Biotechnol. Prog. 1995, 11, 58-63
58
Flow Injection Analysis of Glucose, Fructose, and Sucrose Usling a Biosensor Constmeted with Permeabilized Z y m m o n u s nwbilis and Invertase Je-Kyun Park? Min-Chol Shin, Seung-Goo Lee, and Hak-SungKim* Department of Biotechnology, Korea Advanced Institute of Science & Technology, 373-1, Kusung-dong, Yusung-ku, Taejon, 305-701, Korea
Cells of Zymomonas mobilis possessing glucose-fructose oxidoreductase and gluconolactonase were permeabilized with toluene and co-immobilizedwith invertase within a gelatin membrane. This membrane was coated over a pH electrode, and the resulting biosensor was placed in a flow-through cell to develop a flow injection analysis (FIA) system for the specific determination of glucose, fructose, and sucrose. Peak height resulting from the production of hydrogen ion was correlated with sugar concentration, and the effect of operating variables on the response characteristics of the FLA system was investigated on the basis of theoretical and experimental analyses. Under the optimized conditions, the calibration curves for glucose, fructose, and sucrose were linear up to 8, 80, and 60 g/L, respectively. The FIA system was applied to the online monitoring of glucose production in the enzymatic hydrolysis of cellulose, and the glucose concentrations determined using the FIA system coincided well with those determined by the conventional enzymatic method.
Introduction
Sucrose
There has been much effort directed toward rapid and specific determination procedures for sugars such as glucose, fructose, and sucrose because these sugars are of great significance from the standpoint of clinical and industrial analysis. These sugars generally are analyzed by HPLC or selective enzymatic methods. However, these conventional methods are usually tedious and cost intensive, even though the analyses are accurate. In order to overcome these shortcomings, several attempts have been made to integrate biosensor and flow injection analysis (FLA)techniques (Hall, 1991; Ludi et al., 1990). The enzyme electrode for glucose has been developed using glucose oxidase by coupling with an oxygen probe or a platinum electrode (Hendry et al., 1990; Palleschi et al., 1991). In the case of the sucrose electrode (Satoh et al., 1976; Xu et al., 1989), a multienzyme system involving glucose oxidase, mutarotase, and invertase was employed, and oxygen consumption or hydrogen peroxide production resulting from the sequential enzyme reaction was detected. However, the enzyme electrode based on the glucose oxidase reaction has the problem that the electrode response might fluctuate due to oxygen tension in the sample solution and interfere with other oxidizable substances (Bartlett et al., 1991). For the determination of fructose, fructose dehydrogenase (Xie et al., 1991) or some sequential reaction system utilizing hexokinase and glucose-6-phosphate dehydrogenase (Schubert et al., 1986) was employed. But these methods were reported to suffer from the supplementary need for a cofactor or an electron mediator such as ATP, NADH, or ferricyanide. Even in the case of FIA systems for these sugars using immobilized enzyme columns (De Maria and Townshend, 19921, the requirement for soluble reagents still remained. Recently, it was reported (Zachariou and Scopes, 1986) that a glucose-fructose oxidoreductase (GFOR) of Zy~
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* Author to whom all correspondence should be addressed.
'Present address: Central Research Laboratory, Goldstar Co., Ltd., 16, Woomyeon-Dong, Seocho-Ku, Seoul, 137-140,Korea. 8756-7938/95/3011-0058$09.00/0
1
invertase
1c3rse
Fructose
+
glucose-fructose oxidoreductme
Sorbitol
+
Gluconolactone Gluconate
1+
gluconolactonase
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Figure 1. Schematic diagram of the enzyme system used for construction of the biosensor.
momonas mobilis simultaneously oxidizes glucose to gluconolactone and reduces fructose to sorbitol and that the gluconoladone is further hydrolyzed to gluconate and hydrogen ion by a sequential reaction with gluconolactonase within the same cell. It was also revealed that GFOR contains an NADP(H) cofactor that is tightly bound to the active site. A schematic diagram of the enzyme system is shown in Figure 1. In order to utilize the enzyme system described above without purification, 2.mobilis was permeabilized with organic solventa (Chun and Rogers, 1988). Permeabilization was known to cause the leak of soluble cofactors and high-energy compounds needed for enzyme reactions, except for the cofactor for GFOR (RQ and Kim, 1991). This consequently blocked the action of other enzymes involved in sugar metabolism. In our previous publications (Park and Kim, 1990;Park et al., 1991),we developed new biosensors for the specific determination of glucose, fructose, and sucrose using permeabilized cells of 2. mobilis. The biosensor for glucose and fructose was constructed on the basis of the
0 1995 American Chemical Society and American Institute of Chemical Engineers
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Biotechnol. frog., 1995, Vol. 11, No. 1
Solenoid Valve n
s1
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s2
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-7
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f Immobilized membrane
-
...........................................................................................".."...".....",....-.
