Immobilized cellulase on Fe3O4 nanoparticles as a magnetically recoverable biocatalyst for the decomposition of corncob

Article abstract of DOI:10.1016/S1872-2067(15)61028-2

A magnetically recoverable biocatalyst was successfully prepared through the immobilization of cellulase onto Fe₃O₄ nanoparticles. The magnetic nanoparticles were synthesized by a hydrothermal method in an aqueous system. The support (Fe₃O₄ nanoparticles) was modified with (3-aminopropyl)triethoxysilane, and glutaraldehyde was used as the cross-linker to immobilize the cellulose onto the modified support. Different factors that influence the activity of the immobilized enzyme were investigated. The experimental results indicated that the suitable immobilization temperature and pH are 40 °C and 6.0, respectively. The optimal glutaraldehyde concentration is ～2.0 wt%, and the appropriate immobilization time is 4 h. Under these optimal conditions, the activity of the immobilized enzyme could be maintained at 99.1% of that of the free enzyme. Moreover, after 15 cyclic runs, the activity of the immobilized enzyme was maintained at ～91.1%. The prepared biocatalyst was used to decompose corncobs, and the maximum decomposition rate achieved was 61.94%.

Full text of DOI:10.1016/S1872-2067(15)61028-2

Chinese Journal of Catalysis 37 (2016) 389–397
a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / c h n j c
Article
Immobilized cellulase on Fe3O4 nanoparticles as a magnetically
recoverable biocatalyst for the decomposition of corncob
Qikun Zhang *, Junqing Kang, Bing Yang, Leizhen Zhao, Zhaosheng Hou, Bo Tang
College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging,
Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Normal University, Jinan 250014, Shandong, China
A R T I C L E I N F O
A B S T R A C T
Article history:
A magnetically recoverable biocatalyst was successfully prepared through the immobilization of
cellulase onto Fe3O4 nanoparticles. The magnetic nanoparticles were synthesized by a hydrothermal
method in an aqueous system. The support (Fe3O4 nanoparticles) was modified with (3‐aminopro‐
pyl)triethoxysilane, and glutaraldehyde was used as the cross‐linker to immobilize the cellulose
onto the modified support. Different factors that influence the activity of the immobilized enzyme
were investigated. The experimental results indicated that the suitable immobilization temperature
and pH are 40 °C and 6.0, respectively. The optimal glutaraldehyde concentration is ~2.0 wt%, and
the appropriate immobilization time is 4 h. Under these optimal conditions, the activity of the im‐
mobilized enzyme could be maintained at 99.1% of that of the free enzyme. Moreover, after 15
cyclic runs, the activity of the immobilized enzyme was maintained at ~91.1%. The prepared bio‐
catalyst was used to decompose corncobs, and the maximum decomposition rate achieved was
61.94%.
Received 2 December 2015
Accepted 8 December 2015
Published 5 March 2016
Keywords:
Magnetic nanoparticle
Cellulase
Enzyme immobilization
Corncob
Glutaraldehyde
© 2016, Dalian Institute of Chemical Physics, Chinese Academy of Sciences.
Published by Elsevier B.V. All rights reserved.
1. Introduction
30% hemicellulose) of corncobs into glucose, xylose, furfural,
and ethanol via many decomposition methods [10,11]. One of
The limited reserves of coal, crude oil, and natural gas have
expedited the recycling of natural biological resources [1,2].
Cellulose, as one of the main types of bioresources, is a renew‐
able raw material and potential feedstock employed in the in‐
dustry [3]. Therefore, reusing agricultural byproducts that
mainly consist of cellulosic material is particularly important
[4–7]. However, most of the cellulose resources have not been
efficiently used. For instance, corncobs are only used at ~0.5%
of the total output [8,9].
the most promising strategies is decomposing cellulose with
cellulase [12].
