L-Tryptophan Production by Achromobacter liquidum

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APPLID AND NVIRONMNTAL MICROBIOLOGY, July 1983, P. 1-5 99-224/83/71-5$2./ Copyright 1983, American Society for Microbiology Vol. 46, No.

1 APPLID AND NVIRONMNTAL MICROBIOLOGY, July 1983, P /83/71-5$2./ Copyright 1983, American Society for Microbiology Vol. 46, No. 1 L-Tryptophan Production by Achromobacter liquidum TOSHIHIKO UJIMARU, TOSHIO KAKIMOTO,* AND ICHIRO CHIBATA Research Laboratory ofapplied Biochemistry, Tanabe Seiyaku Co., Ltd., Yodogawa-ku, Osaka 532, Japan Received 12 November 1982/Accepted 2 April 1983 Conditions for the production of tryptophanase from Achromobacter liquidum and for the conversion of L-serine and indole to L-tryptophan were studied. The enzyme could be produced in amnounts as great as.75 U/ml (degradation) and.294 U/ml (synthesis) by shaking cultures at 3 C in a medium containing dextrin, yeast extract, L-tryptophan, and L-glutamic acid. L-Tryptophan was produced most efficiently by shaking the cells at 37 C in a reaction mixture containing 6 mg of L-serine per ml, 6 mg of indole per ml, and.5 mm pyridoxal phosphate. After 3 days, 96 mg of L-tryptophan per ml was formed, and L-tryptophan was easily isolated to 85.4% yield by concentration of the reaction mixture. Tryptophanase is known as a pyridoxal phosphate-dependent (16), multifunctional enzyme which catalyzes the stoichiometric conversion of L-tryptophan to pyruvate, ammonia, and indole (equation 1) ahd catalyzes an a,,b-elimination reaction (equation 2) and a p-replacement reaction (equation 3) for several amino acids (11, 12): L-Tryptophan CH3COCOOH + NH3 + indole (1) RICH2-CH-COOH + H2 NH2 R1H + CH3COCOOH + NH3 (2) RICH2-CH-COOH + RIIH NH2 R1H + RI1CH2-CH-COOH NH2 (3) (Rj, -OH, -SH, -Cl, indolyl radical; RI,, indolyl radical). Since tryptophanase-forming microorganisms were found, a number of microbial production methods (9, 1, 15) have been investigated. No report, however, has appeared on the application of this enzyme for the production of L- tryptophan from L-serine and indole. In this paper, an enzymatic procedure which utilized tryptophanase for the production of L- tryptophan from L-serine and indole is presented. MATRIALS AND MTHODS Organisms. Achromobacter liquidum OUT 812 from the collection in this laboratory was selected for the fermentation experiments. 1 Fermentation. Unless otherwise noted, the medium contained 1% dextrin, 1% yeast extract,.2% L- tryptophan,.1% KH2PO4,.5% Na2HPO4 * 12H2, and.1% MgSO4 * 7H2. The medium was adjusted to ph 7. with NaOH, distributed in 2-ml amounts to 5-ml flasks, and sterilized. Culturing was carried out for 24 h at 3 C with reciprocal shaking (14 cpm, 8-cm stroke). Methods of analyses. The assay of tryptophanase activity was carried out by the method of Morino and Snell (8). Cells were harvested from the 2-ml culture broth, washed with distilled water, and suspended in 2 ml of cold water. The intact cells were used as the source of enzyme. The assay reaction mixture contained 8 mm L-tryptophan,.1 mm pyridoxal phosphate,.1 M potassium phosphate buffer (ph 9.),.5% polyoxyethylene-octylphenyl ether (OP-1), and.5 ml of cell suspension in.5 ml. The reaction mixture was incubated for 1 min at 37 C. The reaction was stopped by the addition of p-dimethylaminobenzaldehyde solution (14.7 g ofp-dimethylaminobenzaldehyde was dissolved in 948 ml of ethanol, and 52 ml of sulfuric acid was added). One unit of tryptophanase activity was defined as that amount which formed 1 pmol of indole per min under the conditions of the assay. Total enzyme activity, i.e., the amount of enzyme formed, was