Mechanism of L-Glutamine Production by an L-Glutamine-

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1981, p /81/165-6$2./ Vol. 42, No. 4 Mechanism of L-Glutamine Production by an L-Glutamine- Producing Mutant of Flavobacterium rigense KOICHI NABE, SHIGEKI YAMADA,* AND ICHIRO CHIBATA Research Laboratory ofapplied Biochemistry, Tanabe Seiyaku Company, Ltd., , Kashima, Yodogawa-ku, Osaka, Japan Received 1 March 1981/Accepted 6 July 1981 Properties of some enzymes involved in L-glutamine biosynthesis in an L- glutamine-producing mutant of Flavobacterium rigense were examined. Glutamate-oxaloacetate transaminase in the mutant was nearly at the same level as that in the parent strain and was the most active among the enzymes participating in glutamate biosynthesis from a-ketoglutarate. Glutamine synthetase formation in the mutant was enhanced by increasing the concentration of (NH4)2-fumarate in the medium, but the activity of this enzyme in the parent strain was very low, and its formation was not influenced by the concentration of (NH4)2-fumarate. Glutaminase formation by both strains was similar and was not influenced by the levels of (NH4)2-fumarate. Glutaminase activity of the mutant was inhibited by ammonia and fumarate. Intracellular amino acids and extracellular free amino acids in the mutant were compared with those of the parent strain. It seems reasonable to conclude that L-glutamine leaks out specifically through the cell membrane of strain 73 and that this specific excretion of L-glutamine probably allows a continuous conversion of L-glutamate to L-glutamine inside the cell. As described in previous papers (6, 7), we found that a penicillin-resistant mutant of Flavobacterium rigense, strain 73, produces 25 g of L-glutamine per liter in a culture medium containing mainly 5% glucose and 7% (NH4)2- fumarate. The existence of.9 to 1.6% ammonia and 5.5% fumaric acid in a medium is essential for the accumulation of large amounts of L-glutamine. The fumarate dependence of L-glutamine productivity was a unique characteristic of our strain. Many studies on microbial production of L-glutamine have exclusively used the socalled general group of glutamic acid-producing bacteria (14), including Corynebacterium sp. (8-1), Brevibacterium sp. (15, 16), Micrococcus sp. (11), and others. These bacteria are known to accumulate large amounts of L-glutamine when ammonium ion is supplied in excess, as compared with the minimum requirement for maximum growth or large production of L-glutamic acid, and the ph of the culture broth is maintained at an acidic value (5.5). So far, no report has described such an effect of fumarate on L- glutamine production. Therefore, it was of interest to investigate the mechanism by which large amounts of L-glutamine can be accumulated with F. rigense strain 73 in the presence of excess fumarate. To elucidate the mechanism by which L-glutamine accumulates in the presence of fumarate, 65 some enzyme activities involved in L-glutamine metabolism were examined in cell-free extracts of strain 73, which was cultured in the presence of fumarate. These enzyme activities were compared with the activity of the parent strain, which has no L-glutamine-producing ability. MATERIALS AND METHODS Microorganisms and cultivation methods. A penicillin-resistant mutant of F. rigense, strain 73 (FERM-P no. 3628) (18), and the parent strain (FERM-P no. 3556) (17) were used. The conditions for stock culture were described previously (6). L-Glutamine production medium (basal medium) contained 5% glucose, 1.5% yeast extract, 7% (NH4)2-fumarate,.1% KH2PO4,.1% K2HPO4,.5% MgSO4. 