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Development of a novel glucose enzyme fuel cell system employing protein engineered PQQ glucose dehydrogenase
Biosensors and Bioelectronics 20 (2005) 2145–2150
Short communication
Development of a novel glucose enzyme fuel cell system employing protein engineered PQQ glucose dehydrogenase Noriko Yuhashia , Masamitsu Tomiyamab , Junko Okudaa , Satoshi Igarashia , Kazunori Ikebukuroa , Koji Sodea,∗ a
Department of Life Science and Biotechnology, Tokyo University of Agriculture and Technology, Nakamachi 2-24-16, Koganei, Tokyo 184-8588, Japan b Genetic Diversity Department, Applied Microbiology Laboratory, National Institute of Agrobiological Sciences, Ibaraki 305-8602, Japan Received 31 May 2004; received in revised form 5 August 2004; accepted 12 August 2004 Available online 15 September 2004
Abstract Glucose dehydrogenase harboring pyrroloquinoline quinone as the prosthetic group (PQQGDH) from Acinetobacter calcoaceticus is an ideal enzyme for the anode of biofuel cell, because of its oxygen insensitivity and high catalytic efficiency. However, the application of PQQGDH for the bioanode is inherently limited because of its instability. Using Ser415Cys mutant whose stability was greatly improved, we constructed the biofuel cell system employing the engineered PQQGDH as the bioanode enzyme and bilirubin oxidase (BOD) as the biocathode, and compared the stability of the biofuel cell with that employing wild-type PQQGDH. The maximum power density was 17.6 W/cm2 at an external optimal load of 200 k. Using Ser415Cys mutant, the lifetime of the biofuel cell system was greatly extended to 152 h, more than six times as that of the biofuel cell employing the wild-type. © 2004 Elsevier B.V. All rights reserved. Keywords: Enzyme fuel cell; PQQ glucose dehydrogenase; Protein engineering; Bilirubin oxidase; Stability
1. Introduction Biofuel cell systems utilize biocatalysts, such as enzymes and microorganisms, as the electrocatalysts, instead of transition metal catalysts that are utilized in the conventional fuel cell systems. Several organic compounds can be utilized in biofuel cells, especially biomass based compounds can be used as the fuels such as sugars and several alcohols, and the higher efficient energy conversion can be achieved compared with transition metal based fuel cells, theoretically. Biofuel cell can be applied not only to the biomass conversion, but also to the alternative energy source in the future implantable devices, such as implantable glucose sensors in the artificial pancreas. Recent trends in the development of biofuel cell is the use glucose as the fuel to be oxidized at the bioanode on ∗
Corresponding author. Tel.: +81 42 388 7021; fax: +81 42 388 7021. E-mail address: [email protected] (K. Sode).
0956-5663/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2004.08.017
which glucose oxidase (GOD) is immobilized, combining with appropriate biocathode, employing several dioxygen reducing enzymes (Chen et al., 2001; Katz et al., 1999; Kim et al., 2003; Mano et al., 2002, 2003). Together with the recent advances in microfabrication technology, miniaturized biofuel cells have also been reported (Chen et al., 2001; Mano et al., 2002, 2003; Soukharev et al., 2004). GOD is an extremely stable enzyme with high substrate specificity and relatively high Km value. Besides, in the field of self-blood glucose monitoring, glucose dehydrogenase harboring pyrroloquinoline quinone as the prosthetic group (PQQGDH) is focused owing to its oxygen insensitivity and high catalytic efficiency (Kost et al., 1998; Tang et al., 2001). Additionally its wide substrate specificity is advantageous for application of this enzyme to bioanode especially for biomass conversion. However, the application of PQQGDH to the bioanode is inherently limited because of its instability. The authors have been engaged in protein engineering of PQQGDH in order to develop the ideal enzyme
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for glucose monitoring (Sode et al., 2000; Takahashi et al., 2000). In those studies, one engineered enzyme, the Ser415Cys mutant (S415C) was obtained, of which stability was greatly improved by protein engineering (Igarashi and Sode, 2003). The S415C is stable even at 70 ◦ C. This is the most stable cofactor-binding GDH so far reported. The alteration of the residue at position 415 did not affect the catalytic efficiency of this enzyme, retaining high catalytic efficiency in glucose oxidation. The engineered PQQGDH therefore appears to be the most suitable enzyme for glucose based biofuel cell anode. In this paper, we evaluated the potential application of the engineered PQQGDH, S415C, as the bioanode enzyme in glucose utilizing biofuel cell systems. We constructed an enzyme glucose biofuel cell system employing S415C in the anodic reaction and bilirubin oxidase (BOD) for the cathodic reaction. It is reported that electrochemical reduction of dioxygen to water precedes very effectively at 0.4 V versus Ag/AgCl in pH 7.0 solution at an ambient temperature mediated by the 2,2 -azino-bis(3-ethylbenzothiazoline6-sulfonate) (ABTS2-)-mediated and catalyzed by bilirubin oxidase (Tsujimura et al., 2001). We then investigated the stability of the bioanode to evaluate the future potential application of the protein engineered enzymes to the biofuel cell system.
