Hydrolysis of phosphoric acid ester across an enzyme membrane: Potato acid phosphatase immobilized on a Na-perfluorocarboxylate ionomer membrane for recovery of reaction product

Article abstract of DOI:10.1246/bcsj.79.343

Acid phosphatase from potato was immobilized onto the surface of the external solution side of a perfluorocarboxylate ionomer membrane using a cross-linking reagent. Based on the distinct difference in membrane permeabilities to 4-nitrophenol and 4-nitrop

Full text of DOI:10.1246/bcsj.79.343

ꢀ
2006 The Chemical Society of Japan
Bull. Chem. Soc. Jpn. Vol. 79, No. 2, 343–347 (2006)
343
Hydrolysis of Phosphoric Acid Ester across an Enzyme Membrane:
Potato Acid Phosphatase Immobilized on a Na–Perfluorocarboxylate
Ionomer Membrane for Recovery of Reaction Product
ꢀ
Kazuo Nomura and Mo Liu
Department of Chemistry, Faculty of Science, Kyushu University, 4-2-1 Ropponmatsu, Chuo-ku, Fukuoka 810-8560
Received June 28, 2005; E-mail: nomura@chem.rc.kyushu-u.ac.jp
Acid phosphatase from potato was immobilized onto the surface of the external solution side of a perfluorocarbox-
ylate ionomer membrane using a cross-linking reagent. Based on the distinct difference in membrane permeabilities to 4-
nitrophenol and 4-nitrophenyl phosphate, a membrane reactor system using the immobilized enzyme has been designed
for the recovery of the reaction product of enzymatic hydrolysis. To observe the recovery rate for the hydrolysis product,
the flux from the external solution side to the internal solution side crossing the membrane was measured in the substrate
ꢂ2
ꢂ3
concentration range below 1 ꢁ 10 mol dm . The recovery rate was found to obey a Michaelis–Menten type equation.
The Michaelis constant for the immobilized enzyme was smaller than that for the free enzyme obtained from the kinetic
properties in the bulk solution. The optimum pH value for the immobilized enzyme membrane was investigated.
Different kinds of residual synthesized organic compounds
in water in the environment can be decomposed by virtue of
enzymes from natural sources. Immobilization of an enzyme
free enzyme, which was obtained from the kinetic properties
in bulk solution. The pH dependence of the recovery rate
has also been investigated.
1
on a membrane enables its activity to be retained for a long
period of time; therefore, many types of membrane reactor
systems have been designed to use enzymes for the elimination
Experimental
Materials. Acid phosphatase (E.C.3.1.3.2) from potato with a
ꢂ1
2,3
and recovery of residual organic compounds in water.
protein concentration of 10 mg mL was obtained as a suspension
ꢂ3
On the other hand, in the rhizosphere region of soil, organo-
phosphates are hydrolyzed by extracellular enzymes such as
urease, acid phosphatase, and alkaline phosphatase secreted
from plant roots. The enzymatic activity of these extracellular
soil enzymes are retained by immobilization on clay or into
in 3.2 mol dm (NH4)2SO4 stabilized with bovine serum albumin
(
CalBiochem, U.S.A.). Disodium 4-nitrophenyl phosphate (Fluka,
Switzerland) was used to prepare the substrate solution. All other
reagents were of guaranteed grade and used without further puri-
fication. Water was purified by double distillation, of which one
was from an alkaline potassium permanganate solution. A per-
fluorocarboxylate polymer membrane (Flemion 230, kindly sup-
plied by the Asahi Glass Co., Tokyo, with an ion exchange capaci-
ty of 1.4 mmol univalent ion/g dry)13,14 was used as the support-
ing material for the enzyme.
4–6
organic substances by adsorption or incorporation.
