Biosensor for Asparagine Using a Thermostable Recombinant

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Anal. Chem. 2002, 74, 3336-3341

Biosensor for Asparagine Using a Thermostable Recombinant Asparaginase from Archaeoglobus fulgidus Ju Li, Jianquan Wang, and Leonidas G. Bachas*

Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055

Asparaginase from the hyperthermophilic microorganism Archaeoglobus fulgidus was cloned and expressed in Escherichia coli as a fusion protein with a polyhistidine tail. After heat treatment to denature most of the native E. coli proteins, the enzyme was purified by an immobilized metal ion affinity chromatography method. The activity of the enzyme was determined by monitoring the change in ammonium concentration in solution. It was found that the enzyme is thermostable at temperatures as high as 85 °C. The KM for L-asparagine was 8 × 10-5 and 5 × 10-6 M at 37 and 70 °C, respectively. The catalytic activity for L-asparagine was 5-fold higher than for D-asparagine. The enzyme was immobilized in front of an ammonium-selective electrode and used to develop a biosensor for asparagine. The biosensor had a detection limit of 6 × 10-5 M for L-asparagine. Unlike a sensor based on asparaginase from E. coli, the biosensor based on recombinant asparaginase from A. fulgidus demonstrated higher stability. Thermophilic organisms live under extreme physicochemical conditions. These microorganisms provide a valuable source for thermostable enzymes that can potentially lead to new advances in biosensors and biocatalysis. Unlike their counterparts from mesophilic organisms, thermostable enzymes can be stored at room temperature for long periods of time without significant loss in their activities.1,2 Another advantage of these enzymes is that they are typically resistant to many chemical denaturants.3 Applications of thermostable enzymes include the polymerase chain reaction (PCR), which was developed with the help of thermostable enzymes including the Taq polymerase from Thermus aquaticus,4 starch bioconversion using enzymes such as R-amylase,5 high-efficiency degradation of cellulose by cellulases,6 and production of high-fructose corn syrup by using glucose isomerases,7 whereas enzymes from mesophilic organisms find limited, if any, use in these temperature-demanding processes. * To whom correspondence should be addressed, Phone: (859) 257-6350. Fax: (859) 323-1069. E-mail: [email protected]. (1) Zentgraf, B.; Ahern, T. Pure Appl. Chem. 1991, 63, 1527-1540. (2) Vieille, C.; Zeikus, J. TIBTECH 1996, 14, 183-190. (3) Cowan, D. A. Essays Biochem. 1995, 29, 193-207. (4) Erlish, H. A.; Gelfand, D. H.; Saiki, R. K. Nature 1988, 331, 461-462. (5) Koch, R.; Spreinat, K.; Lemke, K.; Antranikian, G. Arch. Microbiol. 1991, 155, 572-578. (6) Teeri, T. T.; Koivula, A.; Linder, M.; Wohlfahrt, G.; Divne, C.; Jones, T. A. Biochem. Soc. Trans. 1998, 26, 173-178.

