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A microbial biosensor for 6-aminopenicillanic acid
A microbial biosensor for 6-aminopenicillanic acid E. Galindo,* F. Lagunas,* J. Osuna,† X. Sobero´n,† and J. L. Garcı´a* *Department of Bioengineering †Department of Molecular Recognition and Biostructure, Institute of Biotechnology, National Autonomous University of Mexico, Cuernavaca, Morelos, Mexico A protein-engineered b-lactamase (N132 D) with enhanced selectivity to 6-APA was used to construct a whole-cell immersible biosensor for 6-APA based on the immobilization of b-lactamase-rich cells of Escherichia coli on a flat pH electrode. The response time was between 3.5–7 min. The sensitivity for penicillin was two to five fold lower to that for 6-APA. Selectivity of 6-APA was maximum at pH 7.6 using a 0.05 M phosphate buffer. 6-APA in concentrations up to 1 mg ml21 could be detected. At least 70 analyses could be performed over 11 days with no loss of sensitivity. © 1998 Elsevier Science Inc.
Introduction
Materials and methods
The measurement of 6-aminopenicillanic acid (6-APA) is of importance in the pharmaceutical industry since this compound is the basis of all semisynthetic penicillins. Currently, 6-APA can be quantified by colorimetric,1,2 spectrophotometric,3 titrimetric,4 and chromatographic5 methods; however, alternative, simple, fast, direct, and cheap methods are still needed. In principle, any compound susceptible to undergo a biologically catalyzed reaction can be measured using a biosensor provided the specificity of the biological reaction is high and the reaction can be followed by a transducer. That is, the case of penicillin for which a number of biosensors have been developed using several transducers able to follow the reaction catalyzed by b-lactamase (see review in Garcı´a et al.)6 The detection of penicillin derivatives (such as second and third generation semisynthetic penicillins) or 6-APA had not been possible due to the inability (in the case of derivatives) of the wild b-lactamase in degrading such compounds or in the case of 6-APA, to insufficient selectivity. A protein-engineered Escherichia coli b-lactamase has been used to construct a biosensor for wide-spectrum cephalosporins.6 In this work, we report showing that by using another protein-engineered b-lactamase (contained in whole Escherichia coli cells), a biosensor for 6-APA can also be constructed.
Bacteria E. coli JM101 was transformed with derivatives of plasmid PKGS7 where wildtype (WT) and mutant (N132 D) b-lactamase genes had been cloned. Enzyme N132 D has a substitution of aspartic acid by asparagine at position 132. Details of the kinetic behavior of this mutant enzyme have been reported before.8
Culture conditions E. coli was cultured in baffled 500-ml Erlenmeyer shake flasks using a diluted Luria-Bertani broth as described earlier.6
Analytical techniques 6-APA was measured with the p-dimethylaminobenzaldehyde technique.9 Biomass growth was measured by the optical density at 620 nm. b-lactamase activity was measured as described by Ross and O’Callagham10 and total protein by the Lowry et al.11 method.
Construction of the biosensor Biosensor construction and ancillary equipment were as described before for a biosensor for new-generation cephalosporins6 except that clone N132 D was used. E. coli cells (3.1 mg) were immobilized (containing 0.6 U of b-lactamase mg21 protein). A schematic diagram of the biosensor array is depicted in Figure 1.
Results and discussion Address reprint requests to Dr. E. Galindo, National Autonomous Univ. of Mexico, Institute of Biotechnology, Department of Bioengineering, Adpo: Post. 510-3, 62250 Cuernavaca, Morelos, Mexico. J. L. Garcı´a is currently at the Instituto Mexicano de Tecnologı´a del Agua, Cuernavaca, Morelos, Me´xico. Received 27 February 1998; accepted 10 April 1998.
