Step-Up Synthesis of Periodic Mesoporous Organosilicas with a Tyrosine Framework and Performance in Horseradish Peroxidase Immobilization

Article abstract of DOI:10.1002/asia.201701285

New amino-acid-bridged periodic mesoporous organosilicas (PMOs) were constructed by hydrolysis and condensation reactions under acid conditions in the presence of a template. The tyrosine bissilylated organic precursor (TBOS) was first prepared through a multistep reaction by using tyrosine (a natural amino acid) as the starting material. PMOs with the tyrosine framework (Tyr-PMOs) were constructed by simultaneously using TBOS and tetraethoxysilane as complex silicon sources in the condensation process. All the Tyr-PMOs materials were characterized by XRD, FTIR spectroscopy, N₂ adsorption–desorption, TEM, SEM, and solid-state ²⁹Si NMR spectroscopy to confirm the structure. The horseradish peroxidase (HRP) enzyme was first immobilized on these new Tyr-PMOs materials. Optimal conditions for enzyme adsorption included a temperature of 40 °C, a time of 8 h, and a pH value of 7. Furthermore, the novel Tyr-PMOs materials could store HRP for approximately 40 days and maintained the enzymatic activity, and the Tyr-PMOs–10 % HRP with the best immobilization effect could be reused at least eight times.

Full text of DOI:10.1002/asia.201701285

CHEMISTRY
AN ASIAN JOURNAL
www.chemasianj.org
Accepted Article
Title: Step-up synthesis of periodic mesoporous organosilicas with
the framework of tyrosine (Tyr-PMOs) and performance on
horseradish peroxidase immobilization
Authors: Jianqiang Wang, Wenqi Zhang, Changqing Gu, Wenpei
Zhang, Man Zhou, Zhiwei Wang, Cheng Guo, and Linbing
Sun
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To be cited as: Chem. Asian J. 10.1002/asia.201701285
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10.1002/asia.201701285
Chemistry - An Asian Journal
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Step-up synthesis of periodic mesoporous organosilicas with the
framework of tyrosine (Tyr-PMOs) and performance on
horseradish peroxidase immobilization
Wang Jianqiang*[a], Zhang Wenqi[a], Gu Changqing[a], Zhang Wenpei[a], Zhou Man[a], Wang Zhiwei[a],
Guo Cheng*[a] and Sun Linbing[b]
Abstract:
A
new amino-acid bridged periodic mesoporous
have attracted growing research interest as the promising hosts
for enzyme immobilization due to high surface area, large pore
volume, special pore structure, thermal stability and
biocompatibility. [18] Specially, functional mesoporous silica
materials with various organic groups show extremely important
to immobilize enzyme because of enhancing enzymatic affinity
and activity by adjusting the microenvironment of channels. [19]
organosilicas (PMOs) have been constructed via hydrolysis and
condensation reactions under acid condition in the presence of
template. The tyrosine bissilylated organic precursor (TBOS) was
first prepared through multi-step reaction using tyrosine (natural
amino acid) as starting material. The PMOs with the framework of
tyrosine (Tyr-PMOs) were architected by simultaneously using
TBOS and TEOS as complex silicon source in the condensation
process. All the Tyr-PMOs materials were characterized by XRD,
FT-IR, N2 adsorption-desorption, TEM, SEM and 29Si solid-state
NMR to confirm the structure. The enzyme of horseradish
peroxidase (HRP) is first immobilized on these new Tyr-PMOs
materials. The optimal conditions of enzyme adsorption are as the
It is well known that organic groups are usually incorporated
within the channels of mesoporous materials via three ways:
grafting,
co-condensation
and
periodic
mesoporous
organosilicas (PMOs). [20] The first two methods of modification
can well retain the mesostructure but mesoporous channels are
often narrowed or even completely blocked by organic groups to
hinder diffusion of enzyme molecules. [21] Compared with these
two methods, periodic mesoporous organcosilicas (PMOs) can
effectively reduce these shortcomings by using bissilylated
organic precursors of type (R’O)3Si-R-Si(R’O)3 (R: organic
bridge; R′: methoxy or ethoxy group) to incorporate organic units
in the three-dimensional network structure of the silica matrix,
which organic units are distributed homogeneouly in the
framework and do not occupy pore volume. [22-23] Previous
many studies have confirmed that PMOs as an attractive
countermeasure provide new opportunities for enzyme
immobilization. For example, cytochrome c, xylanase, lysozyme
and lipase have been immobilized on PMOs to show better
adsorption capacities and stability. [24-27] PMOs can be tuned
or be targeted on specific applications due to the different
properties in the part “R” of bissilylated organic precursor. Thus,
it is important to design the content of “R” to construct kinds of
PMOs according to the different requirement. Amino acids are
indispensable compounds for human’s body as a source of
energy, and play vital roles in many fields including peptide
synthesis, biomedical sensors and drug delivery system, etc.
They often are immobilized on nanomaterials as promising
molecules. [28-31] Among 20 natural amino acids, tyrosine is an
aromatic amino acid because of a phenolic group on its side
chain. Moreover, tyrosine is an important amino acid in human
biological system, which acts as the precursors to various
neurotransmitters and thyroxine. [32-33] The phenolic group of
tyrosine is also used as the potential binding points to
immobilize enzyme because of hydrophilic property in the
tyrosine-functionalized materials.
◦
followings: temperature of 40 C, time of 8 h, pH of 7. Furthermore,
the novel Tyr-PMOs materials could store HRP for about 40 days to
keep the enzymatic activity and the Tyr-PMOs-10%-HRP with best
immobilization effect could be reused for 8 times at least.