In this communication, acid phosphatase from potato has
been immobilized onto the surface of an artificial perfluorocar-
boxylate ion-exchange membrane. Potato acid phosphatase is a
typical phosphatase, which catalyzes the hydrolysis of phos-
phoric acid monoesters. On the other hand, in perfluorinated
ionomer membranes, as in inverted micellar systems, there
are dispersed hydrophobic regions formed by fluorocarbon
chains of the polymer backbone and hydrophilic regions
formed by clustering of the ionic groups of the polymer, coun-
ter ions, and water molecules.7 As described in the literature,
immobilization by incorporating an enzyme into a perfluoro-
sulfonate ionomer membrane (Nafion) has already been utiliz-
ed to form the enzyme layer of biosensors.1
Immobilization of Enzyme. The ionomer membrane had
ꢂ3
been immersed in a 0.1 mol dm NaCl solution for over 24 h at
ꢃ
2
5 C before the enzyme immobilization. A mixture of 25 mL of
ꢂ1
the enzyme solution (10 mg mL acid phosphatase) and 25 mL of
the aqueous acetate buffer solution (0.1 mol dm acetate buffer
ꢂ3
ꢂ3
pH 4.8 with 0.1 mol dm NaCl) was stirred and then 30 mL of
it was placed on the surface of one side of the perfluorinated car-
–9
ꢃ
boxylate ionomer membrane. After leaving it at 25 C for one
ꢃ
hour, the membrane with the enzyme was stored at 5 C overnight.
After the membrane covered with the enzyme suspension was
0–12
Based on the distinct difference in membrane permeabilities
to 4-nitrophenol and 4-nitrophenyl phosphate observed in this
study, a membrane reactor system using an immobilized en-
zyme membrane has been designed for the purpose of the re-
covery of the reaction product of enzymatic hydrolysis. From
the substrate-concentration dependence of the recovery flux of
the product from the enzyme-layer side to the other side by
crossing the membrane, the Michaelis constant for the immo-
bilized enzyme was obtained and compared with that for the
returned to room temperature, 50 mL of a mixed solution of a
15–18
cross-linking reagent (2.5 wt % glutaraldehyde
solution) with
an equivalent volume of a phosphate buffer solution (pH 7.4) was
placed into the enzyme suspension on the membrane surface.
After one hour, one side of the membrane, over which the enzyme
was immobilized in the micropores, was washed with a 0.1
ꢂ3
mol dm NaCl solution. The immobilized enzyme membrane
ꢃ
mounted in the cell assembly was stored at 5 C before the flux
measurement.
Published on the web February 10, 2006; DOI 10.1246/bcsj.79.343

3
44 Bull. Chem. Soc. Jpn. Vol. 79, No. 2 (2006)
Hydrolysis across Immobilized Enzyme Membrane
Fig. 1. Schematic diagram of assembled system for the
measurement of 4-nitrophenol amount transported through
the enzyme membrane. The time dependence of the
amount of phenol in the internal solution was obtained
through the measurement of the absorption spectrum using
an immersed probe (P) connected to a fiber UV spectro-
ꢂ4
ꢂ3
Fig. 2. Absorption spectra of 1 ꢁ 10 mol dm 4-nitro-
ꢂ4
ꢂ3
phenol and 1 ꢁ 10 mol dm 4-nitrophenyl phosphate
ꢂ3
in 0.1 mol dm acetate buffer solution (pH ¼ 4:8), which
ꢂ3
contained 0.1 mol dm NaCl: 4-nitrophenol (a); 4-nitro-
phenyl phosphate (b).
photometer. The arrow (
) shows the path of the ultra-
violet radiation. Immobilized-enzyme membrane, magnet-
ic spin bar, and thermostatically controlled water are des-
ignated by M, S, and W, respectively.
Measurement of Flux. The assembled measuring system for
the flux of the hydrolysis product is schematically illustrated in
Fig. 1. The immobilized enzyme membrane was horizontally
mounted in the cell for the measurements. The area of the mem-
brane surface adjacent to the solution (diffusion area) was
2
0.20 cm . The layer of acid phosphatase immobilized at the sur-
face was on the external solution side of the enzyme membrane.