Enzyme and Microbial Technology 23:331–334, 1998 © 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
b-lactamase production The kinetics of b-lactamase N132 D production in shake flasks (data not shown) was similar to that observed for other mutants clones,6 which was slightly lower than the wildtype. b-lactamase reached the maximum specific activ-
0141-0229/98/$19.00 PII S0141-0229(98)00057-X
Papers
Figure 1 Schematic diagram of the experimental array of the 6-APA microbial sensor
Figure 3 Calibration curves for 6-APA and penicillin G as a function of the buffer pH (0.1 M)
Figure 2 Response curves for 6-APA and penicillin G 0.1 M (phosphate buffer, pH 7.6). The numbers represent antibiotic concentrations (mg ml21)
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Enzyme Microb. Technol., 1998, vol. 23, October
Figure 4 Calibration curves for 6-APA and penicillin G as a function of the buffer molarity (pH 7.6)
Biosensor for 6-aminopenicillanic acid: E. Galindo et al. Table 1 Sensitivity of the biosensor to 6-APA and penicillin G as a function of pH and ionic strength of the buffer solution
pH (units)
Phosphate buffer concentration (M)
6.7 7.0 7.3 7.6 7.6 7.6 7.6
0.1 0.1 0.1 0.1 0.05 0.025 0.0125
Sensitivitya (DpH mg21 ml21) 3 103 (a) For 6APA (b) For penicillin G a
b
(a)/(b)
35 46 56 174 214 301 560
12 17 16 31 38 61 154
2.9 2.7 3.5 5.6 5.6 4.9 3.6
Calculated as the slope of the DpH versus the concentration of the antibiotic a
ity (of about 0.6 U mg21 protein) toward the end of the growth phase (after 8 h of culture) and later a slight decrease was observed; therefore, for the construction of the biosensor, cells were harvested after 8 h of cultivation.
Response curves Figure 2 shows a typical response curve of the biosensor when 6-APA or penicillin G were used as substrates. The biosensor responded for 6-APA in about 5 min after which the device reached a steady-state plateau in the measured pH drop. A very weak response was observed when the substrate was penicillin G. Clearly, these data show that the change in specificity of the engineered enzyme allowed to measure 6-APA with minimal interference from penicillin G. As far as we know, this is the first time that a biosensor for 6-APA is reported, exploiting the advantages of a protein-engineered enzyme.
solution between 6.7–7.6. This range was determined by the low penicillin G solubility below 6.7 and the high instability of it for pH above 7.6. Figure 3 shows the calibration curves obtained with the biosensor at four different pH values. In general, a higher sensitivity (either for 6-APA and penicillin G) was observed as the pH was higher; however, at pH 7.6, the maximum sensitivity, as measured by the slope of the calibration curve, was observed. Furthermore at that pH value, the difference in sensitivity as compared to that for penicillin G was also maximal. These results were expected from the kinetic characteristics shown by the purified enzyme8 which were rationalized as the protonated state of the modified enzyme favoring the contact with penicillin with the contact with 6-APA also occurring under alkaline pHs. Ionic strength is known to affect the sensitivity of penicillin electrodes based on the measurement of pH by a glass electrode;12,13 therefore, we decided to study the behavior of the 6-APA electrode in terms of the ionic strength of the phosphate buffer solution. Figure 4 shows the results. The behavior of the response of the electrode built with the N132 D strain was similar to that reported previously for a microbial electrode for penicillins.13 The higher the ionic strength of the buffer solution, the lower the sensitivity of the electrode (either for 6-APA and penicillin G). In addition, the difference in sensitivity (to 6-APA in relation to that for penicillin G) was higher as the ionic strength was lowered. This implies that a compromise has to be established between sensitivity and the linear measurement range. Table 1 summarizes the data regarding the sensitivity of the 6-APA biosensor as a function of pH and ionic strength and shows a comparison with data obtained for penicillin G. The data indicated that the best conditions for measuring 6-APA with minimal interference with penicillin G was achieved at pH 7.6 using a buffer molarity of 0.05 m. Although higher sensitivities for 6-APA can be achieved at lower ionic strengths, the selectivity to 6-APA as well as its linear measurement range decreased.
