Studies on the co-immobilized GOD/CAT on cross-linked chitosan microsphere modified by lysine

Article abstract of DOI:10.1016/j.molcatb.2013.07.009

Glucose oxidase (GOD) and catalase (CAT) were co-immobilized on a novel crosslinked chitosan resin modified by l-lysine. The immobilized system exhibited the best performance when the immobilization process was conducted at 4 C for 3 h with GOD/CAT activity ratio of 1:18 and pH 7.0. Under the optimum pH 4.0 and temperature 40 C, the enzyme system could achieve to the maximum conversion of glucose, which was 68.9%. Acidoresistance and thermotolerance of the enzymes were both strengthened. The Km of the co-immobilized enzyme system obtained in this study was 31.02 mM. The GOD enzyme immobilized in this system could maintain 94% of initial activity after the 12th operational cycle. 95.8% of initial activity was left after 50 weeks' storage at 4 C. GOD achieved better performance in this new immobilized enzyme system due to its effective elimination of H₂O₂, which showed potential applications for various commercial uses.

Full text of DOI:10.1016/j.molcatb.2013.07.009

Journal of Molecular Catalysis B: Enzymatic 97 (2013) 80–86
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Studies on the co-immobilized GOD/CAT on cross-linked chitosan
microsphere modified by lysine
Jie Zhanga,1, Xiaohua Zhoua,∗, Dan Wanga,∗, Yingli Wanga, Xing Zhoub, Honghui Wanga,
Qiang Lic, Shiyu Tana
a College of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044, China
b TCM Pharmacochemistry Institute, Chongqing Academy of Chinese Materia Medica, Chongqing 400065, China
c National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Glucose oxidase (GOD) and catalase (CAT) were co-immobilized on a novel crosslinked chitosan resin
modified by l-lysine. The immobilized system exhibited the best performance when the immobilization
process was conducted at 4 ◦C for 3 h with GOD/CAT activity ratio of 1:18 and pH 7.0. Under the optimum
pH 4.0 and temperature 40 ◦C, the enzyme system could achieve to the maximum conversion of glucose,
which was 68.9%. Acidoresistance and thermotolerance of the enzymes were both strengthened. The Km
of the co-immobilized enzyme system obtained in this study was 31.02 mM. The GOD enzyme immobi-
lized in this system could maintain 94% of initial activity after the 12th operational cycle. 95.8% of initial
activity was left after 50 weeks’ storage at 4 ◦C. GOD achieved better performance in this new immobi-
lized enzyme system due to its effective elimination of H2O2, which showed potential applications for
various commercial uses.
Received 24 May 2013
Received in revised form 12 July 2013
Accepted 13 July 2013
Available online xxx
Keywords:
Crosslinked chitosan microspheres
Co-immobilized GOD/CAT
GOD performance
H2O2
© 2013 Elsevier B.V. All rights reserved.
Lysine
1. Introduction
Consequently, the adjacent atoms of enzyme groups may combine
with pyran cycles of chitosan, leading to narrow slits between
Enzyme immobilization has attracted a wide range of interest
from fundamental academic research to many different industrial
applications in recent years [1,2]. A variety of supports have been
used for immobilization of enzymes such as polyamide [3,4],
gelatin [5,6], alginate [7,8] and chitosan [9–14]. Natural poly-
mers, especially chitosan, have been extensively developed and
explored for enzyme immobilization because of the advantages
of nontoxicity, hydrophilicity, and biocompatibility. However, the
mechanical strength of chitosan particles is relatively low, which
leads to a less effective mass transfer. Methods for improving the
structure and function of chitosan have been focus on modifying
the attached side chains by functionalization with various linkers
[15]. Microspheres of cross-linked chitosan were prepared by
covalently cross-linking using various bifunctional reagents such
as glutaraldehyde, dimethyl suberimidate and dimethyl adipim-
idate [16–20]. The bifunctional glutaraldehyde reacts with the
the proteins and the pyran cycles during immobilization. This
phenomenon finally resulted in the lower mass transfer of sub-
strates and products, and further support modifications should be
executed to fix such a problem.
