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Continuous monitoring of D-glucose and L-lactate by flow injection analysis
Enzyme and Microbial Technology 30 (2002) 129 –133
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Continuous monitoring of D-glucose and L-lactate by flow injection analysis Ahmed Haouza,*, Scott Stiegb a
Laboratoire de Biologie Physico-chimique, Universite´ Denis Diderot Paris7, Paris, France b Lachat Instruments 6645 Mill Road, Milwaukee, WI 53218, USA
Received 20 March 2001; received in revised form 26 September 2001; accepted 15 October 2001
Abstract The D-glucose or the L-lactate oxidases were copolymerized by glutaraldehyde with bovine albumin and mounted on an oxygen electrode to form an enzyme electrode. This enzyme electrode was mounted on a Lachat QuikChem® 8000 flow injection analysis (FIA) system. With this system the glucose and the L-lactate is simultaneously measured in the concentration range 0 to 40 g/liter. The enzymatic films can be used continuously for three months, at a rate of one measurement every two minutes. The stability of the enzyme electrode is greater than one year when stored in the dry state at 4°C. This FIA system can be used for monitoring simultaneously the concentration of D-glucose and L-lactate in the bioprocessing industries. © 2002 Elsevier Science Inc. All rights reserved. Keywords: D-glucose; L-lactate; FIA system; Covalent immobilization; Biosensors
1. Introduction There is currently considerable interest in the development of biochemical-specific methods that could be used to monitor and regulate the concentration of the metabolites in various biotechnological processes. Immobilization of enzymes is often considered a prerequisite for preparative techniques in industrial bioreactors, and for the preparation of enzyme electrodes as analytical devices, or biosensors. An enzyme electrode biosensor can give a continuous, instantaneous, and electrochemical monitoring of an enzyme catalyzed reaction, in which a substrate, coenzyme, or inhibitor is converted into a product by means of an enzyme [1,2]. The electroactive species either produced or consumed by the enzymatic reaction may be detected by a commercial solute-specific potentiometric or amperometric electrode. The development of biosensors is closely associated with the immobilization of enzymes. Numerous methods of enzyme immobilization have been reported [3,4]. The enzyme can be entrapped within a synthetic hydrophilic gel, crosslinks can be formed between the molecules to make mem* Corresponding author. Tel.: ⫹1-33-1-4427-4738; fax: ⫹1-33-14427-6995. E-mail address: [email protected] (A. Haouz).
branes, the enzymes can be chemically bound to the membrane. Such techniques can optimize enzymatic kinetics by varying the diffusion rate of substrate to the active center. Glucose oxidase is an ideal enzyme to describe any technological evolution in biosensors [5]. Numerous biosensors to detect glucose or L-lactate have been described in the literature. All of these biosensors use the same enzymes, but differ essentially in the immobilization method or in the type of the transducer (e.g. electrode or thermal sensor) [6]. Many biosensors have been commercialized which detect substances such as glucose, alcohol, lactate and pesticides [7]. The major problem of these biosensors is the stability of the enzyme activity [8]. In our study we have developed the biosensors to detect simultaneously and continuously glucose and L-lactate in bioreactors. We used glucose oxidase from Aspergillus niger (-D-glucose:oxygen1-oxido-reductase, EC 1.1.3.4) with a molecular mass of 180 kDa [9], and L-lactate oxidase from Pediococcus species (L-lactate:oxygen1-oxido-reductase, EC 1.1.3.2) with a molecular mass of 400 kDa for the enzyme extracted from Geotrichum candidum [10]. The glucose oxidase is a homo-dimeric enzyme. The encoded gene of this enzyme is sequenced [11], and its tridimensional structure was resolved at 2.3 Å [12]. This enzyme catalyses the conversion of the -D-glucose into gluconic acid and is highly specific for -D-glucose. Other hexoses,
0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 1 ) 0 0 4 7 7 - X
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A. Haouz, S. Stieg / Enzyme and Microbial Technology 30 (2002) 129 –133
␣-D-glucose, mannose, galactose, and 2-deoxy-glucose, are poorly oxidized. The origin of this specificity is recently identified by the molecular modeling of all substrates in the active center of glucose oxidase [13]. The reaction involves the transfer of two protons from glucose to the coenzyme [9]. During this reaction the flavin adenine dinucleotide (FAD) is transformed to its reduced form (FADH2). The re-oxidation of flavin implicates the molecular oxygen present in the medium and by this means we can follow the reaction with the oxygen electrode. In the same way, the L-lactate oxidase catalyzes the oxidation of L-lactate;however, the coenzyme of this enzyme is FMN [14]. The gene encoded for this enzyme is sequenced for other species and shown to have high similarity with those of the other enzyme catalyzed oxidations of L-alpha-hydroxy acids [15]. Preliminary X-ray studies of the lactate oxidase from Aerococcus viridans show that the enzyme exists in an octameric form [16]. Our biosensor is used in an FIA QuikChem® 8000 system developed by Lachat Instruments USA [17]. This system consists of a data acquisition system, XYZ sampler, peristaltic pump and a sample processing module consisting of a sample injection valve, chemistry manifold, a flow cell for oxygen electrode and an amperometric detector module.
