nanomaterials

Article
Adsorption of Cellulase on Wrinkled Silica
Nanoparticles with Enhanced Inter-Wrinkle Distance
Aniello Costantini 1 , Virginia Venezia 1 , Giulio Pota 1 , Aurelio Bifulco 1 , Valeria Califano 2, *
and Filomena Sannino 3
1 Department of Chemical Engineering, Materials and Industrial Production, Università degli Studi di Napoli
Federico II, P.le Tecchio 80, 80125 Napoli, Italy; anicosta@unina.it (A.C.); virginia.venezia@unina.it (V.V.);
giulio.pota@unina.it (G.P.); aurelio.bifulco@unina.it (A.B.)
2 Istituto Motori, Italian National Research Council, via G. Marconi 4, 80125 Napoli, Italy
3 Department of Agricultural Sciences, Università degli Studi di Napoli Federico II, Via Università 100,
80055 Portici, Italy; fsannino@unina.it
* Correspondence: v.califano@im.cnr.it; Tel.: +39-081-7177-226




Received: 12 July 2020; Accepted: 7 September 2020; Published: 10 September 2020


Abstract: Mesoporous silica materials offer a unique opportunity for enzyme immobilization thanks
to their properties, such as tuneable pore size, large surface area and easy functionalization. However,
a significant enhancement of cellulase enzyme activity entrapped inside the silica pores still represents
a challenge. In this work, we immobilized cellulase by adsorption on wrinkled silica nanoparticles
(WSNs), obtaining an active and stable biocatalyst. We used pentanol as co-solvent to synthesize WSNs
with enhanced inter-wrinkle distance in order to improve cellulase hosting. The physical-chemical and
morphological characterization of WSNs and cellulase/WSNs was performed by thermogravimetric
(TG), Fourier transform infrared (FT-IR), and transmission electron microscopy (TEM) analyses.
The obtained results showed that this matrix generates a favourable microenvironment for hosting
cellulase. The results of the catalytic assays and operational stability confirmed the key role of size,
morphology and distribution of the pores in the successful outcome of the cellulase immobilization
process. The immobilization procedure used allowed preserving most of the secondary structure of
the enzyme and, consequently, its catalytic activity. Moreover, the same value of glucose yield was
observed for five consecutive runs, showing a high operational stability of the biocatalyst.

Keywords: enzyme immobilization; cellulase; wrinkled silica; mesoporous nanoparticles

1. Introduction
Over the last Century, the scientific community have devolved a huge interest to address the
problems of fossil fuel depletion and climate change [1]. These issues make the alternative sustainable
and renewable energy sources research necessary [2]. In this frame, bioethanol and biodiesel represent
a very good alternative to fossil fuels. Cellulose represents a broad platform to obtain chemicals and
energy. Cellulose is a polymer composed of glucose units linked by 1,4-β-glycosidic bond [3]. It is
present in large amount in the biosphere [4,5] and is the main component of lignocellulosic biomass.
The use agricultural and forestry waste as raw materials is a valid strategy to produce bioethanol.
Enzymatic hydrolysis of cellulose is very advantageous in respect to chemical hydrolysis, due to the
milder operative conditions. Cellulase are a class of enzymes devoted to cellulose hydrolysis. They are
composed by three different classes of enzymes, endoglucanase, exoglucanase and β-glucosidase,
which act synergistically and sequentially on the polymeric chains of cellulose. The use of enzymes
has some drawbacks, due to their high cost and their fragile nature. To address these problems,
one possible solution is their immobilization on insoluble supports.

Nanomaterials 2020, 10, 1799; doi:10.3390/nano10091799 www.mdpi.com/journal/nanomaterials

Nanomaterials 2020, 10, 1799 2 of 14

Mesoporous silica can be used for many applications such as drug delivery, fluorescence biological
probes [6,7] and are suitable supports for enzyme immobilization [8–10]. They have high chemical,
mechanical and thermal stability. Their high surface area and pore volume allow for large enzyme
loadings and a good dispersion inside the pores. In this regard, the matching between the pore
size and the molecular diameter of the enzyme plays a key-role: the pores must be large enough to
host the enzyme and maintain its mobility/flexibility within the cavities. Still there should be space
enough to allow the substrate diffusion of to the active sites of the enzymes. However, too large
pores can give rise to enzyme leaching and the loss of its conformational flexibility due to crowding
effects. Takimoto et al. [11] immobilized cellulase from Trichoderma viride in Santa Barbara Amorphous
15 (SBA-15) with different pore size—5.4, 8.9 and 11 nm. They demonstrated that the activity of
immobilised cellulase were maximized by the matching of the silica pore size and the molecular size
of cellulase. In fact, when the pore size was just enough to host the cellulase, the enzyme remained
close to the pore entrance, allowing an easy accessibly of its active site. Chen et al. [12] synthesized
two mesoporous silica with different pore size of 17.6 nm (MS-17.6) and 3.8 nm (MS-3.8). They used
these mesoporous materials to immobilize cellulase from Acremonium by pure physical adsorption.
MS-3.8 displayed a higher specific activity than MS-17.6. Again, this was attributed to the sticking
of the enzyme at the pore entrance, increasing the accessibility of the active site and preserving the
protein native structure. In MS-17.6, the dense and ordered arrangement inside the pores hampered
the conformational flexibility of cellulase, lowering its activity.
Surface functionalization is often necessary to improve the interactions between the enzyme
and the silica support, and to increase the loading, since cellulase has large hydrophobic areas.
Many researchers have functionalised mesoporous silica with organic compounds such-as vinyl
(CH=CH2 ) [13] and thiol (–SH) in SBA-15 and FDU-12 (FDU coming from Fudan University) [14,15].
FDU-12 materials, due to their big pores and three-dimensional connectivity, represent a valid support
to host bulky enzymes such as cellulase, avoiding mass diffusion limitation. Vinyl functionalized
FDU-12 showed significant improvements of the cellulase activity [16]. The hydrophobic characteristics
within silica materials induced a high loading amount and created a favourable microenvironment for
catalysis. Cellulase immobilized on functionalized mesoporous cellular foam (MCF) showed higher
activity than the free one due to the proven activity of the functional groups on surface modified MCF
toward the hydrolysis of carboxymethylcellulose sodium salt (CMC) [17]. Although there are many
studies on the immobilization of cellulase into mesoporous silica [18], significant enhancement of
immobilized cellulase activity remains a challenge. Actually, adsorption from a mixture is difficult to
control due to the varying kinetics of adsorption, variations in the degree of unfolding, and competitive
binding effects [19]. Furthermore, cellulase establishes a strong interaction with silica. This interaction
inhibits cellulose hydrolysis [20].
The successful immobilization of enzymes on porous silica samples is largely determined by the
physicochemical properties of the porous hosts, such as morphology, particle size, composition and
pore size [21]. For example, Hisamatsu et al. [22] studied the adsorption of α-amylase on mesoporous
silica with different pore sizes, morphology and surface properties. They found that the amount
of encapsulated enzyme increased with increasing pore size of the silica support. The presence of
large pores with disordered arrangements and smaller particles size favoured the enzyme adsorption.
Lei et al. [23] studied the influence of SBA-15 support morphology on the immobilization behaviour
of lysozyme finding that rod-like SBA-15 exhibited higher loadings and a faster adsorption rate
than spherical SBA-15. Zhou et al. [24] investigated the influence of particle morphology on the
immobilization of Candida rugosa lipase (CRL). CRL immobilization on vesicle-like silica possessed
higher thermal stability and reusability compared to CRL immobilized on rod-like silica.
Recently, silica nanoparticles with radial wrinkle structure (WSNs) were synthesized [25,26].
WSNs have wrinkles that widens radially outward enhancing the accessibility of functional materials
inside the pores and reducing pore blocking. They were used for lipase [27] and β-glucosidase [28–30]
immobilization. Lipase immobilized in WSNs showed higher activity than the free enzyme. The better
Nanomaterials 2020, 10, 1799 3 of 14

