- Email: [email protected]
Purification and Characterization of Thermostable Aspartase fromBacillussp. YM55-1
Archives of Biochemistry and Biophysics Vol. 366, No. 1, June 1, pp. 40 – 46, 1999 Article ID abbi.1999.1186, available online at http://www.idealibrary.com on
Purification and Characterization of Thermostable Aspartase from Bacillus sp. YM55-1 Yasushi Kawata,* ,1 Koichi Tamura,* Shigeru Yano,† Tomohiro Mizobata,* Jun Nagai,* Nobuyoshi Esaki,‡ Kenji Soda,‡ ,2 Masanobu Tokushige,† and Noboru Yumoto§ *Department of Biotechnology, Faculty of Engineering, Tottori University, Tottori 680-0945, Japan; †Department of Chemistry, Faculty of Science, Kyoto University, Kyoto 606-8224, Japan; ‡Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan; and §Osaka National Research Institute, AIST, Ikeda, Osaka 563-8577, Japan
Received December 4, 1998, and in revised form February 2, 1999
A thermostable aspartase was purified from a thermophile Bacillus sp. YM55-1 and characterized in terms of activity and stability. The enzyme was isolated by a 5-min heat treatment at 75°C in the presence of 11% (w/v) ammonium sulfate and 100 mM aspartate, followed by Q-Sepharose anion-exchange and AF-Red Toyopearl chromatographies. The native molecular weight of aspartase determined by gel filtration was about 200,000, and this enzyme was composed of four identical monomers with molecular weights of 51,000 determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Unlike Escherichia coli aspartase, the enzyme was not activated by the presence of magnesium ion at alkaline pH. At the optimum pH, the K m and V max were 28.5 mM and 700 units/mg at 30°C and 32.0 mM and 2200 units/mg at 55°C, respectively. The specific activity was four and three times higher than those of E. coli and Pseudomonas fluorescens enzymes at 30°C, respectively. Eighty percent of the activity was retained after a 60-min incubation at 55°C, and the enzyme was also resistant to chemical denaturants; 80% of the initial specific activity was detected in assay mixtures containing 1.0 M guanidine hydrochloride. The purified enzyme shared a high sequence homology in the N-terminal region with aspartases from other organisms. © 1999 Academic Press Key Words: aspartase; aspartate ammonia-lyase; thermophile; purification; characterization; Bacillus species.
1 To whom correspondence should be addressed. Fax: 181– 857– 31–5271. E-mail: [email protected]. 2 Present address: Faculty of Engineering, Kansai University, Suita, Osaka 564-0073, Japan.
40
Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) catalyzes the reversible conversion of L-aspartate to fumarate and ammonium ion and plays an important role in the bacterial nitrogen metabolism. The enzyme has been purified from Escherichia coli (1–3), Pseudomonas fluorescens (4, 5), and Bacillus subtilis (6), and each gene has been cloned and sequenced. Aspartases from E. coli and P. fluorescens were found to have a molecular mass of about 200 kDa and to be composed of four identical 50-kDa subunits. Various studies on the structure–function relationship of E. coli aspartase have been reported (7–13). In particular, the recent elucidation of the three-dimensional structure of aspartase from E. coli (14) provides detailed structural characteristics of this enzyme family, which can be used in further mechanistic studies. Aspartase has been used for the industrial production of L-aspartate from fumarate, which, in turn, is an ingredient in the chemical synthesis of the artificial sweetener aspartame (N-L-a-aspartyl-L-phenylalanine1-methyl ester). In industrial applications, enzymes having stable structures and high catalytic activities are generally preferred. However, although the enzymatic production of L-aspartate using a thermophilic bacterium was reported previously (15), detailed characteristics of a thermostable aspartase have not yet been reported. In our laboratory, we have been studying various thermostable enzymes such as fumarase (16) and catalase (17), which are derived from thermophilic Thermus strains. As part of this effort to increase our understanding of the basis for enzymatic activity and structural stability in thermostable enzymes, we report the purification and characterization of an aspartase from a thermostable bacterium, Bacillus sp. YM55-1. The purified enzyme shared a high sequence homology with various other bacterial aspartases, but 0003-9861/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
PURIFICATION AND CHARACTERIZATION OF THERMOSTABLE ASPARTASE
was much more resistant to thermal and chemical denaturation. MATERIALS AND METHODS Materials. Gdn-HCl 3 (finest grade) was obtained from Nacalai Tesque (Kyoto). Meat extract was obtained from Kyokuto Pharmaceuticals (Tokyo). Q-Sepharose and AF-Red Toyopearl resin were obtained from Pharmacia Biotech (Uppsala, Sweden) and Tosoh Corporation (Tokyo), respectively. Molecular weight standard proteins for gel filtration and SDS–PAGE were purchased from Oriental Yeast Company (Osaka) and Sigma (St. Louis, MO), respectively. Bovine serum albumin was obtained from Sigma. All other reagents were obtained from Wako Fine Chemicals (Osaka) or Nacalai Tesque and used without further purification. Aspartase from E. coli was purified according to the methods reported previously (10). Cultivation of Bacillus sp. YM55-1. Cultivation of Bacillus sp. YM55-1 was performed aerobically in thermophilic bacterial medium (5 g/liter yeast extract, 5 g/liter polypeptone, 2 g/liter meat extract, 2 g/liter glycerol, 5 g/liter sodium L-aspartate, 2 g/liter NaCl, 2 g/liter K 2HPO 4, 2 g/liter KH 2PO 4, 0.1 g/liter MgSO 4 z 7H 2O, and 4 mg/liter biotin at pH 7.2) overnight at 55°C. Enzyme assays and determination of protein concentrations. Aspartase activity was routinely assayed at 30°C by measuring the increase in absorption at 240 nm caused by the production of fumarate. A Hitachi 200 or U-2000A spectrophotometer equipped with a Peltier-type cell temperature control unit (SPR-10) was used in all measurements. The standard assay for activity was performed in 50 mM Taps–NaOH buffer, pH 8.5, containing 0.1 M sodium L-aspartate, 2 mM MgCl 2, and an appropriate quantity of enzyme preparation in a total volume of 1 ml at 30°C. One unit of aspartase was defined as the amount of enzyme that produced 1 mmol of fumarate in 1 min. The molar coefficient of 2.53 3 10 3 M 21 cm 21 at 240 nm for fumarate was used (18). Protein concentration was determined with a protein dye reagent (Protein Assay Kit, Bio-Rad Laboratories, Richmond, CA) using bovine serum albumin as a standard. Purification of aspartase from Bacillus sp. YM55-1. All purification steps were performed at 4°C. Cells (42.1 g wet wt) were suspended in 120 ml of 50 mM potassium phosphate buffer, pH 6.8, containing 0.1 M KCl, 1 mM EDTA, and 5 mM b-mercaptoethanol (Buffer A), and lysed by sonication. After removal of all debris by centrifugation and nucleic acid precipitation using 2.5% streptomycin, the supernatant was fractionated with ammonium sulfate. The fraction corresponding to 35–50% saturated ammonium sulfate was collected, resuspended in Buffer A, and dialyzed extensively against Buffer A. Ammonium sulfate (final concentration: 11% w/v) and 0.1 M sodium L-aspartate were added to the dialyzate, and the mixture was heated at 75°C for 5 min and immediately cooled on ice. Soluble proteins that remained after heat treatment were dialyzed extensively against Buffer B (50 mM potassium phosphate buffer, pH 7.2, containing 1 mM EDTA and 5 mM b-mercaptoethanol). After dialysis, the sample was subjected to a Q-Sepharose anion-exchange column (f 16 3 600 mm) that had been equilibrated with Buffer B, and was eluted with a linear concentration gradient of KCl (0 –1 M). Fractions containing aspartase activity were concentrated using ultrafiltration (Amicon PM30 membrane filter) and the buffer was changed to Buffer C [50 mM Hepes–Tris buffer, pH 6.6, containing 30 mM Mg(CH 3COO) 2, 5 mM b-mercaptoethanol, and 40% (v/v) ethylene glycol] by passage through a PD-10 (Sephadex G-25; Pharmacia) desalting column. The sample was then applied to an AF-Red Toyopearl column (f 22 3 110 mm) equilibrated in Buffer C. After
