Synthesis of mesoporous silica with different pore sizes for cellulase immobilization: Pure physical adsorption

Article abstract of DOI:10.1039/c7nj00441a

To discuss the physical adsorption mechanism of the adsorption process of cellulase, a commercial enzyme cocktail sourced from Acremonium was immobilized in mesoporous silica with various pore sizes by pure physical adsorption in this study. Mesoporous silica materials with 17.6 nm and 3.8 nm pore sizes (hereafter referred to as MS-17.6 nm and MS-3.8 nm, respectively) were synthesized in the manner of a seeded-growth method. Other available mesoporous silica materials denoted as H-32 and diatomite were also used as sorbents. Then, the sorbents were characterized via small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), scanning electron microscopy (SEM), the Barrett-Emmett-Teller (BET) method and the Barrett-Joyner-Halenda (BJH) method to confirm their mesostructure. Furthermore, the adsorption abilities of different sorbents and enzymatic activities of immobilized cellulase were studied. The adsorption amounts exhibited a clear correlation with the pore size of the sorbents; i.e., the adsorption amount of MS-17.6 nm (410 mg g^-1) with the pore size similar to the long axes of cellulase molecules was higher than that of MS-3.8 nm (315 mg g^-1) with the pore size approximated to the short axes of cellulase (which was realized at 50 °C). Besides, the adsorption behavior of diatomite (with a pore size of about 200 nm) revealed a periodicity because the pore size was significantly larger than cellulase molecules. Moreover, the pore size was suggested to be a critical factor for the enzymatic activity of cellulase. When the average pore size of MS-3.8 nm just matched the short axes of cellulase molecules, immobilized cellulase preserved the active sites of cellulase intactly and showed the best activity (i.e. 63.3% of free cellulase activity at 50 °C). Consequently, the pore size of the sorbents had a significant influence on cellulase immobilization.

Full text of DOI:10.1039/c7nj00441a

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PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
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Synthesis of mesoporous silica with different pore size for
cellulase immobilization: pure physical adsorption
Baiyi Chen,a Jianhui Qiu,a,* Haodao Mo,a Yanling Yu,a,b Kazushi Ito,a Eiichi Sakai,a and Huixia Fengc
Received 00th January 20xx,
Accepted 00th January 20xx
To discuss the physical adsorption mechanism of the adsorption process of cellulase, a commercial enzyme
cocktail sauced from Acremonium, was immobilized in mesoporous silica with various pore sizes by pure physical
adsorption in this study. Mesoporous silica materials with 17.6 nm and 3.8 nm pore size (hereafter referred to as
Ms-17.6nm and Ms-3.8nm, respectively) were synthesized in the manner of a seeded-growth method. Other
available mesoporous silica denoted as H-32 and diatomite were also used as sorbents. Then the sorbents were
characterized via Small-angle X-ray scattering (SAXS), transmission electron microscope (TEM), scanning electron
microscope (SEM), Barrett-Emmett-Teller (BET) method and Brarrett-Joyner-Halanda (BJH) method to confirm
their mesostucture. Furthermore, adsorption ability of different sorbents and enzymatic activities of immobilized
cellulase were studied. The adsorption amounts exhibited a clear correlation with the pore size of the sorbents;
i.e., the adsorption amounts of MS-17.6nm (410 mg/g) with the pore size was similar to the long axes of cellulase
molecule was higher than that for MS-3.8nm (315 mg/g) with the pore size approximated to the short axes of
cellulase (which was realized at 50 °C). Besides, the adsorption behavior of diatomite (with pore size about 200
nm) revealed a periodicity because the pore size was significantly larger than cellulase molecules. In the
meantime, the pore size was suggested to be a critical factor for enzymatic activity of the cellulase. When the
average pore size of MS-3.8nm just matched the short axes of cellulase molecules, immobilized cellulase
preserved active sites of cellulase intactly and showed the best activity (i.e. 63.3% of free cellulase activity at 50
°C). Consequently, pore size of the sorbents had a significant influence on cellulase immobilization.
