Process Biochemistry 51 (2016) 229–239

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Immobilization of laccase via adsorption onto bimodal mesoporous

Zr-MOF
Shilong Pang a , Yanwen Wu b , Xiaoqiong Zhang c , Bingning Li b , Jie Ouyang a,∗ ,
Mingyu Ding c,∗∗
a
Department of Food Science and Engineering, College of Biological Sciences and Technology, Beijing Key Laboratory of Forest Food Process and Safety,
Beijing Forestry University, Beijing 100083, China
b
Beijing Center for Physical and Chemical Analysis, Beijing 100089, China
c
Department of Chemistry, Tsinghua University, Beijing 100084, China

a r t i c l e i n f o a b s t r a c t

Article history: A new bimodal micro-mesoporous Zr-metal organic framework (Zr-MOF, MMU) with a particle size
Received 30 August 2015 of approximately 200 nm was synthesized. The Brunauer–Emmett–Teller (BET) surface area and pore
Received in revised form 30 October 2015 diameter of the nanoscale MMU were 453.8 m2 /g and 3.5–7 nm, respectively. Laccase was immobi-
Accepted 30 November 2015
lized onto MMU via physical adsorption. This immobilized system exhibited a large adsorption capacity
Available online 2 December 2015
(221.83 mg/g), broad pH and temperature profiles, and better stability and repeatability than free laccase.
Inactivation of the interaction between the overcrowded laccase at high concentration and pore plugging
Keywords:
were not observed due to the secondary pore structure (3.5 nm). The immobilized laccase showed better
Metal organic framework
Immobilization
stability under extreme conditions than free laccase. The activity of immobilized laccase after utilization
Bimodal mesoporous 10 times remained at approximately 50%, and it remained at 55.4% of its initial activity at the end of 3
Laccase weeks of storage in an aqueous phase. The good stability and retention of enzymatic activity indicated
that the bimodal mesoporous Zr-MOF is a good support for the immobilization of laccase.
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction basic studies focusing on the applications of laccase have been

Enzymes are widely used in many types of industrial applica-
tions as highly efficient biological catalysts. However, the catalytic
ability of enzymes is often hindered in harsh environments, such
as high-acid and high-alkaline solutions, due to transformations of
the enzyme structure. Laccase (EC1.10.3.2, p-benzenediol: oxygen
oxidoreductase, size of 6.5 nm × 5.5 nm × 4.5 nm) [1], an oxidore-
ductase containing 500 amino acids (64–140 kDa, depending on
the type), is widely distributed in plants, fungi and some bacterial
strains. Laccase has a broad range of applications in the printing
and dyeing industry, paper industry, and conversion of aromatic
compounds [2] and removal of phenols, which leads to cancer and
teratogenicity due to waste water [3]. In recent years, exhaustive
Abbreviations: MOF, metal organic framework; MMU, bimodal mesoporous Zr-
MOF; BET, Brunauer–Emmett–Teller; TMB, 1,3,5-trimethylbenzene; DMF, dimethyl
formamide; BSA, bovine serum albumin.
∗ Corresponding author. Fax: +86 10 62338221.
∗∗ Corresponding author. Fax: +86 10 627970 87.
E-mail addresses: ouyangjie@bjfu.edu.cn (J. Ouyang),
dingmy@mail.tsinghua.edu.cn (M. Ding).
conducted. However, the use of laccase in its native form is often
hindered by several limitations, such as its sensitivity to the envi-
ronment, low operational stability and difficulties in recovery and
reuse.
Immobilization technology has been proven to be an effective
method for implementing efficient and continuous applications of
an enzyme [4]. Since the first immobilization experiment in 1916
[5], numerous materials have been applied to immobilize enzymes,
such as agarose, silica, TiO2 , organic gel, chitosan, kaolinite, multi-
walled carbon nanotubes and graphene oxide [6]. Metal–organic
frameworks (MOFs) are compounds consisting of metal ions or
clusters coordinated to organic molecules (ligands) to form one-
, two-, or three-dimensional structures. MOFs with large pores
and high specific surface areas have recently been synthesized,
particularly for applications in catalysis [7], drug delivery [8],
gas storage [9] and separation [10]. Different types of MOFs can
easily be synthesized by changing the central metal ions and lig-
ands [11], and these MOFs possess good Brunauer–Emmett–Teller
(BET) surface areas and excellent dispersity compared with con-
ventional materials such as mesoporous silica materials, carbon
nanotubes and graphene. One of the attractive aspects of MOF
synthesis is that the organic bridging ligand can be synthetically

