Enzyme Shielding in an Enzyme-thin and Soft Organosilica Layer

Abstract

The fragile nature of most enzymes is a major hindrance to their use in industrial processes. Herein, we describe a synthetic chemical strategy to produce hybrid organic/inorganic nanobiocatalysts; it exploits the self-assembly of silane building blocks at the surface of enzymes to grow an organosilica layer, of controlled thickness, that fully shields the enzyme. Remarkably, the enzyme triggers a rearrangement of this organosilica layer into a significantly soft structure. We demonstrate that this change in stiffness correlates with the biocatalytic turnover rate, and that the organosilica layer shields the enzyme in a soft environment with a markedly enhanced resistance to denaturing stresses.

Full text

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Enzyme Shielding in an Enzyme-thin and Soft Organosilica
Layer
M. Rita Correro,[a] Negar Moridi,[a] Hansjörg Schützinger,[a][b]Sabine Sykora,[a] Erik M.
Ammann,[a] E. Henrik Peters,[a] Yves Dudal,[b] Philippe F.-X. Corvini,[a][c] and Patrick
Shahgaldian*[a]
[a] School of Life Science, University of Applied Sciences and Arts Northwestern Switzerland,
Gründenstrasse 40, Muttenz CH-4132 (Switzerland)
[b] INOFEA AG, Hochbergerstrasse 60C, Basel CH-4057 (Switzerland)
[c] School of the Environment
Nanjing University, 210093 Nanjing, (China)
Abstract: The fragile nature of most enzymes is a major hindrance to their use in industrial processes.
Herein, we describe a synthetic chemical strategy to produce hybrid organic/inorganic nanobiocatalysts;
it exploits the self-assembly of silane building blocks at the surface of enzymes to grow an organosilica
layer, of controlled thickness, that fully shields the enzyme. Remarkably, the enzyme triggers a
rearrangement of this organosilica layer into a significantly soft structure. We demonstrate that this
change in stiffness correlates with the biocatalytic turnover rate, and that the organosilica layer shields
the enzyme in a soft environment with a markedly enhanced resistance to denaturing stresses.
Keywords: Nanoparticles • Enzyme Catalysis • Self-assembly • Organosilica
Version: last version submitted
Published: Angew. Chem. Int. Ed. 2016, 55, 6285-6289; Angew. Chem. 2016, 21, 6393-6397
Link to published version: https://doi.org/10.1002/anie.201600590

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Biocatalysis is a major driver of the chemical industry.[1] However, the use of enzymes in industrial
processes is limited by their significant fragility and fast aging in non-physiological environments.
In addition to genetic engineering techniques to improve enzyme stability,[2] a variety of chemical
approaches have been developed to immobilize and shield enzymes on solid carriers.[3] Enzyme
immobilization on solid supports is a valuable approach to address enzyme stability, and has the
additional benefit of allowing the biocatalyst to be retained for continuous operations.
A large number of bio-conjugation strategies have been developed to allow the immobilization and
protection of enzymes on a variety of carrier materials,[4] such as (bio-)polymers, zeolites, noble
metals, and metal- or metalloid oxides (e.g., silica). Recently, sophisticated approaches where
enzymes were confined and protected in materials such as metal-organic frameworks[5] or virus-
like particles[6] have been successfully developed.
Silica is a material of choice to immobilize and protect enzymes as it has advantages such as low
production cost and high thermal and mechanical stability. Sol-gel methods have been extensively
developed to embed enzymes in silica matrices.[7] Reetz et al. reported on the entrapment of a
lipase in chemically modified silica gels. The enzyme was immobilized in a sol-gel matrix containing
a mixtures of tetramethyl orthosilicate and alkylsilanes. A strong dependence of the silane
composition of the matrix on the enzymatic activity was demonstrated.[7c,7d] However the main
focus of sol-gel methods to protect enzymes has been on macroscopic systems that do not allow
controlling the three-dimensional structure of the material produced. Consequently, most
techniques are not amenable to the production of discrete functional nanoparticles that can be
dispersed into fluids, hence severely limiting their use in biomedical applications. Additionally, silica
is inherently negatively charged and cannot create a shell closely surrounding the whole surface
of the protein. We expect that the presence in these silica-based materials of additional functional
groups will increase the number of interaction points with the protein surface. This should allow for
better protection of the enzyme, owing to a better chemical complementarity between the surface
of the enzyme and the protective material. Furthermore, the environment shielding the enzyme
should be designed such that it does not hinder the enzyme’s conformational dynamic mobility,
which is crucial for its biocatalytic activity.[8] This issue has been often neglected in the design of
enzyme protection systems.

