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Redox reaction system conjugating electrochemical reduction of NADP+ and enzymatic reaction across the electron transfer membrane
Redox reaction system conjugating electrochemical reduction of NADP + and enzymatic reaction across the electron transfer membrane Yoshiharu Nakamura, Shuichi Itoh and Shin-ichiro Suye Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Fukui University, Fuku~ Japan
The conjugated redox reaction was driven across the electron transfer membrane prepared from a urethane prepokymer to carry positive charge, where NADP + as electron transfer carrier was adsorbed in the prepared pokyurethane membrane. Glutathione reductase [NAD (P)H: oxidized-glutathione oxidoreductase (EC 1.6.4.1)] was used as the catatystfor production of the reduced form of glutathione (GSH) from the oMdizedform (GSSG) in the objective reaction, and methyl viologen (MV) was used for the electrochemical regeneration of NADPH in the subreaction. The conjugated redox reaction in the separated reactions system, using the three-compartment cell with two membranes, was successful without MV contamination. Under the given conditions, the conversion ratio of G SH from GSSG reached 50% after 4 h of incubation at 30°C and the amount of GSH accumulated was 0.5 gmol ml - 1 of reaction mixture.
Keywords: Electrontransfer;NADPHregeneration;glutathionereductase;conjugatedredoxreaction;separatedreaction system
Introduction Regeneration of pyridine nucleotide NADH and NADPH is important for carrying out enzymatic syntheses that use NAD (P)-linked dehydrogenases. Various biochemical substances, such as amino acids, 1,2 hydroxy acids, 3 and other useful compounds, 4-6 can be produced by coupling an electrochemical or an enzymatic reaction for reduction of NAD(P) + and an NAD(P)-linked dehydrogenase reaction. These reactions are commonly carried out in a onebath system in which an objective reaction (coenzymeconsuming reaction) and a subreaction (coenzymeregeneration reaction) cannot be separated. In these cases the main products are contaminated with substances originating in the subreaction, such as mediator, substrate, byproducts, and enzyme. If redox reactions proceeded separately in a separated reactions system by using an electron transfer membrane, the system would be very useful for
Received28 September1993;accepted27 April 1994 Address reprint requests to Dr. Nakamuraat Fukui University,Department of AppliedChemistryand Biotechnology,3-9-1Bunkyo,Fukui910, Japan
1026
Enzyme Microb. Technol., 1994, vol. 16, December
designing of various enzymatic syntheses, as well as bioreactors, such as artificial kidneys. 1 In our previous papers, 7,8 preparations of the electron transfer membranes containing coenzyme NAD(P)+/ NAD(P)H or kerateine components as an electron carrier, and intercellular components of wool as a channel substance for the proton transport, were reported. Next, the construction of a separated-reactions system, in which twooxidoreductase/redox-reactions successfully proceeded conjugately across the membranes, was presented. In this paper, the separated-reactions system consisting of an electrolytic cell and an enzymatic reaction cell separated by the electron transfer membrane was constructed. In preparing the electron transfer membrane, NADP + as an electron transfer carrier was immobilized in the polyurethane membrane. The electrochemical reduction of NADP + mediated by methyl viologen (MV 2 + ) and ferredoxin NADP +-reductase (FRD) was examined through cyclic voltammetery using a glassy carbon electrode, and constant potential cathodic reduction was performed using a carbon plate electrode. We investigated the conversion of the oxidized form of glutathione (GSSG) to the reduced form (GSH) using glutathione reductase (GR) and FRD with MV 2 + as a mediator. © 1994
Butterworth-Heinemann
Redox reaction across the membrane: Y. Nakamura et al.
Materials and methods
N2 gas
2 gas
Chemicals and apparatus NADP + was obtained from the Oriental Yeast Co. Ltd. MV 2 + was purchased from Nakarai Chemical Co. Ltd., Japan. G R (EC 1.6.4.2, from yeast), FRD (EC 1.18.1.2, from spinach leaves), GSSG, and GSH were obtained from Sigma Co. All other reagents and compounds were analytical grade. A Hokuto Denko potentiostat (HA-301) and function generator (HB-104) or a Yanagimoto polarographic analyzer (P-100) with an X-Y recorder (Nippon Denshi Kagaku Co., model U-335) were used to record voltammograms using a three-electrodes system.
Electrode construction A carbon plate (25 x 25 mm) electrode and glassy carbon electrode (type GC-P2, 3 mm 0) were obtained from Nippon Denkyu Kogyo Co. and Yanaco Co., respectively. A cell combined with a conventional three-electrode system of a working electrode (carbon plate), a reference electrode (saturated calomel electrode, SCE), and counter electrode (carbon fabric electrode) were used. Electrode potential values were recorded in comparison with the SCE. To remove oxygen from the solution in the cell, the solution was bubbled by high-purity nitrogen gas.