expressed in terms of units per milliliter of broth. L-Tryptophan or indole was determined by a modification of the method by Kupfer and Atkinson (6). L-Serine was determined by thin-layer chromatography-densitometry (solvent system, n-butanol-acetic acid-water [4:1:1]). For the estimation of organism growth, the culture broth was diluted with saline to 2 times the original volume, and the optical density was measured at 66 nm with a Hitachi photometer (model 1-12). Dried cell weight was estimated from a standard curve which correlates optical density to the weight of lyophilized cells. Production of L-tryptophan. Unless otherwise noted, L-tryptophan synthetic reactions were carried out by shaking (14 cpm, 3-cm stroke) in a reaction mixture containing 3 mg of L-serine per ml, 3 mg of indole per ml,.5 mm pyridoxal phosphate,.1 M potassium phosphate buffer, and harvested cells from 1 ml of fermentation broth in a total volume of 1 ml (ph 9.), Downloaded from on January 29, 219 by guest

2 2 UJIMARU, KAKIMOTO, AND CHIBATA which was incubated at 37 C for 3 days. Isolation of L-tryptophan. L-Tryptophan was isolated as follows. We diluted the reaction mixture (1 ml) 1 times to dissolve precipitated L-tryptophan, adjusted the ph to 6.5 with HCI, added 1% charcoal, heated the mixture at 8 C for 2 min, and filtered it. The filtrate was concentrated under reduced pressure until it was nearly dry. Crude crystals were collected by filtration and were recrystallized from water to give L-tryptophan. Chemicals. Unless otherwise specified, all chemicals were Katayama (Osaka) certified reagent grade. RSULTS Culture conditions for tryptophanase formation by A. liquidum. Tryptophanase formation has been observed for a number of microorganisms from such genera as scherichia, rwinia, and Proteus (14). Of the tested bacteria, A. liquidum possessed the highest activity and was used for subsequent experiments. To establish the most advantageous culture conditions for the formation of tryptophanase, various culture parameters were investigated. As it was generally noted that tryptophanase formation was subject to catabolite repression in the presence of glucose or another readily available carbon source (1, 5, 7), dextrin was selected as the carbon source for A. liquidum. The effect of dextrin concentration on tryptophanase formation in A. liquidum is shown in Fig. 1. A concentration of 1% dextrin yielded the highest ~~~~~~~~ I%.~~~~~~~~~~~ -C~~~~~~~~~~~~B c.d -, I-.. Yeast extract (Io) FIG. 2. ffect of yeast extract concentration on tryptophanase formation. Symbols:, growth;, tryptophanase activity. xcept for the concentration of yeast extract, all components in the medium were the same as described in the legend to Fig. 1. amount of enzyme formation (.1 U/ml). To select the best nitrogen source for growth and enzyme formation, we examined various 1- (D APPL. NVIRON. MICROBIOL. - L- - cq2. Downloaded from on January 29, 219 by guest 1 2 Dextrin (/) FIG. 1. ffect of dextrin concentration on tryptophanase formation. Symbols:, growth;, tryptophanase activity. In addition to the concentration of dextrin, all media contained 1% yeast extract,.2% L- tryptophan,.1% KH2PO4,.5% Na2HPO4 * 12H2, and.1% MgSO4 * 7H2. L-Tryptophan (/%) FIG. 3. ffect of L-tryptophan concentration on tryptophanase formation. Symbols:, growth;, tryptophanase activity. xcept for the concentration of L-tryptophan, all components in the medium were the same as described in the legend to Fig. 1.

VOL. 46, 1983 organic compounds. Although corn steep liquor, Casamino Acids, and peptone were effective for cell growth, tryptophanase activity on these nitrogen compounds was very low.

