Immobilization of β-glucosidase on mercaptopropyl-functionalized mesoporous titanium dioxide

Article abstract of DOI:10.1016/j.molcatb.2013.07.005

Mesoporous titanium dioxide (M-TiO₂) and mercaptopropyl- functionalized M-TiO₂ (SH-M-TiO₂) were prepared as carriers for immobilization of β-glucosidase (BG). Analyses suggested that in SH-M-TiO₂ the grafted mer

Full text of DOI:10.1016/j.molcatb.2013.07.005

Journal of Molecular Catalysis B: Enzymatic 97 (2013) 303–310
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Immobilization of ␤-glucosidase on mercaptopropyl-functionalized
mesoporous titanium dioxideଝ
Ce Weia,b, Qingshan Lua, Jia Ouyanga, Qiang Yonga, Shiyuan Yua,∗
a Key Laboratory of Forest Genetics and Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing 210037, China
b College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Mesoporous titanium dioxide (M-TiO2) and mercaptopropyl-functionalized M-TiO2 (SH-M-TiO2) were
prepared as carriers for immobilization of ␤-glucosidase (BG). Analyses suggested that in SH-M-TiO2 the
grafted mercaptopropyl groups attributed to about 3.5% of the total weight of the matrices. For the immo-
bilization of BG, SH-M-TiO2 or M-TiO2 powder was added with free BG solution and stirred at 4 ◦C for 8 h,
followed by centrifugation and washing. BG was effectively immobilized on SH-M-TiO2 with an enzyme
activity of 10.9 U/g, which was 92.8% of the total enzyme activity in the starting solution of free BG. BG
immobilized on SH-M-TiO2 (BG-SH-M-TiO2) was more stable in pH, thermal, and storage tests than BG on
M-TiO2 or free BG in solution. The Km and vmax of BG-SH-M-TiO2 were similar to values for free BG in solu-
tion. Batch hydrolysis of cellobiose-containing substrate by BG-SH-M-TiO2 was conducted. Conversion
rates of cellobiose in 10 batches were consistently around 90%. Although the residual enzyme activity
of BG-SH-M-TiO2 decreased gradually, only a small amount of BG activity was detected in supernatants.
This indicated effective adsorption of enzyme molecules to SH-M-TiO2 matrices.
Received 27 January 2013
Received in revised form 21 May 2013
Accepted 8 July 2013
Available online 15 July 2013
Keywords:
␤-Glucosidase
Immobilization
Mesoporous titanium dioxide
Mercaptopropyl group
Enzymatic hydrolysis
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
feedback mechanism by high concentrations of the disaccharide
cellobiose and glucose [10]. This substrate inhibition can be allevi-
The enzyme ␤-glucosidase (BG) is ubiquitous across a broad
range of eukaryotic and prokaryotic species [1,2]. It exhibits wide
substrate specificity and is capable of cleaving the ␤-glucosidic
linkages of conjugated glucosides and some disaccharides [3]. The
substrates of BG include oligosaccharides such as cellotetraose and
cellopentaose [4] as well as cellooligosaccharides. BG also releases
glucose from the disaccharide cellobiose [5] and hydrolyze antho-
cyanins, the main coloring agents in vegetables [6,7]. BG is used in
the food industry to improve the organoleptic qualities of wine and
fruit juices [8].
Extensive production of renewable biofuels and high-value
organic solvents through the enzymatic hydrolysis of lignocellu-
losic wastes is possible. The hydrolysis of lignocellulose requires the
cooperativeaction of exocellulase, endocellulaseand BG, ultimately
producing the fermentable monosaccharide glucose, which is the
major substrate for the production of fuel ethanol, isopropanol
and acetone [9]. However, BG is inhibited through a competitive
ated by elevating the levels of BG in bioreactors [11].
