Enzyme and Microbial Technology 42 (2008) 583–588
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Enzyme and Microbial Technology
journal homepage: www.elsevier.com/locate/emt
Effects of relative humidity on enzyme activity immobilized
in sol–gel-derived silica nanocomposites
Martı́n Federico Desimone a,∗ , Silvia Beatriz Matiacevich b ,
Marı́a del Pilar Buera b , Luis Eduardo Dı́az a
a
National Research Council (CONICET), Cátedra de Quı́mica Analı́tica Instrumental, Facultad de Farmacia y Bioquı́mica,
Universidad de Buenos Aires, Junı́n 956 Piso 3◦ , (1113) Ciudad de Buenos Aires, Argentina
b
National Research Council (CONICET), Departamento de Industrias, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Int. Guiraldes s/n Ciudad Universitaria CP1428EHA, Buenos Aires, Argentina
a r t i c l e
i n f o
Article history:
Received 5 October 2007
Received in revised form 7 March 2008
Accepted 10 March 2008
Keywords:
Sol–gel
Immobilization
Urease
Nanocomposites
Relative humidity
Glycerol
Trehalose
a b s t r a c t
Enzyme immobilization has attracted great interest in biotechnology processes. Herein we report the
immobilization of urease from Canavalia ensiformis (jack bean) in sol–gel-derived silica nanocomposites.
Urease activity, differential scanning calorimetry (DSC), nitrogen and water adsorption isotherms were
used to characterize the effect of storage at various relative humidities on enzyme activity immobilized
in sol–gel-derived silica nanocomposites. In this study, the nanocomposites consist of tetraethoxysilane,
as inorganic silicate precursor, in combination with glycerol or trehalose as organic additives. Entrapped
urease was more stable for all the formulations aged with a relative humidity of 80%. However, significant differences (p < 0.05) in enzyme activity recovered at this relative humidity were observed between
samples with different formulations, reflecting the effect of additives during the immobilization process.
The applications of biocompatible sol–gel-derived matrices can be further extended and utilized in the
development of biosensors with immobilized biomolecules that can be used for long time periods by
taking into account different factors, among which the storage relative humidity has permitted to greatly
improve the stability of the immobilized urease.
© 2008 Elsevier Inc. All rights reserved.
1. Introduction
Enzyme immobilization has attracted great interest in biotechnology processes of biochemical conversion and bioremediation
for its high activity, substrate specificity and easy separation from
products [1]. Nonetheless, industrial application is often hampered
by a lack of long-term storage and operational stability. Improvements in enzyme stability can prolong the lifetime of enzyme
reactors, increase the potential for enzyme reuse, or maintain the
good signal of biosensors [2]. A further benefit of improved enzyme
performance by enhanced stability and repeated use is reflected in
higher catalysis productivity which determines the enzyme cost
per kg of product [3]. Recently, the improvement of enzyme activity, stability and selectivity via immobilization techniques, was
reviewed by Fernandez-Lafuente and co-workers [4].
Occurrence of appropriate environment around entrapped
biomolecules and its long-term stability is one of the important
factors determining the functionality of entrapped proteins and
∗ Corresponding author. Tel.: +54 11 49648254; fax: +54 11 49648254.
E-mail address: desimone@ffyb.uba.ar (M.F. Desimone).
0141-0229/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2008.03.009
enzymes [5]. Previous studies have shown that the degree of protein
hydration and local solvent composition can affect the structure
and dynamics of proteins [6].
Sol–gel science is well developed and applications are increasing [7]. Several industrial applications of sol–gel technology are
well established. The entrapment of a wide variety of biological
species, including active enzymes [8,9] and live microorganisms
[10–13], into different sol–gel-derived nanocomposites, for medicinal, biological and analytical applications have recently been
reviewed [14–17]. Sol–gel enzyme immobilization for biocatalysis
in non-aqueous media has been extensively investigated showing also an improvement of the stability and catalytic activity
of the biomolecule in organic solvents [18,19]. The sol–gel network stabilizing properties derives from the high viscosity and the
restricted mobility inside the matrix. It is generally accepted that
the stabilization process is mediated through protein conformational flexibility limitation and the simultaneous generation of a
protective microenvironment [20–22].
