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Enzymatic production of d -glutamate from l -glutamate by a glutamate racemase
JOURNALOF FERMENTATION AND Vol. 79, No. 1, 70-72. 1995
BIOENGINEERING
Enzymatic Production of D-Glutamate from L-Glutamate by a Glutamate Racemase MAKOTO
YAGASAKI,
MASAKI
AZUMA,
SHUICHI
ISHINO,
AND AK10 OZAKI*
Technical Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., I-I Kyowa-machi, Hofu, Yamaguchi 747, Japan Received 18 July 1994IAccepted 26 September 1994
o-Glutamate was produced from c-glutamate by two successive cellular reactious with a glutamate racemase produced by Escherichiu coli TM93 harboring a plasmid containing a glutamate racemase gene from Lactobacillus brevis ATCC 8287 and a glutamate decarboxylase produced by E. coli ATCC 11246. L-Glutamate was first racemized to DL-glutamate at pH 8.5 and L-glutamate was then decarboxylated at pH 4.2. Starting from 100 g/Z of L-glutamate, 50 g/Z of u-glutamate remained after 15 h reaction. [Key words: racemase, decarboxylase,
D-glutamate,
Lactobacillus, bioconversion]
D-Amino acids are becoming important as starting materials in the production of pharmaceuticals, food additives, and agrochemicals. We have been investigating industrial production systems for D-amino acids and have reported the establishment of efficient enzymatic manufacture of D-alanine from DL-alaninamide (1). We previously reported the enzymatic production of D-glutamate (D-GIu) from L-glutamate (L-Glu) by two successive reactions with a glutamate racemase (EC 5.1.1.3) and a glutamate decarboxylase (EC 4.1 .1.15) from a single strain, Lactobacilfus brevis ATCC 8287 (2). In the first step, L-Glu was converted to DL-Glu by a glutamate racemase at pH 8.5. The pH of the reaction mixture was then shifted to 4.0 and L-Glu in the reaction mixture was decarboxylated by a glutamate decarboxylase to 4amino-n-butyrate (GABA). D-Glu remained in the reaction mixture and could be easily separated from GABA. The advantages of this process in the industrial production of D-Glu are: (i) L-Glu, which is produced industrially by fermentation, is the cheapest starting material for the production of D-Glu, even though the theoretical yield of D-Glu in the process cannot exceed SO%, and (ii) the process can be carried out as a one-pot reaction with a single strain. On the other hand, lactic acid bacteria are not suitable for the industrial process because it is very difficult to obtain a sufficiently large cell mass. To improve this drawback in the D-Glu manufacturing process, we cloned a glutamate racemase gene of L. brevis ATCC 8287 in E. coli TM93, which is defective in a gene for phosphoenolpyruvate carboxylase (EC 4.1.1.3 l), by phenotypic complementation. The glutamate racemase was purified from the recombinant E. colt’ to homogeneity and characterized. Details of the gene cloning, purification, and characterization of the glutamate racemase of L. brevis ATCC 8287 will be reported elsewhere. Here, we report the application of the glutamate racemase from the recombinant E. coli TM93. Plasmid pGAR1 contains a 2.8-kb Hind111 digested DNA fragment derived from L. brevis ATCC 8287 inserted into the Hind111 site of pBR322 (Fig. 1). E. coli TM93 cells harboring pGAR1 from an overnight culture in LB medium (10 g Bacto Tryptone, 5 g Bacto Yeast
Extract, 10 g glucose, and 5 g NaCl in 1 1 of water, pH 7.2) showed a cellular glutamate racemase activity of 1,270 pmol/min/g wet cells. Cells containing plasmid pGAR2, which contains the 2.8-kb Hind111 digested DNA fragment in the opposite direction inserted into the Hind111 site of pBR322, showed activity of 1,390 pmol/min/g wet cells. E. coli TM93 harboring a plasmid containing a 1.4-kb HindIII-EcoRI DNA fragment, into which a glutamate racemase gene was localized, inserted into the multicloning sites of pUCl8 and pUC19, showed cellular activities of 1,480 and 1,810 pmol/min/g wet