measured using 0.6 mM phenazine methosulphate (PMS) and 0.06 mM 2,6-dichlorophenolindophenol (DCIP) following 30 min pre-incubation in 10 mM MOPS buffer (pH 7.0) containing 1 M pyrroloquinoline quinone (PQQ) and 1 mM CaCl2 at room temperature (25 ◦ C). The enzyme activity was determined by measuring the decrease in absorbance of dichlorophenolindophenol (DCIP) at 600 nm. 2.3. Preparation of PQQGDH and bilirubin oxidase electrode An amout of 10 mmol MOPS buffer (pH 7.0) containing 300 U of PQQGDH or 100 U S415C or 5 U of Bilirubin oxidase was mixed with carbon paste (0.5 g graphite powder mixed with 0.3 ml paraffin liquid) and mixture was then lyophilized. The lyophilized mixture was then packed into the end of a carbon electrode (3 mm in diameter, BAS Inc., West Lafayette, USA) and treated with 1% glutaraldehyde solution for 30 min and then washed with 10 mM Tris–HCl buffer (pH 7.0). The PQQGDH or S415C immobilized electrode was then allowed to undergo holo-formation in 10 mM MOPS buffer (pH 7.0) containing 5 M PQQ and 1 mM CaCl2 at 4 ◦ C for at least 30 min and washed with 10 mM MOPS (pH 7.0). BOD electrode was washed with 10 mM MOPS buffer (pH 7.0). All electrodes were stored at 4 ◦ C until use. 2.4. Electrochemical measurements
2. Materials and methods 2.1. Chemicals Calcium chloride, 3-(N-morpholino) propanesulfanic acid (MOPS), 1-methoxyphenazine methosulphate (mPMS), 2,2 -azino-bis(3-ethylbenzothiazolone-6-sulfonic acid) diammonium salt (ABTS Diammonium Salt) and 25% glutaraldehyde were purchased from Wako Chemicals (Osaka, Japan). Pyrroloquinoline quinone (PQQ) was from Mitsubishi Gas Chemical Company, Inc. (Tokyo, Japan). Phenazine methosulfate (PMS), 2,6dichrolophenolindophenol (DCIP), -d-glucose, lactose, galactose, maltose was from Kanto Chemical (Tokyo, Japan). Cellobiose was from SIGMA (St. Louis, US), xylose was from Kishida Chemical Co., Ltd. (Osaka, Japan). Bilirubin oxidase was donated from Amano Enzyme (Aichi, Japan). DC protein assay kit was from Bio Rad Laboratories (California, USA). Carbon paste (0.5 g graphite powder mixed with 0.3 ml paraffin liquid) was from BSA Inc. (West Lafayette, USA). The other reagents were of analytical grade. Ultrafree MC was purchased from Millipore Co., Ltd. (Missouri, USA). 2.2. Enzyme preparation and assay PQQGDH and S415C was prepared as described previously (Igarashi and Sode, 2003). PQQGDH activity was
An Ag/AgCl electrode (Model RE-1, BAS Inc.) and a Pt wire were used as reference and counter electrodes, respectively. The enzyme electrode (3 mm diameter, BAS Inc.) reference electrode, and counter electrode were joined into a 10 ml water-jacket cell (BAS Inc., Model VC-2) through holes in its Teflon cover. The potential was controlled by a potentiostat HA151 (Hokuto-Denko, Tokyo, Japan) in a three-electrode cell and currents were recorded with a chart recorder (Ohkura electric company, Tokyo, Japan). All measurements were carried out at 25 ◦ C in 10 ml of the buffer with magnetic stirring (250 rpm). PQQGDH electrode was immersed in 10 mM MOPS buffer containing 1 mM m-PMS, 1 mM CaCl2 and 20 mM glucose and BOD electrode was immersed in 10 mM MOPS buffer containing 0.5 mM ABTS. The physiologically relevant pH 7 was chosen for both systems. The applied potential for the calibration curve measurements of PQQGDH electrodes was +100 mV versus Ag/AgCl. The calibration curve measurements were carried out with consecutive 100 l injections of glucose solution into the reaction cell. Cyclic voltamogram (CV) measurements of current response were carried out using the same electrode and solution for S415C electrode and BOD electrode. The potential was cycled between −400 and +200 mV for S415C electrode and between +200 and +700 mV for BOD electrode with a sweep rate of 10 mV/s using HZ3000 electrochemical analyzer (Hokuto-Denko, Tokyo, Japan).
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2.5. Biofuel cell construction PQQGDH electrode for anode and BOD electrode for cathode were joined into a 10 ml water-jacket cell (BAS Inc., Model VC-2). In the anode, 10 mM MOPS buffer containing 1 mM m-PMS, 1 mM CaCl2 , and in the cathode, 10 mM MOPS buffer containing 0.5 mM ABTS was used, respectively, under stirring conditions (250 rpm) with a magnetic stirrer at 25 ◦ C. The two compartments were connected by a salt-bridge. Glucose was added into the anodic cell, whereas nothing was added into the cathodic cell, and both cells were kept in the open air. For study of substrate selectivity of the S415C electrode, in the anode, 500 U S415C electrode, 10 mM MOPS (pH 7.0) containing 2 mM m-PMS and 1 mM CaCl2 , and in the cathode, 50 U BOD electrode, 10 mM MOPS (pH 7.0) containing 0.5 mM ABTS were used. 2.6. Biofuel cell operation and characterization The voltage and the current generated by the cell were measured between the two electrodes by applying an external variable load resistance (Model 278620, Yokogawa Electric Corporation, Tokyo, Japan) and using digital multimeter (Advantest, Tokyo, Japan) for the voltage and current measurements. The substrate selectivity was determined using the following six saccharides: glucose, lactose, galactose, maltose, cellobiose and xylose. These saccharides were added into the anode compartment with concentration of 0.2 mM each at the load of 60 k, 25 ◦ C. The operational stability of the cell was investigated by a time course of continuous operation at the load of 200 k, at 25 ◦ C with 20 mM glucose in the anode. 3. Results 3.1. Cyclic voltammetry for anode and cathode Fig. 1 shows the cyclic voltamograms of the S415C immobilized electrode (A) and the BOD immobilized electrode (B). A typical catalytic current was observed by adding 20 mM glucose to the S415C electrode and the increase peak anodic current was observed (Fig. 1A). S415C has the nearly same property as PQQGDH apart from stability (Igarashi and Sode, 2003) and the similar cyclic voltamograms were obtained (data not shown). A typical catalytic current was also observed for BOD electrode, and increased peak cathodic current was observed (Fig. 1B). It was confirmed that the anode and the cathode worked as enzyme electrode in this condition, respectively.