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Enzyme-based biosensors are analytical devices that combine a biological recognition element with a signal transducer. Their response mechanism involves the catalytic transformation of substrates to products, which can be detected by the transducer. One of the limitations of enzyme-based biosensors is the low stability of the enzymes. Enzymes from thermophilic organisms are more stable than those from mesophiles and should result in more stable biosensors. Further, the use of enzymes with high thermostability can improve the applicable temperature range of these biosensors. To date, there have only been a few reports on thermostable biosensors.8-14 Determination of asparagine is very important in food chemistry and biochemistry. For example, the concentration of asparagine can be used as an indicator of freshness for vegetables.15 It can also be used to detect the ripening of certain cheeses.16,17 Asparagine is a good nitrogen source in fermentation reactions.18 Further, some types of tumor cells require L-asparagine as an essential amino acid for protein synthesis. Methods have been developed to prevent these cancerous cells from growing by depleting asparagine from their environment. There are several ways to detect asparagine, such as column chromatography,19 radiometric assays,20 electron spin resonance spectroscopy,21 and enzyme electrodes.22-24 (7) Liu, S. Y.; Wiegel, J.; Gherardini, F. C. J. Bacteriol. 1996, 178, 5938-5945. (8) Rella, R.; Ferrara, D.; Barison, G.; Doretti L.; Lora, S. Biosens. Bioelectron. 1997, 12(5), iii. (9) Kenausis, G.; Chen, Q.; Heller, A. Anal. Chem. 1997, 69, 1054-60. (10) Pasco, N.; Jeffries, C.; Davies, Q.; Downard, A. J.; Roddick-Lanzilotta, A. D.; Gorton, L. Biosens. Bioelectron. 1999, 14, 171-178. (11) Jeffries, C.; Pasco, N.; Baronian, K.; Gorton, L. Biosens. Bioelectron. 1997, 12, 225-232. (12) Metzger, J.; Reiss, M.; Hartmeier, W. Biosens. Bioelectron. 1998, 13, 10771082. (13) Antiochia, R.; Cass, A. E. G.; Palleschi, G. Anal. Chim. Acta 1997, 345, 17-28. (14) D’Auria, S.; Di Cesare, N.; Gryczynski, Z.; Gryczynski, I.; Rossi, M.; Lakowicz, J. R. Biochem. Biophys. Res. Commun. 2000, 274, 727-31. (15) Hurst, P.; Boulton, G.; Lill, R. Food Chem. 1998, 61, 381-384. (16) Izco, J. M.; Torre, P.; Barcina, Y. Food Control 2000, 11, 7-11. (17) Izco, J. M.; Irigoyen, A.; Torre, P.; Barcina, Y. Food Control 2000, 11, 201207. (18) Stetter, K. O. Nature 1982, 300, 258-260. (19) Carlson, A. D.; Riggin, R. M. Anal. Biochem. 2000, 278, 150-155. (20) Sheng, S.; Kraft, J. J.; Schuster, S. M. Anal. Biochem. 1993, 211, 242-249. (21) Jung, K.; Branciamore, S.; Martini, G. Biochim. Biophys. Acta 2000, 1519, 1-5. (22) Tagami, S.; Maisuda, K. Chem. Pharm. Bull. 1990, 38, 153-155. (23) Stein, K.; Shi, R.; Schwedt, G. Anal. Chim. Acta 1996, 336, 113-122. (24) Cullen, D. C.; Sethi, R. S.; Lowe, C. R. Anal. Chim. Acta 1990, 231, 33-40. 10.1021/ac015653s CCC: $22.00