3
We have recently demonstrated that a virus can serve as a template to grow an organosilica layer
on its capsid surface in purely aqueous conditions.[9] Herein, we report a synthetic strategy to grow
a protective layer of controlled thickness at the surface of immobilized enzymes; cf. Scheme 1.
The method consists of a sequential reaction of i) immobilization of an enzyme at the surface of
silica particles; ii) controlled self-assembly and subsequent polycondensation of silanes, resulting
in the growth of an organosilica layer at the surface of the particles.
Scheme 1. Principle of enzyme protection. (a) Step 1: enzyme immobilization at the surface of SNPs
(in black); Step 2 & 3: silane self-assembly and polycondensation.
As carrier material, we chose amino-modified silica nanoparticles (SNPs) produced with a diameter
of 266 ± 1 nm, as measured by field-emission scanning electron microscopy (FE-SEM). The amine
functions allowed the further covalent anchoring of the target enzyme, β-galactosidase (β-gal) from
K. lactis, using glutaraldehyde as a homobifunctional cross-linker. The enzymatic activity, which
was measured using the conventional o-nitrophenyl-β-galactoside (ONPG) assay, showed that
less than 1% of enzyme was left free in the supernatant, a value that was consistent with the total
protein concentration that was under the limit of detection of the method (Supporting Information).
The enzyme activity assay showed a loss of 60% of the initial activity, which could be explained
by the unfavorable oritentation of a fraction of the immobilized enzymes or a partial denaturation
upon immobilization. The particles were subsequently incubated with a mixture of (3-
aminopropyl)triethoxysilane (APTES, 19 mg mL-1) and tetraethylorthosilicate (TEOS, 80 mg mL-1)
in order to grow an organosilica layer at the surface of the enzyme. β-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 tri-axial
ellipsoid with dimensions of 15.9 × 9.3 × 5.3 nm. Assuming that the immobilization strategy used
in the present work 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
1 2 3

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organosilane polycondensation reaction on the SNPs with surface-immobilized β-gal (SNPENZ-OS)
was monitored over time (Figure 1).
Figure 1. Microscopy study. (a) Kinetics of layer growth (mean ± s.e.m), measured on FE-SEM micrographs, at
the surface of the SNPs with (SNPENZ-OS, white square) or without (SNPOS, black squares) surface-immobilized β-
gal. Both systems show a linear diameter increase of 1.2 nm h-1 showing that the presence of the enzyme at the
surface of the SNPs did not significantly influence the kinetics of layer growth (b,c) FE-SEM micrographs of SNPENZ-
OS (b, c) with a protective organosilica layer of 17 nm. In (b) is shown a particle when the protective layer is
damaged; the rounded edge of this layer suggests a soft material. Scale bars represent 100 nm.
The evolution of particle diameter over time was found to be linear, with a linear increase of 1.2
nm h-1. In the last sample collected after 15 hours of polycondensation, the thickness of the
organosilica layer (17 ± 0.6 nm) was sufficient to shield the whole enzyme regardless of its
orientation to the SNP surface. All the particles present a fairly homogeneous and flat surface.
There were only a few sporadic cases where the layer was partially broken and its edges did not
appear sharp, suggesting that this organosilica layer was soft.
When measuring the enzymatic activity of the shielded β-gal (SNPENZ-OS), we noticed that the
enzymatic activity was low when freshly produced SNPENZ-OS was measured right after synthesis,
while the same sample measured after 12 hours of storage at 25°C had a significantly higher
activity. Indeed, before the layer growth, the activity measured was 73 mU mg-1 and dropped after
the layer growth down to 21 mU mg-1. After 12 hours of storage at 20°C, the activity was found to
be 50 U mg-1 which corresponds to a recovery of 68% of the initially immobilized activity. 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 hours 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 2).

