Quantity of immobilized acid phosphatase and reaction rate across enzyme membranes

Article abstract of DOI:10.1246/cl.2010.1102

Acid phosphatase from sweet potato was immobilized on the external-solution side surface of a perfluorocarboxylate ionexchange membrane. Two membranes of different enzyme amounts were prepared without any crosslinking reagents. The effect of the immobiliz

Full text of DOI:10.1246/cl.2010.1102

doi:10.1246/cl.2010.1102
1102
Published on the web September 11, 2010
Quantity of Immobilized Acid Phosphatase and Reaction Rate across Enzyme Membranes
Hideo Takeda, Hiroki Tanaka, and Kazuo Nomura*
Department of Chemistry, Faculty of Science, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581
(Received July 7, 2010; CL-100611; E-mail: nomura@chem.rc.kyushu-u.ac.jp)
Acid phosphatase from sweet potato was immobilized on the
external-solution side surface of a perfluorocarboxylate ion-
exchange membrane. Two membranes of different enzyme
amounts were prepared without any crosslinking reagents. The
effect of the immobilized-enzyme amount on the enzymatic
reaction rate across the membrane was investigated for the
hydrolysis of 4-nitrophenyl phosphate. The flux of the reaction
product, 4-nitrophenol, was found to obey a Michaelis-Menten
equation. As the amount of the immobilized enzyme increased,
the Michaelis constant decreased whereas the maximum flux did
not increase.
kindly supplied by the Asahi Glass Co., Tokyo, with an ion-
exchange capacity of 1.4 mmol univalent ion/g dry) was used as
the supporting material for the enzyme.
¹3
The Michaelis constant for the free enzyme in 0.1 mol dm
acetate buffer solution (pH 4.8) was first determined. The initial
reaction rate was measured at 25 °C by the same method as
mentioned in a previous paper.1 From the Hanes-Woolf plot, the
¹5
Michaelis constant Km was determined to be 6.14 © 10
mol dm¹3. The values of Km reported in the literature are
¹3
¹3
6.8 © 10¹5 mol dm
(in 0.2 mol dm
acetate buffer, 35 °C,
pH 5.8)7 and 1.4 © 10¹4 mol dm
(in 0.1 mol dm
acetate
¹3
¹3
buffer, 40 °C, pH 5.6).2
The dependence of the enzyme activity on the enzyme unit
in bulk solution was investigated at a fixed high concentration of
PNPP in the saturated region of the initial rate vs. the substrate
concentration curve. The initial concentration of PNPP was
5 © 10¹4 mol dm¹3. A linear relationship exists between the
reaction rate and the enzyme unit (correlation coefficient;
R = 0.999). Using this calibration curve, the change in the
amount of the enzyme placed on the membrane after one
immobilization was determined.
Enzymes exhibit their activity not only in the free form but
also in the immobilized state of ionic or covalent-bonding or
physical adsorption on an undissolved carrier. While the
stability of the enzyme is enhanced by immobilization, the
activity of the enzyme is changed compared to that in the free
form.1-5 An immobilized-enzyme membrane reactor is a system
in which membrane transport and enzymatic reaction simulta-
neously take place. The product can be separated, collected, and
concentrated based on the difference between the permeability
of the membrane to a product and that to the reactants and other
products of the enzymatic reaction.
In order to clarify the correlation of the rate of transport of
the product (or the reactant) for an enzymatic reaction with the
reaction rate, information about the amount of the enzyme
immobilized on the membrane is necessary. However, there have
been few reports on the quantitative analysis of coupling in
membrane reactors based on the determination of the amount of
the immobilized enzyme.
The perfluorocarboxylate ion-exchange membrane was cut
and installed in the cell for the measurement. A mixture of 25 ¯L
of the enzyme solution that was prepared by diluting the stock
suspension 10 times by a 1.8 mol dm¹3 (NH4)2SO4 solution with
¹3
a 25 ¯L of 0.1 mol dm acetate buffer was stirred. Thirty
microliters of this mixed suspension was placed on the surface
of the external solution side of the membrane. After leaving it at
25 °C for 1 h, the enzyme suspension on the membrane was
removed and diluted with 0.1 mol dm¹3 acetate buffer to analyze
the enzyme activity. Two enzyme membranes of different
enzyme amounts were prepared. For these enzyme membranes,
the immobilization process mentioned above was carried out
two or three times. The cell for the measurement with an internal
solution of ca. 2 mL of acetate buffer was stored at 5 °C while
the flux measurements were not carried out. The amount of the
enzyme placed on the membrane for the immobilization, E, and
that of the immobilized enzyme on the membrane, En, are in
Table 1. The immobilized enzyme membrane prepared using the
In this study, acid phosphatase from sweet potato was
immobilized on a perfluorocarboxylate ion-exchange membrane.
Hydrolysis of 4-nitrophenyl phosphate (PNPP) across the
enzyme membrane was studied. The apparent pKa of this ion-
¹3
exchange membrane is 1.9 in a 0.1 mol dm NaCl solution.6
Because the dissociable groups in this ion-exchange membrane
is thought to be nearly perfectly ionized in the pH range near the
optimum pH of the acid phosphatase, only 4-nitrophenol (PNP)
of the hydrolysis product of PNPP permeates through the
membrane.1 Two enzyme membranes with different amounts of
the immobilized enzyme were prepared separately. The immo-
bilization procedure in this study involves only adsorption for
the enzyme on the membrane surface without any crosslinking
reagent. The objective is to determine the quantity of the
immobilized enzyme on the membranes and to examine the
enzyme-substrate affinity and the enzymatic reaction rate across
the enzyme membranes that have different amounts of enzymes.
Table 1. The amount of immobilized enzyme onto the cation-
exchange membrane
E/Ua
E1/Ub
E2/Uc
E3/Ud
Etotal/Ue
M-A
M-B
2.13
10.04
0.848
2.627
0.016
0.036
0.021
®
0.88
2.66
aThe amount of enzyme placed on the surface of the cation-
exchange membrane for immobilization. bThe enzyme immo-
Acid phosphatase (E.C.3.1.3.2) from sweet potato with a
¹1
c
protein concentration of 57 mg mL
was obtained as a
bilized by the first immobilization. The enzyme immobilized
¹3
by the second immobilization. dThe enzyme immobilized by the
suspension with 1.8 mol dm aqueous (NH4)2SO4 (Sigma, St.
Luis). A perfluorocarboxylate polymer membrane (Flemion 230,
e
third immobilization. The sum of E1, E2, and E3.
Chem. Lett. 2010, 39, 1102-1103
© 2010 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

