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Subzero Temperature Operating Biosensor Utilizing an Organic Solvent and Quinoprotein Glucose Dehydrogenase Koji Sode", Satoshi Nakasono, Mitsuharu Tanaka, and Tadashi Matsunaga Department of Biotechnology, Faculty of Technology, Tokyo University of Agriculture and Technology, 2-24- 76 Nakacho, Koganei-shi, Tokyo 784, Japan Received July 28, 7992/Accepted January 27, 7993 A subzero temperature operating biosensor was con- structed using immobilized quinoprotein glucose dehy- drogenase (PQQGDH), glassy carbon electrode, soluble electron mediator (ferrocene monocarboxylic acid), and an organic solvent, ethylene glycol, as an antifreezing reagent. Using this biosensor, glucose concentration can be determined even at -7°C. At this temperature, the response was 20% of that obtained at 20°C. This is the first study describing a subzero temperature operating biosensor. 0 1993 John Wiley & Sons, lnc. Key words: biosensor subzero temperature PQQGDH INTRODUCTION Biosensors are generally constructed with biocatalysts and devices; however, the operational stabilities are dominated by biocatalysts themselves. Usually, biosensors are operated at optimum temperature for biocatalytic reaction in order to achieve a rapid and highly sensitive response, although such a condition is not optimum for their stability. The inherent problem with biosensors, which are composed of biocatalysts such as enzymes, antibodies, and whole cells, is their instability under operational conditions. Several approaches have already been reported to overcome this problem, utilizing thermostable bio~atalysts.'.~ Although the application of such thermostable enzymes for biosensors is an attractive technique, it is not possible to substitute all biocatalysts reported in biosensors with thermostable biocatalysts. Another possible approach is to reduce the operational temperature. Besides, the rate of biocatalytic reaction is, like other chemical reactions, strongly dependent upon temperature, principally according to the Arrhenius equation. Therefore, even at subzero temperatures, biocatalysts still possess ac- tivity. Low temperature bioprocesses may have the follow- ing two advantages as compared with general bioprocesses: the lifespan of biocatalysts will be greatly enhanced, and the possibility of contamination by mesophilic microor- ganisms and enzymes derived from these organisms is minimized. Considering these advantages, low tempera- * To whom ;dl correspondence should be addressed. ture operating bioprocesses have been reported in the fields of bacterial l e a ~ h i n g , ~ methane fermentation,8,' and also in the construction of recombinant microorganisms for industrial wastewater treatment." These studies are based on the application of psychrophilic bacteria and their enzymes. Enzyme studies at subzero temperatures have been also reported in the field of affinity chromatography* and enzyme kinetic^.^.^ These results indicate the pos- sible novel area of application of subzero temperature operating enzymatic reactions, such as biosensors. Sub- zero temperature conditions mean that complicating fac- tors need to be considered in using biocatalytic reac- tions in biosensors, such as the freezing of water, the addition and effects of antifreezing reagents, the diffu- sion rate, and electrochemical reactions. If the biosensors are operated at subzero temperatures, which are used for the storage of biocatalysts, an extremely high operational stability will be realized. Furthermore, on-line monitor- ing of bioactive compounds or quality control of foods or unstable compounds in cold rooms can be achieved in situ. In this study, we describe the first report of construc- tion and characterization of subzero temperature operating biosensors system. As the biocatalyst, we have chosen quinoprotein glucose dehydrogenase (PQQGDH). Th' IS en- zyme is known as the membrane-binding protein, which exists in the periplasmic space of various Gram-negative bacteria.' This enzyme is not a heat-labile enzyme but loses its activity drastically at optimum condition for re- action (37°C). In order to achieve the reaction at subzero temperature, the enzyme should survive in the presence of an antifreezing reagent, i.e., an hydrophobic reagent like ethylene glycol. The hydrophobic property of this enzyme is suited to the adaptation in the reaction condition in the presence of the antifreezing reagent. Furthermore, operation of the biosensor at a subzero temperature may result in the reduction of response time and sensitivity which might make it difficult to measure the properties of this sensor system. The reported high turnover number of PQQGDH6 will also be useful at the low temperature operation. Biotechnology and Bioengineering, Vol. 42, Pp. 251 -254 (1993) 0 1993 John Wiley & Sons, Inc. CCC 0006-3592l93lQ2Q251-04

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Page 1: Subzero temperature operating biosensor utilizing an organic solvent and quinoprotein glucose dehydrogenase

Subzero Temperature Operating Biosensor Utilizing an Organic Solvent and Quinoprotein Glucose Deh ydrogenase

Koji Sode", Satoshi Nakasono, Mitsuharu Tanaka, and Tadashi Matsunaga Department of Biotechnology, Faculty of Technology, Tokyo University of Agriculture and Technology, 2-24- 76 Nakacho, Koganei-shi, Tokyo 784, Japan