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Figure 2. Nanomechanical properties assessment. (a) Force-distance curve measurements carried out on bare
silica nanoparticles (black), SNPOS (--) and SNPENZ-OS (− ! −) after 12 hours storage. (b-h) Stiffness distribution
histograms for bare SNPs (b), SNPENZ-OS (c, e and g) and SNPOS (d, f and h) after 2 (c, d), 5 (e, f) and 12 (g, h)
hours of curing.
As expected, bare SNPs were stiff, with a stiffness value of 34 ± 0.11 N m-1 (Figure 2). At the
beginning of the curing reaction, the SNPENZ-OS were also stiff with an average value of 14 ± 0.02
N m-1. After 5 hours of curing, the stiffness value dropped to 6 mN m-1 with a moderately broader
distribution. The softening effect of the organosilica-protein layer continued until the SNPENZ-OS
reached a value as low as 0.5 mN m-1 after 12 hours, which then remained constant for several
days. By contrast, the SNPOS did not exhibit such a trend. The organosilica layer in these reference
particles was soft, with a value of 0.28 N m-1 after termination of the layer growth reaction, which
did not change significantly over the time period of the curing.
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 condensation
0
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Force (nN)
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reaction. In the present case, one could assume that the initial stiff layer was predominantly
stabilized by hydrogen bonds (H-bonds) and ionic interactions of the silanes of short polysiloxanes
with the surface of the protein; this layer became softer through the formation of Si-O-Si bonds.
Regarding the change of enzymatic activity, two hypotheses could explain the recovery of enzyme
activity during the curing/softening of the organosilica layer. The first one was 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 was that the soft environment of the organosilica layer
allowed the protein to acquire a 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 25°C (Figure 3).
Although the maximum velocity of SNPENZ-OS increased over curing time, the apparent Michaelis-
Menten constant Km
app remained relatively unchanged with values averaging 4 mM, similar to that
of the native enzyme (Figure 3). These results allowed us to rule out the possibility that the recovery
of enzyme activity was due to an increase in the porosity of the protective organosilica layer.
Indeed, in that case, the Km
app values would have varied while the Vm
app should have remained
constant. Therefore, our results provide clear evidence that the recovery of enzyme activity was
due to a favorable change in enzyme conformation enabled by the softness of its protective layer.
Figure 3 Recovery of enzymatic activity during the curing period. (a) SNPENZ-OS catalyzed ONPG hydrolysis
reaction velocity; the results obtained for longer curing durations are similar to that obtained after 480 min, and are
omitted for clarity of the figure. (b) Apparent maximum velocity values (Vm
app) were extracted from the kinetic data
using the Lineweaver-Burk plot as a function of the curing time; the insert shows the evolution of the Km
app values
measured for the same ONPG concentrations.
The recyclability of SNPENZ-OS was tested by repetitive cycles of centrifugation/re-suspension in
fresh buffer. No relevant loss of activity was measured after 10 repetitive cycles. We also applied

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a series of stress conditions to SNPENZ-OS (Figure 4). The biocatalytic hydrolytic activity of SNPENZ-
OS, SNPENZ and free β-gal were tested at increasing reaction temperatures. The activity of the
SNPENZ and β-gal exhibited a similar trend, with more than 20% of activity loss at 45°C; 53% at
50°C, 79% at 55°C and as much as 96% at 60°C (Figure 4). In contrast, the behavior of SNPENZ-
OS was very different: the biocatalytic activity increased to 118%, 123% and 114% at 45°C, 50°C
and 55°C, respectively. Moreover, SNPENZ-OS preserved 88% of its catalytic activity at 60 °C. This
gain of activity could be explained by a protective effect of the organosilica layer shielding the
enzyme, along with an increase in the kinetic energy of the substrate molecules, resulting in an
overall increase in the biocatalytic turnover rate.
Figure 4. Physical, chaotropic and biochemical stress tests. (a) Relative activity of SNPENZ-OS (black squares),
soluble β-gal (black triangles) and SNPENZ (white triangles) measured at increasing reaction temperatures. (b)
Relative activity of SNPENZ-OS, soluble β-gal and SNPENZ measured after increasing incubation time at 50°C. (c)
Relative activity of SNPENZ-OS (grey bars), soluble β-gal (black bars) and SNPENZ (white bars) after freeze-thaw
cycles. (d) Relative activity of SNPENZ-OS, soluble β-gal and SNPENZ after ultrasound treatment at increasing
durations. (e) Relative activity of SNPENZ-OS, soluble β-gal and SNPENZ measured after incubation in solutions of
different pH value. (f) Relative activity of SNPENZ-OS, soluble β-gal and SNPENZ measured in presence of 6 M urea
or (g) 1% SDS. (h) Relative activity of SNPENZ-OS, soluble β-gal and SNPENZ after 1 hour protease treatment.
To confirm this hypothesis and to gain further insights into the thermal stability of SNPENZ-OS, we
incubated them at 50°C for increasing durations of time and measured their biocatalytic activity at
40°C. Whereas both SNPENZ and β-gal lost more than 94% and 97% of activity after only 10 min