In our previous study, l-lysine was successfully grafted on
glutaraldehyde semi-crosslinked chitosan to form a novel function-
alized chitosan resin (LMCCR) [21]. Acting as a spacer arm, l-lysine
helps in reducing the effect of steric hindrance and improving the
mass transfer. LMCCR also exhibited considerable adsorption per-
formances for various substances such as acetic acid, alanine, BSA
and Zn2+ [21]. So it might be an ideal support for glucose oxidase
(GOD) immobilization.
GOD catalyzes the oxidation of ␤-d-glucose to gluconic acid,
utilizing molecular oxygen as an electron acceptor with simulta-
neous production of hydrogen peroxide (H2O2) [22–24]. GOD is
often used for the determination of glucose levels in body fluids.
The pH optima of GOD lies in the acidic to neutral range. The opti-
mum temperature for GOD was reported to be 25–30 ◦C. Catalase
(CAT) catalyzes decomposition of H2O2 into molecular oxygen and
water by a two electron transfer mechanism [25]. CAT is potentially
useful for eliminating H2O2 produced in various enzymatic oxida-
tion reactions such as the air oxidation of glucose catalyzed by GOD
[26,27]. It could maintain excellent activity from pH 6 to 8, under
the temperature of 25–40 ◦C.
amino groups of chitosan to form Schiff bases (
C
N
linkage).
∗
Corresponding authors. Tel.: +86 23 65111179; fax: +86 23 65111179.
E-mail addresses: lang19880107@hotmail.com (J. Zhang),
zhou65306590@tom.com (X. Zhou), danwang088@gmail.com (D. Wang).
1
College of Chemistry and Chemical Engineering, Chongqing University, P.O. Box
94#, Shazheng Street 174, Shapingba District, Chongqing 400044, China.
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.07.009

J. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 80–86
81
The bi-enzymatic systems oxidase and CAT were studied by
several investigators. Ene and Maria reported the d-glucose oxi-
dation process in the presence of a bi-enzymatic free system of
pyranose 2-oxidase (P2Ox) and CAT [28]. It indicated an optimum
temperature of 25 ◦C for the catalase activity, even if the P2Ox
reaches its optimal activity at higher temperatures of 30–35 ◦C
when the main reaction rate is more and more inhibited by the large
amounts of catalase. Leitner et al. found that the ratio of CAT/P2Ox
higher than 100 can lead to an acceptable oxidase residual activity
[29]. Ozyilmaz and Tukel found that GOD and CAT simultaneously
co-immobilized onto magnesium silicate in the presence of their
substrates highly improved the activity and reusability of both
enzymes [30]. Godjevargova and Dayal reported the characters of
GOD and CAT immobilized onto three types of ultrafiltration poly-
acrylonitrile membranes with increase pore sizes (PAN 1–PAN 3)
and one microfiltration polyamide membrane [31]. In the study of
Sisak et al. Amberlite UP 900 anion exchange resin was successfully
adopted for the immobilization of commercial GOD containing CAT
(NOVOZYM 771) using adsorption and subsequent cross-linking
with glutaraldehyde [32].
In this study, GOD and CAT were co-immobilized on LMCCR.
The preparation conditions of the novel co-immobilized enzyme
system, including CAT/GOD activity ratios of 0–30, pH 3–8 and
cross-linking time 1–5 h were studied in detail. In enzyme catal-
ysis process, pH varied from 3 to 8, temperature changed from 10
to 60 ◦C, and up to 12 enzyme reusing cycles were investigated
to evaluate its potential for industrial and analytical applications.
The immobilized system can be applied in the process of liquid
foodstuffs deoxygenating, gluconic acid production, glucose con-
centration control in biological compounds, fermentation et al.
and a standard curve was drawn. After mixing PB containing the
extracted enzyme with the assay reagent, the absorbance was mea-
sured by a UV–vis spectrophotometer (Varian CARY 1E) at 595 nm.
