N.H. Abdallah et al. / Journal of Molecular Catalysis B: Enzymatic 108 (2014) 82–88
83
smaller than that of the enzyme to the properties on MPS with pore
diameters larger than the enzyme. The presence of trypsin in the
pores of MPS was demonstrated by the far more rapid digestion of
proteins by MPS immobilised trypsin in comparison to the solution-
based enzyme [8]. Small-angle neutron scattering demonstrated
that cross-linked chloroperoxidase was present in the pores of the
support [9]. Direct observation of an enzyme within the pores was
recently demonstrated in transmission electron microscopy studies
of lipase immobilised onto SBA-12 [10] and lysozyme onto SBA-
adsorbed after the micropores have been filled until after conden-
sation into the mesopores was complete. UV–visible spectroscopy
was performed on a Shimadzu UV1800 spectrophotometer.
Transmission electron microscopy (TEM) was conducted at an
accelerating voltage of 200 kV using a JEOL JEM-2011 microscope.
The sample was placed directly on a formvar-backed carbon-coated
copper grid. Scanning electron microscopy (SEM) was carried out
on a FEI Inspect F instrument operating at 10 kV. Silica samples
were placed on conductive carbon tape prior to analysis. Focussed
ion beam (FIB) was performed using a FEI Helios Nanolab 600 dual-
beam FIB. The electron beam was operated at 5 kV with the ion
beam operating at 30 kV for Pt deposition and thinning. The cross
sections were prepared using a focussed ion beam method [26].
1
5 [11]. These studies provided direct evidence that the enzyme is
adsorbed along the length of the mesopores, the length of which can
extend to over several hundred nanometre. The accessibility and
catalytic efficiency of enzyme molecules adsorbed deeply within
such pores has not been clearly examined, with it being likely that
the catalytic efficiency of the enzyme will decrease due to diffu-
sion constraints, and in particular at high enzyme loadings when
blockage of the pores is more likely.
2.3. Synthesis of mesoporous silica
SBA-15 was prepared using a published procedure [27]. PPS was
In order to investigate this effect, the adsorption and catalytic
efficiency of cytochrome c and CALB on MPS materials with differ-
ent porosities and surface areas have been examined. Cytochrome
c is a small redox protein (12.4 kDa) [12] and has been widely
used as a model system to investigate the adsorption of proteins
on MPS [13]. CALB is a hydrolase enzyme (33 kDa) [14] that is
widely used in biocatalysis [15–17] due to its broad substrate range,
high activity and stability. CALB has been successfully immobilised
by several methods [18–20] onto various solid supports [21–24].
SBA-15 possesses a hexagonal structure with pore diameters of
ca. 7.5 nm which are sufficiently large to accommodate lipase and
cytochrome c. PPS are monodispersed micron sized porous silica
spheres, with an average pore diameter of 7.5 nm. The pore mor-
phology of PPS is continuous and sponge-like. PPS can be utilised
in applications where the facile mass transfer of analytes in to and
out of the pores is required [25]. Both materials possess average
pore diameters (7.5 nm) that are sufficiently large to accommo-
date lipase or cytochrome c. In this study, the immobilisation of
CALB and cytochrome c is examined to determine the influence of
differences in the support on factors such as loading, activity and
stability. The immobilisation of CALB on SBA-15 results in higher
activity and stability when compared to PPS.
prepared using an adaptation of a previously published methods
25,28], Tetraethyl orthosilicate (TEOS) was used as the silica source
and cetyltrimethylammonium bromide (CTAB) acted as the struc-
ture directing agent for pore formation and methanol (MeOH) was
used as a co-solvent. CTAB (1.2 g) was dissolved in deionised water
[
(
2
88 mL) and methanol (MeOH, 500 mL); the solution was stirred for
h. Ammonium hydroxide (32 mL, 32.66% w/w in H O) and TEOS
2
(8 mL) were then added to the solution, the temperature was main-
tained at room temperature and the mixture was stirred for 24 h.
The silica precipitate was separated by centrifugation and dried at
◦
room temperature. The sample was then calcined at 550 C for 8 h.
2.4. Protein immobilisation
The adsorption of cytochrome c was performed in 25 mM
potassium phosphate buffer at pH 7.0 (2 mg/mL silicate). The
concentration of protein was determined using an extinction coef-
−1
−1
ficient of 100,000 M cm at 407 nm [12]. The concentration of
lipase was determined using the Bradford method [29]. The immo-
bilisation of lipase was conducted in phosphate buffer (10 mM, pH
7
.0). A stock solution of lipase was prepared by 1:4 dilution of the
as received enzyme. Varying concentrations were prepared from
the stock solution in 10 mM phosphate buffer at pH 7.0. Adsorption
of the enzyme on MPS was then allowed to proceed for a period
2
. Experimental
.1. Materials
CALB was
◦
of 18 h at 25 C. Lipase loading was calculated by taking 1 mL from
2
the reaction vessel, centrifuging (3000 rpm), measuring the lipase
concentration of the supernatant, and subtracting this value from
the initial concentration.
a
gift from Novozyme. Pluronic P123
(
EO20PO70EO20) was donated from BASF. The following
chemicals were obtained from Sigma–Aldrich and used as
received without further purification: cytochrome (horse
c
2.5. Catalytic activity
heart type VI, >97% purity, HCl, tetraethyl orthosilicate (TEOS),
cetyltrimethylammonium bromide (CTAB), KH PO , K HPO ,
2
4
2
4
The catalytic activity of cytochrome c was determined accord-
ꢀ
2
,2 -azino-bis(3-ethylbenzthiazoline-6-sulphonis acid) (ABTS),
ing to published reports using ABTS as substrate [7,30]. Lipase
activity was determined by measuring the rate of hydrolysis
of 4-nitrophenyl butyrate (4-NPB); typically, a solution contain-
ing 1.9 mL of phosphate buffer (10 mM, pH 7.0), 0.05 mL lipase
ammonium hydroxide solution (32.66%, NH OH), methanol,
4
ethanol, glutaraldehyde (25%), 4-nitrophenylbutyrate, Bradford
assay and 2-propanol. De-ionised water (18.2 Mꢀ cm) was used
for all aqueous solutions.
(
(
either in solution or as a suspension) and 0.05 mL of 4-NPB
1 mM) in 2-propanol was prepared and the increase in absorbance
−
1
−1
2.2. Methods
at 410 nm was recorded (ε4NP = 14,775 M cm ) [31]. Recycling
experiments were performed with a higher concentration of immo-
bilised lipase (8 mg/mL of enzyme). After incubation with 4-NPB
(1 min), the sample was centrifuged (1 min at 3000 rpm) and the
absorbance of an aliquot (0.5 mL diluted to 2 mL with buffer solu-
tion) measured. The immobilised lipase sample was washed twice
with buffer solution (1 mL) and the assay procedure repeated. Sta-
bility tests were performed by measured the catalytic activity of
immobilised lipase on a weekly basis after storage in phosphate
Nitrogen gas sorption isotherms were measured at 77 K using a
Quantachrome Autosorb AS1 system. Samples were pre-treated by
heating under vacuum at varying temperatures until the samples
were no longer out-gassing. The surface area was measured using
the Brunauer–Emmett–Teller (BET) method. The pore size data was
calculated using the Barrett, Joyner and Halenda (BJH) method.
Mesoporous volumes were estimated from the volume of nitrogen