1103
Figure 2. Plot of the Hanes-Woolf type for the immobilized
enzyme membrane. The x intercept is ¹Km of the immobilized
enzyme membrane, and the slope is 1/Jmax. ( ) M-A and ( ) M-B.
Figure 1. Substrate-concentration dependence of the flux for the
immobilized enzyme membrane. ( ) M-A and ( ) M-B.
enzyme solution diluted 10 times was designated as M-A, and
that using the solution diluted two times as M-B.
Table 2. Michaelis constant and maximum flux for immobilized
enzyme membrane
Flux measurements were carried out using the same
apparatus as described in a previous paper.1 The layer of the
immobilized acid phosphatase was on the external solution side
of the membrane. Initially, external and internal solutions
¹1
Km
Jmax
JmaxEtotal
/10¹3 mol dm /10¹13 mol cm¹2 s /10¹13 mol cm¹2 s¹1 U
¹3
¹1
¹1
M-A
M-B
3.00
1.00
9.99
9.25
11.35
3.48
¹3
contained only 50 and 2 mL of a 0.1 mol dm acetate buffer
(pH 4.8), respectively. The reaction was started by injecting a
¹3
small volume of 0.1 mol dm PNPP into the external solution.
The amount of PNP transported into the internal solution was
followed by measuring the time course of the optical absorbance
at 317 nm.
concentration range. From these results, the amount of the
immobilized enzyme is thought to have an effect on the
enzyme-substrate affinity. The value of the maximum flux Jmax
divided by the amount of the immobilized enzyme on the
membrane Etotal can be regarded as the enzyme activity per unit.
As the Etotal increased, the enzyme activity per unit decreased.
Compared to the free state of the enzyme, the immobilized
enzyme on the membrane would be in a dense state. In the
enzymatic reaction, the enzyme forms a complex with the sub-
strate. Thus, the reaction proceeds through a intermediate state
formed by the interaction of the substrate with the active site of
the enzyme. This suggests a possible effect of the dense state of
the enzyme on the formation of the enzyme-substrate complex.
In Figure 1, the dependence of the PNP flux into the internal
solution in a stationary state on the initial substrate concentration
is shown for two enzyme membranes. For both enzyme
membranes, the flux shows the Michaelis-Menten behavior
versus the initial concentration of the substrate. The results show
that the enzymatic reaction on the surface of the enzyme
membrane is coupled to the transport of product PNP into the
internal solution. Thus, the Michaelis-Menten equation can be
applied to the flux of the reaction product so that the flux J is
represented by the Michaelis constant Km, the maximum flux
J
max, and the substrate concentration [S].1
References
J ¼ Jmax½Sꢀ=ðKm þ ½SꢀÞ
By rearranging eq 1, we obtain
½Sꢀ=J ¼ ½Sꢀ=Jmax þ Km=Jmax
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concentration is plotted versus the initial substrate concentration
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slope give ¹Km and 1/Jmax respectively. The determined values
of Km and Jmax are shown in Table 2. In both enzyme
membranes, the enzyme-substrate affinity was apparently lower
than that in the solution. However, the surface of the enzyme
layer immobilized on the membrane with a large amount of
enzyme was saturated by substrate molecules in the low-
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Chem. Lett. 2010, 39, 1102-1103
© 2010 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/