41
the column was washed with 150 ml of Buffer C containing 0.5 mM NAD 1 and 0.5 mM NADP 1 to remove NAD(P) 1-dependent proteins, pure aspartase was selectively eluted with Buffer C containing 5 mM sodium L-aspartate. The aspartase sample was essentially pure after this affinity purification. Determination of molecular weight and amino acid sequence analysis. The molecular weight of native aspartase was determined on a Gilson HPLC system using a Bio-Gel SEC40-XL gel-filtration column (f 7.8 3 300 mm, Bio-Rad Laboratories) at a detection wavelength of 280 nm at 25°C. The buffer used was 50 mM Tris–HCl, pH 7.8, containing 0.2 M NaCl, at a flow rate of 0.5 ml/min. The following proteins were used as the molecular weight standards; bovine thyroglobulin (669,000), horse ferritin (440,000), bovine catalase (232,000), rabbit aldolase (158,000), yeast enolase (94,000). The subunit molecular weight of aspartase was determined by SDS–PAGE. E. coli b-galactosidase (116,000), rabbit phosphorylase b (97,400), bovine albumin (66,000), egg albumin (45,000), and bovine carbonic anhydrase (29,000) were used as molecular weight standards. Amino acid sequence analysis was performed on a Shimadzu PPSQ-10 automated protein sequencer. Enzymology. Experiments to determine the enzymatic constants of the purified aspartase were performed in 50 mM Taps–NaOH buffer, containing 0.1 M sodium L-aspartate and 2 mM MgCl 2. The pH was adjusted to 8.5 for experiments at 30°C and to pH 8.0 for experiments at 55°C. The optimum pH of the reaction was probed at various temperatures by using 50 mM Hepes–NaOH buffer and 50 mM Taps–NaOH buffer. The actual pH of each buffer at the respective temperatures was adjusted for temperature-dependent shifts, by estimating that one degree of temperature increase would result in a shift of 20.015 pH unit for Hepes and 20.027 pH unit for Taps (19). Samples were incubated at the indicated temperatures for 10 min prior to the assay. The concentration of aspartase during the assays was 1.0 mg/ml. Thermal stability. For enzymatic stability measurements, 50 mg/ml purified enzyme was incubated in 50 mM Taps–NaOH buffer, pH 8.0, containing 2 mM MgCl 2 at 55°C. At various intervals, aliquots were taken and assayed for enzyme activity at 30°C. To detect changes in structure, the fluorescence intensity of the enzyme was measured on a Hitachi F-4010 fluorescence spectrophotometer equipped with a thermostatically controlled cell holder at various temperatures. The solution temperature of the cuvette cell containing 50 mg/ml purified enzyme in 50 mM potassium phosphate buffer, pH 7.0, containing 1 mM EDTA and 5 mM b-mercaptoethanol was increased gradually (1°C/min) and the continuous change in fluorescence intensity for tryptophan (excitation wavelength at 295 nm and emission wavelength at 360 nm) or tyrosine (excitation wavelength at 278 nm and emission wavelength at 315 nm) was measured. The temperature of the sample solution was monitored directly with a Sensortek Model BAT-12 thermometer. Stability in Gdn-HCl solution. For stability measurements against Gdn-HCl, aspartase was added to 50 mM Taps–NaOH buffer, pH 8.5, containing 2 mM MgCl 2 and the indicated concentration of Gdn-HCl (concentration of aspartase: 50 mg/ml in the presence of denaturant). After a 24-h incubation at 25°C, the residual activity of the samples was assayed at 30°C by adding aspartate to this mixture. The fluorescence spectra of the samples were also measured at 25°C to determine the changes in aspartase structure caused by various concentrations of Gdn-HCl.
RESULTS AND DISCUSSION
Purification of Aspartase from Bacillus sp. YM55-1 3
Abbreviations used: Gdn-HCl, guanidine hydrochloride; SDS– PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; Taps, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid.
The moderate thermophile Bacillus sp. YM55-1 was selected on the basis of its high aspartase activity in
42
KAWATA ET AL. TABLE I
Purification Table for Bacillus YM55-1 Aspartase
Step
Protein (mg)
Total activity (units)
Specific activity (units/mg)
Purification (-fold)
Yield (%)
Crude extract 35–50% ammonium sulfate precipitation Heat treatment Q-Sepharose AF-Red Toyopearl
3700 650 300 90 1.0
1800 1800 1700 1400 700
0.48 2.8 5.7 15.6 700
1.0 5.8 11.9 32.5 1458
100 100 94 78 39
Note. Activity was measured at pH 8.5 and 30°C.
crude extracts among about 100 thermophile strains stocked in the laboratory. No aspartase activity was detected from any of the extreme thermophilic strains, such as Thermus thermophilus, Thermus aquaticus, and Thermus filiformis. From molecular genetics studies, it was reported recently that T. thermophilus does not possess an aspA gene (20), which agrees with our preliminary screening results. Among the strains tested, Bacillus sp. YM55-1 showed the highest activity in crude extracts and, on this basis, was selected for further studies. Cells of Bacillus sp. YM55-1 were routinely grown in the thermophilic medium with vigorous shaking at 55°C. Overnight cultures yielded an average of 3 g/liter wet cells. Purification was achieved by using several standard chromatographic procedures including a dyeaffinity purification using AF-Red Toyopearl affinity resin and elution by the enzyme substrate (L-aspartate), which was most effective in the purification of other aspartases (21). Table I shows a typical purification of Bacillus sp. YM55-1 aspartase. About 1 mg purified enzyme with a specific activity of 700 U/mg protein (30°C) was obtained from 42.1 g wet cells, with a yield of 39%. This specific activity of the purified enzyme (assayed at 30°C, pH 8.5) was about four times higher than that of the E. coli aspartase (22) and three times higher than that of the P. fluorescens aspartase (Takagi et al., unpublished data) (Table II) and, as such, was the highest specific activity ever detected for an aspartase. TABLE II
Comparison of Enzymatic Properties of Aspartase from Bacillus sp. YM55-1 with Those of Other Enzymes
Specific activity (units/mg) K m or S 1/2 (mM) Cooperativity
Bacillus YM55-1
E. coli
P. fluorescens
700 (30°C) 2200 (55°C) 28.5 (30°C) 32.0 (55°C) None
167 (30°C)
214 (30°C)
2.8 (30°C)
2.3 (30°C)
Yes
Yes
Note. Activity was measured at optimum pH.