Titanium dioxide (TiO2) is an inexpensive, safe and highly stable
nanocrystalline material that is resistant to corrosion in oxidizing
solutions. This material can be used as photo-catalyst, anti-viral
agent, deodorant, and self-cleaning glass [12,13]. TiO2 nanotubes
or nanoparticles are mesoporous, with pores diameter of 10 to
100 nm. The morphology and porosity of TiO2 materials, specifi-
cally the density, size and distribution of the micropores, can be
controlled during fabrication [14,15]. Mesoporous TiO2 (M-TiO2)
forms chemically and thermally robust, highly crystalline particu-
late materials with a high surface-to-volume ratio. These properties
make M-TiO2 an ideal matrix for the immobilization or attachment
of enzymes in nanoscale enzymatic bioreactors. Enzyme immobi-
lization on M-TiO2 is thought to occur via simple adsorption of
proteins to the material surface or via covalent attachment, elec-
trostatic binding or encapsulation [16,17]. The immobilization of
enzymes on M-TiO2 has the potential to improve stability, stor-
age and reuse of enzymes in a number of bioreactor applications
[18,19]. M-TiO2 is a useful support matrix for attachment of BG
enzymes because the oxide layer has a negative charge at phys-
iological pH, allowing quick and stable attachment of BG [20]
without compromising activity or reaction specificity. Most meso-
porous silica materials, such as SBAs, MCM-41 and HMS, were
prepared by “template-growth” process, which is cost prohibitive
and is unlikely to be utilized on an industrial scale [21]. In contrast,
ଝ
This is an open-access article distributed under the terms of the Creative Com-
mons Attribution-NonCommercial-No Derivative Works License, which permits
non-commercial use, distribution, and reproduction in any medium, provided the
original author and source are credited.
∗
Corresponding author. Tel.: +86 25 85427623; fax: +86 25 85424121.
E-mail address: syu@njfu.edu.cn (S. Yu).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.07.005

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C. Wei et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 303–310
M-TiO2 in this work was prepared through a simple synthesis route
at considerably lower cost that might be suitable for commercial
enzyme immobilization. [22].
SH-M-TiO2 was obtained as shown in Fig. 1. M-TiO2 powder
(1 g) was dispersed in 50 ml toluene, then 5 ml (3-mercaptopropyl)
trimethoxysilane (MPTMS, 175617, Sigma-Aldrich) was added. The
mixture was heated under reflux at 110 ◦C for 8 h. After filtration,
the powder was washed with ethanol and dried.
Mesoporous materials are often modified with functional
groups that enhance the capacity for stable attachment of large
amounts of enzyme with activity and specificity that is simi-
lar to or better than soluble enzyme. Bai et al. [23] reported
the immobilization of lipase on mesoporous silica nanotubes and
aminopropyl-grafted mesoporous silica nanotubes. The hydrolysis
activity of the lipase immobilized on aminopropyl-grafted meso-
porous silica nanotubes was almost twice of the activity of the
enzyme on mesoporous silica nanotubes. Yiu et al. [24] modified
SBA-15 with thiol, chloride, amine, and carboxylic acid. Trypsin on
thiol-functionalized SBA-15 was found to be the most promising.
However, no information on the use of functionalized M-TiO2 for
enzyme immobilization could be retrieved.
This work investigated M-TiO2 functionalized with mercap-
topropyl groups by post-synthesis grafting. The functionalized
product SH-M-TiO2 was used as a carrier for BG immobiliza-
tion. The structural properties of SH-M-TiO2 were characterized
by several methods. BG immobilized on SH-M-TiO2 was evalu-
ated for optimum reaction pH and temperature; thermal, pH, and
storage stability; reusability; kinetics; and repeated enzymatic
hydrolysis.
2.2. Structural characteristics of M-TiO2 and SH-M-TiO2
Nitrogen absorption and desorption isotherms of M-TiO2 and
SH-M-TiO2 were obtained (ASAP 2020M Micromeritics, Atlanta,
USA) using liquid nitrogen. Specific surface areas were calcu-
lated by the Brunauer–Emmett–Teller (BET) method with data
in a relative pressure range of P/P0 = 0.1–1.0. Pore size distribu-
tions were determined by analyzing absorption branches with the
Barrett–Joyner–Halenda (BJH) method [26,27].