Introduction
Enzymes are commonly used under mild and environmentally
friendly conditions as a nontoxic biocatalyst.[1] In the field of
green and sustainable chemistry, the utilization of enzymatic
catalysis is one of the most promising and rapidly growing areas
due to high activity as well as high chemo-, region- and stero-
selectivity. [2-4] However, the application of enzymes is inhibited
by many factors to show low operational stability and poor
reusability because they are easy to denature or inactivate by
pH, temperature, organic solvents and detergents. [5-6] In order
to effectively solve these problems, many researchers have
proved that immobilizing enzyme onto solid supports can
overcome these limitations to improve the stability and the
efficiency of the enzymes. [7-9] Subsequently
, different
materials are developed to immobilize enzyme as the solid
supports, such as organic materials including chitosan, chitin,
collagen or cellulose,[10-12] and inorganic materials including
zeolite, ceramics, silica, active carbon or mesoporous
materials.[13-17] Among them, mesoporous silica materials
[a]
A/Prof.Wang, Mrs. Zhang, Mrs. Gu, Mr. Zhang, Mrs. Zhou, Mr.
Wang, Prof. Guo
College of Chemistry and molecular engineering
Nanjing Tech University
30 Puzhu South Road, Jiangsu, Nanjing, 211816, China
E-mail: jqwang@njtech.edu.cn , guocheng@njtech.edu.cn
Prof.Sun
College of Chemical engineering, State Key Laboratory of Materials-
Oriented Chemical Engineering
Nanjing Tech University
To date, the tyrosine as the part of framework on porous
materials has not been reported yet, which spurs us to develop
novel amino acid bissilylated organic precursor and construct
novel PMOs materials based on the skeleton of amino acid.
Thus, in present work, we propose a methodology to architect
PMOs materials based on the framework of amino acid and to
improve the adsorption properties of PMOs materials for enzyme.
This was achieved by co-condensation of tyrosine bissilylated
[b]
Nanjing, 210009, China
Supporting information for this article is given via a link at the end of
the document.
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organic precursors and inorganic silica source in acid medium
using P123 as template. Moreover, tyrosine bissilylated organic
precursors were firstly obtained by selecting tyrosine as starting
compound via multistep reactions. It should be mentioned here
that tyrosine bissilylated organic precursors simultaneously
supply two functionalized groups within the framework (urea
groups, -NH-CO-NH-) and porous channels (phenol groups, -
C6H5O) in our materials. These groups derived from natural
amino acid molecules could potentially improve affinity ability
and immobilization capacity of enzyme. Among the various
types of enzymes, Horseradish peroxidase (HRP) was chosen
as model enzyme because it is widely employed in the oxidative
reactions of many chemicals as an effective biocatalyst. The
potential of novel PMO material based on the framework of
tyrosine as supports for immobilization of HRP was evaluated
and optimized. New bissilylated organic precursors were
observed in the XRD patterns of other materials Tyr-PMOs-7.5%,
Tyr-PMOs-10% and Tyr-PMOs-15%. Moreover, the intensity of
the (100) peak decreased gradually with the increasing
concentration of bissilylated organic precursor TBOS because
the phenolic hydroxyl group of TBOS expending within the
channels reduced the scattering power of channel wall. This
suggests that gradual deterioration of mesostructural order of
Tyr-PMOs with the concentration of TBOS increasing, which is
partly due to the fact that the compound of TBOS with large
molecular size may disturb the formation and self-assembly of
surfactant aggregates during the co-condensation process.
Simultaneously, addition of the high content of TBOS caused a
shift of the (100) diffraction peak to lower 2θ values, as well as
an increasing in d spacing and unit-cell constants. In a word, the
bissilylated organic precursor TBOS as organic building blocks
can successfully form the mesoporous framework in the
presence of template surfactant. Furthermore, the amounts of
TBOS directly affect on the degree of mesostructural ordering of
the PMO framework. Large amounts of TBOS in the pore
framework can diminish the mesoporous order of PMO, even
resulting in a collapse of the mesoporous structures.
1
confirmed by H-NMR, 13C-NMR, ESI-Ms and IR spectroscopy.
The textural properties of all the synthesized PMO materials
were characterized by small-angle X-ray diffraction (XRD), N2
adsorption-desorption
and
Fourier
transform
infrared
spectroscopy (FT-IR). These findings should be useful for
reasonable creation of new periodic mesoporous organosilica
materials according to specific application.
Results and Discussion
Tyr-PMOs-15
Tyr-PMOs-10
Structural characterization of Tyr-PMOs materials
The periodic mesoporous organosilicas (PMOs) materials with
the tyrosine as the part of framework were synthesized by
varying the concentrations of tyrosine-bissilylated organic
(TBOS) precursor in the initial mixture described in the
experimental section (scheme 3). The organic precursor of
TBOS was first prepared by multi-step reaction using tyrosine as
the raw material in scheme 2. All of the compounds including of
the intermediates were confirmed by 1H NMR, FTIR and ESI-MS
(detailed spectra seen in supporting information). Moreover, the
phenolic hydroxyl group in the structure of TBOS was
simultaneously introduced into the channels of Tyr-PMOs
materials, which is clearly indicated in scheme 1. The structural
change and inorganic/organic composition of the Tyr-PMOs-n
were accessed by XRD, TEM, SEM, FTIR, Nitrogen sorption
and 29Si solid-state NMR. The small angle XRD patterns of the
samples Tyr-PMOs-n were indicated in Figure 1. All samples
Tyr-PMOs-7.5
(100)
(110)(200)
Tyr-PMOs-2.5
1.0
1.5
2.0
2.5
2θ/degree
3.0
3.5
4.0
4.5
5.0
Figure.1 Small angle powder X-ray diffraction patterns of periodic mesoporous
organosilicas Tyr-PMOs-n (n=2.5%, 7.5%, 10%, 15%)
TEM and SEM images were used to further elucidate the pore
geometry and morphology of these PMOs materials. Figure 2
shows the TEM (left) and SEM (right) images of Tyr-PMOs-n
with the different concentration of tyrosine-bissilylated organic
precursor. TEM images of all the samples display parallel
aligned mesochannels and hexagonal arranged orifices, which
clearly indicates the p6mm symmetric mesophase. Moreover, All
of the materials Tyr-PMOs showed good long range order
although a less ordered mesostructure was observed as the
content of organic precursor TBOS increasing. These results are
consistent with the XRD patterns. With the increasing amounts
of TBOS in initial mixtures, the peak intensity of Tyr-PMOs
materials decreases, as shown in Figure 1.