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Figure 2. Schematic diagram of the flow injection analysis system and flow-through cell equipped with a biosensor. Symbols are as follows: S1, sample injector; s2, counter injector; P1 and P2, pumps; B, carrier buffer; M, mixing coil; W, wastes; 0-7, solenoid valve control ports.
idea that the production rate of hydrogen ion by the permeabilized cells is dependent on the concentration of glucose and fructose. For the sucrose determination, invertase was co-immobilized with permeabilized cells within the membrane, and the production of hydrogen ion was detected using a pH electrode. From a practical standpoint, the fast and accurate determination of operating parameters such as substrate or product level is a prerequisite for the control and optimization of bioprocesses (Raju and Cooney, 1992). As a solution to this requirement, a flow injection analysis (FIA)system was developed, and its applications have been reported (Garn et al., 1989; Renneberg et al., 1991; Valero et al., 1990). In this article, we have attempted a novel FIA system for the specific determination of glucose, fructose, and sucrose using a single biosensor constructed with permeabilized 2. mobilis and invertase. Cells of 2. mobilis were permeabilized with toluene and co-immobilizedwith invertase within a gelatin membrane. This membrane was coupled with a pH electrode, and the resulting biosensor was placed in a flow-through cell for the construction of a FIA system. The response characteristics of the FIA system were investigated, and its operating conditions were optimized on the basis of theoretical and experimental analyses. The applicability of the FIA system to the on-line monitoring of glucose production in the enzymatic hydrolysis of cellulose was also evaluated. Details are reported here.
Experimental Section Materials. Invertase from yeast (EC 3.2.1.26, 300 unitslmg lyophilysate) was purchased from Boehringer (Mannheim, FRG). Cellulase from Trichoderma uirde
(EC 3.2.1.4, 7.4 unitslmg solid) was also obtained from Sigma. Three-hundred-bloom gelatin from porcine skin and glutaraldehyde (25% aqueous solution) were from Sigma (St. Louis, MO). Teflon tubing (0.8 mm i.d.) was obtained from Alltech Associates (Deefleld, IL). Toluene of analytical grade was obtained from Merck. A diagnostic kit (cat. no. 510A) for glucose analysis was obtained from Sigma. All other reagents were of analytical grade. Stock solutions of sugar were prepared in 2 mM 2-morpholinoethanesulfonic acid (MES) buffer of pH 6.2. The glucose standard solutions especially were allowed to mutarotate for 24 h at 4 "C. Standard samples used for the calibration of sugar concentration were diluted using the same buffer prior to analysis. Microorganism and Culture Conditions. Zymomonas mobilis ZM4 (ATCC 31821) was obtained from the Korea Collection for Type Culture (KCTC, GERI, Korea). The microorganism was cultivated in medium containing 100 g of glucose, 1g of (NH&S04, 1 g of MgSOs7H20, and 5 g of yeast extract per 1 L of distilled water. Cultivation was carried out in a 2 L fermentor (Bioengineering AG, Switzerland)under controlled environmental conditions of 30 "C and pH 5.0. No phosphate salts were added to the final growth medium to minimize the levels of phosphorylated intermediates in the cells. Cultivated cells were harvested at the late exponential growth phase and separated using a centrifuge (DuPont, Wilmington, DE). Permeabilization of the Microorganism. Centrifuged cells of 2. mobilis were suspended in 0.1 M citrate buffer (pH 6.2). Toluene was added to the cell suspension to give a final concentration of 10%(vlv), and this mixture was vortex mixed for 3 min. The solvent-treated cells
Biofechnol. Prog., 1995, Vol. 11, No. 1
60 1.27
(a)
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Figure 3. (a) Initial reaction rate of glucose-fructose oxidoreductase as a function of glucose concentration when the fructose concentration was varied from 0.1 to 0.8 M. (b)Initial reaction rate of glucose-fructose oxidoreductase as a function of fructose concentration when the glucose concentration was varied from 0.01 to 0.8 M.