From SDS–PAGE, the aspartase samples were estimated to be essentially pure after the affinity chromatography step (data not shown). The subunit molecular weight was determined to be 51,000, and the relative molecular weight of native aspartase determined using size-exclusion HPLC was about 200,000. In addition, N-terminal amino acid sequence analysis of the purified aspartase samples (described in detail below) showed that the purified enzyme consisted of a single polypeptide species. These results indicated that the aspartase from Bacillus sp. YM55-1 was composed of four identical subunits with molecular weights of 51,000. Enzymatic Characteristics of Purified Thermostable Aspartase Basic characterization experiments involving the determination of optimum temperature and pH were performed on the newly isolated aspartase. The results are shown in comparison with the values obtained for E. coli. As shown in Fig. 1a, the optimum pH of Bacillus YM55-1 aspartase decreased gradually with increasing temperature; the optimum pH was 8.5 at 30°C and 7.5 at 60°C. Since the activity at each pH and temperature was independent of the incubation time, the decrease in activity at alkaline pH at high temperatures is probably not caused by denaturation of the enzyme. A similar phenomenon was observed for E. coli aspartase (Fig. 1b). This finding suggests that the amino acid residue(s) responsible for catalysis is a residue whose pK a value is affected by temperature, for example, His or Lys (23, 24). The fact that identical results were obtained for both E. coli and Bacillus YM55-1 aspartases suggests that the catalytic residue(s) may be the same in both aspartases. The activities determined at the optimum pH of each temperature are shown in Fig. 1c. The optimum temperatures of Bacillus YM55-1 and E. coli aspartases were 65°C (pH 7.5) and 55°C (pH 8.0), respectively, and the maximum activity was 2500 U/mg for Bacillus YM55-1 aspartase and 800 U/mg for E. coli aspartase at the respective optimum temperatures.
PURIFICATION AND CHARACTERIZATION OF THERMOSTABLE ASPARTASE
43
mM and a V max of 700 U/mg were determined at 30°C and pH 8.5, and a K m of 32.0 mM and a V max of 2200 U/mg at 55°C and pH 7.8. The kinetic constants of Bacillus YM55-1 aspartase are summarized and compared with those of enzymes from E. coli and P. fluorescens in Table II. The K m value of Bacillus YM55-1 aspartase was one order of magnitude higher than those of E. coli and P. fluorescens at 30°C. However, at 55°C, which is the optimum growth temperature of Bacillus YM55-1 aspartase, the K m was almost the same as the value at 30°C while the specific activity was increased by 3.1 times. This suggests that the activity of Bacillus YM55-1 aspartase may be greater than those of E. coli and P. fluorescens enzymes under their respective physiological conditions. Enzymatic Stability The stability of aspartase against heat and chemical denaturation (Gdn-HCl) was monitored in terms of activity. For thermostability experiments, the enzyme was incubated at 55°C and at appropriate intervals the residual activity was measured at 30°C (Fig. 3a). About 80% of the initial activity was retained after a 60-min incubation for Bacillus YM55-1 aspartase, while the E. coli enzyme was completely denatured under the same conditions. In the experiments shown in Fig. 3b, after incubation in the presence of the indicated concentrations of Gdn-HCl at FIG. 1. Optimum conditions for expression of aspartase activity. (a, b) Determination of optimum pH of Bacillus YM55-1 (a) and E. coli (b) aspartases at various temperatures. Sodium L-aspartate (100 mM) and 2 mM MgCl 2 in 50 mM Hepes–NaOH or Taps–NaOH buffer, which had been previously adjusted to the indicated pH at each temperature, were incubated for 10 min at the indicated temperatures, after which enzyme was added. The increase in absorbance at 240 nm was monitored to determine the activity of the sample. The concentration of aspartase during the assay was 1.0 mg/ml. (c) Optimum temperature of aspartase. The activities of aspartase at the optimum pH of each temperature [determined as in (a) and (b)] were plotted. Therefore, in this figure it should be noted that the pH is different for each temperature point in the graph. Closed and open circles represent Bacillus YM55-1 and E. coli aspartases, respectively.
Next, the specific activity of the purified Bacillus YM55-1 enzyme was determined in varying concentrations of the substrate L-aspartate at 30°C and pH 8.5 and at 55°C and pH 7.8 to determine the kinetic constants. As shown in Fig. 2a, the saturation curves at both temperatures were hyperbolic. From the Hill coefficient analysis for the kinetics, Hill constants of 1.2 (30°C) and 1.0 (55°C) were obtained regardless of the presence or absence of Mg 21 ion (data not shown), indicating that the enzyme obeyed Michaelis–Menten rules of kinetics. This behavior was different from that of E. coli aspartase, which is activated cooperatively by the presence of Mg 21 ion at alkaline pH (2, 22, 24). From the Lineweaver–Burk plot (Fig. 2b), a K m of 28.5
FIG. 2. (a) Substrate saturation curves for Bacillus YM55-1 aspartase. The substrate used was L-aspartate. Open circles, pH 8.5 at 30°C; closed circles, pH 8.0 at 55°C. Experiments were performed in 50 mM Taps–NaOH. (b) Lineweaver–Burk plots of the substrate saturation curves shown in (a). The K m and V max values for each experimental condition are given in Table II.
44
KAWATA ET AL.
25°C for 24 h, aliquots of aspartase were assayed for activity at 30°C in the presence of the same concentration of Gdn-HCl. As seen in the figure, more than 50% of the initial activity was retained at Gdn-HCl concentrations as high as 1.1 M for Bacillus YM55-1 aspartase, contrasting sharply with the complete inactivation of E. coli enzyme in the presence of the same concentration of denaturant. Therefore, the present enzyme may be said to possess both of the characteristics ideal for application in industrial processes: high specific activity and robust structural stability. Structural Stability To understand the structural stability of the enzyme in more detail, changes in structure caused by high temperature and Gdn-HCl were probed by monitoring the intrinsic fluorescence. For Bacillus YM55-1 aspartase, a spectrum with a maximum wavelength of 320 nm, which is characteristic of tryptophyl fluorescence, was seen on excitation at 295 nm (0 M Gdn-HCl), indicating that at least one tryptophan residue is included in the primary sequence. On unfolding in 4 M Gdn-HCl, the maximum wavelength was shifted to 350 nm and the intensity was increased (Fig. 4a). Contrariwise, for E. coli aspartase, a spectrum with a maximum at 305 nm was observed at an excitation wavelength of 278 nm, in agreement with the fact that no tryptophan residues are included in the amino acid sequence of the E. coli enzyme (3) (Fig. 4b). On unfolding in 4 M GdnHCl, the intensity of the peak at 305 nm was decreased without wavelength shifts. These fluorescence characteristics on unfolding reflect the structural environments of the fluorescence chromophore. We next used these fluorescence changes as a probe to monitor the structural unfolding of each enzyme. As shown in Fig. 4c, a sharp transition in fluorescence intensity caused by thermal unfolding was observed at 63°C for Bacillus YM55-1 aspartase. A similar transition at 55°C was observed for E. coli enzyme. This finding clearly indicates the high thermostability of Bacillus YM55-1 aspartase relative to the E. coli enzyme, which was also indicated in the results shown in Fig. 3a. Unfolding in Gdn-HCl was also examined for both enzymes (Fig. 4d); the midpoint concentration of the unfolding transition was 1.3 M Gdn-HCl for Bacillus YM55-1 enzyme and 0.8 M Gdn-HCl for E. coli enzyme. This unfolding behavior in Gdn-HCl was consistent with the results shown in Fig. 3b. N-Terminal Amino Acid Analysis of Bacillus YM55-1 Aspartase The N-terminal amino acid sequence of the purified enzyme was determined from Edman analysis and compared with those of other aspartases (3, 4, 6), as shown in Fig. 5. Thirteen of the first forty residues
FIG. 3. Stability of Bacillus YM55-1 and E. coli aspartases against thermal and chemical denaturation. Closed and open circles represent Bacillus YM55-1 and E. coli aspartases, respectively. (a) Thermal denaturation of aspartase at 55°C. Aspartase (50 mg/ml) was incubated in 50 mM Taps–NaOH buffer, pH 8.0, containing 2 mM MgCl 2. At various intervals, aliquots of this sample were taken and assayed for activity at 30°C. (b) Activity of aspartase in the presence of Gdn-HCl. Aspartase samples (50 mg/ml) were incubated at 25°C for 24 h in 50 mM Taps–NaOH buffer, pH 8.5, containing 2 mM MgCl 2 and the indicated concentration of Gdn-HCl. After incubation, the residual activity was assayed at 30°C under the same conditions and plotted against the denaturant concentration.