exhibited
a sharp and relatively intense diffraction peak
assignable to the (100) plane at low 2θ angles suggesting the
formation of hexagonally ordered PMOs. The degree of
mesostructural ordering of the Tyr-PMOs framework depends
mainly on the concentration of TBOS in the initial sol-gel
mixtures. With the increasing amounts of TBOS, the intensity
and form of diffraction peak are largely changed in the XRD
pattern of Tyr-PMOs. For example, the XRD pattern of Tyr-
PMOs-2.5% shows a sharp (100) diffraction peak with 2θ of
0.86o and two broad diffraction (110) and (200) peaks with 2θ of
1.48o and 1.70o, which retains well-ordered mesostructural
organization. In contrast, the (100) diffraction peak is only
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the longitudinal axis. With the increasing amounts of organic
precursor TBOS (Tyr-PMOs-10% and Tyr-PMOs-15%), the
shapes of primary particles transform to curve threads, which
indicates that the organic precursor TBOS improves the
flexibility of framework in the materials Tyr-PMOs. Thus, the
organic precursor TBOS not only produces the micro-
environment of the channels, but also forms flexible and robust
morphology of primary particles.
phenyl -C=C-
(a)
Si-OH
-CH2-
Tyr-PMOs-2.5%
Tyr-PMOs-7.5%
Tyr-PMOs-10%
Tyr-PMOs-15%
-C=O
Si-O-Si
-CH2-
-OH
4000 3500 3000 2500 2000 1500 1000
500
-1
Wavenumber/cm
Figure.2 TEM (left) and SEM (right) images of periodic mesoporous
organosilicas Tyr-PMOs-n: (A) n= 2.5%, (B) n= 7.5%, (C) n= 10%, (D) n=
15%
SEM images display particle shapes of the materials Tyr-PMOs
with the different amounts of organic precursor TBOS. As shown
in figure 2, the morphology is largely affected by the adding
amounts of the precursor TBOS in initial mixtures. Tyr-PMOs-2.5%
shows many twisted morphology aggregating together. However,
the rod-like morphology is still observed in the middle
conspicuous and bigger particle in figure 2A. The similar rod-like
morphology of Tyr-PMOs-7.5% is obviously found to form long
threads. These threads assemble for a fiber-like structure along
Figure.3 (a) FTIR spectra of periodic mesoporous organosilicas Tyr-PMOs-n
(n= 2.5%, 7.5%, 10%, 15%). (b) and (c) Possible assembling formation of
hydrogen bond at the pore wall
In order to confirm the chemical bonds and the presence of
organic precursor TBOS in the mesoporous walls of Tyr-PMOs
materials, FTIR spectra of all the samples were detected after
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extracting the surfactant, as shown in Figure 3a. The absorption
band at 3380-3430 cm-1 can be attributed to O-H stretching
vibration of phenolic hydroxyl group. Moreover, the absorption
band slightly moves to lower wavelength as the content of
organic precursor TBOS increases (For example, the O-H
absorption band of Tyr-PMOs-2.5% is 3430 cm-1 and that of Tyr-
PMOs-15% is 3380 cm-1). Commonly, the change of O-H
absorption band is attributed to the formation of hydrogen bond.
Thus, the result illustrates that the phenolic hydroxyl group of
organic precursor TBOS assembles to form extensive hydrogen
bond at the pore wall of Tyr-PMOs materials with high content of
organic precursor TBOS. These hydrogen bonds were possibly
formed between the adjacent phenolic hydroxyl groups of TBOS
molecule at the same side (Figure 3b) or the conjoint two sides
(Figure 3c) of the pore wall. The existence of the carbonyl group
(C=O) is confirmed by the absorption band located at 1702 cm-1
(stretching vibration mode of C=O). Moreover, the stretching
vibration bands of C=C in phenyl ring are shown at 1615 and
1515 cm-1. The methylene group (CH2) is largely observed in the
structure of TBOS. The C-H vibration of CH2 is exhibited at
absorption bands at 2940 cm-1 (stretching vibration of C-H) and
1458 cm-1 (scissoring vibration of C-H), respectively. All of the
absorption bands that relate to the groups of TBOS indicate that
organic moieties TBOS are successfully anchored within the
materials Tyr-PMOs. The intensity of these peaks becomes
strong with increasing concentration of TBOS in initial
condensation process. Besides, a broad absorption bond at
1080 cm-1 is observed on the spectra of all the materials, which
indicates the formation of the Si-O-Si framework because the
bond is attributed to the asymmetric vibration mode of Si-O.
