Angewandte
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Chemie
enzymatic activity, which was measured by using the conven-
tional o-nitrophenyl-b-galactoside (ONPG) assay, showed
that less than 1% of the enzyme was left free in the
supernatant, a value that was consistent with the total protein
concentration, which was under the limit of detection of the
method (see the Supporting Information). The enzyme-
activity assay showed a loss of 60% of the initial activity.
This decrease in activity could be explained by the unfavor-
able orientation of a fraction of the immobilized enzymes or
partial denaturation upon immobilization. The particles were
subsequently incubated with a mixture of (3-aminopropyl)-
triethoxysilane (APTES, 19 mgmLÀ1) and tetraethylorthosi-
licate (TEOS, 80 mgmLÀ1) to enable the growth of an
organosilica layer at the surface of the enzyme. b-Gal from
K. lactis is a large tetrameric enzyme comprising a dimer of
dimers with two biocatalytic centers located at the interface
within each dimer.[10] This three-dimensional structure can be
approximated as a triaxial ellipsoid with dimensions of 15.9
9.3 5.3 nm3. Assuming that the immobilization strategy used
in the present study did not favor any specific orientation of
the protein with regard to the surface, the protective layer
would need a thickness of at least 16 nm to fully shield the
enzyme. The organosilane polycondensation reaction on the
SNPs with surface-immobilized b-gal (SNPENZ-OS) was moni-
tored over time (Figure 2).
shield the whole enzyme, regardless of its orientation to the
SNP surface. All particles presented a fairly homogeneous
and flat surface. There were only a few sporadic cases in which
the layer was partially broken, and its edges did not appear
sharp, thus suggesting that this organosilica layer was soft.
When measuring the enzymatic activity of the shielded b-
gal (SNPENZ-OS), we noticed that the enzymatic activity was
low for freshly produced SNPENZ-OS right after synthesis,
whereas after storage for 12 h at 258C, the same sample had
significantly higher activity. Indeed, the activity measured
before the layer growth was 73 mUmgÀ1, which dropped to
21 mUmgÀ1 after layer growth. After storage for 12 h at 208C,
the activity was found to be 50 UmgÀ1, which corresponds to
recovery of 68% of the activity of the initially immobilized
enzyme. From our experience with virus-imprinted particles,
we knew that the organosilica layer was not mechanically
stable after the synthesis and had to be stored at room
temperature for 12 h to gain stability.[9a] We decided to
investigate this phenomenon further and to assess possible
changes in the nanomechanical properties of the protective
layer by means of atomic force microscopy (AFM).
The AFM experiments were carried out by measuring
force–distance curves on different SNPs of the same sample
(Figure 3). As expected, bare SNPs were stiff, with a stiffness
value of (34 Æ 0.11) NmÀ1 (Figure 3). At the beginning of the
curing reaction, the SNPENZ-OS particles were also stiff, with an
average value of (14 Æ 0.02) NmÀ1. After curing for 5 h, the
stiffness value dropped to 6 mNmÀ1 with a moderately
broader distribution. The softening effect of the organo-
silica–protein layer continued until the SNPENZ-OS reached
a value as low as 0.5 mNmÀ1 after 12 hours, this value then
remained constant for several days. By contrast, the SNPOS
sample did not exhibit such a trend. The organosilica layer in
these reference particles was soft, with a value of 0.28 NmÀ1
after termination of the layer-growth reaction; this values did
not change significantly over the time period of the curing.
The evolution of the particle diameter over time was
found to be linear, with an increase of 1.2 nmhÀ1. In the last
sample collected after polycondensation for 15 h, the thick-
ness of the organosilica layer (17.0 Æ 0.6 nm) was sufficient to
À À
The formation of covalent siloxane (Si O Si) bonds first
requires the hydrolysis of the ethoxy functions of the silanes
into the corresponding silanols, which further undergo a con-
densation reaction. In the present case, one could assume that
the initial stiff layer was predominantly stabilized by hydro-
gen bonds (H-bonds) and ionic interactions of the silanes of
short polysiloxanes with the surface of the protein; this layer
À À
became softer through the formation of Si O Si bonds.
Regarding the change in enzymatic activity, two hypoth-
eses could explain the recovery of enzyme activity during the
curing/softening of the organosilica layer. The first is that an
increase in porosity of the protective layer resulted in a higher
mass transfer of the substrate to the active site of the enzyme.
The second hypothesis is that the soft environment of the
organosilica layer allowed the protein to acquire sufficient
conformational mobility, known to be of crucial importance
for the catalytic activity of the enzyme.[8] To better understand
the recovery of enzymatic activity during the curing phase, we
performed a kinetic study of the enzymatic activity of freshly
produced samples submitted to the curing reaction at 258C
(Figure 4).
Figure 2. Microscopy study. a) Kinetics of layer growth (meanÆstan-
dard error of the mean) at the surface of SNPs with (SNPENZ-OS, white
squares) or without (SNPOS, black squares) surface-immobilized b-gal,
as measured from FESEM images. For both systems, a linear diameter
increase of 1.2 nmhÀ1 was observed, thus showing that the presence
of the enzyme at the surface of the SNPs did not significantly influence
the kinetics of layer growth. b,c) FESEM images of SNPENZ-OS with
a protective organosilica layer of 17 nm. The particle in (b) has
a damaged protective layer; the rounded edge of this layer suggests
a soft material. Scale bars represent 100 nm.
Although the maximum velocity of SNPENZ-OS increased
over curing time, the apparent Michaelis–Menten constant
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Angew. Chem. Int. Ed. 2016, 55, 6285 –6289