performance was attributed to the radially aligned mesopores of WSNs, allowing dispersion of
dispersing active catalytic sites on large internal surface and pores [27]. The immobilised β-glucosidase
showed better catalytic performance than free enzyme and high thermal and operational stability.
This was attributed to the peculiar morphology of the pores and their hierarchical structure allowing
for optimal hosting of β-glucosidase and avoiding diffusion problems of the substrate [28,29].
WSNs are synthetized by the microemulsion method. The microemulsion generally contains
iso-propanol as co-solvent. It was found that the chain length of the co-solvent influences
the inter-wrinkle distances [25]. In particular, by increasing the chain length of the co-solvent,
the inter-wrinkle distance increases. In our study, pentanol was used as co-solvent to synthesize WSNs
with enhanced inter-wrinkle distance. The aim was to increase the loading and activity of cellulase on
mesoporous silica nanoparticles without the need to functionalize the support with organic compounds.

2. Materials and Methods

2.1. Materials
Tetraethylorthosilicate (TEOS), urea, cetyltrimethylammonium bromide (CTAB), cyclohexane,
pentanol, acetone, 37% hydrochloric acid solution, citric acid, and trisodium citrate were used to
synthesize WSNs with pentanol (WSN-p). WSNs with isopropanol (WSN-ipa) were prepared in the
same way as WSN-p, but using isopropanol instead of pentanol. All chemicals were purchased from
Sigma-Aldrich (Milan, Italy). For enzyme physical adsorption and kinetic measurements, cellulase
from Tricoderma Reesei and glucose oxidase-peroxidase (GOD-POD) assay kit from Sigma-Aldrich
(Milan, Italy) were used. Citrate buffer solution at pH 5.0 was prepared by dissolving 3.9 mg of citric
acid in 205 mL of bidistilled water, and 8.7 mg of trisodium citrate in 295 mL of bidistilled water.
The two solutions were mixed and the volume was brought to 1 L by diluting with bidistilled water.

2.2. Nanoparticle Synthesis

WSNs synthesis route was carried out following the procedure reported in literature [25], using
a water/surfactant/oil ternary system as structural template. CTAB, aqueous urea and pentanol or
isopropanol/cyclohexane were used as surfactant, water phase, co-solvent and oil phase, respectively.
TEOS was chosen as silica precursor. The reaction mixture was vigorously stirred for 30 min at room
temperature, heated up to 70 ◦ C and kept overnight in a closed vessel to avoid solvent evaporation.
Three centrifugations were carried out to recover the nanoparticles. The surfactant was removed by
chemical extraction with ethanol/hydrochloric acid mixture. A certain amount of WSN-ipa was calcined
(WSN-Calc) into a tubular oven (2 ◦ C/min from 25 to 500 ◦ C and isotherm at 500 ◦ C for 4 h) (Memmert,
Buechenbach, Germany) to remove the surfactant that was not eliminated by chemical extraction.

2.3. Chemical and Physical Characterization

The morphology of bare WSN and biocatalyst (cellulase/WSN) was evaluated by transmission
electron microscopy (TEM) (PHILIPS EM208S microscope equipped with a Mega View camera for
digital acquisition of images (FEI, Hillsboro, OR, USA).
The organic content of each sample was estimated by thermogravimetric analysis (TGA)
(TA Instrument, New Castle, DE, USA) using a thermogravimetric apparatus Q600SDT, under
air atmosphere, in a temperature range between 25 and 800 ◦ C and at heating rate of 10 ◦ C/min.
Samples mass were 10 mg, put in a platinum crucible.
A Nicolet Instrument Nexus model equipped with a DTGS KBr (deuterated triglycinesulfate with
potassium bromide windows) detector (Thermo Scientific, Waltham, MA, USA) was used to perform
the FT-IR investigation. IR spectra were recorded in the 4000–400 cm−1 range with a 2 cm−1 resolution.
The IR spectrum of each sample was corrected for the spectrum of blank KBr by mixing 199 mg of KBr
and 1 mg of dried samples powders and pressing into pellets of 13 mm diameter.
Nanomaterials 2020, 10, 1799 4 of 14