;
Figure 1 Apparatus for mixed and conjugated reaction system (two-compartment cell). W: carbon plate; R: SCE; C: carbon fabric; M: cellophane membrane
A N2 gas
N2 gas
C
RW
N2 gas
Synthesis of urethane prepolymer Urethane prepolymer was prepared according to our previous paper. 7 Polyethylene glycol (Mw 600), 46.7 g, and 26.2 g hexamethylene diisocyanate (HMDI) were dissolved in dioxane and heated to 80 °C. The reaction was continued until the amount of the isocyanate (--NCO) group in the reaction mixture was reduced to half its initial value to prevent any further reaction. The - - N C O content was measured by the Sigga-Hanna method. 9 The isocyanate group which did not take part in the reaction with both end groups of polyethylene glycol remained in the product as a free functional group. To protect the end - - N C O group of the produced poly(oxyethylene)bis(6-isocyanatehexylcarbamate), it was reacted with one equivalent imidazole in dioxane at 40°C for 24 h. The resulting compound was obtained as a urethane prepolymer.
Preparation of polyurethane membrane and immobilization of NADP + Three grams of urethane prepolymer and 1.32 mmol of diethylenetriamine (DETA) were mixed together and allowed to stand for 30 min at room temperature. The mixture was cast on a glass plate and allowed to stand for 2 days. The membrane was immersed in water for 1 to 2 weeks to remove dioxane and the remaining imidazole. The membrane was stored in 0.1 M Tris-HCl buffer (pH 7.5) at 4°C, and NADP + was immobilized immediately before use as follows. In 10 ml of 0.25 mM NADP + solution in 0.1 M Tris-HC1 buffer (pH 7.5), one piece of the membrane (diameter 30 mm) was soaked for 24 h at 4°C. The membrane was then washed with a large volume of 0.1 M Tris-HCl buffer (pH 7.5). In this form, the prepared membrane was set between compartments and used as the electron transfer membrane.
Driving of the conjugated redox reaction A conjugated redox reaction in a mixed system was performed by using a two-compartment cell, as shown in Figure 1. Compartments I and II were separated by a cellophane membrane and filled with a 25.0 ml of 0.1 M Tris-HCl buffer (pH 7.5 ). The desired amounts of MV 2+, 12.5 i~mol NADP +, 25.0 i~mol GSSG, 0.5 units FRD, and 0.5 units G R were added to the solution of com-
B
N2 gas
I
N2 gas
N2gos
I
1I Figure 2 Apparatusfor separated and conjugated reaction system (three-compartmentcell).W: carbon plate; R: SCE; C: carbon fabric; M1 : electron transfer membrane; M2: cellophane membrane
partment I, and the desired amounts of MV 2+ were added to the buffer solution of compartment II. The cells were immersed in a water bath and continuously purged with nitrogen gas. The objective oxidoreductase reaction was conjugated with the electrochemical regeneration of NADPH at a constant potential, - 1.2 V vs. SCE and 25°C. A separated and conjugated redox reaction across the membrane was performed by using a three-compartment cell, the electron transfer membrane (M1), and the cellophane membrane (M2), as shown in Figure 2 (type A or B). Compartments I, II, and III were filled with 25.0 ml of 0.1 M Tris-HC1 buffer (pH 7.5). The solution of compartment II for the electrochemical regeneration of NADPH was prepared to contain 25 p,mol MV 2 +, 6.25 ~mol NADP +, and 1.5 units FRD. Furthermore, the solution of compartment III for the objective enzymatic reaction was made to contain 12.5 ~mol GSSG, 25 p,mol NADP +, and 35 units GR. The electrolysis conditions were similar to those described above.
Enzyme Microb. Technol., 1994, vol. 16, December
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Papers Analytical procedure The concentration of GSH in the reaction mixture was analyzed by the differential pulse polarographic method.l° The membrane potential was determined in a cell consisting of two compartments as described previously. 11
1.0 I E
-6
Results and discussion
E
Cyclic voltammograms of M V 2 + and NADP + The electrochemical behavior of MV 2+ and NADP + was investigated using a glassy carbon electrode. The potential scan range was between + 1.2 and - 1.7 V vs. SCE with a rate of 50 mV s - 1. Figure3 shows the cyclic voltammogram for 0.5 mM MV 2+ solution in 0.1 M Tris-HC1 buffer at pH 7.5 (A), (A) plus 2.0 mM NADP + (B), and (A) plus 2.0 mM NADP + and 1.0 unit FRD (C). As already shown by Matsue et al., 12 two cathodic peaks, Epa and Epb, were observed at - 0.72 and - 1.05 V vs. SCE, respectively (A of Figure 3). MV 2+ is effectively reduced to MV + and MV + to MV ° on the glassy carbon electrode. When the solution contained NADP +, the second cathodic wave increased proportionally to the concentration of NADP + (B of Figure 3). In this case, however, the anodic wave of the NADP dimer (at + 0.75 Vvs. SCE), which is inactive for enzymatic reactions, 13,14was also observed. When the solution in the cell was replaced with the buffer solution containing MV 2+, NADP +, and FRD, the anodic peak of the NADP dimer disappeared and the normal anodic peak of N A D P H appeared as shown in C of Figure 3. Contrary to the former case, the second cathodic wave appearing on the glassy carbon electrode is ascribable to the mediated electrochemical reduction of NADP +. According to these results, the cathodic potential for the mediated electrolytic reduction of NADP + was set at - 1.2 V vs. SCE.
