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NOTES An Amperometric Alcohol Sensor Based on Chemically Permeabilized Methylotrophic Microorganisms Jhy-chern Chen, Thomas J. Naglak, and Henry Y. Wang* Department of Chemical Engineering, The University of Michigan, Ann Arbor, Michigan 48109
Chemical permeabilization of the yeast Pichia pastoris NRRL Y-11430 with 1.0% glycine and 0.1 % Triton X-100 does not release intracellular alcohol oxidase but alters the cell envelope structure, allowing alcohol oxidase activity to be detected inside the cells (Naglak, 1990). An alcohol sensor based on the use of chemically permeabilized yeast cells was constructed. The sensor response time was within 2 min. The assay time was less than 30 s by the initial rate method. The calibration curve for ethanol analysis was linear in the range 5-65 mg/L. The sensor response was almost unaffected by pH changes within the range pH 5-9. This alcohol sensor produced the highest sensitivity for methanol; the sensitivity for ethanol was about half that for methanol. The stability and other characteristics of this new alcohol sensor are also discussed in this work.
Introduction Rapid and reliable determination of alcohol is important for various chemicaland industrial applications. Gas chromatography, spectrophotometry, and redox titration methods have traditionally been employed for alcohol analysis. The couplingof biochemical reactionswith electrochemical electrodes for alcohol sensing has also received extensive attention in the literature. All kinds of isolated enzymes and microorganisms were used to construct alcohol biosensors: these included alcohol oxidase (Clark et al., 1972; Guilbault et al., 1974; Nanjo et al., 1975; Verduyn et al., 1983), alcohol dehydrogenase (Malinauskas et al., 1978; Blaedel et al., 1980; Walters et al., 1988; Kitagawa et al., 1989), and yeasts and bacteria (Hikuma et al., 1979; Kitagawa et al., 1987; Mascini et al., 1989). Recently, chemically permeabilized cells appear to be apromising alternative for biosensors (Park and Kim, 1990; Park et al., 1991). Naglak (1990) reported that Pichia pastoris can be chemically permeabilized with selective release of 50% of intracellular protein by treating the cells with 1.0% glycine and 0.1% Triton X-100, but the treatment does not release the alcohol oxidase. The permeabilized cell envelope was found to lose some mass transfer resistance, allowing alcohol oxidase activity to be detected readily inside the cells. This paper concernsthe constructionof an alcohol sensor based on chemically permeabilized cells. The new alcohol sensor consists of an oxygen electrode with immobilized chemically permeabilized yeast cells. The characteristics of this new alcohol sensor, its sensitivity to various alcohols, and the effect of pH on its response were investigated.
Materials and Methods Yeast Strain, Growth Media, and Cell Preparation.
Pichia pastoris NRRL Y-11430 (supplied by Dr. A. J. Lyons of the Northern Regional Research Center, Peoria, IL) was maintained at -20 "C in 50% (v/v) glycerol. Flask
* To whom correspondence should be addressed. 8756-7938/92/3008-0161$03.00/0
cultures were grown on methanol medium at 30 "C in a rotary shaker at 175 rpm for 48 h. The inoculation was at between 1O:l and 1OO:l dilution. The composition of methanol medium is 6.75 g of yeast nitrogen base without amino acids, 12.0 g of KH2P04, and 2.1 g of K2HPOl in 1 L of distilled water. The pH was adjusted to 6.0 prior to autoclaving. Methanol (1% v/v) was aseptically added into the medium immediately prior to inoculation. Chemical Permeabilization of Yeast Cells. Cells from a 48-h flask culture were recovered by centrifugation and twice washed with 0.1 M phosphate buffer at pH 7.5. The washed cells were collected by centrifugation, and they were resuspended in a flask containing 0.1 M phosphate buffer solution, pH 7.5, with 1.0%glycine and 0.1 % Triton X-100. The flask was shaken at 30 "C and 175 rpm for another 14 h. This allowed release of most intracellular components from P. pastoris. Some intracellular componenb are found to cause severe interference with the response of the oxygen electrode. The chemically permeabilized cells were recovered by filtration with a 0.45-pmcellulose acetate filter membrane (HAWP 04700, Millipore Co., Bedford, MA) and washed three times with 0.1 M phosphate buffer, pH 7.5. The wet cake of chemically permeabilized cells was stored at 4 "C until use. Construction of Alcohol Sensor with Chemically Permeabilized Cells. Polargraphic oxygen electrodes were constructed in our laboratory. The cathode was made by placing a segment of gold wire (99.99% ) with a diameter of 1.0 mm and length of 5.0 mm within a glass capillary tube (3.0 mm 0.d.) after having been bonded to a silver wire. The glass around the unattached end was drawn over the gold segment and the tip polished so that only a disk of gold was available. A silver wire (99.9%)of diameter 0.5 mm was wound around the glass tube and used as an anode. The electrodes were covered by a Teflon membrane (Teflon PFA, fluorocarbon film lOOLP, E. I. du Pont de Nemours & Co., Inc.) with thickness of 0.5 mils and sheathed by an outer glass tube with a diameter of 6.0 mm. The space between the membrane inner surface