DOI: 10.1039/x0xx00000x
www.rsc.org/
requirement of high pressure, high temperature or strong acids.
These methods are often energy intensive, and might produce
undesirable by-products.[7–10] On the contrary, enzyme-assisted
Introduction
Fuel energy production from renewable biomass has been
considered as a strategic way to solve the problems of fossil fuel
depletion and environmental pollution. Lignocellulosic biomass,
such as forestry and agricultural waste, is one of the most promising
sources to generate biomass-based energy.[1–5] Cellulose, which
consists of 1, 4-beta glycosidic bond linked glucose units, is the
main component of lignocellulosic biomass. Since the hydrolysate
of cellulose, glucose is easily converted into ethanol, butanol, or
processes for cellulose conversion are usually performed in
relatively mild reaction conditions with high specificity, which has
been considered as an alternative “green” approach.[11,12]
For this “green” approach, cellulase, which is a general term for a
group of enzymes that hydrolyze cellulose, has been intensively
studied due to its significant function in converting cellulosic
biomass to glucose.[13,14]. Therefore enzyme cost reduction is one of
the critical factors to decrease the cost of cellulosic biofuel
production. Since free cellulase has a low stability and cannot be
efficiently recovered and reused, if these problems can be solved,
the cost of enzymatic hydrolyzation may be reduced potentially To
overcome these problems, immobilization methods, ultrafiltration
systems, aqueous two-phase systems and modification of cellulase
have been studied.[15-19] These reports suggested that
immobilization of cellulase may be a promising way of increasing
the efficiency of enzyme utilization.[20,21]
other fuels and fine chemicals, researchers have intensively
[5–8]
investigated the way to convert cellulose into glucose
Three
main methods concluding physical, chemical, and biological process
have been applied to cellulose conversion.[3-5] However, physical
and chemical processes usually require extreme treatments, such as
During the past two decades, extensive research has been carried
out on the immobilization of cellulase on inorganic materials.
Unfortunately, immobilized cellulase on the surface of inorganic
material, such as amorphous silica or magnetic nanoparticles, could
not retain the full activity of free cellulase. Among these various
inorganic materials, mesoporous silica have been intensively
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investigated for their potential application as delivery vehicles for
small-molecule drugs, DNA, and proteins, owing to their uniform
pore size, large surface area, and high accessible pore volume.[22-26]
Chang et al.[27] synthesized two mesoporous silica with different
pore size and surface area, for physical adsorption and chemical
binding of cellulase. Meanwhile, the reaction conditions, including
temperature, time, and amount of cellulase for cellulosic hydrolysis,
were optimized. The results showed that the cellulase chemically
linked to mesoporous silica exhibiting carboxyl groups and a large
pore size could achieve an effective cellulose-to-glucose conversion
exceeding 80% yield and excellent stability.
Experimental
Materials
All chemicals were of analytical grade and used without further
purification. Carboxymethyl cellulose sodium salt (CMC, nacalai
tesque), sodium acetate (NaAc, nacalai tesque), acetic acid (nacalai
tesque), hexadecyltrimethylammonium bromide (CTAB, nacalai
tesque), ethyl acetate (EtAc, nacalai tesque), sodium silicate meta
(nacalai tesque), methanol (nacalai tesque), hydrochloric acid
(nacalai tesque), H-32 (mesoporous silica, AGC Co.), diatomite
(Huali Co.), GLU CⅡ(glucose kit, Wako) and acremonium cellulase
(Meiji Seika Pharma Co.). Deionized water was used throughout the
experiments.