M-TiO2 and SH-M-TiO2 were tested by scanning electron
microscopy and energy dispersive X-ray spectroscopy (SEM/EDS,
S-4800, Hitachi, Japan).
Thermal analysis was with a thermo gravimetric analyzer
(STA409PC, NETZSCH, Germany) with temperature programming
of 10 ◦C/min in flowing N2.
2.3. Immobilization of ˇ-glucosidase
SH-M-TiO2 (or M-TiO2) powder (10 mg) was added to 0.2 ml
of BG solution (0.261 to 1.19 U/ml) in citric acid-Na2HPO4 buffer
(50 mM, pH 4.8). The mixture was stirred at 150 r/min at 4 ◦C for
8 h to establish adsorption equilibrium. BG immobilized on SH-M-
TiO2 (BG-SH-M-TiO2) or BG immobilized on M-TiO2 (BG-M-TiO2)
was collected by centrifugation at 4000 × g for 10 min, and washed
with citric acid-Na2HPO4 buffer until no BG activity was detected
in the supernatant. All supernatants were collected for BG assays.
2. Materials and methods
2.1. Enzyme and carriers
␤-Glucosidase (Novozyme® 188, from Aspergillus niger) was
from Sigma-Aldrich and stored at 2–8 ◦C.
M-TiO2 was provided by Lu [25,26] and prepared as follows:
A mixture with a TiO2/K2O molar value of 1.9 was prepared by
adding reagent grade K2CO3 to TiO2·nH2O and sintering at 810 ◦C
for 2 h. The sintered product was wet-ground and dried at 60 ◦C and
10 g was soaked in 7 ml of distilled water at ambient temperature
in a closed container for 7 days, during which the potassium-rich
nanophase formed. When the product was completely transformed
to amorphous phase, it was suspended in 100 ml of vigorously
stirred 0.1 M HCl solution to remove K+ ions. The product was sep-
arated by filtration and washed with distilled water, followed by
desiccation at 60 ◦C under vacuum. Calcinations of the dried tita-
nium sample were in a muffle oven at elevated temperature for 2 h.
M-TiO2 with an average pore size of about 20 nm was obtained by
adjusting calcination temperature.
2.4. Determination of BG activity
BG activity was measured as p-nitrophenol (pNP) released
by enzymatic degradation of p-nitrophenyl ␤-D-glucopyranoside
(pNPG). BG solution (0.1 ml), BG-M-TiO2 powder (1–10 mg) or BG-
SH-M-TiO2 powder (1–10 mg), which had approximately the same
BG activity, were mixed with 0.9 ml of citric acid-Na2HPO4 buffer
(50 mM, pH 4.8) and 5 mM of pNPG (Sigma) and incubated at 50 ◦C
for 10 min with stirring. Enzymolysis was terminated with 2 ml of
Na2CO3 (1 M). The mixture was centrifuged at 10,621 × g for 30 min
at 4 ◦C to remove the powder and the supernatant was used in
reactions. Product pNP in the supernatant was measured at 400 nm
using an UV spectrophotometer (752PC UV–VIS spectrophotome-
ter, Shanghai Shunyu Hengping, China). One unit (U) of enzyme
activity was defined as the amount of enzyme that released 1 ␮mol
of pNP per minute [27].
2.5. Optimum reaction pH and temperature
The optimum reaction pH was determined for free BG in solu-
tion, BG-M-TiO2 or BG-SH-M-TiO2 by incubating with 5 mM pNPG
in 50 mM citric acid-Na2HPO4 buffer from pH 3.0 to 7.2 at 50 ◦C for
10 min. The amount of pNP was estimated as described in 2.4.
The optimum reaction temperature was determined with free
BG in solution, BG-M-TiO2 or BG-SH-M-TiO2 by incubating with
5 mM pNPG in 50 mM citric acid-Na2HPO4 buffer at 25–70 ◦C for
10 min. Product pNP was estimated as described in 2.4.