0 1992 Amerlcan Chemical Society and American Instltute of Chemical Engineers
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and the face of the electrodes was filled with 2.0 N potassium chloride electrolyte solution. A small amount of permeabilized P. pastoris paste was put on the surface of the cathode and trapped between the Teflon membrane and the porous Teflon membrane with porosity of 0.2 pm (TF-200, Gelman Sciences Inc., Ann Arbor, MI), and the membranes were secured with a silicon tube. The alcohol sensor was stored in the phosphate buffer solution at 4 "C until used. Assay of Alcohol Oxidase Activity. Alcohol oxidase (E.C. 1.1.3.13) activitywasdetermined byamodified ABTS method (Herberg et al., 1985). The ABTS reagent was made by adding 16.0 mg of chromagen (2,2'-azinobis(3ethylbenzothiazoline-6-sulfonicacid), 2.0 mL of methanol, and 1.0 mL of 0.1 mg/mL peroxidase in 100 mL of buffer solution. Unless another buffer is mentioned specifically, the buffer was 0.1 M phosphate buffer at pH 7.5. The assay was started by adding 50 pL of sample to 2.5 mL of ABTS reagent, and the increase in absorbance at 390 nm is recorded as a function of time. The relative unit of alcohol oxidase activity is defined as the rate of absorbance change per minute measured from 0.5 to 3.0 min after sample addition. Measurement of Alcohols. The alcohol sensor was placed in a 50-mL temperature-controlled (k0.l "C) vessel with 20 mL of 0.1 M phosphate buffer solution, pH 7.5. The solution of the vessel was magnetically stirred and air aerated during experiments. A constant voltage of -0.70 V vs the silver anode was applied to the oxygen electrode bya potentiostat (chemical microsensor,Diamond General Development Corp., Ann Arbor, MI). The electrode current was measured and converted into a digital signal by a data acquisition and control interface board (DASH8, Metra Byte Corp., Taunton, MA). A portable personal computer (COMPAQ Corp., Houston, TX) was used to perform signal analysis, initial rate calculation, and calibration of the alcohol sensor. The standard assay of alcohol was carried out by adding 100pL of sample solution into the isothermal air-saturated buffer solution at 30 "C and pH 7.5 and sequentially measuring the decrease of electrode current at samplingrate of 50 times/s. The initial response rate of alcohol sensor was defined as the rate of current change from 5 to 15 s after the addition of sample. Calibration of the alcohol sensor was carried out by measuing the initial response rate against various concentrations of alcohol in 0.1 M phosphate buffer, pH 7.5, at 30 "C. The stability of the alcohol sensor was determined by the time course of sensor response to an ethanol concentration of 39 mg/L at 4 "C and 30 "C. The effect of pH on the alcoholsensor was measured by analyzing the sensor response to an ethanol concentration of 39 mg/L in the following buffer solutions: 0.1 M phosphate (pH 4-91,O.l M Tris-HC1 (pH 10.0), and 0.1 M acetate (pH 3.0).
Results and Discussion Response Speed, Calibration, and Reproducibility of the Alcohol Sensor. Figure 1 shows the response curve of the alcohol sensor with immobilized permeabilized P. pastoris. It took about 2 min for the sensor to reach steady state when changed from ethanol-free buffer solution to 22 mg/L ethanol solution. When the alcohol sensor was removed from the sample solution to an ethanol-freebuffer solution, it took about 3 min for the electrode current to return to its baseline level. In order to shorten the assay time, the initial rate method was employed for the determination of alcohols. The response of the alcohol sensor was measured as the rate of current change from 5 to 15 s after the addition of 100
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pL of sample into 20 mL of air-saturated phosphate buffer solution. Figure 2 shows a typical calibration curve of the sensor for ethanol measurement. A linear relationship was observed for the initial response rate against ethanol concentration below 65 mg/L. The Michaelis constant for alcohol oxidase vs ethanol was determined by different investigators to be 46 mg/L (Janssen et al., 1968) and 84 mg/L (Guilbault et al., 1969). The upper detection limit of this alcohol sensor falls reasonably within this region. The minimum ethanol concentration that can be determined is 5-10 mg/L. The sensitivity of this sensor to ethanol was 0.1-0.3 pA min-l (mg/L)-l and depended on the preparation of sensor. The characteristics of permeabilized cells and the amount of cells employed on to the electrode will affect the performance of the alcohol sensor. This influence has not yet been systematically studied. The reproducibility of one sensor response to ethanol was evaluated using samples of the same concentration. The rate of current decrease was 0.56 f 0.03 pA/min for 20 replicate samples with an ethanol concentration of 39.5 mg/L. Sensitivity and pH Effect. Alcohol oxidase has been shown to catalyze the oxidation of a variety of short-chain primary alcohols (Janssen et al., 