- Email: [email protected]
Formation of γ-glutamylglycyglycine by extracellular glutaminase of Aspergillus oryzae
[J. Ferment. Technol., Vol. 66, No. 3, 299-304. 1988]
Formation of ?-Glutamylglycylglycine by Extracellular Glutaminase of Aspergillus oryzae KENJI TOMITA*, TOSHIHIRO YANO, HIDEHIKO KUMAGAI, and TATSUROKURO TOCHIKURA
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan ~,-Glutamylglycylglycine (y-GluGlyGly) was formed through the y-glutamyltranspeptidase (GGT) reaction catalyzed by glutaminase in a water extract of wheat bran koji obtained with Aspergillus o~yzae MA-27-IM. The yield of y-GluGlyGly was about 18% from L-glutamine in a reaction mixture containing 50 m M L-glutamine, 50 m M glycylglycine, and the extract (0.1 unit ml as GGT activity) in a 100 m M Tris-HC1 buffer solution (pH 7.2), which was incubated for 7 h at 30°C. The y-GluGlyGly formed was purified by ion exchange chromatographies, and the identified by chemical and enzymatic methods as well as by infrared and P M R spectroscopic analyses.
In a previous study, l) we isolated Aspergillus oryzae MA-27-IM as a high glutaminase producer from a commercial koji seed for soy sauce fermentation, and wheat bran koji was prepared with this strain to investigate the properties of its intraceUular and extracellular glutaminases (L-glutamine amidohydrolase, EC 3.5.1.2). T h e results suggested that the two glutaminases might be the same protein. And the following study 2) revealed that the extracellular glutaminase of this strain possessed a considerable y-glutamyltranspeptidase (GGT) activity. The glutaminase from koji mold, Aspergillus oryzae or sojae, is generally regarded as a key enzyme that controls the delicious taste of fermented foods such as soy sauce.S,4) Thus the fact that glutaminase of a koji mold has G G T activity is interesting and significant from the viewpoint of glutamic acid content of food, on which the taste of the food depends. In this study, as the first step to clarify the significance of the G G T reaction in food fermentation involving koji molds, a yglutamyl peptide, y-glutamylglycylglycine (y-GluGlyGly), was formed from natural * Corresponding author --
substrates, L-glutamine and glycylglycine, using the water extract of a wheat bran koji obtained with strain MA-27-IM, and the peptide was also isolated and identified. Materials and Methods l~elicroorgl~mtlsm and ko,jl c u l t u r e A wheat bran koji with AspergiUus oryzae MA-27-IM was prepared as described in our previous paper, t ) Enzyme preparation The wheat bran koji was soaked in twice its weight of distilled water at 4°C for 12 h and then filtered through cotton cloth. The filtrate was centrifuged at 10,000×g for 20rain. Ammonium sulfate was added to the supernatant to achieve 85% saturation. The solution was centrifuged at 10,000 ×g for 20 rain to obtain the precipitate. The latter was suspended in a small amount of potassium phosphate buffer (pH 7.2, 10raM) and then dialyzed. The solution was used as the enzyme preparation. Enzyme assay Glutaminase activity was assayed by a modification of the method of Orlowski and Meister. s) The assay mixture contained 2.5 m M of L-y-glutamyl-p-nitroanilide, 100mM Tris-HC1 buffer (pH7.2), and the enzyme preparation (100/d), in a final volume of 1 ml. If necessary, the enzyme preparation was diluted with 100 m M Tris-HG1 buffer (pH 7.2). After incubation at 30°C for 30 rain, the enzyme reaction was stopped by the addition of 2 ml of 1.5 N acetic acid to the mixture, and then the absorbance at 410 nm was measured. ~,-Glutamyl-
300
TOMITAet al.