Silica gel matrices are highly porous, showing physical rigidity, chemical and biological inertness, high thermal stability and
the particle morphology of such sol–gel made materials can be
controlled by their method of preparation [23]. However, silica
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gels prepared from alkoxide precursors suffer from considerable
shrinkage during the drying process, which leads to fracture of
the material, pore collapse and loss of enzyme activity [24]. Recent
reports suggested that the use of additives might help to overcome
these problems diminishing the internal stress and shrinkage of
the materials [25]. Nanocomposite materials typically consist of an
inorganic or organic silicate precursor in combination with additives such as hydrophobic or hydrophilic polymers, surfactants,
sugars, and dyes [26,27]. In this way, the use of stable and high
glass transition sugars, such as trehalose, as a drying control chemical additive constituted an interesting possibility in the sol–gel
process. Glycerol, although having very low glass transition temperature, has also been reported as protective additive for encapsulated
enzymes, probably by maintaining adequate enzyme conformation
[28]. Therefore, it is very important to study the physicochemical
properties of the internal environment, because even small changes
in the enzyme conformation upon immobilization could drastically
reduce their catalytic activity [29,30].
Urease catalyses the hydrolysis of urea to ammonia and carbon
dioxide. Numerous papers have been published on the applications
of urease in biotechnology, including the determination of urea
for analytical and biomedical purposes [31,32], analysis of heavy
metals [33,34], urea removal from alcoholic beverages [35], determination of creatinine [36], arginine [37], IgG [38] and Escherichia
coli [39].
The microenvironments of the sol–gel-derived urease biosensors in terms of elemental ratio, surface morphology, specific
surface area and pore size were investigated to characterize the
physicochemical properties of poly(vinyl alcohol)-modified sol–gel
materials [40]. In that work, the sol–gel samples were aged under
ambient conditions at 18 ◦ C with relative humidity of 78% for 1
month. However, the effect of relative humidity on enzyme activity in sol–gel-derived materials still remains unclear. This is an
important aspect of biotechnological significance, which needs to
be taken into account in the formulation of the encapsulating matrices to preserve enzyme stability.
Herein we report the immobilization of urease from Canavalia
ensiformis (jack bean), a plant belonging to Leguminosae family,
in sol–gel-derived silica nanocomposites. Urease activity, differential scanning calorimetry (DSC), nitrogen and water adsorption
isotherms were used to characterize the effect of storage at various
relative humidities on enzyme activity immobilized in sol–gelderived silica nanocomposites. In this study, the nanocomposites
consist of tetraethoxysilane, as inorganic silicate precursor, in combination with glycerol or trehalose as organic additives.
2. Materials and methods
2.1. Materials
Tetraethoxysilane (TEOS, 98.0%) was purchased from FLUKA (Buchs, Switzerland). Urease (EC 3.5.1.5) from C. ensiformis (jack bean) 54300 Units/g was purchased
from SIGMA (St. Louis, MO). Trehalose (99.1%) was obtained from Hayashibara
(Tokyo, Japan). Glycerol (99%) was obtained from Aldrich (Milwaukee, WI). All other
reagents were of analytical grade and were used as received without further purification.
In order to achieve various water contents, samples of gels and lyophilized urease
obtained were placed in 1 cm diameter glass vials and stored over saturated salt
solutions at various relative humidities (RH) into vacuum desiccators for 16 days at
20 ◦ C. The following saturated salt solutions were employed: CH3 COOK (RH 23%),
MgCl2 (RH 33%); K2 CO3 (RH 43%); NaBr (RH 59%); NaCl (RH 75%); KBr (RH 80%); KCl
(RH 84%); KNO3 (RH 94%) [43]. All salts were of analytical grade (Mallinckrodt, USA).
2.3. Urease activity
Enzyme activity was assessed using the indophenol’s method [44]. The ammonium released reacts with phenol in alkaline medium producing blue indophenol,
which was quantified spectrophotometrically at 540 nm. Enzymes were incubated in
50 mM phosphate buffer pH 7, and their activities measured at 25 ◦ C. Urease activity
of 4.5 IU represents 100% relative activity.
2.4. Nitrogen adsorption isotherms
Gels obtained with the different formulations and equilibrated at RH 80% at
20 ◦ C were frozen under liquid nitrogen (−190 ◦ C) and freeze-dried in a Heto-Holten
freeze drier (A/S, CT110 model, Heto Lab Equipment, Denmark) which operated at
a temperature of −110 ◦ C in the condenser plate and a minimum chamber pressure
of 4 × 104 mbar to obtain the dried matrices. Nitrogen adsorption isotherms were
performed at 77 K using an automatic gas adsorption analyzer (Sortometer Gemini 2360 V2.00) after outgassing the samples. The specific surface area (SBET ) and
total pore volume (TPV) were estimated by the Brunauer–Emmett–Teller (BET) and
Barrett–Joyner–Halenda (BJH) methods, respectively.