Fig. 1. Cyclic voltamograms of the S415C immobilized electrode (A) and the BOD immobilized electrode (B). The currents of S415C electrode were measured in 10 mM MOPS buffer (pH 7.0) containing 1 mM m-PMS and 1 mM CaCl2 and the currents of BOD electrode were measured in 10 mM MOPS buffer (pH 7.0) containing 0.5 mM ABTS. The measurements were carried out at 25 ◦ C under the air. The sweep rate was 10 mV/s.
of glucose concentration, using 200 k as the external resistance. With the increase the glucose concentration, the observed power increased. The maximum power, 1.6 W, was observed at glucose concentration above 5 mM, and then the current was saturated. Considering the around 20 mM of Km value for S415C, saturation of the current at 5 mM was not resulted from the S415C but from other factor. The rate-limiting factor in this system might be the electron mediator at the anode and/or the catalytic ability of biocathode including BOD performance. 3.3. Current–voltage behavior of the biofuel cell and power generated from biofuel cell at different external loads Fig. 3 shows the current–voltage behavior of the biofuel cell employing S415C as an anode enzyme with different
3.2. Glucose concentration dependence of power Fig. 2 shows the power generated by the biofuel cell system employing S415C as an anode enzyme as the function
Fig. 2. Electrical power generated from the biofuel cell system. Biofuel cell was operated at the load of 200 k( as the function of glucose concentration.
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Fig. 3. Current–voltage behavior of biofuel cell at different external loads.
external loads. The power extracted from the biofuel cell element (W = Vcell Icell ) is shown in Fig. 4 with different external loads. The open circuit voltage of this cell was 577 mV and the maximum current was 4.3 A (61.4 A/cm2 ). The maximum power corresponds to 1.23 W (17.6 W/cm2 ) at the external load of 200 k. The maximum power of 1.6 W was obtained at the experiment investigating the glucose dependence and those value were slightly different but those were resulted from the electrodes fabrication. 3.4. Substrate selectivity Wide substrate selectivity is one of the property and advantage of PQQGDH for the application to the biofuel cell system and it was investigated. Fig. 5 shows the power generated by the biofuel cell system employing S415C as an anode enzyme as the function of fuels using 60 k as the external resistance. With the increase of the glucose concentration, and with the addition of the different saccharides the increased power obtained (Fig. 5A). In this experiment, 0.2 mM of glucose, lactose, galactose, maltose, cellobiose and xylose were added in this order. Fig. 5B shows the rate of power increase of biofuel cell system. Compared to the power increase caused by the addition of 0.2 mM glucose, 50–60% of power was generated by adding each other saccharide. This result shows that various saccharides that were contained in biomass can be simultaneously utilized for anode fuel in the biofuel cell employing PQQGDH. This is the advantage of this biofuel cell employing PQQGDH, and when glucose oxidase electrode is used,
Fig. 4. Electrical power generated from the biofuel cell system at different external loads.
Fig. 5. Electrical power generated from the biofuel cell system. The biofuel cell was operated at the load of 200 k as the function of glucose concentration (open circles) or saccharides concentration (closed circles) (A). Each 0.2 mM saccharides were added in the order corresponding to glucose, lactose, galactose, maltose, cellobiose and xylose. The rates of power increase of the biofuel cell system with various saccharides compared to that with glucose (B). The data in (B) were calculated from the data (A) and error bars in (B) means the errors of three measurements. The biofuel cell was operated at the load of 200 k.
various saccharides cannot be utilized because of their narrow substrate specificity. 3.5. Stability The stability of the biofuel cell employing wild-type PQQGDH or the engineered PQQGDH, S415C as anode enzyme was investigated as a function of time. The operational stability of the cell was investigated by a time course of continuous operation at optimal load 200 k, 25 ◦ C with 20 mM glucose and the linear regression in semi-logarithmic of residual activity against time was obtained (Fig. 6). The
Fig. 6. The residual activity of the biofuel cells against time. Biofuel cell employing PQQGDH as an anode enzyme () open circles or biofuel cell employing S415C as an anode enzyme(䊉) closed circles. The biofuel cell was operated at the load of 200 k and measurements were carried out at 25 ◦ C.