8
and 20 min of incubation, respectively, the decay in the biocatalytic activity of SNPENZ-OS was much
slower, with only 25% and 40% of activity loss after 10 and 20 min of incubation, respectively;
more than 25% activity was preserved even after 60 min of incubation. To investigate further this
thermal protective effect, we submitted the SNPENZ-OS, SNPENZ and β-gal to freezing-thawing
cycles. The results showed that while SNPENZ and β-gal had already lost 45% and 25% of their
activity after the first cycle, respectively, the loss for SNPENZ-OS was only 5%. While both SNPENZ
and β-gal experienced a gradual loss of biocatalytic activity with each cycle, reaching values of
15% and 38%, respectively, after 5 cycles, SNPENZ-OS remained relatively unchanged with a
biocatalytic activity of 95%.
As an additional physical stress test, we submitted the particles to ultrasound. The particles were
incubated in phosphate buffer in an ultrasonic bath at 25°C for increasing durations of time.
Although the biocatalytic activity of both SNPENZ and β-gal decayed quite rapidly over time, the
loss of activity of SNPENZ-OS was very moderate; it conserved 88% of activity after 40 minutes of
treatment (18% activity was conserved for β-gal and 5% for SNPENZ) (Figure 4).
To assess the resistance of the SNPENZ-OS to pH variation, they were incubated for 15 min at
different pH values. For values lower than the optimum pH value of the enzyme (6.5), SNPENZ and
β-gal have a similar trend with a loss of more than 40% activity at pH 5 and 100% at pH 4. By
contrast, SNPENZ-OS was more stable and lost only 29% and 71% of activity for the same pH values.
When stored at pH 3, SNPENZ-OS, SNPENZ and β-gal all lost more than 97% of their initial activity.
At pH values higher than the optimum value, SNPENZ lost more than 45% and 75% of activity at
pH 7 and pH 8, respectively, while SNPENZ-OS lost only 10 and 15% of activity. At these pH values,
the most stable system is the soluble β-gal, which retained 100% of activity. At pH 9 and pH 10,
SNPENZ and β-gal both lost more than 95% of activity, whereas SNPENZ-OS lost only 19% and 64%,
respectively. For higher pH values, the hydrolytic activity of SNPENZ and β-gal was reduced to 0%,
whereas SNPENZ-OS retained 7% of activity. Overall, these results clearly demonstrate that the
organosilica layer had a beneficial effect on the resilience of the enzyme to pH changes.
We also studied the effect of the organosilica layer against chaotropic stress using urea (6M) and
sodium dodecylsulfate (1%). The activity was measured directly in the denaturing conditions. In
the case of urea, neither SNPENZ and β-gal exhibited any biocatalytic activity but 12% of activity

9
could be measured for SNPENZ-OS. Urea acts as a potent H-bond donor and acceptor,[11] it can also
act as an enzyme inhibitor by forming H-bonds with important residues located in the active site of
the enzyme. In the present case, one of the effects of the protective layer could be to conserve the
active conformation of the protein; the loss in enzyme activity may be due in part to inhibition
caused by the high concentration of urea.
In the case of SDS treatment, we showed that, as expected, the free β-gal was completely inactive
in these conditions. While the activity of SNPENZ was as low as 7%, SNPENZ-OS maintained 45% of
activity. The final assay that was performed was the stability against protease digestion. As
expected, after incubation with Proteinase K for 60 min, no more activity could be measured for β-
gal and a loss of 30% of activity was measured for SNPENZ, suggesting that the accessibility of the
immobilized enzyme partially hindered protease digestion. In the case of SNPENZ-OS, no loss of
biocatalytic activity was measured, confirming that the shielded enzyme is not accessible to the
protease.
In order to test the effiency of the developed nanobiocatalysts in a real matrix; we tested the
hydrolytic activity of the shielded β-galactosidase for its natural substrate, lactose, in skim milk
using 14C-radioactively-labeled lactose. The experimental results showed that the shielded
enzyme catalyzed the conversion of lactose into glucose and galactose 30% more efficiently than
the soluble β-gal (Supporting Information).
In order to assess the versatility of the developed shielding strategy, we tested different enzymes;
i.e. acid phosphatase, laccase, alcohol dehydrogenase, glutamate-oxalate transaminase (Table 1
and Supporting Information).
Table 1: Immobilization and shielding of different enzymes
Enzyme
Enzyme size (nm)
Shell
thickness
(nm)
Specific activity*
Acid phosphatase
4.0 × 6.0 × 7.5
12
23.01
Laccase
5.7 × 11.0 × 16.5
19
11.02
Alcohol
dehydrogenase
10.5 × 10 × 5.5
13
1.53
Aspartate
aminotransferase
10.5 × 6.3 × 5.4
18
75.04
**Absolute activity (mU) per mg of nanoparticles measured using the following substrates: 1p-nitrophenyl-phosphate; 22, 2-azino-bis (3-
ethylbenzothiazoline-6-sulphonic acid); 32-amino-benzyl alcohol, NAD+; 4L-aspartate, 2-oxoglutarate.
For all tested enzymes, the shielding strategy turned out to be successful. More specifically,
another hydrolase enzyme, namely acid phosphatase, was protected with an organosilica shell of