2.5. Water adsorption isotherms
After equilibration, water content of systems was determined gravimetrically
using a Mettler Toledo Analytical Balance (Switzerland) by difference in mass before
and after drying the samples in an oven at 95 ± 2 ◦ C for 5 days. The water adsorption
isotherms (water content vs. relative humidities plots) were obtained and fitted
to the Guggenheim-Anderson-de Boer (GAB) equation by using the least square
method for minimizing the absolute differences between measured and calculated
water content values. The GAB model has been considered the best fit model for
water adsorption in many food and biological materials over a wide range of water
activity [45]. The model allows calculating the water content at the monolayer (m0 )
which represents the saturation with one water molecule for each of the more active
polar sites of the adsorbate. The following equation was used to evaluate the sorption
surface area for water (SWGAB ) [46]:
SWGAB =
m0 Na
M
(1)
where SWGAB is the surface area for water adsorption (m2 /g), m0 is the water content
at the monolayer value obtained by GAB equation (gH2 O/g), N is the Avogadro’s
number (6.02 × 1023 molecules/mol); M is the molar mass of water (18 g/mol); a is
the area of one water molecule (10.6 × 10−20 m2 /molecule).
2.6. Thermal analysis
After equilibrium at various relative humidities, the samples (between 5 and
10 mg) were weighted with a precision of ±0.01 mg, sealed into aluminum pans
(40 ␮L of capacity) and loaded into the differential scanning calorimeter (DSCMettler Toledo 822, Switzerland). All experiments were performed in duplicate
following the same protocol. The instrument was calibrated using indium, zinc and
lead. An empty pan was used as a reference.
The DSC thermograms were obtained from −140 to 120 ◦ C at a heating rate of
10 ◦ C/min. The enthalpy (H) of the endothermic transitions was calculated from
the area under the peaks. The glass-to-liquid transition temperature (Tg ) was determined as the onset temperature of the discontinuities in the curves of heat-flow
versus temperature (indicating a change in specific heat). All thermograms were
analyzed using STARe Software v. 6.1 (Mettler Thermal Analysis).
3. Results
2.2. Urease immobilization
Different compositions of sol–gel-derived silica nanocomposites were performed. The sol was prepared, as previously described [41,42], by sonicating
(Transsonic 540, 35 kHz, Elma, Singen, Germany) a mixture of 1 mL TEOS, 0.2 mL
of water (milli Q) and 0.06 mL of 0.04 M HCl for 30 min. Then, 0.15 mL of the sol
obtained were mixed, in a multiwell plate, with the same volume of a solution of
1 mg mL−1 of urease suspended in 50 mM sodium phosphate buffer pH 8 with and
without 20% (v/v) of glycerol or trehalose, respectively. The solutions were allowed
to gelate for 2 min and subsequently storage at various relative humidities.
The obtained gels were T (only TEOS), TT (TEOS + trehalose), TU (TEOS + urease),
TUT (TEOS + urease + trehalose), TG (TEOS + glycerol), TUG (TEOS + urease + glycerol).
The stability of urease was examined after immobilization
in sol–gel silica matrices (TEOS + urease) and in two types of
nanocomposites (TEOS + urease + trehalose) and (TEOS + urease +
glycerol). The samples were aged at 20 ◦ C with various relative
humidities for 16 days, to evaluate the influence of formulation
composition and ambient relative humidity on the preservation
of the enzyme catalytic activity. Fig. 1 shows that the entrapped
urease was more stable for all the formulations equilibrated at RH
80%. However, significant differences (p < 0.05) in enzyme activity
M.F. Desimone et al. / Enzyme and Microbial Technology 42 (2008) 583–588
585
Fig. 1. Effects of relative humidity and formulation composition on the preservation of the enzyme catalytic activity. The samples were aged at 20 ◦ C with
different relative humidities for 16 days. () TUG (TEOS + urease + glycerol), ( )
TUT (TEOS + urease + trehalose) and () TU (TEOS + urease).
Fig. 3. Water adsorption isotherms of the silica nanocomposites. The samples were
aged at 20 ◦ C with relative humidities of 80% for 16 days. (䊉) T (TEOS), () TT
(TEOS + trehalose), () TU (TEOS + urease) and () TUT (TEOS + urease + trehalose).