© 2002 American Chemical Society Published on Web 05/24/2002

Here, we report the cloning of the gene for a thermostable asparaginase from the thermophilic microorganism Archaeoglobus fulgidus and characterization of the recombinant enzyme. A. fulgidus VC-16 is the first sulfur-metabolizing organism to have its genome sequence determined.25,26 The microorganism can be found in subsurface oil field and hydrothermal environments. It can grow between 60 and 95 °C, with the optimal growth temperature at 83 °C. Based on bioinformatic analysis, the genomic DNA of A. fulgidus contains a gene that putatively codes for asparaginase (asnA). In the present work, this gene was isolated by PCR, cloned, and expressed in Escherichia coli as a fusion protein with a polyhistidine tag. A potentiometric method was employed to detect the enzyme-catalyzed reaction by monitoring the change in ammonium concentration. The thermostable recombinant asparaginase was used along with an ammoniumselective electrode to develop a biosensor for asparagine. To our knowledge, this is the first potentiometric biosensor using a thermostable enzyme. EXPERIMENTAL SECTION Reagents. Sodium phosphate was purchased from Fisher Scientific (Pittsburgh, PA). Tris(hydroxymethyl)aminomethane (Tris) was bought from Research Organics (Cleveland, OH). Luria Bertani (LB) broth and LB agar were obtained from Becton Dickinson (Sparks, MD). Imidazole, phenylmethanesulfonyl fluoride (PMSF), L-arabinose, L- and D-asparagine, L-glutamine and E. coli asparaginase were from Sigma (St. Louis, MO). TaqPlus precision polymerase was from Stratagene (La Jolla, CA). PstI and EcoRI restriction enzymes were purchased from Life Technologies (Rockville, MD). PCR primers were custom synthesized by Operon Technologies (Alameda, CA). Talon metal affinity gel was bought from Clontech Laboratories (Palo Alto, CA). The pBADTOPO kit and TOPO 10 competent cells were purchased from Invitrogen (Carlsbad, CA). Apparatus. The Accumet pH Meter (model 915), Sonic Dismembrator (model 550), and Isotemp refrigerated circulator (model 9500) were purchased from Fisher Scientific (Pittsburgh, PA). The ammonium-selective glass electrode GT-DJ-NH4 was bought from Innovative Sensors (Anaheim, CA). Centriprep-10 concentrators (10 000 MW cutoff) were obtained from Millipore (Bedford, MA). The SDS-PAGE PhastSystem was from Pharmacia Biotech (Uppsala, Sweden). The GeneAmp PCR system 2400 was from Perkin-Elmer (Norwalk, CT), and the DNA electrophoresis system EC105 was purchased from E-C Apparatus (Holbrook, CA). Construction of Cloning Vector. The genomic DNA of A. fulgidus was kindly provided by K. O. Stetter, Germany. A pair of oligonucleotide primers was used for the amplification of the asnA (25) Klenk, H. P.; Clayton, R. A.; Tomb, J. F.; White, O.; Nelson, K. E.; Ketchum, K. A.; Dodson, R. J.; Gwinn, M.; Hickey, E. K.; Peterson, J. D.; Richardson, D. L.; Kerlavage, A. R.; Graham, D. E.; Kyrpides, N. C.; Fleischmann, R. D.; Quackenbush, J.; Lee, N. H.; Sutton, G. G.; Gill, S.; Kirkness, E. F.; Dougherty, B. A.; McKenney, K.; Adams, M. D.; Loftus, B.; Peterson, S.; Reich, C. I.; McNeil, L. K.; Badger, J. H.; Glodek, A.; Zhou, L.; Overbeek, R.; Gocayne, J. D.; Weidman, J. F.; McDonald, L.; Utterback, T.; Cotton, M. D.; Spriggs, T.; Artiach, P.; Kaine, B. P.; Sykes, S. M.; Sadow, P. W.; D’Andrea, K. P.; Bowman, C.; Fujii, C.; Garland, S. A.; Mason, T. M.; Olsen, G. J.; Fraser, C. M.; Smith, H.; Woese, C.; Venter, J. C. Nature 1997, 390, 364-370. (26) Stetter, K. O.; Huber, R.; Blochl, E.; Kurr, M.; Eden, R. D.; Fielder, M.; Cash, H.; Vance, I. Nature 1993, 365, 743-745.

Figure 1. Plasmid pBAD-ASN carrying the asparaginase gene asnA. EcoRI and PstI denote sites for the corresponding restriction enzymes, PBAD is the promoter, and PolyHis refers to nucleotides coding for a six-histidine tag. The stop codon is after the PolyHis sequence.

gene: forward 5′-CTG CAG ATG AGG CCA AAA AGC TGG GGG3′ and reverse 5′-GAA TTC ATA CTG TGT CGT CGG CTC TAT3′. The boldface sequences are the cutting sites for the restriction enzymes PstI and EcoRI, respectively. The genomic DNA was first denatured at 96 °C for 5 min. Then, the PCR amplification was carried out using TaqPlus precision polymerase for 25 cycles with denaturation at 95 °C for 50 s, annealing at 55 °C for 60 s, and polymerization at 72 °C for 150 s with a final extension step at 72 °C for 10 min. After amplification, the PCR product was cloned into the pBADTOPO vector to yield the pBAD-ASN vector (Figure 1), which produced a histidine-tagged asparaginase upon expression. The fusion protein contains 16 additional amino acids at the Nterminus. The expressed fusion protein is referred to as recombinant asparaginase throughout the text. The pBAD-ASN vector was transformed into competent E. coli TOPO 10 cells. The E. coli cells were grown overnight at 37 °C on LB-agar plates supplemented with 100 µg/mL ampicillin. Colonies were selected from the plates and grown again overnight in LB-broth solution with 100 µg/mL ampicillin. The plasmid DNA containing the PCR product was minipreped from the E. coli cells and confirmed by using the two restriction enzymes, PstI and EcoRI. The plasmid DNA was sequenced to verify the inserted gene for asparaginase with the forward and reverse sequencing primers provided with the pBAD-TOPO kit at the Macromolecular Structural Analysis Facility at the University of Kentucky. Preparation of Enzyme. To obtain the recombinant asparaginase, E. coli cells harboring the pBAD-ASN vector were grown in LB medium supplemented with 100 µg/mL ampicillin at 37 °C. When the absorbance at 600 nm reached 0.5, L-arabinose was added into the culture at 0.02% (w/v) to induce the expression of recombinant asparaginase. The cells were grown for another 6 h. The culture was centrifuged at 8000g for 10 min. The paste was dissolved in 50 mM Tris-HCl buffer, pH 7.5, and the protease inhibitor PMSF (1 mM) was added. The solution was sonicated for 10 min to disrupt the cells, followed by centrifugation at 8000g for 10 min. The supernatant was incubated at 70 °C for 5 min to Analytical Chemistry, Vol. 74, No. 14, July 15, 2002