transpeptidase (GGT) activity was measured in the same way as glutaminase activity with 50 mM glycylglycine as the 7-glutamyl acceptor in the reaction mixture. One unit of either activity was defined as the amount of enzyme that catalyzed the release of 1/zmole of p-nitroaniline per minute under the conditions described above. The molar extinction coefficient ofp-nitroaniline was 8.3 × 103 M.cm -x. Reaction system 7-GluGlyGly was formed in a reaction mixture of 50 mM L-glutamine, 50 mM glycylglycine, 100 mM Tris-HC1 buffer (pH 7.2), and the enzyme preparation containing 0.1 unit.ml-I of GGT. The reaction was done at 30°C, and stopped by immersing the reaction vessel in boiling water for 3 min. Amino acid analysis ~-GluGlyGly and amino acids or peptides in the reaction mixture were detected and analyzed with an amino acid analyzer (Kyowa Seimitsu Co., Ltd.; KLA-101S) by the method of Speckman et al. s) Isolation and purification of ~--GluGlyGly The reaction mixture was successively chromatograph ed on columns of Dowex 50W-X2 (pyridinium formV) and H form) and Dowex l-X2 (acetate form). Fractions containing y-GluGlyGly were collected and concentrated by a rotary evaporator. After treating the concentrate with ethanol (see Results), the purified y-GluGlyGly was hydrolyzed in two ways, with 2 N HCI at 120°C for 2 h, and with a commercial GGT (from bovine kidney). For the latter, a mixture containing an appropriate amount of the purified y-GluGlyGly and 4 units.m1-1 of the commercial GGT in 100 mM potassium phosphate buffer (pH 6.0) was incubated at 30°C for 6 h. It had been confirmed that the commercial GGT only catalyzed the hydrolysis of the 7-glutamyl linkage under these conditions. After the hydrolysis, the products were analyzed with the amino acid analyzer. Chemicals GGT from bovine kidney was obtained from Sigma Chemical Co., USA. L-~,Glutamyl-p-nitroanilide was purchased from Wako Pure Chemicals Co., Ltd. The ion exchange resins were obtained from Dow Chemicals Co., Ltd.
[J. Ferment. Technol.,
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Formation and detection of ~'-GluGIyGly The enzyme preparation had about 2.4 times h i g h e r G G T a c t i v i t y t h a n g l u t a m i n ase activity. T h i s suggested t h a t the e n z y m e p r e p a r a t i o n h a d a c o n s i d e r a b l e G G T activity. T hu s , we i n v e s t i g a t e d th e f o r m a t i o n o f 7 - G l u G l y G l y . F i g u r e 1 shows t h e course o f y - G l u G l y G l y a n d g l u t a m i c acid f o r m a t i o n
4 6 8 10 12 Reaction tlme (h)
Fig. I. Course of ~-GluGlyOly formation. The reaction was done under the conditions given in Materials and Methods. O, 7-GluGlyGly; ©, glutamic acid. f r o m T.-glutamine a n d glycylglycine u n d e r the conditions g i v e n in Materials and Methods. A t the b e g i n n i n g of the reaction, the rate o f y - G l u G l y G l y f o r m a t i o n was a b o u t 1.5 times as fast as t h a t o f g l u t a m i c a c i d f o r m a t i o n . T h i s showed t h a t the G G T r e a c t i o n exceeded the g l u t a m i n a s e r e a c t i o n u n d e r these conditions. T h e a m o u n t o f y - G l u G l y G l y reached 9 m M at 7 h o f i n c u b a t i o n a n d g r a d u a l l y decreased after 10 h o f i n c u b a t i o n , w h i l e t h a t
0,5
(a) >,~
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(b) Gln GlyGly.
~0,2 ~O,i
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2
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i
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Elution tlme (mln) Fig. 2. Detection of 7-GluGlyGly with an amino acid analyzer. The reaction was done under the conditions given in Materials and Methods for 7 h. (a) The reaction mixture; (b) the reaction mixture after hydrolysis with a commercial GGT (from bovine kidney). The arrow indicates the peak of the y-GluGlyGly.
Vol. 66, 1988]
y-GluGlyGly Formation by
of glutamic acid was still increasing at 13 h of incubation. O n the other hand, the formation of a small amount of glycine, which seemed to be produced from the y-glutamyl acceptor, glycylglycine, by peptidase contaminating the enzyme preparation, was observed as the reaction proceeded (data not shown). Figure 2(a) shows the results of amino acid analysis of the reaction mixture after 7 h of incubation. A peak that seemed to correspond to y-GluGlyGly was eluted at an retention time of about 8 min (indicated by an arrow). On treatment of the reaction mixture with the commercial G G T , the peak indicated by the arrow almost disappeared, and the amount of glutamic acid increased (Fig. 2(b) ). Purification of 7-GluGlyGIy One liter of the reaction mixture after 7 h of incubation was obtained and its p H was adjusted to 2.6 with glacial acetic acid, and then denaturated protein was removed by centrifugation (10,000 ×g, for 20 rain). The supernatant solution was put on a column of Dowex 50W-X2 (pyridinium form, 4X 60 cm), which had been equilibrated with a 0.03 M pyridine-acetic acid buffer solution.
