Brazilian Journal
of Chemical
Engineering
ISSN 0104-6632
Printed in Brazil
www.scielo.br/bjce
Vol. 35, No. 02, pp. 649 - 658, April - June, 2018
dx.doi.org/10.1590/0104-6632.20180352s20160094
CELLULASE IMMOBILIZATION ON
POLY(METHYL METHACRYLATE)
NANOPARTICLES BY MINIEMULSION
POLYMERIZATION
Patrícia Simon1, Janaína S. Lima1, Alexsandra Valério1, Débora de
Oliveira1, Pedro H. H. Araújo1, Claudia Sayer1, Antônio Augusto U. de
Souza1 and Selene M. A. Guelli U. de Souza1*
1
Departamento de Engenharia Química e Engenharia de Alimentos, Universidade
Federal de Santa Catarina, PO Box 476, CEP 88040-900 Florianópolis, SC, Brasil
(Submitted: February 12, 2016; Revised: January 17, 2017; Accepted: January 22, 2017)
Abstract - Cellulases are efficient enzymes for the conversion of cellulose into glucose. Their use in immobilized
form enables them to be reused in successive cycles in many biotechnological processes. Unlike conventional
methods of immobilization by covalent bonding, in miniemulsion polymerization the immobilization of enzyme and
the synthesis of polymer nanoparticles (support) occur simultaneously. Based on these aspects, the immobilization
of cellulose on poly(methyl methacrylate) (PMMA) nanoparticles by miniemulsion polymerization was studied.
The surfactant type (non-ionic and ionic) and latex pH showed great influence on cellulase activity. High activity
values were obtained only when non-ionic surfactant (Lutensol AT50) and buffering agent (NaHCO3) were used
simultaneously. MMA polymerization rate and final monomer conversion were not affected by the presence of
cellulase. The maximum immobilization efficiency (60%) was obtained when 6 wt.% of cellulase was used and
stable PMMA nanoparticles (133 nm) were obtained. The relative activity profile of immobilized cellulase, for
pH as well as temperature, was similar to that reported for the free form. Immobilized enzyme keeps its activity
throughout seven days when stored at 4 ºC and phosphate buffer pH 6.0. Based on the results obtained in this
work, miniemulsion polymerization as a method for cellulase immobilization on PMMA nanoparticles showed
to be a promising technique with high possibility of industrial application.
Keywords: Cellulase, Immobilization, Polymeric nanoparticles, Miniemulsion polymerization.
INTRODUCTION
Due to their biotechnological potential, cellulases
contribute to the improvement of several processes,
including food, textile, paper and cellulose industries,
agriculture and, more recently, second-generation
ethanol production. For this reason, these enzymes
have been studied by several research groups both in
the academic and industrial scope (Kuhad and Singh,
2011). Although the use of cellulase is widespread
in several areas, its use in free form, as well as other
enzymes, has some unfavorable aspects such as low
stability in solution, high isolation and purification
costs, and especially the difficulty of recovery from
*Corresponding author: Tel.: (+55) (48) 3721- 9448; Fax: (+55) (48) 3721-9687. E-mail address: selene.souza@ufsc.br
650
P. Simon, J. S. Lima, A. Valério, D. de Oliveira, P. H. H. Araújo, C. Sayer, A. A. U. de Souza and S. M. A. G. U. de Souza
the reaction medium for subsequent reuse. As a result,
the industrial application of cellulases is still a costly
process (Daoud et al., 2010; Sheldon and Van Pelt,
2013). By biocatalyst immobilization, many of these
drawbacks can be overcome, reflecting in economic
benefits.
Nanoscale supports are very attractive for
immobilization of enzymes since they can improve
significantly biocatalyst efficiency. The reduced size
provides a larger surface area for immobilization,
enabling an increased amount of enzyme per particles
and reducing the diffusion boundaries (Ansari and
Husain, 2012; Jia et al., 2003). The most common
ways to link an enzyme to nanoparticles are through
electrostatic adsorption, covalent attachment to
surface modified nanoparticles, direct conjugation
to the nanoparticle surface and conjugation using
specific affinity of protein (Ahmad and Sardar, 2015;
Sheldon and Van Pelt, 2013; Elnashar, 2010). These
approaches generally involve at least two steps, one
for obtaining the support and another for enzyme
immobilization, which usually requires a lot of time.