However, the potential of such a process is limited as hy‐
drocellulose enzymes cannot be efficiently reused. Specifically,
they lack long‐term stability under processing conditions, and
it is very difficult to recycle and reuse the catalyst from the
reaction system [13]. Immobilizing enzymes on a solid support
offers an attractive protocol to improve the reusability of con‐
ventional biocatalyst systems. Immobilized cellulase has more
advantages than free cellulase for batch treatment or continu‐
ous processes. The immobilized cellulase can be easily re‐
To efficiently use corncobs, research scientists have at‐
tempted to convert the main components (~40% cellulose and
* Corresponding author. Tel: +86‐18663770029; Fax: +86‐531‐86180589; E‐mail: zhangqk@sdnu.edu.cn
This work was supported by the National Basic Research Program of China (973 Program, 2013CB933800), the Natural Science Foundation of Shan‐
dong Province (ZR2013EMM004), the Jinan University Independent Innovation Program (201401245), and the College Students, Innovation Training
Program of Ministry of Education (201410445074).
DOI: 10.1016/S1872‐2067(15)61028‐2 | http://www.sciencedirect.com/science/journal/18722067 | Chin. J. Catal., Vol. 37, No. 3, March 2016

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Qikun Zhang et al. / Chinese Journal of Catalysis 37 (2016) 389–397
moved from the reaction mixture and displays adaptability to
varying engineering designs [14,15].
2. Experimental
Many supports can potentially be used to immobilize cellu‐
lase such as pumice [16], electro‐spun polyacrylonitrile, nano‐
fibrous membranes [17], methacrylate copolymer [18], and
graphene nanoparticles [19]. Generally, any solid support with
surface functional groups that can provide strong chemical
interactions with the enzyme can be used [20,21]. However,
employing nanoparticle supports is of particular interest. Na‐
noscale particles offer larger specific areas and afford a rea‐
sonably high enzyme loading capacity toward potentially
achieving higher enzyme activity. Furthermore, using nanopar‐
ticles may offer a better solution for diffusion problems that are
often encountered in conventional reaction systems. Currently,
nano‐sized magnetic particles are widely used for the immobi‐
lization of proteins, peptides, and enzymes [22]. Of particular
interest, using magnetic nanoparticles as supports for enzyme
immobilization offers several advantages as follows: (1) higher
specific surface area that favors higher binding efficiencies; (2)
lower mass transfer resistance and reduced fouling; (3) the
immobilized enzymes can be selectively separated using an
applied magnetic field, thereby affording reduced operation
costs; and (4) the application of a continuous biocatalysis sys‐
tem [23].
Popular immobilization strategies include chemical and
physical (ionic exchange, hydrophobic adsorption, affinity ad‐
sorption) binding to porous supports, non‐porous nanomateri‐
als, and the immobilization of enzymes in the absence of a
support. Particularly, covalent coupling methods have been
used to immobilize enzymes onto different supports [24]. Ac‐
cordingly, enzymes with amino acid residues can be
site‐directly immobilized via the formation of covalent bonds
between the amino acid residue and an active group on the
support. Comparatively, the covalent immobilization method
can eliminate or significantly reduce enzyme leakage through
increased bond strength.
2.1. Raw materials
All chemicals employed in this paper were of pure grade
and used as received without further purification. Solutions
were prepared with deionized water. Cellulase was purchased
from Novozymes (Denmark). The corncobs were collected from
the nearby countryside. Glutaraldehyde (50%) and ammonia
(NH3·H2O) were obtained from Sinopharm Chemical Reagent
Co., Ltd., China. (3‐Aminopropyl)triethoxysilane (APTS; 96%)
was purchased from Shandong Qufu Wanda Chemicals Co., Ltd.,
China.
2.2. Preparation of Fe3O4 magnetic nanoparticles
Magnetic Fe3O4 nanoparticles were prepared using a hy‐
drothermal method. In a typical process, 4.48 g (0.112 mol)
NaOH was dissolved in 50 mL distilled water at room temper‐
ature, after which 1.02 g Na2S2O3·5H2O was added. The result‐
ing solution was placed into a 250‐mL round‐bottom flask to
form a homogeneous mixture by vigorous stirring. The solution
was purged with N2 for 30 min. Subsequently, 11.12 g (0.03
mol) FeSO4·7H2O was dissolved in 100 mL distilled water and
then immediately poured into the aforementioned solution.
After stirring for 2 h, the solution was transferred to a hydro‐
thermal reactor that was heated at 160 °C for 16 h. After com‐
pletion of the reaction, the upper mixture was washed with
distilled water by magnetic decantation, and thorough dialysis
was performed until solution neutrality was achieved [29]. A
black precipitate was obtained. After separation with a magnet,
the precipitate was redispersed in distilled water. The resulting
Fe3O4 nanoparticle aqueous solution (diluted to 10 mg/mL)
was stored in a sealed container.