http://dx.doi.org/10.1016/j.procbio.2015.11.033
1359-5113/© 2015 Elsevier Ltd. All rights reserved.
230 S. Pang et al. / Process Biochemistry 51 (2016) 229–239
Fig. 1. Transmission electron microscopy (TEM) image of bimodal mesoporous Zr-MOF after activation.
modified to introduce a desired functionality to the framework,
which can still possess good stability compared with conventional
materials such as chitosan and sodium alginate. For the above
reasons, MOFs can be considered a suitable carrier for biocata-
lyst immobilization. Most types of MOFs are microporous (pore
size <2 nm). The small pore size prevents large molecules (such
as enzymes) from accessing the center sites inside MOFs. One
method for increasing the pore size is to expand the ligands:
a longer ligand can lead to either interpenetrating structures or
fragile frameworks that will collapse upon removal of the guests.
Furthermore, it restricts the size of the pore aperture [12,13]. Thus,
the surfactant-templating method used to prepare various meso-
porous silicas can be applied to synthesize mesoporous MOFs by
adding a chelating agent and surfactants to the reaction. The chelat-
ing agent can sufficiently establish the interaction between the
framework building blocks and surfactants. The crystal growth is
thus directed by micelles at the nanoscale. Subsequently, crys-
talline materials with carboxyl groups and two types of pores can be
achieved after removal of the guests [14,15].
There are several methods for enzyme immobilization, includ-
ing enzyme entrapment, absorption, encapsulation in an organic
or inorganic polymer, and covalent binding to the carrier or
self-immobilization [16]. Each method has its own particular
strengths and weaknesses, e.g., entrapment, encapsulation and
covalent binding have a relatively stronger interaction between
the enzyme and surface of the support, leading to a simultane-
ous decrease in activity and enzyme loading. The first studies on
the immobilization of laccase date back to 2011 [17], but there are
few reports on the immobilization of laccase using mesoporous
MOFs other than the reports on microperoxidase-11 immo-
bilized in Tb-mesoMOF [18] and zirconium- metalloporphyrin
as biomimetic catalysts [19].
In view of these problems, the present study primarily aimed to
synthesize mesoporous Zr-MOFs (MMU), which are similar to UIO-
66 (UiO is the University of Oslo), using the surfactant-templating
method [20]. Zirconium was selected as the central metal ion rather
than copper because a series of HKUST (Hong Kong University of
Science and Technology) MOFs were easy to decompose in the
aqueous phase and because the release of copper ions could deac-
tivate the adsorbed laccase. A previous study showed that UIO-66
series materials possess excellent thermal stability, chemical stabil-
ity and mechanical stability due to the use of competitive reagents
and high-symmetry subgroups [12]. Combined with the advantages
of UIO-66 and its own features, MMU was suitable for use in enzyme
immobilization. In this study, MMU was used as the support for
immobilizing laccase, and the quantity of adsorbed laccase, prop-
erties of immobilized laccase, reusability and storage stability were
investigated.
2. Materials and methods
2.1. Materials
Laccase (purified from white rot fungi) and ABTS (2,2 -azino-bis-
(3-ethylbenzothiazoline-6-sulfonic acid)) were purchased from
Sigma–Aldrich (St. Louis, MO, USA) and stored at 0–4 ◦ C before
use. Zirconium chloride (ZrCl4 ) was purchased from Strem Chem-
icals (USA). Terephthalic acid (BDC) was purchased from Tokyo
Chemical Industry Co., Ltd. (Japan). Bovine serum albumin (BSA),
Coomassie brilliant blue (G250), cetyltrimethylammonium bro-
mide (CTAB), dimethyl formamide (DMF), 1,3,5-trimethylbenzene
(TMB) and other chemicals were purchased from Beijing Chemicals
Corporation (Beijing, China).
2.2. Preparation of the support
MMU was synthesized in our laboratory using the hydrothermal
method [20]. In detail, 0.7 g of ZrCl4 , 0.5 g of BDC, 0.324 g of CTAB,
30 mL of DMF, 4 mL of trifluoroacetic acid and 0.124 g of hydrochlo-
ric acid were mixed in a teflon tube. The mixture was sonicated
for 10 min to form a homogeneous solution and then heated at
120 ◦ C for 24 h. Subsequently, the mixture was centrifuged, and
the precipitated as-synthesized MMU was washed 3 times with
DMF to remove unreacted chemicals. To remove the template, the
sediment was washed 3 times with 1 mol/L ammonium nitrate
(dissolved in ethanol/H2 O (1:2, v/v)) at 60 ◦ C for 24 h, followed by
washing with ethanol/H2 O (1:2, v/v). Finally, the mesostructured
MMU was desiccated at 60 ◦ C and activated at 190 ◦ C.
2.3. Characteristics of MMU
Transmission electron microscopy (TEM) images of MMU were
obtained using an H-7650B transmission electron microscope
(Hitachi, Japan) operating at 80 kV. N2 adsorption–desorption and
pore size isotherms were obtained using a Micromeritics TriStar
3020 II system (USA). The desorption branches of the isotherms
were used to derive the pore size distribution based on the BJH
model, and BJH analyses were performed to evaluate the total
pore volume. Thermogravimetric analysis (TGA, Q50, USA) was per-
formed under a nitrogen atmosphere at a rate of 10 ◦ C/min up to
 S. Pang et al. / Process Biochemistry 51 (2016) 229–239
Fig. 2. Nitrogen adsorption–desorption isotherms and distribution of pore diameters of bimodal mesoporous Zr-MOF after activation.
1000 ◦ C. Fourier transform infrared (FTIR) spectra were recorded