Thus, miniemulsion polymerization is emerging as
a promising technique to immobilize enzymes on
polymer nanoparticles in a single-step without the
use of organic solvents. In other words, the synthesis
of polymeric support and immobilization of enzyme
occur concurrently, eliminating the extra steps for
functionalization and immobilization.
Miniemulsion polymerization is defined as a
relatively stable dispersion of oil droplets in water
in a size range of 50-500 nm. The oil nanodroplets
are obtained by application of high shear in a system
containing oil, water, surfactant and costabilizer
(Antonietti and Landfester, 2002; Landfester et al.,
1999). The high stability of the polymer nanoparticles
obtained is a result of the combined effects of surfactant
and cosurfactant, which suppress coalescence and
Ostwald ripening, respectively (Crespy and Landfester,
2010). Thus, the proper choice of surfactant and a
stabilizer as well as their amounts are very important
to obtain stable nanoparticles for long periods.
Cipolatti et al. (2014) reported efficient
immobilization of lipase CalB on PEGylated
nanoparticles of poly(urea-urethane) (PUU) during step
growth miniemulsion polymerization. Immobilized
enzyme showed higher enzymatic activity and higher
thermal stability than free CalB enzyme. Valério et
al. (2015) immobilized lipase CalB on well-defined
PMMA core-shell nanoparticles during free radical
miniemulsion polymerization. The authors showed
that, by applying this technique it was possible to
obtain 75% of immobilization yield. Immobilized
enzyme had a higher operational stability than free
enzyme.
Despite the aforementioned promising works,
this approach still requires further studies, because
the effects of reaction conditions on enzyme activity
are not yet entirely elucidated (Cipolatti et al., 2014;
Valério et al., 2015). For instance, due to the quite
specific actuation of each enzyme, different enzymes
may present distinct behaviors, as well as effects on
miniemulsion polymerization. The few works found in
the literature employ miniemulsion polymerization as
a technique for immobilization of enzymes and they are
restricted to immobilization of lipase (Cipolatti et al,
2014; Valério et al., 2015; Chiaradia et al., 2016). Thus,
aiming to contribute with the development of effective
and innovative cellulase immobilization techniques,
this paper presented a study of immobilization of a
commercial cellulase on PMMA nanoparticles during
miniemulsion polymerization.
MATERIALS AND METHODS
Materials
Free cellulase (Cellusoft CR) was kindly donated
by Novozymes Brasil (Araucária, PR, Brazil). In
the miniemulsion polymerization Crodamol GTCC,
a triglyceride of capric and caprylic acids, used as
costabilizer, was provided by Alfa Aesar and either
Lutensol AT50 from BASF, or sodium dodecyl sulfate
(SDS, Sigma-Aldrich) was used as a surfactant.
Methyl methacrylate (MMA, 99.5%) monomer,
initiator potassium persulfate (KPS, P.A) and buffer
sodium bicarbonate (NaHCO3, P.A) were provided
by Sigma-Aldrich. Sodium hydroxide (NaOH, P.A)
was acquired from Lafan Ltda. Potassium and sodium
tartrate (P.A) were both provided by Synth. Glucose
D(+) anhydrous dextrose (P.A), 3,5-dinitrosalicylic
acid (DNS, P.A), monobasic anhydrous potassium
phosphate (P.A), dibasic sodium dihydrogen phosphate
(P.A), and sodium carboxymethylcellulose (CMC)
were all provided by Sigma-Aldrich. Cellulose acetate
membranes (Unifil, 47 mm, 0.45 µm) and qualitative
filter paper was used to determine enzymatic activity.
Amicon® Ultra Centrifugal Filters (0.5 mL, 100.000Da)
were used in the sample centrifugation. All reagents
were used as received without previous purification.
Cellulase immobilization by miniemulsion
polymerizationon PMMA nanoparticles
The synthesis of PMMA nanoparticles followed
the methodology previously described by Valério
Brazilian Journal of Chemical Engineering
Cellulase immobilization on poly(methyl methacrylate) nanoparticles by miniemulsion polymerization
et al. (2015), with some modifications. The aqueous
phase (distilled water, surfactant (Lutensol AT50
or SDS) and enzyme) and organic phase (Crodamol
and monomer, MMA) were prepared according to
the formulation shown in Table 1. Initiator (KPS)
and sodium bicarbonate (NaHCO3) (when used)
were dissolved in an aliquot of the water phase and
added to the dispersion after sonication, beginning the
polymerization process. Reactions were carried out
under constant magnetic stirring at 70 ºC for 3 h, in a
jacketed borosilicate glass reactor (50 mL).