2. Materials and methods 2.1. Proteins and reagents Lyophilized glucose oxidase, L-lactate oxidase, catalase, and bovine albumin fraction V (SAB), were purchased from Sigma® and used without further purification. Tris[hydroxymethyl]aminomethane, glutaraldehyde, sodium benzoate, D-glucose, L-lactate were purchased from Sigma®. Tween 20 was purchased from Fluka®. All solutions were prepared with deionized water (18 megohm).
Fig. 1. QuikChem FIA system with the general fluidic setup. The enzymatic film, oxygen electrode and the flow cell is mounted in the detector module.
2.3. Fabrication of the enzymatic films For glucose analysis, we prepared solutions of 20 UI of glucose oxidase, 10 UI of catalase, 20 l of 3% (v/v) glutaraldehyde, and 200 l of SAB 30% (w/v), diluted to 1 ml with 10 mM Tris buffer, pH 7.5. 1 ml of this solution was spread homogeneously under an area of 10 cm2 of polypropylene membrane. The film was stored at –20°C for 24 h to complete the covalent immobilization. After defrosting, we cut out the enzymatic film in several 1.5 cm2 squares. The face containing the protein gel of each square piece of enzymatic film was covered with 1.5 cm2 squares of the dialysis membrane. The two membranes were then mounted under the oxygen electrode cap, with the dialysis membrane in contact with the solution to be measured. The polypropylene membrane was thus in contact with the electrode’s electrolyte. For lactate analysis, a solution of 20 UI of L-lactate oxidase, 10 UI of catalase, 20 l of 1%(v/v) glutaraldehyde, and 200 l of SAB 30% (w/v) was prepared, diluted to 1 ml with 10 mM Tris buffer, pH 7.5, and thereafter, used as in the glucose procedure described above.
2.4. Flow injection experiments 2.2. Materials The dialysis membrane was regenerated cellulose, MWCO100000, from Spectra/por®, and the 6 m thick polypropylene film was obtained from Bollore Inc. Paris France. The oxygen electrode used in this study is a commercial electrode from Zellweger Analytics, Noisy-le-Grand, France, which can measure dissolved oxygen concentrations in the mg/liter range. This electrode is composed of a gold cathode and silver anode and filled with a 3 M KCl electrolyte. This electrode is polarized at – 0.7 V and is mounted in a proprietary flow cell of the QuikChem 8000® system. The FIA system is controlled by a computer with Omnion FIA software developed by Lachat instruments. Milwaukee USA.
The carrier (buffer) used in all experiments to measure a baseline was Tris-HCl 10 mM, pH 7.5 containing 10 mg/ liter sodium benzoate and 0.1% (w/v) Tween 20. At 20°C and atmospheric pressure this buffer (100% oxygenated) contains 9 mg/liter of oxygen. This buffer was continuously pumped at a flow rate of one liter/hour. The carrier passed through the injection valve and then to the measurement flow cell containing the enzymatic film mounted on the oxygen electrode, then to the waste (Fig. 1). The injection valve can adopt two positions:in the load position the sample to be measured was loaded into the sample loop, and in the inject position the sample present in this loop was injected into the flow cell (detector). After each measurement 100% of oxygenation was achieved when the baseline was obtained (Fig. 2).
A. Haouz, S. Stieg / Enzyme and Microbial Technology 30 (2002) 129 –133
Fig. 2. Peak response of glucose biosensor after injection of standard solutions of glucose. The first three peaks correspond to injection of 10 g/liter of glucose and the last three peaks correspond to injection of 5 g/liter.