The enzyme protein content was then determined according to
the standard curve. The ID was calculated as follows: The ID was
calculated as follows:
(preliminary enzyme content − enzyme content in the filtrate)
ID =
× 100%
preliminary enzyme content
2.2.3. Activity assays of GOD
Enzymatic activities of free and immobilized GOD were assessed
in terms of the oxidation of ␤-d-glucose to d-gluconic acid car-
ried out under stirring in air. The d-gluconic acid formed was
quantified according to the complex amount with Cu (II)–NaOH
solution. Firstly, soluble GOD or suspension of IGOD or CI-GOD/CAT
was added to a reaction solution containing 8.0 mL of mixed
chromogenic reagents and 2.0 mL of 6.5% (w/v) glucose solu-
tion. The mixture above was freshly prepared, containing 5.0 mL
0.1 M CuSO4, 3.0 mL 0.1 M NaOH and 20 mL 0.06 mol/L phosphate
buffer (KH2PO4–Na2HPO4 solution of pH 6.0). 10 min later, the
absorbance of the blue dye at 660 nm was measured. Under the
assay conditions, one unit (U) of GOD activity was defined as the
amount of enzyme which produces 1.0 ␮mol Cu(II)–gluconic acid
mL−1 min−1. The activity of free GOD was given as U g protein−1
and immobilized GOD activities were expressed as U g immobilized
GOD−1
.
2.2.4. Activity assays of CAT
The CAT activity was determined according to Özlem [26]
and Bergmeyer [38]. CAT activity was measured spectrophoto-
metrically at 240 nm using a specific absorption coefficient of
0.0392 cm2 ␮mol H2O2−1. CAT activity was determined in reaction
mixture containing 2.5 mL of substrate made up of 10 mM H2O2 in
a 50 mM phosphate buffer pH 7.0 and 3.5 mL of free CAT or 2.5 g of
immobilized CAT. Reaction was carried out at 25 ◦C for 2 min. The
rate data were taken from the first linear phase (0–2 min, every 10 s)
by an on-line UV–vis spectrophotometer, and the CAT activity can
be evaluated from the kinetic curve slope. One unit of activity w◦as
defined as the decomposition of 1 mM H2O2 per minute at 25 C
and pH 7.0. And activity of free CAT was given as U mL protein−1
and immobilized CAT activities were expressed as U g immobilized
2. Experimental
2.1. Materials
The GOD (EC. 1.1.3.4) is a flavoprotein with MW = 150,000 Da.
The CAT (EC. 1.11.1.6) is from haematin group with
MW = 250,000 Da. The specific activity of GOD used in this
experiment was 1000 U/g, while the specific activity of CAT was
50000 U/mL. These enzymes were supplied by Fluka (Buchs,
Switzerland). l-lysine was purchased from Merck, and all other
chemicals were of analytical grade.
2.2. Experimental methods
CAT−1
.
2.2.1. Preparation of co-immobilized GOD/CAT
The cross-linked chitosan microsphere was prepared as pre-
viously described in reference [21]. The LMCCR beads, 5 g, were
suspended in 10 mL buffer solution for 60 min at 4 ◦C. The buffer
solution was composed of various volumes of disodium hydrogen
phosphate solution (0.2 M) and citric acid solution (0.1 M), and the
final pH of the solution varied from 3.0 to 8.0. Then they were mixed
with 10 mL 0.1% solution of GOD and CAT with activity ratio ran-
ging from 1:1 to 1:30 for 20 h at 4 ◦C. The cross-linking process
using glutaraldehyde was performed as previous reports [32–36].
Aqueous glutaraldehyde solution of 1.5% (v/v) was dropped in and
the suspension was then incubated for several hours. After the
reaction mixture was filtered and washed with 0.2 mol/L phos-
phate buffer (PB, pH 6.8) to remove excessive glutaraldehyde, the
co-immobilized GOD plus CAT (CI-GOD/CAT) was prepared.
2.2.5. Michaelis constant of the co-immobilized enzyme
The Michaelis constant (Km) of immobilized GOD was
determined according to Lineweaver–Burk [39]. The glucose con-
centration was varied from 12.5 to 100 mM. The reaction rate was
determined at 25 ◦C in 0.1 M phosphate buffer (pH 7.0) according to
the method mentioned in Section 2.2.3. The kinetic parameters Km
for free and immobilized enzymes were determined from double
reciprocal plot.