With the immobilization process ended, the
final colloidal suspension of PMMA-cellulase
(latex) was used to determine the enzyme activity
and thermal stability at 4 ºC and room temperature,
in order to determine the nanoparticles stability
and the immobilized enzyme storage stability. To
determine the immobilization efficiency, the final
colloidal suspension of PMMA-cellulase (latex) was
centrifuged at 11.300 g for 30 min in an Eppendorf
- MiniSpin centrifuge, making use of ultrafiltration
devices (Amicon® filters - 0.5 mL, 100.000Da), and
the permeate was used for the analysis. Fig. 1 presents
schematically the described procedure.
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Enzyme activity assays
The cellulase activity was determined by the DNS
method, following the procedure described: 900 µL
of 4% (m/v) CMC solution, in phosphate buffer (0.05
M, pH 6.0), and 100 µL of PMMA-cellulase colloidal
suspension were incubated at 55 ºC for 30 minutes.
After the incubation time, 1.5 mL of DNS was added.
The resulting solution was heated in a boiling water
bath for 5 minutes and cooled in an ice bath, followed
by water addition for sample dilution. These samples
were then filtered in qualitative filter paper and
cellulose acetate membranes for particulates removal.
Finally, the glucose amount produced was determined
from the absorbancea t540 nm. The relation between
absorbance and concentration was achieved through a
calibration curve, using glucose as standard.
In order to avoid interferences, a control solution
was prepared for all experiments, adding 1.5 mL of
DNS to the CMC and PMMA-cellulase solution at
the beginning of enzymatic hydrolysis. One cellulose
activity (U) unit was defined as glucose amount
generated per minute (µmol/min). All measurements
were performed in duplicate. The relative activity
(AR) (Equation 1), where noted, was expressed as
percentage of enzyme activity at a specific value (AEi)
relative to the maximum activity (AEmax). Maximum
activity is the highest activity among all activities
values obtained for the variable under study.
AEi
(1)
AR =
x100
AEmax
Free enzyme activity was determined in accordance
with procedures and conditions used to determine the
activity of the immobilized enzyme, excluding only
the filtration steps.
Figure 1. Schematic procedure for cellulase immobilization by
miniemulsion polymerization
Table 1. .Experiments performed to study the effect of pH, surfactant type and concentration on enzymatic activity and polymer particle size.
Exp.
SDS (g)
Lutensol AT50
(%)*
NaHCO3(g)
Latex pH
Dp**(nm)
PDI
EA (U/mL)
1
-
2.7
0.01
6.5
131 ± 0.3
0.145
368.9 ± 2.8
2
-
2.7
-
3.0
128 ± 1.1
0.164
16.8 ± 3.1
3
0.060
-
0.01
5.1
***
***
37.8 ± 11.5
4
0.060
-
-
3.0
***
***
1.8 ± 0.26
5
-
1.4
0.01
6.5
193 ± 1.4
0.398
319 ±3.0
6
4.0
0.01
6.3
112 ± 0.7
0.185
386 ±6.0
wt.% related to the total weight of the reaction medium.
** Values expressed as mean ± standard deviation.
*** Unstable emulsion.
All reactions were performed at 70 °C during 3 h under constant magnetic stirring with: 3.09 g MMA, 3.03 g Crodamol, 0.30 g cellulase, 0.03 g KPS, 24 g
water.
Brazilian Journal of Chemical Engineering Vol. 35, No. 02, pp. 649 - 658, April - June, 2018
652
P. Simon, J. S. Lima, A. Valério, D. de Oliveira, P. H. H. Araújo, C. Sayer, A. A. U. de Souza and S. M. A. G. U. de Souza
Immobilization efficiency
Storage stability
The amount of cellulase immobilized (Yimob) on
the PMMA nanoparticles was determined using
Equation 2.
The storage stability of free and immobilized
cellulose at 4 ºC was monitored by measuring the
enzyme activity during 7 days. For this analysis free
enzymes were kept in phosphate buffer (0.05M, pH
6.0), and the final latex was used as prepared (latex
storage).
free
sup erna tan t
(2)
Yimob Q%V =
A
-A
A free
in which Afree is the free enzyme activity before
the immobilization process (U/mL); Asupernatant is the
supernatant activity after immobilization (U/mL).