were separated by centrifugation at 2000g and washed twice with 0.85% (v/v) saline solution. Immobilization of Permeabilized Cells and Invertase. Gelatin as an immobilization matrix was dissolved in 20 mM 2-morpholinoethanesd.fonic acid (MES)buffer (pH 6.2) by incubation in an oven at 50 "C. Permeabilized cells of 2.mobilis (0.2 g of DCW)were suspended in 1mL of 20 mM MES buffer (pH 6.2) in the presence of invertase (2500units), and 1.5 mL of 10% gelatin solution was added. This mixture was spread on a glass plate of 200 cm2 and dried at 25 "C for 2 h. The resulting membrane was treated with 2.5% glutaraldehyde solution for 2 min. The thickness of the membrane was estimated to be 50 pm. Construction of a Biosensor. Immobilized membrane (1 x 1cm) was coated over a pH electrode (Orion Research, Model 81-35, Cambridge,MA) with an O-ring, and the resulting electrode was covered with a nylon net (50-mesh) to protect the membrane. The resulting biosensor was placed into the homemade flow-through cell. The holdup volume of the flow-through cell was about 50 pL, and the biosensor body was inclined 10-15" from horizontal to facilitate the release of interfering bubbles from the inlet. Flow Injection Analysis System. A schematic diagram of the FIA system constructed in this work is shown in Figure 2. This system is composed of twochannel, six-roller peristaltic pumps (Eyela MP3 type, Japan), two sample injectors, a flow-through cell containing a biosensor, and a pH meter (Bioengineering AG, Switzerland). All connections were made with Teflon
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Figure 4. (a) Effect of mixing coil length on the FIA response for 40 giL fructose. The concentration of glucose injected into the sample counter injector was 0.3 M, and the flow rate of carrier solution was 1 mumin. (b) Peak height of the FIA response at different glucose concentrations as a function of flow rate. The fructose concentration was fixed at 0.5 M.
joints in order to minimize the sample dispersion. The injection of samples was realized by an arrangement of solenoid valves (Iso-Latch 3-way miniature Teflon type, General Valve Co., NJ). The volume of each injector was 100 pL, and thus the total volume of sample flowing into the flow-through cell was 200 pL. The FIA manifold has two different sample injectors, S1 and S2, due to the characteristic of the GFOR reaction mechanism in which recycling of the NADRH) cofactor is required for the reaction to proceed. Thus, when glucose was analyzed, a predetermined volume of glucose solution was injected through injector 1(Sl), while a fixed concentration of fructose was supplied simultaneously to counterinjector 2 (52) as a cosubstrate. Alternatively, when fructose was determined, a fxed concentration of glucose as cosubstrate was provided through injector 2. In the case of sucrose analysis, only the sample solution was injected through injector 1. Sampling, injection, and detection of the electrode signal were automatically controlled using a personal computer (IBM PCKT-compatible) equipped with a peripheral interface card (PC-LabCard,PCL812, Advantech Co., Taiwan). The intedace card contained a 12-bit A/D converter and 16 digital YO lines, and its resolution in A D conversion was 0.0025 pH unithit. The operating software was programmed in BASIC and was well modulated into three modes: sampling, injection, and rinsing. The operating mode was selected by changing the directions of the flow streams, which were dependent on the ordoff state of each solenoid valve. The response of the FIA system was detected and recorded every second, and maximum peak height was correlated with the sugar concentration. The flow rate of each pump (Pl,
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61 20-
t P
c
0
Glucose concentration (g/L)
10
20 30 40 50 Sucrore concentration (g/L)
60
Figure 6. Calibration curve for sucrose in the FIA system. The vertical bars represent the standard deviation of six successive measurements.
where g and f denote glucose and fructose, respectively. By rearranging above equation, the reaction rate can be expressed as a function of glucose concentration at each concentration of fructose.
0
20 40 60 Fructose concentration (g/L)
80
Figure 5. (a) Calibration curve for glucose in the FIA system. (b) Calibration curve for fructose in the FIA system. The volumes of sample loops are as follows: 25 p L (A);100 p L (m); 250 ,uL (0).The vertical bars represent the standard deviation of six successive measurements.