Effects of the pH and ionic strength of the buffer The pH of the reaction solution plays an important role in enzyme catalysis. This is particularly relevant when the actual response of the electrode is in terms of a pH decrease; therefore, we investigated the response to 6-APA and penicillin G of the biosensor varying the pH of the buffer
Comparison with the wildtype enzyme In order to assess the benefits of the engineered enzyme, a comparison was made using a biosensor constructed with cells harboring a wildtype TEM1 b-lactamase. The test was conducted using the same enzyme load and under the same
Figure 5 Calibration curves for 6-APA and penicillin G for biosensors constructed with clone N132 D (a) and the wildtype (b)
Enzyme Microb. Technol., 1998, vol. 23, October
333
Papers
Acknowledgments This work was financed in part by CONACyT (grant 0800-N9110 and grant 0104P-N).
References 1.
2.
Figure 6 Operational stability of the microbial biosensor for 6-APA. Numbers indicate consecutive days of operation
3.
4.
pH and molarity of the buffer (pH 7.6 and phosphate 0.1 m). The results are shown in Figure 5. The response for penicillin G was very similar in either strain. Although the wildtype strain was also more sensitive (threefold) to 6-APA than to penicillin G, the 2.2 higher sensitivity to 6-APA of the engineered strain translates into the relative sensitivity (6-APA/penicillin) of 5.5 as compared with 3.1 for the wildtype strain.
5.
6.
7.
Operational stability
8.
As shown in Figure 6, the biosensor can be used for at least 64 determinations performed over a period of 11 days without appreciable loss in sensitivity or response time.
9.
10.
Conclusions An immersible, whole-cell biosensor for 6-APA was constructed by using a protein-engineered b-lactamase which exhibits enhanced selectivity for 6-APA. Manipulating the pH and buffer molarity allowed the achievement of a sensitivity for 6-APA nearly sixfold higher than for penicillin G. Our results are an example where protein engineering has been applied for the enhancement of the biological component of the biosensor.
334
Enzyme Microb. Technol., 1998, vol. 23, October
11.
12.
13.
Boxer, G. E. and Everett, P. M. Colorimetric determination of benzylpenicillin. Colorimetric determination of the total penicillins. Anal. Chem. 1949, 21, 670 – 673 Korany, M. A., Abdel-Hay, M. H., Bedair, M. H., and Gazy, A. A. Colorimetric determination of some penicillins and cefalosphorins with 2-nitrophenylhydrazine hydrocloride. Talanta 1989, 36, 1253– 1257 Goodall, R. R. and Davies, R. Automatic determination of penicillin in fermentation broth. An improved iodometric assay. Analyst 1961, 86, 326 –335 Alicino, J. F. Iodometric method for the assay of penicillin preparations. Ind. Eng. Chem. 1946, 18, 619 – 620 Haginaka, J. and Wakai, J. Liquid chromatographic determination of penicillins by postcolumn degradation with sodium hypocloride. Anal Chem. 1986, 5, 1896 –1889 Garcı´a, R. J., Nu´nez, C. J., Gonza´lez, E. G., Osuna, J., Sobero´n, X., and Galindo, E. A microbial sensor for new-generation cephalosporins based in a protein-engineered b-lactamase. Appl. Biochem. Biotechnol. 1998, 22, 173–186 Kuhn, I., Stephenson, F. H., Boyer, H. W., and Greene, P. J. Positive-selection vectors utilizing lethality of the EcoRI endonuclease. Gene 1986, 44, 253–263 Osuna, J., Viadiu, H., Fink, A. L., and Sobero´n, X. Substitution of Asp for Asn at position 132 in the active site of TEM b-lactamase. J. Biol. Chem. 1995, 270, 775–780 Balasingham, K., Warburton, D., Dunnill, P., and Lilly, D. The isolation and kinetics of penicillin amidase from E. coli. Biochem. Biophys. Acta 1972, 276, 250 –256 Ross, W. and O’Collagham. b-lactamase assay. Meth. Enzymol. 