3. Results and discussion
3.1. Optimization of co-immobilization conditions
The co-immobilization mechanism was shown in Scheme 1. The
free amino groups in l-lysine residues formed Schiff base with
aldehyde groups of glutaraldehyde and the enzymes were immobi-
lized. To achieve the maximum conversion of d-glucose and highest
ID, the optimized preparation conditions were explored, including
activity ratio of the enzymes, pH, and cross-linking time.
2.2.2. Determination of immobilization degree
The immobilization degree (ID) was defined as percentage
of total enzyme protein content immobilized on the support.
Total enzyme protein assay was carried out according to Bradford
method [37]. The absorbances of protein standards were measured

82
J. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 80–86
CH2OH H2N
OH
O
CH2OH
H2N
OH
O
O
O
OH
O
n
O
O
O
O
OH
CH2OH
HO
N
n
O
CH
CH2OH
IGOD if amino group of
GOD here took the place
HO
N
(H2C)3
HC
CH
CAT
(H2C)3
HC
OHC
GOD
(CH2)3
NH2
CHO
NH2
GOD
N
OH
CH2OH
Occasionally
N
CAT
O
N
OH
CH2OH
CH
(CH2)3
CH
(CH2)4N
HO
O
O
O
O
O
n
HO
O
O
O
CH2OH HO
NH
C
O
n
H
C
O
CH2OH HO
NH
H
C
O
N
C
(CH2)4NH2
GOD
CAT
N
C
(H2C)3
CH
NH2
H
Occasionally
ICAT if amino group of
CAT here took the place
LMCCR
CI-GOD/CAT
Scheme 1. Co-immobilization mechanism of the enzymes on the LMCCR. The cross-linker was glutaraldehyde.
3.1.1. Effect of the activity ratio of CAT/GOD (U/U)
As it was shown in Fig. 1, when the activity ratio of CAT/GOD
was below 5, the concentration of H2O2 increased rapidly. But it
decreased when the activity ratio was greater than 5. The concen-
tration of H2O2 remained 7 mM while the activity ratio of CAT/GOD
was higher than 18:1. At the ratio of 0:1 without CAT existed, H2O2,
at a concentration of about 30 mM, exerted a strong inhibitory effect
on GOD activity [40]. A small amount of co-immobilized CAT which
caused larger amount of H2O2 could increase the activity of GOD.
However, the concentration of H2O2 fell when the activity of CAT
was 4 times more than that of GOD. The H2O2 concentration was
stable when the activity ratio reached 18:1. It could be deduced
that the system became balanced when the concentration of H2O2
was lower than 7 mM. Under this condition, 16.7 mg GOD was
immobilized on 1 g of LMCCR and the activity of GOD detected was
H2O2 was considered as the key factor in the system. H2O2, the
substrate of CAT, was generated from reactions catalyzed by GOD,
as seen from Scheme 2. H2O2 content was dependent on the activity
ratio of enzymes, which should be controlled due to its inhibitory
effects of GOD.
To promote the catalyzing efficiency of the enzyme system, the
activity ratio of CAT/GOD was examined. In the process of catalytic
reaction, gluconic acid concentration was maintained at mM level
and the acidity of the microenvironment would not be changed.
Thus the investigation was based on the premise that the pH value
of the co-immobilized enzyme system was stable enough for GOD.
Otherwise, H2O2 could not be self-decomposed due to its low level
and the acid environment [40].
CH2OH
CH2OH
O
O
GOD
HO
OH
H2O2
CAT
HO
O
+
HO
OH
HO
OH
CH2OH
(CH2OH)4
COOH
O2
H2O
+
Scheme 2. Reaction mechanism of the co-immobilized enzyme system. The substrate of the CAT catalyzing reaction, H2O2, was generated from GOD catalyzing reaction.

J. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 80–86
83
140
120
100
80
70
65
60
55
50
45
40
35
30
25
Enzyme activity recovery
Immobilization degree
60
40
20
0
0
5
10
15
20
25
30
1
2
3
4
5
Time (h)
Activity ratio of CAT/GOD
Fig. 3. Effect of the cross-linking time on the immobilization. Immobilization condi-
Fig. 1. Effect of the activity ratio of CAT/GOD (U/U) on the immobilization. The
co-immobilized enzyme catalyzing efficiency was focused on GOD and expressed
by H2O2 production. Immobilization conditions: adsorption time 20 h, 4 ◦C, cross-
linking time 3 h, pH value 6.5.
tions: adsorption time 20 h, 4 ◦C, the activity ratio of CAT/GOD (U/U) 18:1, pH value
6.5.
the higher the degree of nitrogen atoms non-protonated, which
was more favorable for aldehyde-amine condensation reaction.
However, enzyme immobilization would be affected in a solution
with pH higher than 7.0 due to self-condensation of glutaralde-
hyde [43,44]. The lowest enzyme immobilized efficiency at pH 5.0
was possibly caused by the isoelectric point of GOD protein, 4.6,
where the homogeneous immobilization reaction was altered to
heterogeneous immobilization reaction. Under this condition, the
Schiff-base was difficult to form, so, ID and AR decreased. GOD and
CAT enzyme proteins were soluble when the pH varied from 5.0 to
7.0, which facilitated the homogeneous reaction and resulted in the
ID increasing. The results showed that the immobilization process
should be conducted at pH 7.0.
about 0.51 U mg−1. Although the methionine and cysteine residue
of GOD enzyme protein could be oxidized by H2O2, it was safe for
GOD with the H2O2 concentration lower than 7 mmol/L in micro-
environment [32]. Thus 18:1 was the optimum activity ratio of
CAT/GOD for the co-immobilization. At the same time, preliminary
investigations suggest that higher than optimum CAT/GOD ratios
are not indicated, leading to a flattening of the product yield.
In Leitner’s study, the bi-enzymatic free system of P2Ox and
CAT obtained an acceptable oxidase residual activity while the ratio
of CAT/P2Ox was higher than 100 [29]. And the optimal activity
ratio of CAT/GOD was 5:1 when GOD and CAT were immobilized
onto chemically modified acrylonitrile copolymer membranes in
Godjevargova’s study [31]. Krysteva and Bradley reported that for
effective enzyme reaction the activity of CAT could be higher when
theoptimalconditionsfor immobilizationwereestablished[41,42].
Hence, the ratio of CAT/GOD in immobilization process was smaller
than the free system.
3.1.3. Effect of the cross-linking time
The time of cross-linking reaction was optimized in this study
because of its potential impact on behaviors of glutaraldehyde.
As shown in Fig. 3, the ID increased within the first 3 h. But 3 h
later, the ID decreased. The process of immobilization could be
divided into two steps. Firstly, free enzyme diffused toward the
LMCCR until absorption equilibrium was achieved. Secondly, the
glutaraldehyde diffused into the space between GOD and CAT,
and the cross-linking reaction started. Biomacromolecules, like
enzyme, moved slowly in solution, and perhaps 3 h were needed
for the majority of enzyme to reach the reaction sites. It could
be deduced that all the cross-linking sites had been occupied by
enzymes 3 h later, and ID achieved the peak value. If the reac-
tion was not stopped after 3 h, the excess glutaraldehyde might
react with free enzymes and the enzymes might turned into giant
cross-linked protein molecules. The protein bunch then turned into
sedimentation, which resulted in a decline of ID [26]. Based on our
experiments, the co-immobilization reaction process should last
for 3 h.
3.1.2. Effect of the pH
The highest immobilization degree and activity recovery (AR)
point were both obtained at pH 7.0 in Fig. 2. The higher the pH value,
70
Enzyme activity recovery
65
Immobilization degree
60
55
50
45
40
35
30
3.2. Enzyme characteristics of CI-GOD/CAT
3.2.1. Michaelis constant
As shown in Table 1, compared to the free enzyme, Km value
of GOD increased by 21.9% after the co-immobilization, agreeing
with the result of immobilized trypsinase on the carrier modified
by arginine [17]. The enzyme affinity for substrate was decreased
for the reason that due to the steric hindrance of the support, diffu-
sion of substrate molecules to active site of the enzyme was difficult
[45]. Yet the Km value in our study was only 1.4% higher than that of
3
4
5
6
7
8
pH value
Fig. 2. Effect of the pH value on the immobilization. Immobilization conditions:
adsorption time 20 h, 4 ◦C, cross-linking time 3 h, the activity ratio of CAT/GOD (U/U)
18:1.