using a Perkin–Elmer spectrometer in the frequency S3 range
of 4000–500 cm−1 with a resolution of 4 cm−1 . X-ray diffraction
(XRD) patterns were collected using a D8 Advance X-ray diffrac-
tometer (Bruker, Germany) with CuK␣ radiation ( = 1.5406 Å).
Ultraviolet–visible (UV–vis) absorption spectra of proteins were
recorded using a U-3010 UV–vis spectrophotometer (Hitachi,
Japan).
2.4. Adsorption of laccase on MMU
2.4.1. Assay of laccase activity
The activity of free laccase was determined spectrophotometri-
cally at 420 nm [21]. Briefly, 0.5 ␮mol/mL ABTS (2.9 mL, pH 3.0) and
10 mg/mL laccase (0.1 mL, pH 3.0) were mixed in a cuvette at 30 ◦ C
for 20 min. A blank solution was used as a reference. The activity
of immobilized laccase was determined by adding the immobi-
lized laccase (approximately 10 mg) into 0.5 ␮mol/mL ABTS (pH
4.0, 20 mL) at 40 ◦ C. This mixture was centrifuged every 1.5 min,
and the absorbance of 3 mL of the supernatant was measured at
420 nm. One unit (IU) of laccase activity was defined as the par-
ticular specific variation (0.001) of the absorbance per minute. The
specific activity of the immobilized enzyme was calculated using
the following formula:
  A
SA IU/g =
(M × m × t)
where SA is the specific activity (IU/g), A is the absorbance vari-
ation during a certain period of reaction time, M is the adsorption
ability of the support (mg/g), m is the amount of support added to
the solution (g), and t is the reaction time (min). The support with-
out immobilized laccase was also tested. The results revealed that
MMU possessed no catalytic activity.
2.4.2. Optimization of the laccase adsorption concentration
To evaluate the loading capacity of the support, approxi-
mately 10 mg of activated MMU was immersed in 5 mL of sodium
acetate buffer (0.1 mol/L, pH 3.0) with different initial laccase
concentrations (0.05–50 mg/mL, pH 3.0). The adsorption pro-
cess was performed in a shaker at room temperature for 1 h.
231
The precipitate was washed 3 times with sodium acetate buffer
(0.1 mol/L, pH 3.0) and dried at −20 ◦ C. The activities of the immo-
bilized laccases with different adsorption concentrations were
detected. The activity recovery was also determined.
The activity recovery of the immobilized laccase was calculated
using the following formula:
Ri
R (%) =
Rf
where R is the activity recovery of the immobilized laccase (%), Ri
is the activity of immobilized laccase (IU/g), and Rf is the activity of
free laccase (IU/g) under similar experimental conditions.
2.4.3. Optimization of the laccase adsorption time
To evaluate the immobilization time of the support, approxi-
mately 10 mg of activated MMU was immersed in 5 mL of laccase
solution (5 mg/mL dissolved in 0.1 mol/L sodium acetate buffer, pH
3.0). The adsorption process was performed in a shaker at room
temperature for different times (0.5–3 h). The precipitate was sep-
arated and washed 3 times with sodium acetate buffer (0.1 mol/L,
pH 3.0) and then lyophilized. The activities of the immobilized
laccases with different adsorption times were detected, and the
activity recovery was calculated.
2.4.4. Adsorption amount of the laccase
When the adoption process finished, the adsorption amount
was determined at 595 nm with a mixture containing 0.1 mL of
the supernatant, 0.9 mL of distilled water and 5 mL of G250, using
BSA as a reference (Bradford method). The standard curve was
defined (y = 4.7017x − 0.0333, R2 = 0.9946) to calculate the laccase
concentration in the supernatant. The adsorbed amount of MMU
was calculated using the following formula:
  (Y1 − Y2 )
M mg/g = 50 ×
m
where M is the adsorption amount of the support (mg/g), Y1 is the
laccase concentration in the supernatant at a certain time, Y2 is the
laccase concentration in the supernatant at another time later than
Y1 , and m is the quantity of support in the solution (g).
232 S. Pang et al. / Process Biochemistry 51 (2016) 229–239
2.5. Properties of immobilized laccase
2.5.1. Effect of pH and temperature on the activities of free and
immobilized laccases
A series of substrate solutions (0.5 ␮mol/mL ABTS) were pre-
pared with 0.1 mol/L sodium acetate at different pH values
(2.0–7.0). The catalytic activity was measured as described in Sec-
tion 2.4.1.
The effect of pH on immobilized laccase was determined at room
temperature by adding the immobilized laccase (10 mg) to 20 mL
of the substrate at different pH values (2.0–7.0). The effect of pH on
free laccase was determined by adding 0.1 mL of laccase solution
(10 mg/mL) to 2.9 mL of substrate at different pH values (2.0–7.0).
The effect of temperature on the activities of the immobilized
and free laccases was determined in the temperature range from
20 to 70 ◦ C at pH 4.0 and 3.0 using preheated 0.5 ␮mol/mL ABTS
(dissolved in 0.1 mol/L sodium acetate buffer, pH 3.0) as the sub-
strate.
Relative activity is defined as the specific activity of laccase at
one point divided by the highest specific activity of laccase in the
same group of experiments.
2.5.2. Effect of pH and temperature on the stabilities of free and
immobilized laccases
The effect of pH on the stability of laccase was determined by
incubating immobilized and free laccases in solutions with differ-
ent pH values ranging from 2.0 to 7.0 for 1 h at room temperature,
and the catalytic activity was measured under the optimal con-
ditions (free laccase: pH 3.0, 30 ◦ C; immobilized laccase: pH 4.0,
40 ◦ C), as described in Section 2.4.1.
The thermal stability was determined by incubating the immo-