Polymer characterization
The monomer conversion (MMA) was determined
by gravimetric method. Latex samples were taken
at different time intervals and added to previously
pre-weighted aluminum capsules containing 0.2
g of hydroquinone aqueous solution (1 wt.%) to
immediately stop reaction. The capsules were then
dried at 60 ºC in a forced convection oven until
constant weight. Conversion was calculated relating
the polymer mass present in the reactor and the fed
monomer mass.
The intensity average diameter (Dp) of the PMMA
nanoparticles was determined by the dynamic light
scattering technique (DLS - Malvern Instruments,
Zetasizer Nano S); for the measurements, samples
were diluted in distilled water. Scanning electronic
microscopy with field emission (MEV-FEG - JEOL
JSM-6701, LCME UFSC) was used in order to verify
the morphology of PMMA nanoparticles. Sample
preparation was performed by dropping the diluted
latex (1:23 in distilled water) on a stub. After complete
drying, the samples were sputter coated with a thin
gold layer and analyzed.
Attenuated total reflectance Fourier Transform
Infrared Spectroscopy (ATR-FTIR- Bruker TENSOR
27) in the range from 600 to 4000 cm-1 was used to
confirm the chemical structure of the PMMA.
Effect of pH and temperature on enzymatic activity
In order to determine the influence of pH and
temperature on free and immobilized cellulase, the
activity assays were carried out in a range of pH from
4.0 to 8.0, and in a range of temperature from 35 to
75 ºC. Thermal stability was determined by measuring
enzymatic activity of free and immobilized cellulase
using the previously described procedure at different
incubation times (30-300 min).
RESULTS AND DISCUSSION
Influence of the surfactant type and pH on the enzymatic activity
The influence of pH and surfactant type (anionic
- SDS or non-ionic - Lutensol AT50) used in the
miniemulsion polymerization, on the cellulase
enzymatic activity, was evaluated using different
polymerization formulations. The results are shown
in Table 1. For experiments conducted with different
surfactants and at the same pH (experiments 2 and 4),
it was observed that the enzymatic activity was almost
10 times higher when non-ionic surfactant (Lutensol
AT50) was used. The negative influence of ionic
surfactants on cellulase hydrolysis was also reported
by Eriksson et al. (2002) and Ueda et al. (1994).
All reactions were performed at 70 ºC during 3 h
under constant magnetic stirring with: 3.09 g MMA,
3.03 g Crodamol, 0.30 g cellulase, 0.03 g KPS, 24 g
water.
Comparing the experiments performed with the
same surfactant and at different pH (experiments
1 and 2), it is noted that the enzymatic activity was
much greater when NaHCO3was employed. A possible
explanation for these results is the latex pH was under
the enzyme stability range, causing its denaturing.
From experiment 1, the latex pH was in the optimum
range of enzyme activity (determined previously for
free enzyme) upon adding only 0.01 g of buffering
agent (NaHCO3). According to these results, it can
be concluded that the concomitant use of a non-ionic
surfactant and a buffer is necessary to obtain high
enzymatic activities.
Effect of surfactant concentration
The effect of Lutensol AT50 concentration on
nanoparticle diameter was investigated. According to
Table 1, the nanoparticle diameter (Dp) and dispersion
(PDI) decreased upon increasing the surfactant
concentration. The interfacial tension decreases with
Brazilian Journal of Chemical Engineering
Cellulase immobilization on poly(methyl methacrylate) nanoparticles by miniemulsion polymerization
increasing surfactant concentration, facilitating the
dispersion of the monomer droplets during sonication,
thus reducing nanoparticle diameter (Antonietti and
Landfester, 2002; Bechthold et al., 2000; Romio et al.,
2009; Valério et al., 2013). The polymeric nanoparticle
surface area per surfactant molecule (Asurf) was
reduced from approximately 1.77 to 1.02 nm2/
surfactant molecule when increasing the surfactant
concentration from 1.4 to 4.0 wt.%. It means that the
higher surfactant concentration increased the packing
density of surfactant molecules at the particle surface.