2.3. Preparation of APTS‐coated Fe3O4 (APTS@Fe3O4)
Accordingly, in this paper, a biocatalyst was prepared,
whereby the immobilized enzyme was prepared via the cova‐
lent immobilization strategy [25]. In a typical process, magnetic
Fe3O4 nanoparticles coated with (3‐aminopropyl)triethox‐
ysilane were activated via glutaraldehyde, and the activated
magnetic supports were effectively coupled with cellulase with
high enzymatic activity via covalent bonding [26–28]. The im‐
mobilization of cellulase onto the Fe3O4 nanoparticles was in‐
vestigated by Fourier transform infrared spectroscopy (FT‐IR).
The size and surface morphology of the particles before and
after binding cellulase were characterized by transmission
electron microscopy (TEM), and the thermal stability of the
immobilized enzyme was evaluated by thermal gravimetric
analysis (TGA). Furthermore, several interdependent parame‐
ters, i.e., temperature, pH, glutaraldehyde concentration, and
immobilization time, that affect the immobilization efficiency
and enzymatic activity of the biocatalyst were investigated.
Subsequently, the activity of the immobilized cellulase toward
the decomposition of corncob to glucose was examined. The
effect of several parameters on the efficiency of the decomposi‐
tion reaction was also evaluated.
magnetic nanoparticles
The prepared Fe3O4 magnetic nanoparticles were diluted to
a solution consisting of 182 mL ethanol, 10 mL distilled water,
and 8 mL APTS. The solution was transferred to a 500‐mL
three‐neck round‐bottom flask and stirred at 40 °C for 2 h in N2.
The resulting nanoparticles were washed thrice with distilled
water and twice with ethanol, and then dried into a powder at
room temperature under vacuum.
2.4. Immobilization of cellulase on APTS@Fe3O4 magnetic
nanoparticles
Approximately 0.05 g APTS@Fe3O4 nanoparticles were dis‐
persed in 25 mL buffer solution prepared with acetic acid and
sodium acetate. Then, 0.5 mL glutaraldehyde and 0.5 mL cellu‐
lose were added to the buffer solution in a conical flask and
shaken at 40 °C for 3 h. The immobilized cellulase nanoparti‐
cles were then washed with distilled water and separated with
a magnet. The washing process was repeated several times
until no free cellulase was detected in the rinsing solution (wa‐

Qikun Zhang et al. / Chinese Journal of Catalysis 37 (2016) 389–397
391
ter). The residual concentration of cellulase in the rinsing solu‐
tion were determined by the dinitrosalicylic acid (DNS) meth‐
od. The amount of immobilized cellulase was calculated by
thermal gravimetric analysis [30].
dium acetate buffer solution (pH = 6.0) and 50.0 mg immobi‐
lized cellulase were introduced into the flask. The flask was
shaken at 40 °C for a given time. Then, the glucose released into
solution was evaluated according to the method described in
Section 2.5 [33]. The decomposition rate was calculated as fol‐
2.5. Determination of cellulase activity
lows:
m×25
M ×n×1000
(2)
D =
×100%
To evaluate the enzymatic activity of the free and immobi‐
lized enzyme, cellulase activity units (U) are defined as the re‐
quired amount of enzyme to produce 1 µg glucose from the
hydrolysis of sodium carboxymethylcellulose within 1 min at
40 °C and pH = 4.6. In a typical procedure, 4 mL buffer solution
(pH = 4.6) was introduced into a conical flask containing 0.05 g
immobilized cellulase. Then, 1 mL sodium carboxymethyl‐
cellulose solution (1.0 g sodium carboxymethylcellulose dis‐
solved in 100 mL buffer solution) was introduced to the flask,
and the mixture was shaken at 40 °C for 30 min. Cellulase mol‐
ecules catalytically degraded sodium carboxymethylcellulose
molecules to glucose molecules. Subsequently, the magnetic
immobilized cellulase was separated and recovered with an
external magnet. The upper solution without biocatalyst was
introduced into the colorimetric tube. Then, 1.5 mL DNS rea‐
gent solution was added to the upper solution and heated in
boiling water for 5 min to give a colored product, which was
evaluated by measuring the solution absorbance at 540 nm.
