BIOTECHNOLOGY AND BIOENGINEERING, VOL. XIX, PAGES 1493-1501 (1977)
Electric Field Control of Lipase Membrane Activity ISAO KARUBE, YOICHI YUGETA, and SHUICHI SUZUKI, Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo 152, Japan
Summary Lipase (EC 3.1.1.3.,from Pseudomonas sp.) was entrapped in collagen membrane containing liquid crystal (4-methoxybenzilidene-4’-n-butylaniline). The activity of the lipase-liquid crystal membrane a t an applied voltage of 4 V was 3.4 compared to a membrane tested without imposition of an external electric field. A linear relationship was observed between the activity of the l i p a s e liquid crystal membrane and the current. The apparent Michaelis constant (K’m) of the lipase-liquid crystal membrane under electric field was identical to that of the membrane under ordinary condition. Activation of the lipaseliquid crystal membrane was observed repeatedly, i.e., activation in the presence of an electric field and reversion to a basal level upon removal of the field occurred cyclically. Activity control of immobilized enzymes is desirable for switching devices of a bioreactor. Possible mechanisms of the lipase activation by electric field are discussed.
INTRODUCTION Many enzymes have been immobilized by various methods and then applied to industrial production and clinical Activity control of immobilized enzymes has a potential application in a switch device or as a controller of a bioreactor. Activity control of immobilized enzymes with light irradiation was reported previously.4-6 Those papers showed that the property changes of an enzyme carrier with light irradiation induced the activity change of entrapped enzymes. It is well known that molecules of a liquid crystal arrange themselves regularly under an electric field. Therefore, the diffusion of a material through a liquid crystal membrane may be controlled in the presence of an electric field. Activity of enzymes immobilized in liquid crystal can be controlled with an electric field. As immobilization of enzymes in liquid crystal (liquid state under ordinary conditions) is difficult, an enzyme and a liquid crystal were entrapped 1493 @ 1977 by John Wiley & Sons, Inc.
1494
KARUBE, YUGETA, AND SUZUKI
in a collagen membrane. A preliminary report on the properties and the electric field effect of enzyme membranes was described by the authors.’ In this paper, lipase was entrapped in liquid crystal-(collagen) membrane and the possible mechanisms of the lipase activation by an electric field are discussed.
MATERIALS AND METHODS Materials Collagen was obtained from steer hide and was purified by the method previously reported. * Lipase (glycerolester hydrolase, 2900 units/g) was obtained from Amano Pharmaceutical Co. A liquid crystal, 4-methoxybenzilidene-4’-n-butylaniline (MBBA) was purchased from Fuji Color Co. Sorbitan monolaurate poly(oxyethy1ene)ether (Tween 20) was chosen for the substrate of lipase because a soluble substrate is needed for the determination of kinetic parameters. Tween 20 was purchased from Wako Pure Chemicals Ind. Other reagents were commercially available analytical reagents or laboratory grade materials. Deionized water was used in all procedures. Preparation of Lipase-Liquid Crystal Collagen Membrane The preparatory suspension of the lipase-liquid crystal (collagen) membrane contained 50 g of 0.9% collagen fibrils a t pH 4.3,8 ml of 4% MBBA (ethanol solution), and 20 ml of lipase solution (0.64g lipase). The lipase-liquid crystal membrane was prepared by casting the suspension on a Teflon plate and drying a t 20°C. Then, the lipase-liquid crystal membranes were treated with 0.1% glutaraldehyde solution for 30 sec. The membrane was washed with a large volume of deionized water for 12 hr and dried at 20°C. The thickness of the lipase-liquid crystal membrane (6 :3 :4) was approximately 50 wm. The lipase content of the membrane was determined indirectly from the amount of lipase leaked during washing.
Preparation of Lipase-Collagen Membrane Eighty g of 0.9% collagen fibril suspension (pH 4.3) and 20 ml of lipase solution (0.64g lipase) were sufficiently mixed, cast onto a Teflon plate, and dried a t room temperature. The lipase-collagen membranes were treated with 0.1% glutaraldehyde solution for 30 sec and dried a t 20°C.
