Surface functionalization of chitosan-coated magnetic nanoparticles for covalent immobilization of yeast alcohol dehydrogenase from Saccharomyces cerevisiae

Article abstract of DOI:10.1016/j.jmmm.2010.08.008

A novel and efficient immobilization of yeast alcohol dehydrogenase (YADH, EC1.1.1.1) from Saccharomyces cerevisiae has been developed by using the surface functionalization of chitosan-coated magnetic nanoparticles (Fe ₃O₄/KCTS) as support. The magnetic Fe₃O ₄/KCTS nanoparticles were prepared by binding chitosan alpha-ketoglutaric acid (KCTS) onto the surface of magnetic Fe₃O ₄ nanoparticles. Later, covalent immobilization of YADH was attempted onto the Fe₃O₄/KCTS nanoparticles. The effect of various preparation conditions on the immobilized YADH process such as immobilization time, enzyme concentration and pH was investigated. The influence of pH and temperature on the activity of the free and immobilized YADH using phenylglyoxylic acid as substrate has also been studied. The optimum reaction temperature and pH value for the enzymatic conversion catalyzed by the immobilized YADH were 30 °C and 7.4, respectively. Compared to the free enzyme, the immobilized YADH retained 65% of its original activity and exhibited significant thermal stability and good durability.

Full text of DOI:10.1016/j.jmmm.2010.08.008

Journal of Magnetism and Magnetic Materials 322 (2010) 3862–3868
Contents lists available at ScienceDirect
Journal of Magnetism and Magnetic Materials
journal homepage: www.elsevier.com/locate/jmmm
Surface functionalization of chitosan-coated magnetic nanoparticles
for covalent immobilization of yeast alcohol dehydrogenase from
Saccharomyces cerevisiae
a,b
b
a,n
c
c
Gui-yin Li , Zhi-de Zhou , Yuan-jian Li , Ke-long Huang , Ming Zhong
a
Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, Changsha, Hunan 410078, China
b
Biomedical Engineering Research Centre of Guilin University of Electronic Technology, Guilin, Guangxi 541014, China
c
College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A
novel and efficient immobilization of yeast alcohol dehydrogenase (YADH, EC1.1.1.1) from
Saccharomyces cerevisiae has been developed by using the surface functionalization of chitosan-coated
magnetic nanoparticles (Fe /KCTS) as support. The magnetic Fe /KCTS nanoparticles were
prepared by binding chitosan alpha-ketoglutaric acid (KCTS) onto the surface of magnetic Fe
nanoparticles. Later, covalent immobilization of YADH was attempted onto the Fe /KCTS
Received 16 April 2010
Received in revised form
3
O
4
3 4
O
2
8 July 2010
3 4
O
Available online 7 August 2010
3 4
O
Keywords:
nanoparticles. The effect of various preparation conditions on the immobilized YADH process such as
immobilization time, enzyme concentration and pH was investigated. The influence of pH and
temperature on the activity of the free and immobilized YADH using phenylglyoxylic acid as substrate
has also been studied. The optimum reaction temperature and pH value for the enzymatic conversion
catalyzed by the immobilized YADH were 30 1C and 7.4, respectively. Compared to the free enzyme, the
immobilized YADH retained 65% of its original activity and exhibited significant thermal stability and
good durability.
Immobilization
Magnetic nanoparticles
Yeast alcohol dehydrogenase
&
2010 Elsevier B.V. All rights reserved.
1
. Introduction
particles [14,15]. Wu and co-workers [14] used magnetic Fe
3 4
O
-
chitosan nanoparticles for lipase immobilization. The loading
With the rapid development of nanotechnology, magnetic iron
ability of the particles reached 129 mg/g and only lost 12% of
enzyme activity after five batches.