To create more suitable immobilization of cellulase by using
mesoporous silica, it is of great importance to understand the
factors that influence the immobilizing behavior of proteins within
mesoporous silica. It has been found that two factors may greatly
influence the immobilization properties. The first is the surface
characteristics of mesoporous silica and proteins. The surface
charges of sorbents and the proteins must be complementary,
because it is generally accepted that the electrostatic interaction
between protein and mesoporous silica is one of the most
important factors that influence adsorption and desorption.[28] The
second is the size of mesopore, or more specifically, the pore size
with relative to the protein molecule size. To absorb cellulase, the
pore size of the mesochannels should be sufficiently large for
‘‘comfortable’’ entrapment of biomolecules.[29] Kisler et al.[30] have
demonstrated that the adsorption amounts in MCM-41 materials
depended strongly on the adsorbing molecular size relative to the
pore size for a range of biomolecules. Nevertheless, Takimoto et
al.[31] encapsulated the cellulase molecules using mesoporous silica
SBA-15 and the enzymatic activities of cellulase immobilized onto
mesoporous silica of various pore sizes were studied. The
encapsulated cellulase on SBA-15 with a pore size of 8.9 nm gave
the highest enzymatic activity, despite not having largest pore size
and maximum adsorption amout. Herein, a new theory is proposed
for pore size, not the larger the better it gets -- larger pore size may
achieve an excellent initial adsorption amount but following
desorption and perishing operation stability, meanwhile, the
molecular dense arrangement, caused by abundant adsorption
would prevent conformational flexibility of cellulase and finally
result in enzymatic activity lost. Although there have been several
reports focusing on physical adsorption of enzymes with
mesoporous silica as sorbents,[23] it is a lack of information to use
mesoporous silica for cellulase adsorption.
Synthesis of mesoporous silica
In the experimental procedure, mesoporous silica denoted as
MS-17.6 nm was prepared by a seeded-growth method.[32] A mixed
solution was prepared by dissolving 5.0 g CTAB and 6.0 g Na2O·SiO2
in 90 mL of aqueous solution at room temperature (25 °C). Then,
6.4 mL of ethyl acetate was added, and the mixture was stirred for
5 min and allowed to stand at room temperature for 6 h. After this
period of aging, the mixture was stirred at 90 °C for 48 h in an oil
bath. Then, the heating was stopped and the suspension was
cooled naturally to room temperature. Finally, the product was
collected by centrifugation and CTAB templates were completely
extracted by Soxhlet extractor with acidic methanol solution (pH
1.4) for 10 h.
MS-3.8 nm was synthesized as follows. Briefly, 4.2 g CTAB and 3.5
g Na2O·SiO2 were added to 170 mL of aqueous solution and stirred
for 30 s at room temperature. After a clear solution was obtained,
3.2 mL of ethyl acetate was subsequently added to the system. The
reaction was allowed to proceed for 48 h at 80 °C. After removing
the CTAB templates from the as-synthesized materials by heating
them at 550 °C for 3 h, the mesoporous silica was collected.
Immobilization of cellulase on sorbent
The immobilization of cellulase on sorbents was carried out
according to the following procedures. In a typical adsorption
experiment, a certain amount of sorbents were added to the
cellulase solution (100 mg cellulase, 25 mL of 10 mM NaAc buffer
with pH value 5.0) in a vessel covered to prevent evaporation. Then
the mixture was stirred at a given temperature for 24 h to establish
the adsorption equilibrium. The immobilized cellulase on sorbents
was collected by centrifugation at 10000 × g for 10 min following
washed by NaAc buffer for 3 times. The amount of cellulase
molecules remaining in the supernatant was determined by the
method of Bradford[33] using boving serum albumin as the standard.
The amount of cellulase immobilized was calculated using the
formula
To investigating the physical adsorption mechanism of the
adsorption process, we discussed several factors as follow: two
mesoporous silica with different pore size of 17.6 nm and 3.8 nm
(MS-17.6nm and MS-3.8nm, where ‘MS’ means the mesoporous
silica) were synthesized, meanwhile,
a series of available
(x/m)a=(Ci −Ce)×Va/W,
mesoporous silica denoted as H-32 and diatomite were also used as
sorbents. In addition, the effect of morphology of different
sorbents, immobilization temperature and amounts of sorbents on
the adsorption amounts were exhaustively studied. We also
investigated the enzymatic activity of the cellulase immobilized on
different sorbents.