For non-ionic surfactant, steric stabilization is the
main mechanism for the nanoparticle stabilization
and it arises from a physical barrier. The surfactant
molecules adsorbed on the surface of the nanoparticles
extend into the continuous phase, providing a volume
restriction or a physical barrier for particle interactions
that prevents aggregation or coalescence and hence
stabilizes emulsions. The higher the packing density
of surfactant molecules at the particle surface, more
effective is the physical barrier and higher is the
nanoparticle stability, as was observed.
Influence of cellulase concentration
The effect of enzyme concentration on the relative
activity during miniemulsion polymerization,
immobilization efficiency, particle diameter and
monomer conversion was also studied. According to
the results shown in Fig. 2, it can be verified that the
relative activity decreased during the polymerization
for all concentrations of enzymes tested. A similar
behavior was reported by Valérioet al. (2015) using
the miniemulsion polymerization technique for CalB
lipase immobilization.
Due to the immobilization process, part of the
enzyme catalytic activity can be compromised.
However, the relative activity obtained by the
immobilization method used in the current work (6478.7%) is equal to or higher than those reported for
other immobilization methods by covalent bonds
(Jordan et al., 2011; Silva et al., 2012; Abd El-Ghaffar
and Hashem, 2010).
653
Figure 2. Influence of cellulase concentration on relative activity during
miniemulsion polymerization using 2.7 wt.% of Lutensol AT50.
By measuring the supernatant enzyme activity, it
could be observed that the highest immobilization
efficiency was obtained when 6 wt.% of cellulase
was used. At this concentration, approximately 60%
of the initial enzyme activity was retained on PMMA
nanoparticles. As shown in Table 2, at 10 wt.% of
enzyme an expressive reduction in immobilization
efficiency was observed. At low enzyme concentration
the packing factor is lower than at higher enzyme
concentration. However, there is a saturation point
where the surface of the nanoparticle is fully covered
by enzyme and the packing factor is maximum. Thus,
increasing enzyme concentration above this point,
a greater non-immobilized enzyme concentration is
detected in the supernatant, leading to a decrease in
immobilization efficiency.
According to Table 2, different concentrations of
enzyme did not affect the particle size. Furthermore,
there was not any evidence of the formation of
coagulum during the polymerization reaction. These
results suggest that, for all enzyme concentrations
tested, the miniemulsions were stable. The enzyme
concentration did not affect MMA conversion and for
all reactions MMA conversion was above 90%. Similar
behavior was observed by Valério et al. (2015).
Table 2. Influence of cellulase concentration on immobilization yield and nanoparticle diameter (Dp) and dispersion (PDI).
Cellulase Concentration (relative to
MMA, wt.%)
Immobilization Yield (%)
0
Dp (nm)*
PDI
132±1.0
0.154
2
47±2
136±0.6
0.197
6
59±1
133±0.8
0.176
22±2
138±0.5
0.168
10
*values are expressed as mean ± standard deviation
Brazilian Journal of Chemical Engineering Vol. 35, No. 02, pp. 649 - 658, April - June, 2018
654
P. Simon, J. S. Lima, A. Valério, D. de Oliveira, P. H. H. Araújo, C. Sayer, A. A. U. de Souza and S. M. A. G. U. de Souza
Stability of PMMA-cellulase nanoparticles
The average size of PMMA nanoparticles was
monitored during 60 days (Fig. 3). A colloidal
dispersion of PMMA-cellulase nanoparticles was
stored at room temperature and at 4ºC to determine the
effect of temperature on nanoparticle stability.
Figure 3. PMMA-cellulase nanoparticle (synthesized using 2.7 wt.%
of Lutensol AT50 and 6 wt.% of cellulase) stability at 4 °C and at room
temperature.
As observed in Fig. 3, the nanoparticle diameter
slightly increased for both temperatures after 60 days
of storage and the formation of coagulum was not
observed indicating that the dispersion was stable and
could be stored for long periods.
The MEV-FEG image (Fig. 4a) shows that PMMA
nanoparticles had a spherical morphology and the
particle size is in the same range as that obtained by
DLS. As cellulase is predominantly lipophobic and it
is dispersed in the water phase, the interaction between
the lipophilic groups of the enzyme and PMMA occurs
only at the surface of the nanoparticle, resulting in a
core-shell structure where the immobilized enzyme
remains at the nanoparticle surface. The TEM image
(Fig. 4b) shows a polymer nanoparticle with a shadow
around it that could be attributed to cellulase.