2.4. Cellulase Immobilization

The adsorption procedure followed the route described by our research group in a previous
work [29]. Herein, physical immobilization of cellulase into WSN-p was carried out changing
adsorption time, enzyme/support ratio and temperature. In the first case, each sample was prepared
by dissolving 10 mg of enzyme in 8 mL of a 20 mM citric acid/sodium citrate buffer solution at pH 5.0.
Then, in order to obtain enzyme-to-support weight ratio of 0.5, 2 mL of buffer containing 20 mg of
suspended WSN-p were added to the enzyme solution. The final colloidal solutions were gently stirred
at 25 ◦ C for 1 h, 3 h, 6 h and 24 h, respectively. In the second case, physical adsorption was carried
out in the same conditions for 24 h but we fixed four different enzyme-to-support weight ratios: 0.25,
0.50, 0.75, 1. In the third case, the adsorption temperature was set to 25, 30, 40 and 50 ◦ C using an
enzyme-to-support weight ratio of 0.5.
The resulting biocatalysts were recovered by centrifugation at 12,500 rpm for 15 min and washed
twice with the buffer to eliminate the non-adsorbed enzyme molecules.
Further cellulase was adsorbed into WSN-ipa (30 ◦ C, enzyme-to-support weight ratio 0.50, 24 h),
following the same procedure described above. Moreover, the enzyme was immobilized into WSN-Calc
(enzyme-to-support weight ratio 0.50, 24 h).
The TGA curves of all the samples, not reported in this paper, showed a first weight loss below
200 C related to water desorption and a second weight loss starting from 200 ◦ C. The latter can be
◦

attributed to different causes, such as the condensation of surface Si–OH to form Si–O–Si bonds for
WSNs, the degradation of organic component, i.e., the progressive deamination, decarboxylation, and
depolymerisation arising from the breaking of polypeptide bonds for biocatalyst [29]. We evaluated
the amount of immobilized enzyme (IE), subtracting the weight loss of each biocatalyst to the one of
bare support in the range between 200 and 800 ◦ C. Then, we determined the immobilization yield of
cellulase (YI) according to the following equation:

E
YI = (1)
E0

where E0 is the weight of the enzyme used in the adsorption step and E that of the adsorbed enzyme.

2.5. Catalytic Assays

The kinetic behaviour of immobilized cellulase was assessed by measuring the concentration of
glucose produced by the hydrolysis of carboxymethylcellulose sodium salt (CMC), used as substrate.
The specific activity is defined as µmoles of produced glucose over time (minutes) per gram of
immobilized enzyme. CMC was dissolved gradually in 20 mM citric acid/sodium citrate buffer
(at 60 ◦ C, under vigorous stirring) to have a final concentration of 20 mg/mL. Then, a specific volume
of this solution was added to an equal volume of a 20 mg/mL cellulase/WSN-p buffer suspension,
in order to reach a final enzyme and substrate concentration of 1 mg/mL and 10 mg/mL, respectively.
The reaction was carried out at 50 ◦ C, under gentle stirring. Precise volumes of the reaction mixture
were withdrawn at fixed time (30 min, 1 h, 2 h, 6 h and 24 h) put in oven (100 ◦ C, 10 min) to thermally
inactivate the biocatalyst and finally centrifuged (11,500 rpm, 10 min). The supernatant was recovered
and its glucose concentration was determined. A similar procedure was set up for testing the activity
of free cellulase. A specific volume of 20 mg/mL CMC solution was mixed to an equal volume of a
2 mg/mL cellulase buffer solution to obtain the reaction mixture. Just the thermal inactivation of the
biocatalyst was needed for all the withdrawn volumes to be ready for the analysis.
Glucose concentration was evaluated by glucose (GO) assay kit, following the D-glucose
oxidase–peroxidase method [31]. The quantity of glucose originating from the reaction was estimated
by adding 2 mL of glucose-measuring reagent to 1 mL of quenched reaction mixture, previously
diluted. The resulting mixture was kept at 37 ◦ C for 30 min in a thermostatically controlled water
bath. Absorbance (OD) at 540 nm was measured by a spectrometer instrument (Perkin Elmer
Nanomaterials 2020, 10, 1799 5 of 14

Instruments, Lambda 25 UV/Vis, PerkinElmer, Inc., Waltham, MA, USA). Each experiment was
Nanomaterials 2020, 10, x FOR PEER REVIEW 5 of 14
performed in triplicate.
2.6. Operational
2.6. Operational Stability
Stability
The immobilized
The immobilized samplesample cellulase/WSN-p
cellulase/WSN-p was was analysed
analysed inin consecutive
consecutive 2424 h
h reaction
reaction cycles
cycles to
to
evaluate the biocatalyst reusability. Each reaction was carried out under standard assay
evaluate the biocatalyst reusability. Each reaction was carried out under standard assay conditions conditions
(50 ◦ C,
(50 °C,pHpH 5 and
5 and [CMC]
[CMC] 10 mg/mL).
10 mg/mL). Aftercycle,
After each eachthecycle, the was
solution solution was centrifuged
centrifuged and the
and the supernatant
supernatant was analysed by UV-Vis analysis to determine glucose concentration.
was analysed by UV-Vis analysis to determine glucose concentration. The hydrolysis yield after the The hydrolysis
yieldcycle
first afterofthe
thefirst cycle was
reaction of the reaction
used as thewas used as(100%
reference the reference (100% conversion).
conversion).

3. Results
3. Results and
and Discussion
Discussion

3.1.
3.1. Choice of Supports
The
The morphology
morphology of
of the
the two synthesized
synthesized siliceous
siliceous supports
supports is
is illustrated
illustrated in
in the TEM micrographs
micrographs
of
of Figure
Figure 1.
1.

Figure 1.
Figure 1. Transmission
Transmissionelectron
electronmicroscopy
microscopy(TEM)
(TEM) micrographs
micrographs of WSNs
of WSNs withwith isopropanol
isopropanol (WSN-
(WSN-ipa)
ipa) (top)
(top) and WSNs
and WSNs withwith pentanol
pentanol (WSN-p)
(WSN-p) (bottom)
(bottom) at lower
at lower (left)(left)
and and higher
higher (right)
(right) magnification.
magnification.

In both cases, the particles are made of silica fibres that spread radially inside out of the particle.
In
However, the
However, the WSN-p
WSN-p show
show aa lower
lower density
density and
and appear
appear more
morebranched.
branched. The
The inter-wrinkle
inter-wrinkle distance
distance
is greater.
is greater.
Figure 22 shows
Figure showsthe
theTEM
TEMmicrograph
micrographofofWSN-p
WSN-p atat lower
lower magnification.
magnification. It can
It can be observed
be observed thatthat
the
the particles
particles are quite
are quite monodisperse,
monodisperse, withwith
sizessizes ranging
ranging fromfrom
aboutabout 300
300 to to about
about 500 nm.
500 nm.
WSN-ipa were also calcined, with the aim of removing the surfactant not extracted by the acid
treatment and allow for more space between the silica fibres. From the spectra shown in Figure 3, no
particular difference is noted in the silica structure of the three samples. The calcined sample shows
the disappearance of the bands related to organic material, at about 1400 cm−1 and between 2800 and
3000 cm−1 .
 Nanomaterials 2020, 10, 1799 6 of 14
Nanomaterials 2020, 10, x FOR PEER REVIEW 6 of 14

Figure 2. TEM micrographs of WSN-p at low magnification.