Where (x/m)a is the amount of cellulase immobilized per unit
weight of sorbent (mgmg−1), Ci is the initial concentration of protein
(mgmL−1) in cellulase solution, Ce is the equilibrium concentration of
protein (mgmL−1), Va is the solution volume (mL), and W is the
sorbent weight (mg).
To investigate the effect of sorbents morphology on
immobilization, the immobilization was carried out by adding 250
mg of different sorbents to 25 mL of 4 mg/mL cellulase solution and
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stirring for 24 h at 50 °C. Then, the amount of cellulase immobilized
on different sorbents was measured by UV-Vis spectroscopy. To
investigate the effect of temperature on immobilization, 25 mL of 4
mg/mL cellulase solution was added to MS-17.6nm powder (250
mg). The mixture was stirred at 50 °C, 25 °C and 4 °C for 24 h. To
investigate the effect of sorbents amounts on immobilization,
experiments involving adsorption of cellulase into MS-17.6nm and
MS-3.8nm were conducted for a range of initial input of sorbents
from 150 mg to 250 mg in given concentration of cellulase at 50 °C
for 24 h. To keep the concentration of cellulase as a constant, the
mass of cellulase was 100 mg dissolved in 25 mL buffer.
Enzymatic hydrolysis of the cellulose
Fig. 1. Small-angle X-ray scattering patterns for (a) MS-17.6nm and (b) MS-
3.8nm.
Cellulase activity was determined as the amount of released
glucose after cellulose degradation. 200 µL of cellulase immobilized
on sorbents (containing 0.5 mg cellulase) or free cellulase solution
(2.5 mg/mL) was mixed with 200 µL of CMC solution (2%) and then
incubated at 50 °C for 30 min. After that, the mixture was boiling
for 5 min to stop the hydrolysis reaction. One International Unit (IU)
of cellulase activity is defined as the amount of cellulase that
diffraction peak with a d spacing of 38.4 Å, as well as two small
(110) and (200) peaks associated with
a 2D hexagonal
mesostructure (p6mm), confirming the highly ordered
[34]
mesostructure of the silica host.
For the MS-3.8 nm, SAXS
hydrolyzes CMC to produce 1 µmol glucose per minute.
pattern showed a (100) diffraction peak, as you have said, MS-
3.8nm have a similar mesostructure to MS-17.6nm. But, the
intensity of (100) peak is weak, and there are no other two peaks,
indicating that the 2D hexagonal structure of MS-3.8nm is
incomplete. This can be associated with low temperature and dilute
solution or the short hydrocarbon chains of the organic template
during synthesis.[35-37] In addition, a part of 2D hexagonal structure
of MS-3.8nm may be destroyed. So, MS-3.8nm is less ordered
mesostructure than MS-17.6nm.
After hydrolysis, the mixture was centrifugation at 10000 × g for
10 min to remove immobilized cellulase. And then the supernatant
was used in glucose analysis via the glucose kit of GLU C
Ⅱ.
Characterization
Nitrogen (N2) sorption isotherms were measured with
a
Micromeritics Tristar 3000 analyser at 77 k. The Barrett-Emmett-
Teller (BET) method was utilized to calculate the surface areas. The
pore volume and pore size distributions were calculated from the
desorption branch of the isotherms using the Brarrett-Joyner-
Halanda (BJH) method. Solution UV-vis spectra were achieved by
UV spectrophotometer (Quawell Q5000) under 595 nm wave
length. Small Angle X-ray Scattering (SAXS) experiments were
performed on a Kratky compact small-angle system equipped with a
position-sensitive wire detector (OED 50M from MBraun, Graz)
containing 1024 channels of width 53.6 µm. CuKα radiation of
wavelength 1.542 Å was provided by a Seifert ID 3000 X-ray
generator operated at 50 kV and 40 mA. The analysis of size, shape
and particle distribution of the samples was carried out using TEM
(Tecnai G2 F30) at 100 kV and SEM (Hitachi S-4300) at 15 kV. For
this purpose, dispersions of sample nanoparticles were pipetted
onto carbon-coated copper grids.