2.6. Stability
Stability under different pH conditions was determined for
free BG in solution, BG-M-TiO2 and BG-SH-M-TiO2 by measuring
Fig. 1. Formation of mercaptopropyl-functionalized M-TiO2 (SH-M-TiO2). MPTMS,
3-mercaptopropyl trimethoxysilane.

C. Wei et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 303–310
305
residual BG activity after incubation in 50 mM citric acid-Na2HPO4
buffer at pH 3.0 to 7.2, at 25 ◦C for 24 h.
Thermal stability was tested for free BG in solution, BG-M-TiO2
and BG-SH-M-TiO2 by measuring residual BG activity after incuba-
tion in 50 mM, pH 4.8 citric acid-Na2HPO4 buffer at 25 to 70 ◦C for
4 h.
Storage stability was determined for free BG in solution, BG-M-
TiO2 and BG-SH-M-TiO2 by measuring residual BG activity after 5
months in citric acid-Na2HPO4 buffer at 50 mM, pH 4.8. Residual
activity was tested once per month.
Reusability was tested for BG-M-TiO2 and BG-SH-M-TiO2 by
BG assays as described in 2.4. BG-M-TiO2 and BG-SH-M-TiO2 were
recovered by centrifugation and washing three times with 50 mM,
pH 4.8 citric acid-Na2HPO4 buffer.
2.7. Determination of kinetic constants
Michaelis constant (Km) and maximum reaction velocity (vmax
)
of free BG in solution, BG-M-TiO2 and BG-SH-M-TiO2 were deter-
mined by measuring initial reaction rate with pNPG (0.5–8 mmol/l)
in citric acid-Na2HPO4 buffer, 50 mM, pH 4.8 at 50 ◦C for 10 min.
Km and vmax were obtained by Lineweaver–Burk double reciprocal
plots using Michaelis–Menten kinetic equations.
Fig. 3. Representative SEM images of (a) M-TiO2, mesoporous TiO2; (b) SH-M-TiO2,
mercaptopropyl-functionalized M-TiO2.
3. Results and discussion
3.1. Structural characteristics of mesoporous TiO2 and
mercaptopropyl-functionalized M-TiO2
3.1.1. Analysis of N2 absorption and desorption isotherms
N2 absorption and desorption isotherms of M-TiO2 and SH-M-
TiO2 are in Fig. 2a. Although the absorption isotherm curves of
M-TiO2 and SH-M-TiO2 are similar, less N2 absorption was observed
for SH-M-TiO2. After mercaptopropyl functionalization, the meso-
porous structure of M-TiO2 changed slightly. Fig. 2b indicates that
the pore size distribution profile of SH-M-TiO2 shifted to the left
compared to M-TiO2. The number of pores with diameters in the
range of 20–30 nm decreased after M-TiO2 functionalized, probably
because the mercaptopropyl groups occupied pore space. Table 1
shows that the surface area decreased from 62.04 to 53.50 m2/g
and the total pore volume decreased from 0.37 to 0.31 cm3/g after
M-TiO2 was functionalized. Both Bai et al. [23] and Zhang et al. [28]
Table 1
a
Textural property of M-TiO2 and SH-M-TiO2
.
Surface area
(m2/g)
Pore diameter
(nm)
Total pore
volume (cm3/g)
M-TiO2
SH-M-TiO2
62.04
53.50
20.3
18.2
0.37
0.31
Fig. 2. N2 absorption and desorption isotherms (a) and pore diameter distribution
profiles (b) of M-TiO2 and SH-M-TiO2. M-TiO2, mesoporous TiO2, ꢀ; SH-M-TiO2,
mercaptopropyl-functionalized M-TiO2, ꢀ.
a
M-TiO2, mesoporous TiO2; SH-M-TiO2, mercaptopropyl-functionalized M-TiO2.