N. Yuhashi et al. / Biosensors and Bioelectronics 20 (2005) 2145–2150
inactivation proceeded slowly with the biofuel cell employing S415C compared to PQQGDH. After 24 h operation, the wild-type PQQGDH electrode maintained only 40% of the initial response. The calibration graph obtained after 24 h operation showed approximately 30% of the decrease to the original value in current response (data not shown). These data indicate that wild-type PQQGDH is readily and irreversibly inactivated during the continuous operation for 24 h. In contrast, the cell employing the S415C maintained 80% of the initial response after 24 h continuous operation (data not shown). The half-life of biofuel cell employing S415C was calculated as 152 h, which reflects a greater stability than the biofuel cell employing the wild-type enzyme (half-life 52.4 h). The quaternary structure of S415C is greatly stabilized by a disulfide bond introduced in its dimer interface (Igarashi and Sode, 2003) so that S415C should be stable enough to be utilized in biofuel cell. The inactivation of this biofuel cell employing S415C during the continuous operation therefore was likely resulted from the inactivation of mediator.
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with higher substrate affinity have been prepared. The former engineered PQQGDH would be useful for biomass conversion and the latter is for energy source in the implantable devices, such as implantable glucose sensor. The Km value of wild-type PQQGDH is approximately 20 mM. The biofuel cell employing engineered PQQGDH with higher substrate affinity would generate maximum power at 5 mM glucose concentration, which is equivalent to the glucose concentration in blood. For application to glucose based electronic power generation in implantable medical deices, it is preferable to construct the system without mediator in the solution. The immobilization of mediator or improvement of mediator is necessary. Moreover, mediator less type or direct electron transfer type biofuel cell would be preferable. We have already reported the construction of the engineered PQQGDH generating the electric current without mediator (Okuda and Sode, 2004), so that such biofuel cell would be constructed using it. The electric current generated from this mutant with the addition of glucose was small, therefore the improvement is required for this application.
4. Discussion PQQGDH appears to be powerful enzyme for glucose based biofuel cell anode, since various saccharides can be simultaneously utilized for anode fuel in the biofuel cell employing PQQGDH because of its wide substrate specificity. This is advantage for application of this system to biomass conversion. In our biofuel cell, the open circuit voltage was 0.577 V and the maximum current was 4.3 A (61.4 A/cm2 ). The power generated from this cell was 1.23 W (17.6 W/cm2 ) at the external load of 200 k. The power was smaller than that of previous reported GOD based system (Chen et al., 2001; Katz et al., 1999; Kim et al., 2003; Mano et al., 2002, 2003). In these systems, enzyme at anode and cathode were immobilized using redox polymer or in structurally arranged manner through the surface reconstitution of the enzymes on their cofactor on the electrode. It is possible to increase the power density of our biofuel cell by optimizing the amount of enzyme and mediator or the immobilization method of them at anode and cathode. The biofuel cell employing GOD lost 6–20% of its power per day, when they were operated continuously (Chen et al., 2001; Kim et al., 2003; Mano et al., 2002, 2003). Our system employing the S415C maintained 80% of the initial response after 24 h continuous operation. The inactivation of our biofuel cell employing S415C during the continuous operation might be resulted from the inactivation of the mediator; it is possible to extend the lifetime of our system by selection of more stable mediator. We have been engaged in other protein engineering studies of PQQGDH (Sode et al., 2000, 2002; Takahashi et al., 2000), which would be suitable for the application to biofuel cell. For example, PQQGDH with expanded substrate specificity and
5. Conclusions The biofuel cell was constructed using the PQQGDH and its thermostable mutant S415C and the biofuel cell employing the S415C showed high stability. The wide substrate stability enables the biofuel cell to use wide variety of saccharides as fuel, and it is the advantage of the biofuel cell using the PQQGDH. Since many kinds of mutants of PQQGDH bearing different property were already prepared, development of various biofuel cells having different advantages are expected and those are promising to wide range of applications.
References Chen, T., Barton, S.C., Binyamin, G., Gao, Z.Q., Zhang, Y.C., Kim, H.H., Heller, A., 2001. A miniature biofuel cell. J. Am. Chem. Soc. 123, 8630–8631. Igarashi, S., Sode, K., 2003. Stabilization of quaternary structure of watersoluble quinoprotein glucose dehydrogenase. Mol. Biotechnol. 24 (2), 97–103. Katz, E., Willner, I., Kotlyar, A.B., 1999. A non-compartmentalized glucose vertical bar O-2 biofuel cell by bioengineered electrode surfaces. J. Electroanal. Chem. 479, 64–68. Kim, H.H., Mano, N., Zhang, X.C., Heller, A., 2003. A miniature membrane-less biofuel cell operating under physiological conditions at 0.5 V. J. Electrochem. Soc. 150 (2), A209–A213. Kost, G.J., Vu, H.T., Lee, J.H., Bourgeois, P., Kiechle, F.L., Martin, C., Miller, S.S., Okorodudu, A.O., Podczasy, J.J., Webster, R., Whitlow, K.J., 1998. Multicenter study of oxygen-insensitive handheld glucose point-of-care testing in critical care hospital ambulatory patients in the United States and Canada. Crit. Care Med. 26, 581–590. Mano, N., Mao, F., Heller, A., 2002. A miniature biofuel cell operating in a physiological buffer. J. Am. Chem. Soc. 124, 12962–12963. Mano, N., Mao, F., Shin, W., Chen, T., Heller, A., 2003. A miniature biofuel cell operating at 0.78 V. Chem. Commun. (Camb) 4, 518–519.
