- Email: [email protected]
Screening, purification, and identification of the enzyme producing N-(l -α-l -aspartyl)-l -phenylalanine methyl ester from l -isoasparagine and l -phenylalanine methyl ester
Journal of Bioscience and Bioengineering VOL. 108 No. 3, 190 – 193, 2009 www.elsevier.com/locate/jbiosc
NOTE
Screening, purification, and identification of the enzyme producing N-(L-α-L-aspartyl)-L-phenylalanine methyl ester from L-isoasparagine and L-phenylalanine methyl ester Ikuo Kira,1,⁎ Yasuhisa Asano,2 and Kenzo Yokozeki1 AminoScience Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan 1 and Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Imizu Toyama 939-0398, Japan 2 Received 30 January 2009; accepted 27 March 2009
Screening was carried out for microorganisms able to produce N-(L-α-L-aspartyl)-L-phenylalanine methyl ester [APM] from and L-phenylalanine methyl ester hydrochloride. Of the 422 strains examined, 44 strains belonging to the family Enterobacteriaceae were found to produce APM. The enzyme catalyzing APM production was purified and identified as dipeptidase E. © 2009, The Society for Biotechnology, Japan. All rights reserved.
L-isoasparagine
[Key words: N-(L-α-aspartyl)-L-phenylalanine-1-methyl ester; L-isoasparagine; L-phenylalanine methyl ester; Dipeptidase E; Screening]
APM is known as aspartame, an excellent sweetening compound, which has a 200-fold higher sweetness than sucrose. APM is currently produced commercially on a scale of many thousands of tons per year by chemical and enzymatic methods. In the process developed by Ajinomoto, N-formyl-L-aspartic acid anhydride and L-phenylalanine methyl ester (PM) are condensed chemically in an organic solvent to form N-formyl-α-L-aspartyl-L-phenylalanine methyl ester (FAPM), which is subsequently deformylated to obtain the desired APM (Yukawa, T., Kawasaki, H., Nakamura, M., Yamashita, T. and Tsuji, T., US Patent, US-4820861, 1986). In the process developed by NutraSweet/Monsanto, N-formyl-L-aspartic acid anhydride and L-phenylalanine are condensed chemically to form N-formyl-α-L-aspartyl-Lphenylalanine (FAP), which is subsequently deformylated and esterified to obtain APM (Hill, J-B. and Gelman, Y., US Patent, US4946988, 1990). These chemical methods give β-forms of FAPM and FAP. In addition, these methods required deformylation of the precursor of APM. On the other hand, in the process developed by DSM/Tosoh, N-benzyloxycarbonyl-L-aspartic acid is enzymatically coupled to PM using thermolysin to give N-benzyloxycarbonyl-APM, which is subsequently deprotected to APM by hydrogenolysis (Irino, S., Oyama, K., Nakamura, S-I., Van Dooren, T-J., and Quaedflieg, P-J-L., European Patent, EP-0664338, 1994). This enzymatic method required both the introduction of a benzyloxycarbonyl group to L-Asp and deprotection of the precursor of APM. Additional enzymatic methods of synthesizing N-(L-α-aspartyl)-L-phenylalanine (AP) and APM have also been reported. Thomas et al. reported AP production by the coupling reaction of L-Asp and L-Phe using an ATP dependent enzyme (1). Arima et al. reported APM production by a coupling reaction of ⁎ Corresponding author. Tel.: +81 59 346 0138; fax: +81 59 346 0140. E-mail address: [email protected] (I. Kira).
L-Asp
and PM using aminopeptidase from Streptomyces septatus (2). The former reaction requires ATP for formation of the peptide bond and gives β-AP. On the other hand, the latter reaction requires methanol as a reaction solvent for peptide bond formation. From an industrial point of view, β-APM production and the use of solvents are not desirable. Prompted by these observations, the present study screened for enzymes producing APM from L-isoasparagine (isoAsn) and PM (Fig. 1) in order to construct a new route for APM production. There have been no previous reports regarding an isoAsn-transferring enzyme. This study reports the screening, purification, and identification of an isoAsn-transferring enzyme for APM production. A total of 422 strains of bacteria (44 genera) and 310 strains of yeasts (21 genera), which were kept at Toyama Prefectural University, were tested for their ability to produce APM or AP from isoAsn and PM. For the first screening, bacteria and yeasts were grown for 1–2 days at 30 °C on CM2G-agar medium (pH 7.0), which contained 5 g/L glucose, 10 g/L yeast extract, 10 g/L peptone, and 5 g/L NaCl, and YM-agar medium (pH 7.0), which contained 10 g/L glucose, 3 g/L yeast extract, 5 g/L peptone, and 3 g/L malt extract. The reaction mixture consisted of 50 μL of 0.2 M potassium acetate buffer, pH 6.0, 0.2 M potassium phosphate buffer, pH 8.0, or 0.2 M sodium carbonate buffer, pH 10.0, each containing 330 mg isoAsn (Sigma-Aldrich Chemical Co.), 108 mg L-phenyl alanine methyl ester hydrochloride (PM.HCl; Sigma-Aldrich Chemical Co.) and approximately 2.5 mg wet weight of bacterial or yeast cells. The reaction was incubated at 30 °C for 2 h, and the synthesis of APM and AP was analyzed by thin layer chromatography with a solvent system of chloroform–methanol–formic acid–water (20:15:3). AP and APM were detected using ninhydrin reagent. In the first screening, 44 strains of bacteria consisting of Cedecea, Enterobacter, Escherichia, Hafnia, Klebsiella, Morganella, Rahnella, Serratia, and Yersinia showed APM and AP-producing activity at pH 10.0.