4
Aspergillus Glutaminase
301
(pH2.8), and then eluted with the same buffer solution. The elution pattern is shown in Fig. 3. y-GluGlyGly was eluted faster than other components in the reaction mixture. The fractions containing y-GluGlyGly (nos. 106117) were collected and concentrated to a small volume with a rotary evaporator. The concentrate was adjusted to p H 2 . 0 with HCI and put onto a column of Dowex 50W-X2 (H form, 2 × 20 cm) and then the column was washed with deionized water. Eltttion was stepwisely performed with 0.1, 0.25 and 0.5 M NH4OH, y-GluGlyGly being eluted with 0.5 M N H , O H . The y-GluGlyGly fractions were concentrated by a rotary evaporator, and after the p H was adjusted to 9.0 with N a O H , the concentrated fraction was put on a column of Dowex I-X2 (acetate form, 2 x 13 cm), then washed with deionized water. O n stepwise elution with 0.1, 0.25 and 0 . 5 M acetic acid, y-GluGlyGly was eluted with 0.5 M acetic acid. The y-GluGlyGly fraction were concentrated by a rotary evaporator to dryness. The residue was dissolved in a small volume of hot water. Hot ethanol was added to the solution until faint turbidity appeared, and then the mixture was placed in a refrigerator (at --20°C). The weight of the purified y-GluGlyGly, which was obtained as a white
~0.4 x~ ¢-
2
0,3
D
~-GluGlyGly
EJ
g z
~0,2
t-E3 ,.1:21
o E
0
i00
200
300
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Fractlon number (15 ml/tube ) Fig. 3. Dowex 50WoX2 column chromatography of ~-GluGlyGly. Elution was done with 0.03 M pyridine-acetic acid buffer (pH2.8). T h e column size was
4x60 cm. 0, ~,-GluGlyGly;A, glutamine; O, glutamic acid.
i
0
8
16
I
i
24
32
Eluti0n time (min) Fig. 4. A chromatogram of purified ~-GluGlyGly with an amino acid analyzer.
302
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(b)
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.~ 40
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Fig. 6. Infrared spectrum of the purified 7-GluGlyGly (KBr tablet) using Shimadzu Model 435.
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of glutamic acid and glycine, in a molar ratio at 1 : 1.95. On the other hand, hydrolysis of 0 8 16 the peptide with the commercial G G T gave Fig. 5. Identification of the purified y-GluGlyGly two peaks of glutamic acid and glycylglycine, in a molar ratio at 1 : I. 15 in the chromatowith an amino acid analyzer. (a) After hydrolysiswith HC1; (b) after hydrolysis gram using the amino acid analyzer (Fig. with a commercial GGT (from bovine kidney). 5 (b)). With these two kinds of hydrolysis, The hydrolysis conditions are given in Materials the 7-GluGlyGly peak completely disapand Methods. peared. These results strongly suggested that the peptide obtained was y-GluGlyGly. The infrared spectrum of the purified powder, was 50 rag. A chromatogram of the purified 7-GluGlyGly, using an amino acid y-GluGlyGly is shown in Fig. 6. An N - H analyzer, is shown in Fig. 4. The purified stretching vibration of the peptide bond y-GluGlyGly gave a single peak showing the appeared at 3350cm-1 (A), its value being same elution time (about 8 rain) as the peak higher than those of peptide bonds via aindicated in Fig. 2(a), but other components glutamyl linkage, s) Moreover, a C = 0 in the reaction mixture were not detected. stretching vibration, what is called an "amide Identification of r-GluGIyGly As I b a n d " , of the peptide bond appeared at Fig. 5(a) shows, hydrolysis of the purified 1650cm -1 (B), its value being lower than 7-GluGlyGly with 2 N HC1 gave two peaks those of peptide bonds via a-glutamyl linkage, s) 24 3 2 0 8 16 24 Elution time (min)
800
700
600
32
500
400
300
200
lO0
0 Hz
(D) £OOH I (C) H2N-CH-CH2-CH 2
(A)(B) ~ONH!!~2 (D) CONH-CH2-COOH
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Vol. 66, 1988]
7-GluGlyGly Formation by Aspergi!lus Glutaminase
From these findings, it was confirmed that a peptide bond via a 7-glutamyl linkage existed in the peptide. Figure 7 shows the P M R spectrum of the purified y-GluGlyGly. As to the chemical structure of the peptide, peaks (A), (B), (C) and (D) in Fig. 7 were attributed to protons (A), (B), (C) and (D), respectively. From the above results, the peptide was identified as 7-GluGlyGly. Discussion