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denature native E. coli proteins in the solution. The protein solution was dialyzed against 50 mM sodium phosphate buffer, pH 7.0, overnight. The dialyzed solution was passed through a Talon metal affinity column to separate the recombinant asparaginase. After loading, the column was washed with 5 mM imidazole in 50 mM sodium phosphate, pH 7.0. The enzyme was eluted from the column with 150 mM imidazole in 50 mM sodium phosphate, pH 7.0. The eluted fractions were tested with SDSPAGE to confirm protein purity. The protein solution was dialyzed against 50 mM Tris-HCl buffer, pH 7.0, overnight, and then concentrated with a Centriprep-10 concentrator. Enzyme Assay. A buffer solution with 50 mL of Tris-HCl (pH 9.2) was added to a thermostated jacketed beaker. The solution was stirred for 10 min to allow equilibration at a set temperature. Then, 100 µL of 0.100 M L-asparagine and 20 µL of asparaginase solution were added to the chamber. Deamination of asparagine resulted in the formation of ammonium ions, which were monitored by the ammonium-selective glass electrode. The initial rate of the deamination reaction was determined by the potential drop over the first 30 s and expressed as the asparaginase activity (one unit of enzyme can generate 1.0 mmol of ammonium nitrogen from L-asparagine per min at pH 8.6 and 37 °C). Asparagine Biosensor. Asparaginase from E. coli and A. fulgidus was used to assemble two biosensors for asparagine. A solution of 10 µL of asparaginase in 50 mM Tris-HCl, pH 9.2, was placed behind a dialysis membrane (12 000-14 000 MW cutoff) that was held to the bottom of the ammonium-selective glass electrode with a rubber O-ring. The sensors were placed in a solution of the same Tris-HCl buffer. L-Asparagine solutions were used to calibrate the biosensors at 37 and 70 °C. The selectivity of the biosensor was tested using L-glutamine and D-asparagine as substrates. The stability of the biosensors was evaluated in TrisHCl, pH 9.2, at 37 °C. RESULTS AND DISCUSSION Bioinformatic Analysis. The results of a BLAST homology search indicated that the putative asnA gene from A. fulgidus has a 30% identity to the asparaginase gene from E. coli.25 Further, a comparison of the amino acid sequences of asparaginases from E. coli, Helicobacter pylori, Methanobacterium thermoautotrophicum, and the putative one from A. fulgidus was made. The sequences were aligned using the Pileup analysis program.27 Among the consensus amino acids found in all sequences, residues 89-93 of the enzyme from E. coli (corresponding to residues 164-168 of the enzyme from A. fulgidus) are located in the active site of the enzyme.28-30 Because these residues are conserved in the A. fulgidus sequence, the corresponding gene was putatively assigned to an asparaginase gene. Kinetic studies of mutants of E. coli asparaginase confirmed that residues Thr91 and Asp-92 are required for the activity of the enzyme.31,32 (27) Feng, D. F.; Doolittle, R. F. J. Mol. Evol. 1987, 25, 351-360. (28) Peterson, R. G.; Richards, F. F.; Handschumacher, R. J. Biol. Chem. 1977, 252, 2072-2076. (29) Swain, A. L.; Jaskolski, M.; Housset, D.; Rao, J. K.; Wlodawer, A. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 1474-1478. (30) Palm, G. J.; Lubkowski, J.; Derst, C.; Schleper, S.; Ro¨hm, K. H.; Wlodawer, A. FEBS Lett. 1996, 390, 211-216. (31) Ro ¨hm, K. H.; Van Etten, R. L. Arch. Biochem. Biophys. 1986, 244, 128136. (32) Derst, C.; Henseling, J.; Ro ¨hm, K. H. Protein Eng. 1992, 5, 785-789.