WSN-ipa were also calcined, with the aim of removing the surfactant not extracted by the acid
treatment and allow for more space between the silica fibres. From the spectra shown in Figure 3, no
particular difference is noted in the silica structure of the three samples. The calcined sample shows
the disappearance of the bands related to organic material, at about 1400 cm−1 and between 2800 and
3000 cm−1.
Figure
Figure 2. TEM
2. TEM micrographsof
micrographs of WSN-p
WSN-p atat
low magnification.
low magnification.

WSN-ipa were also calcined, with the aim of removing the surfactant not extracted by the acid
treatment and allow for more space between the silica fibres. From the spectra shown in Figure 3, no
particular difference is noted in the silica structure of the three samples. The calcined sample shows
the disappearance of the bands related to organic material, at about 1400 cm−1 and between 2800 and
3000 cm−1.

Figure 3. Fourier transform infrared (FT-IR) spectra of (A) WSN-p, calcined WSN (WSN-calc) (B) and
Figure 3.WSN-ipa
Fourier(C).
transform infrared (FT-IR) spectra of (A) WSN-p, calcined WSN (WSN-calc) (B) and
WSN-ipa (C).
To choose the best support, preliminary adsorption experiments were performed at 25 ◦ C and
the enzyme/support complex examined by FT-IR in the region 1480–1800 cm−1 . In fact, two of
To choose the bestbands
the characteristic support,
of the preliminary adsorption
polypeptide structure experiments
fall in this wereI performed
range, the amide and the amideat 25 °C and
the enzyme/support
II [32]. These bands,complex examined
in particular the amidebyI,FT-IR in the that
have positions region 1480–1800
are very sensitive tocm . In fact, two of the
the−1secondary
structure of the enzyme. In fact, the amide I band originates
characteristic bands of the polypeptide structure fall in this range, the amide I and from the stretching of the carbonyl
the amide II [32].
groups of the peptide bond, and is given by the overlap of a set of vibration modes. Each of these
These bands,
modes
in particular
of vibration
thetoamide
is linked
I, have
a particular
positions
element of of (A)
secondary
that are very
structure of WSN
sensitiveα-helices,
the enzyme:
to the secondary
Figure 3. Fourier transform infrared (FT-IR) spectra WSN-p, calcined (WSN-calc) (B) and
structureβ-sheets,
of the enzyme. In fact, the amide I band originates from the stretching of the carbonyl
WSN-ipa (C).turns, disordered structures and intermolecular aggregates. During adsorption, proteins
groups ofcanthe peptide
undergo bond, and
conformational is given
changes due tobyinteractions
the overlap withoftheamatrix
set ofsurface
vibration modes. Each of these
or protein-protein
modes interactions. The presence of intermolecular aggregates, with vibration modes at lowerof the numbers
wave
To of vibration
choose
(about 1610the cm is−1linked
best ) support,
to apreliminary
is an indication
particular element
adsorption
of the incipient
of secondary
experiments
denaturation
structure
werenative
of the protein performed enzyme:
structureatand
α-helices,
25 °C and
β-sheets, turns,
the enzyme/support disordered
complex
is therefore deleterious
structures
forexamined
and
its catalytic by
intermolecular
FT-IR
activity [33].inThe
theFT-IR aggregates.
region 1480–1800
spectra
During adsorption,
cm . In fact,
−1
of the immobilized
proteins
two of the
and free
can undergo
characteristic conformational
bands
cellulase changes
of the polypeptide
are displayed due to interactions with the matrix surface
in Figure 4. structure fall in this range, the amide I and the amide II [32]. or protein-protein
interactions.
These bands, The presencethe
in particular of amide
intermolecular aggregates,
I, have positions that with vibration
are very modes
sensitive to theat secondary
lower wave
numbers of
structure (about 1610 cm
the enzyme. In−1)fact,
is antheindication
amide I bandof the incipient
originates denaturation
from of the
the stretching protein
of the native
carbonyl
structure
groups andpeptide
of the is therefore
bond, and deleterious forthe
is given by itsoverlap
catalytic
of aactivity [33]. Themodes.
set of vibration FT-IREach
spectra of the
of these
immobilized
modes and free
of vibration cellulase
is linked to aare displayed
particular in Figure
element 4.
of secondary structure of the enzyme: α-helices,
Nanomaterials 2020, 10, x FOR PEER REVIEW 7 of 14

Nanomaterials 2020, 10, 1799 7 of 14

Figure 4. FT-IR spectra of cellulase adsorbed on WSN-p (A), WSN-calc (B) and WSN-ipa (C) and free
cellulase (D).