1975, 43, 69 – 85 Lowry, O., Rosebrough, J., Farr, A., and Rondall, R. Protein measurement with the folinphenol reagent. J. Biol. Chem. 1951, 193, 265–275 Nilsson, H., Mosbach, K., Enfors, S. O., and Molin, N. An enzyme electrode for measurement of penicillin in fermentation broth: A step toward the application of enzyme electrodes in fermentation control. Biotechnol. Bioeng. 1978, 20, 527–539 Galindo, E., Bautista, D., Garcı´a, J., and Quintero, R. Microbial sensor for penicillins using a recombinant strain of E. coli. Enzyme Microb. Technol. 1990, 12, 642– 646
Introduction
Materials and methods
The measurement of 6-aminopenicillanic acid (6-APA) is of importance in the pharmaceutical industry since this compound is the basis of all semisynthetic penicillins. Currently, 6-APA can be quantified by colorimetric,1,2 spectrophotometric,3 titrimetric,4 and chromatographic5 methods; however, alternative, simple, fast, direct, and cheap methods are still needed. In principle, any compound susceptible to undergo a biologically catalyzed reaction can be measured using a biosensor provided the specificity of the biological reaction is high and the reaction can be followed by a transducer. That is, the case of penicillin for which a number of biosensors have been developed using several transducers able to follow the reaction catalyzed by b-lactamase (see review in Garcı´a et al.)6 The detection of penicillin derivatives (such as second and third generation semisynthetic penicillins) or 6-APA had not been possible due to the inability (in the case of derivatives) of the wild b-lactamase in degrading such compounds or in the case of 6-APA, to insufficient selectivity. A protein-engineered Escherichia coli b-lactamase has been used to construct a biosensor for wide-spectrum cephalosporins.6 In this work, we report showing that by using another protein-engineered b-lactamase (contained in whole Escherichia coli cells), a biosensor for 6-APA can also be constructed.
Bacteria E. coli JM101 was transformed with derivatives of plasmid PKGS7 where wildtype (WT) and mutant (N132 D) b-lactamase genes had been cloned. Enzyme N132 D has a substitution of aspartic acid by asparagine at position 132. Details of the kinetic behavior of this mutant enzyme have been reported before.8
Culture conditions E. coli was cultured in baffled 500-ml Erlenmeyer shake flasks using a diluted Luria-Bertani broth as described earlier.6
Analytical techniques 6-APA was measured with the p-dimethylaminobenzaldehyde technique.9 Biomass growth was measured by the optical density at 620 nm. b-lactamase activity was measured as described by Ross and O’Callagham10 and total protein by the Lowry et al.11 method.
Construction of the biosensor Biosensor construction and ancillary equipment were as described before for a biosensor for new-generation cephalosporins6 except that clone N132 D was used. E. coli cells (3.1 mg) were immobilized (containing 0.6 U of b-lactamase mg21 protein). A schematic diagram of the biosensor array is depicted in Figure 1.
Results and discussion Address reprint requests to Dr. E. Galindo, National Autonomous Univ. of Mexico, Institute of Biotechnology, Department of Bioengineering, Adpo: Post. 510-3, 62250 Cuernavaca, Morelos, Mexico. J. L. Garcı´a is currently at the Instituto Mexicano de Tecnologı´a del Agua, Cuernavaca, Morelos, Me´xico. Received 27 February 1998; accepted 10 April 1998.