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FIG. 1. Production of N-(L-α-L-aspartyl)-L-phenylalanine methyl ester from L-isoasparagine and L-phenylalanine methyl ester.
On the other hand, activity was not found at pH 6.0 or pH 8.0. Only bacteria belonging to the family Enterobacteriaceae showed activity among the microorganisms tested. For the second screening, each strain selected from 9 genera, which showed activity in the first screening, was tested for APM- and APproducing activity. The reaction mixture consisted of 500 μL of 0.2 M sodium carbonate buffer, pH 10.0, each containing 3.3 g isoAsn, 1.08 g PM.HCl and approximately 50 mg wet weight of cells, which were grown for 1 day at 30 °C on CM2G-agar medium. The reaction was incubated at 30 °C for 1 h. After completion of the incubation, the reaction mixture was heated at 95 °C for 5 min. The reaction mixture was centrifuged (12,000 ×g, 10 min) and the supernatant was applied to high performance liquid chromatography (HPLC) using an Inertsil ODS column (4.6 × 150 mm, GL Sciences Inc.). The mobile phase was 5 mM potassium phosphate buffer (pH 3.0): methanol (4:1, v:v) and the flow rate was 0.6 ml/min. AP and APM produced were detected at 254 nm. As shown in Table 1, these strains produced 0.7 mM to 2.1 mM of APM and AP from isoAsn and PM.HCl. Because the genome sequence of E. coli has been elucidated, it was thought to be easy to identify an isoAsn-transferring enzyme from E. coli. Thus, E. coli TPU6303 was selected as the representative strain and used for further experiments. In order to confirm the production of APM and AP by the desired reaction, the reaction using various substrate solutions, which were 1) 50 mM of isoAsn and 150 mM of PM.HCl, 2) 50 mM of isoAsn and 150 mM of Phe, 3) 50 mM of Asp and 150 mM of PM.HCl, and 4) 50 mM of Asp and 150 mM of Phe, were performed at pH 10.0 for 1 h. E. coli TPU6303 wet cells were used as enzyme sources. Among these reactions, only the combination of isoAsn and PM gave APM and AP. Judging from APM production from isoAsn and PM.HCl, it was speculated that AP was produced by chemical and/or enzymatic degradation of APM. To confirm the reaction product produced by E. coli TPU6303, purification and identification of the reaction product were performed. One loop-full of cells subcultured on CM2G-agar medium was inoculated into 4 mL of CM2G-broth in 10 mL test tube and cultured aerobically in a reciprocal shaker at 30 °C for 16 h. Then, 0.5 mL of the culture broth was transferred into 50 mL of CM2G-broth in 500 mL sakaguchi flasks and cultured aerobically in a reciprocal shaker at 30 °C for 16 h. The cells were harvested by centrifugation (8000 ×g,
TABLE 1 Strains Cedecea lapagei TPU 5750 Enterobacter aerogenes TPU 6151 Escherichia coli TPU 6303 Hafnia alvei TPU 6440 Klebsiella planticola TPU 6501 Morganella morganii TPU 6706 Rahnella aquatilis TPU 5901 Serratia marcescens TPU 7303 Yersinia aldovae TPU 7650
APM produced (mM)
AP produced (mM)
APM + AP produced (mM)
0.20 0.30 0.41 0.35 0.20 0.72 0.24 0.85 0.81
0.50 0.94 1.09 0.80 0.88 1.36 0.96 1.21 1.07
0.70 1.24 1.50 1.25 1.08 2.08 1.20 2.06 1.88
10 min) and then washed with 0.85% NaCl. The reaction mixture for APM production contained 0.66 g isoAsn and 10.8 g PM.HCl, plus 5 g wet cells in a total volume of 100 mL of 0.2 M sodium carbonate buffer (pH 10.0). The reaction was performed at 30 °C for 6 h and the reaction mixture was boiled for 10 min to stop the enzyme reaction. Then, the reaction mixture was centrifuged (8000 ×g, 10 min) to remove the cells. The resulting supernatant was concentrated at reduced pressure. The residue was purified by thin layer chromatography using a solvent system of chloroform–methanol–formic acid–water (20:15:3). The reaction product produced by E. coli TPU6303 isolated was identified as AP. 1H NMR spectrum: (D2O)δppm 7.16–7.30 (m, 5 H), 4.45 (dd, 1 H, J = 5.61, 8.78 Hz), 4.08 (dd, 1 H, J = 4.63, 8.44 Hz), 3.09–3.14 (m, 1 H), and 2.61–2.94 (m, 3 H). In order to identify an isoAsn-transferring enzyme from E. coli TPU6303, the enzyme from E. coli TPU6303 was purified, and the Nterminal amino acid sequence of the enzyme was analyzed as described below. All purification procedures were performed at 0–4 °C. The washed cells (127 g wet weight) from 4.2 L of E. coli TPU6303 culture broth were suspended in 1.26 L of saline. The cells were disrupted using an ultra sonicator (Kubota Corporation, Japan) at 20 kHz for 10 min. The disrupted cell suspension was