cells, respectively, without the addition of lac promoter inducers. Addition of isopropylP-D-thiogaiactopyranoside to the culture did not affect the enzyme level in recombinant E. coli. Furthermore, no induction or repression of enzyme production by Dor L-Glu was observed. Figure 2 shows the time course of the production of the glutamate racemase by E. coli TM93 harboring the plasmid pGAR1. The cellular activity of the glutamate racemase in recombinant E. coli was more than 150 times that in L. brevis ATCC 8287 (2). The amount of glutamate racemase produced by E. coli TM93/pGARl was calculated as 4.2% of the total soluble protein, based on the specific activity of the purified racemase preparation (216 pmol/min/mg protein). Racemization of L-Glu was examined by the cellular reaction of E. coli TM93/pGARI. Addition of toluene to the reaction mixture was essential for the cellular reaction. Racemization of 100 g/l of L-Glu was completed in 10 h by 2.5 g/l of wet recombinant cells. Even at a concentration of 200 g/l of L-Glu, racemization was completed within 24 h by the same amount of wet recombinant
0.7
0.4 0.3
0.9
0.2 0.3
(kb)
* FIG. 1. Physical map of pGAR1. The open bar indicates the 2.8kb Hind111 fragment with restriction sites: H, HindIII; P, PsfI; B, BarnHI; E, EcoRI. The figures under the open bar indicate the size in kb of the DNA fragment between the restriction sites. The closed bar corresponds to pBR322, and the relative localization of the ampicillin resistance gene (Ap) and tetracycline resistance gene (Tc) are indicated. The arrow indicates the approximate localization and orientation of the glutamate racemase gene.
* Corresponding author. Present address: Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., 3-6-6 Asahi-machi, Machida, Tokyo 194, Japan. 70
NOTES
VOL. 79, 1995
(4
1600
71
(B) 100
t
80
I!
60 40
0 0
I
I
10
20
30
Culture time (h) FIG. 2. Time course of the production of a glutamate racemase by E. co&. E. co/i TM93 harboring plasmid pGAR1 was cultivated at 30°C in a jar fermentor containing 2 1 of a medium consisting of 5 g/l glucose, 5 g/l peptone (Daigo Eiyo Kagaku Co. Ltd., Osaka), 15 g/l Na2HP04. 12H20, 3 g/l KH2P04, 5 g/l NaCl, 1.2 g/l MgS04. 7H20, 0.2 g/l thiamine HCl and 1 ml/l trace element solution (37 mg (NH4)J407024.4H20, 990 mg FeS04.7Hz0, 880 mg ZnS04.7H20, 393 mg CuSO,. 5Hz0, 72 mg MnCl,.4H,O, and 88 mg NaZB407. IOH in 1 I of water), with agitation at 6COrpm and aeration of 1 vvm. Glucose solution (167 g/l of glucose and 167 g/l of peptone) was fed to. the culture to keep the glucose concentration around 1% after the’initial glucose was consumed. The pH of the culture was kept at 7.0. Portions (20ml) of the culture broth were taken as samples to examine the activity. The culture broth was centrifuged at 8,000 rpm at 4°C for 10 min. The pellet of wet cells so obtained was washed twice with 100 mM sodium phosphate buffer, pH 8.5, at 4°C. The glutamate racemase activity was measured in the reaction mixture (1 ml) consisted of 100mM sodium phosphate buffer, pH 8.5, 170 mM L-Glu, 2% (vol./vol.) toluene, and the cells. The reaction was done at 37°C and the amount of D-Glu produced was measured as reported previously (2). The solid line indicates the cellular glutamate racemase activity and the dotted line the cell growth. One unit of enzyme activity was defined as the amount of enzyme which catalyzed the formation of 1 ,timol of the product in 1 min.
cells (Fig. 3A). As to the amount of recombinant E. coli cells, at least 1 g/l of wet cells were required to racemize 100 g/l of L-Glu within 24 h (Fig. 3B). An increase in the amount of recombinant cells to 5 g/f resulted in a decrease in the time needed for completion of racemization to 5 h. On the other hand, more than lOOg/l of wet cells of L. brevis ATCC 8287 were required for the racemization of 100 g/l of L-Glu for about 10 h (2). As is the case with the glutamate racemase, L. brevis TABLE 1.