Received July 28, 7992/Accepted January 27, 7993

A subzero temperature operating biosensor was con- structed using immobilized quinoprotein glucose dehy- drogenase (PQQGDH), glassy carbon electrode, soluble electron mediator (ferrocene monocarboxylic acid), and an organic solvent, ethylene glycol, as an antifreezing reagent. Using this biosensor, glucose concentration can be determined even at -7°C. At this temperature, the response was 20% of that obtained at 20°C. This is the first study describing a subzero temperature operating biosensor. 0 1993 John Wiley & Sons, lnc. Key words: biosensor subzero temperature PQQGDH

INTRODUCTION

Biosensors are generally constructed with biocatalysts and devices; however, the operational stabilities are dominated by biocatalysts themselves. Usually, biosensors are operated at optimum temperature for biocatalytic reaction in order to achieve a rapid and highly sensitive response, although such a condition is not optimum for their stability. The inherent problem with biosensors, which are composed of biocatalysts such as enzymes, antibodies, and whole cells, is their instability under operational conditions. Several approaches have already been reported to overcome this problem, utilizing thermostable bio~atalysts.'.~ Although the application of such thermostable enzymes for biosensors is an attractive technique, i t is not possible to substitute all biocatalysts reported in biosensors with thermostable biocatalysts. Another possible approach is to reduce the operational temperature.

Besides, the rate of biocatalytic reaction is, like other chemical reactions, strongly dependent upon temperature, principally according to the Arrhenius equation. Therefore, even at subzero temperatures, biocatalysts still possess ac- tivity. Low temperature bioprocesses may have the follow- ing two advantages as compared with general bioprocesses: the lifespan of biocatalysts will be greatly enhanced, and the possibility of contamination by mesophilic microor- ganisms and enzymes derived from these organisms is minimized. Considering these advantages, low tempera-

* To whom ;dl correspondence should be addressed.

ture operating bioprocesses have been reported in the fields of bacterial l e a ~ h i n g , ~ methane fermentation,8,' and also in the construction of recombinant microorganisms for industrial wastewater treatment." These studies are based on the application of psychrophilic bacteria and their enzymes. Enzyme studies at subzero temperatures have been also reported in the field of affinity chromatography* and enzyme kinetic^.^.^ These results indicate the pos- sible novel area of application of subzero temperature operating enzymatic reactions, such as biosensors. Sub- zero temperature conditions mean that complicating fac- tors need to be considered in using biocatalytic reac- tions in biosensors, such as the freezing of water, the addition and effects of antifreezing reagents, the diffu- sion rate, and electrochemical reactions. If the biosensors are operated at subzero temperatures, which are used for the storage of biocatalysts, an extremely high operational stability will be realized. Furthermore, on-line monitor- ing of bioactive compounds or quality control of foods or unstable compounds in cold rooms can be achieved in situ.

In this study, we describe the first report of construc- tion and characterization of subzero temperature operating biosensors system. As the biocatalyst, we have chosen quinoprotein glucose dehydrogenase (PQQGDH). Th' I S en- zyme is known as the membrane-binding protein, which exists in the periplasmic space of various Gram-negative bacteria.' This enzyme is not a heat-labile enzyme but loses its activity drastically at optimum condition for re- action (37°C). In order to achieve the reaction at subzero temperature, the enzyme should survive in the presence of an antifreezing reagent, i.e., an hydrophobic reagent like ethylene glycol. The hydrophobic property of this enzyme is suited to the adaptation in the reaction condition in the presence of the antifreezing reagent. Furthermore, operation of the biosensor at a subzero temperature may result in the reduction of response time and sensitivity which might make it difficult to measure the properties of this sensor system. The reported high turnover number of PQQGDH6 will also be useful at the low temperature operation.

Biotechnology and Bioengineering, Vol. 42, Pp. 251 -254 (1993) 0 1993 John Wiley & Sons, Inc. CCC 0006-3592l93lQ2Q251-04

Page 2: Subzero temperature operating biosensor utilizing an organic solvent and quinoprotein glucose dehydrogenase

MATERIALS AND METHODS

Enzyme Preparation

As the construction glucose sensor, quinoprotein glucose dehydrogenase (PQQGDH) was used. PQQGDH was used as the membrane fraction of Acinetobacter calcoaceticus I F 0 12552. A. calcoaceticus cells cultured in L-broth were centrifuged, and their membrane fraction was obtained by French-press (110 MPa X 2), followed by ultracentrifuga- tion (69,800 g, 90 min 4°C).