ELECTRIC CONTROL OF LIPASE MEMBRANE
1495
Procedure
Unless otherwise noted, standard assays of the lipase-liquid crystal membrane were carried out as follows. The lipase-liquid crystal membrane (25 mg; 2 x 4 cm2) was fixed on a platinum electrode (2 x 4 cmz) with epoxy resin (Konishi Co., Osaka). The lipaseliquid crystal membrane-Pt electrode was used as a cathode. A platinum electrode (diam, 0.1 x 0.4 cm2)was also used as a counterelectrode. The electrode distance was fixed to 0.3 cm. Both electrodes were inserted in a reaction mixture of 20 ml, 10% Tween 20 in 0.05M tetramethylammonium chloride solution (pH 5.5), and the reaction was carried out under electric field or ordinary conditions for 30 min at 37°C with stirring. The lipase-liquid crystal membrane activities were measured by the method described previ~usly.~ A potentiostat (Hokuto Denko Co., model PS-500B) was used to apply a constant voltage across the two electrodes.
RESULTS The activities of the lipase-liquid crystal membrane and the lipase-collagen membrane were 2.9 x lo2 and 1.5 X lo2 units/g lipase, respectively. The carrier-to-enayme ratio was constant for both the lipase-liquid crystal membrane and the lipase-collagen membrane. The activity of the lipase-liquid crystal membrane was higher than that of the lipase-collagen membrane. Figure 1 shows the effect of electric field. The activity of the lipase-liquid crystal membrane was increased with an increase in terminal voltage between the two electrodes. A remarkable activation of the lipase-liquid crystal membrane was observed above 2 V. The activation was also observed in the case of the lipase-collagen membrane; however, the degree of activation was lower than that of the lipase-liquid crystal membrane. Table I shows the kinetic parameters of the lipase-liquid crystal membrane. As shown, the apparent Michaelis constant (ITm) of the lipase-liquid crystal membrane under electric field was identical to that of the membrane under ordinary conditions, i.e., without the external electric field. The maximum velocity (Vmax)of the lipaseliquid crystal membrane showed an increase under electric field. Figure 2 shows the pH-activity curves of the lipase-liquid crystal membrane. No significant shift of the optimum pH of the lipaseliquid crystal membrane under electric field was observed. The pH-activity curve of the lipase-liquid crystal membrane under
1496
KARUBE, YUGETA, AND SUZUKI
1
2
0
0
1
0
7
2 3 Voltage( V )
,
4
Fig. Effect of electric field on the activity of the lipase-..juid crystal membrane and the lipase-collagen membrane. Lipase-liquid crystal membrane (25 mg, 2 X 4 cmz) and the lipase-collagen membrane (19 mg, 2 X 4 cmz) were employed for experiments. Enzyme assay was carried out under standard conditions. ( 0 ) Lipase-liquid crystal membrane; ( 0 )lipase-collagen membrane.
electric field was bell-shaped, unlike that of lipase-collagen membrane (without liquid crystal). The effect of ionic strength on the activity of the lipase-liquid crystal membrane is shown in Figure 3. Tetramethylanimonium chloride was used as the electrolyte. The activity was constant in the ionic strength range of 04.05111 under ordinary conditions. However, the activity of the lipase-liquid crystal membrane under electric field increased linearly with an increase in substrate ionic strength. Figure 4 shows the relationship between the activity of the lipaseliquid crystal membrane and the current. The activity was increased linearly with an increase in the current. Therefore, the degree of activation of the lipase-liquid crystal membrane could be correlated with the current. TABLE I Kinetic Parameters of Lipase-Liquid Crystal Membrane
0 4
2.60 2.60
430 1470
ELECTRIC CONTROL OF LIPASE MEMBRANE
4
5
6
7
1497
8
PH Fig. 2 . p H vs. activity curves of electric field and ordinary condition. dard conditions a t various pH. pH HC1 or 0.05N NaOH. Lipase-liquid and under ordinary conditions ( 0 ) .
the lipase-liquid crystal membrane under Enzyme assay was carried out under stanof the substrate was adjusted with 0.05N crystal membrane under electric field (O),
Figure 5 shows the time course of the lipase-liquid crystal membrane activity after activation. Initially the activity was determined under ordinary conditions. Then, the activity was determined under electric field. The activation of 6he lipase-liquid crystal membrane by electric field was observed in this case. Then the activity was determined again under ordinary conditions. As
1
0
1
0.02
005
Ionic strength ( M )
Fig. 3. Effect of ionic strength on the activity of the lipase-liquid crystal membrane. Enzyme assay was carried out under standard conditions at various ionic strengths. Tetramethylammonium chloride was employed for experiments. and under ordinary Lipase-liquid crystal membrane under electric field of 4 V ,).( conditions ( 0 ) .