3 4
oxide (Fe O ) nanoparticles have shown great potential applica-
tions in many biological and medicinal fields [1], such as
bioseparation [2], tumor hyperthermia [3], magnetic resonance
imaging diagnostic contrast agents [4], magnetically guided site-
specific drug delivery agents [5,6], biomolecules (proteins,
peptides and enzymes) immobilization [7,8] and so on. As the
support of immobilized enzymes, magnetic nanoparticles have
many advantages like higher surface area, lower mass transfer
resistance, easier to recover by a magnetic field. Thus, there have
been many reports on using magnetic nanoparticles to immobi-
lize enzymes including proteases, lipase, penicillin G acylase,
glucose oxidase and so on [9–13]. Increasing the loading amount
of the enzymes on the magnetic particles could improve the
stability of immobilized enzymes. Functionalized magnetic
particles with water soluble, biocompatible and reactive func-
tional groups are desired [11]. Chitosan and its derivatives are the
most favorable micromolecules used to functionalize magnetic
Alcohol dehydrogenase, which catalyzes the oxidation of
alcohols and the reduction of carbonyl compounds such as
aldehydes and ketones, has attracted more attention because of
its potential applications in preparing various starting materials
and intermediates [16–19]. YADH is a special and important
alcohol dehydrogenase that exists with large amounts in the
microorganisms and livers of animals. Generally, Saccharomyces
cerevisiae is a good source for this enzyme. In spite of the wide
range of applications and interests, YADH is very sensitive and has
poor stability in aqueous solutions, which limits its application
[20]. YADH immobilization could offer many advantages with the
possibility of continuous processing, reusing of the enzyme, and
reduction of auto-digestion. To the best of our knowledge, YADH
has already been immobilized on various supports such as
platinum surface, silica nanotubes-doped alginate gel, agarose,
magnetic nanoparticles, [13,20–25]. Till now, research conducted
on YADH immobilization onto magnetic nanoparticles is notably
scarce. Liao and Chen [23] immobilized alcohol dehydrogenase
onto the magnetite nanoparticles, the bound yeast dehydrogenase
retained 62% of its original activity and exhibited a 10-fold
improved stability than the soluble enzyme using 2-butanone as
n
Corresponding author. Tel./fax: +86 731 82355078.
E-mail addresses: yuan_jianli@yahoo.com (Y.-j. Li),
klhuang@mail.csu.edu.cn (K.-l. Huang).
0304-8853/$ - see front matter & 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jmmm.2010.08.008

Gui-yin Li et al. / Journal of Magnetism and Magnetic Materials 322 (2010) 3862–3868
3863
substrate. Moreover, our previous study [13] dedicated to the
direct immobilization of YADH onto the magnetic Fe -chitosan
nanoparticles via glutaraldehyde activation. The immobilized
YADH retained 49% activity and exhibited significantly improved
stability than the free enzyme.
phosphate buffer (0.2 M, pH 6.2). The dialyzed sample was
clarified by centrifugation at 12000 ꢀ g for 20 min and the crude
YADH solution was obtained in the supernatant.
3 4
O
Very recently, magnetic Fe
composite (Fe /KCTS) nanoparticles have been prepared by
covalently binding alpha-ketoglutaric acid chitosan (KCTS) onto
Fe magnetic nanoparticles via carbodiimide activation in our
group [26]. The resultant Fe /KCTS nanoparticles are super-
paramagnetic with a diameter of about 26 nm, and a saturation
magnetization of 24.8 emu/g. In the present study, the surface
functionalization of chitosan-coated magnetic nanoparticles
3 4
O /carboxyl functional chitosan
2
.3. Preparation of surface functionalization of chitosan-coated
3 4
O
magnetic nanoparticles
3 4
O
3 4
KCTS was prepared according to reference [27]. Fe O
3 4
O
nanoparticles were prepared by a hydrothermal method with a
ferrous complex using H as an oxidizer. Magnetic Fe /KCTS
nanoparticles were prepared according to the reported method
26]. Typically, Fe nanoparticles (0.2 g) were dispersed in a
2
O
2
3 4
O
[
3 4
O
3 4
(Fe O /KCTS) was used for immobilizing YADH by electrostatic
solution with 30 ml paraffin and 0.5 ml span-80, and then 15.0 ml
solution of KCTS in acetic acid with a concentration of 2% was
added. The mixture was ultrasonic dispersed for 30 min, and then
adsorption and covalent binding. The activity of immobilized
YADH was measured using phenylglyoxylic acid as substrate and
nicotinamide adenine dinucleotide (NADH) as coenzyme. The
immobilization conditions were optimized and the effect of pH
and temperature on the activity of free and immobilized YADH
was determined. This study will prove that magnetic nanomater-
ials play an important role in enzyme immobilization/stabiliza-
tion techniques, which will undoubtedly give rise to novel
applications of immobilized enzymes in the fields of industry
and biomedicine.