10
12 nm sufficient to cover the whole enzyme. It showed a relevant biocatalytic activity of 23 mU/mg
of SNPs. The activity of a shielded laccase, which is a copper-containing oxidase that requires
oxygen as second substrate, demonstrated that the shielding organosilica layer does not prevent
oxygen diffusion and that the protected enzyme is active after protection. In the case of a
nicotinamide adenine dinucleotide (NAD+)-dependant alcohol dehydrogenase, the activity
measured when the enzyme is fully shielded in the protective organosilica layer showed that this
shell does not hinder the diffusion of the cofactor. Finally, the activity recovered for an aspartate
transaminase demonstrated that the amine exchange between aspartic and oxoglutaric acids is
not hampered in the protective organosilica shell.
In summary, we developed a versatile strategy to produce nanobiocatalysts with enhanced stability
that imparts to the enzyme resistance to a large set of denaturing stresses.
Acknowledgements
Financial supports from the Swiss Nanoscience Institute (SNI) through the NanoArgovia program
(NanoZyme project), the Swiss commission for technology and innovation (grant agreement
16437.1 PFEN-NM) the EU-Eurostar program (grant agreement E!6894) and the Swiss State
Secretariat for Education, Research and Innovation (EU-H2020 INMARE project) are gratefully
acknowledged.
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Angew. Chem. 2003, 115, 3458-3459.
[4] a) M. Misson, H. Zhang, B. Jin, J. R. Soc. Interface 2015, 12, 20140891; b) L. Cao, Carrier-bound
immobilized enzymes: principles, application and design, John Wiley & Sons, Weinheim, 2006.
[5] K. Liang, R. Ricco, C. M. Doherty, M. J. Styles, S. Bell, N. Kirby, S. Mudie, D. Haylock, A. J. Hill, C.
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Ciriminna, A. Fidalgo, V. Pandarus, F. Béland, L. M. Ilharco, M. Pagliaro, Chem. Rev. 2013, 113,
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Angew. Chem. 1995, 107, 373-376; d) M. T. Reetz, A. Zonta, J. Simpelkamp, Biotechnol. Bioeng.
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Supporting Information
Content
General .................................................................................................................................................................. 13
β-galactosidase purification and SDS-PAGE ......................................................................................................... 13
Enzyme shielding ................................................................................................................................................... 13
Enzymatic assay and kinetic studies ...................................................................................................................... 13
Atomic Force Microscopy ....................................................................................................................................... 13
Scanning electron microscopy and particle size measurement ............................................................................. 14
Enzymatic assay of the shielded lactase in skim milk ............................................................................................ 14
Physical, chaotropic and biochemical stress tests ................................................................................................. 14
Additional enzyme shielding and activity assays ................................................................................................... 15
Table 1: Immobilization and shielding of different enzymes. Enzymes features, catalytic reactions, reaction
conditions and organosilica layer thickness of the different enzymes. .................................................................. 15
Figure S1 ................................................................................................................................................................ 15
References ............................................................................................................................................................. 16