Furthermore, the absorption bands at 806 and 454 cm-1 that are
assignable to symmetric vibration mode of Si-O and in-plane
bending vibration mode of Si-O-Si are also clearly displayed in
figure 3. The vibration band for the residual Si-OH appears at
absorption band at 954 cm-1. These results indicate that the
expected siloxane frameworks are formed in all of the materials
Tyr-PMOs with the different content of TBOS.
materials Tyr-PMOs-n, which shows that the functional moieties
occupy the mesopore walls. The pore diameter decreased from
7.3 to 5.6 nm with increasing the organosilica loading from 2.5%
to 15%. In short, the results of N2 adsorption-desorption analysis
show that Tyr-PMOs-n materials with the framework of TBOS
are mesoporous materials with uniform pore distributions, which
supplies the possibility of immobilizing HRP molecules.
A
Adsorption
Desorption
Tyr-PMOs-2.5%
Tyr-PMOs-7.5%
Tyr-PMOs-10%
Tyr-PMOs-15%
0.0
6
0.2
0.4
0.6
0.8
1.0
Realtive Pressure (P/P0)
B
Tyr-PMOs-2.5%
5
4
3
2
1
0
Tyr-PMOs-7.5%
Tyr-PMOs-10%
Tyr-PMOs-15%
The N2 adsorption–desorption isotherms and the corresponding
pore size distribution curves of Tyr-PMOs are shown in figure 4.
All the samples with different contents of TBOS exhibited type IV
adsorption isotherms with capillary condensation according to
the IUPAC classification. However, the shape of hysteresis loop
shows the different type, reflecting the distinct effect of TBOS
contents in initial condensation process on the channel structure
of Tyr-PMOs materials. The Tyr-PMOs-n (n=2.5% and 7.5%)
materials loaded with lower amounts of TBOS display type IV
isotherm with H1 hysteresis loop at high relative pressure with
P/P0 range of 0.6-0.9, which is due to the uniform pore size and
structure of these materials. The materials Tyr-PMOs-10% and
Tyr-PMOs-15% show the type IV with a clear H4 type hysteresis
loop, which demonstrates that mesopore and micropore are
simultaneously formed within the channels of these materials.
Based on the N2 adsorption-desorption isotherms, the textural
parameters of all the materials are reported in Table 1. With the
increasing concentration of TBOS in the framework, a decrease
in specific surface area and pore volume was observed in the
2
4
6
8
10
pore diameter (nm)
Figure.4 Nitrogen adsorption-desorption isotherms (A) and pore size
distribution curves (B) of periodic mesoporous organosilicas Tyr-PMOs-n (n=
2.5%, 7.5%, 10%, 15%)
Table.1 Textual parameters of periodic mesoporous organosilicas Tyr-PMOs-n
[a]
[b]
d100
(Ǻ)
α0
(Ǻ)
SBET
Pore volume
(cm3/g)
Pore diameter[c]
(nm)
Sample
(m2/g)
Tyr-PMOs-
2.5%
Tyr-PMOs-
7.5%
Tyr-PMOs-
10%
Tyr-PMOs-
15%
102.7
105.2
107.7
113.3
118.6
549
338
279
212
0.78
0.35
0.15
0.10
7.3
6.4
5.9
5.6
121.5
124.4
130.8
[a] Interplanar spacing of the (100) plane, as obtained from the XRD analysis.
3
[b] The α0 is unit cell parameter, calculated from α0=2d100
/
. [c] Pore
diameter is calculated using the BJH model based on the desorption branch of
the isotherm.
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Figrue 5 displays the 29Si solid-state NMR spectra of the Tyr-
PMOs-n materials with the different concentration of organic
precursor TBOS. Both Q and T sites corresponding to Qn (Qn =
Si(OSi)n(OH)4-n, n=2-4) and Tm (Tm = RSi(OSi)m(OEt)3-m, m=2, 3)
[34] were clearly observed in these spectra. The chemical schifts
of Q4, Q3 and Q2 were designated at -110 ppm, -102 ppm and -
91 ppm, respectively. The T signals arising from the organic
functional parts of bridged organosilane were obtained in the
range of -50 ppm to -70 ppm, which were assigned to the T3 at -
67 ppm and T2 at -59 ppm. Based on normalized peak areas,
the ratio of Tm/(Tm+Qn) is around 0.094, 0.171, 0.214 and 0.227,
which is related to the materials Tyr-PMOs-n (where n = 2.5%,
7.5%, 10%, 15%), respectively.Generally, the Q signals imply
the degree of TEOS cross-linking. And the T signals confirm the
stability of Si-C bond. As the content of TBOS increases, the
intensity of Q signals become weak and the intensity of T signals
become strong. These results indicated that the organic moities
TBOS were incorporated in the silica network of the Tyr-PMOs
materials.
electrostatic attractions were obtained which would enhance the
HRP binding onto all of the Tyr-PMOs materials at pH 7.