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C. Wei et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 303–310
Fig. 5. Thermal gravimetric and differential thermal gravimetric (TG/DTG) curves
of (a) M-TiO2, mesoporous TiO2; (b) SH-M-TiO2, mercaptopropyl-functionalized M-
TiO2.
Fig. 4. EDS spectrums of (a) M-TiO2, mesoporous TiO2; (b) SH-M-TiO2,
mercaptopropyl-functionalized M-TiO2.
3.2. Immobilization of ˇ-glucosidase
BG was immobilized on M-TiO2 or SH-M-TiO2 at initial concen-
trations of 0.261 to 1.19 U/ml free BG in solutions. BG-M-TiO2 and
BG-SH-M-TiO2 were analyzed for enzyme activity. The percentage
of immobilized BG activity to initial BG activity in the solution was
defined as activity recovery. BG activity in the supernatant after
immobilization was also determined.
As shown in Table 2, as initial BG activity in free BG solu-
tion increased, immobilized BG activity on M-TiO2 or SH-M-TiO2
increased. SH-M-TiO2, the mercaptopropyl-functionalized carrier,
showed higher immobilization efficiency than M-TiO2. When initial
free BG activity in solution was 1.19 U/ml, SH-M-TiO2 immobilized
a BG activity of 15.2 U/g, twice as much as M-TiO2 at 7.67 U/g.
Immobilized enzyme attained the most effective activity balance
when the highest activity recovery was obtained. The highest activ-
ity recovery of BG-M-TiO2 was 64.1% as initial BG activity in solution
was 0.364 U/ml. By contrast, the highest activity recovery of BG-SH-
M-TiO2 was 92.8% as initial BG activity in solution was 0.592 U/ml,
with an immobilized BG activity of 10.9 U/g. These results indi-
cated that SH-M-TiO2 had a better capacity than M-TiO2 to load
BG molecules.
reported that surface area and total pore volume decreased after
carriers were functionalized.
3.1.2. Scanning electron microscopy/energy dispersive X-ray
spectroscopy analysis
Scanning electron microscopy (SEM) images of M-TiO2 and
SH-M-TiO2 are in Fig. 3. Both M-TiO2 and SH-M-TiO2 had highly
porous structures with similar distributions. Because of the porous
structure and large surface area, the two carriers might be good
matrices for immobilization of enzyme molecules. Comparisons of
the energy dispersive X-ray spectroscopy (EDS) spectra of M-TiO2
(Fig. 4a) and SH-M-TiO2 (Fig. 4b), shows Si and S elements in SH-
M-TiO2. Analysis of the data presented in the insets of Fig. 4a and
Fig. 4b indicated that, after M-TiO2 was functionalized, the grafted
mercaptopropyl groups attributed to about 3.5% of the total weight
of the matrices.
3.1.3. Thermal analysis
Thermal gravimetric and differential thermal gravimetric
(TG/DTG) curves of M-TiO2 and SH-M-TiO2 are in Fig. 5. A clear
exothermic DTG peak at 355 ◦C with an accompanying weight loss
could be seen for SH-M-TiO2 in Fig. 5b but was not observed for M-
TiO2 (Fig. 5a). The exothermic peak was attributed to the thermal
decomposition of mercaptopropyl groups, consistent with another
report [28]. The results of TG analysis furthermore demonstrated
that about 3.5% mercaptopropyl groups grafted on the total weight
of matrices after M-TiO2 was functionalized.
3.3. Optimum reaction pH and temperature
Free BG in solution, BG-M-TiO2 and BG-SH-M-TiO2 were incu-
bated at 50 ◦C in 5 mM pNPG solution at pH 3.0 to 7.2 for 10 min.
The effects of reaction pH for the three enzyme preparations were
similar, as shown in Fig. 6a. The optimum reaction pH for the three
enzyme preparations was 4.2. BG-SH-M-TiO2 exhibited more stable

C. Wei et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 303–310
307
Table 2
Efficiency of ␤-glucosidase immobilization on M-TiO2 and SH-M-TiO2
a
.