1968). The sensitivity of the sensor toward various alcohols can be ascertained by comparing the slopes of the calibration curves. The results indicate that the alcohol sensor produced the highest sensitivity for methanol. The sensitivity for ethanol was about half the sensitivity for methanol. The sensitivity decreased with any increase of the alcohol chain length. This behavior was also observed by other workers using purified alcohol oxidase enzyme (Guilbault et al., 1974; Nanjo et al., 1975; Sahm, 1976). It has been reported that the activity of alcohol oxidase is rather low at pH values below 7.0 (Guilbault et al., 1974; Nanjo et al., 1975; Sahm, 1976). The study of the effect of pH on the response of our alcohol sensor was found to be different from that in other works. Experiments were conducted using samples with identical ethanol concentrations but different pHs. Figure 3 shows that the rate of electrode response for samples of ethanol concentration 39 mg/L was almost uneffected by changes of pH from 5 to 9. Similar behavior was observed by measuring the alcohol oxidase activity of a chemically permeabilized cell suspension with the ABTS method. The relative activity was found to be almost constant at pH 5-9. P. pastoris has been reported to grown on methanol at low pH (Cregg et al., 1989). It appears that the cell membrane provides protection to the intracellular enzyme, which makes the
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30 "C upon 39 mg/L ethanol and (b) the alcohol oxidase activity of chemically permeabilized cell suspension. The relative activities for the sensor were calculated by dividing the response rate by the rate at pH 7.5. The relative activities for the cell suspension were calculated by dividing the enzyme activity by 2 times the enzyme activity at pH 7.5.
pH tolerance of alcohol oxidase inside the cell greater than in bulk solution. However, when the alcohol sensor was stored at pH 5.0 and 30 "C for 24 h, the rate of sensor response to the sample sample under the standard assay ! of its original level. Since the condition decreased to 25 % pH value of most biological fluids is below 7.5, the pH tolerance of the chemically permeabilized cells makes it possible to apply the alcohol sensor in the biological fluid without using buffer solution for short-term analysis. Stability. Alcohol oxidaseis reported to lose its enzyme activity during conversion of alcohol to aldehyde. The stability of our alcohol sensor was examined under storage and assay conditions. The response of the sensor to samples of ethanol at 39 mg/L was measured periodically by the initial rate method. When the alcohol sensor was stored at 4 O C , a gradual decrease of 0.8%/day in sensor response was observed (Figure 4). The alcohol oxidase activity of chemically permeabilized cells was also stable at 4 "Cfor more than 14 days. When the sensor was held at 30 "C and performed 20 assayslday, its response was stable for the initial 24 h, and then it decreased at an average rate of 4.6%/day. After 300 assays of ethanol over 15days at 30 "C the sensor remained with 35% enzyme activity.
Figure 4. Stability of the alcohol sensor stored at (A)4 O C and
( 0 )30 "C and stability of the alcohol oxidase activity in the chemically permeabilizedcells at 4 "C (D). The relative activities for the sensor were calculated by dividing the response rate by the rate of first day and multiplied by 2 for (a) and 1for (b). The relative rate for the suspension cells was calculated by dividing the enzyme activity by the activity of the first day and multiplying by 0.5.
Murray et al. (1990)explained that the loss of alcohol oxidase activity was due to the catabolite inactivation by aldehyde in the presence of oxygen and the proteolytic degradation. The product inhibition in the chemically permeabilized cells could be reduced since aldehyde can easily diffuse out of the cell membrane. This alcoholsensor was more stable at 4 "C than at 30 "C, indicating the possible presence of protease in the chemically permeabilized cells. The activity of proteases is inhibited at temperatures below 15 "C (Murray et al., 1990). The enzymatic performances of various alcohol biosensors based on alcohol oxidase and oxygen electrodes (i.e., calibration range and the sensitivity to various alcohols) are about the same. However, the stabilities and optimum pH working ranges are quite different and depend on the environment of enzyme on the electrode. The pH tolerance of the alcohol oxidase inside the permeabilized cell is greater than that in the bulk solution. In conclusion, we have demonstrated that alcohol detection can be achieved using a new device which consists of immobilized permeabilized cells and oxygen electrode. There are three distinct features of this new alcoholsensor; the ease of preparation of the biocatalyst, the convenience of the immobilization of biocatalyst on to the electrode,and the extraordinarypH tolerance, which allows alcohol assay without buffer solution. Further studies on the effect of intracellular proteases on the stability of alcohol oxidase activity in the chemically permeabilized cells are being conducted.