ꢂ1
stirred with 1 ml carbodiimide solution (30 g L
in 0.003 M
/KCTS
3 4
phosphate buffer, pH 6.0, 1 M NaCl). After 4 h, the Fe O
nanoparticles were recovered from the reaction mixture by a
permanent magnet with a surface magnetization of 6000 G, and
washed three times with water and ethanol, dried at 50 1C under
vacuum. Scheme 1 shows an illustration for preparation of
magnetic Fe
The resultant Fe
with a diameter of about 26 nm, and a saturation magnetization of
4.8 emu/g.
3
O
4
/KCTS nanoparticles.
3 4
O /KCTS nanoparticles are superparamagnetic
2
2
. Material and methods
2.1. Reagents
3 4
2.4. Covalent immobilization of YADH on magnetic Fe O /KCTS
nanoparticles
Strains (S. cerevisiae, strain no.3) were obtained from our
culture collection. Yeast alcohol dehydrogenase (EC1.1.1.1) from
S. cerevisiae was prepared as described in section 2.2. Phenyl-
3 4
Magnetic Fe O /KCTS nanoparticles (50 mg) were redispersed
in 30 ml 5% glutaraldehyde phosphate buffer solution at pH 6.8
and kept at room temperature for 10 h. The nanoparticles were
separated by magnetic decantation and then washed three times
with deionized water. Finally, 30 ml YADH diluent solution (5 ml
2 2
glyoxylic acid and carbodiimides (cyanamide, CH N ) were
purchased from Sigma Chemical Co., Ltd. Nicotinamide adenine
dinucleotide (NADH) was procured from Roche Ltd. Chitosan (MW
5
4
.9 ꢀ 10 , degree of deacetylation 95%) was procured from Dalian
0
.23 mg/ml YADH solution in phosphate buffer (0.02 M, pH 7.4)
was added to the particles and continuously shaken at 25 1C at
50 rpm for 2 h. The immobilized YADH was recovered by a
Xindie Chitin Co. (Dalian, China). 25% Glutaraldehyde solution
was purchased from Hunan Normal University Chemical Co.
1
(
Hunan, China). Alpha-ketoglutaric acid was purchased from
Qianshan Science and Technology Development Company
Zhuhai, China). Ferrous sulphates heptahydrate and aqueous
ammonia solution were procured from Tianjin No. 3 Chemical
Plant (Tianjin, China). Sodium borohydride (NaBH ) was supplied
magnetic separation, washed with phosphate buffer at pH 7.4 to
remove unbound YADH. The obtained immobilized YADH was
directly used for the measurements of activity and stability. The
supernatant solution was used to detect the amount of unbound
enzyme.
(
4
by Fluka Co. Other chemicals are analytic grade reagents and used
without further purification.
2
.5. Assay of the amount of YADH immobilized on the magnetic
2
.2. Cultivation and purification of YADH from Saccharomyces
Fe /KCTS nanoparticles
3 4
O
cerevisiae
The amount of protein in the YADH solution and in super-
natant after immobilization was determined by a Lab Tech UV
100 spectrophotometer (absorption at 280 nm) with bovine
serum albumin as the standard. The amount of protein immobi-
lized on the magnetic nanoparticles was calculated by mass
balance as the following formula:
S. cerevisiae was cultivated in bottle using culture medium
g/L): glucose 50, peptone 3.0, yeast extract 2.5, K HPO 1.0,
MgSO O 0.5, NaCl 0.5, Fe (SO 0.01, ZnSO 0.01, at pH 6.5.