7H2, and 1% CaCO3 (adjusted to ph 6.4 with KOH). The concentration of (NH4)2-fumarate in the basal medium was changed according to the experimental conditions. Cultivation was carried out in a 5-ml flask containing 5 ml of medium for 48 h at 3 C on a reciprocal shaker operating at 14 strokes per min with a 7-cm amplitude. Cell growth was determined as described previously (6). Analytical methods. Unless otherwise stated, analytical methods were the same as described previously (6, 7). Preparation of cell-free extract. Culture broth (5 ml), cultivated for 48 h at 3 C, was centrifuged at 1, x g for 1 min at 5 C. The cells were washed twice with.9% NaCl, suspended in 1 ml of.2 M phosphate buffer (ph 7.), and disrupted by an ultra- Downloaded from on July 24, 218 by guest

2 66 NABE, YAMADA, AND CHIBATA sonic oscillator. The homogenate was centrifuged at 2, x g for 2 min at 5 C. The clear supernatant was used for the enzyme assay as cell-free extracts (about 6 mg of protein per ml). Assay methods. Protein was determined by the method of Lowry et al. (3) with bovine serum albumin as the standard. Unless otherwise stated, 1 U of enzyme activity was defined as that activity which converted 1 jtmol of substrate to product per min under the conditions of the assay. Total enzyme activity was expressed as units per total weight of cells in 1 ml of broth, and specific activity was expressed as units per milligram of dried cells. (i) Glutamine synthesis. Glutamine synthetase was assayed by measuring the formation of y-glutamyl hydroxamate by the method of Rowe et al. (12). This assay measures the forward reaction of the enzyme to form L-glutamine from L-glutamate and ammonia in the presence of adenosine triphosphate, and the enzyme forms y-glutamyl hydroxamate when ammonia is replaced by hydroxylamine. The reaction mixture (1. ml) contained 8 mm imidazole hydrochloride buffer (ph 7.2), 1 mm MgCl2, 1 mm,8-mercaptoethanol, 1 mm glutamate, 1 mm hydroxylamine, 1 mm adenosine triphosphate, and.25 ml of cell-free extract. The reaction was carried out at 3 C, and was stopped by the addition of 1.5 ml of ferric chloride reagent. After centrifugation, the absorbance at 535 nm was read. (ii) Glutaminase. Glutaminase activity was determined by the procedure of Tsunoda et al. (15), except that cell-free extract was substituted for cell suspension. (iii) Glutamate dehydrogenase. Glutamate dehydrogenase was assayed by determining the rate of glutamate formation from a-ketoglutarate by the method of Takahashi et al. (13). We determined the amount of glutamate formation manometrically by using acetone-dried cells of Escherichia coli Crookes strain 8739 as a specific L-glutamate decarboxylase preparation (16). (iv) Glutamate-oxaloacetate transaminase. Glutamate-oxaloacetate transaminase was assayed by determining the rate of glutamate formation from a- ketoglutarate and L-aspartate by the method of Takahashi et al. (13). The amount of glutamate formed was determined by the above-mentioned method. (v) Glutamate-pyruvate transaminase. Glutamate-pyruvate transaminase was also measured by determining the rate of glutamate formation from a- ketoglutarate and L-alanine by a modification of the method of Takahashi et al. (13). The amount of glutamate formed was determined by the above-mentioned method. (vi) Isocitrate dehydrogenase. Isocitrate dehydrogenase was assayed by monitoring nicotinamide adenine dinucleotide reduction at 34 nm by the method of Ferguson and Sims (2). (vii) a-ketoglutarate dehydrogenase. a-ketoglutarate dehydrogenase activity was measured by the method of Massey (5). (viii) Succinate dehydrogenase. Succinate dehydrogenase activity was determined by the method of Bernath and Singer (1). APPL. ENVIRON. MICROBIOL. (ix) Fumarate hydratase. Fumarate hydratase activity was determined by the method of Massey (4). Analyses of intracellular free amino acids. Preparation of intracellular free amino acid pool samples was as follows. Samples of 5-ml volumes were removed from the culture