Improvement in the catalytic activity of cytochrome c by immobilisation on a novel mesoporous silica sheet

Article abstract of DOI:10.1039/c3ra45861j

Here, we introduce a new synthetic approach to mesoporous silica (MPS) sheets (thickness ≤ 100 nm). By using the carboxylate surfactant (N-palmitoyl-l-alanine) and Pluronic P123 as dual-templating agents, an MPS sheet was synthesised under similar conditions to SBA-15. Cytochrome c immobilised on an MPS sheet showed extremely high relative activity.

Full text of DOI:10.1039/c3ra45861j

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Improvement in the catalytic activity of
cytochrome c by immobilisation on a novel
Cite this: RSC Adv., 2014, 4, 4732
mesoporous silica sheet†
a
b
Kazuma Nakanishi,ab Masahiro Tomita and Katsuya Kato
Received 18th October 2013
Accepted 4th December 2013
*
DOI: 10.1039/c3ra45861j
www.rsc.org/advances
Here, we introduce a new synthetic approach to mesoporous silica aminosilane as a co-structure-directing agent.11 Yu et al. repor-
(MPS) sheets (thickness # 100 nm). By using the carboxylate surfactant ted the preparation of radially oriented MPS nanotubes by using
(N-palmitoyl-L-alanine) and Pluronic P123 as dual-templating agents, a chiral anionic surfactant as a template.12 Li et al. demon-
an MPS sheet was synthesised under similar conditions to SBA-15. strated the convenient preparation of chiral mesoporous
Cytochrome c immobilised on an MPS sheet showed extremely high nanotubes and nanoribbons by using a dual-templating route.13
relative activity.
This approach was used in the self-assembly of chiral amphi-
philes and the F127 tri-block copolymer as templates. On the
basis of these approaches, we demonstrate a new approach to
prepare an MPS sheet by using dual templates, the original
carboxylate surfactant (N-palmitoyl-L-alanine) and the Pluronic
P123 tri-block copolymer. Then, the prepared MPS-sheet was
used as carrier of bio-molecule.
There is great interest in porous materials because they can
interact with atoms, ions, molecules and nanoparticles at their
surfaces as well as throughout the bulk of the material. Meso-
porous silica (MPS) (pore diameter 2–50 nm) has been applied
in many elds including enzyme immobilisation, catalysis and
chromatography because of its mechanical stability, extremely
large surface area and controllable pore size.1–6 In our previous
study, the enhanced activity and stability of enzymes and
proteins on MPS as an effective carrier were reported.7–9
Therefore, the presence of pores in nanostructured materials
for biological applications should be promoted. However, to
obtain a high enzymatic activity, a high adsorption amount of
catalytic enzyme was required. To solve this costly problem, we
focused on the morphologies of host materials in order to
increase the mass transfer of the substrate. The morphologies
have an important role in the activity of the immobilised
enzyme.
N-Palmitoyl-L-alanine (C16-L-Ala) was prepared as a surfactant
according to the procedure reported in the literature.12 Then,
7.2 g (0.08 mmol) of L-alanine was dissolved in a mixture of 11.6
mL of de-ionised water, 17.4 mL of acetone and 3.24 g (0.08
mmol) of NaOH. Furthermore, 25 g (0.09 mmol) of palmitoyl
chloride and 10 M NaOH were simultaneously added drop wise
to the mixture under ice-cooling maintaining pH 12. Aer stir-
ring for 1 h, the reaction mixture was acidied to pH ¼ 1 with
HCl. The mixture was then washed with distilled water to
remove any unreacted amino acid. Aer being neutralised, the
precipitate of crude crystals of N-palmitoyl-L-alanine was
washed out to remove any unreacted palmitoyl chloride by
diethyl ether. The product was ltered in vacuo and the N-pal-
mitoyl-L-alanine was puried by recrystallisation from toluene.