(The DNS reagent solution was prepared by dissolving 182.0 g
sodium potassium tartrate tetrahydrate, 21.0 g sodium hy‐
droxide, 6.3 g 3,5‐dinitrosalicylic acid, 5.0 g phenol, and 5.0 g
sodium sulfate in 1000 mL distilled water and kept in the dark
for 7–10 d before use.) A parallel sodium carboxymethylcellu‐
lose solution sample without enzyme was prepared as a blank
control. The amount of hydrolyzed glucose was calculated from
the normalization equation of the standard curve. The enzy‐
matic activity of the free and immobilized enzymes was calcu‐
lated as follows:
where D, m, n, and M are the decomposition rate (%), mass of
glucose in solution calculated from the normalization equation
of the standard curve (mg), content of cellulose in the corncob
sample (%), and mass of the corncob sample (mg), respectively.
2.8. Characterization
The size and morphology of the nanoparticles were deter‐
mined by TEM (Hitachi H600) at an acceleration voltage of 200
kV. Mass loss measurements were conducted via TGA (Netzsch
TG‐20) in N2 atmosphere from 50 to 600 °C at a heating rate of
20 °C/min. To confirm the immobilization of cellulase on the
surface of the Fe3O4 nanoparticle support, FT‐IR (Bruker Ten‐
sor 27) analysis was conducted within a wavenumber range of
4000–500 cm−1. Powder XRD (Bruker D8 Advance) was used to
investigate the crystal structure of the obtained nanoparticles.
The magnetic properties of the nanoparticles before and after
immobilization were analyzed by vibrating sample magnetom‐
etry (VSM; JDM‐13E, Digital Measurement System, Inc.).
The enzymatic activity of the magnetic biocatalysts was de‐
termined by direct measurement of the absorbance of the glu‐
cose solution at 540 nm using a UV‐visible spectrophotometer
(TU 1900, Shimadzu Corporation, assembled in China).
3. Results and discussion
3.1. TEM analysis of the magnetic nanoparticles
W ×n×1000
(1)
X =
The size and surface morphologies of the bare and coated
Fe3O4 nanoparticles and biocatalysts were examined by TEM.
The average size of each layer was calculated (Table 1) to as‐
sess the change in particle size after each process.
Additionally, as observed from Fig. 1, nearly monodisperse
Fe3O4 nanospheres were successfully prepared by the hydro‐
thermal method. Compared with the bare particles, the surface
of the magnetic biocatalyst was rougher owing to the mem‐
branous layer covering the particle support.
T ×M
where X, W, n, T, and M are the cellulase activity units (U), mass
of glucose in solution calculated from the normalization equa‐
tion of the standard curve (g), dilution factor of the enzyme
solution, reaction time (min), and mass of the enzyme sample
(g), respectively [13].
2.6. Pretreatment of corncob
Prior to use, the corncobs obtained from the nearby coun‐
tryside were air‐dried, ground into a powder, and sieved with
200‐mesh sieve. Then, 1.5 mol/L ammonia was added to the
corncob powder at a mass ratio of 10:1. The mixture was pre‐
treated at 80 °C for 3 h, after which the corncobs were sepa‐
rated from the mixture. The pretreated power was washed
with water to pH = 7.0 and dried at room temperature [31,32].
3.2. FT‐IR analysis
Fig. 2 shows the FT‐IR spectra of cellulase, bare and coated
Fe3O4 nanoparticles, and representative biocatalyst. The band
Table 1
Diameter of the bare and APTS‐coated Fe3O4 nanoparticles and repre‐
sentative biocatalyst.
2.7. Decomposition of corncob
Sample
Bare Fe3O4
Diameter (nm)
15  3
APTS‐coated Fe3O4
Biocatalyst
50  10
110  20
For the decomposition studies, 0.20 g pretreated corncobs
were transferred to a 100‐mL conical flask. Then, 25.0 mL so‐

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Qikun Zhang et al. / Chinese Journal of Catalysis 37 (2016) 389–397
results confirmed that cellulase was successfully immobilized
onto the Fe3O4 nanoparticles.