The cells were incubated at 30 1C and harvested after 24 h. And
the cells were collected from culture medium by centrifugation at
(
2
4
2
4
ꢁ 7H
2
2
4
)
3
4
6
1
687.5 ꢀ g for 15 min at 4 1C. Cell pellets were suspended with
0 ml 0.9% sterile physiological saline per gram of cell pellet and
ðC ꢂC Þ ꢀ V
i
f
disrupted using Ultrasonic vibra cell crusher KS-250F (power
00 W, 120 times, work 8 s, stop 8 s). The disrupted cell mixture
Q ¼
W
4
was centrifuged at 6687.5 ꢀ g for 10 min at 4 1C. The supernatant
was collected and precipitated with ammonium sulfate at a final
concentration of 3 M. The precipitant was collected by centrifuga-
tion at 6687.5 ꢀ g for 15 min at 4 1C and then resuspended in 5 ml
of phosphate buffer (0.2 M, pH 6.8) per 100 mg of precipitant. The
resuspended solution was dialyzed overnight against 500 ml of
where Q is the amount of bound YADH onto supports (mg/g), C
and C are the concentration of the YADH protein initial and
supernatant solution after immobilization (mg/ml), V is the
volume of the medium (ml) and W is the weight of the supports
(g). All data used in this formula are the average of duplicated
experiments.
i
f

3
864
Gui-yin Li et al. / Journal of Magnetism and Magnetic Materials 322 (2010) 3862–3868
NH2
NH=C
CH CH COOH
2
2
O
H
NH CHCOOH
CH CH C
2
2
H
O
HN=C=NH
OH
H
H
O
H
NH CHCOOH
H
H
O
OH
H
H
O
CH OH
O
2
n
H
CH OH
(KCTS)
2
n
O
CH CH C
2
2
O
OH
Fe O )
O
C
H
NH CHCOOH
H
(
3
4
OH
H
H
O
O
+
H
NH2
CH OH
H N
2
2
n
(
Fe O /KCTS)
3 4
Scheme 1. An illustration for preparation of Fe
3
O
4
/KCTS nanoparticles.
H
H
COOH
COOH
CONH2
+
CONH2
+
C
O
+
C
OH
H
N
+ H
N
+
R
Alcohol dehydrogenase
R
NADH
NAD+
Scheme 2. YADH catalyzed resolution of phenylglyoxylic acid to (R)-mandelic acid.
2
.6. Determination of enzymatic activity
and immobilized YADH, the YADH activity was measured at
various temperature (4, 20, 30, 35, 40 and 50 1C) in the presence
of phosphate buffer (0.02 M, pH 7.4), and different pH of 5.8, 6.4,
6.8, 7.0, 7.4 and 8.0 at 30 1C, respectively. The highest activity
under the optimum conditions was considered as 100% for both
free and immobilized YADH, and the relative activity under other
reaction conditions was defined as the proportion of the highest
activity.
The kinetic of YADH catalyzed reduction of phenylglyoxylic acid
+
to (R)-mandelic acid coupling with the oxidation of NADH to NAD
was studied (Scheme 2). According to the reference [21–25], the
enzyme activity was determined by measuring the initial reduction
rate of phenylglyoxylic acid by YADH following the decrease of
NADH concentration at 340 nm at the desired temperature.
Generally, 4 ml phosphate buffer solution (0.02 M, pH 7.0), 1 ml
1
mM NADH solution and 1 ml 0.1 M phenylglyoxylic acid solution
2.8. Thermal stability and reusability of immobilized YADH
were mixed to the test tube for 5 min, then 10 mg YADH-bound
magnetic nanoparticles(5 ml 0.23 mg protein/ml YADH solution
immobilized on 50 mg magnetic nanoparticles)was added and
mixed for several minutes; the solution was separated from the
magnetic nanoparticles via a permanent magnet and the decrease
was measured in absorbance of NADH at 340 nm in 3 min. The
For the thermal stability test, 1 ml of free enzyme (430 U/ml)
and 10 mg immobilized enzyme (32.98 mg/g) were incubated in a
water bath for 60 min at different temperatures ranging from 40
to 70 1C. After each incubation period, the enzyme was quickly
chilled in crushed ice for 5 min. The enzyme was brought to room
temperature and the residual enzyme activity was determined as
described above. The residual activities were expressed as relative
to the original activity assayed without heating.