84
J. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 80–86
Table 1
70
65
Kinetics parameters of free and immobilized GOD.
Free GOD
CI-GOD/CAT
IGOD
Km (mM)
Vmax (U/mg)
26.29
23.81
31.02
20.41
30.60
14.71
60
55
50
45
40
35
30
The Km values are all based on GOD with substrate of glucose.
IGOD while the Vmax was 38.7% higher. The possible reason might
be that without CAT immobilization in the IGOD system, H2O2 was
accumulated to a certain degree and the active sites of GOD was par-
tially oxidized. But co-immobilizing of CAT in the system resulted
in the decomposition of H2O2, and GOD could maintain its active
conformation more easily. Furthermore, Km value of the immobi-
lized form was at least 1.26 times higher than that of the enzyme
immobilized on polyacrylonitrile membranes (PAN 1–PAN 3) car-
riers in Dayal’s study [31]. It indicated that the CI-GOD/CAT system
in our study was more desirable for industrial applications due to
the high affinity between the enzyme and its substrate.
10
20
30
40
ã
50
60
Temperature ( C)
Fig. 5. Optimum temperature of CI-GOD/CAT. Glu represents d-glucose. Conditions:
6.5% (w/v) solution of d-glucose, pH 4.2, reaction time 10 min, enzyme immobilized
with the activity ratio of CAT/GOD (U/U) 18:1, 4 ◦C, pH 7.0 and cross-linking time
3 h.
3.2.2. Optimal pH
The conversion of glucose catalyzed by the CI-GOD/CAT
achieved peak values at pH 4.0 or 7.0, and the lowest value was
obtained at pH 5.0, as shown in Fig. 4. The highest enzyme activity
was achieved at pH 4.0. It meant that the optimum pH shifted to
acidic region after the immobilization, considering that optimum
pH for free GOD and CAT were about 6.0 and 7.5 [45,46], respec-
tively. In other words, acidoresistance of the co-immobilization
enzyme was strengthened in our study.
Since enzyme activity is dependent on the ionization state of
the amino acids in the active site, pH plays an important role
in maintaining the proper conformation of an enzyme [47]. A
shifting toward acid range may result from the charge of the micro-
environment. In our study, the support chitosan was negative
charged, and it was easier to absorb cationic ion in the solution
after the immobilization, including H+. It would demand higher
concentration of anion in the diffusion layer than free enzyme in a
specific microenvironment. Thus, to achieve desirable CI-GOD/CAT
activity, the external solution had to be controlled at lower pH
for balance. Different from our study, the optimum activity of
GOD/CAT co-immobilized onto magnesium silicate was observed
at pH 6.5 in Ozyilmaz’s study [30]. The shift to neutral pH may
be related to the production of gluconic acid, which may cause a
decrease in the pH of the microenvironment of the immobilized
enzyme.
3.2.3. Optimal temperature
Enzymes are known to be sensitive to changes in tempera-
ture. Thermal stabilities of CI-GOD/CAT were investigated as shown
in Fig. 5. It indicated that the optimal temperature of the co-
immobilized system rose to 40 ◦C, which was 10 degrees higher
than free GOD, 30 ◦C, and 15◦ for free CAT, 25 ◦C [46,48]. The results
showed that thermal stability of CI-GOD/CAT increased compared
to the free enzymes. When enzyme was immobilized, flexibility
reduced and steric effect was strengthened. Collision frequency and
reaction rate required higher molecular kinetic energy to overcome
the new energy barrier. Therefore, a higher optimal temperature
was needed. Ozyilmaz and Tukel reported that the maximum activ-
ity was obtained at 30–35 ◦C for GOD/CAT co-immobilized onto
magnesium silicate [30]. The best productivity was achieved at
25 ◦C when GOD and CAT were co-immobilized on Amberlite UP
900 anion exchange resin [32]. The study also reported that the pro-
ductivity declined with the temperature ranging from 25 to 45 ◦C.