20
15
10
5
(d)
(a)
(b)
(c)
3.3. XRD crystal analysis
The change in the crystallinity of the Fe3O4 nanoparticles
before and after coating with APTS and after loading with cel‐
lulase was investigated by XRD (Fig. 3). According to the Joint
Committee on Powder Diffraction Standards (JCPDS) database,
the XRD pattern of a standard Fe3O4 crystal with a spinel
structure has six characteristic peaks at 2θ = 30.1°, 35.5°, 43.1°,
53.4°, 57.0°, and 62.6° that correspond to the (220), (311),
(400), (422), (511), and (440) planes of Fe3O4, respectively. As
observed in Fig. 3, the bare Fe3O4 nanoparticles featured dis‐
tinct crystalline peaks at 2θ = 30.40°, 35.80°, 43.60°, 53.70°,
57.20°, and 62.70°. The XRD patterns of the starting material
(Fe3O4), APTS@Fe3O4, and biocatalysts were consistent with
the pattern exhibited by standard magnetite. Therefore, it could
be concluded that the modification treatment did not cause a
phase change in Fe3O4, and the magnetite nanoparticles modi‐
fied with APTS and cellulase also featured the spinel structure.
Furthermore, as the APTS and cellulase coatings are amor‐
phous, new peaks attributable to these groups were not de‐
tected in the XRD patterns. However, the crystallinity of the
samples decreased after the coating process. This phenomenon
was likely due to the partial destruction or hindering of the
regularity of the Fe3O4 structure after coating with APTS and
cellulase. The most probable structure of the prepared biocata‐
lysts is shown in Fig. 4.
0
12 13 14 15 16 17 18
Diameter (nm)
20
(e)
15
10
5
0
46 48 50 52 54
Diameter (nm)
(f)
15
10
5
0
100
110
120
Diameter (nm)
Fig. 1. TEM images and mean diameters of bare Fe3O4 (a, d), APTS@
Fe3O4 (b, e), and representative biocatalyst (c, f).
at 567 cm−1 was attributed to the vibration of Fe–O bonds in
the crystalline lattice of bare Fe3O4. In contrast, the biocatalyst
sample featured a weaker absorption band (567 cm−1). The
absorption peak at 3450 cm−1 was attributed to O–H in the bare
and coated Fe3O4 samples. In the biocatalyst sample, the ab‐
sorption peak at 1047 cm−1 was attributed to the stretching
vibrations of C–O of carboxyl. The band at 3400 cm−1 could be
associated with N–H stretch, which overlapped with the O–H
stretch absorption peak at 3100 cm−1. The absorption peak at
1650 cm−1 was attributed to the C=O stretch of amide. These
3.4. Magnetic properties
The magnetic properties of the bare and coated Fe3O4 and
biocatalyst nanoparticles were determined by VSM. Fig. 5
shows the corresponding magnetization curves. The curve of
bare Fe3O4 displayed no coercivity features. The mass satura‐
tion magnetization (Ms) of the bare Fe3O4 nanoparticles was
71.96 emu/g, thereby indicating that the prepared particles
exhibit superparamagnetic behavior. Furthermore, changes in
the Ms of the particles after coating with APTS and enzyme
were observed. Ms decreased from 71.96 to 54.74 emu/g (for
(2)
(3)
1.0
(4)
0.9
0.8
0.7
0.6
0.5
0.4
567
(1)
2933
1420
(1)
1650
(2)
(3)
1047
3450
4000 3500 3000 2500 2000 1500 1000
Wavenumber (cm1)
500
20
30
40
50
60
70
2/(o )
Fig. 2. FT‐IR spectra of cellulase (1), bare Fe3O4 (2), APTS@Fe3O4 (3),
Fig. 3. XRD patterns of the bare Fe3O4 (1), APTS@Fe3O4 (2), and repre‐
and representative biocatalyst (4).
sentative biocatalyst (3) nanoparticles.

Qikun Zhang et al. / Chinese Journal of Catalysis 37 (2016) 389–397
393
N
N
N
N
N
N
N
N
Fig. 4. Schematic representation of the preparation of the magnetic biocatalyst.
APTS@Fe3O4) and to 48.81 emu/g (for biocatalyst). This de‐
crease was attributed to the contribution of the non‐magnetic
APTS shell to the total mass of the particles, which apparently
weakened the magnetic properties.
cellulase activity and immobilization efficiency were investi‐
gated. Specifically, the immobilization temperature, buffer so‐
lution pH, concentration of glutaraldehyde, and immobilization
time were considered.