PQQGDH activity was determined spectrophotomet- rically, using phenazine methosulfate (PMS) and 2, 6-dichloroindophenol sodium n-hydrate (DCIP) as the electron mediator. Five microliters of enzyme sample was added into 20 p L of tris-HCI buffer (pH 8.0, 20 mM) containing PMS-DCIP (0.6 mM) and incubated for 3 min at 37°C. The reaction was then initiated by adding 1 p L of 2 M of glucose (final glucose concentration 80 mM) and incubated at 37°C. After 2 min, the decrease in absorbance at 600 nm was measured.

Sensor Construction and Operation

PQQGDH contained within the membrane fraction was immobilized onto a nitrocellulose filter (0.8 p m , advan- tec, Japan) by adsorption. The enzyme-immoblized filter was then set onto a glassy carbon electrode (BAS Co., West Lafayette, In, Model 11-2012) and covered with dial- ysis membrane. An Ag/AgCl reference electrode and a carbon wire counter electrode were used. Ferrocene mono- carboxylic acid (FcCOOH) (Sigma Chemical Co., St. Louis, MO) was used as the soluble electron mediator. FcCOOH was dissolved in 0.1 M tris-HCI buffer (pH 7.5) containing 35% ethylene glycol. The sensor system was immersed in a 50-mL reaction vessel filled with 20 mL of buffer solution containing ethylene glycol and FcCOOH, which was equipped with a water jacket. Measurements were carried out by injecting 10 p L of 400 mM glucose dis- solved in the same buffer containing 35% ethylene glycol, to eliminate freezing of the sample in the reaction vessel.

RESULTS AND DISCUSSION

To achieve a subzero temperature enzymatic reaction, we utilized ethylene glycol as an antifreezing reagent. Be- fore application for the biosensor system, the effect of ethylene glycol on the activity of quinoprotein glucose dehydrogenase (PQQGDH) was investigated (Fig. 1). With the increase of ethylene glycol concentration, PQQGDH activity gradually decreased. At an ethylene glycol concen- tration of 35%, at which temperatures lower than -20°C could be used, more than 60% of PQQGDH activity was observed. Therefore, further experiments were carried out using buffer solution containing 35% ethylene glycol.

In the presence of 35% ethylene glycol, the stability of enzymatic activity was examined. Although at temperatures higher than 30°C, the enzymatic activity of PQQGDH

0 0 1 0 2 0 30 4 0 5 0

Ethylene glycol concentration (%)

Figure 1. Effect of ethylene glycol concentration on PQQGDH activity. Enzyme sample: solubilized membrane fraction from Acinetobacter cal- coaceticus IF0 12552 containing PQQGDH. Reaction temperature: 30°C.

decreased to less than 70% after 1 week of incubation, at -7°C it retained more than 90% of initial activity (measurements were carried out at 30°C).

The addition of ethylene glycol also enhanced the solu- bility of the electron mediator, ferrocene monocaroboxylic acid (FcCOOH). The solubility of FcCOOH was very low (0.1 mM) at 4°C. However, in the presence of 35% ethylene glycol, which increased hydrophobicity of the solvent, FcCOOH concentrations of up to 0.5 mM were obtained even at -20°C. Due to the dissolved FcCOOH, the limiting current of FcCOOH could be measured at -20°C.

A biosensor system was constructed using a glassy carbon electrode with immobilized PQQGDH and a buffer solution containing ethylene glycol equipped with a water jacket. Figure 2 shows the response curves of the sensor at various temperatures. It took 35 min to obtain the steady state current even at 20°C. These curves demonstrate that the PQQGDH was still active in the presence of ethylene glycol at a subzero temperature. Because, in this study, we used a nitrocelluose-immersed enzyme covered with dialysis membrane, the diffusion of substrate as well as mediator might be low, consequently resulting in the slow response. The lowest temperature at which the detectable signal was obtained, with our apparatus, was -7°C. At this temperature, no lag time was observed and, after 55 min, steady state current was obtained.

Figure 3 shows the correlation between injected glucose concentration and current increase at various temperatures. These experiments were carried out by successive injection of 100 p L of 400 mM glucose. A linear correlation was obtained at subzero temperatures. At -7"C, glucose con- centration of lower than 6 mM could be determined using this sensor system. Comparing the slope in Figure 3, the signal at -7°C was approximately 20% of that obtained at 20°C. These results demonstrate that glucose measurement using a biosensor was possible at subzero temperatures.