Glutamate decarboxylase activities in E. coli
Strain L. E. E. E. E.
brevis ATCC 8287 coli ATCC 11246 coli ATCC 13676 coli ATCC 21186 coli TM93
Glutamate decarboxylase activity units/g wet cells
relative activity
8 91 21 16 0
1 11.4 2.6 2 0
E. coli cells were grown at 30°C with shaking in LB medium overnight. L. brevis ATCC 8287 was cultured by the method reported previously (2). Cellular reactions were done in a reaction mixture (1 ml) consisting of 200 mM sodium acetate buffer, pH 4.2, 70 mM L-Glu, 2% (vol./vol.) toluene, and the cells. The reaction was carried out at 37°C for 30 min and the amount of L-Glu was measured as reported previously (2). One unit of enzyme activity was defined as the amount of enzyme which catalyzed the decarboxylation of 1 pmol of L-Glu in 1 min.
I
I
10
20
01
,
F 10
20
Reaction time (h) FIG. 3. Effects of L-Glu concentration (A) and the amount of E. coli TM93/pGARl cells (B) on the racemization of L-Glu. Cellular reactions were done by the methods described previously (2). (A) The reaction mixture consisted of 100 mM sodium phosphate buffer, pH 8.5, 2.5 g/l E. coli TM93/pGARl wet cells, L-Glu and 2% (vol./vol.) toluene. The concentrations of L-Glu were lOOg/l (0), 150 g/l (A), and 200 g/l ( 0 ), respectively. (B) The reaction mixture consisted of 100 mM sodium phosphate buffer, pH 8.5, 100 ~/IL-Glu, E. coli TM93/pGARl wet cells, and 2% (vol./vol.) toluene. The cell amounts were 0.5 g/l ( l ), 1 g/l ( 0 ), and 2.5 g/l (A), and 5 g/l (0), respectively. The reactions were done in 10 ml reaction mixture with shaking at 37°C.
ATCC 8287 is not suitable as a source of glutamate decarboxylase because of the difficulty in obtaining a large cell mass. Therefore, to establish an industrial production process of D-Glu, it is necessary to find a strain which efficiently produces a glutamate decarboxylase. We examined several E. coli strains known to be glutamate decarboxylase producers, and E. co/i ATCC 11246 was selected as a high producer of the enzyme (3, 4). The cellular glutamate decarboxylase activity of E. coli ATCC 11246 was 91 pmol/min/g wet cells, which is more than 11 times that of L. brevis ATCC 8287 (Table 1). Finally, we examined the production of D-Glu from LGlu by two successive cellular reactions with E. coli TM93/pGARl and E. coli ATCC 11246. The racemization reaction was carried out at pH 8.5, at which a glutamate racemase has its maximam activity and a glutamate decarboxylase is inactive. In a reaction system containing lOOg/l of L-Glu and 3 g/l of wet cells of E. coli TM93/pGARl, L-Glu was racemized completely and 5Og/Z of D-Glu was produced after 5 h. The pH of the reaction mixture was then shifted to 4.2, at which a glutamate decarboxylase is active and a glutamate racemase is inactive, and 5 g/l of wet cells of E. coli ATCC 11246 was added into the reaction mixture as a source of glutamate decarboxylase to convert L-Glu to GABA. L-Glu in the reaction mixture was then converted completely to GABA after 10 h and 50 g/l of D-Glu remained in the reaction mixture (Fig. 4). The production of D-Glu by a system employing a recombinant E. coli glutamate racemase and a E. coli glutamate decarboxylase has an advantage over the L. brevis system reported previously (2), in that the growth rate and yield of the cell mass of E. coli are much higher than those of L. brevis. The D-Glu production system reported here is the most efficient among the methods of D-Glu production so far reported (5-7), even though the
12
YAGASAKI ET AL.