were identical with respect to all of the sequences, as shaded in the figure. More specifically, 21 residues (53%) for E. coli, 19 residues (48%) for P. fluorescens, and 26 residues (65%) for B. subtilis were conserved in a one-to-one comparison with Bacillus YM55-1 aspartase. These homology scores are increased if homologous residues such as threonine and serine, lysine and arginine, and isoleucine and leucine are considered. It should be noted that Asp10 and Arg29 (Fig. 5, asterisk), which were reported to be the catalytic residue and substrate binding residue, respectively, in a previous study (25), are conserved in all of the sequences. The high homology, although only in the N-terminal region, with other thermolabile aspartases is interesting when considered together with the unique structural stability observed for Bacillus YM55-1 aspartase. Since the analysis of the amino acid composition of Bacillus YM55-1 aspartase showed that no differences
PURIFICATION AND CHARACTERIZATION OF THERMOSTABLE ASPARTASE
45
FIG. 4. Fluorescence spectra of Bacillus YM55-1 and E. coli aspartases. Tryptophan fluorescence of Bacillus YM55-1 aspartase on excitation at 295 nm (a) and tyrosine fluorescence of E. coli aspartase on excitation at 278 nm (b). Open and closed circles indicate fluorescence at 0 M Gdn-HCl (native state) and 4 M Gdn-HCl (unfolded state), respectively. All spectra were corrected by subtracting the spectrum of the buffer without enzyme. The protein concentration was 50 mg/ml. (c) Thermal unfolding of Bacillus YM55-1 (closed circles, at 360 nm) and E. coli (open circles, at 315 nm) aspartases monitored by fluorescence. The excitation wavelength was 295 nm for Bacillus YM55-1 aspartase and 278 nm for E. coli aspartase. (d) Gdn-HCl unfolding of Bacillus YM55-1 (closed circles) and E. coli (open circles) aspartases monitored by fluorescence. The conditions of the fluorescence measurements were the same as in (c).
in the compositions of the E. coli and P. fluorescens enzymes are evident (data not shown), elucidation of the entire amino acid sequence and tertiary structure of Bacillus YM55-1 aspartase is required for an understanding of the molecular basis of this robust stability. CONCLUDING REMARKS
A new aspartase enzyme was purified from Bacillus sp. YM55-1, a moderate thermophile. Although the enzyme was similar to other mesophilic aspartases in
subunit molecular weight, oligomeric state, and N-terminal amino acid sequence, the structural stability against heat and denaturant of the Bacillus YM55-1 aspartase was remarkable and the specific activity at 30°C was the highest value so far reported. These findings indicate that this newly purified enzyme may be very useful in various industrial processes and also provide a research field in fundamental studies on the structure and function of thermophilic enzymes, especially aspartase.
FIG. 5. Sequence homologies of the first 40 N-terminal amino acids of Bacillus YM55-1 aspartase with the corresponding sequences of various other aspartases. The sequences were aligned manually. Shaded residues indicate residues that were conserved in all of the sequences compared. Asterisks indicate residues that are considered to be involved in the catalysis and substrate binding in E. coli aspartase. The numbers above the sequence correspond to the amino acid residue number of Bacillus YM55-1 aspartase and E. coli aspartase. The P. fluorescens sequence and the B. subtilis sequence are shown beginning from the seventh and fourth residues, respectively.
46
KAWATA ET AL.
REFERENCES 1. Rudolph, F. B., and Fromm, H. J. (1971) Arch. Biochem. Biophys. 147, 92–98. 2. Suzuki, S., Yamaguchi, J., and Tokushige, M. (1973) Biochim. Biophys. Acta 321, 369 –381. 3. Takagi, J. S., Ida, N., Tokushige, M., Sakamoto, H., and Shimura, Y. (1985) Nucleic Acids Res. 13, 2063–2074. 4. Takagi, J. S., Fukunaga, R., Tokushige, M., and Katsuki, H. (1984) J. Biochem. 96, 545–552. 5. Takagi, J. S., Tokushige, M., and Shimura, Y. (1986) J. Biochem. 100, 697–705. 6. Sun, D., and Setlow, P. (1991) J. Bacteriol. 173, 3831–3845. 7. Tokushige, M., Eguchi, G., and Hirata, F. (1977) Biochim. Biophys. Acta 480, 479 – 488. 8. Yumoto, N., and Tokushige, M. (1983) Biochim. Biophys. Acta 749, 101–105. 9. Imaishi, H., Yumoto, N., and Tokushige, M. (1989) Physiol. Chem. Phys. Med. NMR 21, 221–228. 10. Imaishi, H., Yumoto, N., and Tokushige, M. (1990) Biotechnol. Appl. Biochem. 12, 196 –205. 11. Murase, S., Kawata, Y., and Yumoto, N. (1993) J. Biochem. 114, 393–397. 12. Murase, S., Kawata, Y., and Yumoto, N. (1993) Biochem. Biophys. Res. Commun. 195, 1159 –1164. 13. Saribas, A. S., Schindler, J. F., and Viola, R. E. (1994) J. Biol. Chem. 269, 6313– 6319.
14. Shi, W., Dunbar, J., Jayasekera, M. M. K., Viola, R. E., and Farber, G. K. (1997) Biochemistry 36, 9136 –9144. 15. Suzuki, Y., Mino, Y., and Abe, S. (1980) Appl. Microbiol. Biotechnol. 11, 23–27. 16. Mizobata, T., Fujioka, T., Yamasaki, F., Hidaka, M., Nagai, J., and Kawata, Y. (1998) Arch. Biochem. Biophys. 355, 49 – 55. 17. Kagawa, M., Murakoshi, N., Nishikawa, Y., Matsumoto, G., Kurata, Y., Mizobata, T., Kawata, Y., and Nagai, J. (1999) Arch. Biochem. Biophys. 362, 346 –355. 18. Tokushige, M. (1985) Methods Enzymol. 113, 63–72. 19. Dawson, R. M. C., Elliot, D. C., Elliot, W. H., and Jones, K. M. (1986) Data for Biochemical Research, 3rd ed., Clarendon Press, Oxford. 20. Kosuge, T., Umehara, K., and Hoshino, T. (1998) J. Ferment. Bioeng. 86, 125–129. 21. Karsten, W. E., Hunsley, J. R., and Viola, R. E. (1985) Anal. Biochem. 147, 336 –341. 22. Murase, S., Takagi, J. S., Higashi, Y., Imaishi, H., Yumoto, N., and Tokushige, M. (1991) Biochem. Biophys. Res. Commun. 177, 414 – 419. 23. Ida, N., and Tokushige, M. (1984) J. Biochem. 96, 1315–1321. 24. Karsten, W. E., and Viola, R. E. (1991) Arch. Biochem. Biophys. 287, 60 – 67. 25. Jayasekera, M. M. K., Shi, W., Farber, G. K., and Viola, R. E. (1997) Biochemistry 36, 9145–9150.