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Plasmid Construction and Protein Expression. PstI and EcoRI were selected as the restriction endonucleases to clone asnA because there are no recognition sites for these two enzymes in the asnA gene of A. fulgidus. These two cutting sites were incorporated into the forward and reverse primers used for PCR amplification. Because the genomic DNA was from a thermophilic microorganism, it was first heated at 96 °C (higher than the typical temperatures used in PCR of DNA from mesophilic organisms) for 5 min to completely denature the genomic DNA before entering the PCR cycle. Using DNA electrophoresis, it was found that the PCR product contained two major bands when the annealing temperature was under 50 °C. One of these bands was ∼1200 bp, which is the right size for asnA, the other was ∼1000 bp. Experiments were also carried out with the annealing temperature above 50 °C. It was found that when the annealing temperature was 55 °C, the band around 1000 bp disappeared while the band at 1200 bp remained on the DNA electrophoresis gel. If the annealing temperature was increased to higher than 55 °C, there was no PCR product. Thus, 55 °C is an appropriate temperature for the annealing step. The PCR product was used to construct the plasmid pBAD-ASN. DNA sequencing confirmed the presence of the asnA gene in the plasmid. pBAD-ASN was designed to contain a DNA segment that codes for a six-histidine peptide at the C-terminus of asparaginase. The plasmid contains the PBAD promoter upstream from the asnA gene, which allows heterologous gene expression in the presence of L-arabinose. Therefore, asparaginase was expressed under arabinose induction as a fusion protein with a polyhistidine tag. Because the asparaginase is from a thermophilic microorganism, the cell extract was first heated to 70 °C for 5 min to denature most of the native E. coli proteins. This heat treatment method, which simplifies purification, can be used as a first step to separate thermostable proteins expressed in mesophilic organisms.33,34 The polyhistidine tag allowed subsequent purification of the recombinant enzyme using immobilized metal affinity chromatography. The protein was eluted from the column with a solution containing 150 mM imidazole and 50 mM sodium phosphate at pH 7.0. The fractions containing high asparaginase activity were pooled. SDSPAGE analysis of the pooled fractions gave one major band at ∼55 kDa, which is close to the molecular mass of the asparaginase construct (52 kDa). This purified protein was then dialyzed against 50 mM Tris-HCl, pH 9.2, and concentrated. Enzyme Characterization. There are different types of electrodes for detecting ammonium ion or ammonia gas. One of them is the ammonia gas sensor, which is not appropriate for use above 50 °C.35 From the ammonium-selective electrodes, those that are based on plastic membranes are also limited to the same temperature range. However, ammonium-selective glass electrodes are operational at higher temperatures up to 135 °C.36 Therefore, the glass electrode was selected to determine the activity of the thermostable recombinant asparaginase, although it is less selective than the ammonia gas sensor. High concentra(33) Verhagen, M. F.; O’Rourke, T. W.; Menon, A. L.; Adams, M. W. Biochim. Biophys. Acta 2001, 1505, 209-219. (34) Martino, A.; Schiraldi, C.; Fusco, S.; Di Lernia, I.; Costabile, T.; Pellicano, T.; Marotta, M.; Generoso, M.; van der Oost, J.; Sensen, C. W.; Charlebois, R. L.; Moracci, M.; Rossi, M.; De Rosa, M. J. Mol. Catal. B 2001, 11, 787794. (35) ThermoOrion, Laboratory Products Catalog, 2001, p 49. (36) Innovative Sensors, Model GT-DJ-NH4 Product Information.

Figure 2. Effect of pH on recombinant asparaginase activity (in mmol of ammonium nitrogen/min). 50 mM Tris-HCl, 2 mM asparagine, and 0.4 unit of enzyme were used for each condition. Data represent average ( standard deviation (n ) 3).