Various kinds of y-glutamyl peptides have been found, and many 7-glutamyl peptides and G G T in animals, plants and microorganisms (including Basidiomyces) have been studied. Meister et al. 9-11) have studied the properties of G G T isolated from animal tissues in detail and the formation of 7glutamyl peptides by the enzyme. On the basis of the results of these studies, they proposed the "y-glutamyl cycle" hypothesis for the transprotation of amino acids through membranes, where glutathione acts as a 7glutamyl donor and amino acids act as Yglutamyl acceptors. TM Thompson et al. ~2) discussed the formation, breakdown and physiological roles of 7-glutamyl peptides in plants. As for microorganisms, Vitali et al.13) and Hasegawa et al. ~4) isolated several Yglutamyl peptides from glutamic acid fermentation broth. Hasegawa et al. isolated five y-glutamyl peptides, 7-glutamylglutamine, y-glutamylvaline, 7-glutamylleucine, 7-glutamylglutamic acid and 7-glutamyl7-glutamylglutamic acid from the fermentation broth of Corynebacterium glutamicum. T h e y concluded that the formation of these 7-glutamyl peptides was caused by 7-glutamyl transpeptidation or the reverse reaction of the glutaminase produced by the bacterium.15, 16) Bacterial G G T was purified from Proteus mirabilis as a homogeneous protein and its catalytic properties were elucidated. 17) However, for molds, there have been no studies on 7-glutamyl peptides or the G G T other than our previous study, 2) although a good deal of works on the glutaminase has
303
been done, especially relating to soy sauce fermentation, s, 4,1s, 19) In this study, a y-glutamyl peptide, 7GluGlyGly, was synthesized from L-glutamine (7-glutamyl donor) and glycylglycine (7glutamyl acceptor) using a wheat bran koji extract obtained with AspergiUus oryzae MA27-IM. The peptide was isolated and identified. Then, we can suppose that the G G T reaction may occur during the brewing period, using L-glutamine as a donor and L-peptides or L-amino acids as acceptors, which are liberated from material proteins by proteases and peptidases. In the following study, we will investigate the possible formation of many 7-glutamyl compounds by the G G T reaction of this strain. References
1) Yano, T., Ito, M., Tomita, K., Kumagai, H., Tochlkura, T.: J. Ferment. Technol., in preparation. 2) Tomita, K., Ito, M., Yano, T., Kumagai, H., Tochikura, T. : Agric. Biol. Chem., in preparation. 3) Yamamoto, S., Hirooka, H.: J. Ferment. Technol., 52, 570 (1974). 4) Shikata, H., Yasui, T., Ishigarai, Y., Omori, K.: Nippon Shoyu Kenkyusho Zasshi, 4, 48 (1979). 5) Orlowski, M., Meister, A.: J. Biol. Chem., 240, 338 (1965). 6) Speckman, D. H., Stein, W. H., Moore, S.: Anal. Chem., 80, 1190 (1958). 7) Nakai, T., Lai, C.Y., Horecker, L.: Anal. Biochem., 58, 563 (1974). 8) Kasai, T., Sakamura, S.: Nippon Nogeikagaku Kaishi, 48, 521 (1974). 9) Tate, S.S., Meister, A.: J. Biol. Chem., 249, 7593 (1974). 10) Tate, S.S., Meister, A.: J. Biol. Chem., 250, 4619 (1975). 1l) Orlowskl, M., Meister, A.: Proc. Natl. Acad. Sci. U.S.A., 67, 1248 (1970). 12) Thompson, J.F., Morris, C.J., Arnold, W.N., Turner, D.H., Holden, J.T. (ed.): Amino Acid Pools, p. 54, Elsevier Publishixig Company, New York (1962). 13) Vitali, R.A., Inamine, E., Jacob, T.A.: J. Biol. Chem., 240, 2508 (1965). 14) Hasegawa, M., Fukuda, N., Higuchi, H., Noguchi, S., Matsubara, I.: Agric. Biol. Chem., 41, 49 (1977). 15) Hasegawa, M., Matsubara, I.: Agric. Biol. Chem.,
304
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42, 371 (1978). 16) Hasegawa, M., Matsubara, I.: Agric. Biol. Chem., 42, 383 (1978). 17) Nakayama, R., Kumagai, H., Tochikura, T.: J. Bacteriol., 160, 341 (1984). 18) Shikata, H., Yasui, T., Ishigami, Y., Omori, K.:
Nippon Shoyu Kenkyusho Zasski, 5, 21 (1979). 19) Teramoto, A., Ishige, M., Furuya, T., Uchida, K., Yoshino, H.: Nippon Nogeikagaku Kaishi, 59, 245 (1985). (Received January 11, 1988)
Formation of ?-Glutamylglycylglycine by Extracellular Glutaminase of Aspergillus oryzae KENJI TOMITA*, TOSHIHIRO YANO, HIDEHIKO KUMAGAI, and TATSUROKURO TOCHIKURA
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan ~,-Glutamylglycylglycine (y-GluGlyGly) was formed through the y-glutamyltranspeptidase (GGT) reaction catalyzed by glutaminase in a water extract of wheat bran koji obtained with Aspergillus o~yzae MA-27-IM. The yield of y-GluGlyGly was about 18% from L-glutamine in a reaction mixture containing 50 m M L-glutamine, 50 m M glycylglycine, and the extract (0.1 unit ml as GGT activity) in a 100 m M Tris-HC1 buffer solution (pH 7.2), which was incubated for 7 h at 30°C. The y-GluGlyGly formed was purified by ion exchange chromatographies, and the identified by chemical and enzymatic methods as well as by infrared and P M R spectroscopic analyses.