Purification and Characterization of Thermostable Aspartase from Bacillus sp. YM55-1 Yasushi Kawata,* ,1 Koichi Tamura,* Shigeru Yano,† Tomohiro Mizobata,* Jun Nagai,* Nobuyoshi Esaki,‡ Kenji Soda,‡ ,2 Masanobu Tokushige,† and Noboru Yumoto§ *Department of Biotechnology, Faculty of Engineering, Tottori University, Tottori 680-0945, Japan; †Department of Chemistry, Faculty of Science, Kyoto University, Kyoto 606-8224, Japan; ‡Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan; and §Osaka National Research Institute, AIST, Ikeda, Osaka 563-8577, Japan
Received December 4, 1998, and in revised form February 2, 1999
A thermostable aspartase was purified from a thermophile Bacillus sp. YM55-1 and characterized in terms of activity and stability. The enzyme was isolated by a 5-min heat treatment at 75°C in the presence of 11% (w/v) ammonium sulfate and 100 mM aspartate, followed by Q-Sepharose anion-exchange and AF-Red Toyopearl chromatographies. The native molecular weight of aspartase determined by gel filtration was about 200,000, and this enzyme was composed of four identical monomers with molecular weights of 51,000 determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Unlike Escherichia coli aspartase, the enzyme was not activated by the presence of magnesium ion at alkaline pH. At the optimum pH, the K m and V max were 28.5 mM and 700 units/mg at 30°C and 32.0 mM and 2200 units/mg at 55°C, respectively. The specific activity was four and three times higher than those of E. coli and Pseudomonas fluorescens enzymes at 30°C, respectively. Eighty percent of the activity was retained after a 60-min incubation at 55°C, and the enzyme was also resistant to chemical denaturants; 80% of the initial specific activity was detected in assay mixtures containing 1.0 M guanidine hydrochloride. The purified enzyme shared a high sequence homology in the N-terminal region with aspartases from other organisms. © 1999 Academic Press Key Words: aspartase; aspartate ammonia-lyase; thermophile; purification; characterization; Bacillus species.
1 To whom correspondence should be addressed. Fax: 181– 857– 31–5271. E-mail: [email protected]. 2 Present address: Faculty of Engineering, Kansai University, Suita, Osaka 564-0073, Japan.
40
Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) catalyzes the reversible conversion of L-aspartate to fumarate and ammonium ion and plays an important role in the bacterial nitrogen metabolism. The enzyme has been purified from Escherichia coli (1–3), Pseudomonas fluorescens (4, 5), and Bacillus subtilis (6), and each gene has been cloned and sequenced. Aspartases from E. coli and P. fluorescens were found to have a molecular mass of about 200 kDa and to be composed of four identical 50-kDa subunits. Various studies on the structure–function relationship of E. coli aspartase have been reported (7–13). In particular, the recent elucidation of the three-dimensional structure of aspartase from E. coli (14) provides detailed structural characteristics of this enzyme family, which can be used in further mechanistic studies. Aspartase has been used for the industrial production of L-aspartate from fumarate, which, in turn, is an ingredient in the chemical synthesis of the artificial sweetener aspartame (N-L-a-aspartyl-L-phenylalanine1-methyl ester). In industrial applications, enzymes having stable structures and high catalytic activities are generally preferred. However, although the enzymatic production of L-aspartate using a thermophilic bacterium was reported previously (15), detailed characteristics of a thermostable aspartase have not yet been reported. In our laboratory, we have been studying various thermostable enzymes such as fumarase (16) and catalase (17), which are derived from thermophilic Thermus strains. As part of this effort to increase our understanding of the basis for enzymatic activity and structural stability in thermostable enzymes, we report the purification and characterization of an aspartase from a thermostable bacterium, Bacillus sp. YM55-1. The purified enzyme shared a high sequence homology with various other bacterial aspartases, but 0003-9861/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
PURIFICATION AND CHARACTERIZATION OF THERMOSTABLE ASPARTASE
was much more resistant to thermal and chemical denaturation. MATERIALS AND METHODS Materials. Gdn-HCl 3 (finest grade) was obtained from Nacalai Tesque (Kyoto). Meat extract was obtained from Kyokuto Pharmaceuticals (Tokyo). Q-Sepharose and AF-Red Toyopearl resin were obtained from Pharmacia Biotech (Uppsala, Sweden) and Tosoh Corporation (Tokyo), respectively. Molecular weight standard proteins for gel filtration and SDS–PAGE were purchased from Oriental Yeast Company (Osaka) and Sigma (St. Louis, MO), respectively. Bovine serum albumin was obtained from Sigma. All other reagents were obtained from Wako Fine Chemicals (Osaka) or Nacalai Tesque and used without further purification. Aspartase from E. coli was purified according to the methods reported previously (10). Cultivation of Bacillus sp. YM55-1. Cultivation of Bacillus sp. YM55-1 was performed aerobically in thermophilic bacterial medium (5 g/liter yeast extract, 5 g/liter polypeptone, 2 g/liter meat extract, 2 g/liter glycerol, 5 g/liter sodium L-aspartate, 2 g/liter NaCl, 2 g/liter K 2HPO 4, 2 g/liter KH 2PO 4, 0.1 g/liter MgSO 4 z 7H 2O, and 4 mg/liter biotin at pH 7.2) overnight at 55°C. Enzyme assays and determination of protein concentrations. Aspartase activity was routinely assayed at 30°C by measuring the increase in absorption at 240 nm caused by the production of fumarate. A Hitachi 200 or U-2000A spectrophotometer equipped with a Peltier-type cell temperature control unit (SPR-10) was used in all measurements. The standard assay for activity was performed in 50 mM Taps–NaOH buffer, pH 8.5, containing 0.1 M sodium L-aspartate, 2 mM MgCl 2, and an appropriate quantity of enzyme preparation in a total volume of 1 ml at 30°C. One unit of aspartase was defined as the amount of enzyme that produced 1 mmol of fumarate in 1 min. The molar coefficient of 2.53 3 10 3 M 21 cm 21 at 240 nm for fumarate was used (18). Protein concentration was determined with a protein dye reagent (Protein Assay Kit, Bio-Rad Laboratories, Richmond, CA) using bovine serum albumin as a standard. Purification of aspartase from Bacillus sp. YM55-1. All purification steps were performed at 4°C. Cells (42.1 g wet wt) were suspended in 120 ml of 50 mM potassium phosphate buffer, pH 6.8, containing 0.1 M KCl, 1 mM EDTA, and 5 mM b-mercaptoethanol (Buffer A), and lysed by sonication. After removal of all debris by centrifugation and nucleic acid precipitation using 2.5% streptomycin, the supernatant was fractionated with ammonium sulfate. The fraction corresponding to 35–50% saturated ammonium sulfate was collected, resuspended in Buffer A, and dialyzed extensively against Buffer A. Ammonium sulfate (final concentration: 11% w/v) and 0.1 M sodium L-aspartate were added to the dialyzate, and the mixture was heated at 75°C for 5 min and immediately cooled on ice. Soluble proteins that remained after heat treatment were dialyzed extensively against Buffer B (50 mM potassium phosphate buffer, pH 7.2, containing 1 mM EDTA and 5 mM b-mercaptoethanol). After dialysis, the sample was subjected to a Q-Sepharose anion-exchange column (f 16 3 600 mm) that had been equilibrated with Buffer B, and was eluted with a linear concentration gradient of KCl (0 –1 M). Fractions containing aspartase activity were concentrated using ultrafiltration (Amicon PM30 membrane filter) and the buffer was changed to Buffer C [50 mM Hepes–Tris buffer, pH 6.6, containing 30 mM Mg(CH 3COO) 2, 5 mM b-mercaptoethanol, and 40% (v/v) ethylene glycol] by passage through a PD-10 (Sephadex G-25; Pharmacia) desalting column. The sample was then applied to an AF-Red Toyopearl column (f 22 3 110 mm) equilibrated in Buffer C. After