KARUBE, YUGETA, AND SUZUKI
1498
.-
c
300
.->
:
U
0 0
1 2 3 Current (mA )
Fig. 4. Relationship between the activity of the lipase-liquid crystal membrane and the current. Enzyme assay was carried out under standard conditions. Experiments were carried out at constant current (04V) for 30 min.
-
800
.-
zU
200
t I
ov
0
4v
-ov+
1
2
3
4
Time ( h r )
Fig. 5. Time course of the lipase-liquid crystal membrane activity. assay was carried out under standard conditions.
(TJ 4v
ov
4v
ov
4v
ov
Enzyme
4v
Voltage(V)
Fig. 6. Activity reversibility of the lipase-liquid crystal membrane. assay was carried out under standard conditions.
Enzyme
ELECTRIC CONTROL OF LIPASE MEMBRANE
1499
shown in Figure 5 , the activity of the lipase-liquid crystal membrane became lower than the initial activity. However, as the contact time was increased, the activity of the lipase-liquid crystal membrane under ordinary conditions gradually wa.s restored to the initial level. The enzymatic activity of the membrane was determined alternately with and without the influence of an electric field (Fig. 6). An activity increase was observed repeatedly under electric field. Upon removal of the field, the activity decreased to normal, i.e., unactivated level. Such a periodic change in activity occurred over several cycles.
DISCUSSION The activity of lipase immobilized in the liquid crystal (collagen) membrane was higher than that in collagen membrane. However, the reason for the high activity is not now clear. Activation of the membrane under electric field may be caused by the following reasons: 1) the diffusivity change of substrate; 2 ) subtle pH or temperature alterations in the microenvironment of the immobilized enzyme; 3) the migration of reaction products; 4) conformational changes in enz-ymestructure; 5 ) structural changes of the collagen membrane; 6) the effect of the liquid crystal. As described above, molecules of liquid crystal arrange themselves regularly under an electric field. Therefore, it was assumed that diffusivity of the substrate was decreased under an electric field. However, the activity of the lipase-liquid crystal membrane was increased with an increase in terminal voltage (Fig. 1). Furthermore, the apparent Michaelis constant (K’J of the lipase-liquid crystal membrane under electric field was identical to that of the membrane under ordinary conditions. Therefore, the diff usivity change of the substrate did not occur under electric field. As described previously18the vicinity of the cathode WM slightly basic (pH 8.5). However, activation was not observed under alkaline conditions (Fig. 2 ) . On the other hand, the activity of the membrane did not change remarkably around 40°C. Therefore, activation is not caused from subtle pH or temperature alt,erations in the microenvironment of the immobilized enzyme. The hydrolysis of Tween 20 with lipase is shown in the following equation. Lauric acid was produced after hydrolysis of Tween 20. As described above, the vicinity of the cathode was slightly basic. Therefore, a part of the lauric acid dissociates into ions. The migration of these dissociated ions under electric field may increase the
KARUBE, WGETA, AND SUZUKI
1500
velocity of the reaction. The maximum velocity (V,,,) of the lipaseliquid crystal membrane under electric field was larger than that under ordinary conditions.