In a previous study, l) we isolated Aspergillus oryzae MA-27-IM as a high glutaminase producer from a commercial koji seed for soy sauce fermentation, and wheat bran koji was prepared with this strain to investigate the properties of its intraceUular and extracellular glutaminases (L-glutamine amidohydrolase, EC 3.5.1.2). T h e results suggested that the two glutaminases might be the same protein. And the following study 2) revealed that the extracellular glutaminase of this strain possessed a considerable y-glutamyltranspeptidase (GGT) activity. The glutaminase from koji mold, Aspergillus oryzae or sojae, is generally regarded as a key enzyme that controls the delicious taste of fermented foods such as soy sauce.S,4) Thus the fact that glutaminase of a koji mold has G G T activity is interesting and significant from the viewpoint of glutamic acid content of food, on which the taste of the food depends. In this study, as the first step to clarify the significance of the G G T reaction in food fermentation involving koji molds, a yglutamyl peptide, y-glutamylglycylglycine (y-GluGlyGly), was formed from natural * Corresponding author --
substrates, L-glutamine and glycylglycine, using the water extract of a wheat bran koji obtained with strain MA-27-IM, and the peptide was also isolated and identified. Materials and Methods l~elicroorgl~mtlsm and ko,jl c u l t u r e A wheat bran koji with AspergiUus oryzae MA-27-IM was prepared as described in our previous paper, t ) Enzyme preparation The wheat bran koji was soaked in twice its weight of distilled water at 4°C for 12 h and then filtered through cotton cloth. The filtrate was centrifuged at 10,000×g for 20rain. Ammonium sulfate was added to the supernatant to achieve 85% saturation. The solution was centrifuged at 10,000 ×g for 20 rain to obtain the precipitate. The latter was suspended in a small amount of potassium phosphate buffer (pH 7.2, 10raM) and then dialyzed. The solution was used as the enzyme preparation. Enzyme assay Glutaminase activity was assayed by a modification of the method of Orlowski and Meister. s) The assay mixture contained 2.5 m M of L-y-glutamyl-p-nitroanilide, 100mM Tris-HC1 buffer (pH7.2), and the enzyme preparation (100/d), in a final volume of 1 ml. If necessary, the enzyme preparation was diluted with 100 m M Tris-HG1 buffer (pH 7.2). After incubation at 30°C for 30 rain, the enzyme reaction was stopped by the addition of 2 ml of 1.5 N acetic acid to the mixture, and then the absorbance at 410 nm was measured. ~,-Glutamyl-
300
TOMITAet al.
transpeptidase (GGT) activity was measured in the same way as glutaminase activity with 50 mM glycylglycine as the 7-glutamyl acceptor in the reaction mixture. One unit of either activity was defined as the amount of enzyme that catalyzed the release of 1/zmole of p-nitroaniline per minute under the conditions described above. The molar extinction coefficient ofp-nitroaniline was 8.3 × 103 M.cm -x. Reaction system 7-GluGlyGly was formed in a reaction mixture of 50 mM L-glutamine, 50 mM glycylglycine, 100 mM Tris-HC1 buffer (pH 7.2), and the enzyme preparation containing 0.1 unit.ml-I of GGT. The reaction was done at 30°C, and stopped by immersing the reaction vessel in boiling water for 3 min. Amino acid analysis ~-GluGlyGly and amino acids or peptides in the reaction mixture were detected and analyzed with an amino acid analyzer (Kyowa Seimitsu Co., Ltd.; KLA-101S) by the method of Speckman et al. s) Isolation and purification of ~--GluGlyGly The reaction mixture was successively chromatograph ed on columns of Dowex 50W-X2 (pyridinium formV) and H form) and Dowex l-X2 (acetate form). Fractions containing y-GluGlyGly were collected and concentrated by a rotary evaporator. After treating the concentrate with ethanol (see Results), the purified y-GluGlyGly was hydrolyzed in two ways, with 2 N HCI at 120°C for 2 h, and with a commercial GGT (from bovine kidney). For the latter, a mixture containing an appropriate amount of the purified y-GluGlyGly and 4 units.m1-1 of the commercial GGT in 100 mM potassium phosphate buffer (pH 6.0) was incubated at 30°C for 6 h. It had been confirmed that the commercial GGT only catalyzed the hydrolysis of the 7-glutamyl linkage under these conditions. After the hydrolysis, the products were analyzed with the amino acid analyzer. Chemicals GGT from bovine kidney was obtained from Sigma Chemical Co., USA. L-~,Glutamyl-p-nitroanilide was purchased from Wako Pure Chemicals Co., Ltd. The ion exchange resins were obtained from Dow Chemicals Co., Ltd.