The reusability of immobilized YADH was performed by
conducting activity measurement of immobilized YADH at 25 1C
at time intervals of 30 min. After each activity measurement, the
immobilized YADH was separated by magnetization, washed
three times with a phosphate buffer (0.02 M, pH 7.0). Then the
fresh NADH and phenylglyoxylic acid solutions were added to the
immobilized YADH in sequence and the next activity measure-
ment was carried out and compared with the first run (activity
defined as 100%).
concentration of NADH was calculated from
DA340nm. Unless
specially stated, the amount of YADH to 10 mg magnetic nanopar-
ticles was 1 ml 0.23 mg protein/ml YADH solution. The activity of
YADH was determined in phosphate buffer (0.02 M, pH 7.0) at 25 1C,
and the concentrations of NADH and phenylglyoxylic acid in the
reaction mixture were 1 mM and 0.1 M, respectively.
For free YADH, the activity measurement was done following
similar procedures and conditions for the immobilized YADH,
except for free YADH solution used.
One unit of YADH activity was defined as the amount of YADH,
which used 1 nmol NADH per minute at the assay conditions.
The activity recovery (%) was the ratio between the activity of
immobilized YADH and the activity of free YADH.
The relative activity (%) was the ratio between the activity of
every sample and the maximum activity of sample.
3. Results and discussion
2.7. Optimization of the reaction conditions of enzymatic activity
3.1. Effect of immobilization time on immobilized enzyme process
To determine the optimum pH and temperature for the
Fig. 1 shows the effect of immobilization time on the bound of
reduction of phenylglyoxylic acid to (R)-mandelic acid by free
3 4
YADH onto the magnetic Fe O /KCTS nanoparticles. It was found

Gui-yin Li et al. / Journal of Magnetism and Magnetic Materials 322 (2010) 3862–3868
3865
3
3
3
2
2
4
2
0
8
6
36
3
2
2
2
1
2
8
4
0
6
0.05
0.10
0.15
0.20
0.25
0.30
0.35
Initial YADH concentration (mg/ml)
0
30
60
90
120
150
180
Immobilization time (min)
Fig. 2. The effect of concentration of YADH protein on immobilized enzyme
process (Immobilization conditions: at 0.02 M phosphate buffer (pH 7.4) and 25 1C
with 5.0 ml different concentration of YADH protein on 50 mg supports for
Fig. 1. The effect of immobilization time on immobilized enzyme process
Immobilization conditions: at 0.02 M phosphate buffer (pH 7.4) and 25 1C with
.0 ml 0.23 mg/ml YADH protein on 50 mg supports).
(
120 min).
5
3
3
3
2
4
2
0
8
that there was a gradual increase of immobilization YADH protein
until equilibrium. When the immobilization time was lower than
1
20 min, the enzyme loading amount on the particles would keep
pace with the immobilization time. But when the immobilization
time was longer than 120 min, the enzyme loading amount
on the particles would decrease. The time necessary to reach
equilibrium was about 120 min and significant increases in the
adsorbed amounts were not observed after the equilibrium
stage. The possible explanation might be the interaction between
the functional groups on the surface of particles and YADH
enzyme. When these particles were added to the YADH solution,
YADH molecule would be absorbed around the particles by
electrostatic adsorption first, and then be immobilized onto the
particles via a covalent reaction between carboxyl groups of
the particles and the amino groups (or thiol, hydroxyl groups) of
the YADH.