Figure 3. Initial rate of the catalytic reaction as a function of asparagine concentration at 70 °C. The initial reaction rate (in mmol of ammonium nitrogen/min) was determined by the change in the potential of the ammonium-selective electrode over the first 30 s. The enzyme amount used in each reaction was 0.4 unit. Data represent average ( standard deviation (n ) 3).

tions of cations such as potassium and sodium are typical interferences of ammonium glass electrodes.37 To avoid interferences when this electrode was used to monitor enzyme activity, the assay buffer for asparaginase was free of potassium and sodium. The pH effect on enzyme activity was evaluated using a solution containing 0.4 unit of purified enzyme and 2 mM L-asparagine (Figure 2). The initial rate of the reaction was measured at 70 °C. The maximum rate occurred around pH 9.2, while the optimal pH condition for the E. coli enzyme is 8.6.22 Therefore, pH 9.2 was selected for all subsequent experiments. It should be noted that at each pH the recorded electrode potential was converted to total amount of ammonium nitrogen through a calibration plot; the latter was used to calculate the initial rate. (37) Bailey, P. L. Analysis with Ion-Selective Electrodes; Heyden & Son: London, 1976; Chapter 4.

Figure 4. Effect of 5-min exposure at different temperatures on recombinant asparaginase activity from A. fulgidus. The residual activity was converted to the percentage of activity at 70 °C.

Figure 5. Recombinant asparaginase stability as a function of storage time. The enzyme was stored in 50 mM Tris-HCl, pH 9.2, at room temperature. The residual activity was converted to the percentage of the initial enzyme activity. Data represent average ( standard deviation (n ) 3).

To determine the KM for L-asparagine, the enzyme activity was measured at different concentrations of the substrate. For that, the buffer (Tris-HCl, pH 9.2) was equilibrated in the reaction chamber for 10 min to reach the desired temperature. LAsparagine was added to the solution and allowed to equilibrate for an additional 5 min before the enzyme solution (0.4 unit) was added. The changes in potential (at different concentrations of substrate) were monitored by the ammonium-selective electrode (Figure 3). The KM was found to be 5 × 10-6 and 8 × 10-5 M at 70 and 37 °C, respectively. The KM at 70 °C was lower than the value at 37 °C, indicating a stronger affinity for the substrate at the higher temperature. As a comparison, the KM of the enzyme from E. coli was 1.2 × 10-5 M at 37 °C.29 Thermostability of Recombinant Asparaginase. To study the thermostability of recombinant asparaginase from A. fulgidus, enzyme solutions (0.4 unit) were first incubated at temperatures between 70 and 95 °C for 5 min. Then, the solutions were immediately transferred to an ice bath to stop denaturation of the enzyme, and the residual enzyme activity in each of the solutions was determined at 37 °C. Figure 4 shows the residual enzyme Analytical Chemistry, Vol. 74, No. 14, July 15, 2002

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Figure 6. Comparison of the thermostability of asparaginases from A. fulgidus and E. coli. The enzyme activities were measured at 37 °C, and the incubation temperature was 70 °C. Table 1: KM Values of A. fulgidus Recombinant Asparaginase for Different Substrates

Figure 7. Calibration plots of the biosensor for L-asparagine in 50 mM Tris buffer (pH 9.2) at 70 °C using the response at 2 or 10 min after addition of the substrate. Data represent average ( standard deviation (n ) 3). The inset is a typical response at different L-asparagine concentrations: (a) 2.0 × 10-5, (b) 1.0 × 10-4, (c) 5.0 × 10-4, (d) 1.0 × 10-3, (e) 5.0 × 10-3, and (f) 1.0 × 10-2 M.

KM, mM temp, °C

L-asparagine

D-asparagine

L-glutamine

37 70

0.08 0.005

1.8 1.2

0.35 0.20

activity as a percentage of the activity at 70 °C. Recombinant asparaginase retained 70% of its activity after 5-min incubation at 85 °C, and ∼10% of its activity at 95 °C. This is consistent with the optimal growth temperature of A. fulgidus, which is 83 °C. Also, the recombinant asparaginase retained ∼90% of enzyme activity after a four-month storage at room temperature in 50 mM Tris-HCl, pH 9.2 (Figure 5). The stability of recombinant asparaginase from A. fulgidus was also compared with that of asparaginase from E. coli. In this study, both enzymes were subjected to heat treatment for a set period of time at 70 °C. Then, the enzyme activities were measured at 37 °C for each incubation time. The asparaginase from E. coli lost most of its activity after 2-min incubation at 70 °C, while recombinant asparaginase from A. fulgidus showed much higher activity at the same temperature (Figure 6). Substrate Selectivity of Recombinant Asparaginase. LAsparaginases typically demonstrate some catalytic activity for D-asparagine and L-glutamine in addition to their preferred substrate, L-asparagine.38-40 The selectivity of recombinant asparaginase from A. fulgidus for D- and L-asparagine was evaluated at 37 and 70 °C using 0.2 unit of the enzyme. It was found that the enzyme has 5-fold higher activity for L- than for D-asparagine. Further, the enzyme has higher affinity (lower KM) for Lasparagine than for D-asparagine (Table 1). Thus, the enzyme is more selective for L-asparagine than for D-asparagine. Also, the recombinant asparaginase has 4-fold higher activity for L-aspar(38) Distasio, J. A.; Niederman, R. A.; Kafkewitz, D.; Goodman, D. J. Biol. Chem. 1976, 251, 6929-6933. (39) Aghaiypour, K.; Wlodawer, A.; Lubkowski, J. Biochemistry 2001, 40, 56555664. (40) Howard, J. B.; Carpenter, F. H. J. Biol. Chem. 1972, 247, 1020-1030.