3. Results and discussion 3.1. Measurements The 100% oxygenated buffer/carrier formed the baseline for the analysis which gave the largest signal. When the analyte (D-glucose or L-lactate substrate) passed over the dialysis membrane in the flow cell, it reacted with the enzymatic film, and was oxidized by the enzyme, the amount of the dissolved oxygen decreased because oxygen was used during the re-oxidation of the reduced enzyme form. As the analyte left the flow cell, the amount of dissolved oxygen increased again to its baseline concentration. This produced a negative peak whose area was proportional to the concentration of D-glucose or L-lactate substrate. The negative peak was inverted to a positive form by the detector. For each substrate injection the result is the integrated peak area and expressed in V/S (Fig. 2). So that the cycle period of the experiment could be reduced to 120 s, giving a throughput of 30 samples/hour, different MW cutoffs of the dialysis membrane and other immobilization methods were tested. The glucose or lactate oxidases bound on the surface of an affinity membrane (Ultrabind® from Gelman) [18] gave a cycle time period greater than 300 S. The slow rate of diffusion of oxygen and substrate through this membrane appears to be limiting. 3.2. Dosage concentrations of D-glucose and L-lactate To detect the concentration of D-glucose or L-lactate immobilized glucose oxidase or L-lactate oxidase were used as described above. Although the enzymes were covalently immobilized, they can still be characterized by the turnover number, concentration and Michaelis constant (Km) of the enzyme, as is the case for the enzyme free in solution [19]. The differences observed in the corresponding soluble en-
131
zyme is usually due to the diffusional, charge, and boundary layer effects [20] of the substrate to the heterogeneous phase (enzymatic film) According to Michaelis equation the proportionality between the response and the substrate concentration are obtained if the substrate concentration injected is below 1/3 Km. In our study we have not characterized our system by the turnover number or the maximum velocity of the reaction since we are only interested in the linearity range. Our results shown that the linear range of the method depends on the type of loop (large, medium and micro) used in the valve to inject the substrate. Fig. 3 a show the low range:0 to 1 g/liter for the -D glucose and L- lactate. As seen, almost the same response for the two films is observed because the same quantities of enzyme were immobilized. These two enzymes do not support the same quantity of the glutaraldehyde, the reagent used for the covalent immobilization. The glucose oxidase enzyme is not denatured in the presence of glutaraldehyde below 10% (w/v) but the lactate oxidase cannot support a concentration of glutaraldehyde higher than 2%. Also the residual activity of immobilized enzyme depend on others parameters such as the membrane thickness and the volume between active membrane and the pO2 electrode. The best results are obtained with the concentration described in the materials and methods. Fig. 3b shows the two other ranges for the determination of Llactate:1–10 g/liter and 5– 40 g/liter. The response curve in the case of the medium loop is an hyperbola confirming that these enzymes still obey the Michaelis-Menten model. If the concentration of substrate is higher than 40 g/liter, the sample must be diluted in the range. The same results for the -D-glucose are observed. For both substrates’ biosensors, the detection limit is 0.02 g/liter and the relative standard deviation (RDS) is 2%. 3.3. Reproducibility and stability To estimate the repeatability of the measurement, we injected 9.5 g/liter of -D-glucose into the flow cell. Fig. 4 shows that for 318 measurements a Gaussian representation is obtained with the mean at 9.5 g/liter, exactly the same as the injected concentration. The relative standard deviation is 0.1 g/L The immobilized enzymes are more resistant to various denaturation factors (extreme pH and temperature values, high ionic strength, denaturing reagents, proteases, etc) than their corresponding forms in a soluble state. This increase in stability is due to covalent attachment of the enzyme protein, which reduces internal movements which can induce the unfolding of proteins [21]. In our case, the enzymatic films are very stable. After mounting on the electrode they can be used for up to three months, 24 h a day. Any loss of activity can be corrected by the calibration of the FIA system with standards solutions. The calibration frequency is one calibration every week. The high stability of these enzymatic films is due to three factors:(i) if the molecular
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A. Haouz, S. Stieg / Enzyme and Microbial Technology 30 (2002) 129 –133
Fig. 4. Response distribution of 318 injections of 9.5 g/liter glucose. The average value is exactly the injected concentration of D-glucose.
3.4. Validation of the FIA system
Fig. 3. Linearity of response with different sample loops. (a) Large loop: (■) glucose, (䊐) L-lactate. (b) large loop (E) and medium loop (F) for the L-lactate biosensor.
enzyme is released from the film, it is still in contact with electrode by dialysis membrane, (ii) the presence of catalase in the enzymatic film hydrolyzes the hydrogen peroxide which can inactivate the enzyme, (iii) in the carrier sodium benzoate was used to minimise any microbial proliferation. The fabricated film with or without catalase, protected by the dialysis membrane, has a lifetime of less than one week. The conservation of the enzymatic film can be achieved by freeze drying. In this state, the film maintains its activity for over one year.