incubated for 48 h and immediately centrifuged. After the cells were washed twice with.9% NaCl, they were suspended in 5 ml of distilled water and placed in a boiling-water bath for 3 min. Cell debris was separated from the boiledwater extract by centrifugation. After appropriate dilution with.2 N HCl, the amount of amino acid extracted was analyzed by a Hitachi model 835 amino acid analyzer. The analysis of the extracellular free amino acids was done on the same analyzer. Results were expressed as micromoles per gram of cells. From these values, the concentrations of the intracellular amino acids in the native cells were calculated with the assumption that the cell was 8% water in the viable state. RESULTS Formation of enzymes involved in glutamate biosynthesis. The activities of four enzymes in the tricarboxylic acid cycle and three enzymes involved in glutamate biosynthesis from a-ketoglutarate were examined with the extracts prepared from F. rigense strain 73 and the parent cells grown in the basal medium. With respect to four enzymes in the tricarboxylic acid cycle, i.e., isocitrate dehydrogenase, a- ketoglutarate dehydrogenase, succinate dehydrogenase, and fumarate hydratase, there were no differences between strain 73 and the parent strain. No significant differences were observed among the three enzymes of glutamate biosynthesis in strain 73 and those in the parent strain (Table 1). Glutamate-oxaloacetate transaminase activities of strain 73 and of the parent strain were higher than glutamate dehydrogenase activities. Although the data are not included in Table 1, these enzyme activities were not markedly influenced by the concentration of fumarate in the medium. Formation of glutamine synthetase. It is well known that L-glutamine is formed from glutamate by the action of glutamine synthetase. Our previous experiment (7) showed that L-glutamine production by F. rigense strain 73 is enhanced by increasing the concentration of (NH4)2-fumarate in media. Therefore, the effect of the initial concentration of (NH4)2-fumarate in culture medium on glutamine synthetase formation was investigated with both strains. The relationships between the concentration of fumarate in the medium and glutamine synthetase activity of the cells grown in that medium are shown in Fig. 1. The total and specific activities of strain 73 were enhanced by increasing the concentration of fumarate. The activity of the Downloaded from on July 24, 218 by guest

3 VOL. 42, 1981 TABLE 1. Some enzyme activities involved in glutamate biosynthesis by F. rigensea Sp act (U/mg of dried celis) Enzyme Strain 73 Parent strain Glutamate dehydrogenase 1.4 x 1O x 1o-3 Glutamate-pyruvate trans- 9.7 x 1O x 1O-4 aminase Glutamate-oxaloacetate 9.8 x 1O x 1O-3 transaminase a Cultivation was carried out for 48 h at 3C with the basal medium. 2r r- - 11)) "I,, E E E 2i _ I. R -5 CV (NH4)2- fumarate (%) FIG. 1. Effect of (NH4)2-fumarate concentration in a medium on glutamine synthetase formation by F. rigense strain 73 and parent strain. Symbols:, total activity of strain 73;, specific activity of strain 73; O1, cell growth of strian 73; A, total activity ofthe parent strain; A, specific activity of the parent strain; A, cell growth of the parent strain. parent strain was very low (about.1 to.2 U/mg of dried cells) and was not influenced by the concentration of fumarate. Formation of glutaminase. The activity of glutaminase catalyzing the degradation of L-glutamine was assayed with the extracts prepared from cells of both strains grown at various concentrations of fumarate. The specific activities of both strains were similar and were not influenced by the levels of (NH4)2-fumarate (Fig. 2). However, total activity of strain 73 was decreased by increasing the concentration of fumarate. Properties of glutaminase. (i) Effect