D.F.M. Neri et al. / Applied Catalysis A: General 401 (2011) 210–214
211
Here, magnetic POS–PVA (mPOS–PVA) particles were used as
2.5. Reuse of immobilized XOD on POS-PVA magnetic support
a matrix for XOD immobilization. Some properties of the immo-
bilized enzyme were investigated (apparent Km using xanthine
as substrate; optimal pH and temperature and reuse) and the
catalytic action on the 6-mercaptopurine oxidation pathway.
The reuse of the immobilized XOD activity was evaluated incu-
bating the same preparation with fresh 1 mL of xanthine (100 M)
every 10 min for 10 times. The uric acid production was spectropho-
tometrically evaluated as described above.
2
. Experimental procedures
3. Results and discussion
2.1. Xanthine oxidase purification
Xanthine oxidase was extracted from bovine milk and par-
Fresh bovine milk (1 L without any preservative) was unstirred
◦
tially purified by ammonium sulfate precipitation. The fraction
precipitated at 38–50% of ammonium sulfate presented the highest
specific activity (69 mU/mg of protein with purification and yield
of 1.57 and 66%, respectively). Previous report demonstrated that
immobilized XOD using highly purified preparations lost activity
during reuses [6]. Probably, this deleterious effect is due to the
kept at 4 C overnight. The cream milk was separated from the
◦
fresh milk and then was hand churned for 3 h at 4 C. Afterwards,
the overlaid layer was discarded and the buttermilk was filtered
by using cheesecloth. Then casein was precipitated at pH 4.8 by
dropping 1 M HCl, followed by centrifugation at 27,000 × g for
1
1
5 min and immediately increasing pH value up to pH 7.0 by adding
M NaOH. The casein-free buttermilk (65 mL) was 38% saturated
−
release of free radicals (O2 ) and H O produced during XOD activ-
2
2
ity. Addition of catalase and superoxidase dismutase prevented this
effect [18]. Therefore, this partially purified enzyme preparation
was used throughout this work.
by adding ammonium sulfate. The suspension was centrifuged at
2,000 × g for 15 min and the precipitated was discarded. Fur-
1
ther ammonium sulfate was added to the 0–38% ammonium
sulfate supernatant to 50% saturation. The precipitate (38–50% frac-
tion) was collected by centrifugation at 27,000 × g for 30 min and
dissolved in 5 mL of 10 mM Tris–HCl, pH 8.0, containing 5 mM 2-
mercaptoethanol and 100 M EDTA. This suspension was dialyzed
twice, first against the buffer containing 100 M EDTA and later
against buffer containing 30 M EDTA. The dialyzed material was
The scheme of the chemical basis of this immobilization proce-
dure is described elsewhere [13]. The POS–PVA composite degree of
swelling has been described to decrease remarkably with increas-
ing TEOS content [11]. The POS–PVA ratio used in this work allows
a high degree of PVA swelling and a hydrophilic microenvironment
for the enzyme catalytic action. Those hybrid beads were smashed
into small particles (powder) to increase surface area for immobi-
lization. The inconvenience of using small particles was overcome
◦
finally centrifuged at 35,000 × g and the supernatant stored at 4 C
and used throughout this work.
2
+
3+
by magnetization, co-precipitating Fe and Fe ions in ammonia
◦
solution at 100 C, which allowed the particles to be easily collected
2
.2. POS–PVA synthesis, magnetization and XOD immobilization
under a magnetic field.
The immobilization of XOD on mPOS–PVA yielded preparations
containing 9.5 ± 0.5 g of protein per mg of support. This would be
equivalent to 63.5 ± 3.3% of the offered XOD for the 10 mg of the
composite. Furthermore, these immobilized XOD POS-PVA deriva-
tives presented specific activity of 36.3 ± 7.8 mU/mg of protein,
retaining 55.0 ± 11.7% of that found for the free enzyme. The homo-
geneity of the magnetic particles sizes was demonstrated by the
linear relationship achieved between the volume of the magnetic
particles suspension versus their weight and specific enzymatic
activities (data not shown).
The magnetic POS–PVA synthesis and enzyme immobilization
were carried out according to Neri et al. [13,14], except that XOD
preparation (127 g/mL) replaced the -galactosidase and the used
buffer was 10 mM Tris–HCl, pH 8.0, containing 100 M EDTA. The
particles were collected and the supernatant used for protein deter-
mination according to Sedmak and Grossberg [17] using bovine
serum albumin as the standard. The bound protein was calculated
by the difference between the offered protein and that estimated
in the supernatant.
◦
The optimum pH (8.8) and temperature (60 C) found for the
2
.3. Enzyme activity measurements
immobilized XOD on the mPOS–PVA particles were slightly higher
◦
than those established for the free enzyme (8.2 and 55 C, respec-
The soluble XOD (50 L) was incubated with 1000 L of 100 M
tively), as one can see in Fig. 1. Shift to the right for the immobilized
enzyme pH profile compared to the free enzyme one is usually
caused by negatively charged support. Thus the enzyme microen-
vironment would presents higher hydroxonium concentration
attracted by the support demanding for higher hydroxide ions in
the bulk of the reaction, where de enzyme activity is measured.
Here, the immobilized enzymatic preparation is composed of XOD,
a semi-interpenetrated network of polysiloxane and polyvinyl alco-
xanthine, prepared in 50 mM of Tris–HCl, pH 8.2 (from now
on called the buffer) and following the uric acid production at
− −1
1
2
95 nm (ε293 = 9.5 mM cm ). The immobilized enzyme (10 mg)
was mildly stirred with 1000 L of 100 M xanthine prepared in
the buffer and at time intervals of 5 min the magnetic enzymatic
preparation collected by a magnetic field of 0.6 T; the supernatant
absorbance read at 295 nm and immediately reincubated with the
immobilized enzyme allowing the reaction to proceed for another
hol strongly and magnetite (Fe O ). Further studies should be
3
4
5
min. Immobilized XOD activity on 6-mercaptopurine was also
carried out to evidence the presence of negative charge on the
support surface. However, its presence does not seem to change
optimum pH significantly.
The increase in the optimum temperature activity of the immo-
bilized enzyme has been attributed to the more rigid conformation
generated by bonds linking the enzyme to the water insoluble
matrix [19]. The optimal pH values widely vary in the litera-
evaluated by incubating a 650 M solution prepared in the buffer at
◦
2
5 C. Then the supernatant of the enzymatic reaction was simulta-
neously analyzed at 240 nm, 256 nm, 308 nm and 340 nm in 10 min
intervals.
2
.4. Optimal pH and temperature
ture reports: 6–8 for XOD immobilized on CuPtCl /glassy carbon
6
The effect of pH and temperature on the enzyme activity were
chemically modified electrode [20]; 7.3 on poly (mercapto-p-
benzoquinone) [21]; 8.4 on polypyrrole film [22] and 6.0–7.5 on
nanocrystal gold–carbon paste electrode [8]. Lower optimal tem-
◦
investigated in the ranges of 7.6–9.0 and 30–65 C, respectively,
using the above described activity determination procedures and
xanthine as substrate.
◦
peratures have been reported for immobilized XOD such as 42 C