Initially, both the external and internal solutions contained only
ꢂ3
0
.1 mol dm NaCl. The pH’s of both compartments were adjust-
ꢂ3
ed with a 0.1 mol dm acetate buffer. The measurement was start-
ed by injecting the substrate (4-nitrophenyl phosphate) solution
into the external solution. The hydrolysis product (4-nitrophenol)
could permeate through the non-activated region of the ionomer
membrane and cross the membrane–solution interface on the
internal solution side. In the experiments, the amount of 4-nitro-
phenol in the internal buffer solution of NaCl was followed by
measuring the time course of optical absorbance at 317 nm. The
UV spectra for 4-nitrophenol as well as for 4-nitrophenyl phos-
phate at pH 4.8 are shown in Fig. 2. The linearity of the cali-
bration curve for the optical absorbance at 317 nm vs the concen-
tration was confirmed (correlation coefficient; R ¼ 0:999) in an
ꢂ5
ꢂ3
Fig. 3. Absorption spectra of 2 ꢁ 10 mol dm 4-nitro-
ꢂ5
ꢂ3
phenol and 2 ꢁ 10 mol dm 4-nitrophenyl phosphate
ꢂ3
in 0.1 mol dm Tris buffer solution (pH 8.5), which con-
ꢂ3
tained 0.1 mol dm NaCl: 4-nitrophenol (a); 4-nitrophen-
yl phosphate (b).
solution. The enzymatic reaction started with the injection of an
amount of the substrate (4-nitrophenyl phosphate) solution. The
ꢂ4
ꢂ3
experimental concentration range (up to 2:0 ꢁ 10 mol dm ).
ꢃ
A probe of the transmission type with a light-path length of
1
temperature of the solution was maintained at 25.0 C by circula-
cm was immersed in the internal solution and connected to a
tion of the thermostatically controlled water. The solution of each
compartment was stirred by a magnetic stirrer at 650 rpm. At a
certain time interval, 2 mL of the reaction mixture was drawn
dual type fiber optic spectrometer (Model SD-2000, Ocean Optics,
Dunedin, Florida, U.S.A.) by an optical fiber. Throughout the
measurements, the temperature of the membrane system was
ꢂ3
and immediately mixed with 2 mL of 0.035 mol dm NaOH to
ꢃ
maintained at 25.0 C by the circulation of thermostatted water.
terminate the hydrolysis. After adjusting the pH of the solution
with Tris buffer (pH ¼ 8:5), the liberated 4-nitrophenol was deter-
mined by measuring the optical absorbance at 400 nm using the
same spectrometric system used for the flux measurements. The
absorption spectra for 4-nitrophenol as well as for 4-nitrophenyl
Each compartment was stirred by a magnetic stirrer at a speed
of 650 rpm.
Reaction Kinetics of Free Enzyme in Bulk Solution. The
3
ꢂ3
reaction rate was investigated using 50 cm of a 0.1 mol dm
ꢂ3
ꢃ
ꢂ3
buffer solution with 0.1 mol dm NaCl at 25.0 C. Five mL of
the stock solution of acid phosphatase was added to this mixed
phosphate in 0.1 mol dm NaCl at pH 8.5 adjusted with Tris buf-
fer are shown in Fig. 3. The calibration curve at pH 8.5 for the

K. Nomura et al.
Bull. Chem. Soc. Jpn. Vol. 79, No. 2 (2006)
345
Fig. 5. The membrane permeability of the perfluorocarbox-
ylate ionomer membrane in the absence of the enzyme.
The membrane permeability to 4-nitrophenol, PP, and the
flux of 4-nitrophenol, JP, are shown as a function of the
initial concentration of 4-nitrophenol in the external solu-
tion.
Fig. 4. Time course of the concentration in the internal
solution for 4-nitrophenol (a) and that for 4-nitrophenyl
phosphate (b). Two compartments are separated by the
perfluorocarboxylate ionomer membrane without enzyme.
Initially, both the external and the internal solutions
ꢂ3
contained 0.1 mol dm acetate buffer (pH 4.8) and 0.1
ꢂ3
mol dm NaCl. Initial concentration in the external solu-
tion of 4-nitrophenol or that of 4-nitrophenyl phosphate
ꢂ3
JP ¼ ꢂPPꢀCP;
ð1Þ
was 0.01 mol dm
.
where ꢀCP is the concentration difference of 4-nitrophenol
between the external and internal solutions.
absorbance at 400 nm vs the concentration was linear in an exper-
ꢂ5
ꢂ3
imental concentration range up to 4 ꢁ 10 mol dm (correlation
coefficient; R ¼ 0:999).