of ammonia. It has been shown that the glutaminase of the general group of glutamic acid-producing bacteria is inhibited by ammonia. Accordingly, the inhibition of glutaminase from F. rigense strain 73 by ammonia was investigated. As a result, the enzyme activity was reduced by L-GLUTAMINE PRODUCTION BY F. RIGENSE 67 4 _' 3 >1,5 2 C._ -5 * - A - -- s~~~~~~ - 2. E 1.5 O~ 77 or 59% in the presence of.1 or.2 M ammonia, respectively. (ii) Effect of fumarate. Our previous fermentation experiments (7) showed that some organic acids are effective for the production of L-glutamine. Therefore, the effect of several organic acids on glutaminase activity in cell-free extract from strain 73 was investigated (Fig. 3). Acetic acid and tartaric acid had no measurable effect on the enzyme activity, whereas fumaric acid and succinic acid significantly inhibited the activity. When (NH4)2-fumarate was added to the re-.s- 1. (- xa) Q. ( (NH4)2- fumarate (%) FIG. 2. Effect of (NH4)2-fumarate concentration in a medium on glutaminase formation by F. rigense strain 73. Symbols:, total activity of strain 73;, specific activity of strain 73; A, total activity of the parent strain; A, specific activity of the parent strain A --~~~~~~~~ 6 2 z 2_\ Concentration (%) FIG. 3. Effect of organic acid on glutaminase activity of F. rigense strain 73. Symbols:, acetate; A, fumarate; V; malate;, succinate; *, tartarate;, citrate. Downloaded from on July 24, 218 by guest

4 68 NABE, YAMADA, AND CHIBATA action mixture, the highest inhibition was observed (Fig. 4). (iii) Optimum ph. The effect of ph on the enzyme activity was examined. Maximum activity was obtained at ph 8.5 (Fig. 5). There was very little activity over the range of ph 6 to 6.5, which was the ph of the medium during the fermentation. Composition of intracellular amino acid pools and extracellular accumulation of L- glutamine. With the cells grown in the medium containing 4 or 7% (NH4)2-fumarate, the intracellular amino acid pools and the extracellular free amino acids of F. rigense strain 73 were compared with those of the parent strain (Tables 2 and 3). The total amount of the intracellular amino acids as a whole and the amount of intracellular L-glutamine in strain 73 were less than those in the parent strain (Table 2). Significant effects of the concentration of (NH4)2-fumarate were not observed with either strain, except for the increase in the intracellular glutamine level in the parent strain. In strain 73 and the parent strain, the pool of glutamate plus glutamine was approximately 8% of the total pool. On the contrary, the amounts of extracellular glutamate and L-glutamine in strain 73, especially L-glutamine, were higher than those in the parent strain, and these were dramatically increased when the concentration of (NH4)2-fumarate was increased from 4 to 7%. DISCUSSION For enzyme activities involved in glutamate formation, no significant difference was observed between strain 73 and the parent strain. Glutamate-oxaloacetate transaminase showed the > 6 - c, n (N H4)2- fumorate (%) FIG. 4. Effect of (NH4)2-fumarate concentration on glutaminase activity of F. rigense strain ci) I1) 14 APPL. IENVIRON. MICROBIOL ph FIG. 5. Optimum ph ofglutaminase activity. highest activity among the enzymes tested. These data indicate that there are no differences in the biosynthetic route between strain 73 and the parent strain until glutamate is formed through the tricarboxylic acid cycle and glutamate-oxaloacetate transaminase, which plays a leading role in the biosynthetic route. Because it is generally accepted that glutamate dehydrogenase is a dominant enzyme in the glutamate biosynthetic pathway in the so-called glutamic acid-producing bacteria, high glutamate-oxaloacetate transaminase activity in F. rigense is noteworthy. Glutamine synthetase