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Figure 8. Biosensor stability as a function of storage time. The biosensors were stored in 50 mM Tris buffer, pH 9.2, at 37 °C. The residual activity was converted to the percentage of enzyme activity at 0 h: sensor using asparaginase from E. coli (9), sensor using A. fulgidus asparaginase ([).

agine than for L-glutamine. In Table 1, the KM values for L-glutamine were 0.35 and 0.20 mM at 37 and 70 °C, respectively. These values are much larger than those for L-asparagine. As a comparison, the asparaginase from E. coli has 3-4% glutaminase activity compared to its asparaginase activity.41 Asparagine Biosensor. The A. fulgidus recombinant asparaginase was used to develop a biosensor for asparagine that can function at high temperature. In this sensor, the enzyme was trapped behind a semipermeable membrane in front of the ammonium-selective electrode. The sensor was calibrated by adding different concentrations of L-asparagine to Tris-HCl, pH 9.2, at 70 °C (Figure 7). The biosensor has a detection limit of 6 × 10-5 M for L-asparagine. Thus, it could be useful in the determination of asparagine in real samples such as cheese, juices, and asparagus.15,16,42 Calibration plots were prepared using the (41) Arens, A.; Rauenbusch, E.; Irion, E.; Wagner, O.; Bauer, K.; Kaufmann, W. Hoppe Seylers Z. Physiol. Chem. 1970, 351, 197-212.

Figure 9. Response of the biosensor to L-asparagine ([), Lglutamine (9), and D-asparagine (2). Measurements were performed in Tris-HCl, pH 9.2, at 37 °C. Data represent average ( standard deviation (n ) 3).

response obtained 2 or 10 min after addition of the substrate. The plot using the signal after 10 min has higher sensitivity than the 2-min plot. However, in cases where a faster assay is necessary, the 2-min plot can be used for analysis. In addition to being functional at high temperatures, biosensors based on thermophilic enzymes should be quite stable because of the inherent stability of proteins from thermophilic organisms. (42) Stein, K.; Shi, R.; Schwedt, G. Anal. Chim. Acta 1996, 336, 113-122.

To establish this, the storage stability of the biosensor was evaluated at 37 °C. As a comparison, a biosensor based on the mesophilic asparaginase from E. coli was assembled in the same manner. As shown in Figure 8 the biosensor based on the mesophilic enzyme lost 90% of its activity after 96 h, while the biosensor based on the thermophilic enzyme retained more than 80% of its activity during the same period of time. The selectivity of the biosensor based on thermophilic recombinant asparaginase was tested using L-glutamine and D-asparagine (Figure 9). The biosensor has higher sensitivity and lower detection limit for L-asparagine. The sensitivity of the biosensor was lower for L-glutamine and even lower for D-asparagine. This is consistent with the data obtained in solution (see above). In conclusion, the gene for asparaginase from the thermophilic microorganism A. fulgidus was cloned and expressed as a recombinant protein in E. coli. This enzyme can tolerate temperatures as high as 85 °C. The enzyme maintains part of its activity at room temperature. The thermostable recombinant asparaginase was coupled with an ammonium-selective electrode to develop a biosensor for asparagine of increased stability compared to a sensor based on an enzyme from a mesophilic microorganism. ACKNOWLEDGMENT We thank the National Aeronautics and Space Administration (NASA) for funding this research. The authors thank K. O. Stetter for providing the genomic DNA of A. fulgidus. Received for review October 22, 2001. Accepted April 17, 2002. AC015653S

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