Fig. 2. Nitrogen adsorption isotherms of the silica nanocomposites. The samples were aged at 20 ◦ C with relative humidities of 80% for 16 days. ()
TU (TEOS + urease), () TG (TEOS + glycerol), () TT (TEOS + trehalose), (䊉) TUT
(TEOS + urease + trehalose), () TUG (TEOS + urease + glycerol) and (䊉) T (TEOS).
recovered at this RH were observed between samples with different formulations, reflecting the effect of additives during the
immobilization process. Fig. 1 shows that the highest urease activity was retained in the presence of glycerol (approximately 100%).
This activity level was maintained even after 1 month of storage at
20 ◦ C (data not shown). In addition, 5–10% of activity was recovered
in glycerol-containing systems at others RH, while no activity was
detected in the rest of the formulations.
Silica nanocomposites possessed nitrogen adsorption isotherms
similar to the isotherms characteristic of mesoporous materials
(type IV), as it can be seen in Fig. 2. The nitrogen isotherms presented two inflections, the first one, between relative pressures
(P/Po ) = 0–0.05, is due to formation of a monolayer of gaseous adsorbent and the second one, between P/Po = 0.1–0.9, shows the gas
filling and condensing in the mesopores, followed by nitrogen
condensation in the macropores. Table 1 summarizes the different parameters obtained from nitrogen (SBET and TPV) and water
(m0 and SWGAB ) adsorption isotherms, and from remaining relative
enzymatic activity determinations. It was observed that the aged
TUG material presented the lowest surface area and the lowest total
pore volume for nitrogen adsorption. As discussed before in relation
to Fig. 1, urease activity in glycerol matrices remained at the highest
activity level. The TEOS–urease (TU) matrix had the highest surface
area and the highest total pore volume. In this case the immobilized
enzyme retained only 76% of the relative activity under the same
experimental conditions. The trehalose systems had intermediate
values of surface areas and total pore volumes, and the recovered
urease activity in this matrix was 90%.
Glycerol addition leads to a strong decrease in surface area. This
suggests that glycerol is located in the mesoporous of silica gel and
may therefore be in close contact with the encapsulated protein
which is a key factor for its ability to stabilize immobilized urease. This hypothesis was strengthened by cryo-scanning electron
microscopy experiments performed in TEOS–glycerol gels [16,47].
Water sorption resulted in type V isotherms, highlighting the
hydrophobicity of the samples at low relative pressures (Fig. 3).
Similar water sorption isotherms were obtained for all systems
(with no significant differences). No data could be obtained for
water adsorption in the samples containing glycerol. Glycerol evaporation leads to higher gravimetric determination results that
difficult the determination of actual water content. The water fraction in TUG systems was obtained by Gordon and Taylor equation
Table 1
Data obtained from water and nitrogen adsorption isotherms
Sample
TUT
TUG
TT
TG
TU
Urease activitya (%)
90
100
–
–
76
Nitrogen sorption isotherms
Water sorption isotherms
SNBET b [m2 /g]
TPVc [cm3 /g]
m0 d [gH2 O/g]
SWGAB e [m2 /g]
222.57
92.30
224.30
101.67
779.85
0.348
0.133
0.337
0.150
0.871
0.0028
ND
0.0024
ND
0.0028
9.93
ND
8.51
ND
9.93
ND: Not determined. All values are given per gram of adsorbate.
a
Remaining relative urease activity at 80% RH (%).
b
SNBET : Surface area for nitrogen adsorption, as determined by BET equation.
c
TPV: Total pore volume.
d
m0 : Water monolayer value calculated by GAB equation.
e
SWGAB : Surface area for water adsorption, as determined by GAB equation.
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M.F. Desimone et al. / Enzyme and Microbial Technology 42 (2008) 583–588
Fig. 4. Enthalpies of the endothermic transitions corresponding to water fusion,
which are proportional to the amount of frozen water during cooling. () RH 80%,
( ) RH 84% and () RH 94%.
[48], using the Tg value of the sample and the glycerol constant
k = 2.02 [49]. A steep increase in water adsorption by capillary condensation occurred from around RH 80%. Aging of the wet silica
network over a period of days to weeks promotes further condensation and strengthens the network. The relative proportion
of siloxane to silanol groups increase owing to coarsening of the
material [7,23]. Enhanced reactivity of the matrix surface towards
water at RH 80% and above may result from the hydration of silanol
groups or of bulk constituents. Differential scanning calorimeter
confirmed the presence of frozen water only in samples stored at
relative humidity higher than 75%.