centrifuged at 8000 ×g for 10 min. The supernatant was used as the crude extract. The cell-free extract was fractionated by salting out with ammonium sulfate 30% to 60% saturation, followed by dialysis against 20 mM Tris–HCl buffer (pH 8.3). The dialysate was applied to a DEAE–Toyopearl 650 M column (42 mm × 220 mm; Tosoh Corporation, Tokyo, Japan) equilibrated with 20 mM Tris–HCl buffer (pH 8.3). The column was washed with 600 mL of buffer and the enzyme was eluted with a 1.2 L linear gradient of NaCl (0 to 0.2 M) in buffer. The active fractions were collected and ammonium sulfate was added (30% saturation) to the active fraction. The fraction was centrifuged at 8000 ×g for 10 min. The fraction was applied to a Butyl–Toyopearl column (42 mm × 220 mm; Tosoh Corporation) equilibrated with 20 mM Tris–HCl buffer (pH 8.3) containing ammonium sulfate (30% saturation). The column was washed with 600 mL of buffer, and the enzyme was eluted with a 1.2 L linear gradient of ammonium sulfate (30% to 0% saturation) in the buffer. The active fractions were collected and dialyzed against 20 mM Tris–HCl buffer (pH 8.3). The dialyzed enzyme solution was applied to a DEAE–Toyopearl column (32 mm × 250 mm) equilibrated with 20 mM Tris–HCl buffer (pH 8.3). The column was washed with 400 mL of buffer and the enzyme was eluted with an 800 mL linear gradient of NaCl (0 to 0.2 M) in buffer. The active fractions were collected and dialyzed against 10 mM potassium phosphate buffer (pH 7.0). The active fractions were concentrated (136 mL to 20 mL) with Centriprep-10 ultrafiltration unit (Amicon Inc., Beverly, MA, USA). The concentrated enzyme solution was applied to a Gigapite column (24 mm × 44 mm; Toagosei Chemical Industry Co., Ltd, Tokyo, Japan) equilibrated with 10 mM potassium phosphate buffer (pH 7.0). The enzyme was eluted with 50 mL of buffer and the active fractions were collected and dialyzed against 20 mM Tris–HCl buffer (pH 8.3). The dialyzed enzyme solution was applied to a DEAE–Toyopearl column (24 mm × 44 mm) equilibrated with 20 mM Tris–HCl buffer (pH 8.3).
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J. BIOSCI. BIOENG.,
The column was washed with 40 mL of buffer and the enzyme was eluted with a 100 mL linear gradient of NaCl (0 to 0.2 M) in buffer. The active fractions were collected and dialyzed against 10 mM potassium phosphate buffer (pH 7.0). The dialyzed enzyme solution was applied to a MonoQ HR 5/5 column (5 mm × 50 mm; GE Healthcare UK Ltd, Buckinghamshire, UK) equilibrated with 10 mM potassium phosphate buffer (pH 7.0). The enzyme was eluted with a 30 mL linear gradient of NaCl (0 to 0.2 M) in buffer at a flow rate of 1.0 mL/min. The active fractions were concentrated (15.6 mL to 4 mL) using a Centriprep-10 ultrafiltration unit and then applied to a Superdex ™ 200 column (10 mm × 300 mm; GE Healthcare UK Ltd, Buckinghamshire, UK) equilibrated with 20 mM potassium phosphate buffer (pH 7.0) containing 0.2 M NaCl. The enzyme was eluted with the buffer at a flow rate of 0.4 mL/min. The active fraction was used as the purified enzyme. The activity of the isoAsn-transferring enzyme from E. coli TPU6303 was determined by measuring α-aspartyl-hydroxamate formed from isoAsn and hydroxyl amine hydrochloride. The standard assay mixture comprised 0.2 M sodium carbonate buffer (pH 10.0), 50 mM isoAsn, 0.5 M hydroxyl amine hydrochloride, and an appropriate amount of enzyme solution. The substrate solution containing isoAsn and hydroxyl amine hydrochloride was adjusted at pH 10.0 with 6 N NaOH before adding the enzyme. The reaction was performed at 30 °C for 1 h. After the reaction was completed, 750 μL of color-producing reagents for aspartyl-hydroxamate (100 g FeCl3 and 33 g trichloroacetic acid was dissolved in 1 L of 0.7 M HCl) was added to the reaction mixture. Then the reaction mixture was centrifuged (12,000 ×g, 5 min) to remove the insoluble materials. The absorbance of the supernatant was measured at 515 nm in a spectrophotometer. One unit of the enzyme was defined as the amount producing 1 μmol of aspartyl-hydroxamate per minute. As α-aspartyl-hydroxamate was not available, it was postulated that the molar absorbance coefficient of α-aspartyl-hydroxamate was the same as that of β-aspartylhydroxamate (Sigma-Aldrich Chemical Co.). Protein concentrations were measured using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) using bovine serum albumin as the standard (3). The purification of the enzyme is summarized in Table 2. The