J. FERMENT.BIOENG..
theoretical yield of D-Glu in the process cannot exceed SO%, because L-Glu, which is produced industrially by fermentation, is the cheapest starting material for the production of D-Glu and the process can be carried out as a one-pot reaction. REFERENCES
Reaction time (h) FIG. 4. Enzymatic production of D-Glu from L-Glu. E. coli TM93/pGARl was cultivated with shaking by the method described in the legend of Fig. 2. E. coli ATCC 11246 was cultivated in LB medium at 37°C overnight. The culture broth was centrifuged at 8,000 rpm at 4°C for 10 min. The wet cells so obtained was washed twice with 100 mM sodium phosphate buffer, pH 8.5, for E. coli TM93, and 200 mM sodium acetate buffer, pH 4.2, for E. coli ATCC 11246. Cellular reactions were done by the methods described previously (2). The reaction mixture (500 ml) consisted of 100 g/l L-Glu, 3 g/l E. coli TM93/pGARl wet cells, and 2% (vol./vol.) toluene; the pH of the reaction mixture was adjusted to 8.5 with sodium hydroxide. The reaction was carried out at 37°C for 5 h with agitation at 200 rpm and no aeration. After the pH was shifted to 4.2 with hydrochloric acid, 5 g/l E. coli ATCC 11246 wet cells was added to the reaction mixture as a source of glutamate decarboxylase. The reaction continued at 37°C for another 10 h, and the pH was kept between 4.0 and 4.5 with hydrochloric acid. Symbols indicate the amounts of L-Glu (0), D-Glu ( l), and GABA (A). The arrow indicates the time that the pH was shifted to 4.2.
1. Ozakl, A., Kawasaki, H., Yagasaki, M., and Hashimoto, Y.: Enzymatic production of D-alanine from DL-alaninamide by novel o-alaninamide specific amide hydrolase. Biosci. Biotech. Biochem., 56, 1980-1984 (1992). 2. Yagasaki, M., Ozaki, A., and Hashimoto, Y.: Enzymatic production of D-Glu from L-Glu by Lactobacillus brevis ATCC 8287. Biosci. Biotech. Biochem., 57, 1499-1502 (1993). 3. Najjar, V. A. and Fisher, J.: Glutamic acid decarboxylase from E. coli. Fed. Proc., 11, 264 (1952). 4. Najjar, V. A. and Fisher, J.: Studies on L-glutamic acid decarboxylase from Escherichiu coli. J. Biol. Chem., 206, 215219 (1954). 5. Yokozeki, K., Nakamori, S., Yamanaka, S., Eguchi, C., Mitsugi, K., and Yoshinaga, F.: Optimal conditions for the enzymatic production of D-amino acids from the corresponding 5-substituted hydantoins. Agric. Biol. Chem., 51, 715-719 (1987). 6. del Pozo, A.M., Merola, M., Ueno, H., Manning, J. M., Tanizawa, K., Nishimura, K., Soda, K., and Ringe, D.: Stereospecificity of reactions catalyzed by bacterial D-amino acid transaminase. J. Biol. Chem., 264, 17784-17789 (1989). 7. Yonaha, K. and Soda, K.: Applications of stereoselectivity of enzymes: synthesis of optically active amino acids and cu-hydroxy acids, and stereospecific isotope-labeling of amino acids, amines and coenzymes, p. 95-130. In Fiechter, A. (ed.), Advances in biochemical engineering and biotechnology, vol. 33. Springer-Verlag, Berlin (1986).