ACH-CHz--O-COR
CHZ Ho-AH
~H-O-(CH1-CHz-o~,-CH,-CH,-OH
x
lip-
+
AH Tween 20
I
OH
Therefore, the rate of the decomposition of the enzyme-substrate (ES) complex to product under electric field was greater than that under ordinary conditions. Furthermore, the activity was increased linearly with an increase in the current (Fig. 4) or terminal voltage (Fig. 1). It is known that the migration velocity is increased with an increase in terminal voltage. Therefore, the migration of reaction product under an electric field increased the activity of the membrane. This activation phenomenon was also observed in the case of the lipase-collagen membrane (without liquid crystal). Therefore, conformational changes in the enzyme structure or structural changes of the collagen membrane under an electric field may also induce activation of lipase. A remarkable activity increase was observed in the case of the lipase-membrane containing liquid crystal under electric field. Furthermore, the state of the lip-liquid crystal membrane changed after activation with electric field (Fig. 6 ) . The liquid crystal may also play an important role in the activation of lipase. However, the detailed mechanisms of activation are far from being understood. The activity of the lipase-liquid crystal membrane was increased under electric field and then decreased again under ordinary conditions. This electrolytic activation of the lipase-liquid crystal membrane could be observed repeatedly. As reported previously, the activity control was poor in the case of the photosensitive enzymecollagen membrane.5 In this case, irreversible isomerization of
ELECTRIC CONTROL OF LIPASE MEMBRANE
1501
a photochromic compound (spiropyran) bound on collagen fibrils occurred during light irradiation. However, irreversible changes in the lipase-liquid crystal membrane did not occur during electrolysis.
References 1. I. Chibata, T. Tosa, T. Sato, T. Mori, and Y. Matsuo, Proc. ZV IFS: Ferment. Technol. Today, 4, 383 (1972). 2. K. Venkatasubramanian and W. R. Vieth, Biotechnol. Bioeng., 15,583 (1973). 3. Y. Nakamoto, I. Karube, and S. Suzuki, Biotechnol. Bioeng., 17,1387 (1975). 4. I. Karube, Y. Nakamoto, K. Namba, and S. Suzuki, Biochim. Biophys. Acta, 429, 975 (1976). 5. I. Karube, Y. Nakamoto, and S. Suzuki, Biochim. Biophys. Acta, 445. 774 (1976). 6. Y. Nakamoto, I. Karube, S. Terawaki, and S. Suzuki, J. Solid-Phase Biochem., 1, 143 (1976). 7. M. Kubo, I. Karube, and S. Suzuki, Biochem. Biophys. Res. Commun., 69, 731 (1976). 8. I. Karube, I. Mizuguchi, and S. Suzuki, J. Chem. SOC.Jpn., (Ind. Chem. Sec.), 74, 971 (1971).
Accepted for Publication April 22, 1977
Electric Field Control of Lipase Membrane Activity ISAO KARUBE, YOICHI YUGETA, and SHUICHI SUZUKI, Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo 152, Japan
Summary Lipase (EC 3.1.1.3.,from Pseudomonas sp.) was entrapped in collagen membrane containing liquid crystal (4-methoxybenzilidene-4’-n-butylaniline). The activity of the lipase-liquid crystal membrane a t an applied voltage of 4 V was 3.4 compared to a membrane tested without imposition of an external electric field. A linear relationship was observed between the activity of the l i p a s e liquid crystal membrane and the current. The apparent Michaelis constant (K’m) of the lipase-liquid crystal membrane under electric field was identical to that of the membrane under ordinary condition. Activation of the lipaseliquid crystal membrane was observed repeatedly, i.e., activation in the presence of an electric field and reversion to a basal level upon removal of the field occurred cyclically. Activity control of immobilized enzymes is desirable for switching devices of a bioreactor. Possible mechanisms of the lipase activation by electric field are discussed.
INTRODUCTION Many enzymes have been immobilized by various methods and then applied to industrial production and clinical Activity control of immobilized enzymes has a potential application in a switch device or as a controller of a bioreactor. Activity control of immobilized enzymes with light irradiation was reported previously.4-6 Those papers showed that the property changes of an enzyme carrier with light irradiation induced the activity change of entrapped enzymes. It is well known that molecules of a liquid crystal arrange themselves regularly under an electric field. Therefore, the diffusion of a material through a liquid crystal membrane may be controlled in the presence of an electric field. Activity of enzymes immobilized in liquid crystal can be controlled with an electric field. As immobilization of enzymes in liquid crystal (liquid state under ordinary conditions) is difficult, an enzyme and a liquid crystal were entrapped 1493 @ 1977 by John Wiley & Sons, Inc.
1494
KARUBE, YUGETA, AND SUZUKI
in a collagen membrane. A preliminary report on the properties and the electric field effect of enzyme membranes was described by the authors.’ In this paper, lipase was entrapped in liquid crystal-(collagen) membrane and the possible mechanisms of the lipase activation by an electric field are discussed.