enzyme was purified 2527-fold to homogeneity with an overall recovery of 1.2%. SDS-PAGE was performed in a 5–20% gradient acrylamide slab gel (ATTO Corporation, Japan), as described by Laemmli (4), and the subunit molecular weight was estimated to be about 25,000 by SDS-PAGE (Fig. 2). The N-terminal amino acid sequence was analyzed using an automatic protein sequencer (6625 Prosequencer; Millipore Corp., USA), and its N-terminal amino acid sequence was compared with those of other proteins in the SWISS-PROT protein database. The N-terminal 20 amino acid sequence of the enzyme was MELLLLSNSTLPGKA-LE-A, and the amino acid sequence of the enzyme was same as that of dipeptidase E (EC3.4.13.21) from E. coli K-12 (N-terminal amino acid
TABLE 2. Purification of the LisoAsn-transferring enzyme from E. coli TPU6303. Step
Cell-free extract Ammonium sulfate (30–60%) DEAE–Toyopearl Butyl–Toyopearl DEAE–Toyopearl Gigapite DEAE–Toyopearl MonoQ Superdex
Total activity (units) 25.4 20.2 16.1 5.47 2.00 1.63 1.23 0.78 0.31
Total protein (mg) 18926 8434 2315 225 41.2 7.7 5.1 0.5 0.09
Specific activity (units/mg)
Yield (%)
Purification fold
0.00134 0.00240
100 79.7
1 1.8
0.00695 0.0242 0.0484 0.213 0.240 1.66 3.39
63.4 21.5 7.9 6.4 4.8 3.1 1.2
5.2 18.0 36.0 158 179 1240 2527
FIG. 2. SDS-PAGE of the isoAsn-transferring enzyme from Escherichia coli TPU6303. (a) Standards (from top): phosphorylase b (Mr = 94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), and trypsin inhibitor (20,000) (Daiichi Pure Chemicals, Tokyo, Japan). (b) Purified enzyme. The arrow indicates the position of the purified enzyme (Mr = 25,000). The gel was stained with Coomassie Brilliant Blue R-250 for protein.
sequence: MELLLLSNSTLPGKAWLEHA). In addition, the molecular weight of dipeptidase E from E. coli was predicted to be 24,800 from the ORF of pepE, which encodes the dipeptidase E gene (5). Judging from the N-terminal amino acid sequence and molecular weight of the enzyme, isoAsn-transferring enzyme selected through the screening was suggested to be dipeptidase E. The pepE genes from E. coli and Salmonella typhimurium, Citrobacter koseri, and Shigella flexneri among the family Enterobacteriaceae was reported and APM-producing microorganisms found through the screening might possess dipeptidase E, which might catalyze the APM-producing reaction. Characterization of a dipeptidase E from E. coli has not been reported previously, whereas a dipeptidase E from S. typhimurium, which showed 89% amino acid sequence homology with that from E. coli, had been purified and characterized (6). Salmonella dipeptidase E catalyzed the hydrolysis of dipeptides containing N-terminal aspartate residues, but it did not act on peptides with N-terminal Glu, Asn, or Gln, nor did it cleave isoaspartyl peptides. Salmonella dipeptidase E showed hydrolyzing activity toward L-aspartic acid p-nitroanilide at pH 5.5–9.0, whereas APM production was detected at pH 10 but not at pH 6 or pH 8 in the screening. It was speculated that peptide bond formation from an amide residue of isoAsn and an amino residue of PM required nucleophilic attack of the amino residue of PM to the enzyme–isoAsn complex, and in the neutral pH range, the nucleophilicity of the amino residue of PM was lower than that in the alkaline pH range, and the hydrolysis reaction of isoAsn proceeded. Thus, it was speculated that the hydrolyzing activity for isoAsn was higher at pH 6–8 and transferring activity for isoAsn was higher at pH 10. In addition, Salmonella dipeptidase E showed 12 times higher activity for hydrolyzing APM than that of AP
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(6). The enzyme from E. coli produced APM from isoAsn and PM.HCl, whereas this enzyme did not produce AP from isoAsn and Phe. When Phe was used as the substrate, the reaction product, AP, may be hydrolyzed rapidly during the reaction. This is the first report regarding the coupling reaction of isoAsn and PM.HCl by dipeptidase E. From an industrial point of view, in the present study APM accumulation was low. However, it was thought that the conversion yield and accumulation of APM could be improved by modification of the enzyme and optimization of the reaction conditions. Further studies are in progress to optimize the reaction conditions using the enzyme overexpressed in recombinant E. coli. ACKNOWLEDGMENTS We thank Drs. T. Onuki, K. Izawa, H. Yamada, and Y. Kato of our laboratory for their encouragement and helpful suggestions throughout this study.