26
2
2
2
4
2
0
5
.5
6.0
6.5
7.0
pH
7.5
8.0
3.2. Effect of YADH concentration on immobilized enzyme process
Fig. 3. The effect of pH on immobilized enzyme process(Immobilization condi-
tions: at 25 1C with 5 ml 0.23 mg/ml YADH protein on 50 mg supports for
In order to investigate the effect of enzyme concentration,
initial YADH enzyme concentration was changed between 0.08
and 0.34 mg YADH protein/ml with 5 ml on 50 mg magnetic
supports. As seen in Fig. 2, an increase in YADH enzyme
concentration in the medium led to an increase in enzyme
loading, when the initial YADH concentration of 0.23 mg/ml, the
enzyme loading amount reached 32.98 mg/g, then the amount of
YADH adsorbed was little affected when the YADH concentration
is higher than 0.23 mg/ml. It is a well-recognized fact that the
process of protein adsorption on a solid surface is a two-regime
process [28]. At the initial stage, the solid surface is bare, and
the kinetics of adsorption is governed by the diffusion of the
molecules from the bulk solution to the surface. All of the
molecules that arrive at the surfaces are assumed to be
immediately adsorbed. At the later stage, a barrier of adsorbed
molecules exists, and the molecules arriving from solution have to
diffuse across this barrier. We can also know that the YADH
activity declined after the YADH was immobilized. 65% of the
activity of the free enzyme was kept after immobilization when
120 min).
3.3. Effect of pH on the immobilization process
Fig. 3 shows the effect of incubation pH on the immobilization
process. The enzyme loading increased on increasing the pH of the
solution (pH 5.8–7.4); the highest enzyme loading was gained at
pH 7.4. The immobilized behavior showed that adsorption of
protein onto magnetic nanoparticles was governed by hydro-
phobic interaction and by electrostatic interactions [29,30]. The
influence of pH showed decreasing affinity with increasing
electrostatic repulsion between protein and adsorbent and a
maximum plateau value at pH 7.4.
In brief, the optimal conditions of immobilized YADH were:
immobilization time 120 min, immobilization pH 7.4 and 5 ml
3 4
0.23 mg/ml YADH protein on 50 mg magnetic Fe O /KCTS
nanoparticles. Under these conditions, the activity recovery of
the immobilized YADH was 65% and the protein loading was
32.98 mg/g.
5
ml 0.23 mg/ml YADH was immobilized on 50 mg magnetic
3 4
Fe O /KCTS nanoparticles.

3
866
Gui-yin Li et al. / Journal of Magnetism and Magnetic Materials 322 (2010) 3862–3868
3.4. SEM micrographs of the immobilized YADH
100
Free enzyme
Immobilized enzyme
The immobilization of YADH on the magnetic Fe
3
O
4
/KCTS
nanoparticles was investigated by scanning electron microscopy
SEM). Fig. 4a and b shows the SEM images of Fe /KCTS without
9
8
7
0
0
0
(
3 4
O
and with bound YADH enzyme, respectively. Before enzyme
immobilization, the particles remained discrete and had a similar
size. After enzyme immobilization, it was observed in Fig. 4b that
the particles were covered with a certain amount of enzymes and
the particles became uneven with a rough surface. The SEM
images confirmed that the YADH molecules have been substan-
tially immobilized on the magnetic Fe
adsorption.
3 4
O /KCTS nanoparticles by
60
50
3
.5. Effect of pH on the activity of immobilized YADH
5.0
5.5
6.0
6.5
pH
7.0
7.5
8.0
The effect of pH on the activity of the free and immobilized
enzyme for the reduction of phenylglyoxylic acid has been
studied at different pH ranging from 5.8 to 8 at 30 1C, and the
data are shown in Fig. 5. As seen from Fig. 5, the free and
immobilized enzymes showed different behaviors of pH
Fig. 5. Effect of pH on the activity of free and immobilized YADH for the reduction
of phenylglyoxylic acid (Incubated in phosphate buffer (0.02 M, pH 5.8–8.0) at
30 1C for 5 min).