2.20
2.15
2.10
2.05
2.00
1.95
Tyr-PMOs-2.5%
Tyr-PMOs-7.5%
1.90
1.85
Tyr-PMOs-10%
Tyr-PMOs-15%
4
5
6
7
8
9
10
pH
Figure. 6 Effects of immobilization pH of the buffer solution on HRP
immobilization
2.20
2.15
2.10
2.05
2.00
Tyr-PMOs-2.5%
Tyr-PMOs-7.5%
Tyr-PMOs-10%
1.95
Tyr-PMOs-15%
Figure.5 29Si solid-state NMR spectra of periodic mesoporous organosilicas
1.90
0
2
4
6
8
10 12 14 16 18 20 22 24
Tyr-PMOs-n: (a) n= 2.5%, (b) n= 7.5%, (c) n= 10%, (d) n= 15%
Time/h
Figure.7 Effects of immobilization time on HRP immobilization
Effects of different conditional parameters on HRP
immobilization
Figure. 7 depicts the effect of time on HRP adsorption onto Tyr-
PMOs materials (pH 7.0). As can be seen from the Figure 7, the
amounts of absorbed enzyme were increased along with the
extension of time. When the immobilization time was 8h, the
amounts of immobilized enzyme reached the adsorption
equilibrium and no longer increased over time. On the contrary,
when the immobilization time was more than 8 h, the amounts of
immobilized enzyme decreased. Presumably ， the possible
reasons can be explained as follows: Firstly, When the loading
time is relatively short, the enzyme HRP molecules is attached
into the channels of support, but fail to achieve saturation,
causing small amounts of immobilized enzyme HRP. When the
absorption time is long enough, the HRP molecules have been
immobilized onto the surface of supporter channels at saturation
coverage, leading to maximum amount of absorbed enzyme.
However, the long absorption time gives multiple complicated
binding of HRP molecules on the surface of carrier channels,
In addition of the textural parameters of adsorbents, three
important factors of immobilization pH, time and temperature
determine the adsorption amounts of enzyme. Figure.
6
indicates that the pH affects the adsorbed amounts of HRP on
Tyr-PMOs materials. Because pH alters the ionization degree of
the functionals groups of the enzyme and the surface charge of
the adsorbents, the pH directly influences the adsorption
amounts and effectiveness of HRP [35]. Moreover, when the pH
is close to the isoelectric point (pI) of enzyme, the adsorbed
enzyme can come up to the maximum amounts on the
adsorbents [36]. As shown in figure.4, the optimum pH value of
absorbed HRP was 7, and the adsorbed amounts were relatively
low at the other pH value. At the immobilization pH of 7, the net
charge of the enzyme HRP was positive (pI～8.9) while that of
the support surface was negative (pI of silica ～2). Thus, high
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◦
therefore changing the enzyme’s subspace structure, which
leads the declination of adsorbed enzyme amounts. When
binding sites of the carrier near or have achieved saturation, the
total amount of immobilized enzyme can no longer increase due
to constant amount of the carrier. Secondly, the long absorption
time may cause the HRP molecules leach from the sorbents,
thereby decreasing the amount of immobilized enzyme logically.
Thus, the optimized adsorption time of HRP is 8h on the Tyr-
PMOs materials.
60 C，the activity of HRP enzyme decreased. It is well known
that the increase of the temperature is beneficial to the coupling
of the HRP enzyme to the carrier. Besides, an increase of the
temperature may accelerate the molecular motion of the HRP
enzyme. However, too high temperature may induce irreversible
deactivation of enzyme. To summarize, the supporter shows the
best adsorption ability owing to the specific micro-environment
within the channels, which the better adsorption temperature of
◦
HRP enzyme is 40 C on the surface of the Tyr-PMOs materials.
Based on these results, the optimized conditions of HRP
enzyme are immobilized on Tyr-PMOs-n materials at pH of 7.0
for 8h, and the temperature of 40◦C is chosen.
140
Tyr-PMOs-2.5%
Tyr-PMOs-7.5%
Tyr-PMOs-10%
Tyr-PMOs-15%
120
100
80
Besides these immobilization parameters, pore structural
characters of Tyr-PMOs-n materials are also very important
factor of HRP immobilization. As shown in these figures, the
adsorption amounts of HRP relate to the concentrations of
TBOS in the framework of Tyr-PMOs-n materials. In general, it is
clearly seen that the higher immobilization efficiency of Tyr-
PMOs-10% and Tyr-PMOs-15% with high content of precursors
was observed in all figures though their surface area and pore
volume as well as pore size decline. Generally, the size of the
HRP molecule is 3.7 nm *4.3 nm *6.4 nm [37]. However, the
pore size of Tyr-PMOs-10% and Tyr-PMOs-15% are still able to
accommodate HRP molecules in their mesoporous channels
because the HRP molecule is elongated, and more amounts of
HRP are immobilized on the two samples. Thus, we speculate
that the result is probably due to the conformational flexibility of
enzyme molecule. In addition, the organic group of phenolic
hydroxyl group was simultaneously induced into the channels at
the preparation of Tyr-PMOs materials. Moreover, the phenolic
hydroxyl group largely added the binding power through
hydrogen-bonds to prevent the leaching of HRP enzyme, as
shown in scheme 1. The materials of Tyr-PMOs-n (n=2.5%,
7.5%) can absorb HRP molecule easily due to the low diffusion
resistance of broad pore structure. However, the adsorbed
60
40
20
0
10
20
30
40
50
60
Temperature(oC)
Figure.8 Effects of immobilization temperature on HRP immobilization
Adsorption isotherms of the HRP immobilized on Tyr-PMOs
materials at different temperature were showed in Figure. 8. As
we can see from Figure. 8, with an increase of temperature, the
overall trend of immobilized enzyme’s activity showed a slow
rise within the temperature range of 20-40 C, but the trend is up
and down. The optimum adsorption temperature for the
◦
◦
immobilization of HRP was 40 C, at which, the activity of the
enzyme reached maximum. Within the temperature range of 40-
Scheme 1 Possible mechanism of HRP loading in the channel of Tyr-PMOs
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amounts of HRP on the two PMOs materials are lower than one
of the PMOs materials with high concentrations of TBOS
because of less organic group to anchor the HRP enzyme. To
sum up, all the above factors produce complicated impact on the
loading efficiency of HRP enzyme, and they are interdependent
in the whole process.
be caused by the following reasons: One is the slightly release
of enzyme HRP that was loosely immobilized from the support,
and another is the loss of small quantities of enzyme during
reuse and washing operations.