To the best of our knowledge, only a few mesoporous sheet
have been reported because they are difficult to prepare.10
However, chiral MPS was discovered in a templating route that
was based on the self-assembly of a chiral anionic surfactant
and inorganic precursors by using aminosilane or quaternised
1
The structure of the product was identied by H-NMR.
A mesoporous sheet was synthesised using a novel dual-
templating route. 0.31 g of C16-L-Ala (1 mmol) and 0.30 g of
Pluronic P123 (5.4 ꢀ 10ꢁ2 mmol) were dissolved in distilled
water (25.5 mL) and 0.1 M NaOH (10.0 mL) with vigorous stir-
ring at room temperature. 0.1 M HCl (4.0–4.5 mL) was added to
the above solution to partially neutralise (pH 9–10). 1.46 g of
TEOS (7.0 mmol) and 0.28 g of APTES (1.6 mmol) were added to
the mixture with stirring at 4 ꢂC. Aer 3 h stirring, the reaction
mixture was aged for 24 h at 25 ꢂC. The resulting precipitate was
washed repeatedly with distilled water and further hydrother-
mally treated at 100 ꢂC for 24 h. Finally, the sample was washed
aDepartment of Chemistry for Materials, Graduate School of Engineering, Mie
University, 1577 Kurimamachiya-cho, Tsu, Mie 514-8570, Japan. E-mail:
Kazuma-nakanishi@aist.go.jp; Fax: +81 59 231 9430; Tel: +81 59 231 9428
bNational Institute of Advanced Industrial Science and Technology, 2266-78,
Anagahora, Moriyamaku, Nagoya, Aichi 463-8560, Japan. E-mail: katsuya-kato@
aist.go.jp; Fax: +81 52 736 7405; Tel: +81 52 736 7551
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3ra45861j
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with ethanol and acetone to dry and then calcined at 500 ꢂC for sample of 0.30 g of P123 (Fig. 1(e)) and over 100 nm in 0.45 g of
4 h to remove the template. P123 (Fig. 1(f)). To demonstrate the role of surfactants, we
The morphology of the prepared samples was characterised changed P123 to CTAC, the typical template for preparing MPS
using eld emission scanning electron microscopy (FE-SEM) MCM-41 (Fig. 1(g)) and C16-L-Ala to palmitic acid (Fig. 1(h)). In
and transmission electron microscopy (TEM). Fig. 1 shows the both these samples we did not see any sheet morphology. From
FE-SEM image of the samples synthesised at various ratios of these results, the synthetic mechanism of the silica sheet
surfactants and silica sources. The morphology of silica mate- structure was directed by C16-L-Ala and P123 as surfactants.
rials can be adjusted by changing the synthesis gram compo- Also, we suggest that the P123 copolymer interacts with oligo-
sition: C16-L-Ala : P123 : APTES : TEOS ¼ 0.30 : X : Y : 1.46. In silicates by hydrogen binding onto C16-L-Ala bilayers.10 APTES
Fig. 1(a)–(d), the samples were prepared at X ¼ 1.50 and Y ¼ 0, has high hydrolysis reactivity, leading to a silica framework.
21, 0.28 and 0.35, respectively. In nothing or small amount of The TEM images of the C16-L-Ala : P123 : APTES : TEOS ¼
APTES, the prepared materials were not of a sheet morphology 0.31 : 0.30 : 0.28 : 1.46 sample showed the presence of meso-
(Fig. 1(a) and (b)). When the APTES ratios were increased, pores on the sheet structure (Fig. 2). We describe this material
surprisingly, the sheet structure can only be found in (C16-L-Ala : P123 : APTES : TEOS ¼ 0.31 : 0.30 : 0.28 : 1.46) as
APTES : TEOS ratio of 0.28 : 1.46. With further addition of the MPS sheet.
APTES, the silica materials form an aggregated morphology.