The reverse tendency might be caused by the different character-
istics of the carrier. In order to meet industrial demand, our carrier
LMCCR is more favorable.
70
65
60
55
50
45
40
3.2.4. Reuse stability
The reuse stability of CI-GOD/CAT was determined in batch reac-
tors at 40 ◦C. The results of 12 cycles using the same enzymes
were presented in Fig. 6. No significant decrease in the activity
of GOD immobilized was examined in the course of the opera-
tion, and 94% of its initial activity was maintained after the 12th
operation cycle. The activity of GOD remained 90% after 11 cycles
in Sisak’s study [32]. The GOD immobilized on cellulose acetate-
polymethylmethacrylate (CA-PMMA) membrane also showed a
performance of about 90% of its original activity after 12 reuses [49].
The higher reuse stability in our study could reduce operational cost
and thus is more promising.
3
4
5
6
7
8
3.2.5. Storage stability
pH Value
The storage stability of the CI-GOD/CAT was examined at
4 ◦C and the results were shown in Fig. 7. There was still 95.8%
of the initial activity for CI-GOD/CAT after 50 weeks’ storage.
Mislovicova also investigated storage stability of the immobilized
GOD retained after a long-time period storage at 4 ◦C [45]. The
Fig. 4. Optimum pH of CI-GOD/CAT. Glu represents d-glucose. Conditions: 6.5%
(w/v) solution of d-glucose, temperature 40 ◦C, reaction time 10 min, enzyme immo-
bilized with the activity ratio of CAT/GOD (U/U) 18:1, 4 ◦C, pH 7.0 and cross-linking
time 3 h.

J. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 80–86
85
GOD/CAT (U/U) was ascertained to be 1:18 to control the concen-
tration of H2O2 within the safety range.
100
99
98
97
96
95
94
93
A subsequent attention to its characteristics showed that the
newly prepared CI-GOD/CAT was an excellent enzyme system. The
temperature optima of the immobilized enzyme was shifted to
higher values 40 ◦C in comparison with that of free GOD and CAT,
which was more suitable of industrial applications. The Km of CI-
GOD/CAT was determined to be 31.02 mM, only slightly larger than
that of IGOD. Yet the Vmax improved impressively, indicating that
CAT co-existed was effective for elimination of H2O2, which helped
the maintenance of the active conformation of GOD. Specifically,
the remaining activity of the GOD immobilized on LMCCR was
about 94% after 12 cycles and 95.8% after 50 weeks’ storage, respec-
tively, which was more stable than the free one. Such CI-GOD/CAT
enzyme system has great potentials as biocatalysts for the applica-
tion in biotechnological fields, such as the production of gluconic
acid and its derivatives [50], oxygen removal in food preservation
[51], bioelectroanalysis and overcoming the issue of oxygen inter-
ference in important biosensor classes, etc. [52–54]. However, the
efficiency of the enzyme system could be further enhanced, which
will be investigated in our future study.
y = - 0.503 x + 99.925
R2 = 0.998
0
2
4
6
8
10
12
Reuse circles
Fig. 6. Reuse stability of CI-GOD/CAT. Conditions: operation cycles 12, 6.5% (w/v)
solution of d-glucose, temperature 40 ◦C, pH 4.0, reaction time 10 min, enzyme
immobilized with the activity ratio of CAT/GOD (U/U) 18:1, 4 ◦C, pH 7.0 and cross-
linking time 3 h.
Acknowledgement
100
80
60
40
20
This research was supported by the National Natural Science
Foundation of China (Grant 21106191, 21206175), Natural Science
Foundation Project of CQ CSTC (Grant cstcjjA50002), and Funda-
mental Research Funds for the Central Universities (project No.
CDJXS12220003, CDJXS10221138).
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