100
Tyr-PMOs-10%-HRP
Stability of Tyr-PMOs-n after the immobilization of enzyme
HRP
80
60
40
20
0
Figure
9 and Figure 10 show the storage stability and
operational stability of immobilizing HRP enzyme, respectively.
The Stability of Tyr-PMOs-n-HRP is one of the key factors for
large scale applications. Storage stability of Tyr-PMOs-n-HRP
with the biggest HRP immobilization amount at room
temperature was evaluated after different storage times. (see
Figure. 9). The results show little change in the activity of HRP
immobilized on the Tyr-PMOs materials with the increasing time.
However, it is obvious that the denaturation of immobilized HRP
is likely to increase because of long storage time. Specifically,
the activity of immobilized HRP had no changes basically within
10 days. After 10 days, a little amount of enzyme will drain out of
the channels of Tyr-PMOs materials. Accordingly, it is better to
use Tyr-PMOs-n-HRP within 10 days.
1
2
3
4
5
6
7
8
Cycle times
Figure. 10 Operational stability test of Tyr-PMOs-10%-HRP
Conclusions
140
Tyr-PMOs-10%-HRP
120
The experimental results obtained in the present work revealed
some conclusive remarks, as follows:
(1) Tyrosine, a natural amino acid, is first used as starting
material to successfully prepare a novel bridged organosilicas
precursor TBOS by multi-step reactions.
Tyr-PMOs-15%-HRP
100
(2) New periodic mesoporousꢀmaterials with the amino acid as
the framework have been constructed by a one step co-
condensation of TEOS and Tyrosine bissilylated organic
precursorunder the template. Through controlling the proper
proportion of organic precursor in the silica source, the resulting
samples Tyr-PMOs can still possess the well-ordered 2D
hexagonal mesoporous structure. All the Tyr-PMOs materials
with the different contents of TBOS are fully characterized by
XRD, BET, FT-IR, TEM, SEM and 29Si solid-state NMR.
(3) HRP can be absorbed in various Tyr-PMOs-n materials.
Among them, Tyr-PMOs-10% showed the higher immobilization
efficiency. The optimum immobilization conditions are as follows:
temperature of 40℃, time of 8 h, pH of 7. In this way, one could
store enzymes for a longer time (about 40 days) and the Tyr-
PMOs-10%-HRP with best immobilization effect could be reused
for 8 times at least.
80
Tyr-PMOs-2.5%-HRP
Tyr-PMOs-7.5%-HRP
60
40
20
0
10
20
Days
30
40
Figure. 9 Storage stability test of Tyr-PMOs-n-HRP with the biggest HRP
immobilization amount at room temperature
Operational stability test of screened Tyr-PMOs-n-HRP with the
best HRP immobilization effect is demonstrated in Figure. 10.
The immobilized HRP can be reused after recovering the
sample Tyr-PMOs-10%-HRP from the action solution by simple
centrifugation. As shown in Figure 10. That the activity of
immobilized HRP decreased with the number of recycling runs，
but fortunately still kept 70% of their original activity after the
fourth run. The result shows that the immobilized enzyme
possesses appropriate stability. Starting from the sixth run, the
activity decreased sharply, only 30% of initial activity. The
reduction in the activity of HRP in recycling experiments might
The conclusions obtained in this work may play a significant role
in the understanding of enzyme absorption in this kind of new
periodic mesoporous organosilicas (Tyr-PMOs), which the
material will be undoubtedly useful for their future applications in
the fields of biocatalysis or biosensors.
Experimental Section
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Regents and chemicals
The characterization of TBOS: 1H-NMR(DMSO,400Hz,훿 ppm):9.70(s, 1H,
-NH-CO-), 9.20(s, 1H, -OH), 7.33(s, 1H, -NH-CO-),6.99-7.01(d, J=6.8Hz,
2H, ArH-),6.64-6.67 (d, J=6.8Hz, 2H, ArH-),6.26-6.29 (t, J=6.0Hz ,1H, -
NH-CO-),6.14(t, J=6.2Hz, 1H),6.05(d, J=8.0Hz ,1H), 4.17(q, J=2.7Hz,
1H),3.71-3,76 (d, J=10.0Hz ,12H, -CH2-O),2.90-3.00(m, 4H, -CH2-),2.76-
2.81 (q, J=9.0Hz ,1H, Ar-CH2-) ,2.60-2.66 (q, J=12.0Hz ,1H, Ar-CH2-),
1.36-1.44(m, 4H, -CH2-) , 1.12-1.16 (d, J=8.0Hz ,18H, -CH3), 0.48-
0.53(m, 4H, -CH2Si). 13C NMR (400 MHz, CDCl3, ppm) d 172.3, 157.9
157.7, 155.8, 130.0, 127.3, 114.8, 57.6, 53.6, 42.0 41.8, 36.0, 23.4 23.2,
18.1,7.2 7.2; ESI-MS: m/z = 689, m/z = 690 (M+H+), m/z = 712 (M+Na+);
FT-IR (KBr，cm-1): 3298.38 (Bz-OH), 2975.49(-CH3), 2928.40 (C-CH2-
C), 2887.97 (-CH), 1652.09 (-CO-NH), 1567, 1517, 1450(Bz), 1080.23 (-
O-Si-).