P2) was adjusted to 0.5 mumin, and all measurements were carried out at 35 "C. Membrane Bioreactor. For the on-line monitoring of glucose from the bioreactor, the enzymatic hydrolysis of cellulose was conducted in a membrane bioreactor (ultrafiltration kit, Amicon Model 8200) of the stirred tank type with a water jacket. The working volume of the bioreactor was maintained a t 100 mL, and a membrane (polysulfone, Amicon) with a cutoff size of 10 000 was used. The hydrolysis reaction by cellulase was carried out at 50 "C in 50 mM acetate buffer (pH 5.01, and the enzyme loading was 4200 units.
Results and Discussion Optimal Cosubstrate Concentration. Since the response of the FIA system is directly linked to the rate of reaction by permeabilized cells, investigations regarding the effect of cosubstrate concentration on the reaction rate offer some insight to the optimization of cosubstrate concentration for the FIA system. For this purpose, the sequential reaction by GFOR and gluconolactonase of permeabilized 2. mobilis was simulated. The effect of gluconolactonase on the overall reaction was assumed to be negligible because the rate of reaction by this enzyme is known to be very high compared to that of GFOR. Therefore, the production rate of hydrogen ion by permeabilized 2. mobilis is thought to be directly proportional to the rate of reaction by GFOR, and as reported elsewhere (Zachariou and Scopes, 1986), the initial reaction rate of GFOR can be described by ping-pong kinetics as follows:
Similarly, the reaction rate is also given as a function of fructose concentration at each concentration of glucose, as follows:
In order to simulate the enzyme reaction described above, the kinetic constants of Kmg,Kmf,and Vmaxestimated by Zachariou and Scopes (1986)for permeabilized cells were employed: Kmgis 30 mM (5.4 g/L), K,f is 1.4 M (254 g/L), and V, is 2.8 g of sorbitollg of cell/h. Figure 3a shows the profiles of reaction rate as a function of glucose concentration when the fructose concentration is varied from 0.1 to 0.8 M. The initial reaction rate increased significantly with increasing fructose concentration, which implies that the reaction rate is largely dependent on the fructose concentration. Meanwhile, the reaction rate was saturated at a low concentration of glucose over the whole range of fructose concentrations tested. From these results, it is thought that the response of the FIA system to glucose is affected mainly by the concentration of fructose as a cosubstrate, and the linear range of calibration curves for glucose is expected to be narrow. For the sensitive analysis of glucose, the introduction of higher concentrations of fructose was suggested, but this was found to result in both a prolonged rinsing time and fast degradation of the gelatin membrane. In this work, the concentration of fructose as a cosubstrate was determined to be 0.5 M when glucose was analyzed. The initial reaction rate of GFOR as a function of fructose concentration is shown in Figure 3b when the glucose concentration is changed from 0.01 to 0.8 M. The initial reaction rate increased gradually with the increasing concentration of fructose a t each concentration of glucose, implying a wide linear range of measurement for fructose. The increase in the initial reaction rate a t
Biotechnol. Prog., 1995, Vol. 11, No. 1
62 -5.7
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Figure 7. Reproducibility of the FIA system when 60 g/L fructose was injected successively. The flow rate and injection volume of the sample were 1.0 m u m i n and 100pL,respectively.