3 VOL. 46, 1983 organic compounds. Although corn steep liquor, Casamino Acids, and peptone were effective for cell growth, tryptophanase activity on these nitrogen compounds was very low. Yeast extract was much more effective for growth, and more than.1 U of tryptophanase per ml was obtained. The effect of yeast extract concentration was examined. A yeast extract concentration of 2% yielded the highest tryptophanase activity (.55 U/ml) (Fig. 2). As tryptophanase is an L-tryptophan-inducible enzyme, the optimal L-tryptophan concentration for A. liquidum was investigated. In A. liquidum, the amount of enzyme varied with the L-tryptophan concentration (Fig. 3). The most effective induction was observed at an L-tryptophan concentration of.2%. When the effects of various carboxylic acids on tryptophanase formation in A. liquidum were tested, L-glutamic acid was found to increase the activity. The enzyme activity rose to.73 U/ml in the presence of L-glutamic acid, whereas other mono- or dicarboxylic acids examined were less effective: acetic acid repressed the formation of the enzyme, oxalic acid and tartaric acid inhibited cell growth, and the other dicarboxylic acids of the tricarboxylic acid cycle slightly increased cell growth or enzyme activity or both (Table 1). To study the effect of L-glutamic acid in detail, we examined the formation of tryptophanase in medium containing L-glutamic acid-related compounds such as DL-a-aminobutyric acid, -y-aminobutyric acid, L-glutamine, or a-ketoglutaric acid. The addition of DL-a-aminobutyric acid was inhibitory (Table 2). -y-aminobutyric acid and L- glutamine were ineffective. oa-ketoglutaric acid and L-glutamic acid were effective in increasing the activity. A typical fermentation of A. liquidum was TABL 1. ffect of organic acids on tryptophanase formation Organic acida Growth (mg/ml) Total activity (U/ml) None Acetate Oxalate Malonate Succinate Fumarate Malate Tartarate Adipate Citrate L-Glutamate a All media contained the components described in the legend to Fig. 1 in addition to the above organic acids (.5%). L-TRYPTOPHAN PRODUCTION BY A. LIQUIDUM 3 TABL 2. ffect of L-glutamic acid-related compounds on tryptophanase formation Compounda Growth Activity (mg/ml) (U/ml) None DL-a-Aminobutyric acid y-aminobutyric acid L-Glutamic acid L-Glutamine a-ketoglutaric acid a L-Glutamic acid-related compounds were added at a concentration of.5%. xcept for the L-glutamic acid-related compounds, all components in the respective media were the same as described in the legend to Fig. 1. investigated under optimal conditions. A. liquidum was incubated at 3 C in a 5-ml flask containing 2 ml of medium consisting of 1% dextrin, 2% yeast extract,.2% L-tryptophan as an inducer,.1% KH2PO4,.5% Na2HPO4 * 12H2,.1% MgSO4 * 7H2, and.5% L-glutamic acid (ph 7.). Tryptophanase formation paralleled growth, the ph of the medium slowly rose, and maximal growth was attained after 22 h of incubation. Maximal tryptophanase activity (.75 U/ml) was obtained at 26 h. Conditions for production of L-tryptophan from L-serine and indole. For the production of L-tryptophan, L-tryptophan degradative and synthetic activities of A. liquidum tryptophanase were compared by using cells cultured under the optimal conditions described above. The highest degradative activity of L-tryptophan to pyruvate, ammonia, and indole was seen at ph 7.5 (1. U/ml), and the highest synthetic activity producing L-tryptophan from L-serine and indole was obtained at ph 9. (.294 U/ml) (Fig. 4). The highest synthetic activity (.833 U/ml) was obtained in a 1-min reaction at ph 9.. The enzyme, however, was almost inactivated after a few hours at 57 C, whereas it was stable at temperatures lower than 37 C for at least 3 days in the reaction