Figure 4. FT-IR
The spectra
spectra of cellulase
of cellulase adsorbed
immobilized on WSN-p
on WSN-ipa, (A), calcined
either WSN-calc (B) and
or not, showWSN-ipa (C) and free
a displacement
cellulase
of the (D).
amide I band toward lower wavenumber respect to free cellulase. This is an index of
aggregation/denaturation of enzyme molecules, to the detriment of their catalytic activity. Calcination
Thedoes not improve
spectra this situation.
of cellulase By contrast,on
immobilized for WSN-ipa,
cellulase immobilized on WSN-por
either calcined amide
not,I show
band is aabout
displacement
in the same position as free cellulase. This phenomenon can be related to the different morphology of
of the amide I band toward lower wavenumber respect to free cellulase. This is an index of
the particles. In fact, from the chemical point of view, there are no differences between the two types
aggregation/denaturation of enzyme molecules, to the detriment of their catalytic activity.
of nanoparticles (see Figure 3), while morphologically they are significantly different. The greater
Calcination does
distance not improve
between this
the fibres is situation.
likely By adsorption
to favour the contrast, of
forunmodified
cellulasecellulase
immobilized onBased
molecules. WSN-p amide
I band isonabout in the same
these preliminary position
results, WSN-p as were
free chosen
cellulase. This phenomenon
as support can be related to the different
for subsequent studies.
morphology of the particles. In fact, from the chemical point of view, there are no differences between
3.2. Optimization of the Adsorption Conditions
the two types of nanoparticles (see Figure 3), while morphologically they are significantly different.
First, the adsorption time was optimized by conducting the process at various times ranging from
The greater distance between the fibres is likely to favour the adsorption of unmodified cellulase
1 h to 24 h at 25 ◦ C. In this case, the concentrations of enzyme and nanoparticles used was 1 mg/mL
molecules.
and 2Based
mg/mL on these preliminary
respectively. The adsorptionresults, WSN-p
equilibrium wereatchosen
was reached as support
24 h. At this for subsequent
time, the amount
studies.of adsorbed enzyme was 70 mg per gram of support. The adsorption time was therefore set at 24 h.
To favour the economy of the overall process of enzymatic catalysis at an industrial level, it is
important to
3.2. Optimization of minimize the enzyme
the Adsorption waste. The adsorption should take place with the minimum
Conditions
possible amount of enzyme that assures high load and a high yield of immobilization. For this
First, the adsorption
purpose, timewere
adsorption tests wascarried
optimized by conducting
out varying the process from
the enzyme concentration at various
1 mg/mL times
to ranging
0.25 mg/mL. The results are shown in Figure 5. Figure 5a shows the adsorption isotherm
from 1 h to 24 h at 25 °C. In this case, the concentrations of enzyme and nanoparticles used was 1 fitted
with a Langmuir function. Langmuir adsorption isotherm model assumes monolayer adsorption.
mg/mL and 2 mg/mL respectively. The adsorption equilibrium was reached at 24 h. At this time, the
Monolayer adsorption, as the most probable mechanism involved in the adsorption of cellulase on
amountamorphous
of adsorbed enzyme
silica, wasbeen
has already 70 mg per gram
observed of support.
[23]. Figure The adsorption
5b represents time
the adsorption yieldwas therefore set
vs. the
at 24 h. initial concentration of the enzyme in solution.
To favour the economy of the overall process of enzymatic catalysis at an industrial level, it is
important to minimize the enzyme waste. The adsorption should take place with the minimum
possible amount of enzyme that assures high load and a high yield of immobilization. For this
purpose, adsorption tests were carried out varying the enzyme concentration from 1 mg/mL to 0.25
mg/mL. The results are shown in Figure 5. Figure 5a shows the adsorption isotherm fitted with a
Nanomaterials 2020, 10, x FOR PEER REVIEW 8 of 14

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Nanomaterials2020,
2020, 10,
10, x FOR PEER REVIEW
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Figure 5. Adsorption isotherm (squares) and Langmuir fit (line) (a), and adsorption yield vs. the initial
concentration of the enzyme in solution (squares) fitted with an exponential decay function (line) (b).
Figure 5. Adsorption isotherm (squares) and Langmuir fit (line) (a), and adsorption yield vs. the initial
Figure 5. Adsorption
concentration isothermin (squares) and Langmuir fit (line) (a), and adsorption yield (line)
vs. the(b).
initial
The adsorbedofamount
the enzyme solution
increased (squares)
with fittedconcentration
cellulase with an exponential decay
up to function
1 mg/mL, and the onset of
concentration of the enzyme in solution (squares) fitted with an exponential decay function (line) (b).
the adsorption plateau amounted to 90 mg/g of support (at cellulase concentration of 1 mg/mL). The
The adsorbed amount increased with cellulase concentration up to 1 mg/mL, and the onset of
immobilization
the The adsorbed
adsorption yield
plateau decreased
amount amounted with
increasedto 90 increasing
withmg/gcellulaseenzyme (atconcentration.
concentration
of support A concentration
up concentration
cellulase to 1 mg/mL, and
of 1 the of 0.50
onset
mg/mL). of
mg/mL
the was
Theadsorptionchosen
plateau
immobilization for subsequent
amounted
yield decreased experiments
to 90 mg/g
with because
of support
increasing it makes
(at cellulase
enzyme a good compromise
concentration
concentration. between
of 1 mg/mL).
A concentration the
ofThe
immobilization
0.50 mg/mL wasyield
immobilization chosen
yield and forthe
decreased amount
withof
subsequent immobilized
experiments
increasing enzyme.
because
enzyme it makes a good compromise
concentration. between of
A concentration the0.50
mg/mLThewas
effect
immobilization of
chosen temperature
yield and
for on the
the amount
subsequent adsorption
of immobilized
experiments behaviour
enzyme.
because of cellulase
it makes (bulk concentration
a good compromise between0.50
the
mg/mL, adsorption
The effect ofyield
immobilization time
temperature24
and the onh) is shown in
the adsorption
amount Figure 6a.
behaviour
of immobilized of cellulase (bulk concentration 0.50 mg/mL,
enzyme.
adsorption timeof
The effect 24temperature
h) is shown inon Figure 6a.
the adsorption behaviour of cellulase (bulk concentration 0.50
mg/mL, adsorption time 24 h) is shown in Figure 6a.