BIOENGINEERING
Enzymatic Production of D-Glutamate from L-Glutamate by a Glutamate Racemase MAKOTO
YAGASAKI,
MASAKI
AZUMA,
SHUICHI
ISHINO,
AND AK10 OZAKI*
Technical Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., I-I Kyowa-machi, Hofu, Yamaguchi 747, Japan Received 18 July 1994IAccepted 26 September 1994
o-Glutamate was produced from c-glutamate by two successive cellular reactious with a glutamate racemase produced by Escherichiu coli TM93 harboring a plasmid containing a glutamate racemase gene from Lactobacillus brevis ATCC 8287 and a glutamate decarboxylase produced by E. coli ATCC 11246. L-Glutamate was first racemized to DL-glutamate at pH 8.5 and L-glutamate was then decarboxylated at pH 4.2. Starting from 100 g/Z of L-glutamate, 50 g/Z of u-glutamate remained after 15 h reaction. [Key words: racemase, decarboxylase,
D-glutamate,
Lactobacillus, bioconversion]
D-Amino acids are becoming important as starting materials in the production of pharmaceuticals, food additives, and agrochemicals. We have been investigating industrial production systems for D-amino acids and have reported the establishment of efficient enzymatic manufacture of D-alanine from DL-alaninamide (1). We previously reported the enzymatic production of D-glutamate (D-GIu) from L-glutamate (L-Glu) by two successive reactions with a glutamate racemase (EC 5.1.1.3) and a glutamate decarboxylase (EC 4.1 .1.15) from a single strain, Lactobacilfus brevis ATCC 8287 (2). In the first step, L-Glu was converted to DL-Glu by a glutamate racemase at pH 8.5. The pH of the reaction mixture was then shifted to 4.0 and L-Glu in the reaction mixture was decarboxylated by a glutamate decarboxylase to 4amino-n-butyrate (GABA). D-Glu remained in the reaction mixture and could be easily separated from GABA. The advantages of this process in the industrial production of D-Glu are: (i) L-Glu, which is produced industrially by fermentation, is the cheapest starting material for the production of D-Glu, even though the theoretical yield of D-Glu in the process cannot exceed SO%, and (ii) the process can be carried out as a one-pot reaction with a single strain. On the other hand, lactic acid bacteria are not suitable for the industrial process because it is very difficult to obtain a sufficiently large cell mass. To improve this drawback in the D-Glu manufacturing process, we cloned a glutamate racemase gene of L. brevis ATCC 8287 in E. coli TM93, which is defective in a gene for phosphoenolpyruvate carboxylase (EC 4.1.1.3 l), by phenotypic complementation. The glutamate racemase was purified from the recombinant E. colt’ to homogeneity and characterized. Details of the gene cloning, purification, and characterization of the glutamate racemase of L. brevis ATCC 8287 will be reported elsewhere. Here, we report the application of the glutamate racemase from the recombinant E. coli TM93. Plasmid pGAR1 contains a 2.8-kb Hind111 digested DNA fragment derived from L. brevis ATCC 8287 inserted into the Hind111 site of pBR322 (Fig. 1). E. coli TM93 cells harboring pGAR1 from an overnight culture in LB medium (10 g Bacto Tryptone, 5 g Bacto Yeast
Extract, 10 g glucose, and 5 g NaCl in 1 1 of water, pH 7.2) showed a cellular glutamate racemase activity of 1,270 pmol/min/g wet cells. Cells containing plasmid pGAR2, which contains the 2.8-kb Hind111 digested DNA fragment in the opposite direction inserted into the Hind111 site of pBR322, showed activity of 1,390 pmol/min/g wet cells. E. coli TM93 harboring a plasmid containing a 1.4-kb HindIII-EcoRI DNA fragment, into which a glutamate racemase gene was localized, inserted into the multicloning sites of pUCl8 and pUC19, showed cellular activities of 1,480 and 1,810 pmol/min/g wet cells, respectively, without the addition of lac promoter inducers. Addition of isopropylP-D-thiogaiactopyranoside