The phenomenon of drying and shrinkage in porous materials is
relevant in many technological areas [50], since it determines the
microstructure of the generated materials. Capillary condensation
will occur at a certain partial pressure of water, according to the
pore diameters of the solid material. The capillary tension induced
by the condensed water will generate stresses in all solid–fluid
interfaces, resulting in some deformation of the solid phase and
overall shrinkage of the composite. Changes in specific surface
energy in adsorbed water contribute to the overall shrinkage.
At very low relative humidity, where the material has no capillary condensed water, shrinkage on desorption and expansion
on adsorption depend only on the specific surface energy of the
adsorbed water layer. In the high relative humidity region the pores
were fully saturated by capillary condensed water contributing to
generate a relative humidity-dependent microstructure. As shown
in Table 1 surface areas for water adsorption were similar for the
matrices containing trehalose or no additives. They were, however,
lower than those obtained from nitrogen sorption, which suggests
that water was less efficiently packed in the material pores than
nitrogen, and were in accordance with the shape of the water
sorption isotherm. Moreover, the surface for nitrogen adsorption
was dependent on the matrix formulation. These differences can be
related to the specific interaction (strong or weak) of nitrogen with
these surfaces and with the possibilities of water in wetting their
surfaces. Robens et al. [50] indicated that to obtain information
for practical purposes the specific surface area measurements
and pores size distributions which are determined usually from
adsorption isotherms at low temperatures using nitrogen or noble
gases, need to be supplemented by investigations of water vapor
adsorption.
Thermal transitions were determined in the samples in order to
analyze the interactions among additives, enzyme, water and the
TEOS matrix. Fig. 4 shows the enthalpies of the endothermic transitions corresponding to water fusion, which are proportional to
the amount of frozen water during cooling down to −140 ◦ C the
samples aged at RH 80, 84 and 94%. Although there were no signif-
Fig. 5. Enthalpies of endothermal transitions corresponding to trehalose melting
in TT (TEOS + trehalose) samples aged with different relatives humidities, observed
close to 90 ◦ C by DSC.
icant differences in the amount of frozen water for a given material
at those relative humidities, there were differences in the melting
peak of water among different matrices aged at a given RH. The
amount of frozen water was highest in TU system, indicating that
more mobile water was trapped in this system when compared to
TUG or TUT matrices. The presence of polyol or trehalose promoted
a lower amount of frozen water. These results correlate with the
change in pores and surface area, since the matrix with larger pores,
had larger surface area and more water trapped.
Confinement in the sol–gel matrix did not prevent trehalose
crystallization in TEOS–trehalose systems (TT). An endothermal
transition corresponding to trehalose melting was observed by DSC
close to 90 ◦ C in TT samples, even in matrices with low water contents (RH 44%). Fig. 5 shows the enthalpies related to trehalose
melting. However, in the presence of urease no trehalose crystallization was observed in the matrices at any of the RH studied. The
inhibition of sugar crystallization by the presence of proteins has
been previously reported [51,52] and it is due to the impediments
in the formation of the sugar crystalline network caused by the
presence of foreign molecules.
Nanocomposites containing glycerol (TG and TUG) showed a
thermal transition value corresponding to glass transition temperature (Tg ) close to that of pure glycerol (−85 ◦ C). However,
TEOS–trehalose systems showed a glass transition at −60 ◦ C at RH
75%, which is a much lower temperature than that observed in pure
trehalose systems at the same RH (which is −35 ◦ C, according to
Mazzobre et al. [53]), due to the higher retention of water in the
TEOS matrix at same relative humidity.
Endothermic transitions corresponding to denaturation of pure
urease were observed above RH 75%, with an onset at 80 ◦ C, diminishing this onset while RH increased. This was due to an increased
mobility of protein molecules as increasing water content that led
to lower denaturation temperatures. On the other side, the onset
temperature for urease denaturation did not change significantly
when RH increased in encapsulated urease systems. However,
the associated enthalpy diminished in the studied range of relative humidities (Fig. 6). The lowest enthalpy of denaturation was
obtained in TEOS + urease + glycerol (TUG) nanocomposites and the
transition corresponding to enzyme denaturation was not observed
in TU, TUG and TUT at RH 80%, which corresponds to the relative
humidity at which higher urease activity was recovered, as discussed before. It is noteworthy that at this RH the steep change in
water sorption was observed in TU and TUT.
Aging and drying have a profound effect on the pore structure.