Figure 6. Yield of immobilization plotted versus T (squares) and exponential growth fit (line) (a) and
Figure 6. Yield of immobilization plotted versus T (squares) and exponential growth fit (line) (a) and
Arrhenius plot (b).
Arrhenius plot (b).
The adsorbed amount increased from 25 to 50 ◦ C according to an Arrhenius-like trend.
Figure 6. Yield of immobilization plotted versus T (squares) and exponential growth fit (line) (a) and
ThisThe adsorbed
indicates amount
that the increased
adsorption from 25
of cellulase on to 50 °C
WSNs according toinan
in endothermic Arrhenius-like
nature trend. This
[12,34]. Adsorption
Arrhenius plot (b).
indicates
at higherthat the adsorption
temperatures has notof cellulase on WSNs
been analysed in endothermic
because enzymes areinnormally
nature [12,34].prone Adsorption
to thermal at
higher temperatures
deactivation by has
denaturation. not Inbeen
order analysed
to estimate because
the enzymes
The adsorbed amount increased from 25 to 50 °C according to an Arrhenius-likedata
activation energy are ofnormally
adsorption, prone
the to thermal
were
trend. This
deactivation by
treated according denaturation.
to an ArrheniusIn order
plot,toasestimate
shown in the activation
Figure energy
6b. From theoflinear
indicates that the adsorption of cellulase on WSNs in endothermic in nature [12,34]. Adsorption at adsorption, the data
fit, the adsorption were
energy was found to
treated to be 25 kJ/mol, which is
ascomparable to the energy involvedlinearin hydrogen the bonding
higher according
temperatures an hasArrhenius
not beenplot,analysed shown in Figure
because 6b. From
enzymes are the
normally fit, prone adsorption
to thermal
(10–40
energy kJ/mol).
was found Hence, the driving
to be 25 kJ/mol, force
which of cellulase adsorption into WSN is probably hydrogen
deactivation by denaturation. In order to is comparable
estimate to the energy
the activation energy involved in hydrogen
of adsorption, the databonding
were
bonding
(10–40 betweenHence,
kJ/mol). cellulase
thepolar residues
driving andofsilanol
force groups
cellulase on the silica
adsorption surface
into WSN [20].
is This is important,
probably hydrogen
treated according to an Arrhenius plot, as shown in Figure 6b. From the linear fit, the adsorption
as adsorption
bonding betweenfromcellulase
a mixturepolar
of enzymes
residues is difficult to control.
and silanol In on
fact,thecellulase is composed ofThis
at is
energy was found to be 25 kJ/mol, which is comparable togroups
the energy silica in
involved surface
hydrogen [20].bonding
least three as
important, enzymes with different
adsorption from chemical-physical
a mixture ofcellulase
enzymes properties. In particular,
is difficult to control. they havefact, different
(10–40
shapes,
kJ/mol).
isoelectric
Hence,
points
the
and
driving force of
hydrophobic characteristics.
adsorption
If the
into
driving
WSN
force
is In
for
probably
adsorption
cellulase
hydrogen
were
is
composed
bonding of at least
between three enzymes
cellulase polar with different
residues and chemical-physical
silanol groups on the properties.
silicadue In particular,
surface [20]. they
This is
electrostatic
have different interaction,
shapes, there would
isoelectric probably
points and be preferential
hydrophobic adsorption effects
characteristics. If the to the
driving different
force for
important, as adsorption
isoelectric points from a enzymes.
of the cellulolytic mixture of enzymes
Similarly, theishydrophobic
difficult tointeraction
control. Inwould fact, give
cellulase
rise is
adsorption
composed were
of at electrostatic
least three interaction,
enzymes with there would
different probably be preferential
chemical-physical properties. adsorption
In effects they
particular, due
to preferential adsorption due to the different shape and hydrophobic characteristics of the enzymes.
to thedifferent
have different shapes,
isoelectric points ofpoints
isoelectric the cellulolytic enzymes.characteristics.
and hydrophobic Similarly, the hydrophobic
If the driving interaction
force for
would give rise to preferential adsorption due to the different shape and hydrophobic
adsorption were electrostatic interaction, there would probably be preferential adsorption effects due characteristics
to the different isoelectric points of the cellulolytic enzymes. Similarly, the hydrophobic interaction
8
would give rise to preferential adsorption due to the different shape and hydrophobic characteristics
8
Nanomaterials 2020, 10, x FOR PEER REVIEW 9 of 14

of the enzymes. The hydrogen bond instead ensures the adsorption of a uniform layer from a cellulase
enzyme mixture, being all cellulolytic enzymes rich in hydrogen bonding groups.
Nanomaterials 2020, 10, 1799 9 of 14
The data indicate that the highest adsorption in the temperature range examined occurs at 50
°C. However, increased adsorption does not always coincide with increased enzyme activity, which
The hydrogen bond instead ensures the adsorption of a uniform layer from a cellulase enzyme mixture,
can be denatured by thermal
being all cellulolytic or rich
enzymes crowding effects.
in hydrogen bondingFor this reason, the FT-IR spectra of the three
groups.
samples were Therecorded,
data indicateandthat
aretheshown
highest in Figurein7 the
adsorption in temperature
the regionrange
1480–1800
examinedcm −1.
occurs at 50 ◦ C.
However, increased adsorption does not always coincide with increased enzyme activity, which can be
denatured by thermal or crowding effects. For this reason, the FT-IR spectra of the three samples were
recorded, and are shown in Figure 7 in the region 1480–1800 cm−1 .

Figure 7. FT-IR spectra of cellulase adsorbed in WSN-p at 30 (A), 40 (B) and 50 ◦ C (C), and of free
cellulase (D).

Figure 7. FT-IR spectraofof

The spectrum cellulase
cellulase adsorbed at 50 ◦in
adsorbed WSN-p
C shows at 30of(A),
an offset 40 (B)band
the amide and I50 °C (C),
towards and of free
lower
wave
cellulase numbers compared to free cellulase, indicative of denaturation/aggregation of the molecules
(D).
by means of intermolecular hydrogen bonds. The amide band II is also modified. The other spectra
appear instead very similar to those of free cellulase. In particular, the spectrum of cellulase adsorbed
The spectrum of cellulase
at 40 ◦ C shows adsorbed
a similarity with at 50
that of free °C shows
cellulase an offset
in the position of theintensity
and relative amideofband I towards lower
the amide
wave numbers
bands Icompared to free cellulase,
and II. The temperature indicative
chosen to carry of denaturation/aggregation
out the adsorption of the molecules
was then 40 ◦ C, at which the native
structure
by means of of the polypeptide
intermolecular shows little
hydrogen or no The
bonds. modification
amideand bandthe loading (100modified.
II is also mg/g of support)
Theisother spectra
higher than at 25 and 30 ◦ C.
appear instead very similar to those of free cellulase. In particular, the spectrum of cellulase adsorbed
at 40 °C shows a similarity
3.3. Catalytic Assays with that of free cellulase in the position and relative intensity of the amide
bands I and II.The
Thekinetics
temperature chosencatalysed
of CMC hydrolysis to carrybyout theenzyme
the free adsorption
and thewas then cellulase/WSN-p
biocatalyst 40 °C, at which the native
was evaluated. Figure 8 shows the time course of the reaction, reporting
structure of the polypeptide shows little or no modification and the loading (100 mg/gglucose concentration (mM)
of support) is
vs. time (h).
higher than at 25 and 30 °C.