The chemical structure of PMMA wasverified
by ATR-FTIR and the PMMA spectrum is shown
in Fig. 5. The peak at 1147 cm-1is assigned to the
stretching vibration mode of C-O-C of the ester group.
The peak at 1726 cm-1 is attributed to stretching of the
carbonyl group of PMMA. The absorption bands in the
range 3000-2800 cm-1 are attributed to stretching of C
-H bonds of PMMA (Feuser et al., 2015; Matsushita
et al., 2000). The absence of peaks at 1640 cm-1 is an
evidence of complete polymerization of the monomer.
This peak is attributed to C=C groups that are present
in the monomer molecules that are directly involved in
the polymerization (Otsuka and Chujo, 2010).
Effect of pH and temperature on enzyme activity
The effect of pH and temperature on the activity
of free and immobilized enzyme is shown in Figs. 6
and 7. As can be observed, both free and immobilized
enzymes are sensitive to pH and temperature
variations. After immobilization, the relative activity
profile for pH as well as temperature behavior was
similar to that reported for free enzymes. Besides
that, the maximum activity for free and immobilized
enzymes was obtained at pH 6.0 and temperature of
55 ºC (optimal values). Similar observations were
Figure 4. MEV-FEG (a) and TEM (b) images of PMMA-cellulase nanoparticles synthesized using 2.7 wt.% Lutensol AT50 and 6
wt.% cellulase.
Brazilian Journal of Chemical Engineering
Cellulase immobilization on poly(methyl methacrylate) nanoparticles by miniemulsion polymerization
655
reported by Liang and Cao (2012) for immobilized
cellulase on polyacrylate copolymer.
Thermal stability
The thermal stabilities of free and immobilized
cellulase were compared by measuring their activities
over time at constant temperature (55 ºC). Fig. 8 shows
that free and immobilized cellulase activity decreased
gradually with time. Although the immobilized
enzyme is inserted in a medium consisting of various
components (water, ions, surfactant, etc.) that may
affect its activity, its thermal stability was not affected.
Figure 5. FTIR spectrum of PMMA nanoparticles synthesized using 2.7
wt.% of Lutensol AT50.
Figure 8. Thermal stability of free and immobilized cellulase at 55 °C
and pH 6.0 (PMMA-cellulase nanoparticles synthesized using 2.7 wt.%
Lutensol AT50 and 6 wt.% cellulase).
Storage stability
Figure 6. Effect of pH on enzymatic activity of free and immobilized
cellulose at 55 ºC (PMMA-cellulase nanoparticles synthesized using 2.7
wt.% Lutensol AT50 and 6 wt.%).
Figure 7. Effect of temperature on enzymatic activity of free and
immobilized cellulase at pH 6.0 (PMMA-cellulase nanoparticles
synthesized using 2.7 wt.% Lutensol AT50 and 6 wt.% cellulase)
The storage stability of an enzyme is one of the main
factors to evaluate its technological feasibility. Fig. 9
shows the stability of free and immobilized cellulase
stored in phosphate buffer (0.05 M, pH 6.0) at 4 ºC.
It was observed that the relative activity remained
constant during 7 days for free and immobilized
cellulase. According to Cavaco and Gübits (2003),
temperature is a critical factor during enzyme storage,
in both their solid and liquid forms. The results
suggest that the latex did not interfere in the activity
of immobilized cellulase under the tested conditions
and these conditions (phosphate buffer pH 6.0 at 4 ºC)
were suitable for enzyme conservation. Furthermore,
at this temperature the microbial degradation can be
minimized.
The immobilized cellulase was evaluated for
four successive cycles and 22% of the initial activity
was kept, showing the possibility of reusing the
immobilized biocatalyst prepared here.
Brazilian Journal of Chemical Engineering Vol. 35, No. 02, pp. 649 - 658, April - June, 2018
656
P. Simon, J. S. Lima, A. Valério, D. de Oliveira, P. H. H. Araújo, C. Sayer, A. A. U. de Souza and S. M. A. G. U. de Souza
(CNPQ), Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior (CAPES) and Laboratório Central
de Microscopia Eletrônica of Federal University of
Santa Catarina (LCME-UFSC).
REFERENCES
Figure 9. Storage stability of free and immobilized cellulase at 4 °C
(PMMA-cellulase nanoparticles synthesized using 2.7 wt.% Lutensol
AT50 and 6 wt.% cellulase).