In the bioprocess industry and in many research laboratories, animal cells are cultivated in vitro for the production of different bio-molecules such as proteins having therapeutic value. The cell culture medium includes serum, vitamins, growth factors, minerals, a mixture of amino acid as sources of nitrogen, and glucose as a source of carbon. When the levels of either carbon or nitrogen sources is too high or too low they inhibit growth. There is, therefore, an increasing demand for the analytical methods for monitoring and control of cell cultures. Online control is preferred, since the rapid determination of the glucose and L-lactate level in the culture can be used as a feedback loop for the control of the cell’s productivity [22,23] We used our system to follow the concentration of Dglucose and L-lactate during the growth cycle of E. coli in M9 minimal salts medium (Sigma®) containing 4 g/l of glucose. The concentration of glucose and L-lactate in 5 ml of media was measured during 36 h of growth. The results presented in Table 1 show that the concentration of glucose measured in the media without cells is the same as measured Table 1 Change in concentration of b-D-glucose and L-lactate in the media during the growth of E. coli Samples
[-D-Glucose] g/L ⫾ SD
[L-lactate] g/L ⫾ SD
Media Media Media Media
4.1 ⫾ 0.1 4.0 ⫾ 0.1 3.1 ⫾ 0.1 2.2 ⫾ 0.1
0 0 1.1 ⫾ 0.1 1.8 ⫾ 0.1
before inoculation of E. coli after 1 hour of growth after 24 hours of growth after 36 hours of growth
A. Haouz, S. Stieg / Enzyme and Microbial Technology 30 (2002) 129 –133
in the culture media. During growth a decrease of the concentration of -D-glucose and an increase of the concentration of the L-lactate is observed. We can see also that, during the metabolism of glucose, it is converted finally to the L-lactate. 4. Conclusion The goal of this study was to measure continuously the concentration of D-glucose and L-lactate. Our results show that these metabolites can be on-line monitored every 2 min between 0 to 40 g/liter. The enzyme electrode lasts 3 months and the precision of the measurement is less than 2%. Calibration is done automatically by injecting standard solutions. The data system will prepare a calibration curve by plotting response versus standard concentration. Sample concentration is calculated from the regression equation. The QuikChem FIA system can be used to measure simultaneously these metabolites in a bioprocess and, at the same time, the system can also detect other substances like ammonia, phosphate, without any perturbation of the biosensors. References [1] Gough DA, Andrade JD. Enzyme electrodes. Science 1973;180:380– 4. [2] Turner APF. Biosensors sense and sensitivity. Science 2000;290: 1315–7. [3] Silman I, Katchlski E. Water-insoluble derivatives of enzymes, antigens, and antibodies. Annu Rev Biochem 1966;35:873–908. [4] Wingard LB. Enzyme Engineering, New York: Interscience, 1972. [5] Wilson R, Turner APF. Glucose oxidase:an ideal enzyme. Biosens Bioelectron 1992;7:165– 85. [6] Danielsson B, Mosbach K. Analytical applications with emphasis on biosensors. Introduction Methods Enzymol 1988;137:3–14. [7] Luong JHT, Bouvrette P, Male KB. Developments and applications of biosensors in food analysis. Trends Biotechnol 1997;15:369 –77. [8] Guilbault GG. Enzyme electrode probes. Methods Enzymol 1988; 137:14 –29.
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[9] Wellner D. Flavoproteins. Ann Rev Biochem 1967;36:669 –90. [10] Sztajer H, Wang W, Lunsdorf H, Stocker A, Schmid RD. Purification and some properties of a novel microbial lactate oxidase. Appl Microbiol Biotechnol 1996;45:600 – 6. [11] Kriechbaum M, Heilmann HJ, Wientjes FJ, Hahn M, Jany KD, Gassen HG, Sharif F, Alaeddinoglu G. Cloning and DNA sequence analysis of the glucose oxidase gene from Aspergillus niger NRRL-3. FEBS Lett 1989;255:63– 6. [12] Hecht HJ, Kalisz HM, Hendle J, Schmid RD, Schomburg D. Crystal structure of glucose oxidase from Aspergillus niger refined at 2.3 A resolution. J Mol Biol 1993;229:153–72. [13] Wohlfahrt G, Witt S, Hendle J, Schomburg D, Kalisz HM, Hecht HJ. 1.8 and 1.9 A resolution structures of the Penicillium amagasakiense and Aspergillus