41
the column was washed with 150 ml of Buffer C containing 0.5 mM NAD 1 and 0.5 mM NADP 1 to remove NAD(P) 1-dependent proteins, pure aspartase was selectively eluted with Buffer C containing 5 mM sodium L-aspartate. The aspartase sample was essentially pure after this affinity purification. Determination of molecular weight and amino acid sequence analysis. The molecular weight of native aspartase was determined on a Gilson HPLC system using a Bio-Gel SEC40-XL gel-filtration column (f 7.8 3 300 mm, Bio-Rad Laboratories) at a detection wavelength of 280 nm at 25°C. The buffer used was 50 mM Tris–HCl, pH 7.8, containing 0.2 M NaCl, at a flow rate of 0.5 ml/min. The following proteins were used as the molecular weight standards; bovine thyroglobulin (669,000), horse ferritin (440,000), bovine catalase (232,000), rabbit aldolase (158,000), yeast enolase (94,000). The subunit molecular weight of aspartase was determined by SDS–PAGE. E. coli b-galactosidase (116,000), rabbit phosphorylase b (97,400), bovine albumin (66,000), egg albumin (45,000), and bovine carbonic anhydrase (29,000) were used as molecular weight standards. Amino acid sequence analysis was performed on a Shimadzu PPSQ-10 automated protein sequencer. Enzymology. Experiments to determine the enzymatic constants of the purified aspartase were performed in 50 mM Taps–NaOH buffer, containing 0.1 M sodium L-aspartate and 2 mM MgCl 2. The pH was adjusted to 8.5 for experiments at 30°C and to pH 8.0 for experiments at 55°C. The optimum pH of the reaction was probed at various temperatures by using 50 mM Hepes–NaOH buffer and 50 mM Taps–NaOH buffer. The actual pH of each buffer at the respective temperatures was adjusted for temperature-dependent shifts, by estimating that one degree of temperature increase would result in a shift of 20.015 pH unit for Hepes and 20.027 pH unit for Taps (19). Samples were incubated at the indicated temperatures for 10 min prior to the assay. The concentration of aspartase during the assays was 1.0 mg/ml. Thermal stability. For enzymatic stability measurements, 50 mg/ml purified enzyme was incubated in 50 mM Taps–NaOH buffer, pH 8.0, containing 2 mM MgCl 2 at 55°C. At various intervals, aliquots were taken and assayed for enzyme activity at 30°C. To detect changes in structure, the fluorescence intensity of the enzyme was measured on a Hitachi F-4010 fluorescence spectrophotometer equipped with a thermostatically controlled cell holder at various temperatures. The solution temperature of the cuvette cell containing 50 mg/ml purified enzyme in 50 mM potassium phosphate buffer, pH 7.0, containing 1 mM EDTA and 5 mM b-mercaptoethanol was increased gradually (1°C/min) and the continuous change in fluorescence intensity for tryptophan (excitation wavelength at 295 nm and emission wavelength at 360 nm) or tyrosine (excitation wavelength at 278 nm and emission wavelength at 315 nm) was measured. The temperature of the sample solution was monitored directly with a Sensortek Model BAT-12 thermometer. Stability in Gdn-HCl solution. For stability measurements against Gdn-HCl, aspartase was added to 50 mM Taps–NaOH buffer, pH 8.5, containing 2 mM MgCl 2 and the indicated concentration of Gdn-HCl (concentration of aspartase: 50 mg/ml in the presence of denaturant). After a 24-h incubation at 25°C, the residual activity of the samples was assayed at 30°C by adding aspartate to this mixture. The fluorescence spectra of the samples were also measured at 25°C to determine the changes in aspartase structure caused by various concentrations of Gdn-HCl.
RESULTS AND DISCUSSION
Purification of Aspartase from Bacillus sp. YM55-1 3
Abbreviations used: Gdn-HCl, guanidine hydrochloride; SDS– PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; Taps, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid.
The moderate thermophile Bacillus sp. YM55-1 was selected on the basis of its high aspartase activity in
42
KAWATA ET AL. TABLE I
Purification Table for Bacillus YM55-1 Aspartase
Step
Protein (mg)
Total activity (units)
Specific activity (units/mg)
Purification (-fold)
Yield (%)
Crude extract 35–50% ammonium sulfate precipitation Heat treatment Q-Sepharose AF-Red Toyopearl
3700 650 300 90 1.0
1800 1800 1700 1400 700
0.48 2.8 5.7 15.6 700
1.0 5.8 11.9 32.5 1458
100 100 94 78 39
Note. Activity was measured at pH 8.5 and 30°C.
crude extracts among about 100 thermophile strains stocked in the laboratory. No aspartase activity was detected from any of the extreme thermophilic strains, such as Thermus thermophilus, Thermus aquaticus, and Thermus filiformis. From molecular genetics studies, it was reported recently that T. thermophilus does not possess an aspA gene (20), which agrees with our preliminary screening results. Among the strains tested, Bacillus sp. YM55-1 showed the highest activity in crude extracts and, on this basis, was selected for further studies. Cells of Bacillus sp. YM55-1 were routinely grown in the thermophilic medium with vigorous shaking at 55°C. Overnight cultures yielded an average of 3 g/liter wet cells. Purification was achieved by using several standard chromatographic procedures including a dyeaffinity purification using AF-Red Toyopearl affinity resin and elution by the enzyme substrate (L-aspartate), which was most effective in the purification of other aspartases (21). Table I shows a typical purification of Bacillus sp. YM55-1 aspartase. About 1 mg purified enzyme with a specific activity of 700 U/mg protein (30°C) was obtained from 42.1 g wet cells, with a yield of 39%. This specific activity of the purified enzyme (assayed at 30°C, pH 8.5) was about four times higher than that of the E. coli aspartase (22) and three times higher than that of the P. fluorescens aspartase (Takagi et al., unpublished data) (Table II) and, as such, was the highest specific activity ever detected for an aspartase. TABLE II
Comparison of Enzymatic Properties of Aspartase from Bacillus sp. YM55-1 with Those of Other Enzymes
Specific activity (units/mg) K m or S 1/2 (mM) Cooperativity
Bacillus YM55-1
E. coli
P. fluorescens
700 (30°C) 2200 (55°C) 28.5 (30°C) 32.0 (55°C) None
167 (30°C)
214 (30°C)
2.8 (30°C)
2.3 (30°C)
Yes
Yes
Note. Activity was measured at optimum pH.