to the culture did not affect the enzyme level in recombinant E. coli. Furthermore, no induction or repression of enzyme production by Dor L-Glu was observed. Figure 2 shows the time course of the production of the glutamate racemase by E. coli TM93 harboring the plasmid pGAR1. The cellular activity of the glutamate racemase in recombinant E. coli was more than 150 times that in L. brevis ATCC 8287 (2). The amount of glutamate racemase produced by E. coli TM93/pGARl was calculated as 4.2% of the total soluble protein, based on the specific activity of the purified racemase preparation (216 pmol/min/mg protein). Racemization of L-Glu was examined by the cellular reaction of E. coli TM93/pGARI. Addition of toluene to the reaction mixture was essential for the cellular reaction. Racemization of 100 g/l of L-Glu was completed in 10 h by 2.5 g/l of wet recombinant cells. Even at a concentration of 200 g/l of L-Glu, racemization was completed within 24 h by the same amount of wet recombinant
0.7
0.4 0.3
0.9
0.2 0.3
(kb)
* FIG. 1. Physical map of pGAR1. The open bar indicates the 2.8kb Hind111 fragment with restriction sites: H, HindIII; P, PsfI; B, BarnHI; E, EcoRI. The figures under the open bar indicate the size in kb of the DNA fragment between the restriction sites. The closed bar corresponds to pBR322, and the relative localization of the ampicillin resistance gene (Ap) and tetracycline resistance gene (Tc) are indicated. The arrow indicates the approximate localization and orientation of the glutamate racemase gene.
* Corresponding author. Present address: Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., 3-6-6 Asahi-machi, Machida, Tokyo 194, Japan. 70
NOTES
VOL. 79, 1995
(4
1600
71
(B) 100
t
80
I!
60 40
0 0
I
I
10
20
30
Culture time (h) FIG. 2. Time course of the production of a glutamate racemase by E. co&. E. co/i TM93 harboring plasmid pGAR1 was cultivated at 30°C in a jar fermentor containing 2 1 of a medium consisting of 5 g/l glucose, 5 g/l peptone (Daigo Eiyo Kagaku Co. Ltd., Osaka), 15 g/l Na2HP04. 12H20, 3 g/l KH2P04, 5 g/l NaCl, 1.2 g/l MgS04. 7H20, 0.2 g/l thiamine HCl and 1 ml/l trace element solution (37 mg (NH4)J407024.4H20, 990 mg FeS04.7Hz0, 880 mg ZnS04.7H20, 393 mg CuSO,. 5Hz0, 72 mg MnCl,.4H,O, and 88 mg NaZB407. IOH in 1 I of water), with agitation at 6COrpm and aeration of 1 vvm. Glucose solution (167 g/l of glucose and 167 g/l of peptone) was fed to. the culture to keep the glucose concentration around 1% after the’initial glucose was consumed. The pH of the culture was kept at 7.0. Portions (20ml) of the culture broth were taken as samples to examine the activity. The culture broth was centrifuged at 8,000 rpm at 4°C for 10 min. The pellet of wet cells so obtained was washed twice with 100 mM sodium phosphate buffer, pH 8.5, at 4°C. The glutamate racemase activity was measured in the reaction mixture (1 ml) consisted of 100mM sodium phosphate buffer, pH 8.5, 170 mM L-Glu, 2% (vol./vol.) toluene, and the cells. The reaction was done at 37°C and the amount of D-Glu produced was measured as reported previously (2). The solid line indicates the cellular glutamate racemase activity and the dotted line the cell growth. One unit of enzyme activity was defined as the amount of enzyme which catalyzed the formation of 1 ,timol of the product in 1 min.
cells (Fig. 3A). As to the amount of recombinant E. coli cells, at least 1 g/l of wet cells were required to racemize 100 g/l of L-Glu within 24 h (Fig. 3B). An increase in the amount of recombinant cells to 5 g/f resulted in a decrease in the time needed for completion of racemization to 5 h. On the other hand, more than lOOg/l of wet cells of L. brevis ATCC 8287 were required for the racemization of 100 g/l of L-Glu for about 10 h (2). As is the case with the glutamate racemase, L. brevis TABLE 1.