Acknowledgment We acknowledge the partial financial assistance of National Science Foundation for this work. We thank Dr A. J. Lyons of the Northern Regional Research Center, Peoria, IL, for suppling Pichia pastoris NRRL Y-11430 and Miss Mara Liepa for technical assistance in this study. Literature Cited Blaedel, W. J.; Engstrom, R. C. Reagentless Enzyme Electrodes for Ethanol, Lactate, and Malate. Anal. Chem. 1980,52,16911697. Clark, L. C.,Jr. A family of polargraphic electrodes and the measurement of alcohol. In Biotechnology andBioengineer-
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ing Symposium, Enzyme Engineering 3; Wingard, L. B., Jr., Ed.; Wiley-Interscience: New York, 1972; pp 377-394. Cregg, J. M.; Digan, M. E.; Tschopp, J. F. R.; Brierly, A.; Craig, W. S.; Velicelibi, G.; Siegel, R. S.; Thill, G. P. Expression of foreign genes in Pichia pastoris. In Genetics and Molecular Biology of Industrial Microorganisms; Hersberger, C. L., Queener, S. W., Hegeman, G., Eds.; American Society for Microbiology: Washington, DC, 1989; PP 343-352. Guilbault, G. G.; Sadar, S. H. Fluorometric assay of alcohols using alcohol oxidase. Anal. Lett. 1969,2, 41-48. Guilbault, G. G.;Lubrano, G. J. Amperometric enzyme electrodes. Anal. Chim. Acta 1974, 69, 189-194. Herzberg, G. R.; Rogerson, M. Use of alcohol oxidase to measure the methanol produced during the hydrolysis of D-and L-methyl-3-hydroxybutyric acid. Anal. Biochem. 1985,149,354-357. Hikuma, M.; Kubo, T.; Yasuda, T.; Karube, I.; Suzuki, S. Microbial electrode sensor for alcohol. Biotechnol. Bioeng. 1979,21,1845-1853. Hudson, L.; Hay, F. C. Practical Immunology; Blackwell Scientific Publications: Oxford, England, 1980; pp 203-225. Janssen, F. W.; Ruelius, H. W. Alcohol oxidase, a flavoprotein from several basidiomycetes species. Crystallization by fractional precipitation with polyethylene glycol. Biochim. Biophys. Acta 1968,151, 330-342. Kitagawa, Y.; Kitabatake, K. Alcohol sensor based on membranebound alcohol dehydrogenase. Anal. Chim. Acta 1989,218, 61-68. Kitagawa, Y., Tamiya, E., Karube, I. Microbial-FET Alcohol Sensor. Anal. Lett. 1987, 20 (l),81-96. Malinauskas, A.; Kulys, J. Alcohol, lactate and glutamate sensors based on oxidoreductases with regeneration of nicotinamide adenine dinucleotide. Anal. Chim. Acta 1978, 98, 31-37. Mascini, M.; Memoli, A.; Olana, F. Microbial sensor for alcohol. Enzyme Microb. Technol. 1989, 11, 297-301.
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Murray, W. D.; Duff, S. J. B.; Beveridge, T. I. Catabolite Inactivation in the methylotrophic yeastfichiapastoris. Appl. Environ, Microbiol. 1990, 56 (8), 2378-2380. Nanjo, M.; Guilbault, G. G. Amperometric determination of alcohols, aldehydes, and carboxylic acids with an immobilized alcohol oxidase electrode. Anal. Chim. Acta 1975, 75, 169180. Naglak, T. J. Protein release from microorganisms by chemical permeabilization. Ph.D. Thesis, Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan, 1990. Park, J.; Kim, H. A new biosensor for specific determination of glucose or fructose using an oxidoreductase of Zymomonas mobilis. Biotechnol. Bioeng. 1990, 36, 744-749. Park, J.; Ro, H.; Kim, H. A new biosensor for specific determination of sucrose using an oxidoreductase of Zymomonas mobilis and invertase. Biotechnol, Bioeng. 1991,38,217-223. Sahm, H. Metabolism of Methanol by Yeast. In Advances in Biochemical Engineering; Ghose, T. K., Fiechter, A. Blakebrough, N., Eds: Springer-Verlag: Berlin, 1976, Vol6, pp 77103. Verduyn, C.; van Dijken, J. P.; Scheffers, W. A. A simple, sensitive and accurate alcohol electrode. Biotechnol, Bioeng. 1983,25, 1049-1055. Walters, B. S., Nielsen, T. J. Fiber-optic biosensor for ethanol, based on an internal enzyme concept. Talanta 1988,35,151155. Accepted January 6, 1992. Registry No. Methanol, 67-56-1; ethanol, 64-17-5; glycine, 56-40-6; Triton X-100, 9002-93-1; alcohol oxidase, 9073-63-6.
