382 Sun et al.
Asian J. Chem.
In the second step, magnetic beads were prepared by
convention co-precipitation reaction of iron oxide in the beads.
2.5 g FeCl2 was dissolved in 100 mL purified water and then
was added into a reactor containing 5 g ferric-poly(GMA-ST)
beads obtained in the first step in NH3·H2O (20 mL, 25 % w/v).
The reactor was also equipped with a mechanical stirrer,
nitrogen inlet and reflux condenser at three temperatures
(40 ºC, 50 ºC, 90 ºC for 2 h). Then the beads were kept in
50 % ethanol solution for 3 h and then washed with water.
Finally, the magnetic poly (GMA-ST) beads were transferred
in phosphate butter (pH 8.0; 22.5 mL), containing 2.5 mL of
glutaric dialdehyde (50 %). The reaction was carried out at
25 ºC for 6 h under magnetic stirring. The activated beads were
washed with distilled water and acetic acid solution (0.1 M,
100 mL) and then dried under vacuum for 5.0 h. The resulting
magnetic poly(GMA-ST) beads were used for the immobilization
of β-galactosidase.
keV
Fig. 1. X-ray spectroscopy of the magnetic poly(GMA-ST) beads;
(Accelerating voltage: 25 KeV; take off angle: 36.7739 ; live time:
100 sec; dead time: 14.504)
Preparation of enzyme and substrate solution: 0.0500 g
of β-galactosidase was dissolved in 10 mL of 0.1 M citric
acid buffer (pH 4.0) and then kept in the refrigerator at 4 ºC
for use. The substrate solution was obtained by dissolving
0.0150 g ONPG in 10 mL twice distilled water.
Fig. 2. SEM photographs of the magnetic poly(GMA-ST) beads
Immobilization of β-galactosidase: 0.0500 g of polymer
particles was put in 0.5 mL 0.1 M citric acid buffer (pH 4.0)
containing enzyme (5 mg/mL). The reaction was undergone in
ultrasonic cleaning machine at 25 ºC for 3 h. After that, the
immobilized enzyme was filtered and washed with water and
0.1 M citric acid buffer (pH 5.0) until there was no protein.
Activity assays of β-galactosidase:Activities of free and
immobilized β-galactosidase were assayed by the addition of
0.1 mL of free enzyme or 0.0500 g of immobilized β-galacto-
sidase in the citric acid buffer (pH 5.0), using 0.2 mL of ONPG
(1.5 mg/mL) as the substrate5,6. After 15 min of incubation in
a shaking water bath at 55 ºC, the reaction was stopped by
adding 2 mL of Na2CO3 solution (1 M). The absorbance of
medium was recorded at 405 nm. All activity measurement
experiments were carried out three times. One unit of β-
galactosidase activity is defined as the amount of enzyme
that liberated 1 µmol of product per minute under the assay
condition.
Under the optimum conditions, the magnetic beads were
used to immobilize β-galactosidase and the results obtained
were listed in Table-1. According to the data, the activity of
the immobilized enzyme could reach a maximum of 412.09
U/g dry carrier and the activity recovery of the immobilized
β-galactosidase was 61.32 %. The obtained enzyme activity
was higher than values obtained on the non-magnetic
poly(GMA-ST) beads.
TABLE-1
IMMOBILIZATION RESULTS OF β-GALACTOSIDASE ON
THE MAGNETIC BEADS
Immobilized
enzyme activity
(U/g dry carrier)
Activity
yield
(%)
Carrier
Magnetic poly(GMA-ST) beads
412.09
266.15
61.32
39.62
Non-magnetic poly(GMA-ST) beads
Properties of the immobilized β-galactosidase
Effect of pH and temperature on the catalytic activity:
The effect of pH values of free and immobilized enzymes was
determined in 3-9 pH range and the results were shown in
Fig. 3. The maximum value of relative activity was observed
at pH 5.0 for both free and immobilized enzymes. The enzyme
activity was determined by ONPG as substrate, at 55 ºC in
various pH buffers (3-9) for 15 min.
RESULTS AND DISCUSSION
Discussion about the magnetic poly(GMA-ST) beads:
The magnetism of the poly (GMA-ST) beads obtained was
evidenced by magnetic scales and X-ray spectroscopy. From
the results, it could be seen that magnetic susceptibility (Km)
of the beads ould reach 1.25 × 10-4 cm3/g. X-ray spectroscopy
of magnetic poly (GMA-ST) beads presented in Fig. 1 clearly
showed that the beads obtained described above contained Fe,
which also indicated that magnetism was successfully formed
within the structure of poly(GMA-ST) beads.
The effect of temperature (40-65 ºC) on the free and
immobilized enzymes activities were investigated by using
ONPG as substrate as shown in Fig. 4. The optimum tempe-
rature of both free and immobilized enzymes were at 55 ºC.
pH and thermal stability: Free and immobilized enzymes
were exposed to different pH (2.0-9.0) at room temperature
overnight and then the enzyme activities were determined with
ONPG as substrate. Fig. 5 shows that the immobilized enzymes
hold a better adaptability.
Meanwhile, scanning electron microscopy micrographs
were also done to characterize its surface structure and the
result was illustrated in Fig. 2. The photographs showed that
the magnetic beads had a porous surface structure, which
would be suitable for the immobilization of enzymes and also
provide a good transmission for substrate and product during
the enzymatic reaction.
The thermal stability of free and immobilized enzymes
could be seen in Fig. 6. After incubation at 50 ºC for 8 h,