Hydrolysis of 4-Nitrophenyl Phosphate across Immobi-
lized Enzyme Membrane. In this study, the enzyme was
immobilized at the surface region on the external solution side
of the substrate (4-nitrophenyl phosphate). Because the layer
of the ion-exchange polymer membrane exhibits a high perme-
ability only to the reaction product (4-nitrophenol) as already
mentioned, the liberation of 4-nitrophenol in the enzyme layer
induces flux of the product to the internal solution for recov-
ery. After the flux measurement, the pH of the internal solution
was adjusted to pH 8.5. Then, the UV spectrum was measured
to confirm that the absorbance of 4-nitrophenyl phosphate near
310 nm was not observed. Although diffusional flow in the
opposite direction occurs simultaneously, this flow was not fol-
Results and Discussion
Membrane Transport across the Ionomer Membrane
without Enzyme. The sample of the ionomer membrane
ꢂ3
ꢃ
was immersed in 0.1 mol dm NaCl for over 24 h at 25 C be-
fore the flux measurement. Under the same conditions as for
the experiments of the enzyme membrane system, the flux
measurements were carried out for 4-nitrophenol and 4-nitro-
phenyl phosphate across the perfluorocarboxylate ionomer
membrane of the sodium form that was used as the supporting
membrane for the enzyme immobilization. Initially, both the
ꢂ3
external and internal solutions contained only 0.1 mol dm
lowed in this study. The flux of the product, J , was obtained
P
from the slope of the linear portion of the time-course curve
NaCl. The pH’s of both compartments were adjusted with ace-
tate buffer (pH ¼ 4:8). Flux measurement was started by in-
jecting a 4-nitrophenyl phosphate solution or 4-nitrophenol so-
lution into the external solution. The time course of the con-
centration was followed by the setup of the fiber optic spec-
for the amount of the reaction product recovered in the internal
solution. The results are plotted in Fig. 6 as a function of the
initial concentration of the substrate in the external solution.
The concentration dependence displays a saturation curvature
similar to the Michaelis–Menten kinetics for a free enzyme
reaction.
Presuming a mode analogous to the Michaelis–Menten type,
we plotted the inverse of the flux versus the inverse of the sub-
strate concentration. It can be seen that the linear relation holds
between these two quantities:
ꢃ
trometer shown in Fig. 1 at 25 C. Each compartment was
stirred at a speed of 650 rpm. An example of the time course
for the amount of each compound transported across the mem-
brane without the enzyme is shown in Fig. 4. A higher perme-
ability has been observed for 4-nitrophenol in contrast to 4-ni-
trophenyl phosphate, to which this membrane exhibits no sig-
nificant permeability under the conditions of the experiments.
The flux of 4-nitrophenol, JP, was obtained from the slope of
the linear portion after a transient region in the time course
curve. The concentration dependence of JP is shown in
Fig. 5, together with that of the membrane permeability to 4-
nitrophenol, PP, which is calculated according to the following
equation:
CS
max
JP ¼ JP
;
ð2Þ
ꢀ
K
þ CS
where CS denotes the initial concentration of the substrate in
M
max
the external solution. JP is the maximum rate of recovery
and K is the Michaelis constant for the immobilized acid
ꢀ
phosphatase from potato. K and JP could be obtained
M
ꢀ
max
M

3
46 Bull. Chem. Soc. Jpn. Vol. 79, No. 2 (2006)
Hydrolysis across Immobilized Enzyme Membrane
Fig. 7. The pH dependence of the flux of 4-nitrophenol pro-
duced during the hydrolysis of 4-nitrophenyl phosphate
using the immobilized enzyme. Activities (%) relative to
the flux at pH 4.8 are plotted as a function of the pH in
Fig. 6. Concentration dependence of flux across the en-
zyme membrane at pH 4.8. The flux of 4-nitrophenol pro-
duced during the hydrolysis of 4-nitrophenyl phosphate
using immobilized enzyme was determined by measuring
the absorption spectra of the internal solution. The initial
concentration of 4-nitrophenyl phosphate was in the range
ꢂ3
ꢂ3
the solution. The 0.1 mol dm buffer and 0.1 mol dm
NaCl were included in the substrate solution. The initial
concentration of 4-nitrophenyl phosphate in the external
ꢂ4
ꢂ3
ꢂ3
ꢂ3
ꢂ3
of 2 ꢁ 10 mol dm –5 ꢁ 10 mol dm . 0.1 mol dm
solution was 5 ꢁ 10ꢂ3 mol dmꢂ3
.