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Okuda, J., Sode, K., 2004. PQQ glucose dehydrogenase with novel electron transfer ability. Biochem. Biophys. Res. Commun. 314, 793– 797. Sode, K., Ootera, T., Shirahane, M., Witarto, A.B., Igarashi, S., Yoshida, H., 2000. Increasing the thermal stability of the water-soluble pyrroloquinoline quinone glucose dehydrogenase by single amino acid replacement. Enz. Microbiol. Technol. 26, 820–824. Sode, K., Igarashi, S., Morimoto, A., Yoshida, H., 2002. Construction of engineered water-soluble PQQ glucose dehydrogenase with improved substrate specificity. Biocatal. Biotrans. 20, 405–412. Soukharev, V., Mano, N., Heller, A., 2004. A four-electron o(2)electroreduction biocatalyst superior to platinum and a biofuel cell operating at 0.88 V. J. Am. Chem. Soc. 126, 8368–8369.
Takahashi, Y., Igarashi, S., Nakazawa, Y., Tsugawa, W., Sode, K., 2000. Construction and characterization of glucose enzyme sensor employing engineered water soluble (PQQ) glucose dehydrogenase with improved thermal stability. Electrochemistry 68, 907–911. Tang, Z.P., Louie, R.F., Lee, J.H., Lee, D.M., Miller, E.E., Kost, G.J., 2001. Oxygen effects on glucose meter measurements with glucose dehydrogenase- and oxidase-based test strips for point-of-care testing. Crit. Care Med. 29, 1062–1070. Tsujimura, S., Tatsumi, B., Ogawa, J., Shimizu, S., Kano, K., Ikeda, T., 2001. Bioelectrocatalytic reduction of dioxygen to water at neutral pH using bilirubin oxidase as an enzyme and 2,2 -azinobis (3ethylbenzothiazolin-6-sulfonate) as an electron transfer mediator. J. Electroanal. Chem. 496, 69–75.
Short communication
Development of a novel glucose enzyme fuel cell system employing protein engineered PQQ glucose dehydrogenase Noriko Yuhashia , Masamitsu Tomiyamab , Junko Okudaa , Satoshi Igarashia , Kazunori Ikebukuroa , Koji Sodea,∗ a
Department of Life Science and Biotechnology, Tokyo University of Agriculture and Technology, Nakamachi 2-24-16, Koganei, Tokyo 184-8588, Japan b Genetic Diversity Department, Applied Microbiology Laboratory, National Institute of Agrobiological Sciences, Ibaraki 305-8602, Japan Received 31 May 2004; received in revised form 5 August 2004; accepted 12 August 2004 Available online 15 September 2004
Abstract Glucose dehydrogenase harboring pyrroloquinoline quinone as the prosthetic group (PQQGDH) from Acinetobacter calcoaceticus is an ideal enzyme for the anode of biofuel cell, because of its oxygen insensitivity and high catalytic efficiency. However, the application of PQQGDH for the bioanode is inherently limited because of its instability. Using Ser415Cys mutant whose stability was greatly improved, we constructed the biofuel cell system employing the engineered PQQGDH as the bioanode enzyme and bilirubin oxidase (BOD) as the biocathode, and compared the stability of the biofuel cell with that employing wild-type PQQGDH. The maximum power density was 17.6 W/cm2 at an external optimal load of 200 k. Using Ser415Cys mutant, the lifetime of the biofuel cell system was greatly extended to 152 h, more than six times as that of the biofuel cell employing the wild-type. © 2004 Elsevier B.V. All rights reserved. Keywords: Enzyme fuel cell; PQQ glucose dehydrogenase; Protein engineering; Bilirubin oxidase; Stability
1. Introduction Biofuel cell systems utilize biocatalysts, such as enzymes and microorganisms, as the electrocatalysts, instead of transition metal catalysts that are utilized in the conventional fuel cell systems. Several organic compounds can be utilized in biofuel cells, especially biomass based compounds can be used as the fuels such as sugars and several alcohols, and the higher efficient energy conversion can be achieved compared with transition metal based fuel cells, theoretically. Biofuel cell can be applied not only to the biomass conversion, but also to the alternative energy source in the future implantable devices, such as implantable glucose sensors in the artificial pancreas. Recent trends in the development of biofuel cell is the use glucose as the fuel to be oxidized at the bioanode on ∗
Corresponding author. Tel.: +81 42 388 7021; fax: +81 42 388 7021. E-mail address: [email protected] (K. Sode).