b +10.0 Q
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-o
0.5 --1 "13
o CL "F (/3 L0 I
I
1.0
I
I
2.0 Time ( h )
I
I
I
3.0
Figure4 Effects o f M V 2+ concentration on GSH production by m i x e d and conjugated r e d o x reaction system. The r e d o x reactions w e r e carried out using the standard m e t h o d f o r NADPH regeneration at various M V 2+ concentrations. ( t ) 0.5 mM; (A) 1.0 mM; ( I ) 1.5 mM
Enzymic reaction conjugated with electrochemical regeneration of coenzyme in mired system A conjugated reaction of electrochemical reduction of NADP + and GSH production was carried out in a mixed system by using native FRD for more accurate driving of the electrochemical NADPH-regenerating reaction and G R (Scheme 1A). The effects of MV 2 + concentration in the electrolyte on GSH production were investigated. As shown in Figure 4, the conversion of GSH from GSSG was accelerated with increasing M V 2+ concentrations. In all cases (using 1.25, 2.50, and 3.75 ~mol of M V 2 + ), GSSG was completely converted to GSH after 3.5 h.
,,",,
Enzymic reaction conjugated with electrochemical regeneration of coenzyme in separated system (transmembrane type) <:1, 0.0 . . . . .
-10.0 i
+1.0
1
I
I
0.0
i
-1.0
E ( V vs. SCE ) Figure 3 Cyclic v o l t a m m o g r a m (scan rate = 50 mV.s -1) with carbon plate electrode in 0.1 M Tris-HCI buffer (pH 7.5) containing 0.5 mM M Y 2+ (A . . . . ), 0.5 mM i V 2+ + 2.0 mM NADP + (B, - - ) and 0.5 m a M V 2÷ + 2.0 mM NADP ÷ + 1.0 unit FRD (C, ---)
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The separated system for conjugating the objective enzymic reaction and the electrochemical N A D P H regeneration across the membrane (M1) was designed as shown in Scheme 1. In this type of three-compartment cell, a solution (II) for electrochemical regeneration of N A D P H was isolated with the polyurethane membrane from the solution (III) of substrate and enzyme for the objective reaction (the substrate GSSG can be reduced with G R and NADPH). At first, the reaction in the separated system was examined by using a type A three-compartment cell (Figure 2A). The results are shown in Figure 5. In this figure the concentration of GSH produced in the component III is plotted against time. GSH was hardly produced for a long time. From this result, it can be concluded that the enzyme (FRD) of the coenzyme regeneration reaction in compartment II was attracted toward the positively charged counter
Redox reaction across the membrane: Y. Nakamura A
electrod
e -I
I/2MV 0
NADP +
2GSH
I / 2 M V 2+
NADPH
NADP +
NAOPHNAOPNAOH/2GSS
FRD electrode
I
P1/2MvO
GR
NADP+lNADPH
NADP+
NADP
NADP +
GSH
=
Subreaction
electron
transfer
Objective
reaction
membrane
Scheme I Conjugated electrochemical redox reaction system. (A) Mixed and conjugated electrochemical redox reaction system. (B) Separated and conjugated electrochemical redox reaction system across the membrane
electrode in compartment I, so that the enzyme reaction could not take place at the surface of the electron transfer membrane. A smooth progress of the electron transfer reaction on the membrane surface on the coenzyme regeneration reaction side should be necessary to couple the regeneration reaction and the objective reaction. This conclusion was confirmed by the following experiment. Instead of using electrochemical reduction to regenerate NADH, N A D H was added to compartment II and supplied by diffusion to compartment III through the membrane. The rate of production of GSH was much slower than that of the conjugated reaction. Therefore, the type B cell was designed in such a way that the compartment in which the counter electrode was inserted was repositioned adjacent to the compartment in which a working electrode was inserted for the regeneration reaction, as shown in Figure2B. Figure5 shows that the concentration of GSH accumulated in the reaction mixture reached about 0.5 ~mol ml - 1 after 5 h; the conversion ratio on GSSG was 50%. In comparison with the mixed system, the separated system showed a slower rate of GSH production, which appears to be controlled by a slower reduction of NADP +. This could be explained by the electron transfer efficiency of the membrane between the two compartments. G R activity was not found in compartment II. This means that enzyme could not leak out from compartment I through the electron transfer membrane. In addition, MV could not pass through the membrane because of electrostatic repulsion between cationic MV and the membrane. The main reaction and subreaction could be isolated in individual reactions. From the biotechnological point of view, this cell design is effective for continuous operation, since the main reaction product can be recovered readily.
Effect of membrane potential of the polyurethane membrane The effects of the amount of D E T A in the polyurethane membrane on its properties were investigated. The membrane potentials of the electron transfer membranes prepared under standard conditions except for D E T A content (half its standard value) were - 41.9 and - 23.0 mV, respectively. Increasing the amount of D E T A resulted in a more negative membrane potential, that is, the membrane was charged more positively. This result suggests that the
et al.