3.3. Catalytic Assays

The kinetics of CMC hydrolysis catalysed by the free enzyme and the biocatalyst cellulase/WSN-
p was evaluated. Figure 8 shows the time course of the reaction, reporting glucose concentration
(mM) vs. time (h).
 materials 2020, 10, x FOR PEER REVIEW
Nanomaterials 2020, 10, 1799 10 of 14

Figure 8. Glucose concentration versus time for free cellulase (square) and cellulase/WSN-p (circle).
Data are reported with error bars.
Figure 8. Glucose concentration versus time for free cellulase (square) and cellulase/WSN-p (circle
Thewith
Data are reported activity of thebars.
error free enzyme evaluated at 15 min is 267 µmol/g·min. The immobilized enzyme
retained 100% of the free enzyme activity. Achieving total retention of cellulase activity is not trivial.
For example, Zhang et al. [35] covalently immobilized cellulase on silica gel substrate, retaining only 7%
The activityofof
its the free
specific enzyme
activity evaluated
in the hydrolysis of CMC.at 15 etmin
Chen isimmobilized
al. [12] 267 μmol/g∙min. The immobilized e
cellulase on mesoporous
silica with different pore size, obtaining at the best a retention of 63.3% of free cellulase activity.
ned 100% ofTakimoto
the free enzyme
et al. activity.
[11] immobilized Achieving
cellulase in SBA-15 with total retention
different pore size:of thecellulase activity is not
best biocatalyst
example, Zhang et 67.5%
preserved al. [35]of thecovalently immobilized
activity of the free form. In two cases, cellulase on silica
the activity retention gel was
achieved substrate,
more retainin
than 90%, for cellulase immobilized by sol-gel encapsulation in mesoporous silica functionalized with
of its specific
methylactivity
groups, whichin give
thea certain
hydrolysis of CMC.
hydrophobicity [36] and for Chen
cellulaseet al. [12]on immobilized
immobilized commercial cellul
oporous silicafumedwith different
silica, nonporous in pore
nature size,
[37]. obtaining
Finally, in two at casesthethe best
activitya of
retention
immobilizedof 63.3% of free ce
cellulase
was found to be higher than that of the free one [2,17]. In both cases, cellulase was immobilized by
vity. Takimoto et bonding
covalent al. [11] immobilized
to mesoporous cellulase
silica through in SBA-15
amino functionalization of the with different
silica surface followed by pore size: th
atalyst preserved
crosslinking 67.5% of the activity
with glutaraldehyde. The reasonof for the free inform.
this increase In due
activity was two to acases, the activity ret
certain activity
of functional groups on the silica surface toward the hydrolysis of CMC [17]. In our case, the total
eved was more than 90%,
preservation for cellulase
of the enzymatic immobilized
activity indicates that physical by sol-gel encapsulation
immobilization did not change the native in mesoporou
conformation
tionalized with methyl of the groups,
protein, as already
which indicated
givebyaFT-IR analysis,hydrophobicity
certain and that there are no diffusion
[36] and for ce
limitations of the substrate to the active site of the enzyme. This is due to the particular morphology
obilized on ofcommercial
WSNs, with radial fumed silica,
pore channel size nonporous
increasing from the ininterior
nature [37].
to the surface.Finally, in two
This hinders pore cases the a
blocking, allows
mmobilized cellulase was thefound
enzyme to tosettle
be inhigher
the interior of the
than poresof
that where
thethe interactions
free with theIn
one [2,17]. walls
both cases, ce
are maximized, and still there is room for the substrate to easily diffuse. To endorse this hypothesis,
immobilized by 9covalent
Figure shows the TEM bonding
micrographto of mesoporous
WSN before and after silica through
cellulase adsorption.amino functionalization
For BG adsorbed
on WSN-p (BG/WSN-p), it is possible to observe two populations of nanoparticles. One shows an
a surface followed by crosslinking with glutaraldehyde. The reason for this increase in a
increase in electron density in the inner of the pores, due to the presence of the adsorbed enzyme,
due to a certain
whereas activity
the peripheryof functional groups
of the nanoparticles appears on the
quite silica surface
unchanged (Figure 9b).toward the hydrolysis o
Most BG/WSN-p
exhibit this aspect. However, there is also a population of BG/WSN-p that appear fuller and in which it
In our case, the total preservation of the enzymatic activity indicates that ph
is possible to observe the presence of the enzyme also on the external surface Figure 9c).
obilization did not change the native conformation of the protein, as already indicated by
ysis, and that there are no diffusion limitations of the substrate to the active site of the en
is due to the particular morphology of WSNs, with radial pore channel size increasing fr
ior to the surface. This hinders pore blocking, allows the enzyme to settle in the interior
s where the interactions with the walls are maximized, and still there is room for the subst
y diffuse. To endorse this hypothesis, Figure 9 shows the TEM micrograph of WSN befo
Nanomaterials 2020, 10, 1799 11 of 14
Nanomaterials 2020, 10, x FOR PEER REVIEW 11 of 14
Nanomaterials 2020, 10, x FOR PEER REVIEW 11 of 14

Figure 9.
9. TEM micrographs of WSN-p (a) and BG adsorbed on WSN-p (BG/WSN-p) (b,c).
Figure TEM micrographs of WSN-p (a) and BG adsorbed on WSN-p (BG/WSN-p) (b,c).

The of nanoparticles are also shown in TEM images at

at low
low magnification
magnification of
of Figure
Figure 10.
The two
two kinds
kinds of nanoparticles are also shown in TEM images at low magnification of Figure 10.
10.

Figure 10. TEM micrographs of BG/WSN-p at low magnification.

Figure 10.
Figure 10. TEM
TEM micrographs
micrographs of
of BG/WSN-p at low
BG/WSN-p at low magnification.
magnification.