niger glucose oxidases as a basis for modelling substrate complexes. Acta Crystallogr D Biol Crystallogr 1999;D55: 969 –77. [14] Duncan JD, Wallis JO, Azari MR. Purification and properties of Aerococcus viridans lactate oxidase. Biochem Biophys Res Commun 1989;164:919 –26. [15] Maeda-Yorita K, Aki K, Sagai H, Misaki H, Massey V. L-lactate oxidase and L-lactate monooxygenase:mechanistic variations on a common structural theme. Biochimie 1995;77:631– 42. [16] Morimoto Y, Yorita K, Aki K, Misaki H, Massey V. L-lactate oxidase from Aerococcus viridans crystallized as an octamer. Preliminary X-ray studies. Biochimie 1998;80:309 –12. [17] Karmarkar SV. Analysis of wastewater for anionic and cationic nutrients by ion chromatography in a single run with sequential flow injection analysis. J Chromatogr A 1999;850:303–9. [18] Haouz A. Geloso-Meyer A, Burstein C. Assay of dehydrogenases with an O2-consuming biosensor. Enzyme Microb Technol 1994;16: 292–7. [19] Boda´ lo A, Go´ mez JL, Go´ mez E, Bastida J, Iborra JL, Manjo´ n A. Analysis of diffusion effects on immobilized enzymes on porous supports with reversible Michaelis-Menten kinetics. Enzyme Microb Technol 1986;8:433– 8. [20] Chaplin MF, Bucke C. Enzyme technology. Cambridge: Cambridge University Press, 1990. [21] Flory PJ. Theory of elastic mechanisms in fibrous proteins. J Am Chem Soc 1956;78:5222–32. [22] Enfors SO, Cleland N. Enzyme sensors for fermentation monitoring: sample handling. Methods Enzymol 1988;137:298 –307. [23] Huang YL, Foellmer TJ, Choo SB, Yap MGS. Characterization and application of an on-line flow injection analysis/wall-jet electrode system for glucose monitoring during fermentation. Anal Chim Acta 1995;317:223–32.
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Continuous monitoring of D-glucose and L-lactate by flow injection analysis Ahmed Haouza,*, Scott Stiegb a
Laboratoire de Biologie Physico-chimique, Universite´ Denis Diderot Paris7, Paris, France b Lachat Instruments 6645 Mill Road, Milwaukee, WI 53218, USA
Received 20 March 2001; received in revised form 26 September 2001; accepted 15 October 2001
Abstract The D-glucose or the L-lactate oxidases were copolymerized by glutaraldehyde with bovine albumin and mounted on an oxygen electrode to form an enzyme electrode. This enzyme electrode was mounted on a Lachat QuikChem® 8000 flow injection analysis (FIA) system. With this system the glucose and the L-lactate is simultaneously measured in the concentration range 0 to 40 g/liter. The enzymatic films can be used continuously for three months, at a rate of one measurement every two minutes. The stability of the enzyme electrode is greater than one year when stored in the dry state at 4°C. This FIA system can be used for monitoring simultaneously the concentration of D-glucose and L-lactate in the bioprocessing industries. © 2002 Elsevier Science Inc. All rights reserved. Keywords: D-glucose; L-lactate; FIA system; Covalent immobilization; Biosensors
1. Introduction There is currently considerable interest in the development of biochemical-specific methods that could be used to monitor and regulate the concentration of the metabolites in various biotechnological processes. Immobilization of enzymes is often considered a prerequisite for preparative techniques in industrial bioreactors, and for the preparation of enzyme electrodes as analytical devices, or biosensors. An enzyme electrode biosensor can give a continuous, instantaneous, and electrochemical monitoring of an enzyme catalyzed reaction, in which a substrate, coenzyme, or inhibitor is converted into a product by means of an enzyme [1,2]. The electroactive species either produced or consumed by the enzymatic reaction may be detected by a commercial solute-specific potentiometric or amperometric electrode. The development of biosensors is closely associated with the immobilization of enzymes. Numerous methods of enzyme immobilization have been reported [3,4]. The enzyme can be entrapped within a synthetic hydrophilic gel, crosslinks can be formed between the molecules to make mem* Corresponding author. Tel.: ⫹1-33-1-4427-4738; fax: ⫹1-33-14427-6995. E-mail address: [email protected] (A. Haouz).