bilized and free laccases at different temperatures (20–70 ◦ C) for
1 h. After cooling to room temperature, the enzyme activity was
measured under the optimal conditions (free laccase: pH 3.0, 30 ◦ C;
immobilized laccase: pH 4.0, 40 ◦ C), as described in Section 2.4.1.
2.5.3. Reusability and storage stability of immobilized laccase
The reusability of the immobilized laccase was assessed by per-
forming several consecutive operating cycles using 0.5 ␮mol/mL
ABTS as the substrate. At the end of each cycle, the immobilized
laccase was removed from the reaction medium and washed three
times with sodium acetate buffer (pH 4.0); the cycle was repeated
with a fresh preheated aliquot of substrate mixed with the immo-
bilized laccase.
To measure the storage stability, the immobilized laccase was
stored at 4 ◦ C, and the remaining activity was assayed after storing
for 3 weeks. The enzyme activity was measured under the optimal
conditions (pH 4.0, 40 ◦ C), as described in Section 2.4.1.
2.6. Determination of kinetic parameters
The Michaelis–Menten kinetic parameters (Km ) and maximum
rate (Vmax ) of the free and immobilized laccases were determined
by measuring the laccase activity with ABTS as substrate and initial
concentrations of 0.05–0.50 ␮mol/mL under the optimal conditions
(pH 4.0, 40 ◦ C), as described in Section 2.4.1. The data was calculated
according to Lineweaver–Burk plots.
2.7. Statistical analysis
The experimental results were expressed as the mean value ± SD
of three parallel measurements. All statistical analyses were con-
ducted using Microsoft Excel 2010.
3. Results and discussion
3.1. Characteristics of bimodal mesoporous Zr-MOF
High-quality TEM images of MOFs are difficult to obtain because
MOFs are typically sensitive to the electron beam. As shown in
Fig. 1(a), the particle size of MMU is approximately 200 nm, which
is similar to that in a previous report [22]. The well-defined porous
structure can be observed from the magnified view in Fig. 1(b),
which is consistent with other mesoporous MOFs reported by other
researchers [14,15]. This result preliminarily indicated that MMU
was a mesoporous material. The hexagonal structure was not dis-
tinct because of the small pore diameter. However, MMU retained
the structure of UIO-66 [23] and a pore structure similar to a silica
sphere because of the synthesis method of surfactant templating
[24].
The BET surface area of MMU is relatively low (453.8 m2 /g)
compared with other types of MOFs. However, it is similar to
that of a silica sphere due to its synthesis method. The pore size
distribution from the BJH analysis displayed two main peaks of
3.5 and 7 nm, respectively, and the total pore volume of MMU
was 0.25 cm3 /g (Fig. 2). The N2 adsorption–desorption isotherms
showed two obvious hysteresis loops, demonstrating the existence
of two pore systems. Similar hysteresis loops were also reported in
previous studies [25]. The BET and BJH analytical models showed
that there were bimodal systems in MMU, one of which (smaller
one) was the pore of the framework building blocks and the other
was formed by the framework building blocks, template agent, and
chelating agent. Compared with conventional UIO-66, the “micro-
porous” structure expanded to 3.5 nm because of the addition of
TMB. Recent studies regarding mesoporous MOFs are presented
in Table 1 [26–30]. Among these studies, Cu3 (btc)2 (H2 O)3 was the
only MOF with a pore size sufficient to immobilize enzymes [29].
However, Cu3 (btc)2 (H2 O)3 could not be applied due to its poor
hydrological stability. Although the BET surface area of MMU was
lower than that of all other materials (Table 1), MMU with an ade-
quate pore diameter, stability in solution and carboxyl group to
help immobilize laccase was a suitable support for immobilization
compared with other types of MOFs.
Fig. 3 presents the FTIR spectra of activated MMU with lac-
case, activated MMU and UIO-66. The bands at 1500–1600 cm−1 ,
which correspond to the C O of carboxylates, were coordinated
with metal centers by O during the deprotonation process. A
weak peak in the region of 1420–1480 cm−1 corresponded to the
C C in the aromatic compound of the organic linker. The peak at
1667 cm−1 corresponded to DMF and disappeared after MMU was
activated at 190 ◦ C. The strong peak at 1395 cm−1 was attributed to
the C OH group of carboxylic acid. The bands observed in the region
of 750–655 cm−1 were assigned to the Zr O bond in the frame-
work building blocks. The peaks at 1690 cm−1 (amide I band) and
1290 cm−1 (amide III band) in the spectrum of activated MMU with
laccase confirmed that laccase was immobilized in MMU because
the amino group was not introduced during the synthesis process
and MMU with immobilized laccase was activated. The peak broad-
ening and shifting of the spectrum of MMU with laccase indicated
that the internal environment was changed by electrostatic inter-
actions between laccase and the support.
XRD patterns of UIO-66, activated MMU and MMU with laccase
showed that activated MMU and MMU with laccase still presented
the two characteristic peaks (2 = 7.3◦ and 8.5◦ ) of UIO-66 [31,32],
which indicated that MMU retained the UIO-66 structure (Fig. 4).
However, a significant reduction of the peaks in MMU with laccase
indicated that the crystal phase changed because of the immobi-
lization process, which also confirmed that laccase was successfully
immobilized in MMU.
 S. Pang et al. / Process Biochemistry 51 (2016) 229–239 233