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General
Tetraethyl orthosilicate (TEOS, ≥99%), (3-aminopropyl)triethoxysilane (APTES, ≥ 98%), ammonium hydroxide (ACS
grade, 28-30%), ethanol (ACS grade, anhydrous), glutaraldehyde (Grade I, 25% in water), β-galactosidase (β-gal) from
Kluyveromyces lactis and Proteinase K from Tritirachium album were purchased from Sigma-Aldrich (Switzerland). All
chemicals were used without further purification. Nanopure water (resistivity ≥ 18 MΩ.cm) was produced with a Millipore
®Synergy purification system. Silica nanoparticles (SNPs) were prepared using the reported procedure.[1]
β-galactosidase purification and SDS-PAGE
10 mL β-gal were dialysed against 2 L dialysis buffer (100 mM phosphate, 5 mM MgCl2, 100 mM NaCl, pH 6.5) using a
®SnakeSkin dialysis tube (10K MWCO) at 4°C for 24 h. The dialyzed enzyme was analysed by sodium-dodecyl-sulphate
polyacrylamide gel electrophoresis (SDS-PAGE). As reported in the literature, the commercial enzyme solution shows
the main protein band at ca. 100 kDa and additional bands at ca. 50 and 30 kDa. These additional bands have been
shown to react with anti-β-gal antibodies and have been attributed to fragmentation products of the native enzyme.[2] From
this analysis, one can assume that the enzyme represents 100% of the protein content.
Enzyme shielding
All reactions were carried out in 20 mL glass vials under moderate magnetic stirring (400 rpm). To 18 mL of a suspension
of 3.2 mg mL-1 of SNPs, at 20°C, were added 11 µL (0.047 mmol) of APTES. After 90 min of reaction, the suspension
was centrifuged at 3220 g for 10 min, the resulting pellet was suspended in water. This operation (hereinafter called
“washing cycle”) was repeated twice. The so-modified SNPs were then incubated during 30 min in 18 mL of 0.1% (v/v)
aqueous glutaraldehyde and subsequently submitted to two washing cycles in water. The resulting pellet was suspended
in 18 mL of MES buffer (10 mM MES, 5 mM MgCl2, pH 6.2) and reacted, at 20°C, with β-gal (at 0.035 mg mL-1,
corresponding to 601 mU mL-1) for 1 h to produce SNPENZ. Subsequently and without intermediate washing, 86 µL (0.386
mmol) of TEOS were added to the reaction mixture and allowed to react for 1 h. Consequently, 21 µL APTES (0.088
mmol) were added. Aliquots of 1 mL (SNPENZ-OS) were collected at increasing reaction durations (3, 6, 9, 12 and 15 h after
the addition of APTES), washed in nanopure water, suspended in 1 mL of nanopure water and imaged by FE-SEM. In
parallel, a reference sample (SNPOS), was prepared following the same synthetic procedure but omitting the enzyme
addition. The silane polycondensation reaction was stopped after 15 h by washing the SNPENZ-OS and the SNPOS in activity
buffer (AB) containing 100 mM potassium phosphate, 5 mM MgCl2, pH 6.5. SNPs were then suspended in AB, maintained
at 25°C for 24 h and finally stored at 4°C. Particle sizes were measured on micrographs taken at a magnification of
150,000X using the Olympus ®analySIS software package. The catalytic activity of SNPENZ-OS was determined as
described in the following section.
Enzymatic assay and kinetic studies
In a typical β-gal activity assay, O-nitrophenyl-β-D-galactopyranoside (ONPG) was used as substrate. The assay was
performed at 40°C under shaking at 650 rpm in AB. 190 µL of ONPG (21 mM) in AB were equilibrated for 5 min in a
®Thermomixer (Eppendorf) at 40°C. 10 µL of SNPENZ-OS, soluble β-gal or SNPENZ-REF were added to the ONPG solution
and the reactions mixtures were maintained under shaking (650 rpm) at 40°C for 5 min. The ONPG hydrolysis was
stopped by the addition of 200 µL of 1 M Na2CO3 solution. After centrifugation at 16100 g for 90 sec, the amount of the
produced o-nitrophenol (ONP) was determined by measuring the absorbance at 420 nm (OD 420) of 200 µL solution in
a 96 well-plate using a Synergy H1 (BioTek). The catalytic activities were calculated using the molar extinction coefficient
of o-nitrophenol (ɛ420nm = 4300 M-1 cm-1) measured by preparing a standard curve using the same buffer.
The apparent catalytic parameters (Vmax
app and Km
app) of SNPENZ-OS, were measured performing a typical activity assay with
increasing ONPG quantities (at final concentrations 0.5-20 mM respectively). Vmax
app and Km
app were extracted from the
double reciprocal (Lineweaver-Burke) plot.
Atomic Force Microscopy
A solution of SNPs (3.2 mg mL-1) in nanopure water was prepared and a volume of 2 µL was spread on freshly cleaved
mica. Imaging was performed in contact mode in air using a ®NTEGRA Prima (NT-MDT) system equipped with gold-
coated silicon rectangular cantilevers (length 95 μm, width 30 μm, NT-MDT). Cantilever spring constants were determined
experimentally performing thermal fluctuations measurements. Force-distance spectroscopy was obtained by measuring
cantilever deflection as a function of the scanner z-piezo tube extension. Stiffness was measured as the slope of the
linear part of force-displacement curve.