0956-5663/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2004.08.017
which glucose oxidase (GOD) is immobilized, combining with appropriate biocathode, employing several dioxygen reducing enzymes (Chen et al., 2001; Katz et al., 1999; Kim et al., 2003; Mano et al., 2002, 2003). Together with the recent advances in microfabrication technology, miniaturized biofuel cells have also been reported (Chen et al., 2001; Mano et al., 2002, 2003; Soukharev et al., 2004). GOD is an extremely stable enzyme with high substrate specificity and relatively high Km value. Besides, in the field of self-blood glucose monitoring, glucose dehydrogenase harboring pyrroloquinoline quinone as the prosthetic group (PQQGDH) is focused owing to its oxygen insensitivity and high catalytic efficiency (Kost et al., 1998; Tang et al., 2001). Additionally its wide substrate specificity is advantageous for application of this enzyme to bioanode especially for biomass conversion. However, the application of PQQGDH to the bioanode is inherently limited because of its instability. The authors have been engaged in protein engineering of PQQGDH in order to develop the ideal enzyme
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for glucose monitoring (Sode et al., 2000; Takahashi et al., 2000). In those studies, one engineered enzyme, the Ser415Cys mutant (S415C) was obtained, of which stability was greatly improved by protein engineering (Igarashi and Sode, 2003). The S415C is stable even at 70 ◦ C. This is the most stable cofactor-binding GDH so far reported. The alteration of the residue at position 415 did not affect the catalytic efficiency of this enzyme, retaining high catalytic efficiency in glucose oxidation. The engineered PQQGDH therefore appears to be the most suitable enzyme for glucose based biofuel cell anode. In this paper, we evaluated the potential application of the engineered PQQGDH, S415C, as the bioanode enzyme in glucose utilizing biofuel cell systems. We constructed an enzyme glucose biofuel cell system employing S415C in the anodic reaction and bilirubin oxidase (BOD) for the cathodic reaction. It is reported that electrochemical reduction of dioxygen to water precedes very effectively at 0.4 V versus Ag/AgCl in pH 7.0 solution at an ambient temperature mediated by the 2,2 -azino-bis(3-ethylbenzothiazoline6-sulfonate) (ABTS2-)-mediated and catalyzed by bilirubin oxidase (Tsujimura et al., 2001). We then investigated the stability of the bioanode to evaluate the future potential application of the protein engineered enzymes to the biofuel cell system.
measured using 0.6 mM phenazine methosulphate (PMS) and 0.06 mM 2,6-dichlorophenolindophenol (DCIP) following 30 min pre-incubation in 10 mM MOPS buffer (pH 7.0) containing 1 M pyrroloquinoline quinone (PQQ) and 1 mM CaCl2 at room temperature (25 ◦ C). The enzyme activity was determined by measuring the decrease in absorbance of dichlorophenolindophenol (DCIP) at 600 nm. 2.3. Preparation of PQQGDH and bilirubin oxidase electrode An amout of 10 mmol MOPS buffer (pH 7.0) containing 300 U of PQQGDH or 100 U S415C or 5 U of Bilirubin oxidase was mixed with carbon paste (0.5 g graphite powder mixed with 0.3 ml paraffin liquid) and mixture was then lyophilized. The lyophilized mixture was then packed into the end of a carbon electrode (3 mm in diameter, BAS Inc., West Lafayette, USA) and treated with 1% glutaraldehyde solution for 30 min and then washed with 10 mM Tris–HCl buffer (pH 7.0). The PQQGDH or S415C immobilized electrode was then allowed to undergo holo-formation in 10 mM MOPS buffer (pH 7.0) containing 5 M PQQ and 1 mM CaCl2 at 4 ◦ C for at least 30 min and washed with 10 mM MOPS (pH 7.0). BOD electrode was washed with 10 mM MOPS buffer (pH 7.0). All electrodes were stored at 4 ◦ C until use. 2.4. Electrochemical measurements
2. Materials and methods 2.1. Chemicals Calcium chloride, 3-(N-morpholino) propanesulfanic acid (MOPS), 1-methoxyphenazine methosulphate (mPMS), 2,2 -azino-bis(3-ethylbenzothiazolone-6-sulfonic acid) diammonium salt (ABTS Diammonium Salt) and 25% glutaraldehyde were purchased from Wako Chemicals (Osaka, Japan). Pyrroloquinoline quinone (PQQ) was from Mitsubishi Gas Chemical Company, Inc. (Tokyo, Japan). Phenazine methosulfate (PMS), 2,6dichrolophenolindophenol (DCIP), -d-glucose, lactose, galactose, maltose was from Kanto Chemical (Tokyo, Japan). Cellobiose was from SIGMA (St. Louis, US), xylose was from Kishida Chemical Co., Ltd. (Osaka, Japan). Bilirubin oxidase was donated from Amano Enzyme (Aichi, Japan). DC protein assay kit was from Bio Rad Laboratories (California, USA). Carbon paste (0.5 g graphite powder mixed with 0.3 ml paraffin liquid) was from BSA Inc. (West Lafayette, USA). The other reagents were of analytical grade. Ultrafree MC was purchased from Millipore Co., Ltd. (Missouri, USA). 2.2. Enzyme preparation and assay PQQGDH and S415C was prepared as described previously (Igarashi and Sode, 2003). PQQGDH activity was
An Ag/AgCl electrode (Model RE-1, BAS Inc.) and a Pt wire were used as reference and counter electrodes, respectively. The enzyme electrode (3 mm diameter, BAS Inc.) reference electrode, and counter electrode were joined into a 10 ml water-jacket cell (BAS Inc., Model VC-2) through holes in its Teflon cover. The potential was controlled by a potentiostat HA151 (Hokuto-Denko, Tokyo, Japan) in a three-electrode cell and currents were recorded with a chart recorder (Ohkura electric company, Tokyo, Japan). All measurements were carried out at 25 ◦ C in 10 ml of the buffer with magnetic stirring (250 rpm). PQQGDH electrode was immersed in 10 mM MOPS buffer containing 1 mM m-PMS, 1 mM CaCl2 and 20 mM glucose and BOD electrode was immersed in 10 mM MOPS buffer containing 0.5 mM ABTS. The physiologically relevant pH 7 was chosen for both systems. The applied potential for the calibration curve measurements of PQQGDH electrodes was +100 mV versus Ag/AgCl. The calibration curve measurements were carried out with consecutive 100 l injections of glucose solution into the reaction cell. Cyclic voltamogram (CV) measurements of current response were carried out using the same electrode and solution for S415C electrode and BOD electrode. The potential was cycled between −400 and +200 mV for S415C electrode and between +200 and +700 mV for BOD electrode with a sweep rate of 10 mV/s using HZ3000 electrochemical analyzer (Hokuto-Denko, Tokyo, Japan).