Further evidence for mesostructure was provided by the TEM
images as presented in Fig. 2, which were representative of
mesoporous silica prepared with CTAB. According to the TEM image
of MS-17.6nm (Fig. 2c), there was some curved lattice fringes with a
d
spacing of 36.9 Å, which showed well-ordered mesopore
structures viewed along the [001] directions, suggesting that the
mesoporous silica was highly ordered mesostructure.
a
Furthermore, the pores and lattice fringes of MS-3.8nm could be
observed directly from Fig. 2d, the pore size and d spacing was both
Result and discussion
For the physical adsorption mechanism, synthetic mesoporous
silica MS-17.6nm and MS-3.8nm and commercial materials H-32
and diatomite were used as the sorbents. The effect of morphology
of sorbents, immobilization temperature and the adding amount of
sorbents were studied.
Characterization of mesoporous silica
The morphological and structural characterization of different
mesoporous silica and available porous silica were performed by N2
adsorption-desorption analysis, small-angle XRD,TEM and SEM. Fig.
1 represented the small-angle X-ray scattering patterns of MS-
17.6nm and MS-3.8nm, respectively. It can be seen that the SAXS
pattern of MS-17.6nm (Fig. 1a) exhibited a well-pronounced (100)
Fig. 2. SEM and TEM images of mesoporous silica: (a, c) MS-17.6nm and
(b, d) MS-3.8nm.
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Fig. 4. Cellulase adsorption amount on different sorbents which are given in
Fig. 3. N2 adsorption-desorption isotherms and pore size distribution the inset.
curves of mesoporous silica with different pore size: (a, b) MS-17.6nm
and (c, d) MS-3.8nm.
Table 1 Adsorption amounts of cellulase and physicochemical property of
about 38.2 Å. Meanwhile, it can be observed clearly that MS-3.8nm mesoporous silica.
was composed of accumulation by flaky silica. Both of the results
Mesoporous
Pore
diameter
(nm)
25
BET surface
area (m2g-1)
Total pore
volume
(cm3g-1)
0.77
Adsorbed
amounts (mg
g-1)
agreed well with the value of (100) crystal plane calculated by
small-angle XRD analysis respectively. The particle size of
mesoporous silica was shown in Fig. 2a and Fig. 2b: MS-17.6nm (4-
silica
H-32
232.5
105.2
474.7
13.0
301.0
12 μm) and
MS-3.8nm (1-7μm). Besides, the N2 adsorption
MS-3.8nm
MS-17.6nm
Diatomite
3.8
0.30
271.7
isotherms of the materials and their corresponding (BJH) pore size
distributions determined from desorption branch were also
exhibited in Fig. 3. As displayed in Fig. 3a, the adsorption-
17.6
1.51
325.5
200
0.11
311.5
desorption isotherm of MS-17.6nm possessed a type
Ⅳ curve with
H1 hysteresis loop at relative high P/P0 according to the IUPAC
classification,[38] suggesting a mesoporous structure. The specific
surface area, average pore diameter, and total pore volume of MS-
17.6nm were calculated to be 474.7 m2g-1, 17.6nm and 1.5 cm3g-1,
respectively. The pore-size distribution analyzed by BJH method
was shown in the inset, which displayed a sharp peak at 17.6 nm.
For comparison, MS-3.8nm with specific surface area of 105.2 m2g-
1, average pore diameter of 3.8 nm and total pore volume of 0.3
cm3g-1 was also measured by BET method (Fig. 3c). As can be seen,
times more than that adsorbed by MS-3.8nm. As for the MS-3.8 nm,
H-32 and diatomite, the adsorption amount after 24 h was about
271.7, 301.0 and 311.5 mg/g, respectively, slightly lower than that
of MS-17.6nm. Among these sorbents, it was surprising that the
adsorption behavior of diatomite revealed
a periodicity. The
maximum adsorption amount in the circle 320.0 mg/g was obtained
at 8 h, and the minimum adsorption amount in the circle 261.3
mg/g was obtained at 21 h, then the intact adsorption circle was
calculated as 26 h. These situations may be attributed to the
different morphology of sorbents. To further explain, the
adsorption amounts and physicochemical properties of mesoporous
a typical type
Ⅳ curve with H4 hysteresis loop was observed,
indicating the mesoporous structure which was possibly
attributable to the accumulation by flaky silica. These situations
were consistent with the SAXS and TEM results.
silica were shown in Table 1
.