The hydrolysis catalysed by immobilized cellulase exhibits an initial burst: at 30 min, the glucose
hydrolysis catalysed
The hydrolysis catalysed by
by immobilized
immobilized cellulase
cellulaseexhibits
exhibitsan
an initial
initialburst:
burst: at 30 min, the glucose
concentration reaches more than the 90% of the final value. However, the reaction promoted by the
concentration reaches more than the 90% of the final value. However, the reaction promoted by the
immobilized enzyme approaches more slowly the equilibrium value, if compared to the behaviour
immobilized enzyme
immobilized enzyme approaches
approachesmore
moreslowly
slowlythe
theequilibrium
equilibriumvalue,
value,if if compared
compared to to
thethe behaviour
behaviour of
of free cellulase. This evidence can be attributed to product inhibition caused by the accumulation of
of free
free cellulase.
cellulase. This
This evidence
evidence cancan be attributed
be attributed to product
to product inhibition
inhibition caused
caused by the
by the accumulation
accumulation of
of the
the glucose inside the pores of the support [29]. The curves in Figure 8 share the same asymptotic
the glucose inside the pores of the support [29]. The curves in Figure 8 share the same
glucose inside the pores of the support [29]. The curves in Figure 8 share the same asymptotic value asymptotic
value (about 13 mM of glucose concentration), corresponding to CMC percentage conversion of 56%.
value (about
(about 13 mM13ofmM of glucose
glucose concentration),
concentration), corresponding
corresponding to CMCto percentage
CMC percentage conversion
conversion of 56%.
of 56%.
3.4. Reusability Tests
3.4. Reusability Tests
Reusability of
Reusability of cellulase
cellulase immobilized
immobilized by by physical
physical adsorption
adsorption intointo mesoporous
mesoporous silica
silica materials
mesoporous silica materials
materials
was
was notnot generally tested. The reusability of an enzyme catalyst is a very important factor, which
which
not generally
generally tested.
tested. TheThe reusability
reusability of of an enzyme
enzyme catalyst
catalyst is a very important factor,
contributes to
contributes to reducing
reducing overall
overall costs
costs by
by offsetting
offsetting the
the high
high production
production costcost of
of the enzyme.
enzyme. Figure 11
cost of the
the enzyme. Figure 11
shows the results of reusing
shows the results of reusing the biocatalyst in 5 consecutive 24 h cycles. The amount of glucose that
reusing thethe biocatalyst
biocatalyst in 55 consecutive
consecutive 24 24 hh cycles.
cycles. The amount of glucose that
has formed in each cycle is expressed as
has formed in each cycle is expressed as a percentage a percentage of the maximum obtained from the catalytic
percentage of of the
the maximum
maximum obtained from the catalytic catalytic
assay. Cellulase/WSN-p
assay. Cellulase/WSN-p exhibitsaagood good operationalstability:
stability: glucoseproduction
production is is about 100%upup
assay. Cellulase/WSN-pexhibits exhibits a goodoperational
operational stability:glucose
glucose production about
is about100%
100% to
up
to
thethe third
third reuse,
reuse, while
while in
in the the fourth
fourth one,
one,one, it
it showsshows a
a loss loss of
of aboutabout
8% of 8% of conversion, and in the fifth
to the third reuse, while in the fourth it shows a loss of about 8%conversion, and and
of conversion, in theinfifth one,
the fifth
one,loss
the the loss drops of about 20%, which is still enough forSimilar
reuse. Similarwere
results were observed for
one, thedrops of about
loss drops 20%, which
of about is still enough
20%, which for reuse.
is still enough for reuse. results observed
Similar results for cellulase
were observed for
cellulase immobilized
immobilized by physical byadsorption
physical adsorption
on Si wafers on[20],
Si wafers [20], alcohol-co-ethylene)
Poly (vinyl Poly (vinyl alcohol-co-ethylene)
nanofibrous
cellulase immobilized by physical adsorption on Si wafers [20], Poly (vinyl alcohol-co-ethylene)
nanofibrous[38]
membranes membranes [38] and onactivated
and on commercial commercial activated
carbon [39]. carbon [39].
nanofibrous membranes [38] and on commercial activated carbon [39].
The loss of catalytic performance during reuse can be caused by enzyme leaching/desorption,
The loss of catalytic performance during reuse can be caused by enzyme leaching/desorption,
denaturation during repeated use or physical loss of the biocatalyst during separation and washing
denaturation during repeated use or physical loss of the biocatalyst during separation and washing
processes after each cycle.
processes after each cycle.
11
11
Nanomaterials 2020, 10, x FOR PEER REVIEW 12 of 14
Nanomaterials 2020, 10, 1799 12 of 14

100

80

Glucose production (%)

60

40

20

0
1 2 3 4 5
Number of reuse cycles

Figure 11. Histograms showing the glucose production (%) over the number of reuse cycles of
Figure cellulase/WSN-biocatalyst
11. Histograms showing (data are
thereported
glucosewith error bars). (%) over the number of reuse
production cycles of
cellulase/WSN-biocatalyst (data are reported
The loss of catalytic performance duringwith
reuseerror bars).
can be caused by enzyme leaching/desorption,
denaturation during repeated use or physical loss of the biocatalyst during separation and washing
4. Conclusions
processes after each cycle.

Cellulase was immobilized by adsorption on wrinkled silica nanoparticles with enhanced inter-
4. Conclusions
wrinkle distance.
Cellulase The physical-chemical
was immobilized by adsorptioncharacterization of the
on wrinkled silica biocatalyst
nanoparticles withdemonstrated
enhanced the
improvement induced
inter-wrinkle by The
distance. thephysical-chemical
increase of the inter-wrinkle
characterization distance
of the ondemonstrated
biocatalyst hosting cellulase.
the The
improvement
adsorption energyinduced
was foundby the increase
to be of25thekJ/mol,
inter-wrinkle distanceout
pointing on hosting
that thecellulase. The adsorption
driving force of cellulase
adsorption into WSN is probably hydrogen bonding between cellulase polar residuesinto
energy was found to be 25 kJ/mol, pointing out that the driving force of cellulase adsorption and silanol
WSN is probably hydrogen bonding between cellulase polar residues and silanol groups on the silica
groups on the silica surface. The activity of cellulase/WSNs was determined in the hydrolysis of
surface. The activity of cellulase/WSNs was determined in the hydrolysis of carboxymethyl cellulose.
carboxymethyl cellulose.
The immobilized Thehad
cellulase immobilized cellulase
the same activity hadenzyme
as the free the same activitya as
and showed goodthe free enzyme and
operational
showedstability.
a good Therefore,
operational thisstability. Therefore,
study represents, both this study
for the represents,
simplicity both for the
of the biocatalyst simplicity of the
preparation
biocatalyst preparation
and its and its
good performance andgood performance
reusability, a step towardandthereusability, a step toward
large-scale hydrolysis theforlarge-scale
of cellulose
biofuel production.
hydrolysis of cellulose for biofuel production.
Author Contributions: Conceptualization, V.C. and A.C.; methodology, A.B.; investigation, V.V., G.P. and F.S.;
Author writing—original
Contributions:draft
Conceptualization, V.C.
preparation, V.C. and and
A.C. All A.C.; methodology,
authors A.B.; investigation,
have read and agreed V.V., G.P.
to the published version of and F.S.;
the manuscript.
writing—original draft preparation, V.C. and A.C. All authors have read and agreed to the published version of
Funding: This research received no external funding.
the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
Funding: This research received no external funding.
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Conflicts of Interest: The authors declare no conflict of interest.
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