1
00
9
8
7
6
5
4
0
0
0
0
0
0
Free enzyme
Immobilized enzyme
0
10
20
30
40
50
Temperature (°C)
Fig. 6. Effect of temperature on the activity of free and immobilized YADH for the
reduction of phenylglyoxylic acid (Incubated in phosphate buffer (0.02 M, pH 7.4)
for 5 min in the range of 4–50 1C).
dependence, the maximum activity for free YADH can be clearly
seen at pH 6.8, and the optimum pH of the immobilized form was
found at pH 7.4. From pH 6.8 to 7.4, the relative activity of the
immobilized enzyme kept high activity, while the native enzyme
was more sensitive than the immobilized enzyme in the range of
pH 7.0 to 8.0. Similar phenomena were also observed in previous
reports [13,31]. It was presumed that the configuration of YADH
fixed on the surface of carriers might lead to an increasing of the
enzyme’s tolerability to pH variance in surroundings, or the pH of
the carriers’ inner was changed because of the surface charge of
the carriers. It would be ideal if immobilized enzymes could be
functional at a wide range of pH values for many practical
applications.
3.6. Effect of temperature on the activity of immobilized YADH
The effect of temperature on the free and immobilized YADH
activities were investigated at five different temperatures be-
tween 4 and 50 1C in the presence of 0.02 M phosphate buffer (pH
7.4) by using phenylglyoxylic acid as the substrate, and the results
Fig. 4. SEM micrographs of the magnetic nanoparticles without (a) and with (b)
bound YADH.

Gui-yin Li et al. / Journal of Magnetism and Magnetic Materials 322 (2010) 3862–3868
3867
are given in Fig. 6. From Fig. 6, it was found that the optimal
1
00
25
20
15
temperature for both free and immobilized YADH to achieve the
o
highest activity was 30
%
C, which was consistent with that of
90
other research [24,9]. At low temperature section (from 4 to
0 1C), the catalytic activity of free and immobilized YADH
relative activity
8
7
6
0
0
0
3
leakage rate of protein
increased with the rise of the temperature at first and, after a
maximum for both enzyme at 30 1C, decreased at higher
temperature. For free enzyme, activity drops dramatically with
increasing temperature, which was only 46% relative activity at
1
5
0
0
5
0 1C. The activity of immobilized YADH was far less influenced
50
by temperature, which remained 68% relative activity at 501C. The
effect of temperature on the activity of enzyme has two aspects:
raising the temperature can speed up the enzymatic reaction and
raising the temperature can also make the enzyme denatured. The
optimum temperature for enzyme reaction is the comprehensive
results of the reaction rate of enzyme and thermal stability.
Usually after enzyme immobilization, the optimum temperature
will increase with increase in thermal stability, which is a very
beneficial result. Of course, the optimal temperature was reported
unchanged or decreased [32].
4
3
0
0
1
2
3
4
5
6
Times
Fig. 8. Reuse of the immobilized YADH for the reduction of phenylglyoxylic acid
The initial activity of the immobilized YADH is 280.75 U).
(
it is possible that a very intense multi-interaction with the
support may have some negative effect on the subunits assembly,
reducing the enzyme stability even although each monomer can
be more ‘‘rigid’’. Thus, from 40 to 50 1C, the residual activity of
immobilized YADH decreased sharply than that of free YADH.
However, when the incubation temperatures are above 60 1C, the
residual activities of immobilized enzyme are consistently higher
than that of free enzyme. Immobilized YADH holds approximately
3
.7. Thermal stability of immobilized YADH
The thermal stability of the immobilized enzyme is one of the
most important criteria with respect to applications. In order to
test the thermal stability of immobilized YADH, the thermal
stability of free and immobilized YADH was determined by
incubation of the enzymes in different temperatures (shown in
Fig. 7) for 60 min, and their residual activity was measured. The
data in Fig. 7 showed that both free and immobilized YADH
enzymes had diminished relative activities from 40 to 70 1C.