3.5. Enzyme activity
3.6.1. Immobilization temperature
Cellulase can be covalently immobilized onto the Fe3O4
magnetic nanoparticles by forming a Schiff base linkage be‐
tween the aldehyde group of glutaraldehyde and the terminal
amino group of modified Fe3O4 magnetic nanoparticles and
cellulase. Various factors including the coupling temperature,
coupling time, and glutaraldehyde concentration can affect the
interactions.
The immobilized enzyme activities at different immobiliza‐
tion temperatures were evaluated, and the results are shown in
Fig. 6(a). As observed, the immobilization temperature influ‐
enced the activity of the immobilized enzyme. The activity of
the immobilized cellulase increased with increasing tempera‐
tures in the range of 25–40 °C, and then decreased abruptly.
Generally, higher temperatures benefit the immobilization of
cellulase onto the nanoparticles, leading to higher enzyme
loadings. However, excessively high temperatures can acceler‐
ate the deactivation of the enzyme. Therefore, at excessively
high temperatures, the activity of the immobilized cellulase
decreased. Thus, the optimal immobilization temperature was
determined as 40 °C, corresponding to a maximum relative
enzyme activity of 84.56%.
Stability and activity are essential attributes of a highly effi‐
cient immobilized enzyme. The activities of free cellulase and
the prepared biocatalysts were determined by the DNS method.
In a typical process, a 1.0 mg/mL glucose standard solution was
prepared by dissolving 0.2 g glucose in 200 mL distilled water.
A series of glucose solution concentrations were prepared by
dilution of the glucose standard solution. The DNS reagent was
then used as mentioned above to treat the glucose solutions.
The absorbance of the treated solution (measured at 540 nm)
increased linearly with increasing glucose concentrations in the
range of 0–50 mg/L. Using this method, the concentration of
glucose produced upon decomposition of sodium carbox‐
ymethycellulose by the free cellulase and prepared biocatalysts
can be detected and calculated. And the enzymatic activity of
the free and immobilized cellulase was obtained accordingly.
The results are reported in Table 2, which shows that the ob‐
tained immobilization yield was as high as 99.1%.
3.6. Cellulase immobilization
To investigate the optimal conditions of cellulase immobili‐
zation, several interdependent parameters that influence the
3.6.2. Buffer solution pH
The variation in the biocatalyst activity in buffer solution at
different pHs was determined, and the result is shown in Fig.
6(b). The immobilized biocatalysts displayed maximum activity
at pH = 6 (~99.1%). This result agreed with the product speci‐
fication of cellulose, which was purchased from Novozymes
(Denmark). Excessively high or low buffer solution pHs accel‐
erated the deactivation of the enzyme. Therefore, the optimum
buffer solution pH was determined as 6.0.
80
(1)
60
(2)
40
20
(3)
0
-20
-40
-60
Table 2
Enzymatic activity results.
Immobilization
Sample
W/mg
n
T/min M/g
X /(U/g)
-80
yield (%)
100.00
93.03
99.10
84.56
-15000 -10000 -5000
0
5000 10000 15000
Free enzyme 2.92
5
5
5
5
30 0.048 10234.4 ± 100
30 0.050 9521.3 ± 95
30 0.048 10142.3 ± 100
30 0.055 8654.5 ± 90
Magnetic field (Oe)
Biocatalyst
Biocatalyst
Biocatalyst
2.86
2.87
2.86
Fig. 5. Magnetization curves of the bare Fe3O4 (1), APTS@Fe3O4 (2), and
representative biocatalyst (3) nanoparticles.