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2.5. Biofuel cell construction PQQGDH electrode for anode and BOD electrode for cathode were joined into a 10 ml water-jacket cell (BAS Inc., Model VC-2). In the anode, 10 mM MOPS buffer containing 1 mM m-PMS, 1 mM CaCl2 , and in the cathode, 10 mM MOPS buffer containing 0.5 mM ABTS was used, respectively, under stirring conditions (250 rpm) with a magnetic stirrer at 25 ◦ C. The two compartments were connected by a salt-bridge. Glucose was added into the anodic cell, whereas nothing was added into the cathodic cell, and both cells were kept in the open air. For study of substrate selectivity of the S415C electrode, in the anode, 500 U S415C electrode, 10 mM MOPS (pH 7.0) containing 2 mM m-PMS and 1 mM CaCl2 , and in the cathode, 50 U BOD electrode, 10 mM MOPS (pH 7.0) containing 0.5 mM ABTS were used. 2.6. Biofuel cell operation and characterization The voltage and the current generated by the cell were measured between the two electrodes by applying an external variable load resistance (Model 278620, Yokogawa Electric Corporation, Tokyo, Japan) and using digital multimeter (Advantest, Tokyo, Japan) for the voltage and current measurements. The substrate selectivity was determined using the following six saccharides: glucose, lactose, galactose, maltose, cellobiose and xylose. These saccharides were added into the anode compartment with concentration of 0.2 mM each at the load of 60 k, 25 ◦ C. The operational stability of the cell was investigated by a time course of continuous operation at the load of 200 k, at 25 ◦ C with 20 mM glucose in the anode. 3. Results 3.1. Cyclic voltammetry for anode and cathode Fig. 1 shows the cyclic voltamograms of the S415C immobilized electrode (A) and the BOD immobilized electrode (B). A typical catalytic current was observed by adding 20 mM glucose to the S415C electrode and the increase peak anodic current was observed (Fig. 1A). S415C has the nearly same property as PQQGDH apart from stability (Igarashi and Sode, 2003) and the similar cyclic voltamograms were obtained (data not shown). A typical catalytic current was also observed for BOD electrode, and increased peak cathodic current was observed (Fig. 1B). It was confirmed that the anode and the cathode worked as enzyme electrode in this condition, respectively.
Fig. 1. Cyclic voltamograms of the S415C immobilized electrode (A) and the BOD immobilized electrode (B). The currents of S415C electrode were measured in 10 mM MOPS buffer (pH 7.0) containing 1 mM m-PMS and 1 mM CaCl2 and the currents of BOD electrode were measured in 10 mM MOPS buffer (pH 7.0) containing 0.5 mM ABTS. The measurements were carried out at 25 ◦ C under the air. The sweep rate was 10 mV/s.
of glucose concentration, using 200 k as the external resistance. With the increase the glucose concentration, the observed power increased. The maximum power, 1.6 W, was observed at glucose concentration above 5 mM, and then the current was saturated. Considering the around 20 mM of Km value for S415C, saturation of the current at 5 mM was not resulted from the S415C but from other factor. The rate-limiting factor in this system might be the electron mediator at the anode and/or the catalytic ability of biocathode including BOD performance. 3.3. Current–voltage behavior of the biofuel cell and power generated from biofuel cell at different external loads Fig. 3 shows the current–voltage behavior of the biofuel cell employing S415C as an anode enzyme with different
3.2. Glucose concentration dependence of power Fig. 2 shows the power generated by the biofuel cell system employing S415C as an anode enzyme as the function
Fig. 2. Electrical power generated from the biofuel cell system. Biofuel cell was operated at the load of 200 k( as the function of glucose concentration.
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Fig. 3. Current–voltage behavior of biofuel cell at different external loads.
external loads. The power extracted from the biofuel cell element (W = Vcell Icell ) is shown in Fig. 4 with different external loads. The open circuit voltage of this cell was 577 mV and the maximum current was 4.3 A (61.4 A/cm2 ). The maximum power corresponds to 1.23 W (17.6 W/cm2 ) at the external load of 200 k. The maximum power of 1.6 W was obtained at the experiment investigating the glucose dependence and those value were slightly different but those were resulted from the electrodes fabrication. 3.4. Substrate selectivity Wide substrate selectivity is one of the property and advantage of PQQGDH for the application to the biofuel cell system and it was investigated. Fig. 5 shows the power generated by the biofuel cell system employing S415C as an anode enzyme as the function of fuels using 60 k as the external resistance. With the increase of the glucose concentration, and with the addition of the different saccharides the increased power obtained (Fig. 5A). In this experiment, 0.2 mM of glucose, lactose, galactose, maltose, cellobiose and xylose were added in this order. Fig. 5B shows the rate of power increase of biofuel cell system. Compared to the power increase caused by the addition of 0.2 mM glucose, 50–60% of power was generated by adding each other saccharide. This result shows that various saccharides that were contained in biomass can be simultaneously utilized for anode fuel in the biofuel cell employing PQQGDH. This is the advantage of this biofuel cell employing PQQGDH, and when glucose oxidase electrode is used,
Fig. 4. Electrical power generated from the biofuel cell system at different external loads.