MATERIALS AND METHODS Materials Collagen was obtained from steer hide and was purified by the method previously reported. * Lipase (glycerolester hydrolase, 2900 units/g) was obtained from Amano Pharmaceutical Co. A liquid crystal, 4-methoxybenzilidene-4’-n-butylaniline (MBBA) was purchased from Fuji Color Co. Sorbitan monolaurate poly(oxyethy1ene)ether (Tween 20) was chosen for the substrate of lipase because a soluble substrate is needed for the determination of kinetic parameters. Tween 20 was purchased from Wako Pure Chemicals Ind. Other reagents were commercially available analytical reagents or laboratory grade materials. Deionized water was used in all procedures. Preparation of Lipase-Liquid Crystal Collagen Membrane The preparatory suspension of the lipase-liquid crystal (collagen) membrane contained 50 g of 0.9% collagen fibrils a t pH 4.3,8 ml of 4% MBBA (ethanol solution), and 20 ml of lipase solution (0.64g lipase). The lipase-liquid crystal membrane was prepared by casting the suspension on a Teflon plate and drying a t 20°C. Then, the lipase-liquid crystal membranes were treated with 0.1% glutaraldehyde solution for 30 sec. The membrane was washed with a large volume of deionized water for 12 hr and dried at 20°C. The thickness of the lipase-liquid crystal membrane (6 :3 :4) was approximately 50 wm. The lipase content of the membrane was determined indirectly from the amount of lipase leaked during washing.
Preparation of Lipase-Collagen Membrane Eighty g of 0.9% collagen fibril suspension (pH 4.3) and 20 ml of lipase solution (0.64g lipase) were sufficiently mixed, cast onto a Teflon plate, and dried a t room temperature. The lipase-collagen membranes were treated with 0.1% glutaraldehyde solution for 30 sec and dried a t 20°C.
ELECTRIC CONTROL OF LIPASE MEMBRANE
1495
Procedure
Unless otherwise noted, standard assays of the lipase-liquid crystal membrane were carried out as follows. The lipase-liquid crystal membrane (25 mg; 2 x 4 cm2) was fixed on a platinum electrode (2 x 4 cmz) with epoxy resin (Konishi Co., Osaka). The lipaseliquid crystal membrane-Pt electrode was used as a cathode. A platinum electrode (diam, 0.1 x 0.4 cm2)was also used as a counterelectrode. The electrode distance was fixed to 0.3 cm. Both electrodes were inserted in a reaction mixture of 20 ml, 10% Tween 20 in 0.05M tetramethylammonium chloride solution (pH 5.5), and the reaction was carried out under electric field or ordinary conditions for 30 min at 37°C with stirring. The lipase-liquid crystal membrane activities were measured by the method described previ~usly.~ A potentiostat (Hokuto Denko Co., model PS-500B) was used to apply a constant voltage across the two electrodes.
RESULTS The activities of the lipase-liquid crystal membrane and the lipase-collagen membrane were 2.9 x lo2 and 1.5 X lo2 units/g lipase, respectively. The carrier-to-enayme ratio was constant for both the lipase-liquid crystal membrane and the lipase-collagen membrane. The activity of the lipase-liquid crystal membrane was higher than that of the lipase-collagen membrane. Figure 1 shows the effect of electric field. The activity of the lipase-liquid crystal membrane was increased with an increase in terminal voltage between the two electrodes. A remarkable activation of the lipase-liquid crystal membrane was observed above 2 V. The activation was also observed in the case of the lipase-collagen membrane; however, the degree of activation was lower than that of the lipase-liquid crystal membrane. Table I shows the kinetic parameters of the lipase-liquid crystal membrane. As shown, the apparent Michaelis constant (ITm) of the lipase-liquid crystal membrane under electric field was identical to that of the membrane under ordinary conditions, i.e., without the external electric field. The maximum velocity (Vmax)of the lipaseliquid crystal membrane showed an increase under electric field. Figure 2 shows the pH-activity curves of the lipase-liquid crystal membrane. No significant shift of the optimum pH of the lipaseliquid crystal membrane under electric field was observed. The pH-activity curve of the lipase-liquid crystal membrane under
1496
KARUBE, YUGETA, AND SUZUKI
1
2
0
0
1
0
7
2 3 Voltage( V )
,
4
Fig. Effect of electric field on the activity of the lipase-..juid crystal membrane and the lipase-collagen membrane. Lipase-liquid crystal membrane (25 mg, 2 X 4 cmz) and the lipase-collagen membrane (19 mg, 2 X 4 cmz) were employed for experiments. Enzyme assay was carried out under standard conditions. ( 0 ) Lipase-liquid crystal membrane; ( 0 )lipase-collagen membrane.