Horseradish peroxidase was purchased from Aladdin. Triblock
poly(ethylene
oxide)-poly(propylene
oxide)-poly(ethylene
oxide)
copolymer Pluronic P123 (EO20PO70EO20, Mw=5800) was obtained
from Sigma-Aldrich. (3-isocyanato-propyl) triethoxysilane (IPTES) and
tetraethoxysilane （TEOS）were the products of Aladdin. Other reagents
and solvents were commercially available at analytical grade and used
as purchased without further purification.
Synthesis of Tyrosine-bissilylated organic precursor (TBOS)
Preparation of Periodic mesoporous organosilicas materials with
Tyrosine in the framework (Tyr-PMOs)
The materials of Tyr-PMOs were prepared by the co-condensation of
TEOS and TBOS with the template P123 under the acid media, as
shown in Scheme 3. In a typical synthesis, Pluronic P123 (2.0g) was
firstly dissolved in the mixed solution of distilled water (15g) and HCl (60g
2M) at room temperature to obtain a clear solution. Then, on the basis of
the molar ratios, the certain amount of TEOS and TBOS were added to
the above homogenous solution under stirring at 40℃ for 24h. Thereafter,
the mixtures were transferred into
a stainless steel reactor with
polytetrafluorethylene and heated at 100℃ for another 24h. Finally, the
product was collected by filtration, washed with distilled water and dried
in air under ambient conditions to get a yellowish solid powder. The
template was extracted by refluxing 1.0g of as-synthesized materials in
the mixtures of 300 mL ethanol and 10mL hydrochloric acid for 48h.
Finally, the materials obtained were denoted as Tyr-PMOs-n, where n
(n=2.5%, 5%, 7.5%, 10%, 15%) is the molar percent of
TBOS/(TBOS+TEOS).
Scheme 2. Synthesis of tyrosine-bissilylated organic (TBOS) precursor
Characterization
Tyrosine-bissilylated organic (TBOS) precursor was synthesized by the
route of scheme 2. First, a suspension solution was formed by mixing
tyrosine 1 (11.78 g, 65.00 mmol) and methanol solution (250 mL) in an
ice bath. Then, thionyl chloride (5.20 ml, 71.50 mmol) was dropwise
added to the above solution. The solution became clear after the addition
of thionyl chloride. Next, the reaction mixtures were transferred into oil
bath and refluxed overnight at 80 ℃. The solvent was removed under
reduced pressure to give crude product of tyrosine methyl ester
hydrochloride 2, which are yellowish white solids. Compound 2 was
directly used in the following step without further purification. Second,
Compound 2 (5.70 g, 23.20 mmol) was immersed in tetrahydrofurane
(75.00 mL). Triethylamine (2.35 g, 23.2 mmol) was then added. The
mixture was stirred at room temperature for one night. Then, the mixtures
were filtered to eliminate the precipitate of triethylamine chloride. The
tetrahydrofurane was evaporated obtaining L-Tyrosine methyl ester 3
(dark-white colour, yield of 71%). Third, Hydrazine hydrate (80%, 30 mL)
was added to a solution of 3 (2.02g, 10.3 mmol) in methanol (50 mL) at
room temperature. The reaction mixtures were stirred for 24 h at the
same temperature and subsequently concentrated under reduced
pressure. The residue was purified by recrystallization with methanol to
get the pure tyrosine hydrazine 4 with the yield of 71%. Finally,
compound 4 (3.00 g, 15.0 mmol) was dissolved in dried THF (100 mL)
under nitrogen atmosphere at the three-necked round bottom flask. 3-
Isocyanatopropyltriethoxysilane (IPTES, 7.2 g, 30.0 mmol) was then
added to the solution. The reaction mixtures were allowed to keep in a
pre-heated oil bath at 40◦C for about 6 h. During the period, the reaction
was monitored by TLC. The resulting white oil was collected through
rotary evaporation. Then, the white oil was recrystallized by anhydrous
hexane to afford TBOS as a highly viscous solid with the yield of 80%.
The 1H NMR experiment was recorded on
a AC-400 Avance
spectrometer at 25 °C, and DMSO was chosen as the solvent. All
chemical shifts (d) are reported in parts per million (ppm) downfield from
TMS; J values are given in hertz (Hz). The FT-IR spectra were collected
on a Thermo Nicolet iS5 FT instrument in the range of 400-4000 cm-1 at
298 K, and the sample was mixed with KBr powder at a ratio of 10:90
(w/w). High resolution mass spectra were run on Aglient 6550 iFunnel Q-
TOF. Small-angle X-ray diffraction (SAXRD) measurements were carried
out on a ARL XTRA diffractometer equipped with Cu Kα radiation (λ=
1.5418 Å) sources at RT in a 2θ range (scattering angle) between 0.5°
and 8°. The operating voltage and current of the X-ray tube were 40 kV
and 20 mA, respectively. N2 adsorption–desorption isotherms were
obtained by using Micrometrics ASAP2020. Prior to testing, the samples
were pre-treated at 373 K overnight in vacuum. The surface area was
evaluated from Brunauer–Emmett–Teller (BET) method. The pore
diameter and pore volume of samples were derived from the adsorption
isotherm branch data using Barret–Joyner–Halenda (BJH) method. The
total pore volume was determined from the amount adsorbed at a relative
pressure of 0.99. The BJH pore diameter was defined as the maximum
pore diameter on the pore size distribution curve. Transmission electron
microscopic (TEM) images were collected by a JEOL JEM-200CX
electron
microscope.