As the results of previous analysis showed, cellulase molecule
was an elongated object, and the long and short axes were ca. 124
Å and ca. 37 Å.[39] Obviously, the pore diameter of diatomite was
significantly larger than the size of cellulase molecule, cellulase
molecules can be easily adsorbed into the pores with loose and
disordered arrangement due to capillary action. Meanwhile, when
the system established adsorption equilibrium, loose cellulase
molecules without immobilization were washed out from the pores
under stirring. This kind of circulation generated the unique
periodicity of the adsorption behavior for diatomite. The
geometrical dimension of cellulase molecule and sorbent was
another factor affecting immobilization. Takahashi[40] and his co-
workers indicated that the mesoporous material which had the
pore size close to geometrical dimension of cellulase would show
better adsorption capacity. Although the sorbent of MS-3.8nm had
the pore size of 3.8 nm, very approximate to the short axes of
cellulase, the sorbent exhibited the minimum adsorption amount.
This would be attributed to the pore space was blocked with some
Immobilization of cellulase on different sorbents
Since chemical immobilization may extremely likely occupy the
activity sites of cellulase, leading difficulty for cellulase to change
conformation, the cellulase was immobilized on different sorbents
by pure physical adsorption without any chemical bond or covalent
bond. The cellulase catalytic activity is essentially correlated with its
conformation. To obtain a good activity, the cellulase must be
immobilized in such a way that it can easily change conformation.
Nevertheless, there are various factors crucial to the
immobilization, such as morphology of sorbents, reaction
temperature, amount of sorbents, and reaction time. To obtain
appropriate immobilization to let cellulose change its conformation
freely, the effect of morphology of sorbents was determined first.
Effect of morphology
As shown in Fig. 4, the largest adsorption capacity appeared in
the case of MS-17.6nm, 325.5 mg/g (cellulase/ sorbents), almost 1.2
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reached adsorption equilibrium after 11 h. It was because MS-
17.6nm had reached adsorption saturation after 11 h. It was not
favourable for further loading of cellulase due to the extremely high
space hindrance in mesopores. Meanwhile, the adsorption amounts
of cellulase under 25 °C and 4 °C were increasing as a function of
time over the whole time range, the maximum equilibrium
adsorption amount cannot be observed within 24
h in our
experiments. In addition, at the given time, both the adsorption
amounts and rate of cellulose in MS-17.6nm increased along with
temperature increase. This was in accordance with the cellulase
immobilization process we suggested above. Temperature is an
important factor of adsorption efficiency. It is because adsorption
behaviour of cellulase molecules is an endothermic process.[41]
Fig. 5 Comparison of the cellulase adsorption on MS-17.6nm for Therefore, increasing temperature may promote immobilization of
different temperatures which are given in the inset.
cellulase into mesoporous silica with high efficiency, and it may be
anticipated that, the amounts of cellulase immobilized at 25 °C and
4 °C may be further increased if enough long contact time offered.
cellulase molecules. The cellulase molecules that stuck in the Thus, subsequent experiments used a temperature of 50 °C for
entrance of pores made the diffusion of cellulase into the pores immobilization.