degree of membrane potential depends on the content of DETA. When the lower charged membrane was used for the separated reaction system, production of GSH was slight (data not shown). This brings one to consider that the attractive force between totally negatively charged NADP + and the membrane increases when the positive charge of the polyurethane membrane is increased as NADP + could be trapped in the membrane. A more positively charged membrane is suitable for adsorption of NADP + in the membrane. In conclusion, we showed that the polyurethane membrane on which NADP + was adsorbed can be used as an electron transfer membrane. The separated reaction system, constructed by using the three compartments and two membranes (polyurethane and cellophane), successfully drives the conjugated redox reaction. However, the improvement of electron transfer efficiencythrough the membrane is worthy of further investigation. Recently, modified electrodes using viologen derivatives immobilized on the electrode surface by depositing methods have been prepared, 15,16 but deposited polymerized viologen is subject to separation from the electrode. The immobilization of the viologen derivative to the modified electrode by covalent bonds is now being attempted. Recently, many reactor systems with NAD(P)H recycling have been designed. 12,17,18These methods could not separate the main reaction and the subreaction. In contrast, the present method has a great practical advantage, in that the substrate and enzyme for the main reaction are not contaminated by those of the subreaction. Consequently, the separated reaction system and attached electron transfer membrane can be applicable to the construction of various clean bioreactors.
~ 05 f
=
I
;°4 f o2b
~ 0.3
;of . f = L ~ r ' 1
0
J_
1.0
J_
-L
2.0 3.0 Time ( h )
4.0
5.0
Figure 5 Production of GSH using separated and conjugated redox reaction systems. The reactions were performed using type A cell (11) and type B cell (0)
Enzyme Microb. Technol., 1994, vol. 16, December
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Papers References 1
2
3 4
5
6
7 8
Chang, T. M. S. and Malouf, C. Artificial cells microencapsulated multienzyme system for converting urea and ammonia to amino acid using a-ketoglutarate and glucose as substrate. Trans. Am. Soc.Artif. Intern. Organs 1978, 24, 8-20 Suye, S., Kawagoe, M., Inuta, S., Sano M. and Yokoyama, S. Enzymatic production of L-alanine from malic acid with malic enzyme and alanine dehydrogenase with coenzyme regeneration. Can. J. Chem. Eng. 1992, 70, 306-312 Maeda, H. and Kajiwara, S. Malic acid production by an electrochemical reduction system combined with the use ofdiaphorase and methylviologen Biotech. Bioeng. 1985, 27, 596-602 Vandecasteele, J.-P. Enzymatic synthesis of L-carnitine by reduction of an achiral precursor: The problem of reduced nicotinamide adenine dinucleotide recycling.AppL Environ. MicrobioL 1980, 39, 327334 Eguchi, T., Kuge, Y., Inoue, K., Yoshikawa, N., Mochida, K. and Uwajima, T. NADPH regeneration by glucose dehydrogenase from Gluconobacter seleroides for L-leucovorin synthesis. Biosci. Biotech. Biochem. 1992, 56, 701-703 Dicosimo, R., Wong, C. -H., Daniels, L. and Whitesides, G. M. Enzyme-catalyzed organic synthesis: Electrochemical regeneration of NAD(P)H from NAD(P) using methyl viologen and flavoenzymes. J. Org. Chem. 1981, 46, 4622-4623 Nakamura, Y., Kamon, N. and Hori, T. Study on conjugated redox membranes containing an intercellular component of wool as an electron carrier Bull. Chem. Soc. Jpn. 1989, 62, 551-556 Nakamura, Y., Yamada, M., Miura, J., Hirota, K. and Hori, T. Electron transfer function of kerateine gel and conjugated redox reaction by using the kerateine-immobilized membrane. J. Polym. Sci.: Part A: Polymer Chemistry 1989, 27, 2883-2895
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9 10 11 12 13 14
15 16
17
18
Siggia, S. and Hanna, J. G. Determination of organic isocyanates and isothiocyanates. Anal Chem. 1948, 20, 108. Nakamura, Y. and Saito, Y. A polarographic study on the interaction between NADP and oxidizes glutathion. Rev. Polarography 1986, 32, 68 Nakamura, Y., Tabata, I. and Hori, T. Evaluation of a convenient method for determining membrane potential.Mern. Fac. Eng. Fukui Univ. 1988, 36, 183-192 Matsue, T., Chang, H. -C., Uchida, I. and Osa, T. Bioelectrocatalytic reduction of NAD to NADH on diaphorase modified electrodes. Tetrahedron Lett. 1988, 29, 1551-1554 Burnett, R. W. and Underwood, A. L. A dimer of diphosphopyridine nucleotide. Biochemistry 1968, 7, 3328-3333 Jensen, M. A. and Elving, P. J. Nicotineamide adenine dinucleotide (NAD ÷ ): Formal potential of the NAD +/NAD'couple and NAD.dimerization rate. Biochim. Biophys. Acta 1984, 764, 310315 Landrum, H. L., Salmon, R. T. and Hawkridge, F. M. A surfacemodified gold minigrid electrode which heterogeneously spinach ferredoxin. J. Am. Chem. Soc. 1977, 99, 3154-3158 Bowden, E. F. and Hawkridge, F. M. Thin-layer spectrochemical kinetic study of viologen cation radicals reacting at hydrogenenvolving gold and nickel electrode. J. ElectroanaL Chem. lnterfaciaL Electrochem. 1981, 25, 367-386 Wichmann, R., Wandrey, C., Bueckmann, A. F., and Kula, M. R. Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration. Biotech. Bioeng. 1981, 23, 2789-2802 Miyawaki, O., Nakamura, K. and Yano, Y. An affinity chromatographic reactor for highly efficient turnover of dissociable cofactors. Agric. Biol. Chem. 1985, 49, 2063-2070
The conjugated redox reaction was driven across the electron transfer membrane prepared from a urethane prepokymer to carry positive charge, where NADP + as electron transfer carrier was adsorbed in the prepared pokyurethane membrane. Glutathione reductase [NAD (P)H: oxidized-glutathione oxidoreductase (EC 1.6.4.1)] was used as the catatystfor production of the reduced form of glutathione (GSH) from the oMdizedform (GSSG) in the objective reaction, and methyl viologen (MV) was used for the electrochemical regeneration of NADPH in the subreaction. The conjugated redox reaction in the separated reactions system, using the three-compartment cell with two membranes, was successful without MV contamination. Under the given conditions, the conversion ratio of G SH from GSSG reached 50% after 4 h of incubation at 30°C and the amount of GSH accumulated was 0.5 gmol ml - 1 of reaction mixture.