Fig. 5. Electrical power generated from the biofuel cell system. The biofuel cell was operated at the load of 200 k as the function of glucose concentration (open circles) or saccharides concentration (closed circles) (A). Each 0.2 mM saccharides were added in the order corresponding to glucose, lactose, galactose, maltose, cellobiose and xylose. The rates of power increase of the biofuel cell system with various saccharides compared to that with glucose (B). The data in (B) were calculated from the data (A) and error bars in (B) means the errors of three measurements. The biofuel cell was operated at the load of 200 k.
various saccharides cannot be utilized because of their narrow substrate specificity. 3.5. Stability The stability of the biofuel cell employing wild-type PQQGDH or the engineered PQQGDH, S415C as anode enzyme was investigated as a function of time. The operational stability of the cell was investigated by a time course of continuous operation at optimal load 200 k, 25 ◦ C with 20 mM glucose and the linear regression in semi-logarithmic of residual activity against time was obtained (Fig. 6). The
Fig. 6. The residual activity of the biofuel cells against time. Biofuel cell employing PQQGDH as an anode enzyme () open circles or biofuel cell employing S415C as an anode enzyme(䊉) closed circles. The biofuel cell was operated at the load of 200 k and measurements were carried out at 25 ◦ C.
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inactivation proceeded slowly with the biofuel cell employing S415C compared to PQQGDH. After 24 h operation, the wild-type PQQGDH electrode maintained only 40% of the initial response. The calibration graph obtained after 24 h operation showed approximately 30% of the decrease to the original value in current response (data not shown). These data indicate that wild-type PQQGDH is readily and irreversibly inactivated during the continuous operation for 24 h. In contrast, the cell employing the S415C maintained 80% of the initial response after 24 h continuous operation (data not shown). The half-life of biofuel cell employing S415C was calculated as 152 h, which reflects a greater stability than the biofuel cell employing the wild-type enzyme (half-life 52.4 h). The quaternary structure of S415C is greatly stabilized by a disulfide bond introduced in its dimer interface (Igarashi and Sode, 2003) so that S415C should be stable enough to be utilized in biofuel cell. The inactivation of this biofuel cell employing S415C during the continuous operation therefore was likely resulted from the inactivation of mediator.
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with higher substrate affinity have been prepared. The former engineered PQQGDH would be useful for biomass conversion and the latter is for energy source in the implantable devices, such as implantable glucose sensor. The Km value of wild-type PQQGDH is approximately 20 mM. The biofuel cell employing engineered PQQGDH with higher substrate affinity would generate maximum power at 5 mM glucose concentration, which is equivalent to the glucose concentration in blood. For application to glucose based electronic power generation in implantable medical deices, it is preferable to construct the system without mediator in the solution. The immobilization of mediator or improvement of mediator is necessary. Moreover, mediator less type or direct electron transfer type biofuel cell would be preferable. We have already reported the construction of the engineered PQQGDH generating the electric current without mediator (Okuda and Sode, 2004), so that such biofuel cell would be constructed using it. The electric current generated from this mutant with the addition of glucose was small, therefore the improvement is required for this application.
4. Discussion PQQGDH appears to be powerful enzyme for glucose based biofuel cell anode, since various saccharides can be simultaneously utilized for anode fuel in the biofuel cell employing PQQGDH because of its wide substrate specificity. This is advantage for application of this system to biomass conversion. In our biofuel cell, the open circuit voltage was 0.577 V and the maximum current was 4.3 A (61.4 A/cm2 ). The power generated from this cell was 1.23 W (17.6 W/cm2 ) at the external load of 200 k. The power was smaller than that of previous reported GOD based system (Chen et al., 2001; Katz et al., 1999; Kim et al., 2003; Mano et al., 2002, 2003). In these systems, enzyme at anode and cathode were immobilized using redox polymer or in structurally arranged manner through the surface reconstitution of the enzymes on their cofactor on the electrode. It is possible to increase the power density of our biofuel cell by optimizing the amount of enzyme and mediator or the immobilization method of them at anode and cathode. The biofuel cell employing GOD lost 6–20% of its power per day, when they were operated continuously (Chen et al., 2001; Kim et al., 2003; Mano et al., 2002, 2003). Our system employing the S415C maintained 80% of the initial response after 24 h continuous operation. The inactivation of our biofuel cell employing S415C during the continuous operation might be resulted from the inactivation of the mediator; it is possible to extend the lifetime of our system by selection of more stable mediator. We have been engaged in other protein engineering studies of PQQGDH (Sode et al., 2000, 2002; Takahashi et al., 2000), which would be suitable for the application to biofuel cell. For example, PQQGDH with expanded substrate specificity and
5. Conclusions The biofuel cell was constructed using the PQQGDH and its thermostable mutant S415C and the biofuel cell employing the S415C showed high stability. The wide substrate stability enables the biofuel cell to use wide variety of saccharides as fuel, and it is the advantage of the biofuel cell using the PQQGDH. Since many kinds of mutants of PQQGDH bearing different property were already prepared, development of various biofuel cells having different advantages are expected and those are promising to wide range of applications.
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