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References 1. Duerfahrt, T., Doekel, S., Sonke, T., Quaedfieg, P. -J. -L. -M., and Marahiel, M.-A.: Construction of hybrid peptide synthetases for the production of α-L-aspartyl-Lphenylalanine, a precursor for the high-intensity sweetener aspartame, Eur. J. Biochem., 270, 4555–4563 (2003). 2. Arima, J., Uesugi, Y., Uraji, M., Iwabuchi, M., and Hatanaka, T.: Dipeptide synthesis by an aminopeptidase from Streptomyces septatus TH-2 and its application to synthesis of biologically active peptides, Appl. Environ. Microbiol., 72, 4225–4231 (2006). 3. Bradford, M.-M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem., 72, 248–254 (1976). 4. Laemmli, U.-K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227, 680–685 (1970). 5. Conlin, C.-A., Knox, T.-M., and Miller, C.-G.: Cloning and physical map position of an α-aspartyl dipeptidase gene, pepE, from Escherichia coli, J. Bacteriol., 176, 1552–1553 (1994). 6. Carter, T.-H. and Miller, C.-G.: Aspartate-specific peptidases in Salmonella typhimurium: mutants deficient in peptidase, E. J. Bacteriol., 159, 453–459 (1984).
NOTE
Screening, purification, and identification of the enzyme producing N-(L-α-L-aspartyl)-L-phenylalanine methyl ester from L-isoasparagine and L-phenylalanine methyl ester Ikuo Kira,1,⁎ Yasuhisa Asano,2 and Kenzo Yokozeki1 AminoScience Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan 1 and Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Imizu Toyama 939-0398, Japan 2 Received 30 January 2009; accepted 27 March 2009
Screening was carried out for microorganisms able to produce N-(L-α-L-aspartyl)-L-phenylalanine methyl ester [APM] from and L-phenylalanine methyl ester hydrochloride. Of the 422 strains examined, 44 strains belonging to the family Enterobacteriaceae were found to produce APM. The enzyme catalyzing APM production was purified and identified as dipeptidase E. © 2009, The Society for Biotechnology, Japan. All rights reserved.
L-isoasparagine
[Key words: N-(L-α-aspartyl)-L-phenylalanine-1-methyl ester; L-isoasparagine; L-phenylalanine methyl ester; Dipeptidase E; Screening]
APM is known as aspartame, an excellent sweetening compound, which has a 200-fold higher sweetness than sucrose. APM is currently produced commercially on a scale of many thousands of tons per year by chemical and enzymatic methods. In the process developed by Ajinomoto, N-formyl-L-aspartic acid anhydride and L-phenylalanine methyl ester (PM) are condensed chemically in an organic solvent to form N-formyl-α-L-aspartyl-L-phenylalanine methyl ester (FAPM), which is subsequently deformylated to obtain the desired APM (Yukawa, T., Kawasaki, H., Nakamura, M., Yamashita, T. and Tsuji, T., US Patent, US-4820861, 1986). In the process developed by NutraSweet/Monsanto, N-formyl-L-aspartic acid anhydride and L-phenylalanine are condensed chemically to form N-formyl-α-L-aspartyl-Lphenylalanine (FAP), which is subsequently deformylated and esterified to obtain APM (Hill, J-B. and Gelman, Y., US Patent, US4946988, 1990). These chemical methods give β-forms of FAPM and FAP. In addition, these methods required deformylation of the precursor of APM. On the other hand, in the process developed by DSM/Tosoh, N-benzyloxycarbonyl-L-aspartic acid is enzymatically coupled to PM using thermolysin to give N-benzyloxycarbonyl-APM, which is subsequently deprotected to APM by hydrogenolysis (Irino, S., Oyama, K., Nakamura, S-I., Van Dooren, T-J., and Quaedflieg, P-J-L., European Patent, EP-0664338, 1994). This enzymatic method required both the introduction of a benzyloxycarbonyl group to L-Asp and deprotection of the precursor of APM. Additional enzymatic methods of synthesizing N-(L-α-aspartyl)-L-phenylalanine (AP) and APM have also been reported. Thomas et al. reported AP production by the coupling reaction of L-Asp and L-Phe using an ATP dependent enzyme (1). Arima et al. reported APM production by a coupling reaction of ⁎ Corresponding author. Tel.: +81 59 346 0138; fax: +81 59 346 0140. E-mail address: [email protected] (I. Kira).
L-Asp
and PM using aminopeptidase from Streptomyces septatus (2). The former reaction requires ATP for formation of the peptide bond and gives β-AP. On the other hand, the latter reaction requires methanol as a reaction solvent for peptide bond formation. From an industrial point of view, β-APM production and the use of solvents are not desirable. Prompted by these observations, the present study screened for enzymes producing APM from L-isoasparagine (isoAsn) and PM (Fig. 1) in order to construct a new route for APM production. There have been no previous reports regarding an isoAsn-transferring enzyme. This study reports the screening, purification, and identification of an isoAsn-transferring enzyme for APM production. A total of 422 strains of bacteria (44 genera) and 310 strains of yeasts (21 genera), which were kept at Toyama Prefectural University, were tested for their ability to produce APM or AP from isoAsn and PM. For the first screening, bacteria and yeasts were grown for 1–2 days at 30 °C on CM2G-agar medium (pH 7.0), which contained 5 g/L glucose, 10 g/L yeast extract, 10 g/L peptone, and 5 g/L NaCl, and YM-agar medium (pH 7.0), which contained 10 g/L glucose, 3 g/L yeast extract, 5 g/L peptone, and 3 g/L malt extract. The reaction mixture consisted of 50 μL of 0.2 M potassium acetate buffer, pH 6.0, 0.2 M potassium phosphate buffer, pH 8.0, or 0.2 M sodium carbonate buffer, pH 10.0, each containing 330 mg isoAsn (Sigma-Aldrich Chemical Co.), 108 mg L-phenyl alanine methyl ester hydrochloride (PM.HCl; Sigma-Aldrich Chemical Co.) and approximately 2.5 mg wet weight of bacterial or yeast cells. The reaction was incubated at 30 °C for 2 h, and the synthesis of APM and AP was analyzed by thin layer chromatography with a solvent system of chloroform–methanol–formic acid–water (20:15:3). AP and APM were detected using ninhydrin reagent. In the first screening, 44 strains of bacteria consisting of Cedecea, Enterobacter, Escherichia, Hafnia, Klebsiella, Morganella, Rahnella, Serratia, and Yersinia showed APM and AP-producing activity at pH 10.0.