Table 1
Several recent results for mesoporous MOFs.

Compound Name Pore diameter (nm) BET (m2 /g) Ref.

Zn4 O(TPDC)3 Isoreticular metal–organic framework-16 2.9 – [26]

[Cd3 (bpdc)3 (dmf)]·5dmf·18H2 O Jilin University China-48 2.5 × 2.8 – [27]
Zn20 (cbIM)39 (OH) Zeolitic imidazolate frameworks-100 3.56 595 [28]
Tb16 (TATB)16 (DMA)24 Mesoporous MOF 1 3.9/4.7 1783 [29]
Cu3 (btc)2 (H2 O)3 Micro- and mesoporous MOFs 3.8–31.0 533–1225 [15]
Zr6 (O)4 (OH)10 (H2O)6 (TATB)2 Porous coordination network-777 3.8 2008 [12]
M2 (2,5-dioxidoterephthalate) M is Zn2+ , Mg2+ Isoreticular metal–organic framework-74-I to XI 1.4–9.8 1350–2510 [30]

Fig. 3. FTIR spectra of UIO-66 (bottom), bimodal mesoporous Zr-MOF after activation (middle) and bimodal mesoporous Zr-MOF with laccase (top). FTIR spectra were
recorded within the range 4000–500 cm−1 with a resolution of 4 cm−1 .

Fig. 4. XRD patterns of UIO-66 (top), bimodal mesoporous Zr-MOF after activation
(middle) and bimodal mesoporous Zr-MOF with laccase (bottom). Fig. 5. Thermogravimetric analysis (TGA) of bimodal mesoporous Zr-MOF (top) and
bimodal mesoporous Zr-MOF with laccase (bottom). Thermogravimetric analysis
was performed under a nitrogen atmosphere at a rate of 10 ◦ C/min up to 1000 ◦ C.