14
Scanning electron microscopy and particle size measurement
Particles were imaged using a Zeiss SUPRA® 40VP scanning electron microscope. A 2 µL drop of each sample was
placed on freshly cleaved mica substrates, dried under ambient conditions, and sputter-coated with a gold-platinum alloy
for 15 s at 10 mA. Secondary electron micrographs were acquired using the InLens mode with an accelerating voltage of
20 kV. Particle sizes were measured on micrographs acquired at a magnification of 150,000 X using the ®AnalySIS
software package. 100 measurements were carried out for each sample.
Enzymatic assay of the shielded lactase in skim milk
27 µL of radioactive lactose stock (3.7 MBq Lactose [D-glucose-1-14C], Hartmann Analytic GmbH, Germany) were added
to 428 µL of skim milk and equilibrated at 40°C for 30 min in a shaker at 500 rpm (Thermomixer Comfort, Eppendorf AG,
Germany). 45 µL of shielded enzyme (1.44 U mL-1) or soluble β-gal (1.44 U mL-1) or buffer solution (100 mM K2HPO4, 5
mM MgCl2, pH 6.5) were added and incubated at 40°C in a shaker at 500 rpm. 25 µL aliquots were collected every 60
min for 9 hours and after 20 and 24 hours and added to a volume of 50 µL acetonitrile in order to precipitate the soluble
proteins. After centrifugation at 21.500 g for 10 min (Himac CT 15RE, Hitachi Koki Co., Ltd Japan) the supernatant was
transferred into GC-inlet vials (Infochroma AG, Switzerland) for high-performance liquid chromatography (HPLC)
measurement. HPLC measurements of the hydrolysed lactose were performed using an Agilent Technologies HPLC 1200
Series coupled to a radioisotope ®Ramona Star detector (Raytest GmbH, Germany). The separation was carried out on
a GRACE Prevail Carbohydrate ES 5u column (250 mm × 4.6 mm). Elution was performed using acetonitrile/water 75:25
(v/v) and a flow rate of 1.0 ml min-1. The experiment was performed in duplicates and the results are shown in Figure S1.
Physical, chaotropic and biochemical stress tests
Activity assay at increasing temperatures - 190 µL of ONPG (21 mM) in AB were equilibrated for 5 min at 45, 50, 55 and
60°C respectively. 10 µL of SNPENZ-OS, soluble β-gal or SNPENZ-REF were added to the ONPG solution and the reactions
mixtures were maintained under shaking (650 rpm) at the appropriate temperature for 5 min. The reaction was
consequently stopped with 1M Na2CO3 and measured as described above.
Enzyme stability at 50°C - 200 µL of SNPENZ-OS, soluble β-gal and SNPENZ-REF were incubated at 50°C for 60 min under
shaking at 650 rpm. 10 µL aliquots were collected every 10 min, added to 190 µL of ONPG (21 mM) in AB and measured
for hydrolytic activity at 40°C as described above.
Freeze-thaw cycles - 100 µL of SNPREF-OS, soluble β-gal and SNPENZ-REF were stored at -20°C for 60 min. Subsequently,
the samples were warmed at 25°C for 30 minutes; 10 µL aliquots were collected and the ONPG hydrolysis was measured
as described above. Four additional freeze-thaw cycles were repeated.
Ultrasonic treatment - 100 µL of SNPENZ-OS, soluble β-gal and SNPENZ-REF were incubated at 25°C in an ultrasonic bath
(Elma, 37 kHz) for 40 min. 10 µL aliquots were collected every 10 min, added to 190 µL of AB and measured for ONPG
hydrolysis as described above.
Incubation in different pH solutions - 30 µL of SNPENZ-OS and 50 µL of SNPENZ-REF were centrifuged at 16100 g for 90 sec
and 400 g for 3 min respectively [NB: a faster centrifugation of the SNPENZ-REF induced the formation of a hard, compact
pellet that could not be suspended with a mild mixing and was not suitable for SNPs suspension]. The resulting pellets
were suspended in 30 and 50 µL respectively of solutions at different pH values (100 mM citrate buffer, 5 mM MgCl2 for
pH values 3-5; 100 mM phosphate buffer, 5 mM MgCl2 for pH values 6-8; 100 mM ethanolamine buffer, 5 mM MgCl2 for
pH values 9-11). After 15 min incubation at 25°C under shaking at 650 rpm, samples were centrifuged and suspended in
30 and 50 µL respectively of AB. 10 µL aliquots were thus added to 190 µL ONPG and catalytic activities were measured
as described above. Similarly, 10 µL of β-gal were incubated with 90 µL of solutions at different pH values (3-11) for 15
min at 25°C under shaking at 650 rpm. After a 1:100 dilution, the ONPG hydrolysis was measured [NB: with such dilution,
the effects on the activity assay of pH and composition of the buffer used for the stress assay was neglected]. The catalytic
activities were calculated using the molar extinction coefficient of o-nitrophenol (ɛ420nm = 4300 M-1 cm-1).
Activity assay in presence of 6 M urea or 1% SDS - 190 µL of ONPG (21 mM) in AB containing 6.6 M urea or 1.1% SDS
respectively, were equilibrated for 5 min at 40°C. 10 µL of SNPENZ-OS, soluble β-gal or SNPENZ-REF were added and the
catalytic activity was measured as described above. The catalytic activities were calculated using the molar extinction
coefficient of o-nitrophenol (ɛ420nm = 4300 M-1 cm-1).
Proteinase treatment - 250 µL of SNPENZ-OS or SNPENZ-REF were centrifuged at 16100 g for 90 sec and 400 g for 3 min
respectively and resulting pellets suspended in 250 µL of digestion buffer (10 mM potassium phosphate, 5 mM MgCl2, pH
7.5) containing 1 mg mL-1 Proteinase K. Similarly, β-gal was incubated with the proteinase solution. The protease digestion
was allowed during 60 min at 37°C under shaking at 650 rpm. The catalytic activities of 10 µL SNPENZ-OS, SNPENZ-REF and
soluble β-gal were measured.