The mesoporous structure of the MPS materials, conven-
Thus, the gram ratios of APTES have an effect on the prepara- tional MPSSBA-15, MCM-41 and the prepared MPS sheet, were
tion of sheet materials. By increasing the P123 gram ratio from investigated using small-angle X-ray diffractometer (Fig. 3). The
0.15 to 0.45 g, we grew thicker silica morphologies (Fig. 1(c), (e) SBA-15 displayed three well-resolved diffraction peaks corre-
and (f)). The thickness showed approximately 50–100 nm in the sponding to (100), (110) and (200) reections (Fig. 3(a)) and also
MCM-41 showed similar peaks (Fig. 3(b)). These results show
well-ordered two-dimensional (2D)-hexagonal mesostructures.
No peaks at 0.5–7.0ꢂ were observed for the MPS sheet (Fig. 3(c)).
This result suggests that this is due to a disordered pore
structure within the MPS sheet.
Fig. 2 TEM images of MPS sheet material prepared at g ratios of C16-L-
Ala : P123 : APTES : TEOS
(b) ꢀ100k.
¼ 0.31 : 3.0 : 0.28 : 1.46. (a) ꢀ20k and
Fig. 1 FE-SEM micrographs of MPS materials. Synthesised at various
weight ratios (g) of C16-L-Ala : P123 : APTES : TEOS ¼ 0.31 : X : Y : 1.46.
(a) X ¼ 1.5, Y ¼ 0, (b) X ¼ 1.5, Y ¼ 0.21, (c) X ¼ 1.5, Y ¼ 0.28, (d) X ¼ 1.5, Y ¼
0.35, (e) X ¼ 3.0, Y ¼ 0.28 and (f) X ¼ 0.45, Y ¼ 0.28. Prepared at different
surfactant (g) 0.31 g of C16-L-Ala and 0.30 g of CTAC, (h) 0.24 g of Fig. 3 Small-angle X-ray diffraction patterns of MPS materials: (a)
palmitoyl acid and 0.30 g of P123. Scale bar: 1 mm.
SBA-15, (b) MCM-41 and (c) the MPS sheet.
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We analysed the N2 adsorption–desorption isotherms and
corresponding pore-size distributions. Nitrogen adsorption–
desorption isotherms of the MPS sheet sample showed two
small hysteresis loops at relative pressures (P/P0) between 0.45
and 0.98 indicating that these originated from the mesoporous
within the silica sheet-wall (ESI, Fig. S1†). The physicochemical
properties of the MPS materials, as derived from the BET and
BJH methods are summarised in Table 1. The surface area
and pore volume and pore size of MPS sheet were 192.1 m2 gꢁ1
and 0.43 cm3 gꢁ1, respectively. Also, the pore diameter was #10
nm (average pore size 6.6 nm) with a disordered pore size
distribution (ESI, Fig. S2†).
Cytochrome c (cyt c, molecular size of 2.5 ꢀ 2.5 ꢀ 3.7 nm, MW
12 kDa) is a great biocatalyst in vitro, reported to catalyse the
oxidation of a wide variety of organic substrates.14–16 We
compared the catalytic ability of the immobilised cyt c on MPS
morphology (thin sheet MPS, conventional SBA-15 and MCM-
41). Carrier materials (1.5 mg) were suspended in 530 mL of
10 mM phosphate buffer (pH ¼ 7.0) including 30 mg of cyt c for
24 h at 4 ꢂC with stirring. The solid precipitate was prepared by
centrifugation at a speed of 1200 rpm at 4 ꢂC and washed with the
phosphate buffer. The catalytic activity assays on the immobi-
lised and native cyt c were determined by oxidation of phenol and
4-aminoantipyrine with H2O2. 650 mL of 10 mM phosphate
buffer, 20 mL of 1.76% 4-AAP, 20 mL of 6% phenol and 100 mL of
H2O2 solution (1 mL of 30% H2O2 : 500 mL of phosphate buffer)
were mixed with the precipitate and stirred at 25 ꢂC for 30 min.