The thermogravimetric analysis curves are shown in Fig. 5. The
mass loss in the first region of 50–100 ◦ C could be attributed to the
desorption of adsorbed gas from the pore spaces. In the 150–250 ◦ C
region, mass loss occurred due to the removal of guest species
(DMF) and carbonization of the immobilized laccase. The final mass
loss in the region of 500–550 ◦ C was attributed to the disintegration
of MOFs. The decomposition temperatures of MMU and MMU with
laccase confirmed that MMU possesses excellent thermostability
and that the immobilization process had no influence on its ther-
mostability [20,25]. The final residue amounts of MMU and MMU
with laccase were 48.9% and 44.2%, respectively.
3.2. Optimization of the adsorption conditions for immobilized
laccase
3.2.1. Optimization of the immobilization laccase concentration
The maximal activity of immobilized laccase was
18316 ± 53 IU/g when the initial laccase concentration reached
5 mg/mL. Meanwhile, the immobilized laccase exhibited maxi-
mal activity recovery (95.90 ± 0.28%) and the amount of laccase
adsorbed on MMU also reached the maximum (Fig. 6). The activ-
ity and adsorption amount of immobilized laccase increased
with increasing initial adsorption concentration when the
234 S. Pang et al. / Process Biochemistry 51 (2016) 229–239
Fig. 6. Effect of the adsorption concentration on the immobilization of laccase on bimodal mesoporous Zr-MOF. The immobilization was performed in a shaker at room
temperature for 1 h at different initial concentrations. The immobilized laccase activity was assayed using 0.5 ␮mol/mL ABTS (20 mL) as a substrate at room temperature and
pH 3.0.
concentration was less than 5 mg/mL. The increasing amount of
adsorbed laccase could increase the probability of contacting the
substrate, and thus, the activity was improved. When the laccase
concentration reached 5–20 mg/mL, the activity recovery was
essentially constant (greater than 90%). In this case, the adsorption
amount was almost unchanged, and the internal environment for
adsorption and enzymatic reaction was not very crowded. A sig-
nificant decrease in the activity recovery (remaining 75% activity
of free laccase) was observed when the laccase concentration was
as high as 50 mg/mL, whereas the adsorbed amount of laccase
remained the same. This result could be explained by “competitive
adsorption” due to the high concentration of free laccase, which
was unable to completely enter the pores of the support. At the
same time, laccase in the pores had to share a limited number of
carboxyl groups, representing an important way to be adsorbed
onto the support. The adsorption onto MMU was achieved by the
physical adsorption method, primarily relying on the hydrophilic,
electrostatic interactions and intermolecular forces between the
enzyme and MMU. Therefore, the high concentration was not
helpful in improving the activity due to the crowded internal
environment.
3.2.2. Optimization of the immobilization time
MMU was immersed in a 5 mg/mL laccase (19100 ± 210 IU/g)
solution for different times (0.5–3 h). As shown in Fig. 7, an immo-
bilization time of 1 h was considered to be ideal for reaching
the maximum immobilized activity (17928 ± 95 IU/g) and activity
recovery (93.86 ± 0.49%). An increase in the immobilization time
(longer than 1 h) led to a decrease in the activity recovery and the
adsorption amount. This result could be explained by the physical
adsorption based on Fig. 7. The laccase adsorbed on the surface of
the support was easily detached when it was immersed in solution
for a long period of time. Consequently, the substrate was relatively
difficult to contact the laccase inside the pores. Additionally, the
desorbed laccase might affect the contact between laccase and the
carboxyl group of the support, leading to a decrease in activity and
adsorption quantity, consistent with the adsorption amount results
over 1–3 h in Fig. 7. The steady trend of the adsorbed amount after
2 h indicated that most of the laccase adsorbed on the surface of
the support was desorbed, which also confirmed that most of the
laccase was adsorbed inside the pores. This result also confirmed
that immersing MMU in a high concentration of enzymatic solution
for a long period of time did not lead to severe inactivation of the
laccase (87.06 ± 1.55% activity recovery remaining after adsorption
for 3 h), although the pore diameter was not 2–4 times as large as
that of enzyme molecules [33]. This was due to the secondary pore
size that allowed the substrate to enter; the relatively low BET sur-
face area, which made multi-point attachment between the laccase
and support more difficult; and the mass transfer resistance, which
made the laccase molecule difficult to desorb. Therefore, 1 h was
chosen as the optimal immobilization time.
3.2.3. Adsorption amount of laccase
The highest amount of laccase adsorbed on MMU was
221.83 ± 8.74 mg/g after incubation for 1 h in a 5 mg/mL laccase
solution, as shown in Figs. 6 and 7. The adsorption amount was
higher than that of most of the supports applied in the immobiliza-
tion of laccase [25]. There was a slight decrease in the adsorption
amount (approximately 60 mg/g) over 1–3 h without a serious loss
of activity (90% activity recovery remaining), as shown in in Fig. 7.
After incubating for 4 days, the immobilized laccase was com-
pletely separated from MMU, indicating that the desorption likely
led to the activity decrease in during the long immersion in the
high concentration solution rather than the unexpected contrac-
tion between the crowed laccase molecules. To our knowledge, the
excessive adsorbed concentration has three negative effects on the
immobilized laccase activity [32]: (1) inducing the undesired lateral
interaction between the over-crowded laccase molecules, (2) caus-
ing multi-point attachment between the laccase and support, and
(3) lowering the ABTS accessibility to immobilized laccase. How-
ever, in this work, the pore diameter of the support was slightly
larger than that of laccase molecules. This typical diameter could
prevent excessive laccase molecules from entering the channel to
form an undesired interaction. At the same time, the substrate and
degradation products can pass through the secondary pore size
(3.5 nm), even though the 7-nm mesoporous material was blocked.
Consequently, desorption was the main reason for the decrease
in activity, followed by the undesired interaction. In general, the
adsorption time varied (2–24 h), according to the different supports
[32,34,35], and the smaller pore diameter increased the adsorp-
tion quantity but lowered the activity recovery [36,37]. Therefore,
a high laccase concentration was necessary to accelerate the
 S. Pang et al. / Process Biochemistry 51 (2016) 229–239
Fig. 7. Effect of the adsorption time on the immobilization of laccase on bimodal mesoporous Zr-MOF. The immobilization was performed in a shaker at room temperature
and pH 3.0 for different lengths of time. The immobilized laccase activity was assayed using 0.5 ␮mol/mL ABTS (20 mL) as a substrate at room temperature and pH 3.0.
adsorption equilibrium and increase the possibility of laccase
molecules entering the pores. Moreover, there was no significant
decrease in the activity recovery when MMU was incubated in a
relatively high concentration of laccase solution (Fig. 6). Therefore,
5 mg/mL and 1 h were chosen as the optimal adsorption concentra-
tion and adsorption time, respectively.
3.3. Optimal conditions for laccase catalytic reaction
3.3.1. Optimal pH for laccase activity
The optimal pH for immobilized laccase activity depends on
the immobilization method and support used. As shown in Fig. 8,
the activity of free laccase reached the maximum at pH 3.0
(14540 ± 594 IU/g) and then quickly decreased with increasing pH
and was almost fully deactivated at the neutral condition. The activ-
ity variation trend of immobilized laccase vs. pH was similar to
that of free laccase. The relative activity of immobilized laccase
increased to approximately 90% when the pH changed from 2.0 to
3.0, and it reached the maximum level (12861 ± 768 IU/g) at pH 4.0.
Then, it decreased rapidly with increasing pH. Compared with free
laccase, the optimal pH for immobilized laccase shifted from pH 3.0
to 4.0 and showed a significantly higher activity than that of the free
enzyme between pH 3.0 and 7.0, indicating that the immobilized
laccase was more stable than free laccase.
Regarding free laccase, the ionic groups of laccase produced a
strong electrostatic repulsion when the pH value changed, leading
to stretching of the protein molecule, destruction and degeneration
of the enzyme active center, and failure to decompose the substrate
[38]. The decrease in the immobilized laccase activity with increas-
ing pH is due to the weakness of the electrostatic force between the
laccase and support. Compared with free laccase, the restriction of
laccase in the pore site of MMU kept the internal microenvironment
stable. Even at the extreme pH value of 7.0, the immobilized laccase
still retained 15% of its relative activity. The change in the opti-
mal pH of immobilized laccase was because MMU was negatively
charged; when laccase was adsorbed on the negatively charged
support, the optimum pH moved to the alkaline range [39].
3.3.2. Optimal temperature for laccase activity
According to the above result, the optimal pH values of 3.0 and
4.0 were used for the reaction of free laccase and immobilized
235
laccase, respectively. Fig. 9 shows that the activity of immobi-
lized laccase tended to increase from 20 ◦ C until it reached the
maximum activity (15185 ± 502 IU/g) at 40 ◦ C, and then it quickly
decreased. By contrast, free laccase reached the highest activity
(18250 ± 424 IU/g) at 30 ◦ C and declined dramatically above 30 ◦ C.
The internal microenvironment and electrostatic force between the
immobilized laccase and support changed the structure of laccase,
which led to the change in the optimal temperature. It was clear
that the activities of immobilized laccase and free enzyme were
very close at low temperatures from 20 to 40 ◦ C and that the activity
of immobilized laccase was higher than that of free laccase, indi-
cating that immobilized laccase was more stable than free laccase.
This result may be due to the block effect of laccase immobiliza-
tion in the pore size of MMU [40]. The immobilized laccase retained
approximately 70% of its maximum activity at 70 ◦ C, indicating that
it possessed good thermal stability.
3.4. Stability of immobilized laccase
3.4.1. Effect of pH on immobilized laccase stability
In general, a high pH value will affect the stability of an immobi-
lized enzyme, including the process of adsorption and the enzyme
catalytic reaction. Immobilized laccase had a broader range of
applications than free laccase, but a significant decrease in the
activity of immobilized laccase at pH 7.0 was observed (Fig. 10). This
result could also be explained by the desorption of laccase. The elec-
trostatic adsorption of the physical adsorption was achieved below
the isoelectric point (under the acidic condition). Under this con-
dition, laccase was positively charged. Then, the positively charged
laccase could be used to attract the negatively charged MMU
because of the carboxyl group on the surface. When the immo-
bilized laccase was in the alkaline condition, the electrostatic force
was not significant, leading to desorption. The maximum activity
of immobilized laccase (17864 ± 118 IU/g) was slightly higher than
that of free laccase (15100 ± 141 IU/g) after immersion for 1 h due to
the electrostatic force of the microstructures in negatively charged
MMU changing the active structure of laccase [24].
3.4.2. Effect of temperature on immobilized laccase stability
The activities of free and immobilized laccase significantly
changed in the temperature range from 20 to 70 ◦ C after 1 h of
236 S. Pang et al. / Process Biochemistry 51 (2016) 229–239