mixture. Considering the heat stability of the enzyme, it is best to carry out the reaction at 37 C. The effect of substrate concentration was investigated by shaking the reaction mixture for 3 days. The maximal amount of L-tryptophan accumulated was 14 mg/ml in 3 days from 11 mg of L-serine per ml and 11 mg of indole per ml (Fig. 5). When such a high concentration of L- tryptophan accumulated, it precipitated in the reaction mixture without the addition of precipitant (9, 1). With the addition of 6 mg of L-serine per ml and 6 mg indole per ml, % mg of L-tryptophan per ml was produced in 3 days; the conversion Downloaded from on January 29, 219 by guest

4 4 UJIMARU, KAKIMOTO, AND CHIBATA APPL. NVIRON. MICROBIOL. 1* -3 c-r._ -, [.2 :L CL.. 6. I ph FIG. 4. ffect of ph on L-tryptophan degradative () and synthetic () activity. Synthetic activity was studied for 1 h. rate was 82.4% for L-serine and 92.4% for indole. L-Tryptophan production was enhanced 1.3-fold by the addition of pyridoxal phosphate at concentrations above.5 mm. L-Tryptophan production under optimal conditions. A typical L-tryptophan formation is illustrated in Fig. 6. The experiment was conducted L- o -J 15. 1! L-Serine or indole (mg/ml) FIG. 5. ffect of L-serine and indole concentrations on L-tryptophan production. Symbols:, amount of L-tryptophan formed in 1 day; A, amount of L-tryptophan formed in 3 days. 5- ~ o Reaction time (h) FIG. 6. Changes during enzyme reaction. The reaction mixture contained 6 mg of L-serine per ml, 6 mg of indole per ml, and.5 mm pyridoxal phosphate. Symbols:, L-tryptophan;, L-serine; A, indole. by incubating (at 37 C) a mixture containing cells obtained from 1 ml of cultured broth under optimal conditions as the enzyme source, 6 mg of L-serine per ml, 6 mg of indole per ml,.5 mm pyridoxal phosphate, and.1 M potassium phosphate buffer (ph 9.). The amount of L- tryptophan increased with the consumption of L- serine and indole. The reaction mixture was maintained at ph 9., and decomposition of accumulated L-tryptophan was not observed throughout the reaction. Isolation of L-tryptophan. Accumulated L- tryptophan was isolated by the method described above. From the reaction mixture containing 9.6 g of L-tryptophan, 8.2 g of pure crystals (yield, 85.4%) was obtained; the melting point was 266 C (decomposition); and [a]2d = (concentration = 1; H2). No other amino acids were detected by thin-layer chromatography. DISCUSSION Tryptophanase is an L-tryptophan-inducible (although a very few examples of constitutive tryptophanase have been found [2]), pyridoxalphosphate dependent enzyme which has been found in various bacteria. Since the studies by Hall et al. (4) and others (11, 12), it has been demonstrated that tryptophanase is a multifunc- Downloaded from on January 29, 219 by guest

5 VOL. 46, 1983 L-TRYPTOPHAN PRODUCTION BY A. LIQUIDUM 5 tional enzyme. This property of the enzyme provided the basis for the development of useful procedures for the microbial preparation of L- tryptophan. L-Tryptophan synthesis from pyruvate, ammonia, and indole by the reversal of the ca,o-elimination reaction has been clearly established by Nakazawa et al. (9, 1). Tryptophanase in A. liquidum OUT 812 was an inducible enzyme which was formed effectively in the presence of L-tryptophan (Fig. 3). In addition to the effect of inducer, yeast extract markedly increased the amount of the enzyme in A. liquidum. The amount of enzyme increased 5.5-fold with the addition of 2% yeast extract to the medium (Fig. 2). Besides this effect, we noted an effect of L-glutamic acid in A. liquidum. The amount of enzyme formed increased 1.3- fold with the addition of L-glutamic acid to the medium. This effect appeared to increase the specific activity of the