formation in strain 73, but not in the parent strain, was enhanced both in total and specific activities by increasing the concentration of (NH4)2-fumarate in the medium. Although glutaminase formation in the parent strain was not affected by (NH4)2- fumarate in the medium, the formation of this enzyme in strain 73 was reduced in proportion to the decrease of the cell growth because of increasing the concentration of fumarate. Strain 73 seems to be sensitive to (NH4)2-fumarate, and this may be attributable to characteristics of the cell membrane of strain 73. Glutaminase activity in the cell-free extract of strain 73 was inhibited not only by fumaric acid but also by ammonia in the reaction mixture. Display of the enzyme activity was also inhibited at low ph values, such as those of the fermentation broth. These results accounted for the effect of (NH4)2- fumarate on L-glutamine productivity in our previous fermentation experiments, in which addition of 7% (NH4)2-fumarate in the medium was essential for the accumulation of large amounts of extracellular L-glutamine by F. rigense strain 73. It seems that glutamate formed from a- ketoglutarate in the tricarboxylic acid cycle is rapidly converted to L-glutamine by the in- Downloaded from on July 24, 218 by guest

5 VOL. 42, 1981 TABLE 2. L-GLUTAMINE PRODUCTION BY F. RIGENSE 69 Composition of the intracellular free amino acids in F. rigense strain 73 and the parent cells grown in a medium containing 4 or 7% (NH4)2-fumarate Amino acid compositiona at following (NH4)2-fumarate concn (%): Amino acid Strain 73 Parent strain Alanine 6.11 (1.52)b 1.8 (2.7) 6.41 (1.6) (2.82) Aspartic acid (3.11) 3.45 (.86) (6.4) 34.6 (8.65) Glutamic acid (65.83) (51.23) (1.87) (75.95) Glutamine (3.13) 8.77 (2.19) 27.9 (6.77) (14.7) Lysine 7.83 (1.95) 6.58 (1.64) (2.9) 1.67 (2.66) Phenylalanine 7.53 (1.88) 2.24 (.56) 8.71 (2.17) 8.65 (2.16) Valine (2.83) 6.77 (1.69) (6.13) (3.1) Others' NDd (.) to 4.1 ND (.) to 3.4 ND (.) to 5.4 ND (.) to 7.2 (1.2) (.85) (1.35) (1.75) Sum of others 23.4 (5.71) (4.57) 22.2 (5.62) 2.79 (5.16) "Results are expressed as micromoles of amino acid per gram of dried cells. "Numbers in parentheses indicate the concentration of amino acids in living cells expressed as micromoles per milliliter on the assumption that the cells contain 8% water in the viable state. carginine, cysteine, glycine, histidine, isoleucine, leucine, methionine, proline, serine, threonine, and tyrosine. d ND, Not detectable (less than.1 pmol/g of dried cells). TABLE 3. Composition of the extracellular amino acids in F. rigense strain 73 and the parent strain Amino acid composition' at following (NH4)2-fumarate concn (%): Amino acid Strain 73 Parent strain Aspartic acid Glutamic acid NDb ND Glutamine Othersc ND to.25 ND to.86 ND to.34 ND to.34 aresults are expressed as micromoles of amino acid per milliliter of culture broth without cells. 'ND, Not detectable (less than.5 pmol/ml of culture broth without cells). ' Alanine, arginine, cysteine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine. creased glutamine synthetase, and the reverse reaction by glutaminase is markedly inhibited by the presence of (NH4)2-fumarate so that large amounts of L-glutamine are accumulated in the culture broth. With the assumption that the metabolizing cells contained about 8% water, the concentration of the intracellular glutamine was calculated and expressed as micromoles per milliliter (Table 2). When these values were compared with the concentrations of the extracellular amino acids, it was clear that the efflux of amino acids from metabolizing cells did not occur by simple diffusion. Furthermore, the concentration of extracellular L-glutamine of strain 73 was extremely high and that of the parent strain was very low, although intracellular L-glutamine in strain 