fluoride de-encapsulation step presented here can be adapted
used enzyme,
α-chymotrypsin, was submitted to the same to many other systems and will offer new avenues for immobilized
fluoride buffer compatibility test (See Fig. S4). A minor activity enzymes.
decrease (10-20 %) could be measured for both enzymes, in
the three cases where fluoride buffer at pH 4, fluoride buffer
Conclusions
at pH 4 diluted (1:10) and fluoride buffer at pH 5 was used for
incubation.
In summary, this work demonstrates the synthesis of a novel
enzyme-in-silica material with improved operational and
storage stability, that can undergo enzyme release in solution
upon a chemical trigger. For this purpose, we utilized the well-
known advantages of the mesoporous silicas (high surface area
and stability) and the simplicity of enzyme adsorption and
further optimized them according to our needs in order to
obtain high loadings of active β-glucosidase as a model
It is worth highlighting the importance of the second
comparison on Fig. 5, displaying data after 15 days of wet
storage in the fridge. While the free enzyme lost more than
half of its activity, no substantial activity loss was observed for
the entrapped enzyme after the release.
Besides the high mechanical and storage stabilities already
discussed, the encapsulated enzyme shows a high resistance
towards heat treatment. MCF-β-Glu-SiO2 and β-Glu free in
solution were submitted to 1 hour of incubation at room
temperature, 50 °C, 60°C and 70°C followed by a subsequent
release of the enzyme and enzymatic activity measurement.
While β-glucosidase free in solution performed poor (very low
and no activity at 60 and 70°C), β-glucosidase released from its
entrapped state resulted in 100 % activity recovery at 50 and
60°C and a high ~75 % retained activity after the 70°C
treatment (Fig. 6). This resistance to heat can be attributed to
the protective effect of the silica matrix, which prevents the
protein undergoing extensive conformational changes inside
the material.
enzyme. Furthermore, we developed
a procedure to
additionally silica-entrap the previously immobilized enzymes
which led to high mechanical, storage and heat stability of the
biomolecules. Last, we utilized a non-harmful way to dissolve
the support and trigger an immediate release of the enzyme
molecules, giving a possibility to select from both large and
small substrates. In this way, one could store enzymes for a
long time and release them upon need. In the future, one
could pay special attention to sensitive enzymes, which are
very delicate to handle and require low storage temperatures.
The idea of replacing the freezer with the shelves, eliminating
the multiple freeze-thaw cycles and the large number of
sensitive enzymes available on the market, shows a great
application potential and is certainly worth further detailed
experimentation.
The entrapment not only allows for improved operational,
storage and heat stability of enzymes, but one could also take
the advantage of the triggered release to perform enzymatic
reactions with large substrates, thus tackling some of the
biggest problems faced when working with solid support
immobilization techniques. Additional work on this in
Acknowledgements
The authors thank ICB/ETH Zurich, ETH research grant ETH-31
13-1 and the EU/ITN network Mag(net)icfun (PITN-GA-2012-
290248) for financial support, Dr. Frank Krumeich for TEM,
Gediminas Mikutis for SEM imaging and Ofer Hirsch for SAXS
measurement.
Notes and references
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2. M. Alcalde, M. Ferrer, F. J. Plou and A. Ballesteros, Trends
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Fig. 6 Enzymatic activities of the β-Glu free in solution (dense stripes columns) and β-
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the corresponding activities at room temperature.
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