Fig. 8. Effect of pH on the activities of free laccase and immobilized laccase on bimodal mesoporous Zr-MOF. The free laccase (0.1 mL, 10 mg/mL) and immobilized laccase
(approximately 10 mg) activities were assayed using 0.5 ␮mol/mL ABTS (2.9 mL) and 0.5 ␮mol/mL ABTS (20 mL) as substrates, respectively, at room temperature to determine
the optimal pH value for the reaction.

Fig. 9. Effect of the reaction temperature on the activities of free laccase and immobilized laccase on bimodal mesoporous Zr-MOF. The free laccase (0.1 mL, 10 mg/mL)
and immobilized laccase (approximately 10 mg) activities were assayed using 0.5 ␮mol/mL ABTS (2.9 mL, pH 3.0) and 0.5 ␮mol/mL ABTS (20 mL, pH 4.0) as substrates to
determine the optimal reaction temperature.

incubation (Fig. 11). Clearly, the activities of the two types of lac-
case were strongly dependent on temperature, and free laccase
exhibited the highest activity (5400 IU/g) at 30 ◦ C. Compared with
free laccase without 1 h of incubation, the decrease in the maximal
activity was approximately 67% after the free laccase was incubated
for 1 h. Some researchers have reported that laccase extracted from
Trametes versicolor retained 26.5% of its activity after 4 h of incu-
bation at 45 ◦ C [41], 3% after 2 h of incubation at 65 ◦ C [42], and
0% after 1.5 h of incubation at 70 ◦ C [43]. Laccase extracted from
Rhus vernicifera retained 7% of its activity after 2 h of incubation
at 65 ◦ C [44]. Laccase extracted from white rot fungi retained 0%
of its activity after 4 h of incubation at 70 ◦ C [45]. These data indi-
cated that laccase was extremely sensitive to the environment and
easily deactivated after treatment at a relatively high temperature.
The activity of the immobilized laccase (12174 ± 155 IU/g) was 2.25
times greater than that of free laccase after 1 h of incubation, similar
to free laccase without 1 h of incubation. This difference in activity
was also due to a reduction in the molecular mobility and change
in the laccase conformation after adsorption onto MMU [46]. The
activity of immobilized laccase above 50 ◦ C decreased because a
large proportion of laccase on the MMU surface was inactivated due
to the high temperature and immersion. Due to the high adsorption
quantity and the mesoporous material, immobilized laccase exhib-
ited favorable resistance to thermal inactivation. If the temperature
could be controlled at approximately 40 ◦ C, the immobilized laccase
would exhibit good usability.
3.5. Reusability and storage stability of immobilized laccase
The reusability of immobilized laccase was studied because of its
importance in industrial applications to reduce costs. It was deter-
mined through 10 cycles at 40 ◦ C. The results indicated that the
activity of immobilized laccase decreased relatively quickly dur-
ing the first 5 cycles, retaining 65.5% of its initial activity, and
was comparatively stable after 5 cycles (Fig. 12). This could be
explained by the denaturation of laccase on the surface of MMU
 S. Pang et al. / Process Biochemistry 51 (2016) 229–239 237

Fig. 10. pH stabilities of free and immobilized laccases. The enzymes were incubated for 1 h under the optimal temperature and different pH values to investigate stability.
The free laccase activity was assayed at 30 ◦ C using 0.5 ␮mol/mL ABTS (2.9 mL, pH 3.0) as a substrate. The immobilized laccase activity was assayed at 40 ◦ C using 0.5 ␮mol/mL
ABTS (20 mL, pH 4.0) as a substrate.