[J. Ferment. Technol.,
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~-
0
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Formation and detection of ~'-GluGIyGly The enzyme preparation had about 2.4 times h i g h e r G G T a c t i v i t y t h a n g l u t a m i n ase activity. T h i s suggested t h a t the e n z y m e p r e p a r a t i o n h a d a c o n s i d e r a b l e G G T activity. T hu s , we i n v e s t i g a t e d th e f o r m a t i o n o f 7 - G l u G l y G l y . F i g u r e 1 shows t h e course o f y - G l u G l y G l y a n d g l u t a m i c acid f o r m a t i o n
4 6 8 10 12 Reaction tlme (h)
Fig. I. Course of ~-GluGlyOly formation. The reaction was done under the conditions given in Materials and Methods. O, 7-GluGlyGly; ©, glutamic acid. f r o m T.-glutamine a n d glycylglycine u n d e r the conditions g i v e n in Materials and Methods. A t the b e g i n n i n g of the reaction, the rate o f y - G l u G l y G l y f o r m a t i o n was a b o u t 1.5 times as fast as t h a t o f g l u t a m i c a c i d f o r m a t i o n . T h i s showed t h a t the G G T r e a c t i o n exceeded the g l u t a m i n a s e r e a c t i o n u n d e r these conditions. T h e a m o u n t o f y - G l u G l y G l y reached 9 m M at 7 h o f i n c u b a t i o n a n d g r a d u a l l y decreased after 10 h o f i n c u b a t i o n , w h i l e t h a t
0,5
(a) >,~
o,g
~
(b) Gln GlyGly.
~0,2 ~O,i
!
0 Results
2
1
0
i
i
16 24 320
i
8
i
i
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Elution tlme (mln) Fig. 2. Detection of 7-GluGlyGly with an amino acid analyzer. The reaction was done under the conditions given in Materials and Methods for 7 h. (a) The reaction mixture; (b) the reaction mixture after hydrolysis with a commercial GGT (from bovine kidney). The arrow indicates the peak of the y-GluGlyGly.
Vol. 66, 1988]
y-GluGlyGly Formation by
of glutamic acid was still increasing at 13 h of incubation. O n the other hand, the formation of a small amount of glycine, which seemed to be produced from the y-glutamyl acceptor, glycylglycine, by peptidase contaminating the enzyme preparation, was observed as the reaction proceeded (data not shown). Figure 2(a) shows the results of amino acid analysis of the reaction mixture after 7 h of incubation. A peak that seemed to correspond to y-GluGlyGly was eluted at an retention time of about 8 min (indicated by an arrow). On treatment of the reaction mixture with the commercial G G T , the peak indicated by the arrow almost disappeared, and the amount of glutamic acid increased (Fig. 2(b) ). Purification of 7-GluGlyGIy One liter of the reaction mixture after 7 h of incubation was obtained and its p H was adjusted to 2.6 with glacial acetic acid, and then denaturated protein was removed by centrifugation (10,000 ×g, for 20 rain). The supernatant solution was put on a column of Dowex 50W-X2 (pyridinium form, 4X 60 cm), which had been equilibrated with a 0.03 M pyridine-acetic acid buffer solution.