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FIG. 1. Production of N-(L-α-L-aspartyl)-L-phenylalanine methyl ester from L-isoasparagine and L-phenylalanine methyl ester.
On the other hand, activity was not found at pH 6.0 or pH 8.0. Only bacteria belonging to the family Enterobacteriaceae showed activity among the microorganisms tested. For the second screening, each strain selected from 9 genera, which showed activity in the first screening, was tested for APM- and APproducing activity. The reaction mixture consisted of 500 μL of 0.2 M sodium carbonate buffer, pH 10.0, each containing 3.3 g isoAsn, 1.08 g PM.HCl and approximately 50 mg wet weight of cells, which were grown for 1 day at 30 °C on CM2G-agar medium. The reaction was incubated at 30 °C for 1 h. After completion of the incubation, the reaction mixture was heated at 95 °C for 5 min. The reaction mixture was centrifuged (12,000 ×g, 10 min) and the supernatant was applied to high performance liquid chromatography (HPLC) using an Inertsil ODS column (4.6 × 150 mm, GL Sciences Inc.). The mobile phase was 5 mM potassium phosphate buffer (pH 3.0): methanol (4:1, v:v) and the flow rate was 0.6 ml/min. AP and APM produced were detected at 254 nm. As shown in Table 1, these strains produced 0.7 mM to 2.1 mM of APM and AP from isoAsn and PM.HCl. Because the genome sequence of E. coli has been elucidated, it was thought to be easy to identify an isoAsn-transferring enzyme from E. coli. Thus, E. coli TPU6303 was selected as the representative strain and used for further experiments. In order to confirm the production of APM and AP by the desired reaction, the reaction using various substrate solutions, which were 1) 50 mM of isoAsn and 150 mM of PM.HCl, 2) 50 mM of isoAsn and 150 mM of Phe, 3) 50 mM of Asp and 150 mM of PM.HCl, and 4) 50 mM of Asp and 150 mM of Phe, were performed at pH 10.0 for 1 h. E. coli TPU6303 wet cells were used as enzyme sources. Among these reactions, only the combination of isoAsn and PM gave APM and AP. Judging from APM production from isoAsn and PM.HCl, it was speculated that AP was produced by chemical and/or enzymatic degradation of APM. To confirm the reaction product produced by E. coli TPU6303, purification and identification of the reaction product were performed. One loop-full of cells subcultured on CM2G-agar medium was inoculated into 4 mL of CM2G-broth in 10 mL test tube and cultured aerobically in a reciprocal shaker at 30 °C for 16 h. Then, 0.5 mL of the culture broth was transferred into 50 mL of CM2G-broth in 500 mL sakaguchi flasks and cultured aerobically in a reciprocal shaker at 30 °C for 16 h. The cells were harvested by centrifugation (8000 ×g,
TABLE 1 Strains Cedecea lapagei TPU 5750 Enterobacter aerogenes TPU 6151 Escherichia coli TPU 6303 Hafnia alvei TPU 6440 Klebsiella planticola TPU 6501 Morganella morganii TPU 6706 Rahnella aquatilis TPU 5901 Serratia marcescens TPU 7303 Yersinia aldovae TPU 7650
APM produced (mM)
AP produced (mM)
APM + AP produced (mM)
0.20 0.30 0.41 0.35 0.20 0.72 0.24 0.85 0.81
0.50 0.94 1.09 0.80 0.88 1.36 0.96 1.21 1.07
0.70 1.24 1.50 1.25 1.08 2.08 1.20 2.06 1.88
10 min) and then washed with 0.85% NaCl. The reaction mixture for APM production contained 0.66 g isoAsn and 10.8 g PM.HCl, plus 5 g wet cells in a total volume of 100 mL of 0.2 M sodium carbonate buffer (pH 10.0). The reaction was performed at 30 °C for 6 h and the reaction mixture was boiled for 10 min to stop the enzyme reaction. Then, the reaction mixture was centrifuged (8000 ×g, 10 min) to remove the cells. The resulting supernatant was concentrated at reduced pressure. The residue was purified by thin layer chromatography using a solvent system of chloroform–methanol–formic acid–water (20:15:3). The reaction product produced by E. coli TPU6303 isolated was identified as AP. 1H NMR spectrum: (D2O)δppm 7.16–7.30 (m, 5 H), 4.45 (dd, 1 H, J = 5.61, 8.78 Hz), 4.08 (dd, 1 H, J = 4.63, 8.44 Hz), 3.09–3.14 (m, 1 H), and 2.61–2.94 (m, 3 H). In order to identify an isoAsn-transferring enzyme from E. coli TPU6303, the enzyme from E. coli TPU6303 was purified, and the Nterminal amino acid sequence of the enzyme was analyzed as described below. All purification procedures were performed at 0–4 °C. The washed cells (127 g wet weight) from 4.2 L of E. coli TPU6303 culture broth were suspended in 1.26 L of saline. The cells were disrupted using an ultra sonicator (Kubota Corporation, Japan) at 20 kHz for 10 min. The disrupted cell suspension was centrifuged at 