a fmed fructose concentration became insignificant when the glucose concentration was higher than 0.3 M. Thus, the concentration of glucose as a cosubstrate was selected as 0.3 M in the case of fructose analysis. Response Characteristics. To maximize the analytical performance of the FIA system, the effects of various parameters on the response were investigated. As optimized by the simulation, when measuring the concentration of glucose, a 0.5 M fructose solution as cosubstrate was provided through injector 2. Similarly, a 0.3 M glucose solution was supplied to injector 2 when frmctose was to be determined. Accordingly, a mixing coil is required for the proper mixing of two streams before they enter the flow-through cell, and the homogeneity of the solution might be affected by the length of the mixing coil. Figure 4a shows peak height at different lengths of mixing coil. The decrease in the peak height was observed with the increasing length of the mixing coil, and this seems to originate from the sample dispersion. For this work, the length of the mixing coil was selected to be 50 cm (internal volume, 250 pL), taking into consideration the sensitivity and response time of the FIA system. The effect of the flow rate of carrier buffer on the response at different concentrations of glucose is shown in Figure 4b. The peak height increased as the flow rate
decreased as expected, but the peak width was enlarged and the rinsing time required to return the signal to the base line value was prolonged. Higher peak height indicates a higher concentration of hydrogen ion within the membrane, and consequently, the rinsing time using a buffer solution becomes prolonged, resulting in a low frequency of analysis. In this work, the flow rate of the carrier buffer was chosen to be 1.0 mumin for the appropriate sensitivity of analysis and sampling frequency. Because the biosensor constructed here is based on the measurement of the production of hydrogen ion, the buffering capacity of the carrier solution may show a significant effect on the response of the FIA system. Lower response was observed at higher buffer concentrations in the carrier solution due to the greater resistance to pH changes (Park et al., 1991). For the sensitive analysis, 10 mM MES buffer (pH 6.2) was used as a carrier solution. Calibration Curves for Glucose and Fructose. In order to investigate the linear range of measurement for glucose, the peak height of the FIA response was determined as a function of glucose concentration. As shown in Figure 5a, the peak height increased linearly with increasing glucose concentration up to 8 g/L. In the case of fructose, the peak height was linearly proportional to the concentration of 80 g/L. When the calibration curve for fructose was determined at different injection volumes of sample, the slope of the curve increased with increasing injection volume, suggesting an improved sensitivity of analysis (Figure 5b). However, the time to reach a maximum height and the rinsing time to return to the base line values became longer. Thus, the injection volume of the sample was fixed at 100 pL. Typical response characteristics of the FIA system in successive analyses of fructose are shown in Figure 7. As can be seen in the calibration curves (Figure 5a,b) a significant difference in the linear measurement ranges between glucose and fructose was observed, which coincides well with the results of theoretical analysis. This seems to be mainly due to the difference in the affinities of GFOR of 2. mobilis for glucose and fructose: the apparent K, value for glucose was revealed to be 40 times smaller than that for fructose (Zachariou and Scopes, 1986).
Cellulose
w
Buffer Fructose
Solenoid valve
Figure 8. Schematic diagram of the FIA system for the determination of the glucose concentration at the outlet of the membrane bioreactor. 1-13 represent the selection codes for the flow stream. Other symbols are the same as those for Figure 2.
Biotechnol. Prog., 1995, Vol. 11, No. 1
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[fl [gl Kmf
Kmg V V, 8 B
Notation fructose concentration (g/L) glucose concentration (gL) Michaelis-Menten constant for fructose (gL) Michaelis-Menten constant for glucose (gL) initial reaction rate (g/g/h) maximum reaction rate (g/g/h) Literature Cited
2.0 4,0:/
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4
6
8
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18
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Figure 9. Determination of glucose concentration at the outlet of the membrane bioreactor using the FIA system (0)and the conventional enzymatic method (m).
Calibration Curve for Sucrose. The sucrose concentration is determined on the basis of the ideas that glucose and fructose, which are substrates for glucosefructose oxidoreductase, are produced in equimolar amounts from sucrose by co-immobilized invertase and that the production of hydrogen ion from glucose and fructose by the permeabilized cells of 2.mobilis could be directly proportional to the concentration of sucrose. Figure 6 shows the peak heights at different concentrations of sucrose, and a linear relationship was observed up to 60 gL. From the kinetic analysis of GFOR, the reaction rate was shown to be mainly dependent on the fructose concentration, and this might contribute to the wide linear range of the calibration curve for sucrose. Reproducibility of the FIA System. The reproducibility of measurements using the FIA system was tested by successive analyses of fructose. Figure 7 shows the typical pattern of response for 60 g L fructose. The relative standard deviation for 14 successive analyses was found to be lower than 3.7%,and the detection limit was about 0.2 g/L when the signal to noise (S/N) ratio was 3. Application to On-Line Monitoring. To examine whether the FIA system developed in this work could be applied to the on-line monitoring of bioprocesses, glucose produced from the enzymatic hydrolysis of cellulose in the membrane bioreactor was determined. Figure 8 shows the schematic diagram of the bioreactor coupled with the FIA system. The membrane bioreactor was operated a t 5%of the initial cellulose concentration, and a 1%cellulose was continuously fed into the bioreactor a t a dilution rate of 0.3 h-l. The filtrate from the membrane bioreactor was sampled and analyzed automatically every 15 min. At the same time, the glucose concentration was measured using a conventional enzymatic method. As can be seen in Figure 9, the glucose concentrations determined by the FIA system were coincided well within those determined by the enzymatic method. The discrepancy between the two methods became significant aRer 10 h, and this seems to be largely linked with the leak of enzymes from the gelatin membrane due to the deterioration of the membrane at low pH. The hydrolysis of cellulose in the membrane bioreactor was carried out a t pH 5.0. pH adjustment of the sample solution prior to injection into the FIA system and periodic calibration of the biosensor will improve the performance of the FIA system in the on-line monitoring of glucose, fructose, and sucrose.