Glutamate decarboxylase activities in E. coli
Strain L. E. E. E. E.
brevis ATCC 8287 coli ATCC 11246 coli ATCC 13676 coli ATCC 21186 coli TM93
Glutamate decarboxylase activity units/g wet cells
relative activity
8 91 21 16 0
1 11.4 2.6 2 0
E. coli cells were grown at 30°C with shaking in LB medium overnight. L. brevis ATCC 8287 was cultured by the method reported previously (2). Cellular reactions were done in a reaction mixture (1 ml) consisting of 200 mM sodium acetate buffer, pH 4.2, 70 mM L-Glu, 2% (vol./vol.) toluene, and the cells. The reaction was carried out at 37°C for 30 min and the amount of L-Glu was measured as reported previously (2). One unit of enzyme activity was defined as the amount of enzyme which catalyzed the decarboxylation of 1 pmol of L-Glu in 1 min.
I
I
10
20
01
,
F 10
20
Reaction time (h) FIG. 3. Effects of L-Glu concentration (A) and the amount of E. coli TM93/pGARl cells (B) on the racemization of L-Glu. Cellular reactions were done by the methods described previously (2). (A) The reaction mixture consisted of 100 mM sodium phosphate buffer, pH 8.5, 2.5 g/l E. coli TM93/pGARl wet cells, L-Glu and 2% (vol./vol.) toluene. The concentrations of L-Glu were lOOg/l (0), 150 g/l (A), and 200 g/l ( 0 ), respectively. (B) The reaction mixture consisted of 100 mM sodium phosphate buffer, pH 8.5, 100 ~/IL-Glu, E. coli TM93/pGARl wet cells, and 2% (vol./vol.) toluene. The cell amounts were 0.5 g/l ( l ), 1 g/l ( 0 ), and 2.5 g/l (A), and 5 g/l (0), respectively. The reactions were done in 10 ml reaction mixture with shaking at 37°C.
ATCC 8287 is not suitable as a source of glutamate decarboxylase because of the difficulty in obtaining a large cell mass. Therefore, to establish an industrial production process of D-Glu, it is necessary to find a strain which efficiently produces a glutamate decarboxylase. We examined several E. coli strains known to be glutamate decarboxylase producers, and E. co/i ATCC 11246 was selected as a high producer of the enzyme (3, 4). The cellular glutamate decarboxylase activity of E. coli ATCC 11246 was 91 pmol/min/g wet cells, which is more than 11 times that of L. brevis ATCC 8287 (Table 1). Finally, we examined the production of D-Glu from LGlu by two successive cellular reactions with E. coli TM93/pGARl and E. coli ATCC 11246. The racemization reaction was carried out at pH 8.5, at which a glutamate racemase has its maximam activity and a glutamate decarboxylase is inactive. In a reaction system containing lOOg/l of L-Glu and 3 g/l of wet cells of E. coli TM93/pGARl, L-Glu was racemized completely and 5Og/Z of D-Glu was produced after 5 h. The pH of the reaction mixture was then shifted to 4.2, at which a glutamate decarboxylase is active and a glutamate racemase is inactive, and 5 g/l of wet cells of E. coli ATCC 11246 was added into the reaction mixture as a source of glutamate decarboxylase to convert L-Glu to GABA. L-Glu in the reaction mixture was then converted completely to GABA after 10 h and 50 g/l of D-Glu remained in the reaction mixture (Fig. 4). The production of D-Glu by a system employing a recombinant E. coli glutamate racemase and a E. coli glutamate decarboxylase has an advantage over the L. brevis system reported previously (2), in that the growth rate and yield of the cell mass of E. coli are much higher than those of L. brevis. The D-Glu production system reported here is the most efficient among the methods of D-Glu production so far reported (5-7), even though the
12
YAGASAKI ET AL.