electric field was bell-shaped, unlike that of lipase-collagen membrane (without liquid crystal). The effect of ionic strength on the activity of the lipase-liquid crystal membrane is shown in Figure 3. Tetramethylanimonium chloride was used as the electrolyte. The activity was constant in the ionic strength range of 04.05111 under ordinary conditions. However, the activity of the lipase-liquid crystal membrane under electric field increased linearly with an increase in substrate ionic strength. Figure 4 shows the relationship between the activity of the lipaseliquid crystal membrane and the current. The activity was increased linearly with an increase in the current. Therefore, the degree of activation of the lipase-liquid crystal membrane could be correlated with the current. TABLE I Kinetic Parameters of Lipase-Liquid Crystal Membrane
0 4
2.60 2.60
430 1470
ELECTRIC CONTROL OF LIPASE MEMBRANE
4
5
6
7
1497
8
PH Fig. 2 . p H vs. activity curves of electric field and ordinary condition. dard conditions a t various pH. pH HC1 or 0.05N NaOH. Lipase-liquid and under ordinary conditions ( 0 ) .
the lipase-liquid crystal membrane under Enzyme assay was carried out under stanof the substrate was adjusted with 0.05N crystal membrane under electric field (O),
Figure 5 shows the time course of the lipase-liquid crystal membrane activity after activation. Initially the activity was determined under ordinary conditions. Then, the activity was determined under electric field. The activation of 6he lipase-liquid crystal membrane by electric field was observed in this case. Then the activity was determined again under ordinary conditions. As
1
0
1
0.02
005
Ionic strength ( M )
Fig. 3. Effect of ionic strength on the activity of the lipase-liquid crystal membrane. Enzyme assay was carried out under standard conditions at various ionic strengths. Tetramethylammonium chloride was employed for experiments. and under ordinary Lipase-liquid crystal membrane under electric field of 4 V ,).( conditions ( 0 ) .
KARUBE, YUGETA, AND SUZUKI
1498
.-
c
300
.->
:
U
0 0
1 2 3 Current (mA )
Fig. 4. Relationship between the activity of the lipase-liquid crystal membrane and the current. Enzyme assay was carried out under standard conditions. Experiments were carried out at constant current (04V) for 30 min.
-
800
.-
zU
200
t I
ov
0
4v
-ov+
1
2
3
4
Time ( h r )
Fig. 5. Time course of the lipase-liquid crystal membrane activity. assay was carried out under standard conditions.
(TJ 4v
ov
4v
ov
4v
ov
Enzyme
4v
Voltage(V)
Fig. 6. Activity reversibility of the lipase-liquid crystal membrane. assay was carried out under standard conditions.
Enzyme
ELECTRIC CONTROL OF LIPASE MEMBRANE
1499
shown in Figure 5 , the activity of the lipase-liquid crystal membrane became lower than the initial activity. However, as the contact time was increased, the activity of the lipase-liquid crystal membrane under ordinary conditions gradually wa.s restored to the initial level. The enzymatic activity of the membrane was determined alternately with and without the influence of an electric field (Fig. 6). An activity increase was observed repeatedly under electric field. Upon removal of the field, the activity decreased to normal, i.e., unactivated level. Such a periodic change in activity occurred over several cycles.