From SDS–PAGE, the aspartase samples were estimated to be essentially pure after the affinity chromatography step (data not shown). The subunit molecular weight was determined to be 51,000, and the relative molecular weight of native aspartase determined using size-exclusion HPLC was about 200,000. In addition, N-terminal amino acid sequence analysis of the purified aspartase samples (described in detail below) showed that the purified enzyme consisted of a single polypeptide species. These results indicated that the aspartase from Bacillus sp. YM55-1 was composed of four identical subunits with molecular weights of 51,000. Enzymatic Characteristics of Purified Thermostable Aspartase Basic characterization experiments involving the determination of optimum temperature and pH were performed on the newly isolated aspartase. The results are shown in comparison with the values obtained for E. coli. As shown in Fig. 1a, the optimum pH of Bacillus YM55-1 aspartase decreased gradually with increasing temperature; the optimum pH was 8.5 at 30°C and 7.5 at 60°C. Since the activity at each pH and temperature was independent of the incubation time, the decrease in activity at alkaline pH at high temperatures is probably not caused by denaturation of the enzyme. A similar phenomenon was observed for E. coli aspartase (Fig. 1b). This finding suggests that the amino acid residue(s) responsible for catalysis is a residue whose pK a value is affected by temperature, for example, His or Lys (23, 24). The fact that identical results were obtained for both E. coli and Bacillus YM55-1 aspartases suggests that the catalytic residue(s) may be the same in both aspartases. The activities determined at the optimum pH of each temperature are shown in Fig. 1c. The optimum temperatures of Bacillus YM55-1 and E. coli aspartases were 65°C (pH 7.5) and 55°C (pH 8.0), respectively, and the maximum activity was 2500 U/mg for Bacillus YM55-1 aspartase and 800 U/mg for E. coli aspartase at the respective optimum temperatures.
PURIFICATION AND CHARACTERIZATION OF THERMOSTABLE ASPARTASE
43
mM and a V max of 700 U/mg were determined at 30°C and pH 8.5, and a K m of 32.0 mM and a V max of 2200 U/mg at 55°C and pH 7.8. The kinetic constants of Bacillus YM55-1 aspartase are summarized and compared with those of enzymes from E. coli and P. fluorescens in Table II. The K m value of Bacillus YM55-1 aspartase was one order of magnitude higher than those of E. coli and P. fluorescens at 30°C. However, at 55°C, which is the optimum growth temperature of Bacillus YM55-1 aspartase, the K m was almost the same as the value at 30°C while the specific activity was increased by 3.1 times. This suggests that the activity of Bacillus YM55-1 aspartase may be greater than those of E. coli and P. fluorescens enzymes under their respective physiological conditions. Enzymatic Stability The stability of aspartase against heat and chemical denaturation (Gdn-HCl) was monitored in terms of activity. For thermostability experiments, the enzyme was incubated at 55°C and at appropriate intervals the residual activity was measured at 30°C (Fig. 3a). About 80% of the initial activity was retained after a 60-min incubation for Bacillus YM55-1 aspartase, while the E. coli enzyme was completely denatured under the same conditions. In the experiments shown in Fig. 3b, after incubation in the presence of the indicated concentrations of Gdn-HCl at FIG. 1. Optimum conditions for expression of aspartase activity. (a, b) Determination of optimum pH of Bacillus YM55-1 (a) and E. coli (b) aspartases at various temperatures. Sodium L-aspartate (100 mM) and 2 mM MgCl 2 in 50 mM Hepes–NaOH or Taps–NaOH buffer, which had been previously adjusted to the indicated pH at each temperature, were incubated for 10 min at the indicated temperatures, after which enzyme was added. The increase in absorbance at 240 nm was monitored to determine the activity of the sample. The concentration of aspartase during the assay was 1.0 mg/ml. (c) Optimum temperature of aspartase. The activities of aspartase at the optimum pH of each temperature [determined as in (a) and (b)] were plotted. Therefore, in this figure it should be noted that the pH is different for each temperature point in the graph. Closed and open circles represent Bacillus YM55-1 and E. coli aspartases, respectively.
Next, the specific activity of the purified Bacillus YM55-1 enzyme was determined in varying concentrations of the substrate L-aspartate at 30°C and pH 8.5 and at 55°C and pH 7.8 to determine the kinetic constants. As shown in Fig. 2a, the saturation curves at both temperatures were hyperbolic. From the Hill coefficient analysis for the kinetics, Hill constants of 1.2 (30°C) and 1.0 (55°C) were obtained regardless of the presence or absence of Mg 21 ion (data not shown), indicating that the enzyme obeyed Michaelis–Menten rules of kinetics. This behavior was different from that of E. coli aspartase, which is activated cooperatively by the presence of Mg 21 ion at alkaline pH (2, 22, 24). From the Lineweaver–Burk plot (Fig. 2b), a K m of 28.5
FIG. 2. (a) Substrate saturation curves for Bacillus YM55-1 aspartase. The substrate used was L-aspartate. Open circles, pH 8.5 at 30°C; closed circles, pH 8.0 at 55°C. Experiments were performed in 50 mM Taps–NaOH. (b) Lineweaver–Burk plots of the substrate saturation curves shown in (a). The K m and V max values for each experimental condition are given in Table II.
44
KAWATA ET AL.
25°C for 24 h, aliquots of aspartase were assayed for activity at 30°C in the presence of the same concentration of Gdn-HCl. As seen in the figure, more than 50% of the initial activity was retained at Gdn-HCl concentrations as high as 1.1 M for Bacillus YM55-1 aspartase, contrasting sharply with the complete inactivation of E. coli enzyme in the presence of the same concentration of denaturant. Therefore, the present enzyme may be said to possess both of the characteristics ideal for application in industrial processes: high specific activity and robust structural stability. Structural Stability To understand the structural stability of the enzyme in more detail, changes in structure caused by high temperature and Gdn-HCl were probed by monitoring the intrinsic fluorescence. For Bacillus YM55-1 aspartase, a spectrum with a maximum wavelength of 320 nm, which is characteristic of tryptophyl fluorescence, was seen on excitation at 295 nm (0 M Gdn-HCl), indicating that at least one tryptophan residue is included in the primary sequence. On unfolding in 4 M Gdn-HCl, the maximum wavelength was shifted to 350 nm and the intensity was increased (Fig. 4a). Contrariwise, for E. coli aspartase, a spectrum with a maximum at 305 nm was observed at an excitation wavelength of 278 nm, in agreement with the fact that no tryptophan residues are included in the amino acid sequence of the E. coli enzyme (3) (Fig. 4b). On unfolding in 4 M GdnHCl, the intensity of the peak at 305 nm was decreased without wavelength shifts. These fluorescence characteristics on unfolding reflect the structural environments of the fluorescence chromophore. We next used these fluorescence changes as a probe to monitor the structural unfolding of each enzyme. As shown in Fig. 4c, a sharp transition in fluorescence intensity caused by thermal unfolding was observed at 63°C for Bacillus YM55-1 aspartase. A similar transition at 55°C was observed for E. coli enzyme. This finding clearly indicates the high thermostability of Bacillus YM55-1 aspartase relative to the E. coli enzyme, which was also indicated in the results shown in Fig. 3a. Unfolding in Gdn-HCl was also examined for both enzymes (Fig. 4d); the midpoint concentration of the unfolding transition was 1.3 M Gdn-HCl for Bacillus YM55-1 enzyme and 0.8 M Gdn-HCl for E. coli enzyme. This unfolding behavior in Gdn-HCl was consistent with the results shown in Fig. 3b. N-Terminal Amino Acid Analysis of Bacillus YM55-1 Aspartase The N-terminal amino acid sequence of the purified enzyme was determined from Edman analysis and compared with those of other aspartases (3, 4, 6), as shown in Fig. 5. Thirteen of the first forty residues
FIG. 3. Stability of Bacillus YM55-1 and E. coli aspartases against thermal and chemical denaturation. Closed and open circles represent Bacillus YM55-1 and E. coli aspartases, respectively. (a) Thermal denaturation of aspartase at 55°C. Aspartase (50 mg/ml) was incubated in 50 mM Taps–NaOH buffer, pH 8.0, containing 2 mM MgCl 2. At various intervals, aliquots of this sample were taken and assayed for activity at 30°C. (b) Activity of aspartase in the presence of Gdn-HCl. Aspartase samples (50 mg/ml) were incubated at 25°C for 24 h in 50 mM Taps–NaOH buffer, pH 8.5, containing 2 mM MgCl 2 and the indicated concentration of Gdn-HCl. After incubation, the residual activity was assayed at 30°C under the same conditions and plotted against the denaturant concentration.