Initial BG activity in the
free BG solution (U/ml)
BG-M-TiO2
BG-SH-M-TiO2
Activity (U/g)
Activity
recovery (%)
Activity in the
supernatant (%)
Activity
(U/g)
Activity
recovery (%)
Activity in the
supernatant (%)
0.261
0.364
0.474
0.592
0.677
0.790
0.948
1.19
3.51 0.01
4.68 0.03
5.25 0.02
6.40 0.06
6.53 0.06
7.04 0.08
7.59 0.13
7.67 0.09
63.0 0.4
64.1 0.5
56.3 0.4
51.1 1.1
48.2 1.1
44.6 0.7
41.8 0.6
8.45 0.25
28.0 1.8
27.5 1.6
31.2 1.7
34.2 1.9
38.4 2.2
45.8 2.4
49.4 2.2
86.0 4.2
3.79 0.13
5.36 0.06
7.90 0.26
10.9 0.1
12.0 0.3
13.2 0.3
14.8 0.4
15.2 0.5
72.5 0.5
73.6 0.2
83.3 3.3
92.8 0.6
88.7 1.9
83.3 1.7
78.2 0.4
63.7 1.0
26.8 1.6
26.1 1.5
13.7 0.7
6.88 0.35
7.34 0.35
12.9 0.5
17.5 0.7
27.3 1.0
a
BG-M-TiO2, ␤-glucosidase immobilized on mesoporous TiO2; BG-SH-M-TiO2, ␤-glucosidase immobilized on mercaptopropyl-functionalized M-TiO2. Each value is an
average of three parallel replicates and is represented as mean standard deviation.
Fig. 6. Effect of reaction pH (a) at 25 ◦C and temperature at pH 4.2 (b) on relative activity of free BG in solution, BG-M-TiO2 and BG-SH-M-TiO2. Free ␤-glucosidase in solution,
ꢁ; BG-M-TiO2, ␤-glucosidase immobilized on mesoporous TiO2, ꢀ; BG-SH-M-TiO2, ␤-glucosidase immobilized on mercaptopropyl-functionalized M-TiO2, ꢂ. Values are
averages from duplicate experiments.

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C. Wei et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 303–310
Table 3
Apparent kinetic parameters of the three enzyme preparationsa.
Km (mmol/l)
Vmax (mmol/(l min))
Free BG
BG-M-TiO2
BG-SH-M-TiO2
0.89 0.03
2.66 0.15
1.0 0.04
0.41 0.01
0.39 0.02
0.42 0.03
a
Free BG, free ␤-glucosidase in solution; BG-M-TiO2, ␤-glucosidase immo-
bilized on mesoporous TiO2; BG-SH-M-TiO2, ␤-glucosidase immobilized on
mercaptopropyl-functionalized M-TiO2. Each value is an average of three parallel
replicates and is represented as mean standard deviation.
reaction activity between pH 4.2 and pH 5.4 than free BG in solu-
tion or BG-M-TiO2. Free BG, BG-M-TiO2 and BG-SH-M-TiO2 were
incubated at 25–70 ◦C for 10 min in 5 mM pNPG solution at pH 4.2.
Fig. 6b shows that the optimum reaction temperature of the three
enzyme preparations was at 60 ◦C.
3.4. Stability
Stability at different pH values was determined with free BG in
solution, BG-M-TiO2 and BG-SH-M-TiO2 by measuring residual BG
activity after incubation at pH 3.0 to 7.2 for 24 h. As shown in Fig. 7a,
free BG in solution and BG-M-TiO2 were stable, maintaining more
than 90% activity at pH 4.2–4.8 while BG-SH-M-TiO2 retained 90%
activity at pH 4.2–5.4. BG-SH-M-TiO2 was more stable than both
free BG and BG-M-TiO2 at pH less than 4 and above 5. Therefore,
immobilization of BG on mercaptopropyl-functionalized M-TiO2
maintained BG activity in a wider pH range.