ꢂ3
acetate buffer (pH 4.8) and 0.1 mol dm
included in the substrate solution.
NaCl were
according to the following equation for an enzyme membrane
system:
ꢀ
max
1
1
max
P
K
1
M
¼ J
þ
:
ð3Þ
JP
J
CS
P
From the double reciprocal plot of 1=JP vs 1=CS (correla-
ꢀ
max
P
tion coefficient; R ¼ 0:999), we obtained K and J
to be
ꢂ2 ꢂ1
s , respec-
M
ꢂ4
ꢂ3
ꢂ12
1
:92 ꢁ 10 mol dm and 2:29 ꢁ 10 mol cm
tively. The Michaelis constant for the immobilized enzyme
was greater than that for the free enzyme (described in next
section).
The pH dependence of the performance of the enzyme
immobilized membrane reactor was studied. In the pH range
from pH ¼ 3:7 to 6.2, the pH dependence of the recovery flux
of 4-nitrophenol through the enzyme membrane was investi-
gated. The initial concentration of the substrate in the external
solution was 5 ꢁ 10 mol dm . The pH was adjusted using
different buffers (acetate buffer, pH range: 3.7–5.5; citrate buf-
fer, pH range: 5.5–6.2). The results are shown in Fig. 7. The
optimum pH for the immobilized enzyme is in the range 4.7–
Fig. 8. Time dependence of concentration of produced 4-
nitrophenol catalyzed by free phosphatase for the initial
ꢂ3
ꢂ3
ꢂ4
ꢂ3
substrate concentration of 5 ꢁ 10 mol dm
(a), 2 ꢁ
ꢂ4
ꢂ3
ꢂ4
ꢂ3
ꢂ5
1
mol dm (d). The pH of the solution was 4.8.
0
mol dm (b), 1 ꢁ 10 mol dm (c), and 8 ꢁ 10
ꢂ3
5
.0, which is near the optimum pH for the activity of the free
enzyme reported in the literature.4
A perfluorocarboxylate membrane with the immobilized
Burk plot (correlation coefficient; R ¼ 0:991) for the free acid
phosphatase in bulk solution at pH 4.8, the Michaelis constant,
KM, and the maximum velocity, Vmax, are obtained to be 8:1 ꢁ
ꢃ
acid phosphatase was stored at 5 C after each series of flux
ꢂ5
ꢂ3
ꢂ8
ꢂ3 ꢂ1
s , respectively. For
measurements in this study. During the 40-day test, a decrease
in the performance of the recovery rate (flux of the reaction
product) was not entirely observed.
Reaction Kinetics of Free Enzyme in Bulk Solution. Ex-
amples for the time course of the product are shown in Fig. 8.
The initial velocity, V0, was obtained from the initial slope of
these time course curves. The dependence of V0 on the sub-
strate concentration is shown in Fig. 9. From the Lineweaver–
10 mol dm and 3:7 ꢁ 10 mol dm
the potato acid phosphatase, KM values have been reported in
,18,19
the literature4
smaller value has been obtained for KM at 25 C under the con-
as summarized in Table 1. In this study, a
ꢃ
ꢂ3
dition of pH 4.8, adjusted by the 0.1 mol dm acetate buffer
ꢂ3
in the presence of 0.1 mol dm NaCl. Because the enzyme
is present in the reaction mixture after the hydrolysis, it is
necessary to separate it for reuse.

K. Nomura et al.
Bull. Chem. Soc. Jpn. Vol. 79, No. 2 (2006)
347
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1
3
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Table 1. Michaelis Constant for Free Enzyme
i)a)
(ii)b)
(iii)c)
14
28.
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(
4
ꢂ3
ꢂ4
ꢂ4
ꢂ4
KM/mol dm
Reference
4:9 ꢁ 10
4:3 ꢁ 10
6:2 ꢁ 10
1
5
G. G. Gilbault, Analytical Uses of Immobilized Enzymes,
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ꢃ
a) pH 6.0 (maleate buffer), 37 C. b) pH 4.9 (acetate buffer),
ꢃ
ꢃ
3
8 C. c) pH 5.5 (acetate buffer), 38 C.
1
7
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