CONCLUSIONS
In this work, miniemulsion polymerization was
used as a technique for cellulase immobilization
on poly(methyl methacrylate) (PMMA) polymeric
nanoparticles. Polymerizations with non-ionic
surfactant, Lutensol AT50, concomitantly with a
buffering agent, NaHCO3, led to high enzymatic
activity values. From the study of the effect of
surfactant concentration, 2.7 wt.%of Lutensol AT50
was sufficient to obtain stable nanoparticles with a
high enzyme activity value. The increase in enzyme
concentration in the polymerization reactions led
to higher relative activity values at the end of the
reactions. A cellulase concentration study indicated
that the maximum immobilization yield was 60%,
obtained when 6 wt.% of cellulase was added. Both
free and immobilized enzymes presented the same
behavior in relation to thermal stability, having their
relative activity values reduced to 50% after 60 min of
hydrolysis at 55 ºC and pH 6.0. In relation to storage
stability, it was verified that immobilized enzyme
keeps its activity throughout seven days when stored at
4 ºC in phosphate buffer, pH 6.0. Based on the results
presented in this work, the immobilization of cellulase
on PMMA polymeric nanoparticles by miniemulsion
polymerization can be seen as a promising, feasible and
innovative technique, which aims to cooperate with
improvement, in both an economic and environmental
sense, of several productive industrial processes.
ACKNOWLEDGMENTS
The authors would like to thank the Conselho
Nacional de Desenvolvimento Científico e Tecnológico
Abd El-Ghaffar, M.A. and Hashem, M.S., Chitosan
and its amino acids condensation adducts as
reactive natural polymer supports for cellulase
immobilization. Carbohydrate Polymers, 81 507516 (2010).
Ahmad, R.; Sardar, M., Enzyme Immobilization: An
Overview on Nanoparticles as Immobilization
Matrix. Biochemistry & Analytical Biochemistry,
4(2), 1 (2015).
Ansari, S.A. and Husain, Q., Potential Applications
of enzymes immobilized on/in nano material:
A review. Biotechnology Advances, 30 512-523
(2012).
Antonietti, M. and Landfester, K., Polyreactions in
miniemulsions. Progress in Polymer Science, 27
689-757 (2002).
Bechthold, N., Tiarks, F., Willert, M., Landfester, K.
and Antonietti, M., Miniemulsion Polymerization:
Applications and New Materials. Macromolecular
Symposia, 151 549-555 (2010).
Börjesson, J., Peterson, R. and Tjerneld, T., Enhanced
enzymatic conversion of softwood lignocellulose
by poly(ethylene glycol) addition. Enzyme and
Microbial Technology, 40 754-762 (2007).
Cavaco-Paulo, A. and Gübitz, G.M., Textile processing
with Enzymes. 1.ed. England: Woodhead
Publishing Ltd, 2003. 204 p.
Chiaradia, V.; Valério, A., Oliveira, D., Araújo, P.H.H.,
Sayer, C., Simultaneous single-step immobilization
of Candida antarctica lipase B and incorporation
of magnetic nanoparticles on poly(urea-urethane)
nanoparticles by interfacial miniemulsion
polymerization. Journal of Molecular Catalysis B:
Enzymatic, 131 31-35 (2016).
Cipolatti, E.P., Valério, A., Nicoletti, G., Theilacker, E.,
Araújo, P.H.H., Sayer, C., Ninow, J.L. and Oliveira,
D., Immobilization of Candida antarctica lipase B
on PEGylated-poly(urea-urethane) nanoparticles
by step miniemulsion polymerization. Journal of
Molecular Catalysis B: Enzymatic, 109 116-121
(2014).
Crespy, D., Landfester, K., Miniemulsion
polymerization as a versatile tool for the synthesis
of functionalized polymers. Beilstein Journal of
Organic Chemistry, 6, 1132-1148, (2010).
Brazilian Journal of Chemical Engineering
Cellulase immobilization on poly(methyl methacrylate) nanoparticles by miniemulsion polymerization
Daoud, F.B., Kaddour, S. and Sadoun, T., Adsorption
of cellulase Aspergillus niger on a commercial
activated carbon: Kinetics and equilibrium studies.
Colloids and Surfaces B: Biointerfaces, 75 93-99
(2010).
Elnashar, M.M.M., Review Article: Immobilized
Molecules
Using
Biomaterials
and
Nanobiotechnology. Journal of Biomaterials
Nanobiotechnology, 1 61-77 (2010).