Scanning
electron
microscopy
(SEM)
characterisation was performed by using HITACHI TM-3000, and the
samples were coated with Au film to improve the conductivity prior to
imaging. 29Si solid-state NMR experiments were carried out using a
Bruker AVANCE III-500 spectrometer (BrukerBioSpin, Karlsruhe,
Germany) operating at a magnetic field strength of 11.7 T, equipped with
a 4 mm double-resonance MAS probe, which were collected at an 8 kHz
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MAS spinning speed with a π/4 pulse width of 2.3μs and a recycle delay
spectrophotometer (Shimazu, Japan).
of 90s. UV-vis absorption spectra were collected on
a UV 360
Scheme 3. Preparation of Periodic mesoporous organosilicas materials with the frame work of Tyrosine (Tyr-PMOs)
Immobilization of HRP on material of Tyr-PMOs
PMOs-n-HRP, 5mg) was added into the above solutions, and the
reaction was carried out at 30◦C for 1 min. After the reaction, the enzyme
activity was determined by measuring the UV-vis absorbance of the
product (purpurogallin) at 420 nm. The extinction coefficient of pyrogallol
was found to be 0.0126 M-1cm-1at 420 nm. The assay of free HRP was
performed under the same conditions as the materials of immobilized
HPR were replaced with 5mg of free HRP. All of the samples were
analyzed in triplicate. One unit of HRP activity was defined as the amount
of HRP that released 1 mol per minute of oxidized product at pH 6.0, and
30 ◦C.
The typical procedure of immobilizing HRP on Tyr-PMOs is described as
followings: 250 mg of Tyr-PMOs and 5.0 mL phosphate buffer solution of
HRP with the concentration of 1mg mL-1 were shaken for 6h at 4◦C. The
resulting product was filtered and washed with the same buffer solution
to remove unattached enzyme. The obtained immobilized HRP on Tyr-
PMOs was designated as Tyr-PMOs-n-HPR (n=2.5%, 7.5%, 10%, and
15%, where n is the molar percent of TBOS/(TBOS+TEOS)). In order to
study the effects of different conditional parameters on immobilization of
HRP, the experimental conditions including pH, temperature and time
were considered respectively. The experiments were performed by
adjusting one parameter but the other parameters were fixed in the
immobilization of HRP on Tyr-PMOs.
The enzyme activity was calculated according to Eq. (2):
Enzyme activity ∪=1000X
(2)
푡
The adsorbed amounts of HRP (푞푡 ) was calculated from the mass
balance equation as Eq.(1):
where x is absorbance, and t is enzymatic reaction time (min).
Stabilities of immobilized enzyme HRP on Tyr-PMOs
(
)
푉 퐶 −퐶
0
t
푞푡 =
(1)
W
The storage stability was evaluated after all the samples of Tyr-PMOs-n-
HRP with the maximum amounts of immobilized HRP were kept in room
where c0 is the initial concentration of solutions (mg mL-1) containing
HRP, 푐푡is the concentration after immobilizing HRP (mg mL-1), V is the
volume of solutions (mL) and W is the mass of Tyr-PMOs samples (mg).
The HRP concentrations were analyzed in triplicate, and the
experimental errors were within ±5%.
temperature at dried environment for
a specified period of time.
Subsequently, 2 mg of Tyr-PMOs-n-HRP were taken for activity assay.
The operational stability of immobilized HRP was determined by using
screened Tyr-PMOs-n-HRP with maximum loading amounts of enzyme
to catalyze the reaction of the assay solution containing pyrogallol,
phosphate buffer solution and H2O2 for eight cycles. After each cycle of
reaction, Tyr-PMOs-n-HRP were collected and washed with phosphate
buffer (0.1 M, pH 6.0) to remove any residual substrate. Next, Tyr-PMOs-
n-HRP was transferred into fresh reaction medium and the retained
activity was estimated. We considered the initial activity was 100% for
the immobilized enzyme.
Measurement of enzymatic activities of free and immobilized
enzyme
To determine the activity of free and immobilized enzyme HRP, two
substrates of hydrogen peroxide and pyrogallolin were employed in the
model reaction [38]. 0.5 mL of hydrogen peroxide (0.05 M) was mixed
with the solutions of 2.4 mL phosphate buffer solution (pH 6.0) of
pyrogalloland (0.013M). Then, the material of immobilized HRP (Tyr-
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Financial supported by National Natural Science Foundation of
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gratefully acknowledged. The authors would like to thank Prof.
Hailu Zhang of Suzhou Institute of Nano-tech and Nano-bionics,
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Keywords: Periodic mesoporous organosilicas (PMOs) •
Tyrosine bridged organosilicas • HRP • Immobilization of
enzyme • Amino acid
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Entry for the Table of Contents (Please choose one layout)
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Wang Jianqiang*[a], Zhang Wenqi[a], Gu
Changqing[a], Zhang Wenpei[a], Zhou
Man[a], Wang Zhiwei[a], Guo Cheng*[a]
and Sun Linbing[b]
Novel periodic mesoporous
organosilicas with tyrosine framework
(Tyr-PMOs) was constructed.
Incorporation of the tyrosine units in
the framework, and phenolic hydroxyl
group as binding center introduced
within the channels at the same time.
Long storage time and conventional
stability of Tyr-PMOs after
Page No. – Page No.
Step-up synthesis of periodic
mesoporous organosilicas with the
framework of tyrosine (Tyr-PMOs)
and performance on horseradish
peroxidase immobilization
inmmobilizing HRP were found.
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