O. Fernández et al. / Applied Catalysis A: General 450 (2013) 204–210
205
decrease. Thus, the enzyme can reach most of the internal sur-
faces of the particle, and as a result, high values of enzyme loading
and catalytic efficiency have been obtained. However, their average
particle size is not large enough to keep them retained in 100 m
meshes. Based on these materials, the objective of this work is to
preserve high enzyme loading and similar values of catalytic activ-
ity with larger size particles. Most of the work in this field is focused
on the use of isolated large particles, such as immobilization on
and prepared from different precursors [13]. Some works can also
be found in the literature describing the formation of larger par-
ticles, for example, through crosslinking of lipase immobilized on
ordered mesoporous materials with chitosan by means of reaction
with glutaraldehyde [14]. We propose the formation of agglomer-
ates of octyl silica particles by sticking them to each other through
a minimal contact surface as the strategy to increase the final size
of the catalyst
vacuum for 12 h was suspended in a 10 mL solution of octyltri-
ethoxysilane in toluene (1:4, v/v). The suspension was gently stirred
for 48 h at 80 ◦C. After that, the suspension was filtered and washed
twice with dry toluene, and three times with hexane and acetone,
and finally exhaustively vacuum dried. This support is referred to
as octyl-silica (OS). The same procedure was followed for the func-
tionalization of silica-methacrylate (SM) agglomerates to obtain
the composites referred as OSM-10.
2.5. Characterization of the solids
Nitrogen isotherms were measured at the temperature of liquid
N2 with a Micromeritics ASAP 2000 apparatus. Samples were pre-
viously degassed at room temperature for 20 h. The surface areas
were determined following the BET method. Thermogravimetric
analyses (TGA) were carried out on a Mettler Toledo TGA/SDTA
851e apparatus. Typically, 5 mg of the sample was heated from 25
to 800 ◦C at a rate of 20 ◦C/min under air flow (200 ml/min). Scan-
ning electron microscopy (SEM) micrographs were taken with a
Hitachi TM-1000 at 15 kV and without coating.
2. Materials and methods
2.1. Chemicals
2.6. Immobilization of lipase
n-Octyltriethoxysilane (TCI Europe, Belgium), glycidyl
methacrylate (from now GMA) 97%, ethylene dimethacrylate
(from now EGDMA) 98%, 1,1ꢀ-azobis (cyclohexane-carbonitrile)
98%, cyclohexanol, 1-tetradecanol, poly(vinylpolypyrrolidone),
glyceryltributyrate (tributyrin), oleic acid and butanol were
purchased from Aldrich (St. Louis, USA). Ethanol (HPLC grade),
di-sodium hydrogen phosphate and toluene were purchased from
Panreac (Barcelona, Spain). All chemicals were of analytical grade.
MS3030 silica was kindly donated by Silica PQ Corporation (Valley
Forge, PA, USA). Lipase from C. antarctica B (Lypozyme, CaLB) was
donated by Novozymes (Denmark). p-Nitrophenyl acetate (pNPA)
was purchased from Sigma.
Protein content of the enzyme extract (3.5 mg/ml) was
determined according to the Bradford method [16]. SDS-PAGE elec-
trophoresis of this extract showed a unique band so all the protein
can be attributed to the lipase.
Different amounts of the enzyme extract were dissolved in
50 mM phosphate buffer, pH 7.0, up to a total volume of 20 mL.
After assaying the esterasic activity of these solutions, 100 mg of
the corresponding support previously wet with ethanol were added
and maintained in suspension with a helical stirrer. Aliquots from
suspension and supernatant were withdrawn at 10–240 min to
analyze their esterasic activities.
Final time is determined by the lack of activity, or low constant
activity of the supernatant. After that, suspensions were filtered
and washed three times with 10 mL volumes of 200 mM phosphate
buffer. The derivatives were washed twice with 10 mL dry ace-
tone, filtered out and vacuum dried for at least 30 min to ensure
a complete drying of the catalyst.
2.2. Synthesis of organic resin
The organic resin (M) was synthesized as described in the lit-
erature [15]. The main procedure was as follows: the monomer
phase containing the monomer mixture (3.5 g of GMA and 2.3 g of
EGDMA), 0.15 g 1,1ꢀ-azobis (cyclohexane-carbonitrile) as an initia-
tor and 7.5 g of inert component (6.78 g of cyclohexanol and 0.7 g
of tetradecanol), was suspended in the aqueous phase consisting of
40 g of water and 0.6 g of poly (vinyl pyrrolidone) (PVP). The copo-
lymerization was carried out at 70 ◦C for 2 h and then at 80 ◦C for
6 h with a stirring rate of 200 rpm. After completion of the reaction,
the copolymer particles were washed with water and ethanol, kept
in ethanol for 12 h and then dried in a vacuum oven at 45 ◦C for
24 h.
2.7. Determination of enzyme activity. Hydrolytic activity
2.7.1. Esterasic activity
Despite an assay for pNPA hydrolysis activity is not a specific test
for lipase activity, this assay was selected for use as a routine assay
because it is easy to conduct via spectrophotometric measurements
and it provides a rapid assessment of relevant enzymatic activity.
Hydrolysis of p-NPA was followed at 348 nm in an Agilent 8453 UV-
visible spectrophotometer (Agilent Technologies) equipped with
stirring device and constant temperature capability. The cell con-
tained 1.9 mL of substrate solution at 25 ◦C (0.4 mM p-NPA in 50 mM
sodium phosphate buffer, pH 7.0). Aliquots of the suspension were
diluted in different proportions in 50 mM phosphate buffer (pH 7.0)
prior to being added to the cell (50 L) to facilitate the analysis.
Aliquots from the supernatant were not diluted: 50 L were added
directly to 1.9 mL substrate solution. One esterase unit corresponds
to consumption of 1 mol p-NPA/min ( p-NP = 5150 M−1 cm−1).
2.3. Synthesis of hybrid composite
Different amounts of MS3030 silica were added to the aque-
ous phase before polymerization of organic resin. The mixture was
stirred to form a homogeneous suspension. Next, the monomer
phase of organic resin was added to initiate polymerization. The
final octyl-silica-methacrylate composites will be called OSM-x,
being x the grams of silica added to the synthesis mixture.
OSM hybrids were gently crushed in a mortar and sieved to sep-
arate a fraction with diameters ranging between 100 and 200 m.
2.7.2. Tributyrin activity
The hydrolysis of tributyrin measures lipase activity by the liber-
ation of butyric acid. The reaction was monitored titrimetrically in a
Mettler DL50 pH-stat, using 100 mM sodium hydroxide. A 48.5 mL
potassium phosphate buffer solution (10 mM, pH 7.0) was incu-
bated in a thermostated vessel at 25 ◦C and stirred sufficiently.
Then, 1.47 mL tributyrin were added and the pH-stat was started
2.4. Support functionalization
The functionalization of the support was carried out as described
by Blanco et al. [11]. 1 g of silica previously degassed at 80 ◦C under