J. FERMENT.BIOENG..
theoretical yield of D-Glu in the process cannot exceed SO%, because L-Glu, which is produced industrially by fermentation, is the cheapest starting material for the production of D-Glu and the process can be carried out as a one-pot reaction. REFERENCES
Reaction time (h) FIG. 4. Enzymatic production of D-Glu from L-Glu. E. coli TM93/pGARl was cultivated with shaking by the method described in the legend of Fig. 2. E. coli ATCC 11246 was cultivated in LB medium at 37°C overnight. The culture broth was centrifuged at 8,000 rpm at 4°C for 10 min. The wet cells so obtained was washed twice with 100 mM sodium phosphate buffer, pH 8.5, for E. coli TM93, and 200 mM sodium acetate buffer, pH 4.2, for E. coli ATCC 11246. Cellular reactions were done by the methods described previously (2). The reaction mixture (500 ml) consisted of 100 g/l L-Glu, 3 g/l E. coli TM93/pGARl wet cells, and 2% (vol./vol.) toluene; the pH of the reaction mixture was adjusted to 8.5 with sodium hydroxide. The reaction was carried out at 37°C for 5 h with agitation at 200 rpm and no aeration. After the pH was shifted to 4.2 with hydrochloric acid, 5 g/l E. coli ATCC 11246 wet cells was added to the reaction mixture as a source of glutamate decarboxylase. The reaction continued at 37°C for another 10 h, and the pH was kept between 4.0 and 4.5 with hydrochloric acid. Symbols indicate the amounts of L-Glu (0), D-Glu ( l), and GABA (A). The arrow indicates the time that the pH was shifted to 4.2.
1. Ozakl, A., Kawasaki, H., Yagasaki, M., and Hashimoto, Y.: Enzymatic production of D-alanine from DL-alaninamide by novel o-alaninamide specific amide hydrolase. Biosci. Biotech. Biochem., 56, 1980-1984 (1992). 2. Yagasaki, M., Ozaki, A., and Hashimoto, Y.: Enzymatic production of D-Glu from L-Glu by Lactobacillus brevis ATCC 8287. Biosci. Biotech. Biochem., 57, 1499-1502 (1993). 3. Najjar, V. A. and Fisher, J.: Glutamic acid decarboxylase from E. coli. Fed. Proc., 11, 264 (1952). 4. Najjar, V. A. and Fisher, J.: Studies on L-glutamic acid decarboxylase from Escherichiu coli. J. Biol. Chem., 206, 215219 (1954). 5. Yokozeki, K., Nakamori, S., Yamanaka, S., Eguchi, C., Mitsugi, K., and Yoshinaga, F.: Optimal conditions for the enzymatic production of D-amino acids from the corresponding 5-substituted hydantoins. Agric. Biol. Chem., 51, 715-719 (1987). 6. del Pozo, A.M., Merola, M., Ueno, H., Manning, J. M., Tanizawa, K., Nishimura, K., Soda, K., and Ringe, D.: Stereospecificity of reactions catalyzed by bacterial D-amino acid transaminase. J. Biol. Chem., 264, 17784-17789 (1989). 7. Yonaha, K. and Soda, K.: Applications of stereoselectivity of enzymes: synthesis of optically active amino acids and cu-hydroxy acids, and stereospecific isotope-labeling of amino acids, amines and coenzymes, p. 95-130. In Fiechter, A. (ed.), Advances in biochemical engineering and biotechnology, vol. 33. Springer-Verlag, Berlin (1986).