Keywords: Electrontransfer;NADPHregeneration;glutathionereductase;conjugatedredoxreaction;separatedreaction system
Introduction Regeneration of pyridine nucleotide NADH and NADPH is important for carrying out enzymatic syntheses that use NAD (P)-linked dehydrogenases. Various biochemical substances, such as amino acids, 1,2 hydroxy acids, 3 and other useful compounds, 4-6 can be produced by coupling an electrochemical or an enzymatic reaction for reduction of NAD(P) + and an NAD(P)-linked dehydrogenase reaction. These reactions are commonly carried out in a onebath system in which an objective reaction (coenzymeconsuming reaction) and a subreaction (coenzymeregeneration reaction) cannot be separated. In these cases the main products are contaminated with substances originating in the subreaction, such as mediator, substrate, byproducts, and enzyme. If redox reactions proceeded separately in a separated reactions system by using an electron transfer membrane, the system would be very useful for
Received28 September1993;accepted27 April 1994 Address reprint requests to Dr. Nakamuraat Fukui University,Department of AppliedChemistryand Biotechnology,3-9-1Bunkyo,Fukui910, Japan
1026
Enzyme Microb. Technol., 1994, vol. 16, December
designing of various enzymatic syntheses, as well as bioreactors, such as artificial kidneys. 1 In our previous papers, 7,8 preparations of the electron transfer membranes containing coenzyme NAD(P)+/ NAD(P)H or kerateine components as an electron carrier, and intercellular components of wool as a channel substance for the proton transport, were reported. Next, the construction of a separated-reactions system, in which twooxidoreductase/redox-reactions successfully proceeded conjugately across the membranes, was presented. In this paper, the separated-reactions system consisting of an electrolytic cell and an enzymatic reaction cell separated by the electron transfer membrane was constructed. In preparing the electron transfer membrane, NADP + as an electron transfer carrier was immobilized in the polyurethane membrane. The electrochemical reduction of NADP + mediated by methyl viologen (MV 2 + ) and ferredoxin NADP +-reductase (FRD) was examined through cyclic voltammetery using a glassy carbon electrode, and constant potential cathodic reduction was performed using a carbon plate electrode. We investigated the conversion of the oxidized form of glutathione (GSSG) to the reduced form (GSH) using glutathione reductase (GR) and FRD with MV 2 + as a mediator. © 1994
Butterworth-Heinemann
Redox reaction across the membrane: Y. Nakamura et al.
Materials and methods
N2 gas
2 gas
Chemicals and apparatus NADP + was obtained from the Oriental Yeast Co. Ltd. MV 2 + was purchased from Nakarai Chemical Co. Ltd., Japan. G R (EC 1.6.4.2, from yeast), FRD (EC 1.18.1.2, from spinach leaves), GSSG, and GSH were obtained from Sigma Co. All other reagents and compounds were analytical grade. A Hokuto Denko potentiostat (HA-301) and function generator (HB-104) or a Yanagimoto polarographic analyzer (P-100) with an X-Y recorder (Nippon Denshi Kagaku Co., model U-335) were used to record voltammograms using a three-electrodes system.
Electrode construction A carbon plate (25 x 25 mm) electrode and glassy carbon electrode (type GC-P2, 3 mm 0) were obtained from Nippon Denkyu Kogyo Co. and Yanaco Co., respectively. A cell combined with a conventional three-electrode system of a working electrode (carbon plate), a reference electrode (saturated calomel electrode, SCE), and counter electrode (carbon fabric electrode) were used. Electrode potential values were recorded in comparison with the SCE. To remove oxygen from the solution in the cell, the solution was bubbled by high-purity nitrogen gas.
;
Figure 1 Apparatus for mixed and conjugated reaction system (two-compartment cell). W: carbon plate; R: SCE; C: carbon fabric; M: cellophane membrane
A N2 gas
N2 gas
C
RW
N2 gas
Synthesis of urethane prepolymer Urethane prepolymer was prepared according to our previous paper. 7 Polyethylene glycol (Mw 600), 46.7 g, and 26.2 g hexamethylene diisocyanate (HMDI) were dissolved in dioxane and heated to 80 °C. The reaction was continued until the amount of the isocyanate (--NCO) group in the reaction mixture was reduced to half its initial value to prevent any further reaction. The - - N C O content was measured by the Sigga-Hanna method. 9 The isocyanate group which did not take part in the reaction with both end groups of polyethylene glycol remained in the product as a free functional group. To protect the end - - N C O group of the produced poly(oxyethylene)bis(6-isocyanatehexylcarbamate), it was reacted with one equivalent imidazole in dioxane at 40°C for 24 h. The resulting compound was obtained as a urethane prepolymer.