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Qikun Zhang et al. / Chinese Journal of Catalysis 37 (2016) 389–397
(d)
(a)
(b)
100
90
80
70
60
50
40
(c)
100
80
60
40
20
0
100
90
80
70
60
50
80
60
40
20
0
20 25 30 35 40 45 50 55
Temperature (oC)
1 2 3 4 5 6 7 8 9 1011
pH
0
1
2
3
4
0 1 2 3 4 5 6 7 8 9
Time (h)
c(glutaraldehyde) (%)
Fig. 6. Variations in the activity of immobilized cellulase as a function of enzyme immobilization temperature (a), buffer pH (b), glutaraldehyde con‐
centration (c), and immobilization time (d). Immobilization conditions: cellulase amount = 0.5 mL; temperature = 40 °C (except (a)); pH = 6.0 (except
(b)); glutaraldehyde concentration = 1.0% (except (c)); reaction time = 2 h (except (d)); the free cellulase was defined as the 100% activity.
between 240.5 and 500 °C, a rapid and continuous mass loss
3.6.3. Glutaraldehyde concentration
occurred, and by 500–600 °C, no further significant changes
were observed. The first mass loss was attributed to the evap‐
oration of water or small amounts of free APTS adsorbed by the
particles. The subsequent change was attributed to the desorp‐
tion and thermal degradation of cellulase molecules immobi‐
lized on parent Fe3O4.
To determine whether the grafting treatment can stabilize
cellulase, thermal analysis of pure cellulase was also conducted.
Within the temperature range of 0–240.5 °C, the relative mass
of pure cellulase changed from 100% to 36.24%, thus indicat‐
ing that pure cellulase has poor thermal stability and immobi‐
lization on Fe3O4 nanoparticles can stabilize the cellulase.
Additionally, the Fe3O4 content of the prepared magnetic
biocatalyst, calculated from the TGA data, was 78.0%, and the
immobilization loading of cellulase was 161 mg/g. These re‐
sults confirmed that the magnetic biocatalyst possesses high
enzyme content and is expected to display excellent magnetic
properties.
Glutaraldehyde plays a significant role in the immobilization
process. Considerable changes in the immobilized enzyme ac‐
tivity were observed at varying glutaraldehyde concentrations
(Fig. 6(c)). The activity of immobilized cellulase increased with
increasing glutaraldehyde concentrations in the range of
(0.5–2.0) wt%, and then decreased abruptly. In a typical pro‐
cedure, the amino group on the surface of APTS@Fe3O4 and
enzyme react with the aldehyde group on the glutaraldehyde.
Glutaraldehyde connects the enzyme to the aminated support
as a coupling agent. In general, the immobilization degree in‐
creases with increasing glutaraldehyde concentrations. How‐
ever, excessively high glutaraldehyde concentrations accelerate
the deactivation of the enzyme, thereby accelerating the self‐
cross‐linking of the enzyme or the aminated support. There‐
fore, at an excessively high glutaraldehyde concentration, the
activity of immobilized cellulase showed a decreasing tenden‐
cy. Thus, the optimum glutaraldehyde concentration was 2.0%.
3.6.4. Immobilization time
3.8. Decomposition of corncob
The immobilization time greatly influences the activity of
immobilized cellulase. The variation in the enzymatic activity
with immobilization time is presented in Fig. 6(d). As observed,
the activity of immobilized cellulase increased with immobili‐
zation time until equilibrium was reached within 6 h of reac‐
tion. The result indicated that with increasing coupling time
from 0 to 4 h, the enzymatic activity increased with the amount
of immobilized cellulase. The optimal immobilization time was
determined at 4 h.
Prior to use, the corncobs were pretreated using the method
described in Section 2.6 and subsequently decomposed using
the method described in Section 2.7. The results are presented
100
(1)
90
80
70
60
50
40
30
20
10
(2)
(4)
3.7. Thermal stability of the prepared biocatalysts
Fig. 7 shows the TGA curves of the bare and coated Fe3O4
nanoparticles, cellulase, and representative biocatalyst. The
bare and coated Fe3O4 nanoparticles displayed excellent ther‐
mal stability at increasing temperatures up to 600 °C, with a
mass loss of 1% and 6%, respectively. In contrast, the TGA
curve of the biocatalyst displayed two distinct mass loss stages.
The first mass loss (3.2%) was observed within 0–100 °C.
Thereafter, the biocatalyst was stable up to 240.5 °C. However,
(3)
100
200
300
400
500
600
Temperature(oC)
Fig. 7. TGA curves of the bare Fe3O4 (1), APTS@Fe3O4 nanoparticles (2),
cellulase (3), and representative biocatalyst (4).