Fig. 11. Temperature stabilities of free and immobilized laccases. The enzymes were incubated for 1 h under the optimal pH and different temperatures to investigate
stability. The free laccase activity was assayed at 30 ◦ C using 0.5 ␮mol/mL ABTS (2.9 mL, pH 3.0) as a substrate. The immobilized laccase activity was assayed at 40 ◦ C using
0.5 ␮mol/mL ABTS (20 mL, pH 4.0) as a substrate.

and the leakage of laccase from MMU during the oxidation reac-
tion, in addition to the poisoning effect and diffusion limits caused
by the substrate [47]. Some studies have reported that immobilized
laccase possessed better reusability [48,49]. The force of physi-
cal adsorption between the laccase and MMU was weaker than
covalent attachment, leading to a low reusability. However, the
physical immobilization method had minimal negative impacts
on activity. Other researchers using the physical immobilization
method obtained reusability similar to that of the present study
[16,50]. Further improvement of the reusability of MMU is still
necessary if considering the retaining rate. Covalent binding can
provide strong enzyme attachment. Therefore, carbodiimide acti-
vation can be used when the carboxylic group in the surface of MMU
is expected to react with amino group of laccase for preventing the
leaching of laccase from porous, such as in the case of activated
sepharose and cellulose [51].
Storage stability was tested by storing at 4 ◦ C for 3 weeks. The
results showed that immobilized laccase still retained 55.4% of its
initial activity for a period of 21 days. However, free laccase under
the same storage conditions was completely inactivated, indicating
that immobilization could prevent laccase from inactivating and
revealing the good storage stability.
3.6. Kinetic parameters
Km is an important kinetic parameter in probing the bind-
ing ability of enzyme to substrate. The Km and Vmax reflect the
effective characteristics of enzyme including both partition and
238 S. Pang et al. / Process Biochemistry 51 (2016) 229–239
Fig. 12. Operational stability of immobilized laccase. The immobilized laccase activity was assayed at 40 ◦ C using 0.5 ␮mol/mL ABTS (20 mL, pH 4.0) as a substrate. At the
end of each cycle, the immobilized laccase was removed from the reaction medium and washed three times with sodium acetate buffer (pH 4.0), and then the cycle was
repeated with a fresh preheated aliquot of substrate.
diffusion effects. In this study, the Km and Vmax of free enzyme were
0.330 ± 0.009 and 0.083 ± 0.002 mM/min, while those of immo-
bilized laccase were 0.217 ± 0.005 and 0.072 ± 0.009 mM/min,
respectively. Low Km value indicates high affinity between enzyme
and substrate. The affinity of laccase with substrate increased
after immobilization, which was probably caused by structural
changes in the laccase introduced by immobilization procedure
[46]. Similar results have been reported recently [52,53]. The Vmax
value of immobilized laccase was 86.7% that of free laccase. The
decrease in Vmax was in agreement with the previous reports
that comparing free and immobilized laccase [53,54]. It might
attribute to the substantial secondary structural perturbation and
changes on the enzyme surface upon adsorption [46,54]. As a con-
sequence, the catalytic efficiency (Vmax /Km ) of immobilized laccase
(0.330 ± 0.049) was higher than that of free laccase (0.250 ± 0.002),
which proved that the immobilized laccase had relatively high
catalytic efficiency. The catalytic efficiency was usually decreased
upon immobilization in the similar reaction systems [52–54]. The
reason for the increase of the catalytic efficiency might be the
bimodal mesoporous structure and the physical adsorption which
could help avoid enzyme/enzyme interaction and large aggregated
enzyme/support polymer layers.
4. Conclusions
The success of an immobilization process primarily depends
on the support used. In this work, a nanoscale bimodal (3.5
and 7 nm) mesoporous Zr-MOF modified with a carboxyl group
was synthesized using the surfactant-templating method to
immobilize laccase. This material exhibited a high BET surface
area (453.8 m2 /g), a bimodal mesoporous structure, hydrological
stability and good thermal stability to resist 500 ◦ C. The immo-
bilization performed in a 5 mg/mL laccase solution for 1 h led to
the highest adsorption amount (221.83 ± 8.74 mg/g) and favor-
able activity recovery (95.90 ± 0.28%). This excellent adsorption
amount was better than that of most mesoporous materials. The
adsorption amount tended to be smooth and steady after 2 h
(175.00 ± 1.11 mg/g), proving that most of the laccase molecules
were adsorbed inside the pores. Undesired lateral interactions that
might arise from the relatively narrow pore size of MMU and the
high initial adsorption concentration did not occur. The immo-
bilized laccase exhibited higher activity over wider temperature
and pH ranges than free laccase; in particular, the stability against
heat denaturation increased significantly. The immobilized laccase
exhibited good reusability and retained 55.45% of its initial activity
after storing at 4 ◦ C for 3 weeks. The Vmax /Km of laccase increased
after immobilization and showed higher catalytic efficiency. The
overall results show that, MMU can be used as a new support for
immobilization and brings some advantages for biotechnological
applications.
Acknowledgements
The authors are thankful for the support of “the Fundamental
Research Funds for the Central Universities (No. 2015ZCQ-SW-
04)”, “Beijing Science and Technology Innovation Base Cultivation
and Development Projects (IG201307N)” and “Beijing Municipal
Government Priorities and County Government Pretrigger Projects
(z121100000312010)”.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.procbio.2015.11.
033.
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