Preparation of polyurethane membrane and immobilization of NADP + Three grams of urethane prepolymer and 1.32 mmol of diethylenetriamine (DETA) were mixed together and allowed to stand for 30 min at room temperature. The mixture was cast on a glass plate and allowed to stand for 2 days. The membrane was immersed in water for 1 to 2 weeks to remove dioxane and the remaining imidazole. The membrane was stored in 0.1 M Tris-HCl buffer (pH 7.5) at 4°C, and NADP + was immobilized immediately before use as follows. In 10 ml of 0.25 mM NADP + solution in 0.1 M Tris-HC1 buffer (pH 7.5), one piece of the membrane (diameter 30 mm) was soaked for 24 h at 4°C. The membrane was then washed with a large volume of 0.1 M Tris-HCl buffer (pH 7.5). In this form, the prepared membrane was set between compartments and used as the electron transfer membrane.
Driving of the conjugated redox reaction A conjugated redox reaction in a mixed system was performed by using a two-compartment cell, as shown in Figure 1. Compartments I and II were separated by a cellophane membrane and filled with a 25.0 ml of 0.1 M Tris-HCl buffer (pH 7.5 ). The desired amounts of MV 2+, 12.5 i~mol NADP +, 25.0 i~mol GSSG, 0.5 units FRD, and 0.5 units G R were added to the solution of com-
B
N2 gas
I
N2 gas
N2gos
I
1I Figure 2 Apparatusfor separated and conjugated reaction system (three-compartmentcell).W: carbon plate; R: SCE; C: carbon fabric; M1 : electron transfer membrane; M2: cellophane membrane
partment I, and the desired amounts of MV 2+ were added to the buffer solution of compartment II. The cells were immersed in a water bath and continuously purged with nitrogen gas. The objective oxidoreductase reaction was conjugated with the electrochemical regeneration of NADPH at a constant potential, - 1.2 V vs. SCE and 25°C. A separated and conjugated redox reaction across the membrane was performed by using a three-compartment cell, the electron transfer membrane (M1), and the cellophane membrane (M2), as shown in Figure 2 (type A or B). Compartments I, II, and III were filled with 25.0 ml of 0.1 M Tris-HC1 buffer (pH 7.5). The solution of compartment II for the electrochemical regeneration of NADPH was prepared to contain 25 p,mol MV 2 +, 6.25 ~mol NADP +, and 1.5 units FRD. Furthermore, the solution of compartment III for the objective enzymatic reaction was made to contain 12.5 ~mol GSSG, 25 p,mol NADP +, and 35 units GR. The electrolysis conditions were similar to those described above.
Enzyme Microb. Technol., 1994, vol. 16, December
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Papers Analytical procedure The concentration of GSH in the reaction mixture was analyzed by the differential pulse polarographic method.l° The membrane potential was determined in a cell consisting of two compartments as described previously. 11
1.0 I E
-6
Results and discussion
E
Cyclic voltammograms of M V 2 + and NADP + The electrochemical behavior of MV 2+ and NADP + was investigated using a glassy carbon electrode. The potential scan range was between + 1.2 and - 1.7 V vs. SCE with a rate of 50 mV s - 1. Figure3 shows the cyclic voltammogram for 0.5 mM MV 2+ solution in 0.1 M Tris-HC1 buffer at pH 7.5 (A), (A) plus 2.0 mM NADP + (B), and (A) plus 2.0 mM NADP + and 1.0 unit FRD (C). As already shown by Matsue et al., 12 two cathodic peaks, Epa and Epb, were observed at - 0.72 and - 1.05 V vs. SCE, respectively (A of Figure 3). MV 2+ is effectively reduced to MV + and MV + to MV ° on the glassy carbon electrode. When the solution contained NADP +, the second cathodic wave increased proportionally to the concentration of NADP + (B of Figure 3). In this case, however, the anodic wave of the NADP dimer (at + 0.75 Vvs. SCE), which is inactive for enzymatic reactions, 13,14was also observed. When the solution in the cell was replaced with the buffer solution containing MV 2+, NADP +, and FRD, the anodic peak of the NADP dimer disappeared and the normal anodic peak of N A D P H appeared as shown in C of Figure 3. Contrary to the former case, the second cathodic wave appearing on the glassy carbon electrode is ascribable to the mediated electrochemical reduction of NADP +. According to these results, the cathodic potential for the mediated electrolytic reduction of NADP + was set at - 1.2 V vs. SCE.