DISCUSSION The activity of lipase immobilized in the liquid crystal (collagen) membrane was higher than that in collagen membrane. However, the reason for the high activity is not now clear. Activation of the membrane under electric field may be caused by the following reasons: 1) the diffusivity change of substrate; 2 ) subtle pH or temperature alterations in the microenvironment of the immobilized enzyme; 3) the migration of reaction products; 4) conformational changes in enz-ymestructure; 5 ) structural changes of the collagen membrane; 6) the effect of the liquid crystal. As described above, molecules of liquid crystal arrange themselves regularly under an electric field. Therefore, it was assumed that diffusivity of the substrate was decreased under an electric field. However, the activity of the lipase-liquid crystal membrane was increased with an increase in terminal voltage (Fig. 1). Furthermore, the apparent Michaelis constant (K’J of the lipase-liquid crystal membrane under electric field was identical to that of the membrane under ordinary conditions. Therefore, the diff usivity change of the substrate did not occur under electric field. As described previously18the vicinity of the cathode WM slightly basic (pH 8.5). However, activation was not observed under alkaline conditions (Fig. 2 ) . On the other hand, the activity of the membrane did not change remarkably around 40°C. Therefore, activation is not caused from subtle pH or temperature alt,erations in the microenvironment of the immobilized enzyme. The hydrolysis of Tween 20 with lipase is shown in the following equation. Lauric acid was produced after hydrolysis of Tween 20. As described above, the vicinity of the cathode was slightly basic. Therefore, a part of the lauric acid dissociates into ions. The migration of these dissociated ions under electric field may increase the
KARUBE, WGETA, AND SUZUKI
1500
velocity of the reaction. The maximum velocity (V,,,) of the lipaseliquid crystal membrane under electric field was larger than that under ordinary conditions.
ACH-CHz--O-COR
CHZ Ho-AH
~H-O-(CH1-CHz-o~,-CH,-CH,-OH
x
lip-
+
AH Tween 20
I
OH
Therefore, the rate of the decomposition of the enzyme-substrate (ES) complex to product under electric field was greater than that under ordinary conditions. Furthermore, the activity was increased linearly with an increase in the current (Fig. 4) or terminal voltage (Fig. 1). It is known that the migration velocity is increased with an increase in terminal voltage. Therefore, the migration of reaction product under an electric field increased the activity of the membrane. This activation phenomenon was also observed in the case of the lipase-collagen membrane (without liquid crystal). Therefore, conformational changes in the enzyme structure or structural changes of the collagen membrane under an electric field may also induce activation of lipase. A remarkable activity increase was observed in the case of the lipase-membrane containing liquid crystal under electric field. Furthermore, the state of the lip-liquid crystal membrane changed after activation with electric field (Fig. 6 ) . The liquid crystal may also play an important role in the activation of lipase. However, the detailed mechanisms of activation are far from being understood. The activity of the lipase-liquid crystal membrane was increased under electric field and then decreased again under ordinary conditions. This electrolytic activation of the lipase-liquid crystal membrane could be observed repeatedly. As reported previously, the activity control was poor in the case of the photosensitive enzymecollagen membrane.5 In this case, irreversible isomerization of
ELECTRIC CONTROL OF LIPASE MEMBRANE
1501
a photochromic compound (spiropyran) bound on collagen fibrils occurred during light irradiation. However, irreversible changes in the lipase-liquid crystal membrane did not occur during electrolysis.
References 1. I. Chibata, T. Tosa, T. Sato, T. Mori, and Y. Matsuo, Proc. ZV IFS: Ferment. Technol. Today, 4, 383 (1972). 2. K. Venkatasubramanian and W. R. Vieth, Biotechnol. Bioeng., 15,583 (1973). 3. Y. Nakamoto, I. Karube, and S. Suzuki, Biotechnol. Bioeng., 17,1387 (1975). 4. I. Karube, Y. Nakamoto, K. Namba, and S. Suzuki, Biochim. Biophys. Acta, 429, 975 (1976). 5. I. Karube, Y. Nakamoto, and S. Suzuki, Biochim. Biophys. Acta, 445. 774 (1976). 6. Y. Nakamoto, I. Karube, S. Terawaki, and S. Suzuki, J. Solid-Phase Biochem., 1, 143 (1976). 7. M. Kubo, I. Karube, and S. Suzuki, Biochem. Biophys. Res. Commun., 69, 731 (1976). 8. I. Karube, I. Mizuguchi, and S. Suzuki, J. Chem. SOC.Jpn., (Ind. Chem. Sec.), 74, 971 (1971).
Accepted for Publication April 22, 1977