difficultly. However, the sorbent of MS-17.6nm, of which the pore Effect of amounts of sorbents
size was slightly larger than the long axes of cellulase molecule,
From an economic point of view, determining the optimal
showed the maximum adsorption amount. After cellulase being
amount of sorbent makes it possible to use the minimum amounts
adsorbed into the pores, there was appropriate space for molecules
of sorbents to immobilize cellulase while maximizing the glucose
not to escape but adjust as dense and ordered arrangement. As far
yield. Experiments involving adsorption of cellulase into MS-17.6nm
as H-32 (pore size = 25 nm) is concerned, its adsorption amount
and MS-3.8nm were conducted for a range of initial input of
(301 mg g-1) relatively approached that of MS-17.6nm (325.5 mg g-
sorbents from 150 mg to 250 mg at 50 °C for 24 h. To keep the
1), this phenomenon was because the adsorption mechanism of H-
concentration of cellulase as a constant, the mass of cellulase was
32 was same as that of MS-17.6nm.
100 mg dissolved in 25 ml buffer.
Therefore, pore size slightly larger than the long axes of cellulase
As summarized in Fig. 6, adsorption curves of MS-17.6nm
molecule was essential for cellulase immobilization.
towards contact time showed a little difference. The saturated
Effect of temperature
adsorption amounts of cellulase were about 325, 410, 260 mg/g at
The temperature profiles of the adsorption amounts of cellulase contacting time of 17, 8 and 6 h, where the sorbent amounts were
using MS-17.6nm were measured (Fig. 5). As far as we are aware, 250, 200 and 150 mg, respectively. Different conclusion was drawn
there have been no reports on the relationship between when MS-3.8nm was used (Fig. 7). The adsorption amounts were
temperature and adsorption amounts in MS-cellulase system, about 270, 315, 315 mg/g at contacting time of 21, 21 and 17 h,
possibly due to the fact that most of the proteins are easy to be where the sorbent amounts were 250, 200 and 150 mg,
denatured. 25 mL cellulase (4 mg/mL) solution was added to MS- respectively. The results shown that MS-17.6 was more effective to
17.6nm powder (250 mg). The mixture was stirred at 50 °C, 25 °C adsorbed cellulase than MS-3.8, which was due to a regular
and 4 °C for 24h. It was shown that the reaction system at 50 °C microstructure, large pore size and surface area. The kinetically
Fig. 7 Comparison of the cellulase adsorption on MS-3.8nm for different
Fig. 6 Comparison of the cellulase adsorption on MS-17.6nm for
amounts of sorbents which are given in the inset.
different amounts of sorbents which are given in the inset.
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adsorption rate not only depended on the microstructure of
sorbent, but also related to the sorbent amounts.
In the case of physical adsorption, the physical adsorption
process may contain surface adsorption and pore adsorption. The
size of a single cellulase was shown in Fig. 9, and the average pore
size of MS-3.8 was about 3.8 nm. So surface adsorption was
dominated during the adsorption process. Therefore, in a certain
concentration of enzyme solution, the less adsorbent, and the
higher enzyme concentration in the relative microenvironment, the
faster adsorption rate and the more fully adsorption it got. So that,
the adsorbed amount of average per gram of carrier was higher.
Therefore, when the addition amount of MS-3.8 was 150 mg, the
adsorbed amount of average per gram of carrier was the largest,
and it was the smallest when the addition amount was 250 mg.
Compared to MS-3.8, MS-17.6 had a larger pore size (17.6 nm) Fig. 8 The effect of temperature on the specific activity of immobilized
and surface area (474.7 m2/g), hence there were more surface cellulase. Cellulase was immobilized on MS-17.6nm (▼), MS-3.8nm (▲), H-
adsorption and pore adsorption in MS-17.6. When the amount of 32 (●), and diatomite (◆). Free was no immobilized cellulase (■) as positive
adsorbent was 150 mg, the enzyme concentration in the micro- control.
environment was higher and the adsorption rate was faster.