b +10.0 Q
v
-o
0.5 --1 "13
o CL "F (/3 L0 I
I
1.0
I
I
2.0 Time ( h )
I
I
I
3.0
Figure4 Effects o f M V 2+ concentration on GSH production by m i x e d and conjugated r e d o x reaction system. The r e d o x reactions w e r e carried out using the standard m e t h o d f o r NADPH regeneration at various M V 2+ concentrations. ( t ) 0.5 mM; (A) 1.0 mM; ( I ) 1.5 mM
Enzymic reaction conjugated with electrochemical regeneration of coenzyme in mired system A conjugated reaction of electrochemical reduction of NADP + and GSH production was carried out in a mixed system by using native FRD for more accurate driving of the electrochemical NADPH-regenerating reaction and G R (Scheme 1A). The effects of MV 2 + concentration in the electrolyte on GSH production were investigated. As shown in Figure 4, the conversion of GSH from GSSG was accelerated with increasing M V 2+ concentrations. In all cases (using 1.25, 2.50, and 3.75 ~mol of M V 2 + ), GSSG was completely converted to GSH after 3.5 h.
,,",,
Enzymic reaction conjugated with electrochemical regeneration of coenzyme in separated system (transmembrane type) <:1, 0.0 . . . . .
-10.0 i
+1.0
1
I
I
0.0
i
-1.0
E ( V vs. SCE ) Figure 3 Cyclic v o l t a m m o g r a m (scan rate = 50 mV.s -1) with carbon plate electrode in 0.1 M Tris-HCI buffer (pH 7.5) containing 0.5 mM M Y 2+ (A . . . . ), 0.5 mM i V 2+ + 2.0 mM NADP + (B, - - ) and 0.5 m a M V 2÷ + 2.0 mM NADP ÷ + 1.0 unit FRD (C, ---)
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The separated system for conjugating the objective enzymic reaction and the electrochemical N A D P H regeneration across the membrane (M1) was designed as shown in Scheme 1. In this type of three-compartment cell, a solution (II) for electrochemical regeneration of N A D P H was isolated with the polyurethane membrane from the solution (III) of substrate and enzyme for the objective reaction (the substrate GSSG can be reduced with G R and NADPH). At first, the reaction in the separated system was examined by using a type A three-compartment cell (Figure 2A). The results are shown in Figure 5. In this figure the concentration of GSH produced in the component III is plotted against time. GSH was hardly produced for a long time. From this result, it can be concluded that the enzyme (FRD) of the coenzyme regeneration reaction in compartment II was attracted toward the positively charged counter
Redox reaction across the membrane: Y. Nakamura A
electrod
e -I
I/2MV 0
NADP +
2GSH
I / 2 M V 2+
NADPH
NADP +
NAOPHNAOPNAOH/2GSS
FRD electrode
I
P1/2MvO
GR
NADP+lNADPH
NADP+
NADP
NADP +
GSH
=
Subreaction
electron
transfer
Objective
reaction
membrane
Scheme I Conjugated electrochemical redox reaction system. (A) Mixed and conjugated electrochemical redox reaction system. (B) Separated and conjugated electrochemical redox reaction system across the membrane
electrode in compartment I, so that the enzyme reaction could not take place at the surface of the electron transfer membrane. A smooth progress of the electron transfer reaction on the membrane surface on the coenzyme regeneration reaction side should be necessary to couple the regeneration reaction and the objective reaction. This conclusion was confirmed by the following experiment. Instead of using electrochemical reduction to regenerate NADH, N A D H was added to compartment II and supplied by diffusion to compartment III through the membrane. The rate of production of GSH was much slower than that of the conjugated reaction. Therefore, the type B cell was designed in such a way that the compartment in which the counter electrode was inserted was repositioned adjacent to the compartment in which a working electrode was inserted for the regeneration reaction, as shown in Figure2B. Figure5 shows that the concentration of GSH accumulated in the reaction mixture reached about 0.5 ~mol ml - 1 after 5 h; the conversion ratio on GSSG was 50%. In comparison with the mixed system, the separated system showed a slower rate of GSH production, which appears to be controlled by a slower reduction of NADP +. This could be explained by the electron transfer efficiency of the membrane between the two compartments. G R activity was not found in compartment II. This means that enzyme could not leak out from compartment I through the electron transfer membrane. In addition, MV could not pass through the membrane because of electrostatic repulsion between cationic MV and the membrane. The main reaction and subreaction could be isolated in individual reactions. From the biotechnological point of view, this cell design is effective for continuous operation, since the main reaction product can be recovered readily.
Effect of membrane potential of the polyurethane membrane The effects of the amount of D E T A in the polyurethane membrane on its properties were investigated. The membrane potentials of the electron transfer membranes prepared under standard conditions except for D E T A content (half its standard value) were - 41.9 and - 23.0 mV, respectively. Increasing the amount of D E T A resulted in a more negative membrane potential, that is, the membrane was charged more positively. This result suggests that the
et al.
degree of membrane potential depends on the content of DETA. When the lower charged membrane was used for the separated reaction system, production of GSH was slight (data not shown). This brings one to consider that the attractive force between totally negatively charged NADP + and the membrane increases when the positive charge of the polyurethane membrane is increased as NADP + could be trapped in the membrane. A more positively charged membrane is suitable for adsorption of NADP + in the membrane. In conclusion, we showed that the polyurethane membrane on which NADP + was adsorbed can be used as an electron transfer membrane. The separated reaction system, constructed by using the three compartments and two membranes (polyurethane and cellophane), successfully drives the conjugated redox reaction. However, the improvement of electron transfer efficiencythrough the membrane is worthy of further investigation. Recently, modified electrodes using viologen derivatives immobilized on the electrode surface by depositing methods have been prepared, 15,16 but deposited polymerized viologen is subject to separation from the electrode. The immobilization of the viologen derivative to the modified electrode by covalent bonds is now being attempted. Recently, many reactor systems with NAD(P)H recycling have been designed. 12,17,18These methods could not separate the main reaction and the subreaction. In contrast, the present method has a great practical advantage, in that the substrate and enzyme for the main reaction are not contaminated by those of the subreaction. Consequently, the separated reaction system and attached electron transfer membrane can be applicable to the construction of various clean bioreactors.