branes, the enzymes can be chemically bound to the membrane. Such techniques can optimize enzymatic kinetics by varying the diffusion rate of substrate to the active center. Glucose oxidase is an ideal enzyme to describe any technological evolution in biosensors [5]. Numerous biosensors to detect glucose or L-lactate have been described in the literature. All of these biosensors use the same enzymes, but differ essentially in the immobilization method or in the type of the transducer (e.g. electrode or thermal sensor) [6]. Many biosensors have been commercialized which detect substances such as glucose, alcohol, lactate and pesticides [7]. The major problem of these biosensors is the stability of the enzyme activity [8]. In our study we have developed the biosensors to detect simultaneously and continuously glucose and L-lactate in bioreactors. We used glucose oxidase from Aspergillus niger (-D-glucose:oxygen1-oxido-reductase, EC 1.1.3.4) with a molecular mass of 180 kDa [9], and L-lactate oxidase from Pediococcus species (L-lactate:oxygen1-oxido-reductase, EC 1.1.3.2) with a molecular mass of 400 kDa for the enzyme extracted from Geotrichum candidum [10]. The glucose oxidase is a homo-dimeric enzyme. The encoded gene of this enzyme is sequenced [11], and its tridimensional structure was resolved at 2.3 Å [12]. This enzyme catalyses the conversion of the -D-glucose into gluconic acid and is highly specific for -D-glucose. Other hexoses,
0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 1 ) 0 0 4 7 7 - X
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A. Haouz, S. Stieg / Enzyme and Microbial Technology 30 (2002) 129 –133
␣-D-glucose, mannose, galactose, and 2-deoxy-glucose, are poorly oxidized. The origin of this specificity is recently identified by the molecular modeling of all substrates in the active center of glucose oxidase [13]. The reaction involves the transfer of two protons from glucose to the coenzyme [9]. During this reaction the flavin adenine dinucleotide (FAD) is transformed to its reduced form (FADH2). The re-oxidation of flavin implicates the molecular oxygen present in the medium and by this means we can follow the reaction with the oxygen electrode. In the same way, the L-lactate oxidase catalyzes the oxidation of L-lactate;however, the coenzyme of this enzyme is FMN [14]. The gene encoded for this enzyme is sequenced for other species and shown to have high similarity with those of the other enzyme catalyzed oxidations of L-alpha-hydroxy acids [15]. Preliminary X-ray studies of the lactate oxidase from Aerococcus viridans show that the enzyme exists in an octameric form [16]. Our biosensor is used in an FIA QuikChem® 8000 system developed by Lachat Instruments USA [17]. This system consists of a data acquisition system, XYZ sampler, peristaltic pump and a sample processing module consisting of a sample injection valve, chemistry manifold, a flow cell for oxygen electrode and an amperometric detector module.
2. Materials and methods 2.1. Proteins and reagents Lyophilized glucose oxidase, L-lactate oxidase, catalase, and bovine albumin fraction V (SAB), were purchased from Sigma® and used without further purification. Tris[hydroxymethyl]aminomethane, glutaraldehyde, sodium benzoate, D-glucose, L-lactate were purchased from Sigma®. Tween 20 was purchased from Fluka®. All solutions were prepared with deionized water (18 megohm).
Fig. 1. QuikChem FIA system with the general fluidic setup. The enzymatic film, oxygen electrode and the flow cell is mounted in the detector module.
2.3. Fabrication of the enzymatic films For glucose analysis, we prepared solutions of 20 UI of glucose oxidase, 10 UI of catalase, 20 l of 3% (v/v) glutaraldehyde, and 200 l of SAB 30% (w/v), diluted to 1 ml with 10 mM Tris buffer, pH 7.5. 1 ml of this solution was spread homogeneously under an area of 10 cm2 of polypropylene membrane. The film was stored at –20°C for 24 h to complete the covalent immobilization. After defrosting, we cut out the enzymatic film in several 1.5 cm2 squares. The face containing the protein gel of each square piece of enzymatic film was covered with 1.5 cm2 squares of the dialysis membrane. The two membranes were then mounted under the oxygen electrode cap, with the dialysis membrane in contact with the solution to be measured. The polypropylene membrane was thus in contact with the electrode’s electrolyte. For lactate analysis, a solution of 20 UI of L-lactate oxidase, 10 UI of catalase, 20 l of 1%(v/v) glutaraldehyde, and 200 l of SAB 30% (w/v) was prepared, diluted to 1 ml with 10 mM Tris buffer, pH 7.5, and thereafter, used as in the glucose procedure described above.
2.4. Flow injection experiments 2.2. Materials The dialysis membrane was regenerated cellulose, MWCO100000, from Spectra/por®, and the 6 m thick polypropylene film was obtained from Bollore Inc. Paris France. The oxygen electrode used in this study is a commercial electrode from Zellweger Analytics, Noisy-le-Grand, France, which can measure dissolved oxygen concentrations in the mg/liter range. This electrode is composed of a gold cathode and silver anode and filled with a 3 M KCl electrolyte. This electrode is polarized at – 0.7 V and is mounted in a proprietary flow cell of the QuikChem 8000® system. The FIA system is controlled by a computer with Omnion FIA software developed by Lachat instruments. Milwaukee USA.