Thermal stability was tested for free BG, BG-M-TiO2 and BG-SH-
M-TiO2 by measuring residual activity after incubation in pH 4.8 at
25–70 ◦C for 4 h. As shown in Fig. 7b, the three enzyme preparations
showed similar activity at 20 to 50 ◦C. However, at incubation tem-
peratures over 50 ◦C, the thermal stability of BG-SH-M-TiO2 was
better than free BG or BG-M-TiO2.
Storage stability was studied with free BG, BG-M-TiO2 and BG-
SH-M-TiO2 by measuring residual BG activity after storage at 4 ◦C
for 5 months. Fig. 7c shows that after 3 months, the residual activity
of free BG and BG-M-TiO2 declined to below 20% of initial BG activ-
ity, whereas residual activity of BG-SH-M-TiO2 was higher than 95%
at 5 months. These results indicated that immobilization of BG on
SH-M-TiO2 effectively preserved BG activity.
The operational stability of BG-M-TiO2 and BG-SH-M-TiO2 is
important for applications. This stability was tested by repeated
BG assays. Fig. 7d shows that after 6 rounds of BG assays, residual
activity of BG-M-TiO2 was below 50% of initial activity but resid-
ual activity of BG-SH-M-TiO2 was above 80%. After 30 rounds of BG
assays, the residual activity of BG-SH-M-TiO2 remained at about
50%.
These results showed that BG-SH-M-TiO2 offered better stability
than free BG in solution or BG-M-TiO2 at different pH, tempera-
ture, storage and operational conditions. Comparison of BG-M-TiO2
and BG-SH-M-TiO2 for storage and operational stability showed
weak adsorption interaction between M-TiO2 and BG. Interaction
between SH-M-TiO2 and BG molecules was relatively stronger,
which could be attributed to the covalent attachment of disulfide
bonds between the surface of the enzyme and the SH-M-TiO2 mer-
captopropyl groups. The resulting BG-SH-M-TiO2 could be used
repeatedly, similar to the results of Yiu et al. [24], who showed that
trypsin supported on thiol-functionalized SBA-15 was recyclable.
3.5. Kinetics of enzyme preparations
Fig. 7. Stability under different pH (a), temperature (b), storage (c), and re-use (d)
conditions of free BG in solution, BG-M-TiO2 and BG-SH-M-TiO2. Free ␤-glucosidase,
ꢁ; BG-M-TiO2, ␤-glucosidase immobilized on mesoporous TiO2, ꢀ; BG-SH-M-TiO2,
␤-glucosidase immobilized on mercaptopropyl-functionalized M-TiO2, ꢂ. Values
are averages from duplicate experiments.
Km and vmax of free BG in solution, BG-M-TiO2 and BG-SH-M-
TiO2 were calculated using Lineweaver–Burk plots with pNPG as
substrate. Table 3 shows that Km of free BG in solution was the
lowest of the three enzyme preparations, indicating that free BG

C. Wei et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 303–310
309
Fig. 8a shows a batch hydrolysis course by BG-SH-M-TiO2. The
hydrolysis velocity of cellobiose was high for the first hour, then
declined gradually because of product inhibition. After 6 h, the con-
version rate of cellobiose was 90%, which is acceptable in practice.
Fig. 8b presents the results of 10 batches of hydrolysis using
the same BG-SH-M-TiO2. The conversion rates of cellobiose were
around 90% for all 10 batches, indicating a stable performance
by the immobilized enzyme. BG activity in the supernatant rep-
resents the degree of desorption of immobilized enzyme. After
the first batch of hydrolysis, 5% of the initially immobilized BG
activity was lost; thereafter, enzyme desorption gradually slowed.