Then, the supernatant obtained by centrifugation was analysed
with UV-Vis at 500 nm. All the reported values were estimated by
averaging the results obtained by repeating the characterisation
at least four times. Fig. 4 shows the catalytic activity of cyt c, i.e.
oxidation of phenol and 4-AAP with H2O2. The relative activity of
immobilised MCM-41, SBA-15, MPS sheet and native cyt c were
obtained as 81%, 79%, 178% and 100%, respectively. Thus, the
sheet morphology can be suggested as a big inuence for
enzymic activity as a protein carrier. The supernatant aer cyt c
immobilisation solution did not indicate any catalytic activities.
The pore volume of cyt c immobilised on MPS sheet decreased
from 0.43 to 0.39 cm3 gꢁ1 (ESI, Fig. S3†). These results indicated
that all of cyt c (30 mg) was adsorbed on the not only exterior but
pore interior of carrier materials.17 Also, no reaction occurred
using bare MPS (with no cyt c).
Fig. 4 Oxidation activity of immobilised cyt c on MPS. Relative activity
was calculated as follows: (oxidation activity of cyt c immobilised on
carrier)/(oxidation activity of native cyt c) ꢀ 100%.
Table 2 Kinetic parameters for the oxidation of 4-aminoantipyrine
and phenol by cyt c with H2O2
Samples
KM [mM]
Kcat [Sꢁ1
]
Kcat/KM [mMꢁ1 Sꢁ1
]
Native cyt c
Cyt c immobilised
on MPS sheet
0.93
2.07
0.039
0.101
0.041
0.049
analysed. The kinetic parameters, Michaelis constants (KM), the
turnover number (Kcat) and the catalytic efficiency (Kcat/KM) were
determined according to Lineweaver–Burk equation:
1
Km
1
¼
þ
:
V0 Vmax½Sꢃ
Vmax
The maximum velocity (Vmax) equals Kcat/[E], where [E] is
the immobilised enzyme concentration. The Lineweaver–Burk
plot is described in the ESI (Fig. S4†). The values of KM, Kcat and
Kcat/KM are demonstrated in Table 2. Cyt c immobilised on the
MPS sheet sample was indicated to be approximately twice
the value of KM, i.e. 2.07 mM, which signied a lower affinity to
the reducing substrate. However, the Kcat value, i.e. catalytic rate
constant, of cyt c immobilised on MPS sheet was much higher
than that of native cyt c. The Kcat/KM value in the immobilised
sample was similar or higher than the native sample. These
results suggest that the immobilisation of cyt c on the MPS
sheet enhances the enzyme's catalytic activity. Obviously, the
MPS sheet was obtained as the benecial enzyme carrier
material. Thus, this material can increase the immobilised
enzyme activity more than not only conventional MPS materials
but also nanocarbon materials like multi-walled carbon nano-
tubes and graphene oxide nanomaterials.18,19
The catalytic oxidation activity of cyt c immobilised on MPS
sheet with different concentrations of 4-aminoantipyrine was
Table 1 Nitrogen physisorption parameters of MPS materials
Pore sizea
(nm)
Pore volumea
Surface areaa
(cm3 gꢁ1
)
(m2 gꢁ1
)
SBA-15
MCM-41
MPS sheet
7.5
2.7
#10
1.1
1.5
0.4
901
1055
192
Conclusions
a
The Brunauer–Emmett–Teller (BET) surface area, pore volume and
pore-size distribution curves were obtained from the nitrogen
adsorption–desorption isotherms using the Barrett–Joyner–Halenda
(BJH) method.
The MPS sheet (thickness # 100 nm) was synthesised using a
novel approach for the immobilisation of cyt c. In the synthetic
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process, the dual-templating surfactants of C16-L-Ala and P123
had a role in determining the structural morphology of the
sheet material. Also, the weight ratio of the silica sources TEOS
and APTES was an effective factor for the silica morphology.
Silica sheets could be prepared under the gram ratio conditions
of C16-L-Ala : P123 : TEOS : APTES ¼ 0.31 : 0.30 : 1.46 : 0.28. Cyt
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