15
Additional enzyme shielding and activity assays
Different enzymes (acid phosphatase, laccase, alcohol dehydrogenase and aspartate transaminase (AST)) were
immobilized on silica nanoparticles and shielded with an organosilica layer following the general procedure previously
described (representative micrographs are showed in Figure S2). The catalytic activities of the shielded enzymes were
measured by means of established catalytic assays where the reaction products were spectrophotometrically measurable
at different wavelengths (Table 1)[3-6].
Table 2: Immobilization and shielding of different enzymes. Enzymes features, catalytic reactions, reaction conditions
and organosilica layer thickness of the different enzymes.
Enzyme
(EC; organism)
Enzyme Features
(weight, size)
Catalyzed reaction
Reaction
conditions
Ɛproduct
(mM-1cm-1)
Shell
(nm)
Acid phosphatase
(EC 3.1.3.2)
S. tuberosum
Homo-dimer
472 kDa
4.0 x 6.0 x 7.5 nm
p-nitrophenyl-phosphate →
p-nitrophenol[3]
25 mM acetate
pH 5.2; 37°C
16’825a
12
Laccase
EC 1.10.3.2
T. arenaria
Homo-dimer
160 kDa
5.7 x 11.0 x 16.5 nm
ABTS + O2 → ABTS•+ + H2O[4]
80 mM citric acid,
40 mM Na2HPO4,
pH 3; 25°C
16’700b
19
Alcohol
dehydrogenase
EC 1.1.1.1
S. cerevisiae
Homo-tetramer
146 kDa
10.5 x 10 x 5.5 nm
2-amino-benzyl alcohol + NAD+ →
2-amino-benzaldehyde + NADH[5]
21 mM Na2H2P2O7
0.33 mM Na2HPO4
pH 8.8; 25°C
3’826c
13
AST*
EC 2.6.1.1
S. scrofa
Homo-dimer
92.7 kDa
10.5 x 6.3 x 5.4 nm
L-aspartate + 2-oxoglutarate →
oxaloacetate + L-glutamate[6]
80 mM Na2HPO4
pH 7.5; 37°C
920d
18
* Aspartate aminotransferase; reactions measured spectrophotometrically at: a405 nm; b420 nm; c360 nm; d460 nm
Figure S1
Figure S1: Lactose hydrolysis in skim milk catalysed by the shielded β-gal (black square) and the soluble
enzyme (black triangle)

16
Figure S2: FE-SEM micrographs of shielded SNPs. A): Acid phosphatase; layer: 12 nm. B): Laccase; layer 19
nm; C): Alcohol dehydrogenase; layer 13 nm; D): glutamic oxaloacetic transaminase; layer 18 nm. Scale bars
represent 100 nm.
References
[1] A. Cumbo, B. Lorber, P. F. X. Corvini, W. Meier, P. Shahgaldian, Nat. Commun. 2013, 4, 1503.
[2] D. F. M. Neri, V. M. Balcao, M. G. Carneiro-Da-Cunha, L. B. Carvalho, J. A. Teixeira, Catal. Commun. 2008, 9,
2334-2339.
[3] Y. Sugiura, H. Kawabe, H. Tanaka, S. Fujimoto, A. Ohara, J. Biol. Chem. 1981, 256, 664-670.
[4] H. Cabana, J. P. Jones, S. N. Agathos, Eng. Life Sci. 2007, 7, 429-456.
[5] J. H. R. Kagi, B. L. Vallee, J. Biol. Chem. 1960, 235, 3188-3192.
[6] R. Katoch, Analytical techniques in biochemistry and molecular biology, Springer, New York, 2011