The carrier (buffer) used in all experiments to measure a baseline was Tris-HCl 10 mM, pH 7.5 containing 10 mg/ liter sodium benzoate and 0.1% (w/v) Tween 20. At 20°C and atmospheric pressure this buffer (100% oxygenated) contains 9 mg/liter of oxygen. This buffer was continuously pumped at a flow rate of one liter/hour. The carrier passed through the injection valve and then to the measurement flow cell containing the enzymatic film mounted on the oxygen electrode, then to the waste (Fig. 1). The injection valve can adopt two positions:in the load position the sample to be measured was loaded into the sample loop, and in the inject position the sample present in this loop was injected into the flow cell (detector). After each measurement 100% of oxygenation was achieved when the baseline was obtained (Fig. 2).
A. Haouz, S. Stieg / Enzyme and Microbial Technology 30 (2002) 129 –133
Fig. 2. Peak response of glucose biosensor after injection of standard solutions of glucose. The first three peaks correspond to injection of 10 g/liter of glucose and the last three peaks correspond to injection of 5 g/liter.
3. Results and discussion 3.1. Measurements The 100% oxygenated buffer/carrier formed the baseline for the analysis which gave the largest signal. When the analyte (D-glucose or L-lactate substrate) passed over the dialysis membrane in the flow cell, it reacted with the enzymatic film, and was oxidized by the enzyme, the amount of the dissolved oxygen decreased because oxygen was used during the re-oxidation of the reduced enzyme form. As the analyte left the flow cell, the amount of dissolved oxygen increased again to its baseline concentration. This produced a negative peak whose area was proportional to the concentration of D-glucose or L-lactate substrate. The negative peak was inverted to a positive form by the detector. For each substrate injection the result is the integrated peak area and expressed in V/S (Fig. 2). So that the cycle period of the experiment could be reduced to 120 s, giving a throughput of 30 samples/hour, different MW cutoffs of the dialysis membrane and other immobilization methods were tested. The glucose or lactate oxidases bound on the surface of an affinity membrane (Ultrabind® from Gelman) [18] gave a cycle time period greater than 300 S. The slow rate of diffusion of oxygen and substrate through this membrane appears to be limiting. 3.2. Dosage concentrations of D-glucose and L-lactate To detect the concentration of D-glucose or L-lactate immobilized glucose oxidase or L-lactate oxidase were used as described above. Although the enzymes were covalently immobilized, they can still be characterized by the turnover number, concentration and Michaelis constant (Km) of the enzyme, as is the case for the enzyme free in solution [19]. The differences observed in the corresponding soluble en-
131
zyme is usually due to the diffusional, charge, and boundary layer effects [20] of the substrate to the heterogeneous phase (enzymatic film) According to Michaelis equation the proportionality between the response and the substrate concentration are obtained if the substrate concentration injected is below 1/3 Km. In our study we have not characterized our system by the turnover number or the maximum velocity of the reaction since we are only interested in the linearity range. Our results shown that the linear range of the method depends on the type of loop (large, medium and micro) used in the valve to inject the substrate. Fig. 3 a show the low range:0 to 1 g/liter for the -D glucose and L- lactate. As seen, almost the same response for the two films is observed because the same quantities of enzyme were immobilized. These two enzymes do not support the same quantity of the glutaraldehyde, the reagent used for the covalent immobilization. The glucose oxidase enzyme is not denatured in the presence of glutaraldehyde below 10% (w/v) but the lactate oxidase cannot support a concentration of glutaraldehyde higher than 2%. Also the residual activity of immobilized enzyme depend on others parameters such as the membrane thickness and the volume between active membrane and the pO2 electrode. The best results are obtained with the concentration described in the materials and methods. Fig. 3b shows the two other ranges for the determination of Llactate:1–10 g/liter and 5– 40 g/liter. The response curve in the case of the medium loop is an hyperbola confirming that these enzymes still obey the Michaelis-Menten model. If the concentration of substrate is higher than 40 g/liter, the sample must be diluted in the range. The same results for the -D-glucose are observed. For both substrates’ biosensors, the detection limit is 0.02 g/liter and the relative standard deviation (RDS) is 2%. 3.3. Reproducibility and stability To estimate the repeatability of the measurement, we injected 9.5 g/liter of -D-glucose into the flow cell. Fig. 4 shows that for 318 measurements a Gaussian representation is obtained with the mean at 9.5 g/liter, exactly the same as the injected concentration. The relative standard deviation is 0.1 g/L The immobilized enzymes are more resistant to various denaturation factors (extreme pH and temperature values, high ionic strength, denaturing reagents, proteases, etc) than their corresponding forms in a soluble state. This increase in stability is due to covalent attachment of the enzyme protein, which reduces internal movements which can induce the unfolding of proteins [21]. In our case, the enzymatic films are very stable. After mounting on the electrode they can be used for up to three months, 24 h a day. Any loss of activity can be corrected by the calibration of the FIA system with standards solutions. The calibration frequency is one calibration every week. The high stability of these enzymatic films is due to three factors:(i) if the molecular
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Fig. 4. Response distribution of 318 injections of 9.5 g/liter glucose. The average value is exactly the injected concentration of D-glucose.