Qikun Zhang et al. / Chinese Journal of Catalysis 37 (2016) 389–397
395
Time (h)
6 7
100
95
90
85
80
75
70
65
1
2
3
4
5
8
9
10 11 12
70
60
50
40
30
20
0
2
4
6
8
10 12 14 16 18 20 22
Cycle
1
2
3
4
5
6
7
8
9
10 11 12
Fig. 9. Recovery and recycling stability of the immobilized cellulase.
Reaction conditions: temperature = 40 °C; pH = 6.0; initial corncob
concentration = 2.0 mg/mL; reaction time = 5 h; the free cellulase was
defined as the 100% activity.
Initial corncob concentration (mg/mL)
Fig. 8. Variations in the decomposition rate of corncob as a function of
reaction time and initial corncob concentration. Reaction conditions:
temperature = 40 °C; pH = 6.0.
within the first 15 cycles from 99.1% to 91.1%. After 15 cycles,
the activity of the immobilized catalyst decreased further,
however, remained relatively high. After 20 cycles, the enzy‐
matic activity was 66.2%. Hence, the prepared biocatalyst is
more suitable for practical use than free cellulase.
in Fig. 8. The decomposition rate changed with reaction time
and initial corncob concentration. At a given initial corncob
concentration (2.0 mg/mL), the glucose content in the reaction
solution increased from 73.4 to 247.8 mg/L as the reaction
time increased from 0 h to 6 h, and the decomposition rate of
corncob decreased from 18.36% to 61.94%. When the initial
corncob concentration increased from 2.0 to 16.0 mg/mL and
the reaction time was set to 5 h, the glucose content in the reac‐
tion solution increased from 247.8 to 568.8 mg/L. However, the
total decomposition rate of corncob decreased from 61.94% to
23.72%.
Cellulase is a complex mixture that consists of a variety of
hydrolytic enzymes. Cellulase can be divided into three catego‐
ries: C1 enzyme, Cx enzyme, and β‐glucoside enzyme. C1 en‐
zymes are the catalyst that initiates the decomposition of cel‐
lulose. They can destroy the crystalline structure of cellulose.
Cx enzymes are typically involved in the subsequent decompo‐
sitions stages of cellulose, specifically, the β‐1,4 glycosidic
bonds of cellulose. β‐Glucoside enzyme can decompose cello‐
biose, cellotriose, and other low molecular dextrin to glucose.
Reaction conditions are expected to influence the decomposi‐
tion rate of corncob. Accordingly, studying the effects of several
interdependent parameters such as reaction temperature, re‐
action time, pH, the influence of pretreatment and initial corn‐
cob concentration on the total decomposition rate of corncob
deserves further investigations.
4. Conclusions
A magnetically recoverable biocatalyst was successfully
prepared through the covalent immobilization of cellulase onto
aminated Fe3O4 nanoparticles. The mass saturation magnetiza‐
tion of the bare Fe3O4 nanoparticles and representative pre‐
pared biocatalyst were 71.96 and 48.81 emu/g, respectively.
The enzymatic activity of the immobilized cellulase was
10142.3 U/g, which was 99.1% of the free enzyme activity. The
immobilized enzyme exhibited significant thermal stability (up
to 500 °C) and good durability. After 15 cycles, its activity re‐
mained at 91.1%. The prepared biocatalyst was used to de‐
compose corncob, and the maximum decomposition rate at‐
tained was 61.94%.
Acknowledgments
We like to thank Yiling Bei and Guohui Huang for their ex‐
perimental contribution.
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Qikun Zhang et al. / Chinese Journal of Catalysis 37 (2016) 389–397
Graphical Abstract
Chin. J. Catal., 2016, 37: 389–397 doi: 10.1016/S1872‐2067(15)61028‐2
Immobilized cellulase on Fe3O4 nanoparticles as a magnetically
recoverable biocatalyst for the decomposition of corncob
Corncobs
Fe3O4
Qikun Zhang *, Junqing Kang, Bing Yang, Leizhen Zhao,
Zhaosheng Hou, Bo Tang
Shandong Normal University
KH550
cellulase
Glucose
NI MU
A biocatalyst was successfully prepared through the immobilization
of cellulase onto Fe3O4 nanoparticles and employed as a magnetically
recoverable catalyst for the decomposition of corncob.
SI MU
Magnet
Magnetic biocatalyst recycling
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both the English version and extended Chinese abstract of the paper. The
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