were identical with respect to all of the sequences, as shaded in the figure. More specifically, 21 residues (53%) for E. coli, 19 residues (48%) for P. fluorescens, and 26 residues (65%) for B. subtilis were conserved in a one-to-one comparison with Bacillus YM55-1 aspartase. These homology scores are increased if homologous residues such as threonine and serine, lysine and arginine, and isoleucine and leucine are considered. It should be noted that Asp10 and Arg29 (Fig. 5, asterisk), which were reported to be the catalytic residue and substrate binding residue, respectively, in a previous study (25), are conserved in all of the sequences. The high homology, although only in the N-terminal region, with other thermolabile aspartases is interesting when considered together with the unique structural stability observed for Bacillus YM55-1 aspartase. Since the analysis of the amino acid composition of Bacillus YM55-1 aspartase showed that no differences
PURIFICATION AND CHARACTERIZATION OF THERMOSTABLE ASPARTASE
45
FIG. 4. Fluorescence spectra of Bacillus YM55-1 and E. coli aspartases. Tryptophan fluorescence of Bacillus YM55-1 aspartase on excitation at 295 nm (a) and tyrosine fluorescence of E. coli aspartase on excitation at 278 nm (b). Open and closed circles indicate fluorescence at 0 M Gdn-HCl (native state) and 4 M Gdn-HCl (unfolded state), respectively. All spectra were corrected by subtracting the spectrum of the buffer without enzyme. The protein concentration was 50 mg/ml. (c) Thermal unfolding of Bacillus YM55-1 (closed circles, at 360 nm) and E. coli (open circles, at 315 nm) aspartases monitored by fluorescence. The excitation wavelength was 295 nm for Bacillus YM55-1 aspartase and 278 nm for E. coli aspartase. (d) Gdn-HCl unfolding of Bacillus YM55-1 (closed circles) and E. coli (open circles) aspartases monitored by fluorescence. The conditions of the fluorescence measurements were the same as in (c).
in the compositions of the E. coli and P. fluorescens enzymes are evident (data not shown), elucidation of the entire amino acid sequence and tertiary structure of Bacillus YM55-1 aspartase is required for an understanding of the molecular basis of this robust stability. CONCLUDING REMARKS
A new aspartase enzyme was purified from Bacillus sp. YM55-1, a moderate thermophile. Although the enzyme was similar to other mesophilic aspartases in
subunit molecular weight, oligomeric state, and N-terminal amino acid sequence, the structural stability against heat and denaturant of the Bacillus YM55-1 aspartase was remarkable and the specific activity at 30°C was the highest value so far reported. These findings indicate that this newly purified enzyme may be very useful in various industrial processes and also provide a research field in fundamental studies on the structure and function of thermophilic enzymes, especially aspartase.
FIG. 5. Sequence homologies of the first 40 N-terminal amino acids of Bacillus YM55-1 aspartase with the corresponding sequences of various other aspartases. The sequences were aligned manually. Shaded residues indicate residues that were conserved in all of the sequences compared. Asterisks indicate residues that are considered to be involved in the catalysis and substrate binding in E. coli aspartase. The numbers above the sequence correspond to the amino acid residue number of Bacillus YM55-1 aspartase and E. coli aspartase. The P. fluorescens sequence and the B. subtilis sequence are shown beginning from the seventh and fourth residues, respectively.
46
KAWATA ET AL.
REFERENCES 1. Rudolph, F. B., and Fromm, H. J. (1971) Arch. Biochem. Biophys. 147, 92–98. 2. Suzuki, S., Yamaguchi, J., and Tokushige, M. (1973) Biochim. Biophys. Acta 321, 369 –381. 3. Takagi, J. S., Ida, N., Tokushige, M., Sakamoto, H., and Shimura, Y. (1985) Nucleic Acids Res. 13, 2063–2074. 4. Takagi, J. S., Fukunaga, R., Tokushige, M., and Katsuki, H. (1984) J. Biochem. 96, 545–552. 5. Takagi, J. S., Tokushige, M., and Shimura, Y. (1986) J. Biochem. 100, 697–705. 6. Sun, D., and Setlow, P. (1991) J. Bacteriol. 173, 3831–3845. 7. Tokushige, M., Eguchi, G., and Hirata, F. (1977) Biochim. Biophys. Acta 480, 479 – 488. 8. Yumoto, N., and Tokushige, M. (1983) Biochim. Biophys. Acta 749, 101–105. 9. Imaishi, H., Yumoto, N., and Tokushige, M. (1989) Physiol. Chem. Phys. Med. NMR 21, 221–228. 10. Imaishi, H., Yumoto, N., and Tokushige, M. (1990) Biotechnol. Appl. Biochem. 12, 196 –205. 11. Murase, S., Kawata, Y., and Yumoto, N. (1993) J. Biochem. 114, 393–397. 12. Murase, S., Kawata, Y., and Yumoto, N. (1993) Biochem. Biophys. Res. Commun. 195, 1159 –1164. 13. Saribas, A. S., Schindler, J. F., and Viola, R. E. (1994) J. Biol. Chem. 269, 6313– 6319.
14. Shi, W., Dunbar, J., Jayasekera, M. M. K., Viola, R. E., and Farber, G. K. (1997) Biochemistry 36, 9136 –9144. 15. Suzuki, Y., Mino, Y., and Abe, S. (1980) Appl. Microbiol. Biotechnol. 11, 23–27. 16. Mizobata, T., Fujioka, T., Yamasaki, F., Hidaka, M., Nagai, J., and Kawata, Y. (1998) Arch. Biochem. Biophys. 355, 49 – 55. 17. Kagawa, M., Murakoshi, N., Nishikawa, Y., Matsumoto, G., Kurata, Y., Mizobata, T., Kawata, Y., and Nagai, J. (1999) Arch. Biochem. Biophys. 362, 346 –355. 18. Tokushige, M. (1985) Methods Enzymol. 113, 63–72. 19. Dawson, R. M. C., Elliot, D. C., Elliot, W. H., and Jones, K. M. (1986) Data for Biochemical Research, 3rd ed., Clarendon Press, Oxford. 20. Kosuge, T., Umehara, K., and Hoshino, T. (1998) J. Ferment. Bioeng. 86, 125–129. 21. Karsten, W. E., Hunsley, J. R., and Viola, R. E. (1985) Anal. Biochem. 147, 336 –341. 22. Murase, S., Takagi, J. S., Higashi, Y., Imaishi, H., Yumoto, N., and Tokushige, M. (1991) Biochem. Biophys. Res. Commun. 177, 414 – 419. 23. Ida, N., and Tokushige, M. (1984) J. Biochem. 96, 1315–1321. 24. Karsten, W. E., and Viola, R. E. (1991) Arch. Biochem. Biophys. 287, 60 – 67. 25. Jayasekera, M. M. K., Shi, W., Farber, G. K., and Viola, R. E. (1997) Biochemistry 36, 9145–9150.