3.4. Validation of the FIA system
Fig. 3. Linearity of response with different sample loops. (a) Large loop: (■) glucose, (䊐) L-lactate. (b) large loop (E) and medium loop (F) for the L-lactate biosensor.
enzyme is released from the film, it is still in contact with electrode by dialysis membrane, (ii) the presence of catalase in the enzymatic film hydrolyzes the hydrogen peroxide which can inactivate the enzyme, (iii) in the carrier sodium benzoate was used to minimise any microbial proliferation. The fabricated film with or without catalase, protected by the dialysis membrane, has a lifetime of less than one week. The conservation of the enzymatic film can be achieved by freeze drying. In this state, the film maintains its activity for over one year.
In the bioprocess industry and in many research laboratories, animal cells are cultivated in vitro for the production of different bio-molecules such as proteins having therapeutic value. The cell culture medium includes serum, vitamins, growth factors, minerals, a mixture of amino acid as sources of nitrogen, and glucose as a source of carbon. When the levels of either carbon or nitrogen sources is too high or too low they inhibit growth. There is, therefore, an increasing demand for the analytical methods for monitoring and control of cell cultures. Online control is preferred, since the rapid determination of the glucose and L-lactate level in the culture can be used as a feedback loop for the control of the cell’s productivity [22,23] We used our system to follow the concentration of Dglucose and L-lactate during the growth cycle of E. coli in M9 minimal salts medium (Sigma®) containing 4 g/l of glucose. The concentration of glucose and L-lactate in 5 ml of media was measured during 36 h of growth. The results presented in Table 1 show that the concentration of glucose measured in the media without cells is the same as measured Table 1 Change in concentration of b-D-glucose and L-lactate in the media during the growth of E. coli Samples
[-D-Glucose] g/L ⫾ SD
[L-lactate] g/L ⫾ SD
Media Media Media Media
4.1 ⫾ 0.1 4.0 ⫾ 0.1 3.1 ⫾ 0.1 2.2 ⫾ 0.1
0 0 1.1 ⫾ 0.1 1.8 ⫾ 0.1
before inoculation of E. coli after 1 hour of growth after 24 hours of growth after 36 hours of growth
A. Haouz, S. Stieg / Enzyme and Microbial Technology 30 (2002) 129 –133
in the culture media. During growth a decrease of the concentration of -D-glucose and an increase of the concentration of the L-lactate is observed. We can see also that, during the metabolism of glucose, it is converted finally to the L-lactate. 4. Conclusion The goal of this study was to measure continuously the concentration of D-glucose and L-lactate. Our results show that these metabolites can be on-line monitored every 2 min between 0 to 40 g/liter. The enzyme electrode lasts 3 months and the precision of the measurement is less than 2%. Calibration is done automatically by injecting standard solutions. The data system will prepare a calibration curve by plotting response versus standard concentration. Sample concentration is calculated from the regression equation. The QuikChem FIA system can be used to measure simultaneously these metabolites in a bioprocess and, at the same time, the system can also detect other substances like ammonia, phosphate, without any perturbation of the biosensors. References [1] Gough DA, Andrade JD. Enzyme electrodes. Science 1973;180:380– 4. [2] Turner APF. Biosensors sense and sensitivity. Science 2000;290: 1315–7. [3] Silman I, Katchlski E. Water-insoluble derivatives of enzymes, antigens, and antibodies. Annu Rev Biochem 1966;35:873–908. [4] Wingard LB. Enzyme Engineering, New York: Interscience, 1972. [5] Wilson R, Turner APF. Glucose oxidase:an ideal enzyme. Biosens Bioelectron 1992;7:165– 85. [6] Danielsson B, Mosbach K. Analytical applications with emphasis on biosensors. Introduction Methods Enzymol 1988;137:3–14. [7] Luong JHT, Bouvrette P, Male KB. Developments and applications of biosensors in food analysis. Trends Biotechnol 1997;15:369 –77. [8] Guilbault GG. Enzyme electrode probes. Methods Enzymol 1988; 137:14 –29.
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