8000 ×g for 10 min. The supernatant was used as the crude extract. The cell-free extract was fractionated by salting out with ammonium sulfate 30% to 60% saturation, followed by dialysis against 20 mM Tris–HCl buffer (pH 8.3). The dialysate was applied to a DEAE–Toyopearl 650 M column (42 mm × 220 mm; Tosoh Corporation, Tokyo, Japan) equilibrated with 20 mM Tris–HCl buffer (pH 8.3). The column was washed with 600 mL of buffer and the enzyme was eluted with a 1.2 L linear gradient of NaCl (0 to 0.2 M) in buffer. The active fractions were collected and ammonium sulfate was added (30% saturation) to the active fraction. The fraction was centrifuged at 8000 ×g for 10 min. The fraction was applied to a Butyl–Toyopearl column (42 mm × 220 mm; Tosoh Corporation) equilibrated with 20 mM Tris–HCl buffer (pH 8.3) containing ammonium sulfate (30% saturation). The column was washed with 600 mL of buffer, and the enzyme was eluted with a 1.2 L linear gradient of ammonium sulfate (30% to 0% saturation) in the buffer. The active fractions were collected and dialyzed against 20 mM Tris–HCl buffer (pH 8.3). The dialyzed enzyme solution was applied to a DEAE–Toyopearl column (32 mm × 250 mm) equilibrated with 20 mM Tris–HCl buffer (pH 8.3). The column was washed with 400 mL of buffer and the enzyme was eluted with an 800 mL linear gradient of NaCl (0 to 0.2 M) in buffer. The active fractions were collected and dialyzed against 10 mM potassium phosphate buffer (pH 7.0). The active fractions were concentrated (136 mL to 20 mL) with Centriprep-10 ultrafiltration unit (Amicon Inc., Beverly, MA, USA). The concentrated enzyme solution was applied to a Gigapite column (24 mm × 44 mm; Toagosei Chemical Industry Co., Ltd, Tokyo, Japan) equilibrated with 10 mM potassium phosphate buffer (pH 7.0). The enzyme was eluted with 50 mL of buffer and the active fractions were collected and dialyzed against 20 mM Tris–HCl buffer (pH 8.3). The dialyzed enzyme solution was applied to a DEAE–Toyopearl column (24 mm × 44 mm) equilibrated with 20 mM Tris–HCl buffer (pH 8.3).
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The column was washed with 40 mL of buffer and the enzyme was eluted with a 100 mL linear gradient of NaCl (0 to 0.2 M) in buffer. The active fractions were collected and dialyzed against 10 mM potassium phosphate buffer (pH 7.0). The dialyzed enzyme solution was applied to a MonoQ HR 5/5 column (5 mm × 50 mm; GE Healthcare UK Ltd, Buckinghamshire, UK) equilibrated with 10 mM potassium phosphate buffer (pH 7.0). The enzyme was eluted with a 30 mL linear gradient of NaCl (0 to 0.2 M) in buffer at a flow rate of 1.0 mL/min. The active fractions were concentrated (15.6 mL to 4 mL) using a Centriprep-10 ultrafiltration unit and then applied to a Superdex ™ 200 column (10 mm × 300 mm; GE Healthcare UK Ltd, Buckinghamshire, UK) equilibrated with 20 mM potassium phosphate buffer (pH 7.0) containing 0.2 M NaCl. The enzyme was eluted with the buffer at a flow rate of 0.4 mL/min. The active fraction was used as the purified enzyme. The activity of the isoAsn-transferring enzyme from E. coli TPU6303 was determined by measuring α-aspartyl-hydroxamate formed from isoAsn and hydroxyl amine hydrochloride. The standard assay mixture comprised 0.2 M sodium carbonate buffer (pH 10.0), 50 mM isoAsn, 0.5 M hydroxyl amine hydrochloride, and an appropriate amount of enzyme solution. The substrate solution containing isoAsn and hydroxyl amine hydrochloride was adjusted at pH 10.0 with 6 N NaOH before adding the enzyme. The reaction was performed at 30 °C for 1 h. After the reaction was completed, 750 μL of color-producing reagents for aspartyl-hydroxamate (100 g FeCl3 and 33 g trichloroacetic acid was dissolved in 1 L of 0.7 M HCl) was added to the reaction mixture. Then the reaction mixture was centrifuged (12,000 ×g, 5 min) to remove the insoluble materials. The absorbance of the supernatant was measured at 515 nm in a spectrophotometer. One unit of the enzyme was defined as the amount producing 1 μmol of aspartyl-hydroxamate per minute. As α-aspartyl-hydroxamate was not available, it was postulated that the molar absorbance coefficient of α-aspartyl-hydroxamate was the same as that of β-aspartylhydroxamate (Sigma-Aldrich Chemical Co.). Protein concentrations were measured using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) using bovine serum albumin as the standard (3). The purification of the enzyme is summarized in Table 2. The enzyme was purified 2527-fold to homogeneity