Eriksson, T.; Börjeson, J.; Tjerneld, F., Mechanism
ofsurfactant effect in enzymatic hydrolysis of
lignocellulose. Enzyme and Microbial Technology,
31, 353-364, 2002.
Feuser, P. E., Bubniak, L. S., Silva, M.C.S., Viegas,
A.C., Fernandes, A.C., Ricci-Junior, E., Nele,
M., Tedesco, A.C., Sayer, C., Araújo, P.H.H.,
Encapsulation of magnetic nanoparticles in
poly(methyl methacrylate) by miniemulsion
and evaluation of hyperthemia in U87MG cells.
European Polymer Journal, 68 355-365 (2015).
Jia, H., Zhu, G. and Wang, P., Catalytic Behaviors
of Enzymes Attached to Nanoparticles: The
Effect of Particle Mobility. Biotechnology and
Bioengineering, 84 406-414 (2003).
Jordan, J., Kumar, C.S.S.R. and Theegala, C.,
Preparation and characterization of cellulose-bond
magnetite nanoparticles. Journal of Molecular
Catalysis B: Enzymatic, 69 139-146 (2011).
Landfester, K., Bechthold, N., Förster, S. and
Antonietti, M., Evidence for the preservation of the
particle identity in miniemulsion polymerization.
Macromolecules Rapid Communications, 20 81-84
(1999).
Liang, W. and Cao, X., Preparation of a pHsensitive polyacrylate amphiphilic copolymer
and its application in cellulase immobilization.
Bioresource Technology, 116 140-146 (2012).
Matsushita, A., Ren, Y., Matsukawa, K., Inoue, H.,
Minami, Y., Noda, S., Ozaki, Y., Two-dimensional
657
Fourier-transform Raman and near-infrared
correlation spectroscopy studies of poly (methyl
methacrylate) blends1. Immiscible blends of poly
(methyl methacrylate) and atactic polystyrene.
Vibrational Spectroscopy, 24 171-180 (2000).
Otsuka, T., Chujo, Y., Poly(methyl methacrylate)
(PMMA)-based hybrid materials with reactive
zirconium oxide nanocrystals. Polymer Journal, 42
58-65 (2010).
Romio, A.P., Sayer, C., Araujo, P.H.H., Al-Haydari,
M., Wu, L. and Rocha, S.R.P., Nanocapsules by
Miniemulsion Polymerization with Biodegradable
Surfactant and Hydrophobe. Macromolecular
Chemistry and Physics, 210 747-751 (2009).
Sheldon, R.A. and Van Pelt, S., Enzyme immobilization
in biocatalysis: why, what and how. Chemical
Society Reviews, 42 6223-6235 (2013).
Silva, J.A., Macedo, G.P., Rodrigues, D.S., Giordano,
R.L.C. and Gonçalves, L.R.B., Immobilization
of Candida antarctica Lipase B by covalent
attachment on chitosan-based hydrogels using
different support activation strategies. Biochemical
Engineering Journal, 60 16-24 (2012).
Ueda, M., Koo, H. and Wakida, T., Cellulase treatment
of cotton fabrics. Part II: Inhibitory Effect of
Surfactants on Cellulase Catalytic Reaction. Textile
Research Journal, 64 615-618 (1994).
Valério, A., Araújo, P.H.H. and Sayer, C., Preparation
of Poly(Urethane-urea) Nanoparticles Containing
Açaí Oil by Miniemulsion Polymerization.
Polímeros, 23, 451-455 (2013).
Valério, A., Nicoletti, G., Cipolatti, E.P., Ninow,
J.L., Araújo, P.H.H., Sayer, C. and Oliveira, D.
Kinetic Study of Candida antarctica Lipase B
Immobilization Using Poly(Methyl Methacrylate)
Nanoparticles
Obtained
by
Miniemulsion
Polymerization as Support. Applied Biochemistry
and Biotechnology, 175 2961-2971 (2015).
Brazilian Journal of Chemical Engineering Vol. 35, No. 02, pp. 649 - 658, April - June, 2018
658
P. Simon, J. S. Lima, A. Valério, D. de Oliveira, P. H. H. Araújo, C. Sayer, A. A. U. de Souza and S. M. A. G. U. de Souza
Brazilian Journal of Chemical Engineering