Figure 4 shows the correlation between sensor response and temperature. Temperatures were expressed as the recip- rocal absolute temperature (K-' ), and the sensor responses

252 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 42, NO. 2, JUNE 20, 1993

Page 3: Subzero temperature operating biosensor utilizing an organic solvent and quinoprotein glucose dehydrogenase

+ C ? 3

I

3 Q

3 I

0

, 20'C I i

I

i

lOmin T I n iniartinn sampl,

10'C 5'C

-1.5.c -7 'C

I 1 , L I , T i m e

Figure 2. Response curves of the glucose sensor system at various temperaturcs. Solution: 0.1 M tris-HCI buffer (pH 7.5) containing 35% ethylene glycol and 0.5 m M FcCOOH. Glucose concentration: 2mM. Potential: t0.35 V vs. SCE.

were expressed as the logarithms of the steady state current obtained at each temperature to produce an Arrhenius plot. Because the steady state current obtained as the result of enzymatic reaction equilibrated with the diffusion of substrate and mediator, we used the steady state current as the representative of rate constant.

A good linear correlation was observed at temperatures up to 0°C. At subzero temperatures, although the linear correlation was still observed, the rate of decline was different from that observed at temperatures higher than 0°C. Figure 5 shows the correlation between limiting cur- rent obtained from FcCOOH alone and temperature. This result indicates that the electrochemical reaction itself is well fitted to the Arrhenius plots at subzero temperatures. This indicates that the point at which the rate of decline changed, observed in Figure 4, might not be due to the electrochemical reaction but due to the enzymatic character. In order to measure the enzymatic activity as the function of temperature, particularly at subzero temperatures, it requires

Glucose concentration (mM)

Figure 3. Correlation between glucose concentration and response of glucose sensor system under various temperatures. Response values were measured 60 mi, after sample injection. The experiments were carried out by injecting 100 g L of 400 mM of glucose, sequentially, and obtaining the steady state current. Other experimental conditions were the same as those described in Figure 2. (0) 1 5 T , (0) 2"C, (A) -1S"C, (m) -7.O"C.

Temperature ('C)

- 2 0 7

- D 0 -1

-0.4 3 . 2 3.4 3 .6 3.8 4 .0

IIT (X 10-3)

Figure 4. Temperature dependence of the response of the glucose sensor system. Below 2 5 T , the response values (I) were obtained 60 min after sample injection; whereas, at 3 0 T , the response was measured 24 min after sample injection. The measurements were carried out after the injection of glucose sample at a final concentration of 2 mM. Other experimental conditions were same as those described in Figure 2.

complicated procedures and equipment.6 Therefore, our re- sults also suggest a novel approach to determine the enzyme activity at subzero temperatures by the electrochemical method. Further enzyme analyses are essential to elucidate this phenomenon, but it might be possible that this enzyme, PQQGDH, might show different conformation at subzero temperatures.

In conclusion, we report here, the construction and characterization of a subzero temperature operating biosen- sor. This was the first report of the biosensor operation at subzero temperatures. Further optimization of electron

- rn -I

Figure 5.

Temperature ('C)

. .- 3.2 3.4 3 .6 3 . 8 4 . 0

1IT (x 10 - 3 )

Temperature dependence of the limiting current of FcCOOH. The limiting current was obtained by cyclic voltamograms at a scanning rate of 50 mV/s. Solution: 50 mM tris-HCI buffer (pH 7.5) containing 35% ethylene glycol and 0.1 mM FcCOOH.

COMMUNICATIONS TO THE EDITOR 253

Page 4: Subzero temperature operating biosensor utilizing an organic solvent and quinoprotein glucose dehydrogenase

mediators, antifreezing reagents, and analysis of operating stability are being carried out.

The authors thank Dr. J. G. Burgess for help in compiling this data

References

1. Anthony, C. 1992. Int. J. Biochem. 24: 29-39. 2. Balny, C., Douzou P. 1987. Meth. Enzyrnol. 135: 528-537. 3. Douzou, P., Debey, P., Franks, F. 1978. Biochim. Biophys. Acta 523:

1-8.

254

4 . Ferroni, G. D., Leduc, L. G., Todd, M. 1986. J. Gen. Appl. Microbial. 32: 169-175.

5. Frank Jzn, J . , Duine, J . A,, Balny, C. 1991. Biochimie. 73: 611-613. 6. Hauge, J.G. 1964. J. Biol. Chem. 239: 3630-3639. 7. Iida, T., Kawabe, T., Noguchi, F., Mitarnura, T., Nagata, K., Tornita,

K. 1987. Nihon Kagagukai-shi 10: 1817-1821. 8. Kalia, A. K., Kanwar, S. S. 1989. Biol. Wastes 30: 217-224. 9. Karube, I., Yokoyarna, K., Sode, K., Tamiya, E. 1989. Anal. Lett. 22:

791 -802. 10. Kolenc, R. J. Inniss, W.E., Glick, B. R., Robinson, C. W., Mayfield,

C. I . 1988. Appl. Environ. Microbiol. 54: 638-641. 11 . Matsuyama, H. , Izumi, K. 1988. J. Ferm. Technol. 88: 229-233.

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