73 was less than that in the parent strain. Although there is no direct evidence, the most acceptable explanation is that a penicillin-resistant mutant, strain 73, possesses specific permeability for L-glutamine efflux. It is reasonable to assume that glutamate is forned from a-ketoglutarate mainly by glutamate-oxaloacetate transaminase inside the cells, and it is rapidly converted to L-glutamine by glutamine synthetase enhanced by the presence of (NH4)2-fumarate. Subsequently, L-glutamine leaks out specifically through the cell membrane of strain 73, and a large amount of L-glutamine is accumulated in the extracellular medium. The specific excretion of L-glutamine probably allows a continuous conversion of L-glutamate to L- glutamine inside the cell. ACKNOWLEDGMENT The expert technical assistance of J. Ozaki is gratefully acknowledged. LITERATURE CIED 1. Bernath, P., and T. P. Singer Succinic dehydrogenase. Methods Enzymol. 5: Ferguson, A. R., and A. P. Sims The regulation of glutamine metabolism in Candida utilis: the inactivation of glutamine synthetase. J. Gen. Microbiol. 8: Downloaded from on July 24, 218 by guest

6 61 NABE, YAMADA, AND CHIBATA 3. Lowry,. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Massey, V Fumarase. Methods Enzymol. 1: Massey, V The composition of the ketoglutarate dehydrogenase complex. Biochim. Biophys. Acta 38: Nabe, K., T. Ujimaru, N. Izuo, S. Yamada, and I. Chibata Production of L-glutamine by a penicillin-resistant mutant of Flavobacterium rigense. Appl. Environ. Microbiol. 4: Nabe, K., T. Ujimaru, S. Yamada, and L. Chibata Influence of nutritional conditions on production of L-glutamine by Flavobacterium rigense. Appl. Environ. Microbiol. 41: Nakanishi, T Effects of inorganic ions, on the conversion of L-glutamic acid fermentation to L-glutamine fermentation by Corynebacterium glutamicum. J. Ferment. Technol. 53: Nakanishi, T Roles of ammonium and chloride ions in the conversion of L-glutamic acid fermentation to L-glutamine and N-acetyl-L-glutamine fermentation by Corynebacterium glutamicum. J. Ferment. Technol. 56: Nakanishi, T., J. Nakajima, and K. Kanda Conditions for conversion of L-glutamic acid fermentation by Corynebacterium glutamicum into L-glutamine production. J. Ferment. Technol. 53: Oshima, K., K. Tanaka, and S. Kinoshita Studies on L-glutamic acid fermentation. The conversion of L-glutamic acid fermentation to L-glutamine fermenta- APPL. ENVIRON. MICROBIOL. tion. Amino Acids 7: Rowe, W. B., R. A. Ronzio, A. P. Wellner, and A. Meister Glutamine synthetase (sheep brain). Methods Enzymol. 17: Takahashi, H., M. Shibukawa, T. Ohsawa, and K. Miyai On biochemical characteristics of various bacteria pertaining to L-glutamate production. I. Comparison between L-glutamate producing bacteria and L-glutamate non-producing bacteria. Agric. Chem. Soc. Jpn. 41: Takayama, K., S. Abe, and S. Kinoshita Taxonomical studies on glutamic acid producing bacteria. II. Classification of glutamic acid producing bacteria. Agric. Chem. Soc. Jpn. 39: Tsunoda, T., T. Tsuchiya, K. Kinoshita, and A. Kawamoto Studies on the accumulation of L-glutamine in glutamic acid fermentation. II. The mechanism of L-glutamine accumulation. Agric. Chem. Soc. Jpn. 35: Tsunoda, T., T. Tsuchiya, H. Okada, K. Kinoshita, and A. Kawamoto Studies on the accumulation of L-glutamine in L-glutamic acid fermentation. I. Confirmation and determination of L-glutamnine. Agric. Chem. Soc. Jpn. 35: Yamada, S., K. Nabe, T. Ujimaru, N. Izuo, and L Chibata Extracellular accumulation of a new amino acid, -2-hydroxypropylhomoserine, from 1,2- propanediol by Flavobacterium rigense. Appl. Environ. Microbiol. 35: Yamada, S., K. Nabe, T. Ujimaru, N. Izuo, and L Chibata L-Glutamine formation by Flavobacterium rigense. Appl. Environ. Microbiol. 37: Downloaded from on July 24, 218 by guest