As the pore radius decrease, the solvent was expelled, so the internal viscosity of the solvent increased [54]. Increased microviscosity
and more “structured” water through hydrogen bonding with poly-
M.F. Desimone et al. / Enzyme and Microbial Technology 42 (2008) 583–588
587
4. Discussion
Fig. 6. Effects of relative humidity and formulation composition on the enthalpy
associated to urease denaturation. The samples were aged at 20 ◦ C with different relative humidities for 16 days. () TUG (TEOS + urease + glycerol), (♦) TUT
(TEOS + urease + trehalose) and () TU (TEOS + urease).
Fig. 7. Correlation between enzyme activity and the total pore volume of the gels
after urease immobilization, for samples aged at RH 80%. The calculated correlation
coefficient for lineal regression was 0.980.
Biomolecules are highly sensitive and fragile in nature; hence
their vicinity should be mild and closer to the native environment
after immobilization. Since the inactivation of the enzyme involves
the unfolding or denaturation of the native structure, the stabilization observed (mainly in the matrices containing glycerol and
exposed to RH 80%) suggests that the enzyme did not suffer from
the unfolding process inside the matrix at those conditions. We also
confirm the existence of an optimal catalytic efficiency depending in the mesoporous materials obtained after storage at RH 80%.
Thus, studies should be carried out with attention on the relative
humidity at which the immobilized biomolecules are exposed.
During the aging of the wet silica nanocomposites, entrapped
alcohol and water resulting from the initial hydrolysis and condensation reactions will be removed from the matrix, causing the
matrix to shrink. The addition of glycerol or trehalose helps to
avoid the excessive contraction of the matrix and the consequent
pore collapse, avoiding a significant decrease in the enzyme activity
level. Moreover, the addition of polyols can reduce the adsorption
of proteins onto the silica, presumably due to the preferential coating of the silica with the organic additives, and thereby provide
higher overall activity for the entrapped biomolecules [55]. Previous results, also confirm that the presence of a disaccharide, which
can hydrogen bond to subtilisin in the place of lost water, was necessary to inhibit dehydration-induced unfolding of subtilisin [56].
These results indicated that a critical point to preserve enzymatic activity is controlling pore size during the formation of the
sol–gel silica, which can be done by additives like glycerol and trehalose, and controlling the relative humidity to which the systems
are to be exposed. Thus, nitrogen and water sorption isotherms
are critical measurements for characterizing the behavior of these
porous materials [57].
Physical encapsulation within sol–gel glasses could offer new
opportunities for biotechnologies and it is important to be able to
produce biomaterial surfaces with controllable nanopore sizes and
morphologies [58,59].
5. Conclusion
hydroxylated silane and glycerol or trehalose were thought to be
responsible for increase urease stability.
The enhanced stability of immobilized proteins has been related
to a “molecular confinement” process, wherein the protein is
restricted in its ability to undergo conformational changes [24].
However, increased water content in samples aged above RH 80%,
resulted in enhanced mobility for the entrapped protein and higher
protein–silica interactions favoring protein unfolding.
This indicated that there is a critical solvent level that is necessary for the protein to maintain its structure. The intrinsic
properties of the protein such as size, shape, and its interactions
with the surrounding matrix also determine the extent of drying
effects on its stability.
A negative correlation between enzyme activity and the total
pore volume of the gels after urease immobilization seems to exist,
as it can be seen in Fig. 7, for samples aged at RH 80%. The calculated
correlation coefficient was 0.980 for lineal regression. The addition
of glycerol or trehalose decreased the matrix surface area and total
pore volume, allowing a significant increase in the enzyme activity
level. As mentioned before larger pores, had larger surface area and
more water trapped which allow enhanced conformational mobility, leading to enzyme denaturation. The interactions between the
solvent, biomolecules and nanocomposite material determine the
degree to which the biomolecules retains its native properties.
As shown in this work, relative humidity and the presence of
polyols modify pore volumes, surface area, and state transitions
of water and solid components, and are thus important aspects to
take into account in the formulation of the encapsulating matrices to preserve enzyme stability. The applications of biocompatible
sol–gel-derived matrices can be further extended and utilized in
the development of biosensors with immobilized biomolecules or
whole living cells that can be used for long time periods, without changes in sensitivity, by taking into account different factors,
among which the storage relative humidity has permitted to greatly
improve the stability of the immobilized enzyme.
Acknowledgements
The authors acknowledge financial support from Consejo
Nacional de Investigaciones Cientificas y Técnicas (CONICET PIP
5799), Agencia Nacional de Promoción Cientı́fica y Tecnológica
(PICT 20545, 14192, 32916 and 32310) and Universidad de Buenos
Aires (UBACYT EX226, B055 and B817).
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