~ 05 f
=
I
;°4 f o2b
~ 0.3
;of . f = L ~ r ' 1
0
J_
1.0
J_
-L
2.0 3.0 Time ( h )
4.0
5.0
Figure 5 Production of GSH using separated and conjugated redox reaction systems. The reactions were performed using type A cell (11) and type B cell (0)
Enzyme Microb. Technol., 1994, vol. 16, December
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Papers References 1
2
3 4
5
6
7 8
Chang, T. M. S. and Malouf, C. Artificial cells microencapsulated multienzyme system for converting urea and ammonia to amino acid using a-ketoglutarate and glucose as substrate. Trans. Am. Soc.Artif. Intern. Organs 1978, 24, 8-20 Suye, S., Kawagoe, M., Inuta, S., Sano M. and Yokoyama, S. Enzymatic production of L-alanine from malic acid with malic enzyme and alanine dehydrogenase with coenzyme regeneration. Can. J. Chem. Eng. 1992, 70, 306-312 Maeda, H. and Kajiwara, S. Malic acid production by an electrochemical reduction system combined with the use ofdiaphorase and methylviologen Biotech. Bioeng. 1985, 27, 596-602 Vandecasteele, J.-P. Enzymatic synthesis of L-carnitine by reduction of an achiral precursor: The problem of reduced nicotinamide adenine dinucleotide recycling.AppL Environ. MicrobioL 1980, 39, 327334 Eguchi, T., Kuge, Y., Inoue, K., Yoshikawa, N., Mochida, K. and Uwajima, T. NADPH regeneration by glucose dehydrogenase from Gluconobacter seleroides for L-leucovorin synthesis. Biosci. Biotech. Biochem. 1992, 56, 701-703 Dicosimo, R., Wong, C. -H., Daniels, L. and Whitesides, G. M. Enzyme-catalyzed organic synthesis: Electrochemical regeneration of NAD(P)H from NAD(P) using methyl viologen and flavoenzymes. J. Org. Chem. 1981, 46, 4622-4623 Nakamura, Y., Kamon, N. and Hori, T. Study on conjugated redox membranes containing an intercellular component of wool as an electron carrier Bull. Chem. Soc. Jpn. 1989, 62, 551-556 Nakamura, Y., Yamada, M., Miura, J., Hirota, K. and Hori, T. Electron transfer function of kerateine gel and conjugated redox reaction by using the kerateine-immobilized membrane. J. Polym. Sci.: Part A: Polymer Chemistry 1989, 27, 2883-2895
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9 10 11 12 13 14
15 16
17
18
Siggia, S. and Hanna, J. G. Determination of organic isocyanates and isothiocyanates. Anal Chem. 1948, 20, 108. Nakamura, Y. and Saito, Y. A polarographic study on the interaction between NADP and oxidizes glutathion. Rev. Polarography 1986, 32, 68 Nakamura, Y., Tabata, I. and Hori, T. Evaluation of a convenient method for determining membrane potential.Mern. Fac. Eng. Fukui Univ. 1988, 36, 183-192 Matsue, T., Chang, H. -C., Uchida, I. and Osa, T. Bioelectrocatalytic reduction of NAD to NADH on diaphorase modified electrodes. Tetrahedron Lett. 1988, 29, 1551-1554 Burnett, R. W. and Underwood, A. L. A dimer of diphosphopyridine nucleotide. Biochemistry 1968, 7, 3328-3333 Jensen, M. A. and Elving, P. J. Nicotineamide adenine dinucleotide (NAD ÷ ): Formal potential of the NAD +/NAD'couple and NAD.dimerization rate. Biochim. Biophys. Acta 1984, 764, 310315 Landrum, H. L., Salmon, R. T. and Hawkridge, F. M. A surfacemodified gold minigrid electrode which heterogeneously spinach ferredoxin. J. Am. Chem. Soc. 1977, 99, 3154-3158 Bowden, E. F. and Hawkridge, F. M. Thin-layer spectrochemical kinetic study of viologen cation radicals reacting at hydrogenenvolving gold and nickel electrode. J. ElectroanaL Chem. lnterfaciaL Electrochem. 1981, 25, 367-386 Wichmann, R., Wandrey, C., Bueckmann, A. F., and Kula, M. R. Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration. Biotech. Bioeng. 1981, 23, 2789-2802 Miyawaki, O., Nakamura, K. and Yano, Y. An affinity chromatographic reactor for highly efficient turnover of dissociable cofactors. Agric. Biol. Chem. 1985, 49, 2063-2070