However, the average pore size of MS-17.6 was similar to the size of
a single cellulase. It may block a part of pore in a rapid adsorption,
which would hinder the further adsorption of the channel and thus
increase the ratio of surface adsorption. Finally, the adsorbed
amount was not high. When the amount of adsorbent was 200 mg,
the enzyme concentration in the micro-environment was low, and
the probability of clogging of the channel became smaller. The
carrier can fully adsorb cellulase by pore adsorption and surface
adsorption, and finally obtain the largest adsorbed amount. When
the amount of adsorbent was too much (250 mg), the carrier also
could fully adsorb cellulase by pore adsorption and surface
adsorption, but the adsorbed amount would be decreased because
of the lower relative concentration of cellulase. So, for MS-17.6nm,
Fig. 9 Structural model of a cellulase molecule (a) and image models of
immobilized cellulase in MS-17.6nm (b) and MS-3.8nm (c) using a computer
schematic model.
there is an optimal value between sorbent amount and
concentration of cellulase.
Activities of the different immobilized cellulase
The cellulase immobilized on different sorbents all showed
activities toward cellulose hydrolysis. Significant differences
between relative enzymatic activities have been nevertheless found
depending on the type of sorbents.
axes of cellulase molecules, then the molecules stucked in the
entrance of pores. Therefore, active sites of cellulase molecules
were preserved.
Fig. 8 showed the effect of temperature on the specific activity of
the immobilized cellulase using different sorbents. Each
immobilized or free cellulase used for the reaction had the same
cellulase content 0.5 mg, in comparing the activities of the cellulase.
Intriguingly, the shape of the temperature profile of cellulase
immobilized on MS-3.8nm was very similar to the specific activity
curve of free cellulase and displayed a high specific activity (i.e.
63.3% of free cellulase activity at 50 °C) which could be concluded
that the acremonium cellulase immobilized on MS-3.8nm maintain
preserved the original morphology of free cellulase extremely. In
addition, cellulase immobilized on MS-3.8nm retained a higher level
of specific activity at elevated temperature (i.e. 67.0% of free
cellulase activity at 60 °C) in comparison to the other hydrolysis
temperature. This result illustrated that the immobilization of
cellulase on MS-3.8nm increased the stability of free cellulase with
regard to higher temperature. As described above, sorbent of MS-
3.8nm had the pore size of 3.8 nm, very approximate to the short
As the adsorption amounts increased, specific activity decreased
gradually, cellulase immobilized on H-32 and MS-17.6nm exhibited
specific activity 35.8% and 26.6% of free cellulase activity at 60 °C,
respectively. This may be attributed to the cellulase immobilization
process we suggested previously, sorbents MS-17.6 nm which have
average mesopores size matched long axes of cellulase molecules
showed the maximum adsorption amount. When cellulase was
adsorbed into the pore, there was appropriate space for molecules
not to escape but adjust as dense and ordered arrangement.
However, the dense and ordered arrangement would prevent
conformational flexibility of cellulase, as we know the cellulase
molecules need conformational change in the interaction process
between cellulase and substrate. So, MS-17.6nm revealed a lower
specific activity than H-32. And the image models of adsorption
mechanism for MS-17.6nm and MS-3.8nm was exhibited in Fig.
9.Meanwhile, both of MS-17.6nm and H-32 retained a higher level
of specific activity at elevated temperature in comparison to the
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other hydrolysis temperature, by contrast, it was obvious that
specific activity of free cellulase decreased when temperature
higher than 50 °C.
Acknowledgements
This work was supported by the contribution of Eurus Akita Port
Wind Farm to Akita Prefectural University in Japan and the
International Collaboration Project funded by Shenzhen
Government (GJHZ20150316160614843).
In the case of diatomite, a significant difference in the specific
activity was observed, which revealed the lowest specific activity
i.e. 13.5% of free cellulase activity at 60 °C. This may be contributed
by the lamellar structure of diatomite: immobilized cellulase
molecules were embedded between the lamellas, which prevented
cellulase from contacting with substrate. However, it was still
obvious that the specific activity of cellulase immobilized on
diatomite demonstrated better thermal stability: as temperature
increasing specific increased.
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Studying the physical adsorption mechanism and with high adsorption
amount in the mesoporous silica immobilized cellulase process.