Enzyme and Microbial Technology 23:331–334, 1998 © 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
b-lactamase production The kinetics of b-lactamase N132 D production in shake flasks (data not shown) was similar to that observed for other mutants clones,6 which was slightly lower than the wildtype. b-lactamase reached the maximum specific activ-
0141-0229/98/$19.00 PII S0141-0229(98)00057-X
Papers
Figure 1 Schematic diagram of the experimental array of the 6-APA microbial sensor
Figure 3 Calibration curves for 6-APA and penicillin G as a function of the buffer pH (0.1 M)
Figure 2 Response curves for 6-APA and penicillin G 0.1 M (phosphate buffer, pH 7.6). The numbers represent antibiotic concentrations (mg ml21)
332
Enzyme Microb. Technol., 1998, vol. 23, October
Figure 4 Calibration curves for 6-APA and penicillin G as a function of the buffer molarity (pH 7.6)
Biosensor for 6-aminopenicillanic acid: E. Galindo et al. Table 1 Sensitivity of the biosensor to 6-APA and penicillin G as a function of pH and ionic strength of the buffer solution
pH (units)
Phosphate buffer concentration (M)
6.7 7.0 7.3 7.6 7.6 7.6 7.6
0.1 0.1 0.1 0.1 0.05 0.025 0.0125
Sensitivitya (DpH mg21 ml21) 3 103 (a) For 6APA (b) For penicillin G a
b
(a)/(b)
35 46 56 174 214 301 560
12 17 16 31 38 61 154
2.9 2.7 3.5 5.6 5.6 4.9 3.6
Calculated as the slope of the DpH versus the concentration of the antibiotic a
ity (of about 0.6 U mg21 protein) toward the end of the growth phase (after 8 h of culture) and later a slight decrease was observed; therefore, for the construction of the biosensor, cells were harvested after 8 h of cultivation.
Response curves Figure 2 shows a typical response curve of the biosensor when 6-APA or penicillin G were used as substrates. The biosensor responded for 6-APA in about 5 min after which the device reached a steady-state plateau in the measured pH drop. A very weak response was observed when the substrate was penicillin G. Clearly, these data show that the change in specificity of the engineered enzyme allowed to measure 6-APA with minimal interference from penicillin G. As far as we know, this is the first time that a biosensor for 6-APA is reported, exploiting the advantages of a protein-engineered enzyme.
solution between 6.7–7.6. This range was determined by the low penicillin G solubility below 6.7 and the high instability of it for pH above 7.6. Figure 3 shows the calibration curves obtained with the biosensor at four different pH values. In general, a higher sensitivity (either for 6-APA and penicillin G) was observed as the pH was higher; however, at pH 7.6, the maximum sensitivity, as measured by the slope of the calibration curve, was observed. Furthermore at that pH value, the difference in sensitivity as compared to that for penicillin G was also maximal. These results were expected from the kinetic characteristics shown by the purified enzyme8 which were rationalized as the protonated state of the modified enzyme favoring the contact with penicillin with the contact with 6-APA also occurring under alkaline pHs. Ionic strength is known to affect the sensitivity of penicillin electrodes based on the measurement of pH by a glass electrode;12,13 therefore, we decided to study the behavior of the 6-APA electrode in terms of the ionic strength of the phosphate buffer solution. Figure 4 shows the results. The behavior of the response of the electrode built with the N132 D strain was similar to that reported previously for a microbial electrode for penicillins.13 The higher the ionic strength of the buffer solution, the lower the sensitivity of the electrode (either for 6-APA and penicillin G). In addition, the difference in sensitivity (to 6-APA in relation to that for penicillin G) was higher as the ionic strength was lowered. This implies that a compromise has to be established between sensitivity and the linear measurement range. Table 1 summarizes the data regarding the sensitivity of the 6-APA biosensor as a function of pH and ionic strength and shows a comparison with data obtained for penicillin G. The data indicated that the best conditions for measuring 6-APA with minimal interference with penicillin G was achieved at pH 7.6 using a buffer molarity of 0.05 m. Although higher sensitivities for 6-APA can be achieved at lower ionic strengths, the selectivity to 6-APA as well as its linear measurement range decreased.
Effects of the pH and ionic strength of the buffer The pH of the reaction solution plays an important role in enzyme catalysis. This is particularly relevant when the actual response of the electrode is in terms of a pH decrease; therefore, we investigated the response to 6-APA and penicillin G of the biosensor varying the pH of the buffer
Comparison with the wildtype enzyme In order to assess the benefits of the engineered enzyme, a comparison was made using a biosensor constructed with cells harboring a wildtype TEM1 b-lactamase. The test was conducted using the same enzyme load and under the same
Figure 5 Calibration curves for 6-APA and penicillin G for biosensors constructed with clone N132 D (a) and the wildtype (b)
Enzyme Microb. Technol., 1998, vol. 23, October
333
Papers
Acknowledgments This work was financed in part by CONACyT (grant 0800-N9110 and grant 0104P-N).