Bartlett, P. N.; Tebbutt, P.; Whitaker, G. Kinetic aspects of the use of modified electrodea and mediators in bioelectrochemistry. Prog. React. Kinet. 1991, 16, 55-155. Chun, U. H.; Rogers, U. L. The simultaneous production of sorbitol from fructose and gluconate from glucose using a n oxidoreductase of Zymomonas mobilis. Appl. Microbiol. Biotechnol. 1988,29, 19-24. De Maria, C. G.; Townshend, A. Sequential determination of glucose, fructose and sucrose by flow-injection analysis with immobilized enzyme reactors and spectrophotometric detection. Anal. Chim. Acta 1992,261, 137-143. Garn, M.; Gisin, M.; Thomemen, C.; Cevey, P. A flow injection analysis system for fermentation monitoring and control. Biotechnol. Bioeng. 1989, 34, 423-428. Hall, E. A. H. Flow injection analysis with immobilized reagents. Curr. Opin. Biotechnol. 1991, 2, 9-16. Hendry, S. P.; Higgins, I. J.; Bannister, J. V. Amperometric biosensors. J . Biotechnol. 1990, 15, 229-238. Ludi, H.; Garn, M. B.; Bataillard, P.; Widmer, H. M. Flow injection analysis and biosensors: application for biotechnology and environmental control. J . Biotechnol. 1990,14,7179. Palleschi, G.; Faridnia, M. H.; Lubrano, G. J.; Guibault, G. G . Ideal hydrogen peroxide-based glucose biosensor. Appl. Biochem. Biotechnol. 1991, 31, 21-35. Park, J. M.; Kim, H. S. A new biosensor for specific determination of glucose and fructose using an oxidoreductase of Zymomonas mobilis. Biotechnol. Bioeng. 1990,36,744-749. Park, J. K.; Ro, H. S.; Kim, H. S. A new biosensor for specific determination of sucrose using an oxidoreductase of Zymomonus mobilis and invertase. Biotechnol. Bioeng. 1991, 38, 217-223. Raju, G. K.; Cooney, C. L. Fermentation monitoring. Curr. Opin. Biotechnol. 1992, 3, 40-44. Renneberg, R.; Trott-Kriegeskorte, G.; Lietz, M.; Jager, V.; Pawlowa, M.; Kaiser, G.; Wollenberger, U.; Schubertt, F.; Wanger, R.; Schumid, R. D.; Scheller, F. W. Enzyme sensors-FIA system for on-line monitoring for glucose, lactate, and glutamine in animal cell culture. J . Biotechnol. 1991,21,173-186. Ro, H. S.; Kim, H. S. Continuous production of gluconic acid and sorbitol from sucrose using invertase and an oxidoreductase of Zymomonas mobilis. Enzyme Microb. Technol. 1991, 13, 920-924. Satoh, I.; Karube, I.; Suzuki, S. Enzyme electrode for sucrose. Biotechnol. Bioeng. 1976, 18, 269-272. Schubert, F.; Kirstein, D.; Scheller, F. Enzyme electrode for fructose, glucose-6-phosphate and ATP determination. Anal. Lett. 1986,205, 2155-2167. Valero, F.; Lafuente, M.; Sola, C.; Araujo, A. N.; Lima, J. L. F. C. On-line fermentation monitoring using flow injection analysis. Biotechnol. Bioeng. 1990, 36, 647-651. Xie, X.; Kuan, S. S.; Guilbaut, G. G. A simplified fructose biosensor. Biosens. Bioelectron. 1991, 3, 49-54. Xu, Y.; Guilbault, G. G.; Kuan, S. S. Sucrose enzyme electrode. Anal. Chem. 1989, 61, 782-784. Zachariou, M.; Scopes, R. K. Glucose-fructose oxidoreductase, a new enzyme isolated from Zymomonas mobilis that is responsible for sorbitol production. J . Bacteriol. 1986, 167, 863-869. Accepted July 27, 1994.@ Abstract published in Advance ACS Abstracts, September 15, 1994. @