with an overall recovery of 1.2%. SDS-PAGE was performed in a 5–20% gradient acrylamide slab gel (ATTO Corporation, Japan), as described by Laemmli (4), and the subunit molecular weight was estimated to be about 25,000 by SDS-PAGE (Fig. 2). The N-terminal amino acid sequence was analyzed using an automatic protein sequencer (6625 Prosequencer; Millipore Corp., USA), and its N-terminal amino acid sequence was compared with those of other proteins in the SWISS-PROT protein database. The N-terminal 20 amino acid sequence of the enzyme was MELLLLSNSTLPGKA-LE-A, and the amino acid sequence of the enzyme was same as that of dipeptidase E (EC3.4.13.21) from E. coli K-12 (N-terminal amino acid
TABLE 2. Purification of the LisoAsn-transferring enzyme from E. coli TPU6303. Step
Cell-free extract Ammonium sulfate (30–60%) DEAE–Toyopearl Butyl–Toyopearl DEAE–Toyopearl Gigapite DEAE–Toyopearl MonoQ Superdex
Total activity (units) 25.4 20.2 16.1 5.47 2.00 1.63 1.23 0.78 0.31
Total protein (mg) 18926 8434 2315 225 41.2 7.7 5.1 0.5 0.09
Specific activity (units/mg)
Yield (%)
Purification fold
0.00134 0.00240
100 79.7
1 1.8
0.00695 0.0242 0.0484 0.213 0.240 1.66 3.39
63.4 21.5 7.9 6.4 4.8 3.1 1.2
5.2 18.0 36.0 158 179 1240 2527
FIG. 2. SDS-PAGE of the isoAsn-transferring enzyme from Escherichia coli TPU6303. (a) Standards (from top): phosphorylase b (Mr = 94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), and trypsin inhibitor (20,000) (Daiichi Pure Chemicals, Tokyo, Japan). (b) Purified enzyme. The arrow indicates the position of the purified enzyme (Mr = 25,000). The gel was stained with Coomassie Brilliant Blue R-250 for protein.
sequence: MELLLLSNSTLPGKAWLEHA). In addition, the molecular weight of dipeptidase E from E. coli was predicted to be 24,800 from the ORF of pepE, which encodes the dipeptidase E gene (5). Judging from the N-terminal amino acid sequence and molecular weight of the enzyme, isoAsn-transferring enzyme selected through the screening was suggested to be dipeptidase E. The pepE genes from E. coli and Salmonella typhimurium, Citrobacter koseri, and Shigella flexneri among the family Enterobacteriaceae was reported and APM-producing microorganisms found through the screening might possess dipeptidase E, which might catalyze the APM-producing reaction. Characterization of a dipeptidase E from E. coli has not been reported previously, whereas a dipeptidase E from S. typhimurium, which showed 89% amino acid sequence homology with that from E. coli, had been purified and characterized (6). Salmonella dipeptidase E catalyzed the hydrolysis of dipeptides containing N-terminal aspartate residues, but it did not act on peptides with N-terminal Glu, Asn, or Gln, nor did it cleave isoaspartyl peptides. Salmonella dipeptidase E showed hydrolyzing activity toward L-aspartic acid p-nitroanilide at pH 5.5–9.0, whereas APM production was detected at pH 10 but not at pH 6 or pH 8 in the screening. It was speculated that peptide bond formation from an amide residue of isoAsn and an amino residue of PM required nucleophilic attack of the amino residue of PM to the enzyme–isoAsn complex, and in the neutral pH range, the nucleophilicity of the amino residue of PM was lower than that in the alkaline pH range, and the hydrolysis reaction of isoAsn proceeded. Thus, it was speculated that the hydrolyzing activity for isoAsn was higher at pH 6–8 and transferring activity for isoAsn was higher at pH 10. In addition, Salmonella dipeptidase E showed 12 times higher activity for hydrolyzing APM than that of AP
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(6). The enzyme from E. coli produced APM from isoAsn and PM.HCl, whereas this enzyme did not produce AP from isoAsn and Phe. When Phe was used as the substrate, the reaction product, AP, may be hydrolyzed rapidly during the reaction. This is the first report regarding the coupling reaction of isoAsn and PM.HCl by dipeptidase E. From an industrial point of view, in the present study APM accumulation was low. However, it was thought that the conversion yield and accumulation of APM could be improved by modification of the enzyme and optimization of the reaction conditions. Further studies are in progress to optimize the reaction conditions using the enzyme overexpressed in recombinant E. coli. ACKNOWLEDGMENTS We thank Drs. T. Onuki, K. Izawa, H. Yamada, and Y. Kato of our laboratory for their encouragement and helpful suggestions throughout this study.
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