References 1.
2.
Figure 6 Operational stability of the microbial biosensor for 6-APA. Numbers indicate consecutive days of operation
3.
4.
pH and molarity of the buffer (pH 7.6 and phosphate 0.1 m). The results are shown in Figure 5. The response for penicillin G was very similar in either strain. Although the wildtype strain was also more sensitive (threefold) to 6-APA than to penicillin G, the 2.2 higher sensitivity to 6-APA of the engineered strain translates into the relative sensitivity (6-APA/penicillin) of 5.5 as compared with 3.1 for the wildtype strain.
5.
6.
7.
Operational stability
8.
As shown in Figure 6, the biosensor can be used for at least 64 determinations performed over a period of 11 days without appreciable loss in sensitivity or response time.
9.
10.
Conclusions An immersible, whole-cell biosensor for 6-APA was constructed by using a protein-engineered b-lactamase which exhibits enhanced selectivity for 6-APA. Manipulating the pH and buffer molarity allowed the achievement of a sensitivity for 6-APA nearly sixfold higher than for penicillin G. Our results are an example where protein engineering has been applied for the enhancement of the biological component of the biosensor.
334
Enzyme Microb. Technol., 1998, vol. 23, October
11.
12.
13.
Boxer, G. E. and Everett, P. M. Colorimetric determination of benzylpenicillin. Colorimetric determination of the total penicillins. Anal. Chem. 1949, 21, 670 – 673 Korany, M. A., Abdel-Hay, M. H., Bedair, M. H., and Gazy, A. A. Colorimetric determination of some penicillins and cefalosphorins with 2-nitrophenylhydrazine hydrocloride. Talanta 1989, 36, 1253– 1257 Goodall, R. R. and Davies, R. Automatic determination of penicillin in fermentation broth. An improved iodometric assay. Analyst 1961, 86, 326 –335 Alicino, J. F. Iodometric method for the assay of penicillin preparations. Ind. Eng. Chem. 1946, 18, 619 – 620 Haginaka, J. and Wakai, J. Liquid chromatographic determination of penicillins by postcolumn degradation with sodium hypocloride. Anal Chem. 1986, 5, 1896 –1889 Garcı´a, R. J., Nu´nez, C. J., Gonza´lez, E. G., Osuna, J., Sobero´n, X., and Galindo, E. A microbial sensor for new-generation cephalosporins based in a protein-engineered b-lactamase. Appl. Biochem. Biotechnol. 1998, 22, 173–186 Kuhn, I., Stephenson, F. H., Boyer, H. W., and Greene, P. J. Positive-selection vectors utilizing lethality of the EcoRI endonuclease. Gene 1986, 44, 253–263 Osuna, J., Viadiu, H., Fink, A. L., and Sobero´n, X. Substitution of Asp for Asn at position 132 in the active site of TEM b-lactamase. J. Biol. Chem. 1995, 270, 775–780 Balasingham, K., Warburton, D., Dunnill, P., and Lilly, D. The isolation and kinetics of penicillin amidase from E. coli. Biochem. Biophys. Acta 1972, 276, 250 –256 Ross, W. and O’Collagham. b-lactamase assay. Meth. Enzymol. 1975, 43, 69 – 85 Lowry, O., Rosebrough, J., Farr, A., and Rondall, R. Protein measurement with the folinphenol reagent. J. Biol. Chem. 1951, 193, 265–275 Nilsson, H., Mosbach, K., Enfors, S. O., and Molin, N. An enzyme electrode for measurement of penicillin in fermentation broth: A step toward the application of enzyme electrodes in fermentation control. Biotechnol. Bioeng. 1978, 20, 527–539 Galindo, E., Bautista, D., Garcı´a, J., and Quintero, R. Microbial sensor for penicillins using a recombinant strain of E. coli. Enzyme Microb. Technol. 1990, 12, 642– 646