4
Aspergillus Glutaminase
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(pH2.8), and then eluted with the same buffer solution. The elution pattern is shown in Fig. 3. y-GluGlyGly was eluted faster than other components in the reaction mixture. The fractions containing y-GluGlyGly (nos. 106117) were collected and concentrated to a small volume with a rotary evaporator. The concentrate was adjusted to p H 2 . 0 with HCI and put onto a column of Dowex 50W-X2 (H form, 2 × 20 cm) and then the column was washed with deionized water. Eltttion was stepwisely performed with 0.1, 0.25 and 0.5 M NH4OH, y-GluGlyGly being eluted with 0.5 M N H , O H . The y-GluGlyGly fractions were concentrated by a rotary evaporator, and after the p H was adjusted to 9.0 with N a O H , the concentrated fraction was put on a column of Dowex I-X2 (acetate form, 2 x 13 cm), then washed with deionized water. O n stepwise elution with 0.1, 0.25 and 0 . 5 M acetic acid, y-GluGlyGly was eluted with 0.5 M acetic acid. The y-GluGlyGly fraction were concentrated by a rotary evaporator to dryness. The residue was dissolved in a small volume of hot water. Hot ethanol was added to the solution until faint turbidity appeared, and then the mixture was placed in a refrigerator (at --20°C). The weight of the purified y-GluGlyGly, which was obtained as a white
~0.4 x~ ¢-
2
0,3
D
~-GluGlyGly
EJ
g z
~0,2
t-E3 ,.1:21
o E
0
i00
200
300
ou'J0 , 1
Fractlon number (15 ml/tube ) Fig. 3. Dowex 50WoX2 column chromatography of ~-GluGlyGly. Elution was done with 0.03 M pyridine-acetic acid buffer (pH2.8). T h e column size was
4x60 cm. 0, ~,-GluGlyGly;A, glutamine; O, glutamic acid.
i
0
8
16
I
i
24
32
Eluti0n time (min) Fig. 4. A chromatogram of purified ~-GluGlyGly with an amino acid analyzer.
302
TOMITAet al. (a)
(b)
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0,5
GIyG y
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100 80
60
GIu
0,4
[J. Ferment. Technol.,
.~ 40
20
'-
GIy
go,3
[.6 %.~ 3:6 2'.~ ,. ~B),
1'.4 [.2
~,0
0,8 0,6 0.4
Wove number (X]O3 cm-1)
Fig. 6. Infrared spectrum of the purified 7-GluGlyGly (KBr tablet) using Shimadzu Model 435.
o,2
o
~o,1 i
of glutamic acid and glycine, in a molar ratio at 1 : 1.95. On the other hand, hydrolysis of 0 8 16 the peptide with the commercial G G T gave Fig. 5. Identification of the purified y-GluGlyGly two peaks of glutamic acid and glycylglycine, in a molar ratio at 1 : I. 15 in the chromatowith an amino acid analyzer. (a) After hydrolysiswith HC1; (b) after hydrolysis gram using the amino acid analyzer (Fig. with a commercial GGT (from bovine kidney). 5 (b)). With these two kinds of hydrolysis, The hydrolysis conditions are given in Materials the 7-GluGlyGly peak completely disapand Methods. peared. These results strongly suggested that the peptide obtained was y-GluGlyGly. The infrared spectrum of the purified powder, was 50 rag. A chromatogram of the purified 7-GluGlyGly, using an amino acid y-GluGlyGly is shown in Fig. 6. An N - H analyzer, is shown in Fig. 4. The purified stretching vibration of the peptide bond y-GluGlyGly gave a single peak showing the appeared at 3350cm-1 (A), its value being same elution time (about 8 rain) as the peak higher than those of peptide bonds via aindicated in Fig. 2(a), but other components glutamyl linkage, s) Moreover, a C = 0 in the reaction mixture were not detected. stretching vibration, what is called an "amide Identification of r-GluGIyGly As I b a n d " , of the peptide bond appeared at Fig. 5(a) shows, hydrolysis of the purified 1650cm -1 (B), its value being lower than 7-GluGlyGly with 2 N HC1 gave two peaks those of peptide bonds via a-glutamyl linkage, s) 24 3 2 0 8 16 24 Elution time (min)
800
700
600
32
500
400
300
200
lO0
0 Hz
(D) £OOH I (C) H2N-CH-CH2-CH 2
(A)(B) ~ONH!!~2 (D) CONH-CH2-COOH
(C)(B)
t
9.0
8,0
f
7,0
i
6.0
i
5,0
i
4,0
3.0
t
2,0
l,O
PPm
Fig. 7. PMR spectrum of the purified ~-OluOlyOly (in D~O containing DC1) using Hitachi Model R-22 (90MHz).