enzyme rather than the total activity, whereas the addition of a-ketoglutaric acid increased the enzyme activity by increasing cell growth (Table 2). Such an effect of L-glutamic acid on microbial enzyme formation has also been reported in the case of aspartate-,- decarboxylase formation by Pseudomonas dacunhae or A. liquidum (13). As it was observed that the addition of a- ketoglutaric acid, malic acid, fumaric acid, or citric acid had little effect on increasing the amount of the enzyme among the tested L- glutamic acid-related compounds (Tables 1 and 2), we suggest that tricarboxylic acid cycle regulation might be closely involved in tryptophanase formation by A. liquidum. The concentrations of the substrates, L-serine and indole, had the most significant effects on the synthesis of L-tryptophan by A. liquidum tryptophanase through the a-replacement reaction (Fig. 5). The higher the concentrations of both substrates, the more L-tryptophan was produced. The synthesis appeared to depend more upon L-serine concentration than upon indole concentration. As we could obtain L-senne from inexpensive glycine (3), this method may provide a practical method for producing L-tryptophan. ACKNOWLDGMNTS We thank T. Shibatani and K. Nabe for their cooperation on the initial step of this study. LITRATUR CITD 1. Botsford, J. L., and R. D. DeMoss Catabolite repression of tryptophanase in scherichia coli. J. Bacteriol. 15: DeMoss, R. D., and K. Moser Tryptophanase in diverse bacterial species. J. Bacteriol. 98: ma, M., T. Kakimoto, and I. Chibata Production of L-serine by Sarcina albida. Appl. nviron. Microbiol. 37: Hall, A. N., J. A. Lesson, H. N. Rydon, and J. C. Tweedie The degradation of some Bz-substituted tryptophan by scherichia coli tryptophanase. Biochem. J. 74: Happold, F. C., and L. Hoyle The coli-tryptophanindole reaction. I. nzyme preparations and their action on tryptophan and some indole derivatives. Biochem. J. 29: Kupfer, D., and D.. Atkinson Quantitative method for determination of indole, tryptophan and anthranillic acid in the same aliquot. Anal. Chem. 8: Magasanilk, B., F. C. Neidhardt, and A. P. Levin Metabolic regulation of enzyme biosynthesis in bacteria, p In C. Ladd (ed.), Physiological adaptation. American Physiological Society, Washington, D.C. 8. Morino, Y., and.. Sneli Tryptophanase (. coli B). Methods nzymol. 17A: Nakazawa, H., H. nei, S. Okumura, and H. Yamada Synthesis of L-tryptophan from pyruvate, ammonia and indole. Agric. Biol. Chem. 36: Nakazawa, H., H. nel, S. Okumura, H. Yoshida, and H. Yamada nzymatic preparation of L-tryptophan and 5-hydroxy-L-tryptophan. FBS Lett. 25: Newton, W. A., Y. Morino, and.. Sneil Properties of crystalline tryptophanase. J. Biol. Chem. 24: Newton, W. A., and.. Snell Catalytic properties of tryptophanase, a multifunctional pyridoxal phosphate enzyme. Proc. Natl. Acad. Sci. U.S.A. 51: Shibatani, T., T. Kakimoto, and I. Chibata Stimulation of L-aspartate-3-decarboxylase formation by L-glutamate in Pseudomonas dacunhae and improved production of L-alanine. AppI. nviron. Microbiol. 38: Sneil, Tryptophanase: structure, catalytic properties, and mechanism of action. Adv. nzymol. 42: Watanabe, T., and.. Sneli Reversibility of the tryptophanase reaction: synthesis of tryptophan from indole, pyruvate and ammonia. Proc. NatI. Acad. Sci. U.S.A. 69: Wood, W. A., I. C. Gunsalus, and W. W. Umbrelt Function of pyridoxal phosphate: resolution and purification of the tryptophanase enzyme of scherichia coli. J. Biol. Chem. 17: Downloaded from on January 29, 219 by guest

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1981, p. 65-61 99-224/81/165-6$2./ Vol. 42, No. 4 Mechanism of L-Glutamine Production by an L-Glutamine- Producing Mutant of Flavobacterium rigense KOICHI NABE,