The residual BG activity of BG-SH-M-TiO2 after each batch also
decreased slowly; after 10 batches of hydrolysis, 81% of the ini-
tially immobilized BG activity remained. After repeated hydrolysis
with immobilized enzyme at 60 ◦C, such as in this experiment, pro-
teins are expected to irreversibly deactivate. The loss of residual BG
activity from the BG-SH-M-TiO2 could not be attributed mainly to
desorption of enzyme molecules. Instead, BG activity in the later
supernatants was low. Thus, the results in Fig. 8b show effective
adsorption of enzyme molecules to mercaptopropyl-functionalized
mesoporous TiO2. Since enzyme deactivation at high temperature
is inevitable, the addition of fresh immobilized enzyme is necessary
during enzymatic hydrolysis.
4. Conclusion
For immobilization of BG on an inorganic material, M-TiO2,
a nanostructured crystalline TiO2, was determined as the carrier
since it is inexpensive, safe, highly stable, and easily prepared at
large scale. To enhance the loading and stability of enzyme, M-TiO2
was functionalized with mercaptopropyl group by post-synthesis
grafting; our analysis confirmed the success of this modification.
The resulting SH-M-TiO2 immobilized BG effectively and BG-SH-M-
TiO2 offered better stability in pH, temperature, and storage tests
than BG-M-TiO2 or free BG in solution. When BG-SH-M-TiO2 was
used in repeated batches of enzymatic hydrolysis, the conversion
rate of cellobiose was high, while desorption of enzyme activity was
minor. Although the mechanisms of enzyme attachment to SH-M-
TiO2 require further investigation, effective adsorption of enzyme
molecules to SH-M-TiO2 matrices is proposed. For large-scale tests,
however, M-TiO2 should be fabricated to a specific size and shape to
construct a practical enzymatic bioreactor. During fabrication, the
morphology and porosity of M-TiO2 might differ from the powder
used in this study. More work on the fabrication of M-TiO2 particles
or tubes and immobilization of BG is required.
Fig. 8. Enzymatic hydrolysis of cellobiose-containing substrate by BG-SH-M-TiO2.
(a): Course of batch hydrolysis; (b): repeated hydrolysis of 10 batches. Substrate
contained 26.93 g/l cellobiose and 16.88 g/l glucose. BG-SH-M-TiO2, ␤-glucosidase
immobilized on mercaptopropyl-functionalized mesoporous TiO2, was added at a
total BG activity of 20 U/g sugar. Batch hydrolysis was conducted at pH4.8 and 60 ◦
for 6 h. Values are averages from duplicate experiments.
C
had the highest affinity for the substrate pNPG. Vmax of BG-M-TiO2
was slightly lower than for free BG, probably due to diffusion lim-
itation and steric hindrance [29]. Free BG and BG-SH-M-TiO2 had
similar values for Km and vmax. Therefore, BG-SH-M-TiO2 also had
good affinity for the substrate pNPG.
Acknowledgments
3.6. Repeated enzymatic hydrolysis of cellobiose-containing
substrate by BG-SH-M-TiO2
We thank Professor Xiaohua, Lu and Dr. Wei Zhuang for M-TiO2.
This research was supported by the China Governmental Public
Industry Research Special Funds (Grant No. 201004001) and the Pri-
ority Academic Program Development of Jiangsu Higher Education
Institutions (PAPD).
Previously, we proposed a three-stage enzymatic hydrolysis
model for steam-exploded corn stover with a solid content of 30%
[30]. A high yield of saccharification took only 30 h and the resulting
sugar solution contained a substantial portion of cellobiose. Since
the sugar solution is separated from solid residues, further conver-
sion of cellobiose to glucose is easily achieved by repeated batch
mode or a continuous process with immobilized BG.
A repeated batch hydrolysis was designed to test the practical
efficiency of BG-SH-M-TiO2. A sugar solution containing 26.93 g/l
cellobiose and 16.88 g/l glucose was used as the substrate. BG-SH-
M-TiO2 with a total BG activity of 20 U/g sugar was applied in batch
hydrolysis at pH 4.8 and 60 ◦C for 6 h. Residual immobilized BG
activity was determined and the supernatant was checked for sugar
concentration and lost enzyme activity. BG-SH-M-TiO2 was reused
for another batch of hydrolysis.
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