Vol. 66, 1988]
7-GluGlyGly Formation by Aspergi!lus Glutaminase
From these findings, it was confirmed that a peptide bond via a 7-glutamyl linkage existed in the peptide. Figure 7 shows the P M R spectrum of the purified y-GluGlyGly. As to the chemical structure of the peptide, peaks (A), (B), (C) and (D) in Fig. 7 were attributed to protons (A), (B), (C) and (D), respectively. From the above results, the peptide was identified as 7-GluGlyGly. Discussion
Various kinds of y-glutamyl peptides have been found, and many 7-glutamyl peptides and G G T in animals, plants and microorganisms (including Basidiomyces) have been studied. Meister et al. 9-11) have studied the properties of G G T isolated from animal tissues in detail and the formation of 7glutamyl peptides by the enzyme. On the basis of the results of these studies, they proposed the "y-glutamyl cycle" hypothesis for the transprotation of amino acids through membranes, where glutathione acts as a 7glutamyl donor and amino acids act as Yglutamyl acceptors. TM Thompson et al. ~2) discussed the formation, breakdown and physiological roles of 7-glutamyl peptides in plants. As for microorganisms, Vitali et al.13) and Hasegawa et al. ~4) isolated several Yglutamyl peptides from glutamic acid fermentation broth. Hasegawa et al. isolated five y-glutamyl peptides, 7-glutamylglutamine, y-glutamylvaline, 7-glutamylleucine, 7-glutamylglutamic acid and 7-glutamyl7-glutamylglutamic acid from the fermentation broth of Corynebacterium glutamicum. T h e y concluded that the formation of these 7-glutamyl peptides was caused by 7-glutamyl transpeptidation or the reverse reaction of the glutaminase produced by the bacterium.15, 16) Bacterial G G T was purified from Proteus mirabilis as a homogeneous protein and its catalytic properties were elucidated. 17) However, for molds, there have been no studies on 7-glutamyl peptides or the G G T other than our previous study, 2) although a good deal of works on the glutaminase has
303
been done, especially relating to soy sauce fermentation, s, 4,1s, 19) In this study, a y-glutamyl peptide, 7GluGlyGly, was synthesized from L-glutamine (7-glutamyl donor) and glycylglycine (7glutamyl acceptor) using a wheat bran koji extract obtained with AspergiUus oryzae MA27-IM. The peptide was isolated and identified. Then, we can suppose that the G G T reaction may occur during the brewing period, using L-glutamine as a donor and L-peptides or L-amino acids as acceptors, which are liberated from material proteins by proteases and peptidases. In the following study, we will investigate the possible formation of many 7-glutamyl compounds by the G G T reaction of this strain. References
1) Yano, T., Ito, M., Tomita, K., Kumagai, H., Tochlkura, T.: J. Ferment. Technol., in preparation. 2) Tomita, K., Ito, M., Yano, T., Kumagai, H., Tochikura, T. : Agric. Biol. Chem., in preparation. 3) Yamamoto, S., Hirooka, H.: J. Ferment. Technol., 52, 570 (1974). 4) Shikata, H., Yasui, T., Ishigarai, Y., Omori, K.: Nippon Shoyu Kenkyusho Zasshi, 4, 48 (1979). 5) Orlowski, M., Meister, A.: J. Biol. Chem., 240, 338 (1965). 6) Speckman, D. H., Stein, W. H., Moore, S.: Anal. Chem., 80, 1190 (1958). 7) Nakai, T., Lai, C.Y., Horecker, L.: Anal. Biochem., 58, 563 (1974). 8) Kasai, T., Sakamura, S.: Nippon Nogeikagaku Kaishi, 48, 521 (1974). 9) Tate, S.S., Meister, A.: J. Biol. Chem., 249, 7593 (1974). 10) Tate, S.S., Meister, A.: J. Biol. Chem., 250, 4619 (1975). 1l) Orlowskl, M., Meister, A.: Proc. Natl. Acad. Sci. U.S.A., 67, 1248 (1970). 12) Thompson, J.F., Morris, C.J., Arnold, W.N., Turner, D.H., Holden, J.T. (ed.): Amino Acid Pools, p. 54, Elsevier Publishixig Company, New York (1962). 13) Vitali, R.A., Inamine, E., Jacob, T.A.: J. Biol. Chem., 240, 2508 (1965). 14) Hasegawa, M., Fukuda, N., Higuchi, H., Noguchi, S., Matsubara, I.: Agric. Biol. Chem., 41, 49 (1977). 15) Hasegawa, M., Matsubara, I.: Agric. Biol. Chem.,
304
TOmTA et al.
42, 371 (1978). 16) Hasegawa, M., Matsubara, I.: Agric. Biol. Chem., 42, 383 (1978). 17) Nakayama, R., Kumagai, H., Tochikura, T.: J. Bacteriol., 160, 341 (1984). 18) Shikata, H., Yasui, T., Ishigami, Y., Omori, K.:
Nippon Shoyu Kenkyusho Zasski, 5, 21 (1979). 19) Teramoto, A., Ishige, M., Furuya, T., Uchida, K., Yoshino, H.: Nippon Nogeikagaku Kaishi, 59, 245 (1985). (Received January 11, 1988)