YADH is a tetrameric metalloprotein of molecular mass 150 kDa
and contains two zinc atoms per subunit, one in the active site
54% at 70 1C, while free enzyme holds less than 22% at 65 1C and
no activity at 70 1C. These observations were in agreement with
the findings of some earlier investigators [9,11,12]. Clearly at high
temperatures, loss in the YADH activity of soluble form was due to
denaturation of enzyme. Thermal stability of YADH improved
3 4
after immobilization on the magnetic Fe O /KCTS nanoparticles,
(catalytic zinc) and one in a loop (conformational zinc). The
which is due to either the creation of conformational limitation on
the enzyme movement or a low restriction in the diffusion of the
substrate at high temperature. The results indicate that the
immobilized YADH enzyme had a good thermal stability.
presence of conformational zinc helps keep the conformation of
the active site in a strained state [33]. Residues 40–60 in YADH
may function as an intramolecular chaperone and prevent
complete unfolding of YADH during thermal stress [34]. It has
d
been reported that the thermal denaturation temperature T for
3
.8. Reusability of immobilized YADH
YADH was 63 1C, which was caused by the irreversible thermo-
inactivation of the protein such as thiol group oxidation,
aggregation and deamidation of the protein [35]. From Fig. 7,
the free YADH had more enzyme activity below 50 1C, but less
activity above 65 1C. Due to the tetrameric nature of this enzyme,
To investigate the reusability of immobilized YADH, several
repetitive uses of immobilized YADH were operated. After each
cycle, the immobilized YADH was recovered by magnetic separation
and recycled for the reduction of phenylglyoxylic acid. The super-
natant solution was used to detect the leakage of protein. The
activity of the first batch was taken as 100% and the leakage rate of
protein is 0. The assay condition remained the same for all batches.
Fig. 8 shows variation of activity of the immobilized YADH and the
leakage rate of protein after multiple reusing by magnetic separa-
tion. Within 5 cycles of usage, the relative activity is about 100%,
100
8
6
4
2
0
0
0
0
0
Free enzyme
7
6%, 68%, 55% and 32% of the first run, while the leakage rate of
Immobilized enzyme
protein is 0%, 22%, 23%, 23% and 25%. The decrease in activity may be
caused by the leakage of protein from the carriers and the enzyme
denaturation. Although the immobilized YADH activity decreased
significantly, the immobilized YADH had a good durability and could
be readily recovered by magnetic separation. This revealed the
magnetic Fe
immobilization of enzymes.
3 4
O /KCTS nanoparticles could be practically used for the
4
. Conclusions
30
40
50
Temperature (°C)
60
70
The surface functionalization of chitosan-coated magnetic
(
Fe
3 4
O /KCTS) nanoparticles was used to immobilize YADH by
Fig. 7. Thermal stability of free and immobilized YADH (incubated in phosphate
buffer (0.02 M, pH 7.0) for 60 min in a range of temperature 40–70 1C).
electrostatic adsorption and covalent binding. The process was

3
868
Gui-yin Li et al. / Journal of Magnetism and Magnetic Materials 322 (2010) 3862–3868
simple and effective. The optimal conditions of immobilized
YADH were: immobilization time 120 min, immobilization pH 7.4
and 5 ml 0.23 mg/ml YADH protein on 50 mg supports. Under the
conditions, the activity recovery of the immobilized YADH was
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6
5% and the protein loading was 32.98 mg/g. For the reduction of
[
phenylglyoxylic acid, the maximal activity of the immobilized
YADH appeared at 30 1C and pH 7.4, and the immobilized YADH
retained much of its activity in wider ranges of temperature and
pH than that of the free form. In addition, the immobilized
enzyme maintained a greater rigidity and was more resistant to
unfolding at higher temperatures than its free form. The
immobilized YADH had good durability and could be readily
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3 4
recovered by magnetic separation. Using magnetic Fe O /KCTS
[
[
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to R-(-)-mandelic acid by Saccharomyces cerevisiae FD11b, Enzyme Microb.
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be helpful for the practical application of YADH.
[
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[
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Acknowledgements
This work was supported by the Postdoctoral Research Fund of
China (No. 20090461026) and the Postdoctoral Science Founda-
tion of Central South University (200910).
1
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